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

Introduction: Pathogenic variants in canonical transforming growth factor β (TGFβ) signaling genes predispose patients to thoracic aortic aneurysm and dissection (TAAD),predominantly in aortic root. Although TAAD pathogenesis associated with TGFβ receptor defects is well characterized,distinct and redundant mechanisms of TGFβ isoforms in TAAD incidence and severity remain elusive. Objective: Here we examined the biological role of TGFB2 in smooth muscle cell (SMC) differentiation and investigated how TGFB2 defects can lead to regional TAAD manifestations. Methods: To characterize the role of TGFB2 in SMC differentiation and function,we employed human-induced pluripotent stem cell (hiPSC)-derived SMC differentiation,CRISPR/Cas9 gene editing,three-dimensional SMC constructs,and human aortic tissue samples. Results: Despite the similar effects of different TGFβ isoforms on hiPSC-derived SMC differentiation,siRNA experiments revealed that TGFB2 distinctively displays TGFBR3 dependence for signal transduction,an understudied TGFβ receptor in TAAD. Molecular evaluation of different thoracic aorta regions suggested TGFB2 and TGFBR3 enrichment in the aortic root tunica media. TGFB2 haploinsufficiency (TGFB2KO/+) and TGFB2 neutralization impaired the differentiation of second heart field-derived SMCs. TGFBR3KO/KO prevented the molecular rescue of TGFB2KO/+ by TGFB2 supplementation indicating the involvement of TGFBR3 in TGFB2-mediated SMC differentiation. Lastly,a missense TGFB2 variant (TGFB2G276R/+) caused mechanical defects in SMC tissue ring constructs that were rescued by TGFB2 supplementation or genetic correction. Conclusion: Our data suggests the distinct regulation and action of TGFB2 in SMCs populating the aortic root,while redundant activities of TGFβ isoforms provide implications about the milder TAAD aggressiveness of pathogenic TGFB2 variants. View Publication

Idorn et al. characterized a mouse strain harboring a mutation identified in an HSE patient. Defective IFN-driven antiviral responses led to hyperactivation of inflammatory responses,which contributed to disease development. The study identifies immunopathology as an important contributor to HSE pathogenesis. View Publication

The COVID-19 pandemic has created a global health crisis,with challenges arising from the ongoing evolution of the SARS-CoV-2 virus,the emergence of new strains,and the long-term effects of COVID-19. Aiming to overcome the development of viral resistance,our study here focused on developing broad-spectrum pan-coronavirus antiviral therapies by targeting host protein quality control mechanisms essential for viral replication. Screening an in-house compound library led to the discovery of three candidate compounds targeting cellular proteostasis. The three compounds are (1) the nucleotide analog cordycepin,(2) a benzothiozole analog,and (3) an acyldepsipeptide analog initially developed as part of a campaign to target the mitochondrial ClpP protease. These compounds demonstrated dose-dependent efficacy against multiple coronaviruses,including SARS-CoV-2,effectively inhibiting viral replication in vitro as well as in lung organoids. Notably,the compounds also showed efficacy against SARS-CoV-2 delta and omicron strains. As part of this work,we developed a BSL2-level cell-integrated SARS-CoV-2 replicon,which could serve as a valuable tool for high-throughput screening and studying intracellular viral replication. Our study should aid in the advancement of antiviral drug development efforts. Subject terms: High-throughput screening,Small molecules View Publication

We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ,MMEJ),and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1),affecting several key DNA repair proteins,such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling,gene therapy,chimeric antigen receptor immunotherapy,and cancer treatment. DNA repair pathways shape CRISPR editing outcomes. Here,authors identified FDA approved drugs that can be repurposed as repair modulators or to induce synthetic lethality,and uncovered new roles for ESR2 and AOX1 in DNA repair,enhancing editing and offering potential therapeutic applications. View Publication

5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However,because of 5-azacytidine's general toxicity,other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that,when present in DNA,it inhibited DNA methylation,led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here,the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed. View Publication

There is evidence that neutrophil production is a balance between the proliferative action of granulocyte-colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth,and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3,elastase,azurocidin,and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells,elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase,G-CSF,but not GM-CSF or SCF,was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF. View Publication

Using a high-throughput chemical screen,we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action,we discovered an essential role of E-cadherin signaling for ESC survival. Specifically,we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling,which then leads to a fatal perturbation of integrin signaling. Furthermore,we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally,we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology,growth factor requirement,and sensitivity to enzymatic cell dissociation. View Publication

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