A very-low-calorie ketogenic diet normalises obesity-related enhanced levels of erythropoietin compared with a low-calorie diet or bariatric surgery.

PURPOSE: Nutritional ketosis synergistically with body-weight loss induced by a very-low-calorie ketogenic diet (VLCKD) has proven to be effective in improving obesity-related pathophysiology. Recently, growing attention has been focused on the relation between erythropoietin (EPO) and obesity. Thus, this study aims to investigate whether nutritional ketosis and weight loss induced by a VLCKD modify the circulating levels of EPO in patients with obesity in comparison with the effect of low-calorie diet (LCD) or bariatric surgery (BS). METHODS: EPO levels, iron status and body composition parameters were evaluated in 72 patients with overweight or obesity and 27 normal-weight subjects at baseline and after the three different weight-reduction therapies (VLCKD, LCD and BS) in 69 patients with excess body weight. ß-hydroxybutyrate levels were also measured in the VLCKD group. The follow-up was established at 2-3 months and 4-6 months. RESULTS: It was found that EPO levels were higher in morbid obesity and correlated with higher basal weight, fat mass (FM) and fat-free mass (FFM) in the overall sample. High baseline EPO levels were also correlated with higher impact on the course of weight loss and changes in FM and FFM induced by the three weight-loss interventions. Furthermore, the VLCKD induced a decrease in EPO levels coinciding with maximum ketosis, which was maintained over time, while statistically significant changes were not observed after LCD and BS. CONCLUSION: The obesity-related increased EPO levels are restored after VLCKD intervention at the time of maximum ketosis, suggesting a potential role of the nutritional ketosis induced by the VLCKD. Baseline EPO levels could be a biomarker of response to a weight-loss therapy.

Pituitary surgery in elderly patients: a safe and effective procedure.

PURPOSE: The incidence of pituitary adenoma (PA) increases with age. Transsphenoidal surgery (TSS) in elderly patients is often considered to have greater risk compared to the younger population. The aim of this study is to compare surgical results, evolution and postoperative complications between elderly and young patients undergoing TSS. METHODS: Retrospective review of patients undergoing TSS between 2011 and 2018 in our institution. Patients were divided into two cohorts: elderly (≥65 years) and non-elderly (<65 years). Characteristics and outcomes of both groups were compared at diagnosis, before surgery and for an average of 5.9 years of postoperative follow-up. RESULTS: One hundred and twenty-five patients were included, 53 patients were ≥65 years (42%). The elderly patients were more likely to have non-functioning PA (NFPA) (90.5% vs. 45.8%, p: <0.01), a higher proportion of macroadenomas (92.4% vs. 77.8%, p = 0.029) and greater extrasellar extension (88.7% vs. 68.1%, p = 0.007). The elderly group also had more compressive symptoms (54.7% vs. 34.7%, p = 0.035) and hypopituitarism (66% vs. 47.2%, p = 0.029). Overall, surgical and endocrinological outcomes between the two groups were similar. Inpatient mortality in the elderly group was 1.8%. Regarding long-term outcomes, elderly patients had more postoperative hypopituitarism (67.9% vs. 45.8%, p = 0.03) with no differences in permanent diabetes insipidus, less residual tumours (24.5% vs. 40.3%, p = 0.019) and a higher rate of remission after surgery (71.7% vs. 52.8%, p = 0.034). When only NFPA cases were compared, the only significant difference was a higher frequency of macroadenomas in the elderly group. CONCLUSIONS: Our results support the safety and efficacy of TSS in elderly patients with PA. Age should not be considered an exclusion criterion for TSS given that successful results can be achieved if an experienced pituitary team is available.

Early versus conventional nutritional intervention in head and neck cancer patients before radiotherapy: benefits of a fast-track circuit.

BACKGROUND/OBJECTIVES: Radiotherapy (RT) is a component of therapy for head and neck cancer (HNC) with a negative nutritional impact. Our aim was to compare an early versus a conventional nutritional intervention. SUBJECTS AND METHODS: Retrospective study of HNC patients undergoing RT. Evolution before and after the establishment of a fast-track circuit was evaluated. A conventional group (CG) made up of patients submitted to the nutrition unit during RT after nutritional deterioration, was compared to an early group (EG) represented by patients included in a fast-track circuit, starting nutritional follow-up before the beginning of RT. Only patients with preserved oral intake were involved. Demographic, nutritional and clinical variables were analyzed. Data of hospitalizations and deaths were collected up to three months after RT. RESULTS: 135 subjects constituted the EG and 39 the CG. At baseline, the prevalence of malnutrition was lower in the EG (31.9% vs 69.5%, p = 0.0001), as was the need for nutritional supplements (40% vs 79.5%, p = 0.0001) or nasogastric tube (0% vs 12.8%, p = 0.0001) in comparison to the CG. Three months after RT, there were less patients with oral nutritional support in the EG (79.1% vs 96.9%, p = 0.018), and the number of emergency visits (0.75 vs 1.1 episodes per patient, p = 0.021) and hospitalizations was also lower in this group (29% vs 59%, p = 0.044). CONCLUSIONS: The fast-track approach made early intervention possible. Therefore, patients maintained a better nutritional status, needed less nutritional support and their evolution improved, with a significant decrease in hospitalizations.

Correlates of children's physical activity during the COVID-19 confinement in Portugal.

OBJECTIVES: The aim of the study was to understand the role of household variables on the percentage of physical activity (%PA) during the coronavirus disease 2019 (COVID-19) confinement in Portugal. STUDY DESIGN: A cross-sectional study design using an anonymous online survey was launched to assess how Portuguese families with children aged younger than 13 years adjusted their daily routines to the confinement. METHODS: Separate analyses of variance were performed to investigate how factors such as the number of children, age, sex, the housing characteristics, and the adults' job situation can affect the percentage of time for PA (%PA). RESULTS: Findings, based on data from 2159 children, indicate that (1) boys and girls did not differ in the %PA on any of the age-groups; (2) children with an outdoor space and who had other children in the household were significantly more active (P < .001); (3) children from families with all adults working from home showed lower levels of %PA; and (4) being younger, having a big outdoor space, having other children in the household, and having at least one adult free from working from home were significant positive predictors of children's %PA, explaining 21% of the overall variance. CONCLUSION: Time allocated for PA during this period is reduced compared with what is usually reported on normal days. It is necessary to find strategies to increase children's PA, especially in families in which both parents are working and have no outdoor space.

Vitamin D levels in a pediatric population of a primary care centre: a public health problem?

OBJECTIVE: Vitamin D deficiency is a public health problem that occurs more frequently than expected. The aim of this study is to evaluate the vitamin D levels of children attending the paediatrics unit of the Bertamiráns primary care centre (A Coruña NW Spain). This is an observational study carried out during 1 year on a random sample of the pediatric population aged between 5 and 15 years. The levels of vitamin D (25(OH)D) were determined by immunoassay (ADVIA Centaur Vitamin D®). The results were classified as sufficient (> 20 ng/ml), insufficient (10-20 ng/ml) and deficient (< 10 ng/ml). RESULTS: 153 analyses of vitamin D were carried out (58.2% in girls and 41.8% in boys), distributed in two age groups: 5-10 (62) and 10-15 (91). 66% of the total of the sample presented some degree of vitamin D deficit (60.1% insufficient (92) and 5.9% (11) deficient). In Galicia, there is a high prevalence of vitamin D deficiency/insufficiency in the healthy population, which increases if the patients present some kind of chronic pathology, thus leading to a public health problem. It is advisable to increase the consumption of fortified foods and/or to reconsider the administration of vitamin supplements.

Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy.

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.

Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap.

Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant on this approach. (J Histochem Cytochem 49:803-808, 2001)

Phenotypic analysis of peripheral CD4+ CD8+ T cells in the rat.

Among peripheral T cells, the expression of CD4 and CD8 is almost mutually exclusive. However, here we show, using flow cytometric analysis, that ex vivo approximately 6% of rat T cells stained for both CD4 and CD8. These double positive cells were also detected by confocal microscopy. Only around 50% of double positive cells expressed the CD8beta chain, the remaining cells expressed the CD8alpha chain alone. Double positive cells were blast-like with a phenotype, distinct from that of either CD4 or CD8 single positive cells, suggestive of an activated state. Previous reports of double positive T cells have also suggested that coexpression of CD4 and CD8 is linked to the activation state of the cell. There was an indication that priming animals with a hapten-carrier complex increased the ratio of CD8alphaalpha : alphabeta expressing double positive T cells, although we did not detect an increase in the frequency of double positive T cells following priming. We also show that the frequency of double positive cells was reduced following thymectomy and with age. In conclusion, these studies show that peripheral T cells expressing both CD4 and CD8 can be detected in the rat and that they are phenotypically distinct from CD4 and CD8 single positive T cells.

Specialized transcription factories within mammalian nuclei.

Recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes is reviewed.

The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

The control of RNA synthesis from protein-coding genes is fundamental in determining the various cell types of higher eukaryotes. The activation of these genes is driven by promoter complexes, and RNA synthesis is performed by an enzyme mega-complex-the RNA polymerase II holoenzyme. These two complexes are the fundamental components required to initiate gene expression and generate the primary transcripts that, after processing, yield mRNAs that pass to the cytoplasm where protein synthesis occurs. But although this gene expression pathway has been studied intensively, aspects of RNA metabolism remain difficult to comprehend. In particular, it is unclear why >95% of RNA polymerized by polymerase II remains in the nucleus, where it is recycled. To explain this apparent paradox, this review presents a detailed description of nuclear RNA (nRNA) metabolism in mammalian cells. We evaluate the number of active transcription units, discuss the distribution of polymerases on active genes, and assess the efficiency with which the products mature and pass to the cytoplasm. Differences between the behavior of mRNAs on this productive pathway and primary transcripts that never leave the nucleus lead us to propose that these represent distinct populations. We discuss possible roles for nonproductive RNAs and present a model to describe the metabolism of these RNAs in the nuclei of mammalian cells.-Jackson, D. A., Pombo, A., Iborra, F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III.

Mammalian nuclei contain three different RNA polymerases defined by their characteristic locations and drug sensitivities; polymerase I is found in nucleoli, and polymerases II and III in the nucleoplasm. As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved 'physiological' buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br-UTP, before the resulting Br-RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that approximately 10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only approximately 2000 sites (diameter approximately 40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or 'factories', suggesting that different regions of the nucleus specialize in the transcription of different types of gene.

Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy.

The resolution of conventional light microscopy is limited to approximately 200 nm in the x- and y-axes and >500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100-200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of approximately 40 nm. (J Histochem Cytochem 47:471-480, 1999)

Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response.

Dendritic cells (DC) are thought to initiate Ab synthesis by activation of T cells, which then provide cytokine and cell-bound "help" to B cells. Here, we provide evidence that DC can capture and retain unprocessed Ag in vitro and in vivo, and can transfer this Ag to naive B cells to initiate a specific Ab response. The response is skewed with 4- to 13-fold higher titers of IgG than IgM, and the predominant subclasses of Ab produced in naive animals are those associated with Th2-type responses. Ag retention and the skew in class switching is a physiologic phenomenon because DC loaded with Ag in vivo and isolated 24 h later initiated a class-switched, Ag-specific Ab response in naive animals. In vitro studies confirmed that DC provide naive B cells with signals that are essential for the synthesis of class-switched Ab. Taken together, these observations show that DC have an important role in the initiation of Ab synthesis by direct interaction with B cells.

Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle.

PTF (PSE-binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter approximately 1.3 micron) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription 'factories' where Br-UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only approximately 30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes.

Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells.

In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of approximately 750 replication sites. The majority of these replication "foci" were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles. The majority of replication forks activated at the onset of S phase terminated 45-60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.

The topology of transcription by immobilized polymerases.

Current models for RNA synthesis involve an RNA polymerase that tracks along a static template. However, research on chromatin loops suggests that the template slides past a polymerase immobilized in a large transcription factory. The evidence for immobilized polymerases is reviewed, and a model for transcription by such fixed enzymes is presented. According to the model, gene activation would involve reducing gene-factory distance and increasing the affinity of a promoter for a factory. Locus controlling regions and enhancers would attach to a factory and increase the chances that a promoter could bind to a polymerase; after transcriptional termination, the gene would detach from the factory. As some RNA processing occurs cotranscriptionally, processing sites are also likely to be associated with the factory.