The escalating global demand for meat products has intensified ecological concerns, underscoring the need for sustainable meat alternatives. Although current methods effectively imitate ground meat, mimicking whole cuts, which constitute 54% of the global market, remains challenging due to the lack of scalable technology. Injection molding is a massively scalable manufacturing technology developed for the polymer industry. Here, we introduce two injectable metamaterials: a thermally irreversible fat composite we named proteoleogel, and a multi-scaled meat analog produced by low-temperature extrusion. Viscoelastic screening of plant proteins identifies mung bean for its ability to stabilize complex oleogel structures, mimicking the mechanics of adipose tissue. Mechanical analysis reveals that low-temperature extrusion produces microscale isotropic fibers and mesoscale anisotropic structures mimicking muscle and fascia. These metamaterials can be injection-molded into various whole cuts, from chops to T-bones. Blinded taste tests indicate a 43% preference for our plant-based steak analog. Moreover, technical economic analysis shows injection molding is more cost-effective than 3D printing, costing $9/kg compared to $38/kg. This research represents a step in sustainable food production, offering cost-effective and scalable solutions for the entire meat market.

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 × 106 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. © The Author(s), under exclusive licence to Springer Nature Limited 2024.

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. © 2023, Ehrlich et al.

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.

Cellular agriculture could meet growing demand for animal products, but yields are typically low and regulatory bodies restrict genetic modification for cultured meat production. Here we demonstrate the spontaneous immortalization and genetic stability of fibroblasts derived from several chicken breeds. Cell lines were adapted to grow as single-cell suspensions using serum-free culture medium, reaching densities of 108 × 106 cells per ml in continuous culture, corresponding to yields of 36% w/v. We show that lecithin activates peroxisome proliferator-activated receptor gamma (PPARγ), inducing adipogenesis in immortalized fibroblasts. Blending cultured adipocyte-like cells with extruded soy protein, formed chicken strips in which texture was supported by animal and plant proteins while aroma and flavour were driven by cultured animal fat. Visual and sensory analysis graded the product 4.5/5.0, with 85% of participants extremely likely to replace their food choice with this cultured meat product. Immortalization without genetic modification and high-yield manufacturing are critical for the market realization of cultured meat. © 2022, 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.

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 TC50, 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. © 2022 The Royal Society of Chemistry.

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. © 2022 National Academy of Sciences. All rights reserved.

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. © Copyright 2021 by Annual Reviews.

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 se-cretion, 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 mi-tochondrial labile iron and ROS levels, and restored glucose-stimulated insulin secretion. Addition-ally, 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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

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_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 vali-dating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and β-catenin are affected by nsp2, nsp5_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. © Rauti et al.

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

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. © The Royal Society of Chemistry 2020.

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, HA and HB, and two templates TA and TB 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 Zn2+-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 HC and HD, 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 HE and HF, 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 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. © 2019 by Annual Reviews All rights reserved.

Main Text: (Stem Cell Reports 5, 918–931; November 10, 2015) The karyotype pictures of iPSC clones iMCL#3 and iMCL#11 shown in Figure S1D in the original Supplemental Material were incorrect and have been replaced online. This graphical error does not affect the results and conclusions of the article. The authors apologize for this error. © 2018

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 107 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. © 2018, The Author(s).

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. © 2018 The Royal Society of Chemistry.

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 (CB1R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB1R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB1R blockade or genetically inactivating CB1Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB1R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB1R 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 CB1R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN. Copyright © 2018 by the American Society of Nephrology

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. © 2018 Elsevier B.V.