Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata. RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents' toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.
BACKGROUND Administering lower total product volumes with high nucleated cell (NC) concentrations may have the potential benefit of decreasing volume‐ and dimethyl sulfoxide (DMSO)‐related patient complications, while maximizing the laboratoryʼs freezer storage capacity. Our study is a retrospective investigation of the effect of HPC(A) products with cell concentrations greater than 3 × 108 NC/mL on clinical and product outcomes in patients undergoing autologous peripheral blood stem cell (PBSC) transplantation. STUDY DESIGN AND METHODS A total of 113 consecutive patients with hematological malignancies who underwent autologous PBSC transplantation were included in this retrospective analysis. The primary outcomes were days to initial absolute neutrophil count (ANC) recovery and initial platelet recovery. The secondary outcomes included the storage duration, segment thaw viability, and dose of viable CD34+ cells/kg administered. RESULTS Of 92 patients and 176 apheresis procedures, 81 patients received HPC(A) products with high NC concentration (4.1 × 108 NC/mL), and 11 patients received low NC concentration products (2.4 × 108 NC/mL). There were no observed differences in clinical outcomes with respect to ANC recovery (14 vs. 14 vs. 12 days) and platelet recovery (16 vs. 16 vs. 15 days) when very high NC (5.2 × 108 NC/mL) and high NC (4.1 × 108 NC/mL) groups were compared to the low NC group (2.4 × 108 NC/mL). CONCLUSION Our retrospective investigation provides further supporting evidence that HPC(A) products with cell concentration greater than 3 × 108 NC/mL did not show detrimental effects on the clinical outcomes in patients undergoing autologous PBSC transplantation.
Cells utilize protein-protein interaction (PPI) networks to receive, transduce, and respond to stimuli. Interaction network rewiring drives devastating diseases like cancers, making PPIs attractive targets for pharmacological intervention. Kinases are druggable nodes in PPI networks but high-throughput proteomics approaches to quantify disease-associated kinome PPI rewiring are lacking. We introduce kinobead competition and correlation analysis (Ki-CCA), a chemoproteomics approach to simultaneously map hundreds of endogenous kinase PPIs. We identified 2,305 PPIs of 300 kinases across 18 diverse cancer lines, quantifying the high plasticity of interaction networks between cancer types, signaling, and phenotypic states; this database of dynamic kinome PPIs provides deep insights into cancer cell signaling. We discovered an AAK1 complex promoting epithelial-mesenchymal transition and drug resistance, and depleting its components sensitized cells to targeted therapy. Ki-CCA enables rapid and highly multiplexed mapping of kinome PPIs in native cell and tissue lysates, without epitope tagged baits, protein labeling, or antibodies.
Background: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. Results: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. Conclusions: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions. Keywords: CryoGrid for tissues cryostoring and sampling, proteinase K, PIXUL RNA extraction, RNA sequencing, transcriptomics
Background Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. Results To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. Conclusions RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.
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