CALR but Not JAK2 Mutations Are Associated with an Overexpression of Retinoid X Receptor Alpha in Essential Thrombocythemia.

Essential thrombocythemia (ET) is a blood cancer caused by mutations in JAK2 and CALR. It is widely recognized that both mutations lead to the constitutive activation of JAK2/STAT signaling, although other JAK/STAT-independent pathogenic mechanisms triggered by these alterations have also been described in ET. In an attempt to study JAK2/STAT-independent mechanisms derived from CALR mutations, our research group created a C. elegans model with patient-like mutations in calreticulin that lacks JAK counterparts. The introduction of patient-like mutations in the calreticulin of C. elegans leads to an increase in the transcriptional expression of nhr-2, independently of JAK2/STAT activation. In the present study, we aim to verify if this mechanism is conserved in patients with ET harboring CALR mutations. To do so, we evaluated the expression of potential orthologs of nhr-2 in human cell lines of interest for the study, as well as in bone marrow (BM) or peripheral blood (PB) mononuclear cells from patients with CALR or JAK2 mutations. The results revealed that this mechanism is conserved in CALR-mutated ET patients, since CALR, but not JAK2 mutations, were associated with an overexpression of RXRA in patients with ET. The use of drugs targeting the activation or blockade of this target in the analyzed cell lines did not result in changes in cell viability. However, RXRA might be relevant in the disease, pointing to the need for future research testing retinoids and other drugs targeting RXRα for the treatment of ET patients.

Monitoring Caenorhabditis elegans molting in a conventional luminometer.

Molting is an essential developmental process in Caenorhabditis elegans. However, the study of molting in the worm has been limited by the lack of automated techniques that allow monitoring the process in a simple way. In 2015, Olmedo et al. published an automated method to monitor the timing of each larval stage and molt in C. elegans using bioluminescence. This new method has greatly contributed to the study of molting in this organism but requires the use of a high-sensitivity luminometer, which many laboratories do not have. We have adapted the method to a conventional luminometer, so that it can be used by most laboratories that work with C. elegans and do not have high-sensitivity equipment.•A customization of a method to study molting in C. elegans using a conventional luminometer instead of a high-sensitivity one.•This adaptation allows most laboratories to use their routine luminometers to study molting in C. elegans.•Although the use of a high-sensitivity luminometer, as proposed by Olmedo et al., remains the gold standard for studying molting, this adaptation is suitable for studying significant differences in molting and the duration of larval stages between different strains of C. elegans.

Optimizing simple calreticulin upregulation strategies in Caenorhabditis elegans.

Calreticulin (CALR) is a multifunctional calcium-binding protein whose expression levels have been correlated with detection, clinical phase of disease, metastasis, and survival of various types of cancer. Therefore, the study of the regulation of the cellular levels of CALR may be important to understand the neoplastic process. Caenorhabditis elegans, which has a CALR ortholog (CRT-1), has been used as a model organism for the characterization of CALR, and several conditions promoting the upregulation of crt-1 have been studied and established to understand the molecular control of crt-1 transcription and assess the function of the protein. Here, we propose several modifications of previously published crt-1 upregulation strategies that improve the reproducibility of the assay and allow to achieve higher levels of overexpression. First, the manipulation of synchronized populations of worms instead of mixed-stage animals and the use of solid culture medium in all experimental conditions are proposed. Likewise, we evaluate four new experimental approaches that attempt to promote a higher crt-1 upregulation [short-term exposure to 30 µg/ml tunicamycin at 25°C, short-term exposure to 7% ethanol (EtOH) at 25°C, short-term exposure to 30°C of worms grown at 25°C, and a long-term exposure to 7% EtOH]. Our results not only validate previously published methods, but also point to a new experimental approach that increases previously achieved levels of crt-1 upregulation. More specifically, a 6-h exposure of synchronized worms grown at 25°C to 7% EtOH on solid medium promotes almost a 7-fold upregulation of crt-1.

Transcriptomic Analysis Reveals JAK2/MPL-Independent Effects of Calreticulin Mutations in a C. elegans Model.

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research.

Importance of doctor-patient communication strategies / Importancia de las estrategias de comunicación entre médico y paciente

When we have a fluid and close communication with the patients, we can get better results. A bad doctor-patient communication contributes to increasing conflicts, reflected on complaints, demands and bad practice. The doctor patient interview determines the type of relationship you stablish, because when you get the trust of the patient the relationship is strong and efficient. The goal of the doctor patient relationship is to improve patient health and medical attention. This review focuses on the importance of the doctor-patient communication, and we offer some suggestions to improve it.

Establecer una comunicación efectiva con los pacientes favorece que se logren mejores resultados, pues una mala comunicación médico-paciente contribuye a que existan conflictos, manifestándose en quejas, demandas y mala praxis. La entrevista médico-paciente determina el tipo de relación que se establecerá, ya que al ganarse la confianza del paciente se logrará establecer una relación sólida, perdurable y productiva. El objetivo de dicha comunicación es mejorar la salud y la atención médica. Así pues, este artículo se enfoca en la importancia de la comunicación entre el doctor y el paciente, y hace sugerencias para mejorarla.

Analysis of genes encoding epigenetic regulators in myeloproliferative neoplasms: Coexistence of a novel SETBP1 mutation in a patient with a p.V617F JAK2 positive myelofibrosis.

In recent years it has been shown that the causes of chronic myeloproliferative neoplasms (MPNs) are more complex than a simple signaling aberration and many other mutated genes affecting different cell processes have been described. For instance, mutations in genes encoding epigenetic regulators are more frequent than expected. One of the latest genes described as mutated is SET binding protein 1 (SETBP1). In silico tools have revealed that there are several human SETBP1 paralogous to nuclear receptor binding SET domain protein 1 (NSD1), NSD2 and NSD3, for example, which are also involved in the development of other hematological malignancies. Therefore, the present study analyzed the mutational status of NSD1, NSD2, NSD3 and SETBP1 in BCR-ABL1 negative MPNs with or without Janus kinase 2 (JAK2) p.V617F mutation. The present study revealed that the NSD genes are not frequently mutated in MPNs. However, a novel SETBP1 mutation was identified in a patient with p.V617F JAK2 positive primary myelofibrosis. These results provide further insight into the genetic complexity of MPNs.

Borago officinalis seed oil (BSO), a natural source of omega-6 fatty acids, attenuates fat accumulation by activating peroxisomal beta-oxidation both in C. elegans and in diet-induced obese rats.

Obesity is a medical condition with increasing prevalence, characterized by an accumulation of excess fat that could be improved using some bioactive compounds. However, many of these compounds with in vitro activity fail to respond in vivo, probably due to the sophistication of the physiological energy regulatory networks. In this context, C. elegans has emerged as a plausible model for the identification and characterization of the effect of such compounds on fat storage in a complete organism. However, the results obtained in such a simple model are not easily extrapolated to more complex organisms such as mammals, which hinders its application in the short term. Therefore, it is necessary to obtain new experimental data about the evolutionary conservation of the mechanisms of fat loss between worms and mammals. Previously, we found that some omega-6 fatty acids promote fat loss in C. elegans by up-regulation of peroxisomal fatty acid ß-oxidation in an omega-3 independent manner. In this work, we prove that the omega-6 fatty acids' effects on worms are also seen when they are supplemented with a natural omega-6 source (borage seed oil, BSO). Additionally, we explore the anti-obesity effects of two doses of BSO in a diet-induced obesity rat model, validating the up-regulation of peroxisomal fatty acid ß-oxidation. The supplementation with BSO significantly reduces body weight gain and energy efficiency and prevents white adipose tissue accumulation without affecting food intake. Moreover, BSO also increases serum HDL-cholesterol levels, improves insulin resistance and promotes the down-regulation of Cebpa, an adipogenesis-related gene. Therefore, we conclude that the effects of omega-6 fatty acids are highly conserved between worms and obesity-induced mammals, so these compounds could be considered to treat or prevent obesity-related disorders.

Dihomo-gamma-linolenic acid induces fat loss in C. elegans in an omega-3-independent manner by promoting peroxisomal fatty acid ß-oxidation.

Bioactive compounds, including some fatty acids (FAs), can induce beneficial effects on body fat-content and metabolism. In this work, we have used C. elegans as a model to examine the effects of several FAs on body fat accumulation. Both omega-3 and omega-6 fatty acids induced a reduction of fat content in C. elegans, with linoleic, gamma-linolenic and dihomo-gamma-linolenic acids being the most effective ones. These three FAs are sequential metabolites especially in omega-6 PUFA synthesis pathway and the effects seem to be primarily due to dihomo-gamma-linolenic acid, and independent of its transformation into omega-3 or arachidonic acid. Gene expression analyses suggest that peroxisomal beta oxidation is the main mechanism involved in the observed effect. These results point out the importance of further analysis of the activity of these omega-6 FAs, due to their potential application in obesity and related diseases.

Tú en tu casa, yo en la mía: parejas lat -- / You in your house, I in mine: couples lat -

El texto que se presenta, da cuenta de algunas de las características propias de las parejas LAT, como posibilidad relacional-vincular en el mundo de la pareja; los autores consideran, de valor académico, la lectura y reflexión, referidos a la comprensión en terapia familiar sistémica, y cómo este modelo relacional es cada vez más evidente en las nuevas conformaciones de pareja en el mundo. El artículo está lejos de plantear recetas, recomendaciones y puntos para la intervención; su intención primera es la generación de preguntas y discusiones, a propósito de esta dinámica para vivir en pareja, en pareja LAT.

The text that is presented, accounts for some of the characteristics of LAT couples, such as relational-link possibility in the world of the couple; the authors consider, of academic value, the reading and reflection, referred to the understanding in systemic family therapy, and how this relational model is increasingly evident in the new conformations of couple in the world. The article is far from proposing recipes, recommendations and points for intervention; his first intention is the generation of questions and discussions, about this dynamic to live as a couple, as a LAT couple.

A simple approach for classifying new mutations as somatic or germinal in DNA samples lacking paired tissue.

When studying mutations in DNA samples, determining whether novel sequence changes are somatic mutations or germline polymorphisms can be difficult. Here we describe a novel and very simple approach for identification of somatic mutations and loss of heterozygosity (LoH) events in DNA samples where no matched tissue sample is available. Our method makes use of heterozygous polymorphisms that are located near the putative mutation to trace both germinal alleles.

A new KRT16 mutation associated with a phenotype of pachyonychia congenita.

Pachyonychia congenita is a rare, autosomal dominant genetic disease characterized by painful palmoplantar keratoderma and hypertrophic nail dystrophy. This disorder is caused by mutations in any one of five cytoskeletal keratin proteins, K6a, K6b, K6c, K16 and K17. Here, we describe a new p.Leu421Pro (c.1262T>C) mutation in the highly conserved helix termination motif of K16 in a large Spanish family. Bioinformatic analyses as well as previous descriptions in the literature of homologous mutations in other keratin-coding genes show that this mutation is probably causative of the disease.

CBL mutations in myeloproliferative neoplasms are also found in the gene's proline-rich domain and in patients with the V617FJAK2.

BACKGROUND: Despite the discovery of the p.V617F in JAK2, the molecular pathogenesis of some chronic myeloproliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617FJAK2-negative patients, mainly located in the RING finger domain. In order to determine the frequency of CBL mutations in these diseases, we studied different regions of all CBL family genes (CBL, CBLB and CBLC) in a selected group of patients with myeloproliferative neoplasms. We also included V617FJAK2-positive patients to check whether mutations in CBL and JAK2 are mutually exclusive events. DESIGN AND METHODS: Using denaturing high performance liquid chromatography, we screened for mutations in CBL, CBLB and CBLC in a group of 172 V617FJAK2-negative and 232 V617FJAK2-positive patients with myeloproliferative neoplasms not selected for loss of heterozygosity. The effect on cell proliferation of the mutations detected was analyzed on a 32D(FLT3) cell model. RESULTS: An initial screening of all coding exons of CBL, CBLB and CBLC in 44 V617FJAK2-negative samples revealed two new CBL mutations (p.C416W in the RING finger domain and p.A678V in the proline-rich domain). Analyses performed on 128 additional V617FJAK2-negative and 232 V617FJAK2-positive samples detected three CBL changes (p.T402HfsX29, p.P417R and p.S675C in two cases) in four V617FJAK2-positive patients. None of these mutations was found in 200 control samples. Cell proliferation assays showed that all of the mutations promoted hypersensitivity to interleukin-3 in 32D(FLT3) cells. CONCLUSIONS: Although mutations described to date have been found in the RING finger domain and in the linker region of CBL, we found a similar frequency of mutations in the proline-rich domain. In addition, we found CBL mutations in both V617FJAK2-positive (4/232; 1.7%) and negative (2/172; 1.2%) patients and all of them promoted hypersensitivity to interleukin-3.

La función endotelial es afectada por la lipemia posprandial: presentación de dos casos / Endothelial function is affected by postprandial lipemia: report of two cases

El consumo de comidas con alto contenido graso induce cambios agudos en los lípidos circulantes y disfunción endotelial horas después de la comida, condición que precede el desarrollo del proceso aterosclerótico. Aunque se desconoce exactamente el mecanismo responsable de la disminución de la función endotelial ocasionada por la lipemia post-prandial, se considera que la elevación de las lipoproteínas ricas en triglicéridos, sus remanentes y un estado de estrés oxidativo, son los principales mecanismos que determinan este hecho. Se presenta un reporte de dos adultos saludables de 30 años, a los cuales se les suministró un menú que aportaba 1049 calorías, 31 g de proteína, 79 g de grasa (31 g de grasa saturada), 666 mg de colesterol y 69 g de carbohidratos, con el propósito de describir el efecto que induce la lipemia post-prandial sobre la función endotelial, y su relación con algunos aspectos clínicos y bioquímicos asociados con este estado metabólico...

High-fat meal consumption induces acute changes in circulating lipid and then induces endothelial dysfunction after breakfast time, this dysfunction is associated with atherosclerotic process development. Although the explain mechanism that is responsible to induce endothelial function decrease, by post-prandial lipemia, is unknown. The elevation of lipoproteins high triglycerides and their remnants, and oxidative stress are the main mechanisms. In the present study it is report two healthy adults with median age 30 years, whom take a menu that contain: 1049 calories, 31 g protein, 79 g fat (31 g saturated fat), 666 mg cholesterol, 69 g carbohydrates. Thus, it is described the effect induced by post-prandial lipemia on endothelial function and the relationship of this metabolic state with clinical and biochemical features associated...

Quantification of PDGFRA alternative transcripts improves the screening for X-PDGFRA fusions by reverse transcriptase-polymerase chain reaction.

Hematological malignancies with eosinophilia are often associated with fusions in PDGFRA, PDGFRB, or FGFR1 genes. RT-PCR has proved to be useful for finding new PDGFRA gene fusions, but some studies have shown overexpression of the TK domain which cannot be explained by the existence of such aberrations. This fact could be related to the expression of alternative PDGFRA transcripts. We show that quantification of the expression of three different PDGFRA fragments discriminates between PDGFRA alternative transcripts and fusion genes, and we have tested this novel methodological approach in a group of eosinophilia cases. Our data show that alternative PDGFRA transcripts should be taken into account when screening for PDGFRA aberrations, such as gene fusions, by RT-PCR. Expression from an internal PDGFRA promoter seems to be a frequent event, in both normal and leukemic samples, and is probably related to physiological conditions, but it could have a role in other tumors. Even so, we show that our RQ-PCR methodology can discriminate expression of alternative transcripts from the presence of X-PDGFRA fusion genes.

A new potential oncogenic mutation in the FERM domain of JAK2 in BCR/ABL1-negative and V617F-negative chronic myeloproliferative neoplasms revealed by a comprehensive screening of 17 tyrosine kinase coding genes.

BCR/ABL1-negative chronic myeloproliferative neoplasms (CMPNs) are a heterogeneous group of clonal hematological malignancies. Over recent years, some genetic events in tyrosine kinase (TK) genes have been described as causal events of these diseases. To identify new genetic aberrations underlying these diseases, we used denaturing high performance liquid chromatography and fluorescence in situ hybridization (FISH) to analyze 17 genes from two receptor-TK families (III and IV) and from three cytoplasmic-TK families (Syk, Abl, and Jak) on samples from 44 BCR/ABL1-negative and JAK2(V617F)-negative CMPN patients with different clinical phenotypes. Although screening by FISH did not reveal novel chromosomal aberrations, several sequence changes were detected. None of them were frequent events, but we identified a new potential activating mutation in the FERM domain of JAK2(R340Q). None of the germline JAK2(V617F) single-nucleotide polymorphisms detected differed in distribution between patients and control subjects. In summary, data presented here show that these genes are not frequently mutated or rearranged in CMPNs, suggesting that molecular events causing these disorders must be located in other genes.