How wine tourism affects brand loyalty: a TripAdvisor review analysis in the case of the Ribera del Duero

Treball Final de Màster Universitari en Direcció d'Empreses / Master in Management. Codi: SRO011. Curs 2022-202

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions

Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor

Background : The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had. Methods : OCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed. Results : OCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4. Conclusions : Using a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications.This work was fnancially supported by the Ministry of Science and ICT (NRF2021R1A2C2008821). The Institute of Engineering Research at Seoul National University provided research facilities for this work

NGL-1/LRRC4C Deletion Moderately Suppresses Hippocampal Excitatory Synapse Development and Function in an Input-Independent Manner

Netrin-G ligand-1 (NGL-1), also known as LRRC4C, is a postsynaptic densities (PSDs)-95-interacting postsynaptic adhesion molecule that interacts trans-synaptically with presynaptic netrin-G1. NGL-1 and its family member protein NGL-2 are thought to promote excitatory synapse development through largely non-overlapping neuronal pathways. While NGL-2 is critical for excitatory synapse development in specific dendritic segments of neurons in an input-specific manner, whether NGL-1 has similar functions is unclear. Here, we show that Lrrc4c deletion in male mice moderately suppresses excitatory synapse development and function, but surprisingly, does so in an input-independent manner. While NGL-1 is mainly detected in the stratum lacunosum moleculare (SLM) layer of the hippocampus relative to the stratum radiatum (SR) layer, NGL-1 deletion leads to decreases in the number of PSDs in both SLM and SR layers in the ventral hippocampus. In addition, both SLM and SR excitatory synapses display suppressed short-term synaptic plasticity in the ventral hippocampus. These morphological and functional changes are either absent or modest in the dorsal hippocampus. The input-independent synaptic changes induced by Lrrc4c deletion involve abnormal translocation of NGL-2 from the SR to SLM layer. These results suggest that Lrrc4c deletion moderately suppresses hippocampal excitatory synapse development and function in an input-independent manner

Comparative Analyses Define Differences Between Bhd-Associated Renal Tumour and Sporadic Chromophobe Renal Cell Carcinoma

BACKGROUND: Birt-Hogg-Dubé (BHD) syndrome, caused by germline alteration of folliculin (FLCN) gene, develops hybrid oncocytic/chromophobe tumour (HOCT) and chromophobe renal cell carcinoma (ChRCC), whereas sporadic ChRCC does not harbor FLCN alteration. To date, molecular characteristics of these similar histological types of tumours have been incompletely elucidated. METHODS: To elucidate renal tumourigenesis of BHD-associated renal tumours and sporadic renal tumours, we conducted whole genome sequencing (WGS) and RNA-sequencing (RNA-seq) of sixteen BHD-associated renal tumours from nine unrelated BHD patients, twenty-one sporadic ChRCCs and seven sporadic oncocytomas. We then compared somatic mutation profiles with FLCN variants and RNA expression profiles between BHD-associated renal tumours and sporadic renal tumours. FINDINGS: RNA-seq analysis revealed that BHD-associated renal tumours and sporadic renal tumours have totally different expression profiles. Sporadic ChRCCs were clustered into two distinct clusters characterized by L1CAM and FOXI1 expressions, molecular markers for renal tubule subclasses. Increased mitochondrial DNA (mtDNA) copy number with fewer variants was observed in BHD-associated renal tumours compared to sporadic ChRCCs. Cell-of-origin analysis using WGS data demonstrated that BHD-associated renal tumours and sporadic ChRCCs may arise from different cells of origin and second hit FLCN alterations may occur in early third decade of life in BHD patients. INTERPRETATION: These data further our understanding of renal tumourigenesis of these two different types of renal tumours with similar histology. FUNDING: This study was supported by JSPS KAKENHI Grants, RIKEN internal grant, and the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute (NCI), Center for Cancer Research

The telomere maintenance mechanism spectrum and its dynamics in gliomas

Background : The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation and/or loss indicates an alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues. Methods : Telomerase repeated amplification protocol (TRAP) and C-circle assays were used to profile and characterize the TMM cross-sectionally (n = 412) and temporally (n = 133) across glioma samples. WES, RNA-seq, and NanoString analyses were performed to identify and validate the genetic characteristics of the TMM groups. Results : We show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined solely by the combination of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation, and ATRX loss. Moreover, we observed that a considerable proportion of gliomas lacked both telomerase activity and ALT. This telomerase activation-negative and ALT negative group exhibited evidence of slow growth potential. By analyzing a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed and can change with glioma progression. Conclusions : This study suggests that the TMM is dynamic and reflects the plasticity and oncogenicity of tumor cells. Direct measurement of telomerase enzyme activity and evidence of ALT should be considered when defining TMM. An accurate understanding of the TMM in glioma is expected to provide important information for establishing cancer management strategies.This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Ministry of Science & ICT (NRF-2018M3A9H3021707), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI21C0239)

Perinatal Epigenome Associated with Birth Outcomes and Exposure

Birth outcomes such as gestational age, or environmental exposures like mercury, serve either as one of the strongest predictors for neonatal, adolescent, and adult morbidity and mortality or associated with common diseases such as cancer, cardiovascular disease, and neurological disorders through unknown mechanisms. Identification of genomic loci undergoing epigenetic changes, specifically DNA methylation, would increase our understanding of these unknown mechanisms. To address this, we performed CHARM 2.0, a genome-wide array-based analysis of DNA methylation, in 141 newborns collected in Baltimore, Maryland using bump-hunting based novel statistical methodology to identify genomic regions associated with gestational age. Through this analysis, we identified three DMRs at genome-wide significance levels associated with gestational age near three genes (NFIX, RAPGEF2 and MSRB3) and one DMR commonly associated with total and methyl mercury exposure (TCEANC2). All of the three regions associated with gestational age were validated, and the region associated with both mercury exposure types were replicated by bisulphite pyrosequencing. Of the genes near or containing the DMRs, RAPGEF2 and TCEANC2 gene showed an inverse correlation between DNA methylation level and its expression level. For all of the gestational age DMRs, the DNA methylation levels at these regions appear similar or more extreme than those of the latest gestational ages in a heterogeneous population of adults. Together, the existence of gestational age DMRs suggests that epigenetic changes can occur not only during embryogenesis, but also during later stages of gestation. Also, The existence of mercury DMRs raise the possibility that environmental exposures, particularly heavy metals during pregnancy, would serve as inducing/mediating factors for epigenetic changes in neonates

Perinatal Epigenome Associated with Birth Outcomes and Exposure

Birth outcomes such as gestational age, or environmental exposures like mercury, serve either as one of the strongest predictors for neonatal, adolescent, and adult morbidity and mortality or associated with common diseases such as cancer, cardiovascular disease, and neurological disorders through unknown mechanisms. Identification of genomic loci undergoing epigenetic changes, specifically DNA methylation, would increase our understanding of these unknown mechanisms. To address this, we performed CHARM 2.0, a genome-wide array-based analysis of DNA methylation, in 141 newborns collected in Baltimore, Maryland using bump-hunting based novel statistical methodology to identify genomic regions associated with gestational age. Through this analysis, we identified three DMRs at genome-wide significance levels associated with gestational age near three genes (NFIX, RAPGEF2 and MSRB3) and one DMR commonly associated with total and methyl mercury exposure (TCEANC2). All of the three regions associated with gestational age were validated, and the region associated with both mercury exposure types were replicated by bisulphite pyrosequencing. Of the genes near or containing the DMRs, RAPGEF2 and TCEANC2 gene showed an inverse correlation between DNA methylation level and its expression level. For all of the gestational age DMRs, the DNA methylation levels at these regions appear similar or more extreme than those of the latest gestational ages in a heterogeneous population of adults. Together, the existence of gestational age DMRs suggests that epigenetic changes can occur not only during embryogenesis, but also during later stages of gestation. Also, The existence of mercury DMRs raise the possibility that environmental exposures, particularly heavy metals during pregnancy, would serve as inducing/mediating factors for epigenetic changes in neonates

The Effects of Nurses’ Perception of the Older Adults and Work Stress on Nursing Competency of Nurses Who Care for Older Adult Patients at General Hospital

The older adult population is rapidly increasing in South Korea, and hospitalization at general hospitals is increasing too. Therefore, nurses working at general hospitals need the nursing competency for older adult patients. The study was conducted to examine the effects of nurses’ perception of the older adults and work stress on the nursing competency of nurses at a general hospital, South Korea. A cross-sectional, descriptive correlational design was employed. Participants were a total of 136 nurses working at a general hospital located in Seoul, South Korea. Measures used in the study were the study participants’ general characteristics survey, Korean version of the Attitude Toward Old People Scale (KAOPS), the work stress scale, and the nursing competency scale. Data were collected from February to March, 2021. The regression model was statistically significant, and the explanatory power of the regression model was 33%. The significant factors affecting nursing competency were education level, perception of the older adults, and work stress. The greatest affecting factor was education level, followed by perception of the older adults and work stress in order. Nurses caring for older adult patients at general hospitals should pay attention to affecting factors to help improve the nursing competency in clinical practice. Managers should improve relevant policies to ensure that nurses have more opportunities to participate in the practical training of older adult care and explore effective training methods to improve the nurses’ perception of older adults