Project description:Manufactured nanomaterials (MNMs) are increasingly incorporated into consumer products that are disposed into sewage. In wastewater treatment, MNMs adsorb to activated sludge biomass where they may impact biological wastewater treatment performance, including nutrient removal. Here, we studied MNM effects on bacterial polyhydroxyalkanoate (PHA), specifically polyhydroxybutyrate (PHB), biosynthesis because of its importance to enhanced biological phosphorus (P) removal (EBPR). Activated sludge was sampled from an anoxic selector of a municipal wastewater treatment plant (WWTP), and PHB-containing bacteria were concentrated by density gradient centrifugation. After starvation to decrease intracellular PHB stores, bacteria were nutritionally augmented to promote PHB biosynthesis while being exposed to either MNMs (TiO2 or Ag) or to Ag salts (each at a concentration of 5 mg L-1). Cellular PHB concentration and PhyloChip community composition were analyzed. The final bacterial community composition differed from activated sludge, demonstrating that laboratory enrichment was selective. Still, PHB was synthesized to near-activated sludge levels. Ag salts altered final bacterial communities, although MNMs did not. PHB biosynthesis was diminished with Ag (salt or MNMs), indicating the potential for Ag-MNMs to physiologically impact EBPR through the effects of dissolved Ag ions on PHB producers.
Project description:To investigate the locolization between DIS3 and circRNA in fractions form sucrose density gradient centrifugation (SDGC) assay for 293T cells
Project description:Blood was extracted from embryonic hearts at E4 and E6 and non-red blood was separated by density gradient centrifugation Keywords: Cell type comparison
Project description:Manufactured nanomaterials (MNMs) are increasingly incorporated into consumer products that are disposed into sewage. In wastewater treatment, MNMs adsorb to activated sludge biomass where they may impact biological wastewater treatment performance, including nutrient removal. Here, we studied MNM effects on bacterial polyhydroxyalkanoate (PHA), specifically polyhydroxybutyrate (PHB), biosynthesis because of its importance to enhanced biological phosphorus (P) removal (EBPR). Activated sludge was sampled from an anoxic selector of a municipal wastewater treatment plant (WWTP), and PHB-containing bacteria were concentrated by density gradient centrifugation. After starvation to decrease intracellular PHB stores, bacteria were nutritionally augmented to promote PHB biosynthesis while being exposed to either MNMs (TiO2 or Ag) or to Ag salts (each at a concentration of 5 mg L-1). Cellular PHB concentration and PhyloChip community composition were analyzed. The final bacterial community composition differed from activated sludge, demonstrating that laboratory enrichment was selective. Still, PHB was synthesized to near-activated sludge levels. Ag salts altered final bacterial communities, although MNMs did not. PHB biosynthesis was diminished with Ag (salt or MNMs), indicating the potential for Ag-MNMs to physiologically impact EBPR through the effects of dissolved Ag ions on PHB producers. 18 samples; Triplicate PHB-enriched bacterial communities recovered from activated sludge were exposed to nanoparticle (TiO2 or Ag) or AgNO3 (as a silver control) or were not exposed to an nanoparticles (control) to determine if the naoparticles affected PHB production.
Project description:How ferritin regulates neutrophil leukocyte function remains unknown. To address this question, we injected mice with ferritin. bone marrow-derived neutrophils (BMDN) were isolated by density gradient centrifugation. Then we displayed RNA-seq of isolated BMDNs.
Project description:RNA from MCF-7 cells was fractionated by sucrose density gradient centrifugation to separate RNA associated with membrane-bound polysomes from RNA associated with free polysomes. These two populations were hybridized in triplicate to U133A microarrays. Keywords: repeat sample
Project description:Pancreatic islet isolation for single-cell RNA sequencing (scRNA-seq) requires extensive enzymatic and mechanical processing that may affect cell recovery and transcriptional state. We evaluated a practical gradient-free workflow for murine pancreatic islets from diabetic BKS.Cg-Dock7^m +/+ Lepr^db/J mice. The protocol combined intraductal collagenase perfusion, controlled digestion, stereomicroscope-guided manual islet selection without density-gradient purification, and gentle Accutase/EDTA dissociation. Compared with Ficoll-based purification, the gradient-free workflow omitted density-gradient preparation, gradient centrifugation, additional wash centrifugations, and associated transfer steps while yielding viable cells suitable for scRNA-seq. The resulting library met the applied QC criteria and retained the major endocrine populations. Stress-associated transcriptional differences between the two pooled libraries were descriptive only. Overall, the gradient-free workflow represents a feasible alternative to Ficoll-based purification for preparing murine pancreatic islets for scRNA-seq.
Project description:The goal of this study was to analyze the transcriptome of circulating neutrophils from healthy human donors by high-throughput sequencing of total RNA samples obtained from neutrophils purified directly from whole blood (without density-gradient centrifugation or red blood cell lysis) by immunomagnetic negative selection.
Project description:To characterize the transcriptional program that governs terminal granulocytic differentation in vivo, we performed comprehensive microarray analysis of human bone marrow population highly enriched for promyelocytes, myelocytes / metamyelocytes and neotrophils. Bone marrow and peripheral blood samples were collected from healthy individuals in parallel. Cell populations representing successive stages of terminal granulocytic differentation were isolated from human bone marrow samples by 2-layer density gradient centrifugation, neutrophils were collected from peripheral blood using 1-layer density gradient centrifugation. All populations were depleted of nongranulocytic cells by immunomagnetic sorting.
Project description:Fewer than 50% of patients develop vascular and valvular calcification, implying differential pathogenesis. Tissue-entrapped extracellular vesicles (EVs) are found in mineralization but their contents and functions are unstudied. Tissue EVs were isolated from normal and diseased human carotid arteries and aortic valves by enzymatic digestion, serial (ultra)centrifugation and density-gradient separation. Mass spectrometry characterized the density-gradient separation and ability to exclude iodixanol from the density gradient.