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Cardiovascular-related pathologies are the single leading cause of death in patients with chronic kidney disease (CKD). Previously, we found that a 5/6th nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle, targeting peroxisome proliferator-activated receptor-α and altering the expression of numerous transcripts involved with fatty acid oxidation and glycolysis. In the present study, we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content, lipid peroxidation, and mitochondrial respiration in H9C2 cells. Cells were transfected with anti-miR-21-5p (40 nM), pre-miR-21-5p (20 nM), or the appropriate scrambled oligonucleotide controls before lipid treatment in culture or as part of the Agilent Seahorse XF fatty acid oxidation assay. Overexpression of miR-21-5p attenuated the lipid-induced increase in cellular lipid content, whereas suppression of miR-21-5p augmented it. The abundance of malondialdehyde, a product of lipid peroxidation, was significantly increased with lipid treatment in control cells but attenuated in pre-miR-21-5p-transfected cells. This suggests that miR-21-5p reduces oxidative stress. The cellular oxygen consumption rate (OCR) was increased in both pre-miR-21-5p- and anti-miR-21-5p-transfected cells. Levels of intracellular ATP were significantly higher in anti-mR-21-5p-transfected cells. Pre-miR-21-5p blocked additional increases in OCR in response to etomoxir and palmitic acid. Conversely, anti-miR-21-5p-transfected cells exhibited reduced OCR with both etomoxir and palmitic acid, and the glycolytic capacity was concomitantly reduced. Together, these results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. This likely occurs by reducing cellular lipid uptake and utilization, shifting cellular metabolism toward reliance on the glycolytic pathway. NEW & NOTEWORTHY Both overexpression and suppression of miR-21-5p augment basal and maximal mitochondrial respiration. Our data suggest that reliance on glycolytic and fatty acid oxidation pathways can be modulated by the abundance of miR-21-5p within the cell. miR-21-5p regulation of mitochondrial respiration can be modulated by extracellular lipids.

Cardiovascular events are the leading cause of death in patients with chronic kidney disease (CKD), although the pathological mechanisms are poorly understood. Here we longitudinally characterized left ventricle pathology in a 5/6 nephrectomy rat model of CKD and identify novel molecular mediators. Next-generation sequencing of left ventricle mRNA and microRNA (miRNA) was performed at physiologically distinct points in disease progression, identifying alterations in genes in numerous immune, lipid metabolism, and inflammatory pathways, as well as several miRNAs. MiRNA miR-21-5p was increased in our dataset and has been reported to regulate many identified pathways. Suppression of miR-21-5p protected rats with 5/6 nephrectomy from developing left ventricle hypertrophy and improved left ventricle function. Next-generation mRNA sequencing revealed that miR-21-5p suppression altered gene expression in peroxisome proliferator-activated receptor alpha (PPARα) regulated pathways in the left ventricle. PPARα, a miR-21-5p target, is the primary PPAR isoform in the heart, importantly involved in regulating fatty acid metabolism. Therapeutic delivery of low-dose PPARα agonist (clofibrate) to rats with 5/6 nephrectomy improved cardiac function and prevented left ventricle dilation. Thus, comprehensive characterization of left ventricle molecular changes highlights the involvement of numerous signaling pathways not previously explored in CKD models and identified PPARα as a potential therapeutic target for CKD-related cardiac dysfunction., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.

Mass spectrometry (MS) based proteomic technologies enable the identification and quantification of membrane proteins as well as their post-translational modifications. A prerequisite for their quantitative and reliable MS-based bottom-up analysis is the efficient digestion into peptides by proteases, though digestion of membrane proteins is typically challenging due to their inherent properties such as hydrophobicity. Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach. Multiple parameters, including the number of peptides and proteins identified, total protein sequence coverage, and digestion specificity were used to evaluate transmembrane protein digestion performance. A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage. Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.

High product titer is considered a strategic advantage of fed-batch over perfusion cultivation mode. The titer difference has been experimentally demonstrated and reported in the literature. However, the related theoretical aspects and strategies for optimization of perfusion processes with respect to their fed-batch counterparts have not been thoroughly explored. The present paper introduces a unified framework for comparison of fed-batch and perfusion cultures, and proposes directions for improvement of the latter. The comparison is based on the concept of "equivalent specific perfusion rate", a variable that conveniently bridges various cultivation modes. The analysis shows that development of economically competitive perfusion processes for production of stable proteins depends on our ability to dramatically reduce the dilution rate while keeping high cell density, i.e., operating at low specific perfusion rates. Under these conditions, titer increases significantly, approaching the range of fed-batch titers. However, as dilution rate is decreased, a limit is reached below which performance declines due to poor growth and viability, specific productivity, or product instability. To overcome these limitations, a strategy referred to as "push-to-low" optimization has been developed. This approach involves an iterative stepwise decrease of the specific perfusion rate, and is most suitable for production of stable proteins where increased residence time does not compromise apparent specific productivity or product quality. The push-to-low approach was successfully applied to the production of monoclonal antibody against tumor necrosis factor (TNF). The experimental results followed closely the theoretical prediction, providing a multifold increase in titer. Despite the medium improvement, reduction of the specific growth rate along with increased apoptosis was observed at low specific perfusion rates. This phenomenon could not be explained with limitation or inhibition by the known nutrients and metabolites. Even further improvement would be possible if the cause of apoptosis were understood. In general, a strategic target in the optimization of perfusion processes should be the decrease of the cell-specific perfusion rate to below 0.05 nL/cell/day, resulting in high, batch-like titers. The potential for high titer, combined with high volumetric productivity, stable performance over many months, and superior product/harvest quality, make perfusion processes an attractive alternative to fed-batch production, even in the case of stable proteins.

Protein hydrolysates as substitutes for serum havebeen employed by many in cell culture mediumformulation, especially with the shift to low proteinor protein-free media. More recently, vegetablehydrolysates have also been added as nutritionalsupplements to fortify the amino acid content in smallpeptide form for batch and fed-batch fermentations. Several of these new hydrolysates (peptones of soy,rice, wheat gluten etc.) were tested as protein-freemedium supplements for the production of a recombinanttherapeutic protein. Multiple peptone-supplemented,continuous perfusion bioreactor experiments wereconducted, varying dilution rates and basal mediumcomposition over the various runs. Cell specificrates and product quality studies were obtained forthe various peptones and compared with peptone-freemedium. The potential for peptones to decreaseintrinsic and proteolytic degradation of the productwas also investigated.It was found that peptones confer a nutritionalbenefit, especially at low dilution rates, for therecombinant BHK cell line used in this investigation.The specific productivity increased 20-30% comparedto the peptone-free controls. However, this benefitwas also fully delivered by using fortified medium inplace of the peptone-enriched media. Therefore, whilepeptones may be considered as useful medium additiveswhen development time is limited, their addition maybe avoided by systematic medium development ifpermitted by the time line of the project.

Temperature is a key environmental variable whose potential in animal cell fermentor optimization is not yet fully utilized. The scarce literature data suggests that reduced fermentor temperature results in an improved viability and shear resistance, higher cell density and titer in batch cultures, and reduction in glucose/lactate metabolism. Due to the arrest of the cells in the G1 phase, the specific growth rate was found to decrease at temperatures below 37.0 degrees C. The response of the specific production rate was cell line dependent: in some cases it increased 2-to-3-fold, but decreased in other cases. The controlable slowdown of cell metabolism at lower temperature can be used in optimization of perfusion mammalian cell cultures with several potential advantages, including higher cell density in oxygen limited reactors, lower perfusion rate, improved product quality, simplified pH control, and others. To evaluate this strategy, a series of long-term experiments in 15 L perfusion bioreactors culturing recombinant hamster cells at 20.0 x 10(6) cells/mL were conducted. The temperature was changed over a range of set points, and maintained at each of these for a long period of time. Steady state process data was collected and analyzed. The effect of temperature on the following characteristics of the perfusion process was studied: cell growth, glucose/lactate metabolism, glutamine/ammonia metabolism, cell respiration, cell density at constant oxygen transfer rate, proteolytic activity, and product quality (glycosylation and molecule fragmentation). The results suggest that temperature is a variable with a significant potential in optimization of perfusion cultures. Properly selected temperature set point will contribute to the overall improvement of process performance. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 328-338, 1997.

The accurate, on-line measurement of cell concentration in animal-cell cultures is an on-going problem in bioprocess engineering, and the development of new monitoring techniques is an area of intensive and fruitful research. This article summarizes the recent advances, trends and problems in this field and focuses, in particular, on optical sensors, including the latest laser and infrared probes. Alternative methods, such as multiple-extinction fluorimetry, real-time imaging and particle-size analysis, are also discussed. Although many of these techniques are still at an experimental stage, we believe that some of them have been developed sufficiently that we advocate their routine use in bioprocess monitoring and control.