The metabolic properties of omega-6 fatty acid consumption are being increasingly accepted. We had previously observed that supplementation with a borage seed oil (BSO), as a source of linoleic ...(18:2n-6; LA) and gamma-linolenic (18:3n-6; GLA) acids, reduces body weight and visceral adiposity and improves insulin sensitivity in a diet-induced obesity model of Wistar rats. Here, it was investigated whether the anti-obesogenic properties of BSO could be maintained in a pre-obese model of rats, and if these effects are enhanced by a combination with low doses of quercetin, together with its potential role in the regulation of the adipocyte biology. The combination of BSO and quercetin during 8 weeks was able to ameliorate glucose intolerance and insulin resistance, and to improve liver steatosis. Although no effects were observed on body weight, animals supplemented with this combination exhibited a lower proportion of visceral adiposity. In addition, in vitro differentiation of epididymal adipose-precursor cells of the BSO-treated animals exhibited a down-regulation of Fasn, Glut4, Pparg and Srebp1 genes, in comparison with the control group. Finally, in vitro evaluation of the components of BSO demonstrated that the anti-adipogenic activity of quercetin was significantly potentiated by the combination with both LA and GLA through the down-regulation of different adipogenesis-key genes in 3T3-L1 cells. All these data suggest that omega-6 fatty acids LA and GLA, and their natural sources such as BSO, could be combined with quercetin to potentiate their effects in the prevention of the excess of adiposity and the insulin resistance.
There is growing evidence that
-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery,
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
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
model that naturally lacks
and
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
-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
. 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
strain with patient-like mutations is proposed as a potential model for leukemia research.
Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and ...acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.
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
(
).
tools have revealed that there are several human
paralogous to
(
),
and
, for example, which are also involved in the development of other hematological malignancies. Therefore, the present study analyzed the mutational status of
and
in
negative MPNs with or without
(
) p.V617F mutation. The present study revealed that the NSD genes are not frequently mutated in MPNs. However, a novel
mutation was identified in a patient with p.V617F
positive primary myelofibrosis. These results provide further insight into the genetic complexity of MPNs.
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.
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.
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.
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.
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•Origanum vulgare L. hydroalcoholic extract shows antioxidant activity in vitro and in vivo.•Oral formulation of oregano preserves antioxidant activity from gastrointestinal ...digestion.•Encapsulation improve antioxidant activity of O. vulgare in C. elegans.•Rosmarinic acid and 3,4-dihydroxybenzoic acid seem to be the major bioactive compounds of O. vulgare, even after digestion.
Aging-related diseases can be triggered by multiple factors such as oxidative stress. Oxidative stress is an imbalance between free radicals and antioxidants, so today, compounds capable of reducing or neutralizing free radicals are being studied for a therapeutic use. Origanum vulgare L. is a traditional medicinal plant used for a wide number of health problems due to its antimicrobial, carminative and antioxidant activities. However, when administered orally, gastrointestinal digestion can modify some of therapeutical properties. To avoid this, two different solid oral formulations have been designed for an O. vulgare extract evaluating their antioxidant behaviours in vitro and in vivo after a simulation of gastrointestinal digestion. The results showed that the divided powder has a lower antioxidant activity both in vitro and in vivo than the encapsulated extract. The quantitative difference of polyphenols found on HPLC-DAD (especially luteolin, apigenin and caffeic acid) may explain the differences in pharmacological activity. Thus, we propose that the best form to administrate O. vulgare extracts to maintain the antioxidant properties is the encapsulated form, that is, two capsules of 250 mg of a hydroalcoholic extract of O. vulgare with a minimum of 33 % of rosmarinic acid as a daily dose.
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The aim of this work was to prepare and evaluate cyclodextrins-modified poly(anhydride) nanoparticles to enhance the oral administration of glibenclamide. A conjugate polymer was ...synthesized by incorporating hydroxypropyl-β-cyclodextrin to the backbone of poly(methylvinyl ether-co-maleic anhydride) via Steglich reaction. The degree of substitution of anhydride rings by cyclodextrins molecules was calculated to be 4.9% using H-NMR spectroscopy. A central composite design of experiments was used to optimize the preparative process. Under the optimal conditions, nanoparticles displayed a size of about 170 nm, a surface charge of −47 mV and a drug loading of 69 µg GB/mg. X-ray diffraction studies confirmed the loss of the crystalline structure of GB due to its dispersion into the nanoparticles, either included into cyclodextrin cavities or entrapped in the polymer chains. Glibenclamide was mainly release by Fickian-diffusion in simulated intestinal fluid. GB-loaded nanoparticles produced a hypolipidemic effect over C. elegans N2 wild-type and daf-2 mutant. The action mechanism included daf-2 and daf-28 genes, both implicated in the insulin signaling pathway of C. elegans. In summary, the covalent linkage of cyclodextrin to the poly(anhydride) backbone could be an interesting strategy to prepare nanoparticles for the oral administration of glibenclamide.
The oral administration of therapeutic proteins copes with important challenges (mainly degradation and poor absorption) making their potential therapeutic application extremely difficult. The aim of ...this study was to design and evaluate the potential of the combination between mucus-permeating nanoparticles and permeation enhancers as a carrier for the oral delivery of the monoclonal antibody bevacizumab, used as a model of therapeutic protein. For this purpose, bevacizumab was encapsulated in PEG-coated albumin nanoparticles as a hydrophobic ion-pairing complex with either sodium deoxycholate (DS) or sodium docusate (DOCU). In both cases, complex formation efficiencies close to 90% were found. The incorporation of either DS or DOCU in PEG-coated nanoparticles significantly increased their mean size, particularly when DOCU was used. Moreover, the diffusion in mucus of DOCU-loaded nanoparticles was significantly reduced, compared with DS ones. In a
C. elegans
model, DS or DOCU (free or nanoencapsulated) disrupted the intestinal epithelial integrity, but the overall survival of the worms was not affected. In rats, the relative oral bioavailability of bevacizumab incorporated in PEG-coated nanoparticles as a complex with DS (B-DS-NP-P) was 3.7%, a 1000-fold increase compared to free bevacizumab encapsulated in nanoparticles (B-NP-P). This important effect of DS may be explained not only by its capability to transiently disrupt tight junctions but also to their ability to increase the fluidity of membranes and to inhibit cytosolic and brush border enzymes. In summary, the current strategy may be useful to allow the therapeutic use of orally administered proteins, including monoclonal antibodies.
Graphical Abstract
Abstract
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.
Abstract 4683
Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 ...fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors.
No relevant conflicts of interest to declare.
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