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Cellular agriculture aims to meet the growing demand for animal products. However, current production technologies result in low yields, leading to economic projections that prohibit cultivated meat scalability. Here we use tangential flow filtration for continuous manufacturing of cultivated meat to produce biomass of up to 130 × 10 6 cells per ml, corresponding to yields of 43% w/v and multiple harvests for over 20 days. Continuous manufacturing was carried out in an animal-component-free culture medium for US$0.63 l -1 that supports the long-term, high density culture of chicken cells. Using this empirical data, we conducted a techno-economic analysis for a theoretical production facility of 50,000 l, showing that the cost of cultivated chicken can drop to within the range of organic chicken at US$6.2 lb -1 by using perfusion technology. Whereas other variables would also affect actual market prices, continuous manufacturing can offer cost reductions for scaling up cultivated meat production., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

One of the key challenges in diagnosing thyroid cancer lies in the substantial percentage of indeterminate diagnoses of thyroid nodules that have undergone ultrasound-guided fine-needle aspiration (FNA) biopsy for cytological evaluation. This delays the definitive diagnosis and treatment plans. We recently demonstrated that hydroxyapatite microcalcifications (MCs) aspirated from thyroid nodules may aid nodule diagnosis based on their composition. In particular, Zn-enriched MCs have emerged as potential cancer biomarkers. However, a pertinent question remains: is the elevated Zn content within MCs a consequence of cancer, or do the Zn-enriched MCs encourage tumorigenesis? To address this, we treated the human thyroid cancer cell line MDA-T32 with synthetic MC analogs comprising hydroxyapatite crystals with varied pathologically relevant Zn fractions and assessed the cellular response. The MC analogs exhibited an irregular surface morphology similar to FNA MCs observed in cancerous thyroid nodules. These MC analogs displayed an inverse relationship between Zn fraction and crystallinity, as shown by X-ray diffractometry. The zeta potential of the non-Zn-bearing hydroxyapatite crystals was negative, which decreased once Zn was incorporated into the crystal. The MC analogs were not cytotoxic. The cellular response to exposure to these crystals was evaluated in terms of cell migration, proliferation, the tendency of the cells to form multicellular spheroids, and the expression of cancer markers. Our findings suggest that, if thyroid MCs play a role in promoting cancerous behavior in vivo , it is likely a result of the interplay of crystallinity with Zn and carbonate fractions in MCs.

Pathological calcifications, especially calcium phosphate microcalcifications (MCs), appear in most early breast cancer lesions, and their formation correlates with more aggressive tumors and a poorer prognosis. Hydroxyapatite (HA) is a key MC component that crystallizes in the tumor microenvironment. It is often associated with malignant breast cancer lesions and can trigger tumorigenesis in vitro . Here, we investigate the impact of additives on HA crystallization and inhibition, and how precancerous breast cells respond to minerals that are deposited in the presence of these additives. We show that nonstoichiometric HA spontaneously crystallizes in a solution simulating the tumor microenvironmental fluids and exhibits lump-like morphology similar to breast cancer MCs. In this system, the effectiveness of poly(aspartic acid) and poly(acrylic acid) (PAA) to inhibit HA is examined as a potential route to improve cancer prognosis. In the presence of additives, the formation of HA lumps is associated with the promotion or only minimal inhibition of mineralization, whereas the formation of amorphous calcium phosphate (ACP) lumps is followed by inhibition of mineralization. PAA emerges as a robust HA inhibitor by forming spherical ACP particles. When precancerous breast cells are exposed to various HA and ACP minerals, the most influential factors on cell proliferation are the mineral phase and whether the mineral is in the form of discrete particles or particle aggregates. The tumorigenic effects on cells, ranging from cytotoxicity and suppression of proliferation to triggering of proliferation, can be summarized as HA particles < HA aggregates < ACP particles < ACP aggregates. The cellular response to minerals can be attributed to a combination of factors, including mineral phase, crystallinity, morphology, surface texture, aggregation state, and surface potential. These findings have implications for understanding mineral-cell interactions within the tumor microenvironment and suggest that, in some cases, the byproducts of HA inhibition can contribute to disease progression more than HA itself.

The study of cardiac physiology is hindered by physiological differences between humans and small-animal models. Here we report the generation of multi-chambered self-paced vascularized human cardiac organoids formed under anisotropic stress and their applicability to the study of cardiac arrhythmia. Sensors embedded in the cardiac organoids enabled the simultaneous measurement of oxygen uptake, extracellular field potentials and cardiac contraction at resolutions higher than 10 Hz. This microphysiological system revealed 1 Hz cardiac respiratory cycles that are coupled to the electrical rather than the mechanical activity of cardiomyocytes. This electro-mitochondrial coupling was driven by mitochondrial calcium oscillations driving respiration cycles. Pharmaceutical or genetic inhibition of this coupling results in arrhythmogenic behaviour. We show that the chemotherapeutic mitoxantrone induces arrhythmia through disruption of this pathway, a process that can be partially reversed by the co-administration of metformin. Our microphysiological cardiac systems may further facilitate the study of the mitochondrial dynamics of cardiac rhythms and advance our understanding of human cardiac physiology., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The combination of gene therapy and immunotherapy concepts, along recent advances in DNA nanotechnology, have the potential to provide important tools for cancer therapies. We present the development of stimuli-responsive microcapsules, loaded with a viral immunogenetic agent, harnessing the immune response against the Coronavirus Disease 2019, COVID-19, to selectively attack liver cancer cells (hepatoma) or recognize breast cancer or hepatoma, by expression of green fluorescence protein, GFP. The pH-responsive microcapsules, modified with DNA-tetrahedra nanostructures, increased hepatoma permeation by 50 %. Incorporation of a GFP-encoding lentivirus vector inside the tumor-targeting pH-stimulated miRNA-triggered and Alpha-fetoprotein-dictated microcapsules enables the demonstration of neoplasm selectivity, with approximately 5,000-, 8,000- and 50,000-fold more expression in the cancerous cells, respectively. The incorporation of the SARS-CoV-2 spike protein in the gene vector promotes specific recognition of the immune-evading hepatoma by the COVID-19-analogous immune response, which leads to cytotoxic and inflammatory activity, mediated by serum components taken from vaccinated or recovered COVID-19 patients, resulting in effective elimination of the hepatoma (>85 % yield)., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Viral infection is associated with a significant rewire of the host metabolic pathways, presenting attractive metabolic targets for intervention., Methods: We chart the metabolic response of lung epithelial cells to SARS-CoV-2 infection in primary cultures and COVID-19 patient samples and perform in vitro metabolism-focused drug screen on primary lung epithelial cells infected with different strains of the virus. We perform observational analysis of Israeli patients hospitalized due to COVID-19 and comparative epidemiological analysis from cohorts in Italy and the Veteran's Health Administration in the United States. In addition, we perform a prospective non-randomized interventional open-label study in which 15 patients hospitalized with severe COVID-19 were given 145 mg/day of nanocrystallized fenofibrate added to the standard of care., Results: SARS-CoV-2 infection produced transcriptional changes associated with increased glycolysis and lipid accumulation. Metabolism-focused drug screen showed that fenofibrate reversed lipid accumulation and blocked SARS-CoV-2 replication through a PPARα-dependent mechanism in both alpha and delta variants. Analysis of 3233 Israeli patients hospitalized due to COVID-19 supported in vitro findings. Patients taking fibrates showed significantly lower markers of immunoinflammation and faster recovery. Additional corroboration was received by comparative epidemiological analysis from cohorts in Europe and the United States. A subsequent prospective non-randomized interventional open-label study was carried out on 15 patients hospitalized with severe COVID-19. The patients were treated with 145 mg/day of nanocrystallized fenofibrate in addition to standard-of-care. Patients receiving fenofibrate demonstrated a rapid reduction in inflammation and a significantly faster recovery compared to patients admitted during the same period., Conclusions: Taken together, our data suggest that pharmacological modulation of PPARα should be strongly considered as a potential therapeutic approach for SARS-CoV-2 infection and emphasizes the need to complete the study of fenofibrate in large randomized controlled clinical trials., Funding: Funding was provided by European Research Council Consolidator Grants OCLD (project no. 681870) and generous gifts from the Nikoh Foundation and the Sam and Rina Frankel Foundation (YN). The interventional study was supported by Abbott (project FENOC0003)., Clinical Trial Number: NCT04661930., Competing Interests: AE is registered as an investor in a PCT regarding the use of metabolic regulators for COVID. The author has a patent on the use of PPAR agonists to treat COVID. The author has no other competing interests to declare, KI, MN, IA, YD, NA, LK, NR, MH, SS, IH, CP, MC, AG, AB, MM, SM No competing interests declared, AC has received personal honoraria for statistical consultation from Recipharm, and personal honoraria for manuscript writing from both Sharper Srl and Fidia Pharmaceuticals. The author has no other competing interests to declare, CS is President of Fondazione (totally supported by family). The author has no other competing interests to declare, JC received funding from National Institutes of Health (1R01HL157108-01A1,1R01AG074989-01) . The author has no other competing interests to declare, JC has received consulting honoraria from Sanifit, Bristol Myers Squibb, Merck, Edwards Lifesciences, Bayer, JNJ, Fukuda-Denshi, NGM Bio, Mayo institute of technology and the University of Delaware, and research grants from the National Institutes of Health, Abbott, Microsoft, Fukuda-Denshi and Bristol Myers Squibb. He has received compensation from the American Heart Association and the American College of Cardiology for editorial roles, and visiting speaker honoraria from Washington University, Emory University, University of Utah, the Japanese Association for Cardiovascular Nursing and the Korean Society of Cardiology. The author is named as inventor in a University of Pennsylvania patent for the use of inorganic nitrates/nitrites for the treatment of Heart Failure and Preserved Ejection Fraction and for the use of biomarkers in heart failure with preserved ejection fraction. The author has participated on the Advisory board for Bristol-Myers Squibb Data safety monitoring board for studies by the University of Delaware and UT Southwestern, and is Vice President of North American Artery Society. The author has received research device loans from Atcor Medical, Fukuda-Denshi, Unex, Uscom, NDD Medical Technologies, Microsoft, and MicroVision Medical. The author has no other competing interests to declare, LD is affiliated with BioStats Statistical Consulting Ltd where they work as a Biostatistician. The authors has received payment for statistical work for the manuscript and consulting fees from Tissue Dynamics Ltd. The author has no other competing interests to declare, OS has received consulting honoraria from Sanofi, Roche and Neopharm, and lectures honoraria from Roche . He is the chairmen of the Israel Association for the study of the liver. The author has no other competing interests to declare, YN is registered as an investor in a PCT regarding the use of metabolic regulators for COVID and has a patent on the use of PPAR agonists to treat COVID. The author has no other competing interests to declare, (© 2023, Ehrlich et al.)

Aminoglycosides are an important class of antibiotics that play a critical role in the treatment of life-threatening infections, but their use is limited by their toxicity. In fact, gentamicin causes severe nephrotoxicity in 17% of hospitalized patients. The kidney proximal tubule is particularly vulnerable to drug-induced nephrotoxicity due to its role in drug transport. In this work, we developed a perfused vascularized model of human kidney tubuloids integrated with tissue-embedded microsensors that track the metabolic dynamics of aminoglycoside-induced renal toxicity in real time. Our model shows that gentamicin disrupts proximal tubule polarity at concentrations 20-fold below its TC 50 , leading to a 3.2-fold increase in glucose uptake, and reverse TCA cycle flux culminating in a 40-fold increase in lipid accumulation. Blocking glucose reabsorption using the SGLT2 inhibitor empagliflozin significantly reduced gentamicin toxicity by 10-fold. These results demonstrate the utility of sensor-integrated kidney-on-chip platforms to rapidly identify new metabolic mechanisms that may underly adverse drug reactions. The results should improve our ability to modulate the toxicity of novel aminoglycosides.

Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron-sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron-sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT-VDAC1-mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5&#95;c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein-protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While validating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and β-catenin are affected by nsp2, nsp5&#95;c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19., Competing Interests: RR, MS, YL, RN, EP, RT, VM, TB, KS, AE, KI, YN, RS, UA, BM No competing interests declared, (© 2021, Rauti et al.)

Decreased insulin secretion, associated with pancreatic β-cell failure, plays a critical role in many human diseases including diabetes, obesity, and cancer. While numerous studies linked β-cell failure with enhanced levels of reactive oxygen species (ROS), the development of diabetes associated with hereditary conditions that result in iron overload, e.g., hemochromatosis, Friedreich's ataxia, and Wolfram syndrome type 2 (WFS-T2; a mutation in CISD2 , encoding the [2Fe-2S] protein NAF-1), underscores an additional link between iron metabolism and β-cell failure. Here, using NAF-1-repressed INS-1E pancreatic cells, we observed that NAF-1 repression inhibited insulin secretion, as well as impaired mitochondrial and ER structure and function. Importantly, we found that a combined treatment with the cell permeant iron chelator deferiprone and the glutathione precursor N-acetyl cysteine promoted the structural repair of mitochondria and ER, decreased mitochondrial labile iron and ROS levels, and restored glucose-stimulated insulin secretion. Additionally, treatment with the ferroptosis inhibitor ferrostatin-1 decreased cellular ROS formation and improved cellular growth of NAF-1 repressed pancreatic cells. Our findings reveal that suppressed expression of NAF-1 is associated with the development of ferroptosis-like features in pancreatic cells, and that reducing the levels of mitochondrial iron and ROS levels could be used as a therapeutic avenue for WFS-T2 patients.

Circadian rhythms describe physiological systems that repeat themselves with a cycle of approximately 24 h. Our understanding of the cellular and molecular origins of these oscillations has improved dramatically, allowing us to appreciate the significant role these oscillations play in maintaining physiological homeostasis. Circadian rhythms allow living organisms to predict and efficiently respond to a dynamically changing environment, set by repetitive day/night cycles. Since circadian rhythms underlie almost every aspect of human physiology, it is unsurprising that they also influence the response of a living organism to disease, stress, and therapeutics. Therefore, not only do the mechanisms that maintain health and disrupt homeostasis depend on our internal circadian clock, but also the way drugs are perceived and function depends on these physiological rhythms. We present a holistic view of the therapeutic process, discussing components such as disease state, pharmacokinetics, and pharmacodynamics, as well as adverse reactions that are critically affected by circadian rhythms. We outline challenges and opportunities in moving toward personalized medicine approaches that explore and capitalize on circadian rhythms for the benefit of the patient.

The kidney plays a critical role in fluid homeostasis, glucose control, and drug excretion. Loss of kidney function due to drug-induced nephrotoxicity affects over 20% of the adult population. The kidney proximal tubule is a complex vascularized structure that is particularly vulnerable to drug-induced nephrotoxicity. Here, we introduce a model of vascularized human kidney spheroids with integrated tissue-embedded microsensors for oxygen, glucose, lactate, and glutamine, providing real-time assessment of cellular metabolism. Our model shows that both the immunosuppressive drug cyclosporine and the anticancer drug cisplatin disrupt proximal tubule polarity at subtoxic concentrations, leading to glucose accumulation and lipotoxicity. Impeding glucose reabsorption using glucose transport inhibitors blocked cyclosporine and cisplatin toxicity by 1000- to 3-fold, respectively. Retrospective study of 247 patients who were diagnosed with kidney damage receiving cyclosporine or cisplatin in combination with the sodium-glucose cotransporter-2 (SGLT2) inhibitor empagliflozin showed significant ( P < 0.001) improvement of kidney function, as well as reduction in creatinine and uric acid, markers of kidney damage. These results demonstrate the potential of sensor-integrated kidney-on-chip platforms to elucidate mechanisms of action and rapidly reformulate effective therapeutic solutions, increasing drug safety and reducing the cost of clinical and commercial failures., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Inspired by nature, where dynamic networks control the levels of gene expression and the activities of transcribed/translated proteins, we introduce nucleic acid-based constitutional dynamic networks (CDNs) as functional modules mimicking native circuits by demonstrating CDNs-guided programmed synthesis of genes, controlled transcription of RNAs, and dictated transcription/translation synthesis of proteins. An auxiliary CDN consisting of four dynamically equilibrated constituents AA', AB', BA', and BB' is orthogonally triggered by two different inputs yielding two different compositionally reconfigured CDNs. Subjecting the parent auxiliary CDN to two hairpins, H A and H B , and two templates T A and T B and a nicking/replication machinery leads to the cleavage of the hairpins and to the activation of the nicking/replication machineries that synthesize two "genes", e.g., the histidine-dependent DNAzyme g1 and the Zn 2+ -ion-dependent DNAzyme g2. The triggered orthogonal reconfiguration of the parent CDN to the respective CDNs leads to the programmed preferred CDN-guided synthesis of g1 or g2. Similarly, the triggered reconfigured CDNs are subjected to two hairpins H C and H D , the templates I'/I and J'/J, and the RNA polymerase (RNAp)/NTPs machinery. While the cleavage of the hairpins by the constituents associated with the parent CDN leads to the transcription of the broccoli aptamer recognizing the DFHBI ligand and of the aptamer recognizing the malachite green (MG) ligand, the orthogonally triggered CDNs lead to the CDNs-guided enhanced transcription of either the DFHBI aptamer or the MG aptamer. In addition, subjecting the triggered reconfigured CDNs to predesigned hairpins H E and H F , the templates M'/M and N'/N, the RNAp/NTPs machinery, and the cell-free ribosome t-RNA machinery leads to the CDNs-guided transcription/translation of the green fluorescence protein (GFP) or red fluorescence protein (RFP).

The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the respective hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrices. In addition, loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concentrations of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The liver is the central hub of xenobiotic metabolism and consequently the organ most prone to cosmetic- and drug-induced toxicity. Failure to detect liver toxicity or to assess compound clearance during product development is a major cause of postmarketing product withdrawal, with disastrous clinical and financial consequences. While small animals are still the preferred model in drug development, the recent ban on animal use in the European Union created a pressing need to develop precise and efficient tools to detect human liver toxicity during cosmetic development. This article includes a brief review of liver development, organization, and function and focuses on the state of the art of long-term cell culture, including hepatocyte cell sources, heterotypic cell-cell interactions, oxygen demands, and culture medium formulation. Finally, the article reviews emerging liver-on-chip devices and discusses the advantages and pitfalls of individual designs. The goal of this review is to provide a framework to design liver-on-chip devices and criteria with which to evaluate this emerging technology.

The hepatitis C virus (HCV) non-structural protein 3 (NS3) is essential for HCV maturation. The NS3/4A protease is a target for several HCV treatments and is a well-known target for HCV drug discovery. The protein is membrane associated and thus probably interacts with other membrane proteins. However, the vast majority of known NS3 host partners are soluble proteins rather than membrane proteins, most likely due to lack of appropriate platforms for their discovery. Utilization of an integrated microfluidics platform enables analysis of membrane proteins in their native form. We screened over 2800 membrane proteins for interaction with NS3 and 90 previously unknown interactions were identified. Of these, several proteins were selected for validation by co-immunoprecipitation and for NS3 proteolytic activity. Bearing in mind the considerable number of interactions formed, together with the popularity of NS3/4A protease as a drug target, it was striking to note its lack of proteolytic activity. Only a single protein, Neuregulin1, was observed to be cleaved, adding to the 3 known NS3/4A cleavage targets. Neuregulin1 participates in neural proliferation. Recent studies have shown its involvement in HCV infection and hepatocellular carcinoma. We showed that NS3/4A triggers an increase in neuregulin1 mRNA levels in HCV infected cells. Despite this increase, its protein concentration is decreased due to proteolytic cleavage. Additionally, its EGF-like domain levels were increased, possibly explaining the ErbB2 and EGFR upregulation in HCV infected cells. The newly discovered protein interactions may provide insights into HCV infection mechanisms and potentially provide new therapeutic targets against HCV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Microfluidic sorting offers a unique ability to isolate large numbers of cells for bulk proteomic or metabolomics studies but is currently limited by low throughput and persistent clogging at low flow rates. Recently we uncovered the physical principles governing the inertial focusing of particles in high-Reynolds numbers. Here, we superimpose high Reynolds inertial focusing on Dean vortices, to rapidly isolate large quantities of young and adult yeast from mixed populations at a rate of 10 7 cells/min/channel. Using a new algorithm to rapidly quantify budding scars in isolated yeast populations and system-wide proteomic analysis, we demonstrate that protein quality control and expression of established yeast aging markers such as CalM, RPL5, and SAM1 may change after the very first replication events, rather than later in the aging process as previously thought. Our technique enables the large-scale isolation of microorganisms based on minute differences in size (±1.5 μm), a feat unmatched by other technologies.

Drug development is currently hampered by the inability of animal experiments to accurately predict human response. While emerging organ on chip technology offers to reduce risk using microfluidic models of human tissues, the technology still mostly relies on end-point assays and biomarker measurements to assess tissue damage resulting in limited mechanistic information and difficulties to detect adverse effects occurring below the threshold of cellular damage. Here we present a sensor-integrated liver on chip array in which oxygen is monitored using two-frequency phase modulation of tissue-embedded microprobes, while glucose, lactate and temperature are measured in real time using microfluidic electrochemical sensors. Our microphysiological platform permits the calculation of dynamic changes in metabolic fluxes around central carbon metabolism, producing a unique metabolic fingerprint of the liver's response to stimuli. Using our platform, we studied the dynamics of human liver response to the epilepsy drug Valproate (Depakine™) and the antiretroviral medication Stavudine (Zerit™). Using E6/E7LOW hepatocytes, we show TC50 of 2.5 and 0.8 mM, respectively, coupled with a significant induction of steatosis in 2D and 3D cultures. Time to onset analysis showed slow progressive damage starting only 15-20 hours post-exposure. However, flux analysis showed a rapid disruption of metabolic homeostasis occurring below the threshold of cellular damage. While Valproate exposure led to a sustained 15% increase in lipogenesis followed by mitochondrial stress, Stavudine exposure showed only a transient increase in lipogenesis suggesting disruption of β-oxidation. Our data demonstrates the importance of tracking metabolic stress as a predictor of clinical outcome.

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe Grx1-roGFP2. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach, we defined a redox-dependent heterogeneity of yeast cells and characterized growth, as well as proteomic and transcriptomic profiles of distinctive redox subpopulations. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis., Competing Interests: MR, RF, OY, WB, NS, JG, SI, YG, ST, YN, TR, NF, MS, DR No competing interests declared, (© 2018, Radzinski et al.)

Altered glucose reabsorption via the facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CB 1 R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB 1 R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB 1 R blockade or genetically inactivating CB 1 Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB 1 R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB 1 R or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CB 1 R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN., (Copyright © 2018 by the American Society of Nephrology.)

The facilitative glucose transporter (GLUT) family plays a key role in metabolic homeostasis, controlling the absorption rates and rapid response to changing carbohydrate levels. The facilitative GLUT2 transporter is uniquely expressed in metabolic epithelial cells of the intestine, pancreas, liver, and kidney. GLUT2 dysfunction is associated with several pathologies, including Fanconi-Bickel syndrome, a glycogen storage disease, characterized by growth retardation and renal dysfunction. Interestingly, GLUT2 activity is modulated by its cellular localization. Membrane translocation specifically regulates GLUT2 activity in enterocytes, pancreatic β-cells, hepatocytes, and proximal tubule cells. We have established a system to visualize and quantify GLUT2 translocation, and its dynamics, by live imaging of a mCherry-hGLUT2 fusion protein in polarized epithelial cells. This system enables testing of putative modulators of GLUT2 translocation, which are potential drugs for conditions of impaired glucose homeostasis and associated nephropathy.

Lipid droplets (LDs) are regulated neutral lipid storage organelles having a central role in numerous cellular processes as well as in various pathologies such as metabolic disorders, immune responses and during pathogen infection. Due to the growing significance of LDs, extensive efforts are made to study the mechanism and the dynamics of their formation and life history and how are these diverted or modified by pathogens. Real-time visualization of lipid droplet biogenesis can assist in clarifying these and other important issues and may have implications towards understanding the pathogenesis of the associated diseases. Typically, LDs are post-experimentally stained using lipophilic dyes and are visualized under a microscope. Alternatively, overexpression of LD-associated proteins or immunofluorescence analyses are used to identify and follow LDs. These experimental approaches only examine a single end point of the experiment and cannot answer questions regarding LD dynamics. Here, we describe a simple and novel experimental setting that allows real-time fluorescence staining and detection of LDs in cultured living as well as infected cells. This method is quick and simple and is not restricted to a specific dye or cell line. Using this system, the biogenesis of LDs and their growth is demonstrated in cells infected with hepatitis C virus (HCV), confirming the strength of this method and the wide range of its applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Gradients of diffusible signaling molecules play important role in various processes, ranging from cell differentiation to toxicological evaluation. Microfluidic technology provides an accurate control of tempospatial conditions. However, current microfluidic platforms are not designed to handle multiple gradients and cell populations simultaneously. Here, we demonstrate a rapidly adaptable microfluidic design able to expose multiple cell populations to an array of chemical gradients. Our design is based on pressure-equilibrated concentric channels and a pressure-dissipating control layer, facilitating the seeding of multiple cell populations in a single device. The design was numerically evaluated and experimentally validated. The device consists of 8 radiating stimuli channels and 12 circular cell culture channels, creating an array of 96 different continuous gradients that can be simultaneously monitored over time.

Viruses lack the basic machinery needed to replicate and therefore must hijack the host's metabolism to propagate. Virus-induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host-pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)-responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4α, ketogenesis by PPARα and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4α reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARα or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a host's metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host.

Prediction of drug-induced toxicity is complicated by the failure of animal models to extrapolate human response, especially during assessment of repeated dose toxicity for cosmetic or chronic drug treatments. In this work, we present a 3D microreactor capable of maintaining metabolically active HepG2/C3A spheroids for over 28 days in vitro under stable oxygen gradients mimicking the in vivo microenvironment. Mitochondrial respiration was monitored using two-frequency phase modulation of phosphorescent microprobes embedded in the tissue. Phase modulation is focus independent and unaffected by cell death or migration. This sensitive measurement of oxygen dynamics revealed important information on the drug mechanism of action and transient subthreshold effects. Specifically, exposure to antiarrhythmic agent, amiodarone, showed that both respiration and the time to onset of mitochondrial damage were dose dependent showing a TC50 of 425 μm. Analysis showed significant induction of both phospholipidosis and microvesicular steatosis during long-term exposure. Importantly, exposure to widely used analgesic, acetaminophen, caused an immediate, reversible, dose-dependent loss of oxygen uptake followed by a slow, irreversible, dose-independent death, with a TC50 of 12.3 mM. Transient loss of mitochondrial respiration was also detected below the threshold of acetaminophen toxicity. The phenomenon was repeated in HeLa cells that lack CYP2E1 and 3A4, and was blocked by preincubation with ascorbate and TMPD. These results mark the importance of tracing toxicity effects over time, suggesting a NAPQI-independent targeting of mitochondrial complex III might be responsible for acetaminophen toxicity in extrahepatic tissues.

Microfluidic organ-on-a-chip technology aims to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity, as well as pluripotency and disease processes. However, current methods to evaluate mitochondrial activity still rely on end-point assays, resulting in limited kinetic and prognostic information. Here, we present a liver-on-chip device capable of maintaining human tissue for over a month in vitro under physiological conditions. Mitochondrial respiration was monitored in real time using two-frequency phase modulation of tissue-embedded phosphorescent microprobes. A computer-controlled microfluidic switchboard allowed contiguous electrochemical measurements of glucose and lactate, providing real-time analysis of minute shifts from oxidative phosphorylation to anaerobic glycolysis, an early indication of mitochondrial stress. We quantify the dynamics of cellular adaptation to mitochondrial damage and the resulting redistribution of ATP production during rotenone-induced mitochondrial dysfunction and troglitazone (Rezulin)-induced mitochondrial stress. We show troglitazone shifts metabolic fluxes at concentrations previously regarded as safe, suggesting a mechanism for its observed idiosyncratic effect. Our microfluidic platform reveals the dynamics and strategies of cellular adaptation to mitochondrial damage, a unique advantage of organ-on-chip technology.

Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.

Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expression of the human papilloma virus (HPV) genes E6 and E7 coupled with inhibition of epithelial-to-mesenchymal transition. We show that E6 and E7 expression upregulates the OSM receptor gp130 and that OSM stimulation induces hepatocytes to expand for up to 40 population doublings, producing 10 13 to 10 16 cells from a single human hepatocyte isolate. OSM removal induces differentiation into metabolically functional, polarized hepatocytes with functional bile canaliculi. Differentiated hepatocytes show transcriptional and toxicity profiles and cytochrome P450 induction similar to those of primary human hepatocytes. Replication and infectivity of hepatitis C virus (HCV) in differentiated hepatocytes are similar to those of Huh7.5.1 human hepatoma cells. These results offer a means of expanding human hepatocytes of different genetic backgrounds for research, clinical applications and pharmaceutical development.

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Microfluidic applications range from combinatorial synthesis to high throughput screening, with platforms integrating analog perfusion components, digitally controlled micro-valves and a range of sensors that demand a variety of communication protocols. Currently, discrete control units are used to regulate and monitor each component, resulting in scattered control interfaces that limit data integration and synchronization. Here, we present a microprocessor-based control unit, utilizing the MS Gadgeteer open framework that integrates all aspects of microfluidics through a high-current electronic circuit that supports and synchronizes digital and analog signals for perfusion components, pressure elements, and arbitrary sensor communication protocols using a plug-and-play interface. The control unit supports an integrated touch screen and TCP/IP interface that provides local and remote control of flow and data acquisition. To establish the ability of our control unit to integrate and synchronize complex microfluidic circuits we developed an equi-pressure combinatorial mixer. We demonstrate the generation of complex perfusion sequences, allowing the automated sampling, washing, and calibrating of an electrochemical lactate sensor continuously monitoring hepatocyte viability following exposure to the pesticide rotenone. Importantly, integration of an optical sensor allowed us to implement automated optimization protocols that require different computational challenges including: prioritized data structures in a genetic algorithm, distributed computational efforts in multiple-hill climbing searches and real-time realization of probabilistic models in simulated annealing. Our system offers a comprehensive solution for establishing optimization protocols and perfusion sequences in complex microfluidic circuits.

Unlabelled: The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins., Conclusion: Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology., (© 2015 by the American Association for the Study of Liver Diseases.)

Nutrient sensing pathways adjust metabolism and physiological functions in response to food intake. For example, sugar feeding promotes lipogenesis by activating glycolytic and lipogenic genes through the Mondo/ChREBP-Mlx transcription factor complex. Concomitantly, other metabolic routes are inhibited, but the mechanisms of transcriptional repression upon sugar sensing have remained elusive. Here, we characterize cabut (cbt), a transcription factor responsible for the repressive branch of the sugar sensing transcriptional network in Drosophila. We demonstrate that cbt is rapidly induced upon sugar feeding through direct regulation by Mondo-Mlx. We found that CBT represses several metabolic targets in response to sugar feeding, including both isoforms of phosphoenolpyruvate carboxykinase (pepck). Deregulation of pepck1 (CG17725) in mlx mutants underlies imbalance of glycerol and glucose metabolism as well as developmental lethality. Furthermore, we demonstrate that cbt provides a regulatory link between nutrient sensing and the circadian clock. Specifically, we show that a subset of genes regulated by the circadian clock are also targets of CBT. Moreover, perturbation of CBT levels leads to deregulation of the circadian transcriptome and circadian behavioral patterns., (© 2015 The Authors.)