Project description:Accidental spills of chemical or oil tankers such as Levoli Sun or Prestige have recently impacted the European seas. The analysis of the genes regulated by exposure to spilled chemical substances could help elucidating adaptatory and compensatory pathways that may participate in pathogenesis in marine biota. Hepatic expression profiles of male juvenile S. maximus exposed to a nº2 fuel-oil (FO) and to styrene were studied employing a turbot custom microarray containing 2715 3â-UTR biassed single-sequences, many of them unannotated. Hybridisations identified 169 and 293 genes significantly regulated after 3 and 14 days of exposure to FO, respectively. Typical PAH responsive genes such as AhRR, ARNT2, CYP1A1 and GST were significantly upregulated. Exposure significantly affected genes involved in protein metabolism, synthesis being the most regulated pathway with ribosomal proteins and various translation factors overexpressed. Protein synthesis could participate in compensatory pathways after FO induced protein damage. Additionally, two weeks of exposure resulted in the upregulation of ras pathway genes. Regarding styrene, gene expression was very variable within experimental groups and only 2 genes were significantly regulated after 3 days of exposure. After 1 week of exposure styrene modified the expression levels of 44 genes. Most highly regulated genes, that may constitute new biomarkers of exposure to styrene, included those coding for estrogen-responsive finger protein, transmembrane 9 superfamily member 4 or selenoprotein-T, but the only pathway found to be significantly regulated was once again that of protein synthesis. Funded EU project PRAGMA, Spanish MEC (CANCERMAR), Basque Government (Consolidated Research Groups GIC07/26-IT-393-07). Turbots were exposed to fuel-oil and to seawater control: 7 turbots in Control tank and 8 in Oiled tank. After 3 days of exposure turbots were in situ dissected and liver was extracted, inmersed in RNAlater and frozen in liquid N2
Project description:Accidental spills of chemical or oil tankers such as Levoli Sun or Prestige have recently impacted the European seas. The analysis of the genes regulated by exposure to spilled chemical substances could help elucidating adaptatory and compensatory pathways that may participate in pathogenesis in marine biota. Hepatic expression profiles of male juvenile S. maximus exposed to a nº2 fuel-oil (FO) and to styrene were studied employing a turbot custom microarray containing 2715 3â-UTR biassed single-sequences, many of them unannotated. Hybridisations identified 169 and 293 genes significantly regulated after 3 and 14 days of exposure to FO, respectively. Typical PAH responsive genes such as AhRR, ARNT2, CYP1A1 and GST were significantly upregulated. Exposure significantly affected genes involved in protein metabolism, synthesis being the most regulated pathway with ribosomal proteins and various translation factors overexpressed. Protein synthesis could participate in compensatory pathways after FO induced protein damage. Additionally, two weeks of exposure resulted in the upregulation of ras pathway genes. Regarding styrene, gene expression was very variable within experimental groups and only 2 genes were significantly regulated after 3 days of exposure. After 1 week of exposure styrene modified the expression levels of 44 genes. Most highly regulated genes, that may constitute new biomarkers of exposure to styrene, included those coding for estrogen-responsive finger protein, transmembrane 9 superfamily member 4 or selenoprotein-T, but the only pathway found to be significantly regulated was once again that of protein synthesis. Funded EU project PRAGMA, Spanish MEC (CANCERMAR), Basque Government (Consolidated Research Groups GIC07/26-IT-393-07). Turbots were exposed to fuel-oil and to seawater control: 7 turbots in Control tank and 8 in Oiled tank. After 14 days of exposure turbots were in situ dissected and liver was extracted, inmersed in RNAlater and frozen in liquid N2
Project description:With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. A total number of 204 juvenile turbot were divided into 3 groups, two of them containing 72 fish and the last one 60 fish. Turbot were anaesthetized by immersion in 50 mg/ml buffered tricaine methanesulfonate (MS-222; Sigma) and then, fish from the first two groups were intramuscularly (i.m.) injected with 50 µl of PBS containing 2 µg of pMCV1.4 or pMCV1.4-G860. Turbot from the last batch were i.m. inoculated with 50 µl of PBS. At 8, 24 and 72 h after injection, 12 fish were removed from the first two tanks and, at 8 h after PBS inoculation, other 12 fish were taken from the last tank. These turbot were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for each treatment and time point (3 turbot/replicate). The remaining fish (36 in the plasmid-injected groups and 48 in the PBS-inoculated tank) were maintained during one month and then, 12 fish from the PBS injected group were separated to another tank. This new group of fish was intraperitoneally (i.p.) injected with 50 µl of MEM + penicillin and streptomycin + 2% FBS (PBS - MEM group), whereas the other turbot were i.p. infected with a dose of VHSV860 of 5 x 105 TCID50/fish (pMCV1.4 - VHSV and pMCV1.4-G860 - VHSV groups). At 8, 24 and 72 hours after infection, 12 fish were removed from the VHSV-infected tanks, and at 8 h after MEM injection the 12 fish were taken from the non-infected tank. The fish were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for treatment and time point (3 turbot/replicate)
Project description:Arctic charr is an especially attractive aquaculture species given that it features the desirable tissue traits of other salmonids, but can be bred and grown at inland freshwater tank farms year round. It is therefore of interest to develop upper temperature tolerant (UTT) strains of Arctic charr to increase the robustness of the species in the face of climate change, as well as to enable production in more southern regions. We conducted an acute temperature trial to identify temperature tolerant and intolerant Arctic charr individuals. Specifically, approximately 200 fish were transferred to an experimental tank (diameter: 1.86 m, depth 50 cm) and left to acclimate for 48 h at ambient temperature. After acclimation, 10 fish were removed to act as a control group, then water that had been diverted through a heat exchanger was added to the flow-through system to increase the water temperature in the tank by 6°C/h until it reached 22°C, then 0.5°C every 30 min until the water reached 25°C, the observed lethal temperature for these fish. When the water temperature reached 25°C, the temperature was held constant and the fish were closely monitored for signs of stress. The first and last 10 individuals to show loss of balance were quickly removed from the tank for sampling, thus representing the 5% least and most temperature tolerant fish, respectively. A reference design microarray study was then performed with the cGRASP 32K microarray using six samples from each group (Intolerant, Tolerant, Control) to identify genes differentially expressed between groups. The results of this study will feed into an ongoing Arctic charr marker-assisted selection based broodstock development program, and may contribute to population-based conservation initiatives for salmonids in general.
Project description:Through 8 generations of selection, our group has developed a strain of rainbow trout that exhibits high growth rates on an economically and environmentally sustainable all plant protein, high-soy diet. The selected strain also shows superior performance in bacterial and viral disease challenges compared to commercial trout strains, and even a strain specifically selected over many generations for viral and bacterial disease resistance. The selection criteria was strictly focused on performance on plant-based diets, and therefore the physiological mechanisms responsible for the strain’s superior disease resistance remain unresolved. To better characterize the physiological mechanism behind the superior performance of the selected strain we compared the intestinal gene expression of the select strain to that of a commercial control line of trout during an experimental bacterial infection with Flavobacterium psychrophilum (Fp) (CSF 259-93), the causative agent of bacterial cold water disease (BCWD) in salmonids. At 65 days post hatch, all female rainbow trout from the select and commercial strain were stocked separately into four 150L tanks each, at a density of 45 fish per tank. For both strains of trout, three tanks of fish were experimentally infected with Fp by intramuscular injection and one control tank was mock challenged by sham injection. Sampling was conducted at 5 days post challenge (dpc) (Early Infection) and 21 dpc (Late/Recovered Infection). Two intestinal samples from each tank were pooled and two pools from each tank were utilized for RNAseq library preparation. The select strain of trout showed significantly better survival rates (Log-Rank Test, p < 0.0001) over the 21 day infection period, with 70 and 95 % mortality among the select and commercial strain, respectively. Reads from the RNAseq samples were quantified at the transcript level prior to evaluating differential transcript usage and differential gene expression between the strains of trout, infection time points, and disease status.
Project description:Through 8 generations of selection, our group has developed a strain of rainbow trout that exhibits high growth rates on an economically and environmentally sustainable all plant protein, high-soy diet. The selected strain also shows superior performance in bacterial and viral disease challenges compared to commercial trout strains, and even a strain specifically selected over many generations for viral and bacterial disease resistance. The selection criteria was strictly focused on performance on plant-based diets, and therefore the physiological mechanisms responsible for the strain’s superior disease resistance remain unresolved. To better characterize the physiological mechanism behind the superior performance of the selected strain we compared the intestinal gene expression of the select strain to that of a commercial control line of trout during an experimental viral infection using the CSF 220-90 isolate of Infectious Hematopoietic Necrosis virus (IHNv). At 65 days post hatch, all female rainbow trout from the select and commercial strain were stocked separately into four 150L tanks each, at a density of 45 fish per tank. For both strains of trout, three tanks of fish were experimentally infected with IHNv by static bath and one tank was mock challenged by static bath (Sham). Sampling was conducted at 4 days post challenge (dpc) (Early Infection) and 20 dpc (Late/Recovered Infection). Two intestinal samples from each tank were pooled and two pools from each tank were utilized for RNAseq library preparation. Infections were mild with mortality rates near 50% over the 21 day trial and no significant difference in mortality rates between the two strains of trout. Reads from the RNAseq samples were quantified at the transcript level prior to evaluating differential transcript usage and differential gene expression between the strains of trout, infection time points, and disease status.
Project description:NK-92 a continuously growing cell line bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) CD19.TaNK recognizing CD19+ B cells represents as potential “off the shelf” therapy candidate for B cell malignancies. The goal of this study was to establish the mechanistic rationale for CD19.TaNK therapy in B-cell NHL (bNHL) and to determine the therapeutic potency in vitro against a host of bNHL cell lines, patient derived primary cell lines (including anti-CD20 antibody resistant cell lines), and in in vivo mouse models. We utilized bNHL cell lines SU-DHL10, SU-DHL4, SU-DHL2 (DLBCL), HF-1 (follicular) and Raji (Burkitt’s) and Rituximab- (RR) and obinutuzumab (OR)-resistant bNHL cell lines (SU-DHL2, SU-DHL4, SU-DHL10), and patient derived primary cells (EL-5 and KSC) were investigated the cytolytic activity of CD19.TaNK. We observed significantly increased CD19.TaNK mediated cytolytic activity at E:T ratios (1:1-10:1) via LDH release in all bNHL cell lines and CD20 resistant bHNL cells. Further, the dynamic efficacy of CD19.TaNK determined using droplet based single cell microfluidics analysis of cell interactions (1:1) between CD19.TaNK and anti-CD20 sensitive or resistant bNHL cell showed that vast majority of the cells were killed by single contact >80% (SU-DHL 4, SUD-HL 4 -OR), 40% (SU-DHL10-RR), >60% (SU-DHL10, SUD-HL-10-OR) and 40% (SU-DHL10-RR) within first 40 minutes, while the remainder were killed through events requiring multiple contacts. Thus, suggesting that CD19.TaNK indiscriminately kills both anti-CD20 sensitive and resistant cells. Global transcriptome analysis performed using flow sorted bNHL co-cultured with CD19.TaNK at 1:1 ratio for two hours, revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, immunoregulatory or chemokine signaling pathways in these bNHL cells. Using proximity extension assay based 92-plex cytokine panel we observed increased secretion of various cytokines, granzymes and decreased secretions of ADA, HO-1, CD5, CD28, CD70, CD244, IFN and TNF consistently with anti-CD20 sensitive and resistant cells. Altogether these results demonstrate that CD19.TaNK inflicts mechanistically conserved killing activity against different bNHL cell lines, including in anti-CD20 refractory bNHL. Finally, in SCID mice experiments we observed marked reduction in the volume of SU-DHL10 derived tumor xenografts with infusion of CD19.TaNK compared to control. Overall, we observed potent anti-lymphoma activity with CD19.TaNK involving biologically conserved mechanisms indicating that CD19.TaNK could be equally active under untreated or refractory bNHL in the clinical settings.
Project description:NK-92 a continuously growing cell line bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) CD19.TaNK recognizing CD19+ B cells represents as potential “off the shelf” therapy candidate for B cell malignancies. The goal of this study was to establish the mechanistic rationale for CD19.TaNK therapy in B-cell NHL (bNHL) and to determine the therapeutic potency in vitro against a host of bNHL cell lines, patient derived primary cell lines (including anti-CD20 antibody resistant cell lines), and in in vivo mouse models. We utilized bNHL cell lines SU-DHL10, SU-DHL4, SU-DHL2 (DLBCL), HF-1 (follicular) and Raji (Burkitt’s) and Rituximab- (RR) and obinutuzumab (OR)-resistant bNHL cell lines (SU-DHL2, SU-DHL4, SU-DHL10), and patient derived primary cells (EL-5 and KSC) were investigated the cytolytic activity of CD19.TaNK. We observed significantly increased CD19.TaNK mediated cytolytic activity at E:T ratios (1:1-10:1) via LDH release in all bNHL cell lines and CD20 resistant bHNL cells. Further, the dynamic efficacy of CD19.TaNK determined using droplet based single cell microfluidics analysis of cell interactions (1:1) between CD19.TaNK and anti-CD20 sensitive or resistant bNHL cell showed that vast majority of the cells were killed by single contact >80% (SU-DHL 4, SUD-HL 4 -OR), 40% (SU-DHL10-RR), >60% (SU-DHL10, SUD-HL-10-OR) and 40% (SU-DHL10-RR) within first 40 minutes, while the remainder were killed through events requiring multiple contacts. Thus, suggesting that CD19.TaNK indiscriminately kills both anti-CD20 sensitive and resistant cells. Global transcriptome analysis performed using flow sorted bNHL co-cultured with CD19.TaNK at 1:1 ratio for two hours, revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, immunoregulatory or chemokine signaling pathways in these bNHL cells. Using proximity extension assay based 92-plex cytokine panel we observed increased secretion of various cytokines, granzymes and decreased secretions of ADA, HO-1, CD5, CD28, CD70, CD244, IFN and TNF consistently with anti-CD20 sensitive and resistant cells. Altogether these results demonstrate that CD19.TaNK inflicts mechanistically conserved killing activity against different bNHL cell lines, including in anti-CD20 refractory bNHL. Finally, in SCID mice experiments we observed marked reduction in the volume of SU-DHL10 derived tumor xenografts with infusion of CD19.TaNK compared to control. Overall, we observed potent anti-lymphoma activity with CD19.TaNK involving biologically conserved mechanisms indicating that CD19.TaNK could be equally active under untreated or refractory bNHL in the clinical settings.
Project description:In large-scale production processes, metabolic control is typically achieved by limited supply of essential nutrients like ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programs which result in production yield losses. This study is focused on transcriptional changes due to fluctuating ammonia supply, while sampling a continuously running two-compartment bioreactor system comprising a stirred tank reactor (STR) and a plug flow reactor (PFR). A previously created mutant E.coli SR was used to limit the reaction to environmntal influences via knock-out of stringent response. E. coli WT revealed highly diverging short-term transcriptional responses in ammonia fluctuation compared E. coli SR.