Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production.

BACKGROUND: Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries. RESULTS: In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica. CONCLUSIONS: The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

Inactivation of HAP4 Accelerates RTG-Dependent Osmoadaptation in Saccharomyces cerevisiae.

Mitochondrial RTG (an acronym for ReTroGrade) signaling plays a cytoprotective role under various intracellular or environmental stresses. We have previously shown its contribution to osmoadaptation and capacity to sustain mitochondrial respiration in yeast. Here, we studied the interplay between RTG2, the main positive regulator of the RTG pathway, and HAP4, encoding the catalytic subunit of the Hap2-5 complex required for the expression of many mitochondrial proteins that function in the tricarboxylic acid (TCA) cycle and electron transport, upon osmotic stress. Cell growth features, mitochondrial respiratory competence, retrograde signaling activation, and TCA cycle gene expression were comparatively evaluated in wild type and mutant cells in the presence and in the absence of salt stress. We showed that the inactivation of HAP4 improved the kinetics of osmoadaptation by eliciting both the activation of retrograde signaling and the upregulation of three TCA cycle genes: citrate synthase 1 (CIT1), aconitase 1 (ACO1), and isocitrate dehydrogenase 1 (IDH1). Interestingly, their increased expression was mostly dependent on RTG2. Impaired respiratory competence in the HAP4 mutant does not affect its faster adaptive response to stress. These findings indicate that the involvement of the RTG pathway in osmostress is fostered in a cellular context of constitutively reduced respiratory capacity. Moreover, it is evident that the RTG pathway mediates peroxisomes-mitochondria communication by modulating the metabolic function of mitochondria in osmoadaptation.

SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

Optimization of Microwave-Assisted Extraction of Antioxidants from Bamboo Shoots of Phyllostachys pubescens.

Bamboo is a well-known medicinal plant in Southeast Asia that recently has attracted attention for its high polyphenol content and its medical and nutraceutical applications. In this work, polyphenols have been recovered for the first time by microwave-assisted extraction (MAE) from an unusual Italian cultivar of Phyllostachys pubescens bamboo shoots. The effects of three independent variables, such as extraction time, temperature, and solid/liquid ratio, on polyphenol recovery yield were investigated and successfully optimized through the response surface methodology. We demonstrated that MAE is an excellent polyphenols extraction technique from bamboo shoots because the total phenolic content obtained under microwave irradiation optimal conditions (4 min at 105 °C with 6.25 mg/mL ratio) was about eight-fold higher than that obtained with the conventional extraction method. Furthermore, higher total flavonoid content was also obtained under MAE. Consistent with these results, MAE enhanced the extract antioxidant properties with significant improved DPPH, ABTS, and FRAP scavenging ability. Therefore, this innovative extraction process enhances the recovery of biologically active compounds from Phyllostachys pubescens bamboo shoots with a dramatic reduction of time and energy consumption, which paves the way for its industrial application in functional food production.

Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts.

Systematic screening for the biocatalytic hydration of fatty acids from different oily substrates by Elizabethkingia meningoseptica oleate hydratase through a Design-of-experiments approach.

The edible plant oils production is associated with the release of different types of by-products. The latter represent cheap and available substrates to produce valuable compounds, such as flavours and fragrances, biologically active compounds and bio-based polymers. Elizabethkingia meningoseptica Oleate hydratases (Em_OhyA) can selectively catalyze the conversion of unsaturated fatty acids, specifically oleic acid, into hydroxy fatty acids, which find different industrial applications. In this study, Design-of-experiment (DoE) strategy was used to screen and identify conditions for reaching high yields in the reaction carried out by Escherichia coli whole-cell carrying the recombinant enzyme Em_OhyA using Waste Cooking Oils (WCO)-derived free fatty acids (FFA) as substrate. The identified reaction conditions for high oleic acid conversion were also tested on untreated triglycerides-containing substrates, such as pomace oil, sunflower oil, olive oil and oil mill wastewater (OMW), combining the triglyceride hydrolysis by the lipase from Candida rugosa and the E. coli whole-cell containing Em_OhyA for the production of hydroxy fatty acids. When WCO, sunflower oil and OMW were used as substrate, the one-pot bioconversion led to an increase of oleic acid conversion compared to the standard reaction. This work highlights the efficiency of the DoE approach to screen and identify conditions for an enzymatic reaction for the production of industrially-relevant products.

Process scale-up simulation and techno-economic assessment of ethanol fermentation from cheese whey.

BACKGROUND: Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy's Apulia region, designed to process 539 m3/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data. RESULTS: The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (YE/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a YE/L = 0.45 g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model's dependence on the price or credit associated with cheese-whey handling. CONCLUSIONS: This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol.

Lack of Mitochondrial DNA Provides Metabolic Advantage in Yeast Osmoadaptation.

Alterations in mitochondrial function have been linked to a variety of cellular and organismal stress responses including apoptosis, aging, neurodegeneration and tumorigenesis. However, adaptation to mitochondrial dysfunction can occur through the activation of survival pathways, whose mechanisms are still poorly understood. The yeast Saccharomyces cerevisiae is an invaluable model organism for studying how mitochondrial dysfunction can affect stress response and adaptation processes. In this study, we analyzed and compared in the absence and in the presence of osmostress wild-type cells with two models of cells lacking mitochondrial DNA: ethidium bromide-treated cells (ρ0) and cells lacking the mitochondrial pyrimidine nucleotide transporter RIM2 (ΔRIM2). Our results revealed that the lack of mitochondrial DNA provides an advantage in the kinetics of stress response. Additionally, wild-type cells exhibited higher osmosensitivity in the presence of respiratory metabolism. Mitochondrial mutants showed increased glycerol levels, required in the short-term response of yeast osmoadaptation, and prolonged oxidative stress. The involvement of the mitochondrial retrograde signaling in osmoadaptation has been previously demonstrated. The expression of CIT2, encoding the peroxisomal isoform of citrate synthase and whose up-regulation is prototypical of RTG pathway activation, appeared to be increased in the mutants. Interestingly, selected TCA cycle genes, CIT1 and ACO1, whose expression depends on RTG signaling upon stress, showed a different regulation in ρ0 and ΔRIM2 cells. These data suggest that osmoadaptation can occur through different mechanisms in the presence of mitochondrial defects and will allow us to gain insight into the relationships among metabolism, mitochondria-mediated stress response, and cell adaptation.

Keratinous and corneous-based products towards circular bioeconomy: A research review.

Keratins and corneous proteins are key components of biomaterials used in a wide range of applications and are potential substitutes for petrochemical-based products. Horns, hooves, feathers, claws, and similar animal tissues are abundant sources of α-keratin and corneous ß-proteins, which are by-products of the food industry. Their close association with the meat industry raises environmental and ethical concerns regarding their disposal. To promote an eco-friendly and circular use of these materials in novel applications, efforts have focused on recovering these residues to develop sustainable, non-animal-related, affordable, and scalable procedures. Here, we review and examine biotechnological methods for extracting and expressing α-keratins and corneous ß-proteins in microorganisms. This review highlights consolidated research trends in biomaterials, medical devices, food supplements, and packaging, demonstrating the keratin industry's potential to create innovative value-added products. Additionally, it analyzes the state of the art of related intellectual property and market size to underscore the potential within a circular bioeconomic model.

Industrial Production of Proteins with Pichia pastoris-Komagataella phaffii.

Since the mid-1960s, methylotrophic yeast Komagataella phaffii (previously described as Pichia pastoris) has received increasing scientific attention. The interest for the industrial production of proteins for different applications (e.g., feed, food additives, detergent, waste treatment processes, and textile) is a well-consolidated scientific topic, and the importance for this approach is rising in the current era of environmental transition in human societies. This review aims to summarize fundamental and specific information in this scientific field. Additionally, an updated description of the relevant products produced with K. phaffii at industrial levels by a variety of companies-describing how the industry has leveraged its key features, from products for the ingredients of meat-free burgers (e.g., IMPOSSIBLE™ FOODS, USA) to diabetes therapeutics (e.g., Biocon, India)-is provided. Furthermore, active patents and the typical workflow for industrial protein production with this strain are reported.

Regio- and stereoselective biocatalytic hydration of fatty acids from waste cooking oils en route to hydroxy fatty acids and bio-based polyesters.

The development of biorefinery approaches is of great relevance for the sustainable production of valuable compounds. In accordance with circular economy principles, waste cooking oils (WCOs) are renewable resources and biorefinery feedstocks, which contribute to a reduced impact on the environment. Frequently, this waste is wrongly disposed of into municipal sewage systems, thereby creating problems for the environment and increasing treatment costs in wastewater treatment plants. In this study, regenerated WCOs, which were intended for the production of biofuels, were transformed through a chemo-enzymatic approach to produce hydroxy fatty acids, which were further used in polycondensation reaction for polyester production. Escherichia coli whole cell biocatalyst containing the recombinantly produced Elizabethkingia meningoseptica Oleate hydratase (Em_OhyA) was used for the biocatalytic hydration of crude WCOs-derived unsaturated free fatty acids for the production of hydroxy fatty acids. Further hydrogenation reaction and methylation of the crude mixture allowed the production of (R)- 10-hydroxystearic acid methyl ester that was further purified with a high purity (> 90%), at gram scale. The purified (R)- 10-hydroxystearic acid methyl ester was polymerized through a polycondensation reaction to produce the corresponding polyester. This work highlights the potential of waste products to obtain bio-based hydroxy fatty acids and polyesters through a biorefinery approach.

In Vitro Effects of Low-energy Ultrasound Treatment on Healthy CD3/CD8+ Lymphocytes, Red blood cells, Acute Myeloid leukemia cells, and Jurkat cell line.

The study of the biological effects of low-energy ultrasound and its applications is a rapidly expanding research area. Low-energy ultrasound could be used as anti-tumoral therapy with or without the pharmacological combination even if the second situation has been scarcely investigated up to now. Very little information is available about the ultrasound effects on healthy red blood cells, CD3, and mainly CD8 subset lymphocytes which is the main subset cell having cytotoxic function towards cancer cells. In this study, we investigated in vitro the bioeffects of low energy ultrasound on red blood cells and PBMCs isolated from healthy donors as well as on two myeloid leukemia cell lines (OCI- AML-3 MOLM-13) and lymphoblastic Jurkat cell line. Using low-energy ultrasound (US), a study was conducted to determine how it affects CD3/CD8 lymphocytes and leukemia cells, as well as its potential role in treating blood cancers, by analyzing changes in mitochondrial membrane potential, phosphatidylserine asymmetry, morphological changes for myeloid AML cell lines, proliferation and cytotoxic activation of healthy lymphocytes, and apoptosis for RBCs after US exposure. Overall, we demonstrated that CD3/CD8 lymphocytes proliferation/activation and cytotoxic functions are fully preserved after ultrasound treatments, whereas leukemia cell lines undergo apoptosis and stop proliferating suggesting a potential method of treating blood cancer.

Mitochondrial Role in Intrinsic Apoptosis Induced by a New Synthesized Chalcone in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the fourth cause of cancer-related deaths worldwide. Presently, a few drugs are available for HCC treatment and prevention, including both natural and synthetic compounds. In this study, a new chalcone, (E)-1-(2,4,6-triethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (ETTC), was synthesized and its effects and mechanisms of action over human hepatoma cells were investigated. Cytotoxic activity was revealed in HCC cells, while no effects were observed in normal hepatocytes. In HCC cells, ETTC caused subG1 cell cycle arrest and apoptosis, characterized by nuclear fragmentation. The activation of caspases 3/7 and 9, the increase in pro-apoptotic BAX, and the decrease in anti-apoptotic BCL-2 suggest the activation of the intrinsic pathway of apoptosis. ETTC mitochondrial targeting is confirmed by the reduction in mitochondrial membrane potential and Complex I activity together with levels of superoxide anion increasing. Our outcomes prove the potential mitochondria-mediated antitumor effect of newly synthesized chalcone ETTC in HCC.

A comparative screening of laccase-mediator systems by white-rot fungi laccases for biocatalytic benzyl alcohol oxidation.

Production of value-added compounds from waste materials is of utmost importance for the development of a sustainable society especially regarding their use as catalysts in industrially relevant synthetic reactions. Herein, we show the production of laccases from four white-rot fungi, which were grown on agricultural residues, specifically Trametes versicolor 11269, Pleurotus ostreatus 1020, Panus tigrinus 707 and Lentinula edodes SC-495. The produced laccases were tested on a laccase-mediator system (LMS) for the biocatalytic oxidation of the model substrate benzyl alcohol into benzaldehyde. The LMS was carried out in the presence both of tetrahydrofuran as co-solvent and of the mediator 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) due to its high redox potential and its ability to perform the oxidation. Tolerance studies showed that the dialyzed solutions were able to tolerate 1% (99:1 v/v) of co-solvent, whereas a concentration of 10% v/v had a detrimental activity. Performances in the biocatalytic oxidation of laccase solutions from different purification steps were compared. Similar conversion was observed for laccase in dialysis (raw) and gel filtration (GF) product versus commercial T. versicolor laccase. The latter oxidized almost 99% of substrate while the other laccase solutions were able to reach a conversion from 91% for the laccase solution from P. tigrinus 707 after dialysis, to 50% for the laccase solution from P. ostreatus 1020 after gel filtration. This work highlights the potential of unpurified laccase solutions to be used as catalysts in synthetic reactions.

Identification of a novel Sp1 splice variant as a strong transcriptional activator.

The transcription factor Sp1 regulates expression of numerous genes involved in many cellular processes. Different post-transcriptional modifications can influence the transcriptional control activity and stability of Sp1. In addition to these modifications, alternative splicing isoforms may also be the basis of its distinct functional activities. In this study, we identified a novel alternative splice isoform of Sp1 named Sp1c. This variant is generated by exclusion of a short domain, which we designate α, through alternative splice acceptor site usage in the exon 3. The existence of this new isoform was confirmed in vivo by Western blotting analysis. Although at very low levels, Sp1c is ubiquitously expressed, as seen in its full-length Sp1. A preliminary characterization of Sp1c shows that: (a) Sp1c works as stronger activator of transcription than full-length Sp1; (b) percentage of HEK293 Sp1c-overexpressing cells is higher in G1 phase and lower in S phase than percentage of HEK293 Sp1-overexpressing cells.

Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis.

The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

RTG Signaling Sustains Mitochondrial Respiratory Capacity in HOG1-Dependent Osmoadaptation.

Mitochondrial RTG-dependent retrograde signaling, whose regulators have been characterized in Saccharomyces cerevisiae, plays a recognized role under various environmental stresses. Of special significance, the activity of the transcriptional complex Rtg1/3 has been shown to be modulated by Hog1, the master regulator of the high osmolarity glycerol pathway, in response to osmotic stress. The present work focuses on the role of RTG signaling in salt-induced osmotic stress and its interaction with HOG1. Wild-type and mutant cells, lacking HOG1 and/or RTG genes, are compared with respect to cell growth features, retrograde signaling activation and mitochondrial function in the presence and in the absence of high osmostress. We show that RTG2, the main upstream regulator of the RTG pathway, contributes to osmoadaptation in an HOG1-dependent manner and that, with RTG3, it is notably involved in a late phase of growth. Our data demonstrate that impairment of RTG signaling causes a decrease in mitochondrial respiratory capacity exclusively under osmostress. Overall, these results suggest that HOG1 and the RTG pathway may interact sequentially in the stress signaling cascade and that the RTG pathway may play a role in inter-organellar metabolic communication for osmoadaptation.

Bioisosteric Modification of To042: Synthesis and Evaluation of Promising Use-Dependent Inhibitors of Voltage-Gated Sodium Channels.

Three analogues of To042, a tocainide-related lead compound recently reported for the treatment of myotonia, were synthesized and evaluated in vitro as skeletal muscle sodium channel blockers possibly endowed with enhanced use-dependent behavior. Patch-clamp experiments on hNav1.4 expressed in HEK293 cells showed that N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine, the aryloxyalkyl bioisostere of To042, exerted a higher use-dependent block than To042 thus being able to preferentially block the channels in over-excited membranes while preserving healthy tissue function. It also showed the lowest active transport across BBB according to the results of P-glycoprotein (P-gp) interacting activity evaluation and the highest cytoprotective effect on HeLa cells. Quantum mechanical calculations and dockings gave insights on the most probable conformation of the aryloxyalkyl bioisostere of To042 in solution and the target residues involved in the binding, respectively. Both approaches indicated the conformations that might be adopted in both the unbound and bound state of the ligand. Overall, N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine exhibits an interesting toxico-pharmacological profile and deserves further investigation.

KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.

CaCO3 as an Environmentally Friendly Renewable Material for Drug Delivery Systems: Uptake of HSA-CaCO3 Nanocrystals Conjugates in Cancer Cell Lines.

Chemical and biochemical functionalization of nanoparticles (NPs) can lead to an active cellular uptake enhancing their efficacy thanks to the targeted localization in tumors. In the present study calcium carbonate nano-crystals (CCNs), stabilized by an alcohol dehydration method, were successfully modified by grafting human serum albumin (HSA) on the surface to obtain a pure protein corona. Two types of CCNs were used: naked CaCO3 and the (3-aminopropyl)triethoxysilane (APTES) modified CaCO3-NH2. The HSA conjugation with naked CCN and amino-functionalized CCN (CCN-NH2) was established through the investigation of modification in size, zeta potential, and morphology by Transmission Electron Microscopy (TEM). The amount of HSA coating on the CCNs surface was assessed by spectrophotometry. Thermogravimetric analysis (TGA) and Differential scanning calorimetry (DSC) confirmed the grafting of APTES to the surface and successive adsorption of HSA. Furthermore, to evaluate the effect of protein complexation of CCNs on cellular behavior, bioavailability, and biological responses, three human model cancer cell lines, breast cancer (MCF7), cervical cancer (HeLa), and colon carcinoma (Caco-2) were selected to characterize the internalization kinetics, localization, and bio-interaction of the protein-enclosed CCNs. To monitor internalization of the various conjugates, chemical modification with fluorescein-isothiocyanate (FITC) was performed, and their stability over time was measured. Confocal microscopy was used to probe the uptake and confirm localization in the perinuclear region of the cancer cells. Flow cytometry assays confirmed that the bio-functionalization influence cellular uptake and the CCNs behavior depends on both cell line and surface features.