搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Metabolic imaging of brain tumors using (13)C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-(13)C] pyruvate is a promising neuroimaging strategy which,after a decade of preclinical success in glioblastoma (GBM) models,is now entering clinical trials in multiple centers. Typically,the presence of GBM has been associated with elevated hyperpolarized [1-(13)C] lactate produced from [1-(13)C] pyruvate,and response to therapy has been associated with a drop in hyperpolarized [1-(13)C] lactate. However,to date,lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation,which,in addition to initiating tumor development,also induces metabolic reprogramming. In particular,mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1,MCT4),three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of (13)C MRS of hyperpolarized [1-(13)C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA,MCT1 and MCT4,and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-(13)C] lactate compared to GBM,consistent with their metabolic reprogramming. Furthermore,hyperpolarized [1-(13)C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors,in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas,which,when combined with other clinically available imaging methods,could be used to detect the presence of the IDH1 mutation in vivo. View Publication

Activation-induced cytidine deaminase (AID) mediates immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM),critical processes for maturation of the antibody response. Epigenetic factors,such as histone deacetylases (HDACs),would underpin B cell differentiation stage-specific AID expression. Here,we showed that NAD+-dependent class III HDAC sirtuin 1 (Sirt1) is highly expressed in resting B cells and down-regulated by stimuli inducing AID. B cell Sirt1 down-regulation,deprivation of NAD+ cofactor,or genetic Sirt1 deletion reduced deacetylation of Aicda promoter histones,Dnmt1,and nuclear factor-$\kappa$B (NF-$\kappa$B) p65 and increased AID expression. This promoted class-switched and hypermutated T-dependent and T-independent antibody responses or led to generation of autoantibodies. Genetic Sirt1 overexpression,Sirt1 boost by NAD+,or allosteric Sirt1 enhancement by SRT1720 repressed AID expression and CSR/SHM. By deacetylating histone and nonhistone proteins (Dnmt1 and NF-$\kappa$B p65),Sirt1 transduces metabolic cues into epigenetic changes to play an important B cell-intrinsic role in modulating antibody and autoantibody responses. View Publication

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma-null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34(+) human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks),human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses,as documented by the presence of CD4(+) CD8(+) T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation,human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses,the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune/inflammatory disease characterized by autoantibody production and abnormal T cells that infiltrate tissues through not well-known mechanisms. We report that SLE T lymphocytes display increased levels of CD44,ezrin,radixin,and moesin (ERM) phosphorylation,stronger actin polymerization,higher polar cap formation,and enhanced adhesion and chemotactic migration compared with T cells from patients with rheumatoid arthritis and normal individuals. Silencing of CD44 by CD44 small interfering RNA in SLE T cells inhibited significantly their ability to adhere and migrate as did treatment with Rho kinase and actin polymerization inhibitors. Forced expression of T567D-ezrin,a phosphorylation-mimic form,enhanced remarkably the adhesion and migration rate of normal T cells. Anti-CD3/TCR autoantibodies present in SLE sera caused increased ERM phosphorylation,adhesion,and migration in normal T cells. pERM and CD44 are highly expressed in T cells infiltrating in the kidneys of patients with lupus nephritis. These data prove that increased ERM phosphorylation represents a key molecular abnormality that guides T cell adhesion and migration in SLE patients. View Publication

Charge-coupled device detectors are vulnerable to cosmic rays that can contaminate Raman spectra with positive going spikes. Because spikes can adversely affect spectral processing and data analyses,they must be removed. Although both hardware-based and software-based spike removal methods exist,they typically require parameter and threshold specification dependent on well-considered user input. Here,we present a fully automated spike removal algorithm that proceeds without requiring user input. It is minimally dependent on sample attributes,and those that are required (e.g.,standard deviation of spectral noise) can be determined with other fully automated procedures. At the core of the method is the identification and location of spikes with coincident second derivatives along both the spectral and spatiotemporal dimensions of two-dimensional datasets. The method can be applied to spectra that are relatively inhomogeneous because it provides fairly effective and selective targeting of spikes resulting in minimal distortion of spectra. Relatively effective spike removal obtained with full automation could provide substantial benefits to users where large numbers of spectra must be processed. View Publication

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable,reproducible and controllable cultivation conditions. Up to now,some devices have been developed for the cultivation of cells under submersed conditions. However,systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose,a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts),which can be adjusted for the medium level (ultrasonic pulse-echosensor),time and volume-dependent medium exchange,and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells. View Publication

With a high global incidence of over three million new cases in 2020 and a high mortality of over two million fatalities,ovarian cancer is one of the most common malignant tumors in gynecology. Exosomes can control the immunological condition of the tumor microenvironment (TME) by participating in intercellular interactions. Therefore,we aimed to construct an exosome-related prognostic model to predict the clinical outcomes of ovarian cancer patients. In this research,expression patterns of exosome-related genes were examined in multiple single-cell RNA-sequencing and bulk RNA-sequencing datasets. In addition,a novel exosome-related prognostic model was established by the least absolute shrinkage and selection operator (LASSO) regression method. Then,the correlations between risk score and immunological characteristics of the TME were explored. Moreover,SERPINB1,a gene in the prognostic signature,was further analyzed to reveal its value as a novel biomarker. In the current study,combined with single-cell and bulk omics datasets,we constructed an exosome-related prognostic model of four genes (LGALS3BP,SAT1,SERPINB1,and SH3BGRL3). Moreover,the risk score was associated with worse overall survival (OS) in ovarian cancer patients. Further analysis found that patients with high-risk score tended to shape a desert TME with hardly infiltration of immune cells. Then,SERPINB1,positively correlated with the favorable OS and negatively with the risk score,was chosen as the representative biomarker of the model. Moreover,SERPINB1 was positively correlated with the infiltration of immune subpopulations in both public and in-house cohort. In addition,the high-resolution analysis found that SERPINB1 + tumor cells communicated with microenvironment cells frequently,further explaining the potential reason for shaping an inflamed TME. To sum up,we established a novel exosome-related prognostic model (LGALS3BP,SAT1,SERPINB1,and SH3BGRL3) to predict the prognosis of patients with ovarian cancer and identify the immunological characteristics of the TME. In addition,SERPINB1 was identified as a promising biomarker for prognostic prediction in ovarian cancer. The online version contains supplementary material available at 10.1186/s13048-025-01589-3. View Publication

The use of antiretroviral therapy (ART) has remarkably decreased the morbidity associated with HIV-1 infection,however,the prevalence of HIV-1-associated neurocognitive disorders (HAND) is still increasing. The blood-brain barrier (BBB) is the major impediment for penetration of antiretroviral drugs,causing therapeutics to reach only suboptimal level to the brain. Conventional antiretroviral drug regimens are not sufficient to improve the treatment outcomes of HAND. In our recent report,we have developed a poloxamer-PLGA nanoformulation loaded with elvitegravir (EVG),a commonly used antiretroviral drug. The nanoformulated EVG is capable of elevating intracellular drug uptake and simultaneously enhance viral suppression in HIV-1-infected macrophages. In this work,we identified the clinical parameters including stability,biocompatibility,protein corona,cellular internalization pathway of EVG nanoformulation for its potential clinical translation. We further assessed the ability of this EVG nanoformulation to cross the in vitro BBB model and suppress the HIV-1 in macrophage cells. Compared with EVG native drug,our EVG nanoformulation demonstrated an improved BBB model penetration cross the in vitro BBB model and an enhanced HIV-1 suppression in HIV-1-infected human monocyte-derived macrophages after crossing the BBB model without altering the BBB model integrity. Overall,this is an innovative and optimized treatment strategy that has a potential for therapeutic interventions in reducing HAND. View Publication

Gamma-glutamylcysteine synthetase (gammaGCS),a rate-limiting enzyme in glutathione biosynthesis,plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer. We have determined the crystal structures of Escherichia coli gammaGCS unliganded and complexed with a sulfoximine-based transition-state analog inhibitor at resolutions of 2.5 and 2.1 A,respectively. In the crystal structure of the complex,the bound inhibitor is phosphorylated at the sulfoximido nitrogen and is coordinated to three Mg2+ ions. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure,an open space exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon inhibitor binding,the side chains of Tyr-241 and Tyr-300 turn,forming a hydrogen-bonding triad with the carboxyl group of the inhibitor's cysteine moiety,allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). Based on this crystal structure,the cysteine-binding sites of mammalian and parasitic gammaGCSs were predicted by multiple sequence alignment,although no significant sequence identity exists between the E. coli gammaGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel gammaGCS inhibitors. View Publication

Thyromimetics that specifically target TR$\beta$ have been shown to reduce plasma cholesterol levels and avoid atherosclerosis through the promotion of reverse cholesterol transport in an animal model. We designed novel thyromimetics with high receptor (TR$\beta$) and organ (liver) selectivity based on the structure of eprotirome (3) and molecular modeling. We found that indane derivatives are potent and dual-selective thyromimetics expected to avoid hypothyroidism in some tissues as well as heart toxicity. KTA-439 (29),a representative indane derivative,showed the same high human TR$\beta$ selectivity in a binding assay as 3 and higher liver selectivity than 3 in a cholesterol-fed rat model. View Publication

Human genome engineering has been transformed by the introduction of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system found in most bacteria and archaea. Type II CRISPR/Cas systems have been engineered to induce RNA-guided genome editing in human cells,where small RNAs function together with Cas9 nucleases for sequence-specific cleavage of target sequences. Here we describe the protocol for Cas9-mediated human genome engineering,including construct building and transfection methods necessary for delivering Cas9 and guide RNA (gRNA) into human-induced pluripotent stem cells (hiPSCs) and HEK293 cells. Following genome editing,we also describe methods to assess genome editing efficiency using next-generation sequencing and isolate monoclonal hiPSCs with the desired modifications for downstream applications. View Publication

The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved,we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel,mouse embryonic fibroblast feeders,and Matrigel replated on feeders. At the outset,we profiled and quantified their differentiation efficiency,transcriptome,transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome,thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1,FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs,we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly,the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus,we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency. View Publication