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 modu-lated 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 os-mostress. 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

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

Obese mice exposed to psychosocial stress display cardiac and hippocampal dysfunction associated with local brain-derived neurotrophic factor depletion

Introduction: Obesity and psychosocial stress (PS) co-exist in individuals of Western society. Nevertheless, how PS impacts cardiac and hippocampal phenotype in obese subjects is still unknown. Nor is it clear whether changes in local brain-derived neurotrophic factor (BDNF) account, at least in part, for myocardial and behavioral abnormalities in obese experiencing PS. Methods: In adult male WT mice, obesity was induced via a high-fat diet (HFD). The resident-intruder paradigm was superimposed to trigger PS. In vivo left ventricular (LV) performance was evaluated by echocardiography and pressure-volume loops. Behaviour was indagated by elevated plus maze (EPM) and Y-maze. LV myocardium was assayed for apoptosis, fibrosis, vessel density and oxidative stress. Hippocampus was analyzed for volume, neurogenesis, GABAergic markers and astrogliosis. Cardiac and hippocampal BDNF and TrkB levels were measured by ELISA and WB. We investigated the pathogenetic role played by BDNF signaling in additional cardiac-selective TrkB (cTrkB) KO mice. Findings: When combined, obesity and PS jeopardized LV performance, causing prominent apoptosis, fibrosis, oxidative stress and remodeling of the larger coronary branches, along with lower BDNF and TrkB levels. HFD/PS weakened LV function similarly in WT and cTrkB KO mice. The latter exhibited elevated LV ROS emission already at baseline. Obesity/PS augmented anxiety-like behaviour and impaired spatial memory. These changes were coupled to reduced hippocampal volume, neurogenesis, local BDNF and TrkB content and augmented astrogliosis. Interpretation: PS and obesity synergistically deteriorate myocardial structure and function by depleting cardiac BDNF/TrkB content, leading to augmented oxidative stress. This comorbidity triggers behavioral deficits and induces hippocampal remodeling, potentially via lower BDNF and TrkB levels. Fund: J.A. was in part supported by Rotary Foundation Global Study Scholarship. G.K. was supported by T32 National Institute of Health (NIH) training grant under award number 1T32AG058527. S.C. was funded by American Heart Association Career Development Award (19CDA34760185). G.A.R.C. was funded by NIH (K01HL133368-01). APB was funded by a Grant from the Friuli Venezia Giulia Region entitled: “Heart failure as the Alzheimer disease of the heart; therapeutic and diagnostic opportunities”. M.C. was supported by PRONAT project (CNR). N.P. was funded by NIH (R01 HL136918) and by the Magic-That-Matters fund (JHU). V.L. was in part supported by institutional funds from Scuola Superiore Sant'Anna (Pisa, Italy), by the TIM-Telecom Italia (WHITE Lab, Pisa, Italy), by a research grant from Pastificio Attilio Mastromauro Granoro s.r.l. (Corato, Italy) and in part by ETHERNA project (Prog. n. 161/16, Fondazione Pisa, Italy). Funding source had no such involvement in study design, in the collection, analysis, interpretation of data, in the writing of the report; and in the decision to submit the paper for publication

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

Optimization of RT-QuIC for detection of seeding activity in preclinical blood samples from prion-infected sheep.

Contrasting the knowledge about prion diseases or TSEs in sheep, only a very limited number of strain typing studies are available in goats. Two cases deriv- ing from the zoonotic bovine BSE epidemic were however detected in goats. During 2004–2012, over 70 TSE goat brain samples were collected from seven European countries and evaluated for TSE type/strain variation. A selection of these materials was chosen for in-depth analysis based on various criteria: tissue quality, genotype, broad geographical distribution, potential type variation...

Ultra-Efficient PrPSc Amplification Highlights Potentialities and Pitfalls of PMCA Technology

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrPSc in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrPSc in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrPSc in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrPSc was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrPSc to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro

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

In Situ Photodegradation of Incorporated Polyanion Does Not Alter Prion Infectivity

Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrPSc aggregates in vivo [1], [2]. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrPSc, we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrPC substrate, PC-oligo was incorporated into physical complexes with PrPSc molecules that were resistant to benzonase digestion. Exposure of these nuclease-resistant prion complexes to long wave ultraviolet light (315 nm) induced degradation of PC-oligo into 5 base fragments. Light-induced photolysis of incorporated PC-oligo did not alter the infectivity of in vitro-generated prions, as determined by bioassay in hamsters and brain homogenate sPMCA assays. Neuropathological analysis also revealed no significant differences in the neurotropism of prions containing intact versus degraded PC-oligo. These results show that polyanions >5 bases in length are not required for maintaining the infectious properties of in vitro-generated scrapie prions, and indicate that such properties are maintained either by short polyanion remnants, other co-purified cofactors, or by PrPSc molecules alone