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

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work,further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR,SGX94,has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies,this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells,resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels,thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation,IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections. View Publication

Serotonin receptor affinity and photelectron spectral data were obtained on a number of substituted N,N-dimethyltryptamines. Evidence is presented that electron-donating substituents in the 5-position lead to enhanced behavioral disruption activity and serotonin receptor affinity as compared to unsubstituted N,N-dimethyltryptamine and analogues substituted in the 4- or 6-position. Some correlation was found between ionization potentials and behavioral activity,which may have implications concerning the mechanism of receptor binding. View Publication

The sympathetic (SNS) and parasympathetic (PNS) branches of the autonomic nervous system have been implicated in the modulation of the renewal of many tissues,including the intestinal epithelium. However,it is not known whether these mechanisms are direct,requiring an interaction between autonomic neurotransmitters and receptors on proliferating epithelial cells. To evaluate the existence of a molecular framework for a direct effect of the SNS or PNS on intestinal epithelial renewal,we measured gene expression for the main autonomic neurotransmitter receptors in this tissue. We separately evaluated intestinal epithelial regions comprised of the stem,progenitor,and mature cells,which allowed us to investigate the distinct contributions of each cell population to this proposed autonomic effect. Notably,we found that the stem cells expressed the receptors for the SNS-associated alpha2A adrenoreceptor and the PNS-associated muscarinic acetylcholine receptors (M1 and M3). In a separate experiment,we found that the application of norepinephrine or acetylcholine decreases the expression of cyclin D1,a gene necessary for cell cycle progression,in intestinal epithelial organoids compared with controls (P {\textless} 0.05). Together,these results provide evidence of a direct mechanism for the autonomic nervous system influence on intestinal epithelial stem cell proliferation. View Publication

Acute liver failure (ALF) is a hepatology emergency with rapid hepatic destruction,multiple organ failures,and high mortality. Despite decades of research,established ALF has minimal therapeutic options. Here,we report that the small bioactive compound SCM-198 increases the survival of male ALF mice to 100%,even administered 24?hours after ALF establishment. We identify adiponectin receptor 2 (AdipoR2) as a selective target of SCM-198,with the AdipoR2 R335 residue being critical for the binding and signaling of SCM-198-AdipoR2 and AdipoR2 Y274 residue serving as a molecular switch for Ca2+ influx. SCM-198-AdipoR2 binding causes Ca2+ influx and elevates the phosphorylation levels of CaMKII and NOS3 in the AdipoR2-CaM-CaMKII-NOS3 complex identified in this study,rapidly inducing nitric oxide production for liver protection in murine ALF. SCM-198 also protects human ESC-derived liver organoids from APAP/TAA injuries. Thus,selectively targeting the AdipoR2-CaM-CaMKII-NOS3 axis by SCM-198 is a rapid-acting therapeutic strategy for advanced ALF. Late-stage acute liver failure (ALF) has limited therapies. The authors show that the bioactive compound SCM-198 extends the ALF treatment window from 3 to 24?hours in mice by selectively targeting the identified AdipoR2-CaM-CaMKII-NOS3-NO axis. View Publication

Chronic lymphocytic leukemia is a malignant lymphoproliferative disorder for which primary or acquired drug resistance represents a major challenge. To investigate the underlying molecular mechanisms,we generate a mouse model of ibrutinib resistance,in which,after initial treatment response,relapse under therapy occurrs with an aggressive outgrowth of malignant cells,resembling observations in patients. A comparative analysis of exome,transcriptome and proteome of sorted leukemic murine cells during treatment and after relapse suggests alterations in the proteasome activity as a driver of ibrutinib resistance. Preclinical treatment with the irreversible proteasome inhibitor carfilzomib administered upon ibrutinib resistance prolongs survival of mice. Longitudinal proteomic analysis of ibrutinib-resistant patients identifies deregulation in protein post-translational modifications. Additionally,cells from ibrutinib-resistant patients effectively respond to several proteasome inhibitors in co-culture assays. Altogether,our results from orthogonal omics approaches identify proteasome inhibition as potentially attractive treatment for chronic lymphocytic leukemia patients resistant or refractory to ibrutinib. The molecular mechanisms underlying resistance to therapy in Chronic lymphocytic leukemia (CLL) remain to be explored. Here,the authors perform multi-omics analysis in a mouse model of ibrutinib resistance and suggest proteasome inhibition for overcoming it. View Publication

Pygopus 2 (Pygo2) is a coactivator of Wnt/$\beta$-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer,we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller,differentiated,and less metastatic tumors,due,in part,to decreased canonical Wnt/$\beta$-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGF$\beta$ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically,the Pygo2-histone interaction potentiated Wnt/$\beta$-catenin signaling,in part,by repressing the expression of Wnt signaling antagonists. Furthermore,Pygo2 and $\beta$-catenin regulated the expression of miR-29 family members,which,in turn,repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively,these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors,and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer,as its histone-binding capability promotes $\beta$-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation,EMT,and metastasis. View Publication

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment,tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors,whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms),suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However,CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically,the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer,tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly,in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion,the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress. View Publication

Small non‐coding microRNAs (miRNAs) play essential roles in Alzheimer's disease (AD) pathogenesis. Repressor element 1‐silencing transcription factor (REST) is involved in AD,though its regulation remains unclear. We performed real‐time quantitative polymerase chain reaction (qPCR) in autopsied brain tissues to determine miR‐153‐3p and AD associations. A reporter‐based assay measured the activity of REST mRNA 3′‐untranslated region (3′‐UTR). Induced pluripotent stem cells (iPSC)‐derived neurons and human cell lines were applied to determine miR‐153‐3p regulation of endogenous proteins. Elevation of miR‐153‐3p is associated with a reduced probability of AD,while elevated REST is associated with a greater probability of AD. The 3′‐UTR functional assay pinpointed the miR‐153‐3p binding sites. miR‐153‐3p treatment reduced REST,amyloid precursor protein (APP),and α‐synuclein (SNCA) 3′‐UTR activities and protein levels. miR‐153‐3p treatment altered REST and neuronal differentiation in iPSC‐derived neuronal stem cells. RNA‐sequencing and proteomics revealed miR‐153‐3p‐associated networks. miR‐153‐3p reduces REST,APP,and SNCA expression,pointing toward its therapeutic and biomarker potential in neurodegenerative diseases. With the increased emphasis on comorbidities of Alzheimer's disease (AD) and other neurodegenerative diseases,we identified that miR‐153‐3p,as a master regulator,reduced a group of neurodegeneration related proteins: REST,amyloid precursor protein (APP) and α‐synuclein (SNCA) levels. The elevation of miR‐153‐3p levels is associated with reduced probability of AD in posterior cingulate cortex (PCC),while REST,by contrast,is associated with a greater probability of AD. miR‐153‐3p treatment alters REST protein levels and neuronal differentiation in induced pluripotent stem cells (iPSC) derived neuronal cells. RNA sequencing proteomics and interactome analysis revealed the role of miR‐153‐3p in axonal guidance. View Publication

5-Ethynyl-2′-deoxyuridine (EdU) has revolutionized DNA replication and cell cycle analyses through fast,efficient click chemistry detection. However,commercial EdU kits suffer from high costs,proprietary formulations,limited antibody multiplexing capabilities,and difficulties with larger biological specimens. Here,we present OpenEMMU (Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics),an optimized,affordable,and user-friendly click chemistry platform utilizing off-the-shelf reagents. OpenEMMU enhances efficiency,brightness,and multiplexing capabilities of EdU staining with both non-conjugated and conjugated antibodies across diverse cell types,including T cell activation and proliferation assays. We validated its effectiveness for the fluorescent imaging of nascent DNA synthesis in developing embryos and organs,including embryonic heart,forelimbs,and 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled the deep-tissue 3D imaging of DNA synthesis in zebrafish larvae and under replication stress in embryos at high spatial resolution. This approach opens new avenues for understanding organismal development,cell proliferation,and DNA replication dynamics with unprecedented precision and flexibility. Subject areas: Biochemistry,Cell biology,Developmental biology,Computational bioinformatics View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication