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

Protein disulfide isomerase (PDI,PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies,although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4,CHOP,and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors. View Publication

Although a link between the gut microbiota and alcohol-related liver diseases (ALDs) has previously been suggested,the causative effects of specific taxa and their functions have not been fully investigated to date. Here,we analyze the gut microbiota of 410 fecal samples from 212 Korean twins by using the Alcohol Use Disorders Identification Test (AUDIT) scales to adjust for host genetics. This analysis revealed a strong association between low AUDIT scores and the abundance of the butyrate-producing genus Roseburia. When Roseburia spp. are administered to ALD murine models,both hepatic steatosis and inflammation significantly improve regardless of bacterial viability. Specifically,the flagellin of R. intestinalis,possibly through Toll-like receptor 5 (TLR5) recognition,recovers gut barrier integrity through upregulation of the tight junction protein Occludin and helps to restore the gut microbiota through elevated expression of IL-22 and REG3$\gamma$. Our study demonstrates that Roseburia spp. improve the gut ecosystem and prevent leaky gut,leading to ameliorated ALDs. View Publication

During viral infections viruses express molecules that interfere with the host-cell death machinery and thus inhibit cell death responses. For example the viral FLIP (vFLIP) encoded by Kaposi's sarcoma-associated herpesvirus interacts and inhibits the central cell death effector,Caspase-8. In order to analyze the impact of anti-apoptotic viral proteins,like vFlip,on liver physiology in vivo,mice expressing vFlip constitutively in hepatocytes (vFlipAlbCre+) were generated. Transgenic expression of vFlip caused severe liver tissue injury accompanied by massive hepatocellular necrosis and inflammation that finally culminated in early postnatal death of mice. On a molecular level,hepatocellular death was mediated by RIPK1-MLKL necroptosis driven by an autocrine TNF production. The loss of hepatocytes was accompanied by impaired bile acid production and disruption of the bile duct structure with impact on the liver-gut axis. Notably,embryonic development and tissue homeostasis were unaffected by vFlip expression. In summary our data uncovered that transgenic expression of vFlip can cause severe liver injury in mice,culminating in multiple organ insufficiency and death. These results demonstrate that viral cell death regulatory molecules exhibit different facets of activities beyond the inhibition of cell death that may merit more sophisticated in vitro and in vivo analysis. View Publication

H7 avian influenza viruses represent a major public health concern,and worldwide outbreaks raise the risk of a potential pandemic. Understanding the memory B cell response to avian (H7) influenza virus infection in humans could provide insights in the potential key to human infection risks. We investigated an epizootic of the highly pathogenic A(H7N7) in the Netherlands,which in 2003 led to infection of 89 persons and one fatal case. Subtype-specificity of antibodies were determined for confirmed H7N7 infected individuals (cases) (n = 19),contacts of these cases (n = 21) and a comparison group controls (n = 16),by microarray,using recombinant hemagglutinin (HA)1 proteins. The frequency and specificity of memory B cells was determined by detecting subtype-specific antibodies in the culture supernatants from in vitro stimulated oligoclonal B cell cultures,from peripheral blood of cases and controls. All cases (100{\%}) had high antibody titers specific for A(H7N7)2003 (GMT {\textgreater} 100),whereas H7-HA1 antigen binding was detected in 29{\%} of contacts and 31{\%} of controls,suggesting that some of the H7 reactivity stems from cross reactive antibodies. To unravel homotypic and heterotypic responses,the frequency and specificity of memory B cells were determined in 2 cases. Ten of 123 HA1 reactive clones isolated from the cases bound to only H7- HA1,whereas 5 bound both H7 and other HA1 antigens. We recovered at least four different epitopal reactivities,though none of the H7 reactive antibodies were able to neutralize H7 infections in vitro. Our study serologically confirms the infection with H7 avian influenza viruses,and shows that H7 infection triggers a mixture of strain -specific and cross-reactive antibodies. View Publication

The rare,accelerated aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is commonly caused by a de novo c.1824 C?>?T point mutation of the LMNA gene that results in the protein progerin. The primary cause of death is a heart attack or stroke arising from atherosclerosis. A characteristic feature of HGPS arteries is loss of smooth muscle cells. An adenine base editor (ABE7.10max) corrected the point mutation and produced significant improvement in HGPS mouse lifespan,vascular smooth muscle cell density,and adventitial fibrosis. To assess whether base editing correction of human HGPS tissue engineered blood vessels (TEBVs) prevents the HGPS vascular phenotype and to identify the minimum fraction of edited smooth muscle cells needed to effect such changes,we transduced HGPS iPSCs with lentivirus containing ABE7.10max. Endothelial cells (viECs) and smooth muscle cells (viSMCs) obtained by differentiation of edited HGPS iPSCs did not express progerin and had double-stranded DNA breaks and reactive oxygen species at the same levels as healthy viSMCs and viECs. Editing HGPSviECs restored a normal response to shear stress. Normal vasodilation and viSMC density were restored in TEBVs made with edited cells. When TEBVs were prepared with at least 50% edited smooth muscle cells,viSMC proliferation and myosin heavy chain levels significantly improved. Sequencing of TEBV cells after perfusion indicated an enrichment of edited cells after 5?weeks of perfusion when they comprised 50% of the initial number of cells in the TEBVs. Thus,base editing correction of a fraction of HGPS vascular cells improves human TEBV phenotype. View Publication

BackgroundTimely resolution of innate immune responses activated by surgical intervention is crucial for patient recovery. While cytokines and innate immune cells are critical in inflammation resolution,the specific role of IL-18 in these processes remains controversial and underexplored.MethodsWe investigate determinants of successful recovery using peripheral blood samples from orthopedic surgery (ORT) patients (n?=?33) at T0 (before surgery),T1 (24 h after surgery) and T2 (3 days after surgery). Monocytes from ORT patients underwent immunophenotyping together with bulk transcriptomic analysis. We found that IL-18 strongly defines the recovery immune signature. These results were further validated in vitro by comparing IL-18 and TNF-? effects on monocytes,and in 3D human intestine organoids together with single cell (sc)-RNAseq analysis.ResultsTranscriptomics of ORT monocytes revealed upregulation of ITG family integrins,namely ITGB3 and ITGB5,CXCL family chemokines,notably CXCL1-3,CXCL5,and SCL/TAL1 factor controlling differentiation and migration,but not pro-inflammatory genes. Similar changes were observed in IL-18 stimulated healthy donor monocytes in vitro,including an increase in CD11b,CD64,and CD86 levels,accompanied by increased phosphorylation of Akt but not NF?B. These changes were attenuated in the presence of TNF-?,thus showing a unique role of IL-18 when acting alone without its most frequent paired cytokine TNF-?. We further confirmed that IL-18 induces monocyte-macrophage transition and migration using human intestinal organoids. Finally,TNF-?/IL-18 ratio showed a high predictive value of clinical severity in septic patients.ConclusionsWe propose a novel role of IL-18 on monocyte migration and macrophage transition characterizing successful orthopedic surgery recovery,as well as the ratio of IL-18/TNF-? as a novel marker of inflammation resolution,with potential implications for patient monitoring and therapeutic strategies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06652-7. View Publication

High-throughput massively parallel reporter assays (MPRAs) and phenotype-rich in vivo transgenic mouse assays are two potentially complementary ways to study the impact of noncoding variants associated with psychiatric diseases. Here,we investigate the utility of combining these assays. Specifically,we carry out an MPRA in induced human neurons on over 50,000 sequences derived from fetal neuronal ATAC-seq datasets and enhancers validated in mouse assays. We also test the impact of over 20,000 variants,including synthetic mutations and 167 common variants associated with psychiatric disorders. We find a strong and specific correlation between MPRA and mouse neuronal enhancer activity. Four out of five tested variants with significant MPRA effects affected neuronal enhancer activity in mouse embryos. Mouse assays also reveal pleiotropic variant effects that could not be observed in MPRA. Our work provides a catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays. MPRAs and in vivo transgenic mouse assays are two potentially complementary ways to assay the impact of noncoding variants. Here,authors find a strong and specific correlation between the assays in neural cells. Mouse assays also reveal pleiotropic effects not observed in MPRA. View Publication

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants,including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons,which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS,and TDP-43 modulates several mitochondrial transcripts. In this study,we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons,linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected,which correlated with downregulation of the motor protein complex,DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport,which can be partially rescued by DCTN1 overexpression. Dafinca et al. show that mutations in TDP-43 lead to decreased mitochondrial oxidative phosphorylation,partially due to interactions with the ATP production machinery and COX5A. These have direct effects on axonal transport,which is reduced in amyotrophic lateral sclerosis motor neurons,and overexpression of dynactin-1 significantly increases retrograde mitochondrial dynamics. View Publication

SummaryCardiovascular disease (CVD) is associated with both genetic variants and environmental factors. One unifying consequence of the molecular risk factors in CVD is DNA damage,which must be repaired by DNA damage response proteins. However,the impact of DNA damage on global cardiomyocyte protein abundance,and its relationship to CVD risk remains unclear. We therefore treated induced pluripotent stem cell-derived cardiomyocytes with the DNA-damaging agent Doxorubicin (DOX) and a vehicle control,and identified 4,178 proteins that contribute to a network comprising 12 co-expressed modules and 403 hub proteins with high intramodular connectivity. Five modules correlate with DOX and represent distinct biological processes including RNA processing,chromatin regulation and metabolism. DOX-correlated hub proteins are depleted for proteins that vary in expression across individuals due to genetic variation but are enriched for proteins encoded by loss-of-function intolerant genes. While proteins associated with genetic risk for CVD,such as arrhythmia are enriched in specific DOX-correlated modules,DOX-correlated hub proteins are not enriched for known CVD risk proteins. Instead,they are enriched among proteins that physically interact with CVD risk proteins. Our data demonstrate that DNA damage in cardiomyocytes induces diverse effects on biological processes through protein co-expression modules that are relevant for CVD,and that the level of protein connectivity in DNA damage-associated modules influences the tolerance to genetic variation. View Publication

Background: Pulmonary fibrosis is a major disease that leads to the progressive loss of lung function. The disease manifests early,resulting in type 2 respiratory failure. This is likely due to the bronchocentric fibrosis around the major airways,which causes airflow limitation. It affects approximately three million patients worldwide and has a poor prognosis. Skin fibroblasts isolated from patients offer valuable insights into understanding the disease mechanisms,identifying the genetic causes,and developing personalized therapies. However,the use of skin fibroblasts to study a disease that exclusively impacts the lungs is often questioned,particularly since lung fibrosis primarily affects the alveolar epithelium. Method: We report the reprogramming of skin fibroblasts from patients with an atypical early-onset form of lung fibrosis into induced pluripotent stem cells (iPSCs) and subsequently into alveolar epithelial cells. This was achieved using a Sendai virus approach. Results: We show that the reprogrammed cells carry mutations in the calcium-binding protein genes S100A3 and S100A13,leading to diminished protein expression,thus mimicking the patients’ cells. Additionally,we demonstrate that the generated patient iPSCs exhibit aberrant calcium and mitochondrial functions. Conclusions: Due to the lack of a suitable animal model that accurately resembles the human disease,generating patient lung cells from these iPSCs can provide a valuable “disease in a dish” model for studying the atypical form of inherited lung fibrosis. This condition is associated with mutations in the calcium-binding protein genes S100A3 (NM_002960) and S100A13 (NM_001024210),aiding in the understanding of its pathogenesis. View Publication

Deleterious germline DDX41 variants constitute the most common inherited predisposition disorder linked to myeloid neoplasms (MNs),yet their role in MNs remains unclear. Here we show that DDX41 is essential for erythropoiesis but dispensable for other hematopoietic lineages. Ddx41 knockout in early erythropoiesis is embryonically lethal,while knockout in late-stage terminal erythropoiesis allows mice to survive with normal blood counts. DDX41 deficiency induces a significant upregulation of G-quadruplexes (G4),which co-distribute with DDX41 on the erythroid genome. DDX41 directly binds to and resolves G4,which is significantly compromised in MN-associated DDX41 mutants. G4 accumulation induces erythroid genome instability,ribosomal defects,and p53 upregulation. However,p53 deficiency does not rescue the embryonic death of Ddx41 hematopoietic-specific knockout mice. In parallel,genome instability also activates the cGas-Sting pathway,impairing survival,as cGas deficiency rescues the lethality of hematopoietic-specific Ddx41 knockout mice. This is supported by data from a DDX41-mutated MN patient and human iPSC-derived bone marrow organoids. Our study establishes DDX41 as a G4 resolvase,essential for erythroid genome stability and suppressing the cGAS-STING pathway. Germline DDX41 mutations are linked to myeloid neoplasms,but their roles in the disease is unclear. Here,the authors show that DDX41 resolves G-quadruplex structures to maintain erythroid genome stability and prevent cGAS-mediated cell death. These functions are lost in disease-associated variants. View Publication