Metabolic Profiling Based Quantitative Evaluation of Hepatocellular Metabolism in Presence of Adipocyte Derived Extracellular Matrix

The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0–4], pre-stable stage [day 5–7] and the stable stage [day 8–10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine

Inertial Focusing of Particles in Curved Micro-channels

Inertial focusing is the migration of particles in flow laterally across a channel into well-defined equilibrium positions. In microfluidic channels, inertial focusing takes advantage of hydrodynamic interactions even at high flow speeds. Particle isolation through inertial focusing is a high throughput method of processing biological samples for point-of-care diagnostics. While photos provide qualitative analyses of inertial focusing, we desired quantitative characterization of these systems. In this study, we ran flow experiments, first with fluorescent polystyrene beads and later with cells in solution, through curved micro-channels at controlled rates using a syringe pump. Our results from polystyrene bead experiments confirmed previous studies on flow through curved micro-channels, in which particles are focused along both sides of the channel at low flow rates and transition towards the center of the channel as the flow rate increases. FWHM analysis also showed that the streamline width is minimized at an intermediate flow rate, indicating inertial focusing is optimized under that condition. As this method of analysis was confirmed with polystyrene beads, we further used this analysis method to characterize the focusing of cells in solution. To maximize both throughput and purity, microfluidic devices must be designed to operate at the highest flow rate at which effective separation from bulk fluid can occur. The device presented in this report indeed isolates the desired target cells to be studied in downstream characterization.http://deepblue.lib.umich.edu/bitstream/2027.42/169578/1/Honors_Capstone_Anna_Kaehr.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169578/2/Kaehr_Anna_Capstone_Poster.pptxhttp://deepblue.lib.umich.edu/bitstream/2027.42/169578/3/Capstone_Presentation_Video_Anna_Kaehr.mp

Global sector-specific Scope 1, 2, and 3 analyses for setting net-zero targets: agriculture, forestry, and processing harvested products

The aim of this research was the development of global 1.5 °C net-zero pathways for specific industries as classified under the Global Industry Classification System (GICS). In this article, we described the analysis of the Agriculture & Food and Forestry & Wood Products categories to determine their industry-specific Scope 1, 2, and 3 emissions on a global level. The accounting methodologies for Scope 3 emissions were developed for entity-level accounting and reporting. However, we suggested an alteration of the methodology for industry-wide Scope 3 analyses because of poor data availability and to avoid counting emissions twice. In this article, we described the calculation method and the key results for net-zero pathways for these two industry sectors. We showed that the decarbonization of the energy supply is possible for both sectors globally by 2050. We also described the land-use-related Scope 3 emissions for the agriculture and forestry sectors. The agricultural sector is unlikely to reach net-zero emissions by 2050, whereas the forest industry can become carbon negative

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110045/1/smll201400719.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110045/2/smll201400719-sup-0001-S1.pd

Antibiotic prescriptions for Oral Diseases in India: Pattern and Practices

Introduction: The key objective of this research was to describe the prescription rate of various antibiotics for dental problems in India and to study the relevance of the prescriptions by analysing antibiotic type associated with different dental diagnoses, using a large scale nationally representative dataset. Methods: We used a 12-month period (May 2015 to April 2016) medical audit dataset from IQVIA (formerly IMS Health). We coded the dental diagnosis provided in the medical audit data to International Statistical Classification of Diseases and Related Health Problems (ICD-11) and the prescribed antibiotics for the diagnosis to Anatomic Therapeutic Chemical (ATC) -2020 classification of World Health Organization. The primary outcome measure was the medicine prescription rate per 1,000 persons per year (PRPY1000). Results: Our main findings were -- 403 prescriptions per 1,000 persons per year in the year 2015 -2016 for all dental ailments. Across all ATC level 1 classification, ‘Diseases of hard tissues’ made up the majority of the prescriptions. ‘Beta-lactam’, ‘Penicillin,’ and ‘Cephalosporins’ were the most commonly prescribed antibiotics for dental diagnoses followed by ‘Macrolides’ and ‘Quinolones’. ‘Dental caries’ , ‘Discoloration of tooth’, and ‘Toothache’ were the most common reasons for ‘Beta-Lactams’ and ‘Penicillin’ prescriptions. Conclusion: To conclude our study reports first ever country (India) level estimates of antibiotic prescription by antibiotic classes, age groups, and ICD-11 classification for dental ailments

Profiling Heterogeneous Circulating Tumor Cells (CTC) Populations in Pancreatic Cancer Using a Serial Microfluidic CTC Carpet Chip

Although isolation of circulating tumor cells (CTCs) from pancreatic adenocarcinoma patients is feasible, investigating their clinical utility has proven less successful than other cancers due to the limitations of epithelial cellular adhesion molecule (EpCAM)‐only based CTC assays. An integrated technology‐ and biology‐based approach using a microfluidic “Carpet Chip” is presented to study the biological relevance of heterogeneous CTC populations. Both epithelial CTCs (EpCs) and epithelial‐to‐mesenchymal transition (EMT)‐like CTCs (EMTCs) are isolated simultaneously from the whole blood of pancreatic cancer (PaCa) patients (n = 35) by separately targeting two surface markers: EpCAM and CD133. Recovery of cancer cell lines spiked into whole blood is ≥97% with >76% purity. Thirty‐four patients had ≥5 EpCs mL−1 and 35 patients had ≥15 EMTCs mL−1. Overall, significantly higher numbers of EMTCs than EpCs are recovered, reflecting the aggressive nature of PaCa. Furthermore, higher numbers of EMTCs are observed in patients with lymph node involvement compared to patients without. Gene expression profiling of CTCs from 17 patients reveals that CXCR1 is significantly upregulated in EpCs, while known stem cell markers POU5F1/Oct‐4 and MYC are upregulated in EMTCs. In conclusion, successful isolation and genomic profiling of heterogeneous CTC populations are demonstrated, revealing genetic signatures relevant to patient outcomes.“Carpet Chip” uses sequential immunoaffinity‐based microfluidics to study the biological relevance of heterogeneous circulating tumor cell (CTCs). Both epithelial (EpCs) and epithelial‐to‐mesenchymal transition (EMT)‐like CTCs (EMTCs) are detectable from the blood of pancreatic cancer patients. Based on our observations of EMTCs and patient lymph node involvement, individualizing therapies targeting genes involved in EMT could reduce metastasis, thereby improving patient survival.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147173/1/adbi201800228-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147173/2/adbi201800228.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147173/3/adbi201800228_am.pd

Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer

BACKGROUND: Circulating tumour cells (CTCs) offer a non-invasive approach to obtain and characterise metastatic tumour cells, but their usefulness has been limited by low CTC yields from conventional isolation methods. METHODS: To improve CTC yields and facilitate their molecular characterisation we compared the Food and Drug Administration-approved CellSearch Epithelial Kit (CEK) to a simplified CTC capture method, CellSearch Profile Kit (CPK), on paired blood samples from patients with metastatic breast (n=75) and lung (n=71) cancer. Molecular markers including Human Epidermal growth factor Receptor 2 (HER2) were evaluated on CTCs by fluorescence in situ hybridisation (FISH) and compared to patients' primary and metastatic cancer. RESULTS: The median cell count from patients with breast cancer using the CPK was 117 vs 4 for CEK (P<0.0001). Lung cancer samples were similar; CPK: 145 cells vs CEK:4 cells (P<0.0001). Recovered CTCs were relatively pure (60-70%) and were evaluable by FISH and immunofluorescence. A total of 10 of 30 (33%) breast cancer patients with HER2-negative primary and metastatic tissue had HER2-amplified CTCs. CONCLUSION: The CPK method provides a high yield of relatively pure CTCs, facilitating their molecular characterisation. Circulating tumour cells obtained using CPK technology demonstrate that significant discordance exists between HER2 amplification of a patient's CTCs and that of the primary and metastatic tumour

High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients.

(1) Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers and current methods for isolation of clusters may fall short. (2) Methods: We applied an inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs/CTC clusters from patients with metastatic non-small-cell lung cancer (NSCLC). (3) Results: Using Labyrinth, CTCs (PanCK+/DAPI+/CD45-) were isolated from patients (n = 25). Heterogeneous CTC populations, including CTCs expressing epithelial (EpCAM), mesenchymal (Vimentin) or both markers were detected. CTCs were isolated from 100% of patients (417 +/- 1023 CTCs/mL). EpCAM- CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of \u3e/=2 CTCs were observed in 96% of patients-of which, 75% were EpCAM-. CTCs revealed identical genetic aberrations as the primary tumor for RET, ROS1, and ALK genes using fluorescence in situ hybridization (FISH) analysis. (4) Conclusions: The Labyrinth device recovered heterogeneous CTCs in 100% and CTC clusters in 96% of patients with metastatic NSCLC. The majority of recovered CTCs/clusters were EpCAM-, suggesting that these would have been missed using traditional antibody-based capture methods