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Retinitis pigmentosa (RP) is a group of inherited retinal disorders that lead to photoreceptor loss. RP has been reported to be related to oxidative stress, autophagy, and inflammation. (-)-Epigallocatechin gallate (EGCG), the most abundant catechin-based flavonoid in green tea leaves, has significant antioxidant, anti-carcinogenic, antimicrobial, and neuroprotective properties. EGCG, given its low molecular weight and hydrophilic properties, can cross the blood-retinal barrier and is able to reach different ocular tissues such as the lens, cornea, and retina. EGCG has been shown to provide retinal protection against ischemia; sodium nitroprusside-, N-methyl-D-aspartate-, lipopolysaccharide-, light-, sodium iodate-, or H2O2-induced damage and diabetic retinopathy. This suggests that systemic EGCG administration has the potential to protect against retinal degenerative or neurodegenerative diseases such as RP. The aim of this work was to investigate whether EGCG can protect against RP progression in the animal P23H line 1, the model of RP. Albino P23H rats were crossed with pigmented Long Evans rats to produce offspring exhibiting the clinical features of RP. Pigmented P23H rats were treated via intraperitoneal injection with saline or EGCG at a dose of 25 mg/kg every week from P100 to P160 and then compared to wild-type Long Evans rats. Rats treated with EGCG showed better visual and retinal electrical function with increased contrast sensitivity and b-wave values compared with those observed in P23H rats treated with vehicle. EGCG reduced lipid peroxidation and increased total antioxidant capacity and catalase and superoxide dismutase activities. No differences were observed in visual acuity, nitrate levels, nitrite levels or glutathione S-transferase activity. In conclusion, EGCG not only reduced the loss of visual function in P23H rats but also improved the levels of antioxidant enzymes and reduced oxidative damage. This study was approved by the Institutional Animal Care and Use Committee (CEICA) from the University of Zaragoza under project license PI12/14 on July 11, 2014. This study was supported by the Instituto de Salud Carlos III (project PI13/01124 and Ocular Pathology National Net RETICS-Oftared RD16/ 0008) the Fondo Europeo de Desarrollo Regional (FEDER) funds: “Una manera de hacer Europa”; and the Government of Aragon (Group B08_17R and Predoctoral Grant L. Perdices, C060/2014).

The generation of functional regulatory T cells (Tregs) is essential to keep tissue homeostasis and restore healthy immune responses in many biological and inflammatory contexts. Cannabinoids have been pointed out as potential therapeutic tools for several diseases. Dendritic cells (DCs) express the endocannabinoid system, including the cannabinoid receptors CB1 and CB2. However, how cannabinoids might regulate functional properties of DCs is not completely understood. We uncover that the triggering of cannabinoid receptors promote human tolerogenic DCs that are able to prime functional FOXP3+ Tregs in the context of different inflammatory diseases. Mechanistically, cannabinoids imprint tolerogenicity in human DCs by inhibiting NF-κB, MAPK and mTOR signalling pathways while inducing AMPK and functional autophagy flux via CB1- and PPARα-mediated activation, which drives metabolic rewiring towards increased mitochondrial activity and oxidative phosphorylation. Cannabinoids exhibit in vivo protective and anti-inflammatory effects in LPS-induced sepsis and also promote the generation of FOXP3+ Tregs. In addition, immediate anaphylactic reactions are decreased in peanut allergic mice and the generation of allergen-specific FOXP3+ Tregs are promoted, demonstrating that these immunomodulatory effects take place in both type 1- and type 2-mediated inflammatory diseases. Our findings might open new avenues for novel cannabinoid-based interventions in different inflammatory and immune-mediated diseases.

Neuroinflammation is a process related to the onset of neurodegenerative diseases; one of the hallmarks of this process is microglial reactivation and the secretion by these cells of proinflammatory cytokines such as TNFα. Numerous studies report the relationship between neuroinflammatory processes and exposure to anthropogenic air pollutants, but few refer to natural pollutants. Volcanoes are highly inhabited natural sources of environmental pollution that induce changes in the nervous system, such as reactive astrogliosis or the blood-brain barrier breakdown in exposed individuals; however, no neuroinflammatory event has been yet defined. To this purpose, we studied resting microglia, reactive microglia, and TNFα production in the brains of mice chronically exposed to an active volcanic environment on the island of São Miguel (Azores, Portugal). For the first time, we demonstrate a proliferation of microglial cells and an increase in reactive microglia, as well an increase in TNFα secretion, in the central nervous system of individuals exposed to volcanogenic pollutants. This research was supported by the Universidad de Alicante VIGROB-186.

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes.

The anatomical and histological features of the foveae in a gull, Larus michahellis, were investigated combining immunofluorescence and transmission electron microscopy. In an area of higher acuity, which extends in a band across the field of view, a central convexiclivate deep fovea was observed near of the pecten oculi. Moreover, a shallow fovea was present in the dorsonasal region. The central deep fovea was characterized by a remarkably and well-excavated pit containing extremely thin cones and a radial displacement of the cone axons and inner retinal layers. Immunofluorescence demonstrated the absence of rods and the presence of violet and green/red sensitive cone opsins. Ultrastructural analyses confirmed the lack of double cones, and specialized Müller cell processes forming a network that stabilizes the foveal structure. This deep central fovea, which provides the highest resolution and colour discrimination, may be used for monocular sideways vision of distant objects and a binocular fixation. The dorsonasal fovea was shallower and all retinal layers were present at the pit. This fovea showed the same composition of photoreceptors than central fovea and could be implied in magnification of a wide monocular part of the retinal image. The bifoveate condition of yellow-legged gull retina would allow the formation of three separate and distinct visual fields: two wide lateral monocular fields mediated by the dorsonasal and the central foveae, and a small binocular field mediated by the central deep fovea depending on ocular movements. This research was supported by the University of Alicante VIGROB-186.

Spermatozoa capacitation is a time-dependent physiological process essential for acquiring the fertilizing capacity. In this context, reorganization of spermatozoa surface sugars and tyrosine phosphorylation are some of the most important biochemical changes involved in capacitation. However, the relationship between these 2 biomarkers remains poorly defined. By cytofluorescence we simultaneously characterized the head concanavalin A (ConA)-binding sites and the flagellar tyrosine phosphorylation before capacitation, during different capacitation times (1 and 4 h), and after acrosome reaction induction in human spermatozoa. The results showed a strong connection between ConA-label patterns and tyrosine phosphorylation according to the spermatozoa capacitation time and acrosomal status. Specifically, the spermatozoa subpopulation with phosphotyrosine presented proper sugar location (label in acrosomal and postacrosomal region) just after 1 h of capacitation, while cells without phosphotyrosine needed 4 h to do it. Moreover, after induction of spermatozoa acrosome reaction, phosphorylation was significantly correlated (p < 0.05) with the relocation of ConA-binding residues to the equatorial region, regardless of capacitation time. Overall, these observations provide novel insights regarding spermatozoa subpopulations based on essential physiological events like capacitation and acrosome reaction, which could have potential implications in the improvement of spermatozoa selection techniques. This research was funded by the Human Fertility Cathedra of the University of Alicante and the VIGROB-186.

The health effects of mercury vapor exposure on the brain in volcanic areas have not been previously addressed in the literature. However, 10% of the worldwide population inhabits in the vicinity of an active volcano, which are natural sources of elemental mercury emission. To evaluate the presence of mercury compounds in the brain after chronic exposure to volcanogenic mercury vapor, a histochemical study, using autometallographic silver, was carried out to compare the brain of mice chronically exposed to an active volcanic environment (Furnas village, Azores, Portugal) with those not exposed (Rabo de Peixe village, Azores, Portugal). Results demonstrated several mercury deposits in blood vessels, white matter and some cells of the hippocampus in the brain of chronically exposed mice. Our results highlight that chronic exposure to an active volcanic environment results in brain mercury accumulation, raising an alert regarding potential human health risks. These findings support the hypothesis that mercury exposure can be a risk factor in causing neurodegenerative diseases in the inhabitants of volcanically active areas. info:eu-repo/semantics/publishedVersion

Los Platyhelminthes, comúnmente llamados gusanos planos, es un filo de animales que comprende especies de vida libre (como turbelarios o planarias) y parásitos de gran importancia en medicina o veterinaria (duelas, tenias o monogéneos). En investigación, el microturbalario marino Macrostomum lignano (Platyhelminthes, Rhabditophora) es un modelo emergente de laboratorio de fácil cultivo y manejo, de genoma secuenciado y múltiples herramientas moleculares al alcance para su estudio. Se trata de un organismo bilateral basal con un verdadero Sistema Nervioso Central, el cual consiste en un cerebro compartimentado y dos cordones nerviosos principales laterales. Aquí, se presentan por primera vez los tipos celulares, la ultraestructura y los modelos tridimensionales de las células que forman parte del complejo glandular frontal de la especie. Esto es, el conjunto de receptores sensoriales que conectan anteriormente con el cerebro y las glándulas que lo atraviesan. Se reportan dos nuevas células glandulares; un modelo 3D de su fotorreceptor ciliado; la localización de los cuerpos celulares de los receptores sensoriales en el córtex cerebral; y el lugar donde se establece su límite superior. Se describe también la ultraestructura de las neuronas del córtex cerebral; sus sinapsis; el crecimiento del cerebro; y los procesos de neurogénesis que tienen lugar durante el desarrollo postembrionario a partir de células madre llamadas neoblastos.

Summary RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations—revealed by RNA structural probing—indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.

Heavy metals are endocrine disruptors which interfere with processes mediated by endogenous hormones of the organism, negatively affecting endocrine functions. Some studies have correlated heavy metal exposure with male infertility. However, the number of studies conducted on humans are limited. Therefore, the aim of this study is to summarize the current knowledge on how heavy metals influence human male fertility. Hence, three distinct databases were consulted—PubMed, Scopus and Web of Science—using single keywords and combinations of them. The total number of identified articles was 636. Nevertheless, by using the inclusion and exclusion criteria, 144 articles were finally included in this work. Results display that the development of adequate instruments for heavy metal assessment may play an important function in human male fertility diagnosis and treatment. Furthermore, clinical trials could be useful to confirm the role of heavy metals in human male fertility diagnosis. Overall, further research is required to fully understand the molecular and cellular basis of the influence of environmental and occupational exposure to heavy metals on human male infertility and reproductive outcomes. This study was funded by Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (UGP-19-042 and UGP-20-039).

Xylella fastidiosa releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of X. fastidiosa OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce’s disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce X. fastidiosa diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF (X. fastidiosa DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of X. fastidiosa OMVs in intercellular signaling and host–pathogen interactions.

Pregnancy is a unique immunological situation in which a fetus-bearing paternal histocompatibility antigens can survive in a maternal environment without apparent rejection. To face this challenge, cells of the uterine immune system show characteristic changes in absolute number and composition during pregnancy. Particularly relevant to this process are uterine natural killer (uNK) cells and their cell surface receptors, killer immunoglobulin-like receptors (KIRs). The main purpose of this review is to outline the current body of knowledge on the involvement of KIRs in the complications of pregnancy. Implantation depends on the invasion of embryonic trophoblast cells into maternal uterine tissue and remodeling of the uterine spiral arterioles, which is essential for placental perfusion and successful pregnancy. The proper interaction between maternal KIRs and their ligands human leukocyte antigen (HLA) class I molecules, expressed by the extravillous trophoblast cells, is crucial in this process. KIRs are a complex family that includes both activator and inhibitory receptors. The activation profile is genetically determined in each individual and leads to diverse levels of functionality for NK and T cells on engagement with specific HLA class I molecules. An association between different KIR alleles and HLA molecules has been reported in pregnancy complications, supporting the idea of a relevant role of these receptors in successful pregnancy. Sin financiación 2.829 JCR (2019) Q2, 21/82 Obstetrics & Ginecology, 14/29 Reproductive Biology, 88/177 Genetics & Heredity 1.140 SJR (2019) Q1, 405/2754 Medicine (miscellaneous), 35/185 Obstetrics and Gynecology, 16/76 Reproductive Medicine; Q2, 29/87 Developmental Biology, 120/346 Genetics, 40/99 Genetics (clinical) No data IDR 2019 UEM

Mercury accumulation has been proposed as a toxic factor that causes neurodegenerative diseases. However, the hazardous health effects of gaseous elemental mercury exposure on the spinal cord in volcanic areas have not been reported previously in the literature. To evaluate the presence of volcanogenic inorganic mercury in the spinal cord, a study was carried out in São Miguel island (Azores, Portugal) by comparing the spinal cord of mice exposed chronically to an active volcanic environment (Furnas village) with individuals not exposed (Rabo de Peixe village), through the autometallographic silver enhancement histochemical method. Moreover, a morphometric and quantification analysis of the axons was carried out. Results exhibited mercury deposits at the lumbar level of the spinal cord in the specimens captured at the site with volcanic activity (Furnas village). A decrease in axon calibre and axonal atrophy was also observed in these specimens. Given that these are relevant hallmarks in the neurodegenerative pathologies, our results highlight the importance of the surveillance of the health of populations chronically exposed to active volcanic environments. This research was supported by the University of Alicante VIGROB-186. info:eu-repo/semantics/publishedVersion

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities. This work was supported by Institute of Health Carlos III/ERDF (European Regional Development Fund), Spain [PI16/00409 (DL), PI20/01119 (DL), CP18/00033 (DL), PI15/00227 (MC), CPII16/00037 (SE), and PI18-00286 (SE)], Platform for Proteomics, Genotyping and Cell Lines; PRB3 of ISCIII (PT17/0019/0024); National Science Foundation GACR 18-04393S and the project “Centre of Reconstructive Neuroscience”, registration number CZ.02. 1.01/0.0./0.0/15_003/0000419PI15/00227; Spanish Ministry of Economy and Competitiveness grant BES-2016-076994 (ÁA-L); and Academy of Finland (HS).

Over the last few years an intense activity in the areas of advanced microscopy and quantitative cell biology has put the focus on the morphogenetic events that shape embryos. The interest in these processes is taking place against the backdrop of genomic studies, particularly of global patterns of gene expression at the level of single cells, which cannot fully account for the way cells build tissues and organs. Here we discuss the need to integrate the activity of genes with that of cells and propose the need to develop a framework, based on cellular processes and cell interactions, that parallels that which has been created for gene activity in the form of Gene Regulatory Networks (GRNs). We begin to do this by suggesting elements for building Cell Regulatory Networks (CRNs). In the same manner that GRNs create schedules of gene expression that result in the emergence of cell fates over time, CRNs create tissues and organs i.e. space. We also suggest how GRNs and CRNs might interact in the building of embryos through feedback loops involving mechanics and tissue tectonics.

The classical dogma states that brown adipose tissue (BAT) plays a major role in the regulation of temperature in neonates. However, although BAT has been studied in infants for more than a century, the knowledge about its physiological features at this stage of life is rather limited. This has been mainly due to the lack of appropriate investigation methods, ethically suitable for neonates. Here, we have applied non-invasive infrared thermography (IRT) to investigate neonatal BAT activity. Our data show that BAT temperature correlates with body temperature and that mild cold stimulus promotes BAT activation in newborns. Notably, a single short-term cold stimulus during the first day of life improves the body temperature adaption to a subsequent cold event. Finally, we identify that bone morphogenic protein 8B (BMP8B) is associated with the BAT thermogenic response in neonates. Overall, our data uncover key features of the setup of BAT thermogenesis in newborns., Brown adipose tissue (BAT) in infants has been studied for more than a century, however, the knowledge about its physiological features is limited. Here, the authors investigate the link between BAT thermogenesis and the regulation of temperature in human new-borns with non-invasive infrared thermography.

[EN] This review compiles the most relevant advances made in the production of doubled haploid plants in eggplant, the application of doubled haploid lines in breeding programs, and the future perspectives for the development of alternative technologies for doubled haploid generation in this species. Eggplant is a solanaceous crop cultivated worldwide for its edible fruit. Eggplant breeding programs are mainly aimed to the generation of F1 hybrids by crossing two highly homozygous, pure lines, which are traditionally obtained upon several self crossing generations, which is an expensive and time consuming process. Alternatively, fully homozygous, doubled haploid (DH) individuals can be induced from haploid cells of the germ line in a single generation. Several attempts have been made to develop protocols to produce eggplant DHs principally using anther culture and isolated microspore culture. Eggplant could be considered a moderately recalcitrant species in terms of ability for DH production. Anther culture stands nowadays as the most valuable technology to obtain eggplant DHs. However, the theoretical possibility of having plants regenerated from somatic tissues of the anther walls cannot be ruled out. For this reason, the use of isolated microspores is recommended when possible. This approach still has room for improvement, but it is largely genotype-dependent. In this review, we compile the most relevant advances made in DH production in eggplant, their application to breeding programs, and the future perspectives for the development of other, less genotype-dependent, DH technologies., This research was funded by the Valencian Government, grant number CDEIGENT 2018/023 to RMM and by the Spanish MICINN, grant number PID2020-115763RB-I00 to JMSS. ACS is the recipient of a predoctoral contract from the FPU program of the Spanish Government.

An increased risk of developing colorectal cancer (CRC) and other types of tumor is associated to Lynch syndrome (LS), an inherited condition caused by germline mutations in mismatch repair genes. We selected a cohort of LS patients that had developed CRC and had undergone surgical resection. Formalin-fixed paraffin embedded (FFPE) tissue blocks from matched colorectal and normal mucosa were used for genomic DNA extraction with a commercial kit and sequenced by high-throughput sequencing. A metagenomic approach enabled the taxonomic and functional identification of the microbial community and associated genes detected in the specimens. Slightly lower taxonomic diversity was observed in the tumor compared to the non-tumor tissue. Furthermore, the most remarkable differences between tumors and healthy tissue was the significant increase in the genus Fusobacterium in the former, in particular the species F. nucleatum, as well as Camplylobacter or Bacteroides fragilis, in accordance with previous studies of CRC. However, unlike prior studies, the present work is not based on directed detection by qPCR but instead uses a metagenomic approach to retrieve the whole bacterial community, and addresses the additional difficulty of using long-term stored FFPE samples., We want to particularly acknowledge the patients and the Biobank IBSP-CV (PT17/0015/0017) integrated in the Spanish National Biobanks Network and in the Valencian Biobanking Network, especially A. Ahicart, D. Molina and J. Martínez, for their collaboration. This research was funded by grants to AM from the Fundación Científica de la Asociación Española contra el Cancer (project AECC 2017-1485), including a post-doctoral contract to VPB first and to SRR later. GD is recipient of a PhD fellowship from the Junta Asociada Provincial de Valencia AECC. Action co-financed by the European Union through the Operational Program of European Regional Development Fund (ERDF) of Valencia Region (Spain) 2014-2020.

Background Lynch syndrome (LS) is a hereditary condition characterized by a high risk of colorectal cancer, endometrial cancer, and other neoplasia associated with germline alterations in DNA mismatch repair genes. The classical genetic diagnostic strategy for LS consists of the Sanger sequencing of genes associated with the suspected syndrome. Next‐generation sequencing (NGS) enables the simultaneous sequencing of a large number of hereditary cancer genes. Here, we aimed to study whether other germline pathogenic variants of hereditary cancer genes are present in patients with LS. Methods A cohort of 84 probands with a previous genetic diagnosis of LS by Sanger sequencing was reanalyzed using NGS via a commercial panel of 94 hereditary cancer genes by hybrid capture. The American College of Medical Genetics and Genomics criteria were used to classify the clinical significance of the variants. The findings of NGS were confirmed by Sanger sequencing. When possible, genetic analyses of the new findings in the proband's relatives were also performed by Sanger sequencing. Results We identified five families (6%), out of 84, with at least two germline pathogenic variants conferring to high or moderate risk in different dominant cancer‐predisposing genes: [MLH1‐BRCA2‐NBN], [MLH1‐BRCA1], [MSH2‐ATM], [MSH6‐NF1], and [MLH1‐FANCA]. Interestingly, only one out of these five families exhibited a clinical phenotype associated with the new pathogenic variants. The family with three pathogenic variants of the [MLH1‐BRCA2‐NBN] genes showed a high aggregation of tumors associated with LS and breast and ovarian cancer syndrome. Conclusions Our results showed that the co‐occurrence of more than one pathogenic variant in cancer‐predisposing genes was remarkable among cases of LS. In most cases, no clinicial manifestations were associated with the secondary pathogenic variants. Further studies are needed to confirm these findings and elucidate their clinical impact. Reanalysis of LS families should be considered only in families with mixed clinical phenotypes., We aimed to explore whether other pathogenic variants for hereditary cancer genes are present in Lynch syndrome patients, to have a more comprehensive view of the genetic architecture for cancer risk. We found five families (6%) with at least two pathogenic variant in high‐ and/or moderate‐risk‐associated cancer genes. However, in most cases, no clinical manifestations associated with the secondary pathogenic variants were evidenced.

Signaling mediated by cytokines and chemokines is involved in glaucoma-associated neuroinflammation and in the damage of retinal ganglion cells (RGCs). Using multiplexed immunoassay and immunohistochemical techniques in a glaucoma mouse model at different time points after ocular hypertension (OHT), we analyzed (i) the expression of pro-inflammatory cytokines, anti-inflammatory cytokines, BDNF, VEGF, and fractalkine, and (ii) the number of Brn3a+ RGCs. In OHT eyes, there was an upregulation of (i) IFN-γ at days 3, 5, and 15, (ii) IL-4 at days 1, 3, 5, and 7 and IL-10 at days 3 and 5 (coinciding with downregulation of IL1-β at days 1, 5, and 7), (iii) IL-6 at days 1, 3, and 5, (iv) fractalkine and VEGF at day 1, and (v) BDNF at days 1, 3, 7, and 15. In contralateral eyes, there were (i) an upregulation of IL-1β at days 1 and 3 and a downregulation at day 7, coinciding with the downregulation of IL4 at days 3 and 5 and the upregulation at day 7, (ii) an upregulation of IL-6 at days 1, 5, and 7 and a downregulation at 15 days, (iii) an upregulation of IL-10 at days 3 and 7, and (iv) an upregulation of IL-17 at day 15. In OHT eyes, there was a reduction in the Brn3a+ RGCs number at days 3, 5, 7, and 15. OHT changes cytokine levels in both OHT and contralateral eyes at different time points after OHT induction, confirming the immune system involvement in glaucomatous neurodegeneration.

Sperm glycocalyx and plasma membrane undergo outstanding modifications during fertilization. However, it is unclear how in vitro capacitation time and acrosome reaction affect the specific location of boar sperm glycoconjugates. This study aimed to identify lectin binding patterns and to describe the sequential changes during different in vitro capacitation times (1 and 4 h) and acrosome reaction in boar spermatozoa. With Aleuria aurantia agglutinin (AAA), most uncapacitated cells were labelled in the postacrosomal region. Nevertheless, after 1 h of in vitro capacitation and the acrosome reaction, most AAA binding sites were in the acrosomal region. With Concanavalin A (ConA), most sperm were labeled in the postacrosomal region before and after capacitation. After the acrosome reaction induction, this pattern changed to a highly stained acrosomal and postacrosomal regions. Peanut agglutinin (PNA) binding sites were in the acrosomal region in uncapacitated and capacitated sperm. In acrosome reacted sperm after 4h capacitation, the most frequent pattern showed remaining positive labeling in the central area of the head. With Pisum sativum agglutinin (PSA), most uncapacitated cells showed a postacrosomal region staining. Nevertheless, faint stained all over the head and highly acrosomal region labelling was observed in the major part of capacitated and acrosome reacted sperm respectively. With Wheat germ agglutinin (WGA), the most representative pattern in uncapacitated, capacitated and acrosome reacted sperm was labelled in the acrosomal region. Regarding capacitation time, the most significant changes in the most representative pattern were observed in acrosome reacted spermatozoa after 4 h of in vitro capacitation.Highlights Lectins allow detailed descriptions of eight different populations in boar sperm. Changes in lectin binding patterns during capacitation are time dependent. Lectin binding patterns significantly change after 4 h of in vitro capacitation.

The cave bear ( Ursus spelaeus s.l .) was an iconic extinct bear that inhabited the Pleistocene of Eurasia. The cause of extinction of this species is unclear and to identify the actual factors, it is crucial to understand its feeding preferences. Here, we quantified the shape descriptor metrics in three-dimensional (3D) models of the upper teeth (P 4 –M 2 ) of the cave bear to make inferences about its controversial feeding behaviour. We used comparative samples, including representatives of all living bear species with known diets, as a template. Our topographic analyses show that the complexity of upper tooth rows in living bears is more clearly associated with the mechanical properties of the items consumed than with the type of food. Cave bears exhibit intermediate values on topographic metrics compared with the bamboo-feeder giant panda ( Ailuropoda melanoleuca ) and specialists in hard mast consumption ( Ursus arctos and Ursus thibetanus ). The crown topography of cave bear upper teeth suggests that they could chew on tough vegetal resources of low quality with high efficiency, a characteristic that no living bear currently displays. Our results align with a climate-driven hypothesis to explain the extinction of cave bear populations during the Late Pleistocene.

[EN] Cancer is the second leading cause of death, accountable for about 1 in 6 deaths globally. There is massive investment in the development of novel therapeutic strategies, although brand-new chemotherapy faces several handicaps such as lack of clinical efficacy, pharmacokinetic problems, or the appearance of resistance mechanisms. In this work, we focused on the design of novel compounds aimed to interact with the protein tubulin, which is a validated target in cancer chemotherapy and arguably one of the most successful ones. i) It has proven effective as a therapeutic target since some of the antitumor drugs currently at the forefront in clinical use (e.g. paclitaxel), act by binding to tubulin. ii) It is an essential protein, barely susceptible to relevant mutations that would affect its proper functioning. Microtubules are built up by the lateral association of protofilaments, which in turn, are formed by the head-to-tail polymerization of α,β-tubulin. Microtubules are highly dynamic structures that fulfill a vast range of pivotal functions, such as chromosome segregation during cell division, vesicle transport, or maintenance of cell shape. These roles make them an appealing target in cancer chemotherapy since minor disturbance of their dynamics leads to an antimitotic effect and eventually cell death. Many tubulin binding drugs also exert antivascular activities hampering the growth of solid tumors by shutting down the supply of oxygen and nutrients. Among the several binding pockets described in microtubules to date, we have focused on the colchicine domain. Although some colchicine site ligands are quite advanced in clinical trials, none has yet reached clinical approval. The ligands that bind to this pocket mostly show simple chemical structures, so constructing an extensive library of compounds to explore the impact of structural changes on the antitumor activity is attainable. The colchicine domain has been considered as an ensemble of three consecutive zones (1-2-3) based on the X-ray structures of protein-ligand complexes. Most of the known ligands bind to two zones, and only a few of them interact simultaneously with the three zones of the domain. The primary goal of this work is to obtain compounds with advantageous properties that are able to interact with the colchicine domain, as promising pharmacological agents in cancer therapy. To this end, the design has been approached by parts considering the data provided by PDB structures and structure-activity relationship data of compounds with no X-ray reports, under the presumption of their binding mode. We have designed compounds directed to zones 1-2, 2-3, and 1-2-3, leveraging the common zone 2 in the different approaches by designing common structural elements for this zone that can be later combined with residues in zones 1 and 3. This work has yielded a series of indole-based compounds with two aromatic rings connected by sulfonamide or tetrazole groups that preferentially adopt folded conformations that are required for the interaction with zones 1-2 of the colchicine domain. These compounds bind to the colchicine domain in β-tubulin and preclude the in vitro polymerization of microtubules, inducing disorganization of the tubulin cytoskeleton. Our results expose that these compounds exert antimitotic and antivascular activities. The antimitotic mechanism of action leads to antiproliferative potencies in the submicromolar or nanomolar range against several tumor and non-tumorigenic cell lines. We have studied the mechanism of action and the induction of cell death in the human epithelial cervical carcinoma HeLa cells caused by some representative lead compounds and compared their behavior with other cell lines. This research contributes to further unraveling the molecular processes, both reversible or irreversible, that are involved in the transition from microtubule depolymerization to cell death.

Background: The gut microbiome is known to influence the pathogenesis and progression of neurodegenerative diseases. However, there has been relatively little focus upon the implications of the gut microbiome in retinal diseases such as retinitis pigmentosa (RP) that leads to photoreceptor degeneration and is the main worldwide cause of complete blindness in adulthood. Here, we investigated changes in gut microbiome composition linked to RP, by assessing both retinal degeneration and gut microbiome in the rd10 mouse model of RP as compared to control C57BL/6J mice.Results: In rd10 mice, retinal responsiveness to flashlight stimuli was deteriorated with respect to observed in age-matched control mice, with decreased amplitudes in the a- and b-wave responses of the electroretinogram and concomitant reduction of the visual acuity. This functional decline in dystrophic animals was accompanied by photoreceptor loss, evidenced by decreased outer nuclear layer thickness, and morphologic anomalies in photoreceptor cells. Changes in retinal neurons were paralleled to induction of reactive gliosis in retina, with increased microglial cell numbers and higher Müller cell reactivity in rd10 mice. Furthermore, 16S rRNA gene amplicon sequencing data showed a microbial gut dysbiosis with differences in alpha and beta diversity at the genera, species and amplicon sequence variants (ASV) levels between dystrophic and control mice. A taxonomic partitioning of ASV was observed since unique ASVs present in only one group had a cumulative relative microbial abundance between 17.6% (rd10 mice) and 26.7% (C57BL/6J mice). Remarkably, four fairly common ASV in healthy gut microbiome belonging to -Rikenella spp., Muribaculaceace spp., Prevotellaceae UCG-001 spp., and Bacilli spp.- were absent in the gut microbiome of retinal disease mice, while Bacteroides caecimuris was significantly enriched in mice with retinitis pigmentosa. Conclusions: Our results indicate that retinal degenerative changes in retinitis pigmentosa are linked to relevant gut microbiome changes. The findings suggest that microbiome shifting could be considered as potential biomarker and therapeutic target for retinal degenerative diseases.

Human fertilization success depends on the ability of the spermatozoa to undergo capacitation. Even though this process can be conducted in vitro, the optimal time for a sperm cell to complete capacitation in vitro is still under discussion due to the lack of proper capacitation biomarkers. Here, we evaluated the influence of in vitro capacitation time on HSPA2 distribution over human sperm head testing this chaperone as a potential capacitation biomarker. The chaperone was assessed in human spermatozoa from 16 normozoospermic donors using indirect immunofluorescence in uncapacitated, one and four-hour capacitated spermatozoa. The percentage of HSPA2 immunofluorescent cells before and after one hour of capacitation did not differ significantly. However, after four hours of capacitation, we observed a significantly higher percentage of HSPA2 labelled cells. In fluorescent cells analysed before capacitation, we could not identify a predominant distribution pattern. Meanwhile, after capacitation, most sperm showed a highly labelled equatorial band accompanied by a homogeneous fluorescence throughout the acrosomal region. Our findings suggest that HSPA2 needs more than one hour of in vitro capacitation for being correctly distributed in the anterior region of the sperm head. In conclusion, the present study provides solid evidences for the utility of HSPA2 as a biomarker of human sperm in vitro capacitation. Due to its importance during egg-sperm recognition, the use of HSPA2 as a biomarker before an artificial reproduction technique may be suggested, in addition to a longer capacitation time during sperm preparation. This work was supported by the Human Fertility Cathedra of the University of Alicante and R&D&I projects financed by competitive public entities (ViGrob-186, UAIND17-03 and AGL2015).

The objective of the present study was to evaluate whether gender determination in two amphibian species (Kaloula pulchra and Xenopus laevis) can be reliably carried out by means of magnetic resonance imaging (benchtop magnetic resonance imaging, BT-MRI) or ultrasound (high-resolution ultrasound, HR-US) techniques. Two species of healthy, sexually mature anurans have been used in the present study. Eight Kaloula (blind study) and six Xenopus were used as controls. Magnetic resonance imaging experiments were carried out on a low-field (1 Tesla) benchtop-MRI (BT-MRI) system. HR-US examination was performed with high-resolution equipment. Low-field BT-MRI images provided a clear and quantifiable identification of all the sexual organs present in both genders and species. The HR-US also allowed the identification of testes and ovaries in both species. Results indicate that BT-MRI allowed a very precise sex identification in both anuran species, although its use is limited by the cost of the equipment and the need for anesthesia. HR-US allowed an accurate identification of ovaries of both species whereas a precise identification of testes is limited by the ultrasonographer experience. The main advantages of this technique are the possibility of performing it without anesthesia and the higher availability of equipment in veterinary and zoo institutions.

22q11.2 deletion syndrome (22q11DS)—a neurodevelopmental condition caused by a hemizygous deletion on chromosome 22—is associated with an elevated risk of psychosis and other developmental brain disorders. Prior single-site diffusion magnetic resonance imaging (dMRI) studies have reported altered white matter (WM) microstructure in 22q11DS, but small samples and variable methods have led to contradictory results. Here we present the largest study ever conducted of dMRI-derived measures of WM microstructure in 22q11DS (334 22q11.2 deletion carriers and 260 healthy age- and sex-matched controls; age range 6–52 years). Using harmonization protocols developed by the ENIGMA-DTI working group, we identified widespread reductions in mean, axial and radial diffusivities in 22q11DS, most pronounced in regions with major cortico-cortical and cortico-thalamic fibers: the corona radiata, corpus callosum, superior longitudinal fasciculus, posterior thalamic radiations, and sagittal stratum (Cohen’s d’s ranging from −0.9 to −1.3). Only the posterior limb of the internal capsule (IC), comprised primarily of corticofugal fibers, showed higher axial diffusivity in 22q11DS. 22q11DS patients showed higher mean fractional anisotropy (FA) in callosal and projection fibers (IC and corona radiata) relative to controls, but lower FA than controls in regions with predominantly association fibers. Psychotic illness in 22q11DS was associated with more substantial diffusivity reductions in multiple regions. Overall, these findings indicate large effects of the 22q11.2 deletion on WM microstructure, especially in major cortico-cortical connections. Taken together with findings from animal models, this pattern of abnormalities may reflect disrupted neurogenesis of projection neurons in outer cortical layers. Sin financiación 15.992 JCR (2020) Q1, 10/295 Biochemistry & Molecular Biology 5.071 SJR (2020) Q1, 5/85 Cellular and Molecular Neuroscience No data IDR 2020 UEM

Sperm capacitation includes the reorganization of plasma membrane components and the outstanding modification of the glycocalyx. The α-mannose presence and location during in vitro capacitation have been commonly described in human spermatozoa using Concanavalin A (Con A) lectin. However, it is still unclear to date how in vitro capacitation time affects the α-mannose residues and their topographic spatial distribution on sperm membrane. Here, we characterized the α-mannose density and specific membrane domain locations before and after in vitro capacitation (1–4 h) using high-resolution field emission scanning electron microscopy (FE-SEM). Results showed that α-mannose residues were present preferably on the acrosome domains for all physiological conditions. Uncapacitated sperm comparatively exhibits significant highest labeling densities of α-mannose residues. In addition, as in vitro capacitation takes place, significant and progressive decreasing of sugar residues was combined with their relocation mostly affecting acrosomal domain apical areas. Our findings reveal that combined approach using FE-SEM and gold nanoparticle topographical mapping offers new human sperm biomolecular and structural details during capacitation events. The current study was funded by the Cátedra Human Fertility and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186).

The seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was examined among 105 healthcare workers (HCWs) exposed to four patients who were laboratory confirmed with coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection. These HCWs were immediately under quarantine for 14 days as soon as they were identified as close contacts. The nasopharyngeal swab samples were collected on the first and 14th day of the quarantine, while the serum samples were obtained on the 14th day of the quarantine. With the assay of enzyme immunoassay (EIA) and microneutralization assay, 17.14% (18/105) of HCWs were seropositive, while their swab samples were found to be SARS-CoV-2 RNA negative. Risk analysis revealed that wearing face mask could reduce the infection risk (odds ratio [OR], 0.127, 95% confidence interval [CI] 0.017, 0.968), while when exposed to COVID-19 patients, doctors might have higher risk of seroconversion (OR, 346.837, 95% CI 8.924, 13479.434), compared with HCWs exposed to colleagues as well as nurses and general service assistants who exposed to patients. Our study revealed that the serological testing is useful for the identification of asymptomatic or subclinical infection of SARS-CoV-2 among close contacts with COVID-19 patients.

While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation. Research in Beato's lab is supported by funds from the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC Synergy grant agreement 609989 (4DGenome), from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’ and Plan Nacional (SAF2016-75006-P), as well as support of the CERCA Program/Generalitat de Catalunya. L.B was supported by the Forschungskredit 2019 of the University of Zurich and K.G by a grant of the Oncosuisse (Nr. KFS-3740-08-2015-R). ADP-ribosylation research in the laboratory of MOH is funded by the Kanton of Zurich and the Swiss National Science Foundation, Switzerland (grant 31003A_176177).

Spermatozoa motility is a key parameter during the fertilization process. In this context, spermatozoa tyrosine protein phosphorylation and an appropriate cytoskeleton α‐tubulin distribution are some of the most important physiological events involved in motility. However, the relationship between these two biomarkers remains poorly defined. Here, we characterized simultaneously by immunocytochemistry the α‐tubulin (TUBA4A) distribution and the tyrosine phosphorylation at flagellum before capacitation, during different capacitation times (1 and 4 hr), and after acrosome reaction induction in human spermatozoa. We found that the absence of spermatozoa phosphorylation in tyrosine residues positively and significantly correlated (p

T-cell lymphoblastic lymphoma is a haematological disease with an urgent need for reliable prognostic biomarkers that allow therapeutic stratification and dose adjustment. The scarcity of human samples is responsible for the delayed progress in the study and the clinical management of this disease, especially compared with T-cell acute lymphoblastic leukaemia, its leukemic counterpart. In the present work, we have determined by immunohistochemistry that S194-P-FADD protein is significantly reduced in a cohort of 22 samples from human T-cell lymphoblastic lymphoma. Notably, the extent of such reduction varies significantly among samples and has revealed determinant for the outcome of the tumour. We demonstrate that Fas-associated protein with death domain (FADD) phosphorylation status affects protein stability, subcellular localization and non-apoptotic functions, specifically cell proliferation. Phosphorylated FADD would be more stable and preferentially localized to the cell nucleus; there, it would favour cell proliferation. We show that patients with higher levels of S194-P-FADD exhibit more proliferative tumours and that they present worse clinical characteristics and a significant enrichment to an oncogenic signature. This supports that FADD phosphorylation may serve as a predictor for T-cell lymphoblastic lymphoma aggressiveness and clinical status. In summary, we propose FADD phosphorylation as a new biomarker with prognostic value in T-cell lymphoblastic lymphoma.

ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR5) PG-901 (10−10M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR1 agonist BMS-470539 (10−5 M) or with the mixed MCR3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGF-containing exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10−10M) but not by the MCR3/4 agonist MTII (0.30 nmol) or the MCR1 agonist BMS-470539 (10−5 M). The MCR5-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR5 agonist in the ARPE-19 cells. Altogether, these data suggest that MCR5 is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo. Sin financiación 3.699 JCR (2019) Q2, 95/195 Cell Biology 1.319 SJR (2019) Q1, 318/2754 Medicine (miscellaneous); Q2, 106/300 Cell Biology, 23/87 Developmental Biology, 138/414 Molecular Biology No data IDR 2019 UEV

The purpose of this study was to investigate effects of low maternal folic acid (FA) status on many eye structures in embryonic mice using multifractal analysis, a method for describing complex structural patterns. To do this, we used the FracLac plugin for ImageJ to analyse binary digital images of cornea, lens, retina, and vitreous after immunohistochemical analysis using anti-collagen IV or anti-laminin-1 antibodies. The box-counting and gliding box methods were used to generate metrics including the fractal dimension, lacunarity, divergence, and multifractal spectrum distance metrics for each ocular structure. Results were analysed using bootstrap resampling techniques and the Kruskal-Wallis significance test. For all structures and staining methods studied, differences between embryos from control vs FA-deficient females were detectable in lacunarity plots and multifractal spectra, and statistically significant in cornea, lens, retina and vitreous. Multifractal analysis detected even slight differences in tissue textures presumably attributable to altered expression of extracellular matrix molecules.

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G &gt, T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in M&uuml, ller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.

Laminopathies are causally associated with mutations on the Lamin A/C gene (LMNA). To date, more than 400 mutations in LMNA have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models included different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy, LMNA-associated congenital muscular dystrophy (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that Lmna exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the Lmna p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the Lmna exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies. Ministerio de Ciencia e Innovación (Acción estratégica en Salud intramural PI16III/00017-TPY1348/16). Fundación Andrés Marcio, niños contra la laminopatía (TPY-259/19). 6.600 JCR (2020) Q2, 53/195 Cell Biology 1.220 SJR (2020) Q1, 51/254 Biochemistry, Genetics and Molecular Biology (miscellaneous) No data IDR 2020 UEM

Objective: Considering the demonstrated implication of the retina in Parkinson disease (PD) pathology and the importance of dopaminergic cells in this tissue, we aimed to analyze the state of the dopaminergic amacrine cells and some of their main postsynaptic neurons in the retina of PD. Methods: Using immunohistochemistry and confocal microscopy, we evaluated morphology, number, and synaptic connections of dopaminergic cells and their postsynaptic cells, AII amacrine and melanopsin‐containing retinal ganglion cells, in control and PD eyes from human donors. Results: In PD, dopaminergic amacrine cell number was reduced between 58% and 26% in different retinal regions, involving a decline in the number of synaptic contacts with AII amacrine cells (by 60%) and melanopsin cells (by 35%). Despite losing their main synaptic input, AII cells were not reduced in number, but they showed cellular alterations compromising their adequate function: (1) a loss of mitochondria inside their lobular appendages, which may indicate an energetic failure; and (2) a loss of connexin 36, suggesting alterations in the AII coupling and in visual signal transmission from the rod pathway. Interpretation: The dopaminergic system impairment and the affection of the rod pathway through the AII cells may explain and be partially responsible for the reduced contrast sensitivity or electroretinographic response described in PD. Also, dopamine reduction and the loss of synaptic contacts with melanopsin cells may contribute to the melanopsin retinal ganglion cell loss previously described and to the disturbances in circadian rhythm and sleep reported in PD patients. These data support the idea that the retina reproduces brain neurodegeneration and is highly involved in PD pathology. This work was supported by the Michael J. Fox Foundation for Parkinson’s Research. I.O.-L. and X.S.-S. acknowledge financial support from the Ministry of Education, Spain (FPU 14/03166; FPU 16/04114). N.C. acknowledges financial support from the Ministry of Economy and Competitiveness, Spain (MINECO-FEDER-BFU2015-67139-R), Carlos III Institute of Health (RETICS-FEDER RD16/0008/0016), Retina Asturias Association, and Generalitat Valenciana-European Regional Development Fund (IDIFEDER/2017/064). The Brain and Body Donation Program has been supported by the NIH National Institute of Neurological Disorders and Stroke (U24 NS072026), the NIH National Institute on Aging (P30 AG19610), the Arizona Department of Health Services, the Arizona Biomedical Research Commission, and the Michael J. Fox Foundation for Parkinson’s Research.

The morphology of the retina of the adult Yellow-legged Gull, Larus michahellis, was examined in transverse sections under light microscopy in order to study the retinal adaptations to their specific photic environment that determines their behaviour. We identified rods, single cones and unequal double cones. Although it is a duplex retina, cones are preponderant and coloured oil droplets are present in their inner segments. As several colours in oil droplets are observed, it seems reasonable to conclude that several types of cones are present. Moreover, more cones per unit area are found in the central regions of the retina than in peripheral regions. A probable area centralis is observed. In the inner nuclear layer, two types of horizontal cells, and bipolar and amacrine cells can be recognised. Also, ganglion cells, characterised by prominent nuclei and nucleoli, vary in size and abundance among different regions in the retina. Comparisons are made with the retinae of other marine birds. The morphological characteristics of this retina indicate that Larus michahellis possesses: a good ability to discriminate colour; complex visual processing in the inner retina in order to mediate contrast and motion perception; and an elevated acuity in areas of high ganglion cell density.

We review recent developments in the understanding of the biomechanics of apicomedial actomyosin and how its contractility can tense and deform tissue. Myosin pulses are driven by a biochemical oscillator but how they are modulated by the mechanical context remains unclear. On the other hand, the emergence of tissue behaviour is highly dependent on the material properties of actin, on how strongly components are connected and on the influence of neighbouring tissues. We further review the use of constitutive equations in exploring the mechanics of epithelial apices dominated by apicomedial Myosin contractility. Sin financiación 5.288 JCR (2018) Q1, 22/174 Genetics & Heredity, 47/193 Cell Biology 3.954 SJR (2018) Q1, 5/87 Developmental Biology, 18/351 Genetics No data IDR 2018 UEM

The stem cell field has grown very rapidly during the last decade, offering the promise of innovative therapies to treat disease. Different stem cell populations have been isolated from various human adult tissues, mainly from bone marrow and adipose tissue, but many other body tissues harbor a stem cell population. Adult tissue stem cells are invariably found in discrete microenvironments termed niches, where they play key roles in tissue homeostasis by enabling lifelong optimization of organ form and function. Some diseases are known to strike at the stem cell population, through alterations in their specific microenvironments, making them non-viable. Furthermore, it has been shown that a transformed stem cell population could prompt the development of certain cancers. This review focuses on the potential negative aspects of a range of diseases on the activity of stem cells and how their potential use in cell therapies may be affected. Sin financiación No data JCR 2018 1.292 SJR (2018) Q2, 51/188 Physiology No data IDR 2018 UEM

Purpose: OCT has been a technological breakthrough in the diagnosis, treatment, and follow-up of many ocular diseases, especially retinal and neuro-ophthalmologic pathologic conditions. Until now, several controversies have arisen over the specific cell types that the bands observed in the OCT represent, especially over the 4 outer retinal bands. Design: To correlate the 4 outer hyperreflective bands observed in the OCT with the histologic structures using human retinal sections and immunocytochemistry at the fovea level. Participants: Eyes from human donors. Methods: Vertical cryosections of human retinas were immunostained with antibodies specific for cones photoreceptors, bipolar cells, mitochondria, Müller cells, and retinal pigment epithelium (RPE) cells and were visualized using confocal microscopy. Main Outcome Measures: Morphological correlation between histology and OCT at the fovea level. Results: Triple immunolabeling allowed distinguishing between cells types and different cell compartments. Immunostaining with guanine nucleotide-binding protein β 3 (GNB3) and cellular retinaldehyde-binding protein (CRALBP) antibodies showed all retinal layers at the foveola, especially the separation between the outer nuclear layer and the Henle fiber layer. CRALBP and cytochrome C (Cyt C) immunolabeling revealed that hyperreflective bands 1 and 2, observed in the OCT, correspond to the outer limiting membrane and the cone ellipsoids, respectively, separated by the cone myoids. CRALBP, cytochrome C, and GNB3 showed that the RPE interdigitations extend along the entire external segment of the cones, we do not believe them to be the structure responsible for forming the third band. However, the identification of small fragments of cone outer segments within the RPE led us to characterize the third band as the cone phagosomes located in the top of the RPE. Finally, we propose that the fourth band corresponds to the accumulation of mitochondria at the basal portion of the RPE, as identified by cytochrome C immunoreactivity, and that the hyporeflective band between bands 3 and 4 corresponds to the RPE nuclei and melanosomes zone. Conclusions: This study proposes a new interpretation of the outer retinal bands that leads to a more accurate interpretation of OCT images, providing information about the health of cones and their relationship with the RPE, and could help to form a better understanding of retinal disease diagnosis and prognosis. Supported by the Ministerio de Economía y Competitividad (grant no.: MINECO-FEDER-BFU2015-67139-R); Instituto Carlos III (grant no.: ISCIII RETICS-FEDER RD12/0034/0010); Generalitat Valenciana (grant no.: prometeo 2016/158 [N.C.]); and the Ministerio de Educación (grant no.: FPU 14/03166 [I.O.-L.]).

The modification of sperm glycocalyx is an essential process during sperm capacitation. The presence and redistribution of terminal and linked fucose have been described during in vitro capacitation in humans. However, the influence of the capacitation time on the quantification and localization of terminal and linked fucose is still unknown. In this study, the quantitative and qualitative changes in fucosyl residues during different in vitro capacitation times (1 and 4 h), are simultaneously characterized by using Aleuria aurantia (AAA) lectin–gold labelling and high-resolution field emission scanning electron microscopy (FE-SEM) in human sperm. A significant decrease was found in the number of terminal fucose registered in the whole sperm head during the in vitro capacitation. Nevertheless, the quantification of fucose residues after 1 h of in vitro capacitation was very similar to those found after 4 h. Therefore, the changes observed in terminal and linked fucose during capacitation were not time-dependent. Furthermore, the comprehensive analysis of the topographic distribution showed the preferential fucosyl location in the acrosomal region and the presence of distinct clusters distributed over the head in all the studied conditions. Overall, these findings corroborate the validity of FE-SEM combined with gold labelling to register changes in surface molecules during in vitro sperm capacitation. This research was funded by the Cátedra Human Fertility and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186).

The retinal structure of the booted eagle (Aquila pennata) was investigated using light and electron microscopy. Particular attention is paid to the main ultrastructural features of the receptor cells. This study reveals six distinct varieties of cones. Unequal double cones differ in shape, structure, and length and are comprised by principal long and accessory short members. Principal member contains a green oil droplet and accessory member contains a paraboloid and a pale green droplet. Four types of single cones are distinguished on the basis of their morphology and oil droplets: red, green, blue, and ultraviolet. Cones outnumber rods in all regions. Two types of horizontal cells and several morphological types of amacrine cells are abundant. A large number of bipolar cells are divided into long longitudinal rows by Müller cell processes, a prominent feature of this retina. These processes extend through the external limiting membrane to reach the ellipsoid region of the cones. Moreover, thick processes divide the inner nuclear and plexiform layers and surround the myelinated ganglion cell axons at fairly regular intervals. In the ganglion cell layer and optic nerve fibre layer, abundant oligodendrocytes are present, close to the myelinated axons. The morphological characteristics of this retina indicate that A. pennata have good colour discrimination, a complex visual processing to mediate contrast and motion and an elevated acuity in areas of high cell densities. This research was supported by the University of Alicante VIGROB-186 and UAUSTI16-06.

Using light microscopy, we examined the retina of benthopelagic fish Nezumia sclerorhynchus. Although the retina is typical of other vertebrates, having three nuclear and two synaptic layers, it presents some features associated with the animal's deep-sea habitat. A stratum argenteum containing iridescent crystals is located in the choroid. The pigment cell layer shows bulky cells filled with melanin granules but without the typical apical processes. The visual cells, consisting of a big population of rods, are arranged in several banks. No cones were observed. The outer segments are very long and cylindrical, and the inner segments are constituted by a small ellipsoid at the proximal end. The outer nuclear layer contains several rows of oval nuclei, and the spherules in the outer plexiform layer have less regular outlines than nuclei. The inner retina is characterized by very large horizontal cells, and presumable bipolar and amacrine cells separated by large spaces that are occupied by neuronal processes. Finally, the low density of ganglion cells produces a thin nerve fibre layer. The results of this study suggest that the retina of Nezumia sclerorhyncus exhibits high visual sensitivity and that vision is a sense that plays an important role in its behaviour. This work was supported by VIGROB-186 (University of Alicante).

Catalase (CAT) stands out as one of the most efficient natural enzymes when catalysing the split of H2O2 into H2O and O2; H2O2 is one of the reactive oxygen species (ROS) involved in oxidative stress, a process closely related to aging and several health disorders or diseases like male infertility. Some studies have correlated H2O2 with male infertility and catalase with fertility restoration. However, the number of studies conducted with human beings remains scarce. Considering the use of CAT as a molecular target for biochemical analysis, this review summarises the current knowledge on how CAT influences human beings’ male fertility. Thus, three different databases were consulted—Scopus, PubMed and WOS—using single keywords and combinations thereof. A total of 40,823 articles were identified. Adopting inclusion and exclusion criteria, a final database of 197 articles served to conduct this work. It follows from this analysis that CAT could play an important role in male fertility and could become a good target for male infertility diagnosis and monitoring. However, that potential role of CAT as a tool in diagnosis must be confirmed by clinical trials. Finally, guidelines are suggested to reinforce the use of CAT in daily clinical tests for male fertility diagnosis and monitoring. This research was funded by the Human Fertility Chair, the Department of Biotechnology (VIGROB-186) and the Applied Biochemistry research group of the University of Alicante (VIGROB-309).

Epidermal Growth Factor receptor (EGFR) is a tyrosine kinase receptor widely expressed on the surface of numerous cell types, which activates several downstream signalling pathways involved in cell proliferation, migration and survival. EGFR alterations, such as overexpression or mutations, have been frequently observed in several cancers, including glioblastoma (GBM), and are associated to uncontrolled cell proliferation. Here we show that the inhibition of mammalian target of Rapamycin (mTOR) mediates EGFR delivery to lysosomes for degradation in GBM cells, independently of autophagy activation. Coherently with EGFR internalisation and degradation, mTOR blockade negatively affects the mitogen activated protein/extracellular signal-regulated kinase (MAPK)/ERK pathway. Furthermore, we provide evidence that Src kinase activation is required for EGFR internaliation upon mTOR inhibition. Our results further support the hypothesis that mTOR targeting may represent an effective therapeutic strategy in GBM management, as its inhibition results in EGFR degradation and in proliferative signal alteration.

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques. Cátedra Human Fertility of University of Alicante and VIGROB-186.

Summary Little is known about the intrinsic specification of postnatal cerebellar neural stem cells (NSCs) and to what extent they depend on information from their local niche. Here, we have used an adapted cell preparation of isolated postnatal NSCs and live imaging to demonstrate that cerebellar progenitors maintain their neurogenic nature by displaying hallmarks of NSCs. Furthermore, by using this preparation, all the cell types produced postnatally in the cerebellum, in similar relative proportions to those observed in vivo, can be monitored. The fact that neurogenesis occurs in such organized manner in the absence of signals from the local environment, suggests that cerebellar lineage progression is to an important extent governed by cell-intrinsic or pre-programmed events. Finally, we took advantage of the absence of the niche to assay the influence of the vesicular nucleotide transporter inhibition, which dramatically reduced the number of NSCs in vitro by promoting their progression toward neurogenesis., Graphical Abstract, Highlights • We present a preparation that allows monitoring the behavior of cerebellar NSCs • Isolated NSCs maintain their neurogenic nature in absence of niche factors • The model enables monitoring the three postnatal cerebellar niches simultaneously • VNUT influences the balance between quiescence and activation of cerebellar NSCs, In this article, Ortega and colleagues describe a cell preparation that allows, in combination with live imaging, monitoring the behavior of NSCs from the three postnatal cerebellar niches. The authors provide evidence that, despite the absence of niche signals, isolated NSCs maintain their neurogenic nature and suggest a role for VNUT in the decision between quiescence and activation of cerebellar NSCs.

Signal transduction through the RAF-MEK-ERK pathway, the first described mitogen-associated protein kinase (MAPK) cascade, mediates multiple cellular processes and participates in early and late developmental programs. Aberrant signaling through this cascade contributes to oncogenesis and underlies the RASopathies, a family of cancer-prone disorders. Here, we report that de novo missense variants in MAPK1, encoding the mitogen-activated protein kinase 1 (i.e., extracellular signal-regulated protein kinase 2, ERK2), cause a neurodevelopmental disease within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects. Pathogenic variants promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling in vitro and in vivo. Two variant classes are identified, one of which directly disrupts binding to MKP3, a dual-specificity protein phosphatase negatively regulating ERK function. Importantly, signal dysregulation driven by pathogenic MAPK1 variants is stimulus reliant and retains dependence on MEK activity. Our data support a model in which the identified pathogenic variants operate with counteracting effects on MAPK1 function by differentially impacting the ability of the kinase to interact with regulators and substrates, which likely explains the minor role of these variants as driver events contributing to oncogenesis. After nearly 20 years from the discovery of the first gene implicated in Noonan syndrome, PTPN11, the last tier of the MAPK cascade joins the group of genes mutated in RASopathies. Sin financiación 11.025 JCR (2020) Q1, 11/176 Genetics & Heredity 6.661 SJR (2020) Q1, 11/340 Genetics No data IDR 2020 UEM