Identification in Saccharomyces cerevisiae of two isoforms of a novel mitochondrial transporter for 2-oxoadipate and 2-oxoglutarate.

The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism

Identification of the Yeast Mitochondrial Transporter for Oxaloacetate and Sulfate

Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase

A competitive polymerase chain reaction-based approach for the identification and semiquantification of mitochondrial DNA in differently heat-treated bovine meat and bone meal

The risk of bovine spongiform encephalopathy propagation was drastically reduced after the European Union (EU) Health Authorities adopted restrictions involving a ban on animal-derived proteins in the diet of farm animals. Currently, the EU's officially recommended method for controlling meat and bone meal (MBM) in animal feed is the microscopic method, which involves the identification of bone fragments on the basis of their morphological characteristics. Recently, we demonstrated that a polymerase chain reaction (PCR)-based assay can be used for the detection of taxon-specific DNA in MBM and animal feeds. To ensure the safe rendering of animal by-products, the EU Council requires that this material be treated at 133degreesC at 300 kPa for 20 min. Here we investigate the relationship between DNA degradation, PCR amplification, and MBM heat treatment. With a competitive PCR-based approach, we compare the amplification efficiency of bovine mitochondrial DNA target sequences of different lengths in several heat-treated MBM samples. For our method, a synthetic competitive DNA is used as an internal control for both DNA extraction and PCR reaction. A correlation between an increase in treatment temperature and a reduction in the size of the target sequences suitable for amplification was observed, suggesting progressive DNA fragmentation due to the temperature. We show that short amplicons (147 bp) can be used to detect the presence of bovine mtDNA in MBM samples treated according to the current European regulations. The use of such a competitive approach to compare amplification efficiency levels of targets of different lengths might represent a useful tool for the determination of both the amount of MBM in animal feeds and its proper heat treatment

HIPEC after Interval Debulking Surgery as Best Clinical Practice in Ovarian Cancer Patients: Case Series and Literature Review

Background: Hyperthermic intraperitoneal chemotherapy (HIPEC) combined with interval debulking surgery (IDS) constitutes an adjunctive treatment strategy in advanced ovarian cancer (AOC). This approach is based on the concept of perfusing chemotherapy targeting directly the site of residual tumor after optimal surgical debulking. It improves patients' outcome in terms of overall survival (OS) and disease free survival (DFS). The correct selection of patients eligible for IDS + HIPEC is crucial: in particular, they must have shown a good response to neoadjuvant chemotherapy (NACT) and have a good performance status (PS). The application of HIPEC at the end of debulking does not seem to increase neither the rate of intra/postoperative complications nor the time of hospitalization. Clinical Cases: After approving an internal protocol for the application of HIPEC in our hospital, we have submitted four patients to IDS + HIPEC in the past 12 months. One of these patients underwent a minimally invasive procedure. No intra- or postoperative complications were observed. Results: All patients underwent IDS + HIPEC after being assessed as eligible and after showing a good response to NACT. In the course of IDS in all cases complete debulking was achieved. No patient developed intra- or postoperative complications. Conclusions: The addition of HIPEC to interval debulking surgery should be offered to all eligible patients, considering that the association of HIPEC to IDS seems to improve patients' outcomes in terms of OS and DFS, without increasing post-operative morbidity

Oxidized alginate dopamine conjugate: In vitro characterization for nose-to-brain delivery application

Background: The blood–brain barrier (BBB) bypass of dopamine (DA) is still a challenge for supplying it to the neurons of Substantia Nigra mainly affected by Parkinson disease. DA prodrugs have been studied to cross the BBB, overcoming the limitations of DA hydrophilicity. Therefore, the aim of this work is the synthesis and preliminary characterization of an oxidized alginatedopamine (AlgOX-DA) conjugate conceived for DA nose-to-brain delivery. Methods: A Schiff base was designed to connect oxidized polymeric backbone to DA and both AlgOX and AlgOX-DA were characterized in terms of Raman, XPS, FT-IR, and1H-NMR spectroscopies, as well as in vitro mucoadhesive and release tests. Results: Data demonstrated that AlgOX-DA was the most mucoadhesive material among the tested ones and it released the neurotransmitter in simulated nasal fluid and in low amounts in phosphate buffer saline. Results also demonstrated the capability of scanning near-field optical microscopy to study the structural and fluorescence properties of AlgOX, fluorescently labeled with fluorescein isothiocyanate microstructures. Interestingly, in SH-SY5Y neuroblastoma cell line up to 100 µg/mL, no toxic effect was derived from AlgOX and AlgOX-DA in 24 h. Conclusions: Overall, the in vitro performances of AlgOX and AlgOX-DA conjugates seem to encourage further ex vivo and in vivo studies in view of nose-to-brain administration

Possible human-to-dog transmission of SARS-CoV-2, Italy, 2020

We detected severe acute respiratory syndrome coronavirus 2 in an otherwise healthy poodle living with 4 family members who had coronavirus disease. We observed antibodies in serum samples taken from the dog, indicating seroconversion. Full-length genome sequencing showed that the canine and human viruses were identical, suggesting human-to-animal transmission

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