Microarray of R.cellulolyticum wild type strain and engineered strain grown on cellulose
Ontology highlight
ABSTRACT: R. cellulolyticum WT strains and engineered strain (WT-BGLA) were cultivated in defined VM medium with 20 g/L cellulose.Each strain had four biological replicates and 20 ml cell samples were collected during the exponential phase.
Project description:R. cellulolyticum strains (the parent strain, ΔccpA, ΔccpB and Δcph) were cultivated in defined VM medium with cellulose (10 g/L).Since ΔccpA and Δcph could not grow on cellulose, we performed an incubation experiment to understand how ΔccpA and Δcph respond to cellulose. Specifically, the parent strain, ΔccpA and Δcph were grown to an OD600 of 0.5-0.6 in 50 ml defined VM media with cellobiose as the carbon source. Each strain had three biological replicates. Bacterial cells from each biological replicate were then collected by centrifugation at 4000 g and washed twice with the defined VM medium (no carbon added). Finally, washed cells from each biological replicate were inoculated into the defined VM medium with 10 g/L cellulose. During shaking incubation at 34°C, samples were collected at five time points (0,1, 3, 6, 12 hours). After centrifugation at 4°C, 5000×g for 10 min, cell pellets were immediately flash frozen with liquid nitrogen and then stored at -80°C for further use. For ΔccpB, ΔccpB and the parent strain were cultivated with six biological replicates and collected at mid-exponential growth phase.
Project description:R. cellulolyticum strains (the parent strain, ΔccpA, ΔccpB, Δcph and cph_S46D) were cultivated in defined VM medium with carbon mixture.On carbon mixture, each strain had three biological replicates and 10 ml cell samples were collected for three times during the exponential phase.
Project description:We have implemented an integrated Systems Biology approach to analyze overall transcriptomic reprogramming and systems level defense responses in the model plant Arabidopsis thaliana during an insect (Brevicoryne brassicae) and a bacterial (Pseudomonas syringae pv. tomato strain DC3000) attack. The main aim of this study was to identify the attacker-specific and general defense response signatures in the model plant Arabidopsis thaliana while attacked by phloem feeding aphids or pathogenic bacteria. Defense responses and networks, unique and specific for aphid or Pseudomonas stresses were identified. Our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and thus opened up a new direction to conduct large-scale targeted experiments to explore detailed regulatory links among them. The presented results provide a first comprehensive understanding of Arabidopsis - B. brassicae and Arabidopsis - P. syringae interactions at a systems biology level. Arabidopsis thaliana (ecotype Colombia-0) seeds were sown into 6-cm-diameter pots filled with a sterile soil mix (1.0 part soil and 0.5 part horticultural perlite). Plants were kept in growth chambers VM-CM-6tsch VB 1514 (VM-CM-6tch Industrietechnik GmbH, Germany) with a 16/8 h (light/dark) photoperiod at 22/18 M-BM-0C, 40/70% relative humidity, and 70/0 mmol m-2 s-1 light intensity. The Pseudomonas syringae pv. tomato strain DC3000 culture was grown overnight in 10 ml of Kings B solution supplemented with antibiotics rifampicin (50 M-NM-<g mlM-bM-^HM-^R1) and kanamycin (25 M-NM-<g mlM-bM-^HM-^R1). Overnight culture was washed once in 10 mM MgCl2 and final cell densities were adjusted to approximately 0.20 at 600 nm (approximately 1.5 M-CM-^W 108 cfu mlM-bM-^HM-^R1) in 10 mM MgCl2. Plants were mock-challenged with 10 mM MgCl2 or inoculated with DC3000 strain, 3-4 leaves were infiltrated on the abaxial surface with a needleless 1-ml syringe.Whole rosettes were cut at the hypocotyls and harvested from Pseudomonas infested and mock-infected plants after 72 hours treatment. 4 biological replicates were prepared from each treatment, each containing rosettes from 15 individual plants. Differences in transcriptional responses were measured by comparing genes expression of Pseudomonas infected plants against mock-infected control plants.
Project description:Ventral midbrain (VM) dopaminergic progenitor cells derived from human pluripotent stem cells have the potential to replace endogenously lost dopamine neurons and are currently in preclinical and clinical development for treatment of Parkinson’s Disease (PD). However, one main challenge in the quality control of the cells is that rostral and caudal VM progenitors are extremely similar transcriptionally though only the caudal VM cells give rise to dopaminergic neurons with functionality in PD. Therefore, it is critical to develop assays which can rapidly and reliably discriminate rostral from caudal VM cells during clinical manufacturing. Here, we applied shotgun proteomics to search for novel secreted biomarkers specific for caudal VM progenitors compared to rostral VM progenitors and validated key hits by ELISA. From this, we identified novel secreted markers (CPE, LGI1 and PDGFC) significantly enriched in caudal versus rostral VM progenitor cultures, whereas the markers CNTN2 and CORIN were significantly enriched in rostral VM cultures. With this data, we suggest and test in clinical grade samples a panel of coupled ELISA assays that can be applied as a quality control tool for assessing the correct patterning of cells during clinical manufacturing.
Project description:Purpose: we want to see gene expression changes during in vitro expansion of VM-derived NSCs (VM-NSCs) with cell passges in the absence or presence of Lin28a overexpression. changes upon Lin28 overexpression in P1 and P3 stages of Neural stem cells. RNA-seq, sRNA-seq, and Polysome-seq with/without Lin28 overexpression in P1 and P3 stages of Neural stem cells.
Project description:Aberrant expression of histone deacetylases (HDACs) and their activity are associated with a broad range of tumor development. However, based on cell or tissue types, class IIA HDACs such as HDAC4 and HDAC5 may facilitate or inhibit cancer progression. The goal of this project is to examine the gene expression changes caused by HDAC5 expression. Here, we studied the effects of stable expression of HDAC5 (that is normally downregulated or have a weak basal expression) in four urothelial carcinoma (UC) cell lines (RT112, VM-Cub-1, SW1710, and UM-UC-3) by rRNA-depleted RNA-sequencing in comparison to their vector controls. We observed that HDAC5 expression in VM-Cub-1 triggered a drastic phenotype change from an epitheloid to a mesenchymal (i.e., epithelial-mesenchymal transition, EMT) and altogether diminished cell proliferation of the other three cell lines. Our RNA-seq data are in line with the phenotypic transformation of VM-Cub-1. In addition, we also performed a gene expression profiling of HBLAK, a spontaneously immortalized from primary human bladder epithelial cells that can be directly compared with the four UC vector cells. HBLAK vector cells only were included for RNA-seq as the cells failed to express HDAC5 after lentiviral transduction and selection.
Project description:Meningitis is a life-threatening condition characterized by the inflammation of the leptomeningeal membranes surrounding the brain and spinal cord. The term meningitis is an umbrella term and includes several different etiologies. The majorities of meningitis cases are caused by viruses (viral meningitis; VM) and are often associated with low mortality rates and low risk of developing neurological. In contrast, meningitis caused by some viral infections, such as tick-borne encephalitis (TBE), can be life-threatening when left untreated with increased risk of developing neurological sequelae. Acute bacterial meningitis (ABM), however, is one of the leading causes of death due to infectious diseases worldwide and is associated with rapid disease progression, high mortality rates and increased risk of long-term neurological sequelae in survivors. As meningitis is caused by numerous different pathogens, the host-response is typically highly variable and it is currently unknown if different pathogens can introduce specific proteome changes in the cerebrospinal fluid (CSF). In this study we applied DIA-MS to provide novel insights for in-depth understanding of central nervous system functioning and host response during meningitis in a cohort of patients with differential diagnosis of meningitis, to account for variability contributed by different disease-causing pathogens. The results reveal drastic changes in the CSF proteome during meningitis, where in particular a massive increase of neutrophil derived proteins in the CSF correlated with ABM, suggesting that activated neutrophils play a particular role in ABM. Additionally both ABM and VM result in marked reduction of brain-specific proteins in the CSF, which could be indicative of pathophysiological mechanisms leading to brain damage. Furthermore, generation of lasso regression model enables separation of ABM with high sensitivity and specificity, demonstrating that several proteins are required to confidently discriminate between ABM, VM and BM.
Project description:Advances in large-scale proteomics analysis have been very useful in understanding pathogenesis of diseases and elaborating therapeutic strategies. Proteomics has been employed to study Parkinson’s disease (PD), however, sparse studies reported proteome investigation after cell therapy approaches. In this study, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins in a mouse model of PD after cell therapy. Proteins were extracted from five nigrostriatal-related brain regions of mice previously lesioned with 6-hydroxydopamine (6-OHDA) in the substantia nigra (SN). Protein expression was compared in non-grafted brain to 1 and 7 days after intranigral grafting of E12.5 embryonic ventral mesencephalon (VM). We found a total of 277 deregulated proteins after transplantation, which are known to be involved in lipid metabolism, oxidative phosphorylation and PD; thus, confirming that our animal model is similar to human PD and that the presence of grafted cells modulates the expression of these proteins. Notably, 7 proteins were commonly downregulated in all selected brain regions after engraftment, including Acta1, Atp6v1e1, Eci3, Lypla2, Pip4k2a, Sccpdh and Sh3gl2. These are known to be involved in the formation of lipids and recycling of dopamine (DA) vesicle at the synapse. Moreover, intranigral transplantation of VM cells decreased the expression of proteins related to oxidative stress, especially in the nigrostriatal pathway containing the DA grafted neurons. In the same regions, an upregulation of several proteins including alpha-synuclein and tyrosine hydroxylase were observed, whereas expression of tetraspanin 7 was shut down. Overall, these results suggest that intranigral transplantation of VM tissue in an animal model of PD may induce a decrease of oxidative stress in the nigrostriatal pathway and a restoration of the machinery of neurotransmitters, particularly dopamine release to promote DA transmission through a decrease of D2 dopamine receptors endocytosis. Identification of new mechanistic elements involved in the nigrostriatal reconstruction process is a promising approach to enhance the repair of this pathway in PD patients undergoing cell therapy.
Project description:We have implemented an integrated Systems Biology approach to analyze overall transcriptomic reprogramming and systems level defense responses in the model plant Arabidopsis thaliana during an insect (Brevicoryne brassicae) and a bacterial (Pseudomonas syringae pv. tomato strain DC3000) attack. The main aim of this study was to identify the attacker-specific and general defense response signatures in the model plant Arabidopsis thaliana while attacked by phloem feeding aphids or pathogenic bacteria. Defense responses and networks, unique and specific for aphid or Pseudomonas stresses were identified. Our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and thus opened up a new direction to conduct large-scale targeted experiments to explore detailed regulatory links among them. The presented results provide a first comprehensive understanding of Arabidopsis - B. brassicae and Arabidopsis - P. syringae interactions at a systems biology level. Arabidopsis thaliana (ecotype Colombia-0) seeds were grown in 6-cm-diameter pots filled with a sterile soil mix (1.0 part soil and 0.5 part horticultural perlite), 3 plants per pot. Plants were kept in growth chambers VM-CM-6tsch VB 1514 (VM-CM-6tsch Industrietechnik GmbH, Germany) under the following conditions: a 8/16 h (light/dark) photoperiod at 22M-BM-0C/18M-BM-0C, 40%/70% relative humidity, and 70/0 M-NM-<mol m-2s-1 light intensity. After 32 days plants had 8 fully developed leaves. Each plant was infested with 32 wingless aphids [Brevicoryne Brassicae] (4 per leaf), which were transferred to leaves with a fine paintbrush. Infested plants and aphid-free controls were kept in plexiglass cylinders. Plants were harvested 72 h after infestation between the 6th and 8th hour of the light photoperiod. Four biological replicates were prepared from aphid infested and control plants, each sampled from 15 individual plants. Whole rosettes were cut at the hypocotyls and aphids were removed by washing with Milli-Q-filtered water. Differences in transcriptional responses were measured by comparing genes expression of aphid infested plants against non-infested control plants.
Project description:A study of differential gene expression in the hippocampus of CV mice (C57Bl x VM/Dk) inoculated with ME7 scrapie to determine the molecular events that accompany the scrapie-associated hippocampal neuropathology. Analysis was performed at 170 days post infection, a timepoint that coincides with severe hippocampal neuronal loss.