Enhancing adipose stem cell chondrogenesis: A study on the roles of dexamethasone, transforming growth factor β3 and ascorbate supplements and their combination
Published on: 31st July, 2017
OCLC Number/Unique Identifier: 7317597470
Varied exogenous chondrogenic factors (CFs) are implicated in promoting differentiation of stem cells along a chondrocyte lineage in the field of regenerative tissue engineering for articular cartilage repair. The effects of dexamethasone, transforming growth factor β3 (TGF-β3), ascorbate, and their combinations, on mRNA expression in micromass-cultured human adipose derived stem cells (hADSCs) were investigated as a function of time. Indices include chondrogenic, hypertrophic, angiogenic, fibrogenic and osteogenic markers along with mechanical properties, assessed by atomic force microscopy. Early in the culture, i.e., at day three, no significant differences in mRNA expression of SOX9, aggrecan, lubricin, Col XI, Col X, vascular endothelial growth factor, Col I, and alkaline phosphatase were observed among samples treated with different CFs. However, significant differences in mRNA expression levels of pre-mentioned markers among samples treated with each CF exist when samples were supplied with the CFs for more than three days. A new indexing scheme summing expression of chondrogenic and subtracting non-chondrogenic angiogenic, fibrogenic and osteogenic marker levels shows dexamethasone is the overall leading CF among the factors and their combinations. Based on this scheme, we have projected not only the possible signaling pathways which might be affected by addition of CFs but also hypothetical indexes that may occur upon temporal variation of growth factor regimens.
Cytokine Modulatory Effects of Sesamum Indicum Seeds Oil Ameliorate Mice with Experimental Autoimmune Encephalomyelitis
Published on: 30th October, 2017
OCLC Number/Unique Identifier: 7347013998
Background: Multiple sclerosis (MS) is an autoimmune disorder of the central nerve system (CNS), which affects the brain and spinal cord. Experimental autoimmune encephalomyelitis (EAE) is the most commonly applied experimental model for studying the MS. The aim of this study was to determine the effects of Sesamum indicum seeds oil on Experimental Autoimmune Encephalomyelitis (EAE) in mice.
Methods: Sesame oil was administrated intraperitoneally three days before immunization. IFN-γ, IL-10, IL-17 and TGF-β levels and mRNA expression in supernatant of and within cultured mononuclear cells were assessed.
Results: According to our results, sesame oil treated mice demonstrated significant disease severity reduction (P=0.01 and 0.001, respectively). Treated EAE mice also represented statistically significant delay in the onset of symptoms in comparison with control group. The average IFN-γ levels and mRNA of sesame oil treated EAE mice were less than untreated EAE group. IL-10 and TGF-β levels and mRNA did not differ significantly in sesame oil treated EAE mice in comparison to untreated EAE group. IL-17 levels and mRNA were also found to be decreased significantly in treated mice in comparison to untreated mice.
Conclusions: Even thoughTH1 and TH17 cells through secretion of IFN-γ and IL-17, respectively, are involved in the pathogenesis of multiple sclerosis and EAE, but IL-10 has been shown to exhibit suppressive effects on these disorders. It can be concluded that sesame oil is able to induce TH2 and TH17-related immune responses and suppress TH1 type in EAE
Prediction of protein Post-Translational Modification sites: An overview
Published on: 2nd March, 2018
OCLC Number/Unique Identifier: 7355974901
Post-translational modification (PTM) refers to the covalent and enzymatic modification of proteins during or after protein biosynthesis. In the protein biosynthesis process, the ribosomal mRNA is translated into polypeptide chains, which may further undergo PTM to form the product of mature protein [1]. PTM is a common biological mechanism of both eukaryotic and prokaryotic organisms, which regulates the protein functions, the proteolytic cleavage of regulatory subunits or the degradation of entire proteins and affects all aspects of cellular life. The PTM of a protein can also determine the cell signaling state, turnover, localization, and interactions with other proteins [2]. Therefore, the analysis of proteins and their PTMs are particularly important for the study of heart disease, cancer, neurodegenerative diseases and diabetes [3,4]. Although the characterization of PTMs gets invaluable insight into the cellular functions in etiological processes, there are still challenges. Technically, the major challenges in studying PTMs are the development of specific detection and purification methods.
Role of toll-like receptors and their ligands in adipocyte secretion
Published on: 8th April, 2021
OCLC Number/Unique Identifier: 9031345560
Background: Adipose tissue is one of the main sites of energy homeostasis that regulates whole body metabolism with the help of adipokines. Disruption in its proper functioning results in adipose tissue remodeling (primarily hypertrophy and hyperplasia) which directly influences the secretion of said adipokines. Obesity characterized as chronic low-grade inflammation of the adipose tissue is one such condition that has far reaching effects on whole body metabolism. Inflammation in turn results in immune cells infiltrating into the tissue and further promoting adipocyte dysfunction.
Purpose: In our study we explored this adipose tissue-innate immunity axis by differentiating adipose tissue derived stem cells (ADSCs) into white and beige adipocytes. We further stimulated our cultures with lipopolysaccharide (LPS), flagellin, or meteorin-like, glial cell differentiation regulator (METRNL) to trigger an inflammatory response. We then evaluated Toll-like receptor (TLR) mRNA expression and secretion of interleukin (IL-6), interleukin-8 (IL-8), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) in these cultures.
Results: We found that TLR2 is the highest expressed receptor in adipocytes. Further, LPS and METRNL are strong activators of TLR2 in white and beigeBMP7(-) adipocytes. TLR4 was not significantly expressed in any of our cultures despite LPS stimulation. TLR9 expression is upregulated in ADSCs upon LPS and METRNL stimulation. IL-6 and IL-8 secretion is increased upon LPS stimulation in white adipocytes. METRNL activates both IL-6 and IL-8 expression in adipocyte cultures. Lastly, BDNF and NGF is secreted by all adipocyte cultures with beigeBMP7(-) and beigeBMP7(+) secreting slightly higher amounts in comparison to white adipocytes.
Conclusion: ADSCs and adipocytes alike are capable of expressing TLRs, but white adipocytes remain the highest expressing in both control and stimulated cultures. TLR2 is highly expressed in white and beige adipocytes whereas TLR4 showed no significant expression. LPS and METRNL trigger IL-6 and IL-8 secretion in adipocytes. Products of white adipocyte “browning” are capable of secreting higher amounts of BDNF and NGF in comparison to white adipocytes.
Expression of C-type Natriuretic Peptide and its Specific Guanylyl Cyclase-Coupled Receptor in Pig Ovarian Granulosa Cells
Published on: 22nd August, 2018
OCLC Number/Unique Identifier: 7844580667
Background: C-type natriuretic peptide (CNP) was isolated from porcine brain and is a 22-amino acid peptide which belongs to the natriuretic peptide (NP) family. Even though this peptide shares structural similarity to other endogenous NPs including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) its receptor selectivity is different from other NPs. The present study was undertaken to investigate the expression of C-type natriuretic peptide (CNP) and its specific guanylyl cyclase (GC)-coupled receptor in the granulosa cells of the pig ovarian follicle.
Results: Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the granulosa cell layer of the ovarian follicle with an apparent dissociation constant (Kd>) and a maximal binding capacity (Bmax) of 1.41±0.39 nM and 2.75±0.65 fmol/mm2 respectively. Binding of 125I-[Tyr0]-CNP(1-22) to these sites was also prevented by atrial natriuretic peptide (ANP(1-28)), brain natriuretic peptide (BNP(1-26)) and des[Gln18,Ser19,Gly20, Leu21,Gly22] ANP(4-23) (C-ANP). Production of 3’,5’-cyclic guanosine monophosphate (cGMP) by particulate GC in the granulosa cell membranes was stimulated by natriuretic peptides (NPs) with a rank order of potency of CNP(1-22)>>BNP(1-26)>ANP(1-28). HS-142-1, a selective antagonist of the two recognized GC-coupled NPRs, inhibited CNP(1-22)-stimulated cGMP production in granulosa cell membranes in a dose-dependent manner. Also mRNAs for all three recognized NPRs were detected in granulosa cells using reverse transcriptase-polymerase chain reaction (RT-PCR). Serial dilution curves of granulosa cell extracts were parallel to the standard curve of synthetic CNP.
Conclusion: These results indicate that CNP and its specific receptor are expressed in the granulosa cells of the pig ovary, and suggest that CNP may be a local autocrine and/or paracrine regulator via activation of its specific GC-coupled receptor, NPR-B.
Pseudoephedrine protects mice from infection of H1N1 virus
Published on: 28th February, 2020
OCLC Number/Unique Identifier: 8561620214
Ephedra, an ancient herb, is applied to treat common cold and influenza for such a long time in China. Pseudoephedrine is a main active ingredient from Ephedra which is used for relieving nasal congestion clinically. We previously reported that pseudoephedrine showed a potent anti-inflammatory effect other than sympathomimetic effects. In the present study, we aimed to investigate whether pseudoephedrine could protect mice from the H1N1 virus infection. The mice were infected with a 20% LD50 influenza A virus (IAV) suspension via intranasal administration to establish a virus infection model. Further, the mice were orally administered pseudoephedrine or oseltamivir for 4 days from one day after infection. Our results showed that pseudoephedrine improved lung pathological damage during the IVA infection period, and it dramatically increased the survival rate and attenuated loss of body weight compared with the virus-infected control group. In addition, pseudoephedrine inhibited the cytokine storms and mRNAs expression of the TLR7 signaling pathway. Surprisingly, pseudoephedrine showed an inhibitory effect on the replication of IAV. These results give clear evidence that pseudoephedrine is a potential anti-influenza drug by blunting cytokine storms and inhibition of replication of IAV, and following these results, we speculate that it should be tested in the novel coronavirus pneumonia (COVID-19, a severe epidemic in China currently) in which the cytokine storms play a key role to damage bronchi and lung in the early stage.
Collecting duct PGE2 responses reduce water loss with empagliflozin in mice with type 2 diabetes mellitus
Published on: 8th April, 2021
OCLC Number/Unique Identifier: 9022394723
Introduction: Sodium-glucose cotransporter 2 inhibitors such as empagliflozin (EMPA) protect against diabetic kidney disease. Prostaglandin E2 (PGE2) the main renal product of cyclooxygenase-2, inhibits vasopressin (AVP)-water reabsorption in the collecting duct (CD). The novelty of this study is that for the first time, we examined if EMPA affects the renal PGE2/EP receptor system and determined if CD responses to EMPA prevent water loss.
Methods: Four groups of adult male mice were studied after 6 weeks of treatment: control (db/m), db/m+EMPA (10 mg/kg/day in chow), type 2 diabetic diabetic/dyslipidemia (db/db), and db/db+EMPA. Tubules were microdissected for quantitative polymerase chain reaction (qPCR) and CD water transport was measured in response to AVP, with or without PGE2.
Results: Hyperglycemia and albuminuria were attenuated by EMPA. Renal mRNA expression for COX, PGE synthase, PGE2 (EP) receptor subtypes, CD AVP V2 receptors and aquaporin-2 was elevated in db/db mice, but unchanged by EMPA. Urine PGE2 levels increased in db/db but were unchanged by EMPA. AVP-water reabsorption was comparable in db/m and db/m+EMPA, and equally attenuated to 50% by PGE2. In db/db mice, AVP-water reabsorption was reduced by 50% compared to non-diabetic mice, and this reduction was unaffected by EMPA. In db/db mice, AVP-stimulated water transport was more significantly attenuated with PGE2 (62%), compared to non-diabetic mice, but this attenuation was reduced in response to EMPA, to 28%.
Conclusion: In summary, expression of renal PGE2/EP receptors is increased in db/db mice, and this expression is unaffected by EMPA. However, in diabetic CD, PGE2 caused a greater attenuation in AVP-stimulated water reabsorption, and this attenuation is reduced by EMPA. This suggests that EMPA attenuates diabetes-induced excess CD water loss.
SARS-CoV-2, the emperor’s new clothes and medical tyranny
Published on: 10th March, 2022
SARS-CoV-2 revisits a children’s fairy tale, the Emperor’s New Clothes. The swindler- salesmen are Biden, Fauci, et al. The magical clothes are their deliberate “pandemic of fear,” and the duped emperor is the American public.Extensive evidence is presented here of a great scam. The data details the true and low health risks of SARS-CoV-2; viral biology of natural immunity and the immune response from experimental mRNA gene therapy; side effects of the “jab;” and the draconian consequences of federal mandates. Differences between official pronouncements and scientific data are highlighted.The goal of the SARS-CoV-2 Big Con or scam is the nullification of the U.S. Bill of Rights in order to restore tyranny over the American public. We the People can fight for freedom with ballots and dollars.
New insights of liquid biopsy in ovarian cancer
Published on: 29th September, 2022
Through the development of new analysis technologies, many issues regarding the approach to tumoral diseases have been elucidated. With analytical assays developed in the last years, various omics technologies have evolved in such a manner that the characteristics of tumor cells and products can be evaluated (assessed) in the bloodstream of cancer patients at different times. Ovarian Cancer (OC) is one of the most difficult to diagnose umors, with low survival rates due to the high heterogeneity of these diseases that are distinct in terms of etiology and molecular characteristics, but which simply share an anatomical appearance. Recent findings have indicated that several types of ovarian cancer classified into different histotypes are in fact derived from non-ovarian issues and share few molecular similarities. Within this context, ovarian cancer screening and diagnosis can be made through the evaluation of circulating tumor cells in peripheral blood using liquid biopsy technologies. Advances in the study of various molecules analyzed by liquid biopsy have shown that elucidation of intratumoural and intertumoural heterogeneity and spatial and temporal tumor evolution could be traced by serial blood tests rather than by histopathological analyses of tissue samples from a primary tumor. Therefore, evaluation of some molecules such as circulating tumor cells (CTC), circulating tumor DNA (ctDNA), circulating cell-free RNA (non-coding and mRNA, extracellular vesicles), tumor-educated platelets or different miRNAs using liquid biopsy could lead to improvement of patient management.
A relevant article, white papers and other documents concerning the mRNA vaccine: an interesting collection useful to better understand some phenomena and to generate hypotesys
Published on: 13th October, 2022
The aim of this work is to report some interesting references and documents related to the mRNA vaccine. Every cited reference can act as an instrument to better understand what some independent researchers find: this work is mainly focused on the public debate related presence/absence of graphene derivates in some vaccine vials for COVID-19. Relevant classic articles White Papers or other kinds of a document are useful instruments to better clarify some crucial aspects of this infectious disease, its epidemiology and the efficacy of the measure adopted (preventive, therapeutic, or vaccine).If the reviewed article show an intrinsic recognized international value other kinds of source like a White Paper or other documents can be of interest to generate hypotheses or to open public discussion on crucial topics. For this reason, it is useful to use this method also in the discussion related to impurity profiles in new innovative bio-pharmaceuticals like the mRNA vaccine. The fact that this product was introduced with an emergency authorization, whit reduced the time of experimentation and due to the lack of official quality information related to some raw materials used authorizes us to use also this method.Even if the international official regulatory agency does not find this substance in the control for release of the lots it is interesting to more deeply investigate what funded by an independent researcher in some vials of vaccine or sample patient’s blood after vaccination. This reference even if not a smoking gun the same can stimulate the reasoning about the general concept involved. It is needed, to read this work, to consider the intrinsic limitation of some of the study research reported (White Paper) or the other documents.
SARS-CoV-2 antibody response to third dose vaccination in a healthy cohort
Published on: 23rd November, 2022
Determining the extent of immunity induced by booster doses of COVID-19 vaccinations is crucial for informing recommendations for booster dose regimens as well as constant adjustments of immunization strategies amongst different groups of people within the population. The study involved 31 healthy volunteers (majority were healthcare professionals) who completed either vaccination course with Pfizer or Moderna mRNA vaccines and received a third dose of the vaccine. Here we report results on the evaluation of an antibody response to four different SARS-CoV-2 antigens: RBD, S1, S2 and nucleocapsid prior to third dose and two and four weeks after a booster vaccination. We detected a peak of high titers of antibodies after the third dose with a gradual decline after four weeks. No significant differences were seen between the two vaccines in terms of antibody response. There were no gender discrepancies between the two vaccines. Our results suggest that: third doses are necessary due to the emergence of different SARS-CoV-2 variants and postvaccination antibody testing continues be essential in determining possible standardization of SARS-CoV-2 vaccines regimens.
Design and optimization of mRNAs encoding an Anti-TIGIT antibody with therapeutic potential for cancer in TIGIT-humanized BALB/c Mice
Published on: 7th April, 2023
mRNA drugs are synthesized using cell-free systems without complex and stringent manufacturing processes, which makes their preparation simple, efficient, and economical. Over the past few years, mRNAs encoding antibodies have been one of the research frontiers of antibody drug development. In cancer immunotherapy, mRNAs encoding immune checkpoint antibodies may be advantageous regarding antibody persistence and durability of the anti-tumor immune response of patients. In our previous study, a candidate antibody—AET2010—targeting the novel immune checkpoint TIGIT was reported. Its anti-tumor activity was also investigated using adoptive transfer of NK-92MI cells in a xenograft mouse model, but the limitations of the model did not facilitate precise evaluation. In the present study, we further investigated the therapeutic potential of AET2010 for cancer in TIGIT-humanized BALB/c mice. Next, we explored the design, synthesis, and optimization of mRNAs encoding AET2010 and ultimately obtained a candidate mRNA (mRNA-BU) with favorable in vitro and in vivo expression levels of active AET2010. Particularly, lipid-nanoparticle-encapsulated mRNA-BU delivered to mice produced AET2010 with significantly higher peak concentration and expression duration than an equivalent dose of original AET2010. This study provides a sound basis for developing novel drugs targeting TIGIT.
A Water-soluble Form of Dihydroquercetin Reduces LPS-induced Astrogliosis, Vascular Remodeling, and mRNA VEGF-A Levels in the Substantia Nigra of Aged Rats
Published on: 16th February, 2024
Background: The age-dependent sporadic form of PD is characterized by the degeneration of dopaminergic (DA) neurons in the Substantia Nigra (SN), gliosis, and vascular changes. Vascular changes may contribute to the onset of the disease and exacerbate the neurodegenerative process, as some vascular changes occur before the onset of neuronal loss. To demonstrate the anti-neuroinflammatory efficacy of a new compound, a water-soluble form of dihydroquercetin (DHQ-WF), we studied the structural changes of microcirculatory vasculature, astroglial GFAP, and vascular endothelial growth factor –A (VEGF-A) mRNA expression in the SN of young and old rats after unilateral nigral treatment by lipopolysaccharide (LPS) and oral administration of DHQ-WF.Materials and methods: The experiments were performed on 18 young (8 weeks - 10 weeks old; 250 g - 320 g) and 18 old (18 months - 19 months old; 390 g - 450 g) male Vistar rats. Young and adult rats from the experimental groups were stereotactically injected with 2 μL LPS solution (LPS from Escherichia coli; 0,01 μL/mL) into one side of the SN. Control young and old rats were similarly injected with 2 μL sterile saline. Half of the animals in both the control and experimental groups (6 animals in each group) received a 2 ml solution containing DHQ-WF at a concentration of 3 mg/ml orally every day. After 8 weeks, brains were harvested and serial cryostat sections were prepared for histochemical (FITC-labeled tomato lectin), immunohistochemical (anti-GFAP Antibody, Cy3 Conjugate) staining, and real-time PCR (mRNA VEGF-A).Results: Eight weeks after LPS injection into the SN, a significant excess of areas occupied by cell bodies and processes of astroglial cells, the density of microcirculatory vessels, and mRNA VEGF-A expression was observed in old animals compared to control old animals and young LPS-treated rats. Oral administration of DHQ-WF to LPS-treated rats resulted in a significant reduction of these parameters in old animals.Conclusion: Injection of LPS into rat SN induces neuroinflammation and vascular angiogenesis, maximally expressed in old animals. Administration of DHQ-WF for 8 weeks significantly reduces these LPS-induced changes. DHQ-WF may be an effective treatment for reducing the effects of neuroinflammation in the aging brain.
Effect of TAK242 on MCP-1 and TGF-β in COPD Rats
Published on: 13th March, 2024
Objective: To investigate the mechanism of MCP-1 and TGF-β regulation by TAK242 in COPD rats. Methods: Thirty-six SD rats were randomly divided into normal, COPD control, and TAK242 groups. The normal group was freely fed, and the other groups used the method of fumigation plus lipopolysaccharide tracheal drip to establish an experimental animal model of COPD. After successful modeling, each experimental group received 0.9% NaCl solution and corresponding drugs by intraperitoneal injection for 7 d. After drug administration, lung function was examined; pathological changes in lung tissue were observed by light microscopy with hematoxylin-eosin staining; mRNA expression of MCP-1 and TGF-β was detected by q-PCR; and protein expression of MCP-1 and TGF-β in lung tissue was detected by Western blot and IHC, TGF-β protein expression in rat lung tissue. Results: Compared with the normal group, rats in the COPD control group showed signs and symptoms of COPD, decreased lung function, and increased expression of MCP-1 and TGF-β. The TAK242 group showed decreased expression of MCP-1 and TGF-β compared to the COPD control group. Conclusion: MCP-1, and TGF-β played a crucial role in the early stage of COPD fibrosis. TAK242 could ameliorate airway inflammation and inhibit the progression of COPD lung fibrosis in pre-existing rats in COPD model rats.
Next Generation Tools in mRNA Purification: The Role of Continuous Raman Spectroscopy Testing with Pretreatment of the Sample
Published on: 29th March, 2024
In the biopharmaceutical production field, the purification process is a crucial step in order to obtain Drugs with an impurity profile according to the regulatory agency requirement.The aim of this work is to verify some relevant and recent literature and after analysis to submit to the researcher new Solutions in order to improve global safety and the toxicological profile: Submit a project related to the continuous testing of the purified materials using Raman spectroscopy – with pre-treatment of the sample: using solvents.Nanolipis Payload of Biopharmaceutical is not efficiently detected by direct Raman spectroscopy allowed by the regulatory agency for PAT process analytical technology.
Assessment of Redox Patterns at the Transcriptional and Systemic Levels in Newly Diagnosed Acute Leukemia
Published on: 29th April, 2024
Background: Acute leukemia is the result of clonal transformation and proliferation of a hematopoietic progenitor giving rise to poorly differentiated neoplastic cells. Reactive oxygen species play a role in maintaining the quiescence, self-renewal, and long-term survival of hematopoietic stem cells, but it is unclear how they would affect disease onset and progression. The aim is to evaluate, at the transcriptional and systemic level, the oxidative-inflammatory status in newly diagnosis acute leukemia patients. Methods: Seventy acute leukemia patients [26 acute lymphoblastic leukemia (ALL), 13 Acute Promyelocytic Leukemia (APL), and 31 Acute Myeloid Leukemia (AML)] and forty-one healthy controls were analyzed. Malondialdehyde and catalase activity were evaluated. Gene expression of NRF2, SOD, PRDX2, CAT, IL-6, and TNF-α was analyzed by real-time PCR.Results: Malondialdehyde concentration was similar in all groups studied. Catalase activity was significantly higher in AML and APL patients compared to controls, while ALL showed similar activity to the healthy group. NRF2, CAT, and PRDX2 expression levels were similar between groups, SOD expression was downregulated in all acute leukemia patients. TNF-α expression was lower in AML groups than in healthy individuals, and IL-6 mRNA expression was downregulated in ALL and APL.Conclusion: This is the first report that correlates transcriptional and systemic parameters associated with the oxidative inflammatory status in newly diagnosed acute leukemia. Some of the parameters evaluated could be used as biomarkers in the selection of an effective therapeutic strategy and will open new directions for the follow-up and evolution of this disease.
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