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

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders,resulting in early-onset,therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy,severe developmental delay,progressive microcephaly,visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent,homozygous variant (chr2:64083454A {\textgreater} G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform,which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells,leading to altered glycogen metabolism,upregulated unfolded protein response and premature neuronal differentiation,as modeled during pluripotent stem cell differentiation in vitro. In contrast,the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly,it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease,even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies. View Publication

The stimulator of interferon genes (STING) pathway has been proposed as a key regulator of gastrointestinal homeostasis and inflammatory responses. Although STING reportedly protects against gut barrier damage and graft-versus-host disease (GVHD) after major histocompatibility complex (MHC)-mismatched allogeneic hematopoietic stem cell transplantation (aHSCT),its effect in clinically relevant MHC-matched aHSCT is unknown. Studies here demonstrate that STING signaling in nonhematopoietic cells promoted MHC-matched aHSCT-induced GVHD and that STING agonists increased type I interferon and MHC I expression in nonhematopoietic mouse intestinal organoid cultures. Moreover,mice expressing a human STING allele containing three single-nucleotide polymorphisms associated with decreased STING activity also developed reduced MHC-matched GVHD,demonstrating STING's potential clinical importance. STING-/- recipients experienced reduced GVHD with transplant of purified donor CD8+ T cells in both MHC-matched and MHC-mismatched models,reconciling the seemingly disparate results. Further examination revealed that STING deficiency reduced the activation of donor CD8+ T cells early after transplant and promoted recipient MHC class II+ antigen-presenting cell (APC) survival. Therefore,APC persistence in STING pathway absence may account for the increased GVHD mediated by CD4+ T cells in completely mismatched recipients. In total,our findings have important implications for regulating clinical GVHD by targeting STING early after aHSCT and demonstrate that an innate immune pathway has opposing effects on the outcome of aHSCT,depending on the donor/recipient MHC disparity. View Publication

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of creeping fat" (CrF) defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria." View Publication

BACKGROUND The nonsense mutation,c.3846G{\textgreater}A (aka: W1282X-CFTR) leads to a truncated transcript that is susceptible to nonsense-mediated decay (NMD) and produces a shorter protein that is unstable and lacks normal channel activity in patient-derived tissues. However,if overexpressed in a heterologous expression system,the truncated mutant protein has been shown to mediate CFTR channel function following the addition of potentiators. In this study,we asked if a quadruple combination of small molecules that together inhibit nonsense mediated decay,stabilize both halves of the mutant protein and potentiate CFTR channel activity could rescue the functional expression of W1282X-CFTR in patient derived nasal cultures. METHODS We identified the CFTR domains stabilized by corrector compounds supplied from AbbVie using a fragment based,biochemical approach. Rescue of the channel function of W1282X.-CFTR protein by NMD inhibition and small molecule protein modulators was studied using a bronchial cell line engineered to express W1282X and in primary nasal epithelial cultures derived from four patients homozygous for this mutation. RESULTS We confirmed previous studies showing that inhibition of NMD using the inhibitor: SMG1i,led to an increased abundance of the shorter transcript in a bronchial cell line. Interestingly,on top of SMG1i,treatment with a combination of two new correctors developed by Galapagos/AbbVie (AC1 and AC2-2,separately targeting either the first or second half of CFTR and promoting assembly,significantly increased the potentiated channel activity by the mutant in the bronchial epithelial cell line and in patient-derived nasal epithelial cultures. The average rescue effect in primary cultures was approximately 50{\%} of the regulated chloride conductance measured in non-CF cultures. CONCLUSIONS These studies provide the first in-vitro evidence in patient derived airway cultures that the functional defects incurred by W1282X,has the potential to be effectively repaired pharmacologically. View Publication

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here,we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further,we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2,Vaccinia virus,and Zika virus through a type I interferon (IFN)-independent mechanism. In addition,4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion,NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses,including SARS-CoV2. View Publication

Tumor patients often exhibit limited responses to immunotherapy owing to the low immunogenicity and immunosuppressive environment of tumors. Our previous studies revealed that cryo-thermal therapy caused tumor cell rupture due to mechanical compression,notably causing the release of a substantial amount of DAMPs (danger-associated molecular patterns),such as heat shock protein 70,calreticulin and high-mobility group box protein 1; the release of these DAMPs increased myeloid cell maturation,thereby reshaping the systemic immune environment and ultimately inducing durable CD4+ T helper type 1 (Th1) cell-dominated antitumor immunity. In fact,under conditions of mechanical stress and rapid temperature changes,the disruption of tumor cells caused by cryo-thermal therapy results in extensive deoxyribonucleic acid (DNA) damage and the rapid release of substantial amounts of DNA. Consequently,tumor-derived DNA,which potently activates innate immunity by engaging multiple DNA sensors,plays a pivotal role in orchestrating antitumor immunity. We hypothesized that cryo-thermal therapy induces the transient release of high levels of DNA,which modulates CD11b+ myeloid cell function,subsequently influencing CD4+ Th1-cell dominated antitumor immune responses. In this study,a B16F10 melanoma model was established,and DNA concentrations were measured at different time points after cryo-thermal therapy. Deoxyribonuclease I (DNase I) was subsequently administered immediately following cryo-thermal therapy to deplete extracellular DNA,allowing an investigation of the role of DNA in regulating CD11b+ myeloid cell function and CD4+ T cell differentiation. The phenotype and function of CD11b+ myeloid cells and CD4+ T cells were assessed by flow cytometry,RNA sequencing,and cell culture in vitro. Our studies confirmed that cryo-thermal therapy triggered a transient release of high levels of DNA,which was internalized by CD11b+ myeloid cells via C-type lectin receptors and subsequently sensed by inflammasomes. Then,the intracellular sensing of DNA induced the production of the mature form of interleukin (IL)-18,ultimately promoting the Th1 differentiation of CD4+ T cells. This study highlights the pivotal role of DNA release after cryo-thermal therapy in driving CD4+ Th1 cell-dominant antitumor immunity. View Publication

MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening,we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover,we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program,as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets,reduces ribosome biogenesis,and impairs protein synthesis. Mechanistically,CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci,facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore,combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo,leading to more pronounced tumor growth suppression. Overall,our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation,suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB. MYC-driven medulloblastoma is an aggressive pediatric tumor with limited treatment options. Here,the authors show that CDK8 regulates ribosome biogenesis and that combined inhibition of CDK8 and mTOR demonstrates therapeutic efficacy in mouse models of this cancer. View Publication

Lens transparency relies on proper intercellular communication. Exosomes are crucial mediators of intercellular communication and play a key role in organ homeostasis and development. However,their presence and dynamics in the lens remain unclear. Here,we report endogenous exosomes in the zebrafish lens using cryaa-driven Cd63-AcGFP labeling. Live imaging revealed dynamic exosome movement within lens cells and their potential transfer to adjacent tissues. Additionally,we found that the biogenesis of Cd63+ exosomes in the lens is regulated by the Syntenin-a pathway. And Syntenin-a knockdown delayed lens development by impairing lens cell differentiation,highlighting the potential role of lens cell–derived exosomes. Furthermore,ROR1+ lens progenitor cell-derived extracellular vesicles promoted lentoid differentiation in vitro,with proteomic analysis suggesting underlying mechanisms. Overall,our study addresses the gap in direct observation of endogenous lens exosomes,providing foundational insights into lens pathophysiology and a potential strategy for modulating the lens microenvironment. Visualization of endogenous exosomes in the zebrafish lens reveals their dynamic roles in intercellular communication and development,with Syntenin-a–regulated exosome biogenesis influencing lens cell differentiation. View Publication

Propofol is a commonly used anesthetic for sedation during surgery. This drug is reported to exhibit nonanaesthetic immunomodulatory and anti-inflammatory effects. Herein,we investigated the impact of topical propofol delivery with the aim of mitigating psoriatic inflammation. The antipsoriatic potency of propofol was evaluated in a cell-based study in which keratinocytes,macrophages,and neutrophils were used as models. A significant reduction in the proinflammatory effectors interleukin (IL)-6,IL-8,and CXC motif chemokine ligand (CXCL)1 was found in activated keratinocytes (HaCaT) treated with propofol. This reduction could enable baseline control. Immunoblotting suggested that the antioxidant enzymes nuclear factor erythroid 2-related factor (Nrf)2 and heme oxygenase (HO)-1 were involved in the protective effect of propofol on keratinocyte stimulation. The increase in Nrf2 and HO-1 was mediated by kelch-like ECH-associated protein (KEAP)1 downregulation. Propofol presented scavenging activity and decreased 2,2-diphenyl-1-picrylhydrazyl (DPPH) by 47%. The downregulation of cytokines/chemokines in activated macrophages (differentiated THP-1) and mouse neutrophils was also found after propofol treatment. Macrophage migration triggered by the conditioned medium of activated keratinocytes could be blocked with the intervention of propofol. The absorption level of propofol (3 mM) into intact pig skin was 1.2 nmol/mg. Skin deposition was increased to 3.7 nmol/mg after SC lipid removal to mimic psoriasiform skin. In silico molecular docking demonstrated the facile interaction of propofol with ceramides in the stratum corneum (SC). The treatment of imiquimod (IMQ)-sensitized mice with topical propofol suppressed erythema,acanthosis,and macrophage/neutrophil infiltration. Propofol also dramatically decreased cytokine/chemokine levels and epidermal thickness in the lesion. In summary,propofol exhibits anti-inflammatory and antioxidant properties to treat psoriasiform lesions. Topical propofol delivery is useful as an ideal route to accomplish antipsoriatic therapy and avoid systemic effects. View Publication

Directed differentiation of human induced pluripotent stem cells (iPSCs) into anterior foregut endoderm (AFE) and lung progenitors (LPs) has wide-ranging implications for lung developmental biology,disease modeling,and regenerative medicine. We expand on a previously developed mathematical modeling framework and apply it to the directed differentiation of AFE into LPs. A model-based approach guides experimental design,followed by a multistage model inference process: maximum likelihood estimation based on in vitro data and identifiability analyses to eliminate unidentifiable candidates,thereby guiding model selection. To the authors’ knowledge,this is the first mathematical model of the population dynamics of directed differentiation of AFE into LPs. The model suggests that the overall dynamics are primarily driven by AFE proliferation and differentiation into LPs. In silico experiments predict that daily media change nearly doubles LP yields compared to cultures without media replenishment. Moreover,the model suggests that higher split ratios on day 10 enhance yield per input cell,a measure of differentiation efficiency,by 26%. This work provides a blueprint for refining iPSC-based lung lineage differentiation protocols by combining empirical data and mathematical modeling. View Publication

Specific antibody opsonization significantly enhances the level of phagocytosis of Candida in the absence of complement. Furthermore,we have described a system using a recombinant human antibody single-chain variable fragment that allows a comparative study of phagocytosis of multiple Candida species opsonized via a common antigen. View Publication

Lysophosphatidic acid (LPA),the smallest and structurally simplest phospholipid,is a platelet-derived serum factor that evokes a wide range of biological effects,including stimulation of fibroblast proliferation,platelet aggregation,cellular motility,tumour cell invasiveness and neurite retraction. This review summarizes recent insights into the mode of action of LPA. LPA appears to activate its own G-protein-coupled receptor(s) to initiate both classic and novel signal cascades. Of particular interest is LPA's ability to activate the Ras pathway and to stimulate protein tyrosine phosphorylation in concert with remodelling of the actin cytoskeleton. View Publication