Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia.

Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia

Modelling pulmonary microthrombosis coupled to metastasis: distinct effects of thrombogenesis on tumorigenesis

Thrombosis can cause localized ischemia and tissue hypoxia, and both of these are linked to cancer metastasis. Vascular micro-occlusion can occur as a result of arrest of circulating tumour cells in small capillaries, giving rise to microthrombotic events that affect flow, creating localized hypoxic regions. To better understand the association between metastasis and thrombotic events, we generated an experimental strategy whereby we modelled the effect of microvascular occlusion in metastatic efficiency by using inert microbeads to obstruct lung microvasculature before, during and after intravenous tumour cell injection. We found that controlled induction of a specific number of these microthrombotic insults in the lungs caused an increase in expression of the hypoxia-inducible transcription factors (HIFs), a pro-angiogenic and pro-tumorigenic environment, as well as an increase in myeloid cell infiltration. Induction of pulmonary microthrombosis prior to introduction of tumour cells to the lungs had no effect on tumorigenic success, but thrombosis at the time of tumour cell seeding increased number and size of tumours in the lung, and this effect was strikingly more pronounced when the micro-occlusion occurred on the day following introduction of tumour cells. The tumorigenic effect of microbead treatment was seen even when thrombosis was induced five days after tumour cell injection. We also found positive correlations between thrombotic factors and expression of HIF2$\alpha$ in human tumours. The model system described here demonstrates the importance of thrombotic insult in metastatic success and can be used to improve understanding of thrombosis-associated tumorigenesis and its treatment.Research was supported through a Wellcome Trust Principal Research Fellowship to R.S.J. (RG59596). C.B. is supported through a Scientific Fellowship from Breast Cancer Now (2014MaySF275). C.E.E. received a Pump-Priming Grant from the University of Cambridge British Heart Foundation Centre of Research Excellence (RG68639)

The two enantiomers of 2-hydroxyglutarate differentially regulate cytotoxic T cell function

2-Hydroxyglutarate (2HG) is a byproduct of the tricarboxylic acid (TCA) cycle and is readily detected in the tissues of healthy individuals. 2HG is found in two enantiomeric forms: S-2HG and R-2HG. Here, we investigate the differential roles of these two enantiomers in cluster of differentiation (CD)8+ T cell biology, where we find they have highly divergent effects on proliferation, differentiation, and T cell function. We show here an analysis of structural determinants that likely underlie these differential effects on specific α-ketoglutarate (αKG)-dependent enzymes. Treatment of CD8+ T cells with exogenous S-2HG, but not R-2HG, increased CD8+ T cell fitness in vivo and enhanced anti-tumor activity. These data show that S-2HG and R-2HG should be considered as two distinct and important actors in the regulation of T cell function. Proteomic

An HIF-1α/VEGF-A Axis in Cytotoxic T Cells Regulates Tumor Progression.

Cytotoxic T cells infiltrating tumors are thought to utilize HIF transcription factors during adaptation to the hypoxic tumor microenvironment. Deletion analyses of the two key HIF isoforms found that HIF-1α, but not HIF-2α, was essential for the effector state in CD8+ T cells. Furthermore, loss of HIF-1α in CD8+ T cells reduced tumor infiltration and tumor cell killing, and altered tumor vascularization. Deletion of VEGF-A, an HIF target gene, in CD8+ T cells accelerated tumorigenesis while also altering vascularization. Analyses of human breast cancer showed inverse correlations between VEGF-A expression and CD8+ T cell infiltration, and a link between T cell infiltration and vascularization. These data demonstrate that the HIF-1α/VEGF-A axis is an essential aspect of tumor immunity

Neutrophils fuel effective immune responses through gluconeogenesis and glycogenesis

Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-13C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia. Neutrophils from patients with chronic obstructive pulmonary disease have reduced glycogen cycling, resulting in impaired function. Metabolic specialization of neutrophils may therefore underpin disease pathology and allow selective therapeutic targeting

S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate.

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic-epigenetic axis, to immune fate and function

Εκτίμηση της έκθεσης και της απορρόφησης της ηλεκτρομαγνητικής ακτινοβολίας χαμηλών συχνοτήτων και ραδιοσυχνοτήτων στους ανθρώπινους ιστούς: έκθεση από νέες ασύρματες τεχνολογίες

Electromagnetic fields (EMFs) are among the top issues of public concern. Non-ionizing EMF radiation becomes an essential area of investigation as wireless technologies evolve continuously. The penetration of mobile communications is enormous, with a vast number of applications and services available. The scope of this Ph.D. work was to measure (and then evaluate) both Low-Frequency (LF) and Radiofrequency (RF) radiation levels (up to 3000 MHz) in areas of sensitive land use in Greece. This project was a major part of the National EMF research program (EDBM34) with the participation of the Medical Physics Lab of the University of Thessaly (Faculty of Medicine). A thousand (1000) spots were selected for the RF part, while 247 were selected for the LF part. We evaluated human safety by comparing the exposure values with the latest, updated safety limits of ICNIRP issued in 2020 and the adopted Greek limits, which are stricter than the ICNIRP’s guidelines. Spectrum analyzers were used in both projects providing detailed information on the emissions present in the environment. During the current Ph.D. thesis, 5G networks started to be deployed in Greece at 700 MHz, 2100 MHz, and 3500 MHz (known as C-band). Our study in 5G was based on a few indicative measurements. Moreover, we reviewed the most important international 5G surveys, investigating the possible increase in human exposure caused by 5G technologies, including the evolution of the Internet of Things (IoT). Based on all the experimental and theoretical data, this Ph.D. thesis concluded on human safety in LF and RF spectrum area, considering the legacy and the new wireless technologies that are expected to exhibit wide use in Greece in the forthcoming years. The current state of knowledge, derived from this Ph.D. work, supports that the new wireless technologies are not expected to considerably change the EMF background in residential and outdoor environments. All measurements results were well below the strictest Greek safety limits for all the spectrum bands investigated. A review of the scientific literature was also a part of the current thesis, including the updated safety guidelines by ICNIRP (2020). In the following years, re-evaluating the RF exposure levels, at the locations already measured, will provide the scientific community with more detailed information on the RF environment due to 5G emissions (more 5G antennas will be installed, more subscribers will use these services). The focus should also be given to the new spectrum that will be used in 5G, above 24 GHz, known as millimeter wave (mmWave) radiation.Τα ηλεκτρομαγνητικά πεδία (EMF) είναι μεταξύ των κορυφαίων ζητημάτων που απασχολούν το κοινό. Η μη ιονίζουσα ακτινοβολία EMF αποτελεί ουσιαστικό τομέα έρευνας καθώς οι ασύρματες τεχνολογίες εξελίσσονται συνεχώς. Η διείσδυση των κινητών επικοινωνιών είναι μεγάλη, με έναν σημαντικό αριθμό εφαρμογών και υπηρεσιών. Ο σκοπός αυτής της διδακτορικής διατριβής ήταν μέτρηση (και στη συνέχεια την αξιολόγηση) τόσο των επιπέδων ακτινοβολίας χαμηλής συχνότητας (LF) όσο και των επιπέδων ραδιοσυχνοτήτων (μέχρι 3000 MHz) σε περιοχές ευαίσθητων χρήσεων γης στην Ελλάδα. Το έργο αυτό αποτέλεσε σημαντικό μέρος του Εθνικού ερευνητικού προγράμματος EMF (EDBM34) με τη συμμετοχή του Εργαστηρίου Ιατρικής Φυσικής του Πανεπιστημίου Θεσσαλίας (Ιατρική Σχολή). Χίλια (1000) σημεία επιλέχθηκαν για το τμήμα RF, ενώ επιλέχθηκαν 247 για το τμήμα LF. Αξιολογήσαμε την ανθρώπινη ασφάλεια συγκρίνοντας τις τιμές έκθεσης με τα πιο πρόσφατα, ενημερωμένα όρια ασφαλείας του ICNIRP που εκδόθηκαν το 2020 καθώς και τα εγκεκριμένα ελληνικά όρια, τα οποία είναι αυστηρότερα από τις κατευθυντήριες γραμμές του ICNIRP. Και στα δύο έργα χρησιμοποιήθηκαν αναλυτές φάσματος παρέχοντας λεπτομερείς πληροφορίες σχετικά με τις εκπομπές που υπάρχουν στο περιβάλλον. Κατά την τρέχουσα Ph.D. διατριβή, τα δίκτυα 5G άρχισαν να αναπτύσσονται στην Ελλάδα στα 700 MHz, 2100 MHz και 3500 MHz (C-band). Η μελέτη μας στο 5G βασίστηκε σε μερικές ενδεικτικές μετρήσεις. Επιπλέον, εξετάσαμε τις πιο σημαντικές διεθνείς έρευνες 5G, διερευνώντας την πιθανή αύξηση της ανθρώπινης έκθεσης που προκαλείται από τις τεχνολογίες 5G, συμπεριλαμβανομένης της εξέλιξης του Διαδικτύου των Πραγμάτων (IoT). Με βάση όλα τα πειραματικά και θεωρητικά δεδομένα, η διατριβή κατέληξε σε συμπεράσματα σχετικά με την ανθρώπινη ασφάλεια στην περιοχή του φάσματος LF και RF, λαμβάνοντας υπόψη την προυπάρχουσα τεχνολογία καθώς και τις νέες ασύρματες τεχνολογίες που αναμένεται να παρουσιάσουν ευρεία χρήση στην Ελλάδα τα επόμενα χρόνια. Η τρέχουσα κατάσταση γνώσης, που προέρχεται από αυτό το Ph.D., υποστηρίζει ότι οι νέες ασύρματες τεχνολογίες δεν αναμένεται να αλλάξουν σημαντικά το ηλεκτρομαγνητικό υπόβαθρο σε οικιακά και εξωτερικά περιβάλλοντα. Όλα τα αποτελέσματα των μετρήσεων ήταν πολύ κάτω από τα αυστηρότερα ελληνικά όρια ασφαλείας για όλες τις ζώνες φάσματος που ερευνήθηκαν. Η ανασκόπηση της επιστημονικής βιβλιογραφίας ήταν επίσης μέρος της τρέχουσας διατριβής, συμπεριλαμβανομένων των ενημερωμένων κατευθυντήριων γραμμών για την ασφάλεια από το ICNIRP (2020). Τα επόμενα χρόνια, η επαναξιολόγηση των επιπέδων έκθεσης σε ραδιοσυχνότητες, στις τοποθεσίες που έχουν ήδη μετρηθεί, θα παρέχει στην επιστημονική κοινότητα πιο λεπτομερείς πληροφορίες για το περιβάλλον ραδιοσυχνοτήτων λόγω των εκπομπών 5G (θα εγκατασταθούν περισσότερες κεραίες 5G, περισσότεροι συνδρομητές θα χρησιμοποιούν αυτές τις υπηρεσίες ). Η εστίαση θα πρέπει επίσης να δοθεί στο νέο φάσμα που θα χρησιμοποιηθεί για το 5G, πάνω από 24 GHz, γνωστό ως ακτινοβολία κυμάτων χιλιοστών (mmWave)

5G and human health

The considerable characteristics of 5G technology (Fifth Generation of telecommunication) is the very high amount of data that can be transmitted in the time unit (data speed: Megabits per second - throughput) and the very low delay in data exchange (latency). ElectroMagnetic Fields (EMFs) are used for decades for communication reasons and broadcasting. 700 MHz and lower frequencies are currently being used in Digital TV. Low frequencies (800, 900 MHz) are also being used in previous (but still existing) technologies 2G, 3G, 4G, 4G+. 5G will exploit both low and high frequencies. 5G will be operation mainly in a low band (700 MHz) and a high band (3.5 GHz). In the near future mmWave bands will also be used above 6 GHz (for example 24 GHz, 28 GHz and above). Theoretical models and live measurements have consistently shown that the actual maximum power is always less than the 25% of the maximum peak power of a Massive-MIMO antenna. ICNIRP has published in 1998 “Guidelines for Limiting Exposure to Electromagnetic Fields (100 kHz to 300 GHz)”. The current revision (2020) is based on the same criteria, but it exhibits more accuracy in dosimetry calculations, considering details and based on better biological rationale. mmWave bands (> 6 GHz) is a controversial issue for the population. Reports did not show adverse health effects in daily life under the safety limits. WHO is currently preparing a review about health risks assessment of RF exposure (including mm-Waves), which will be completed by 2022. There is no evidence of adverse health effects at exposure levels below the basic restrictions as described in the ICNIRP (1998) and ICNIRP (2020) guidelines and no evidence of an interaction mechanism that would predict that adverse health effects could occur due to radiofrequency EMF exposure below restriction levels. The new Guidelines provide protection against all adverse health effects, regardless of whether they are due to acute or chronic exposures, regardless of age or health status. Radio and Microwave Frequencies, where mobile technology and Wi-Fi operate, are used in Medicine for therapeutic or diagnostic purposes. These bands are used for various application as Microwave Diathermy (same band as 2G, 3G and Wi-Fi technologies), Microwave induced thermoacoustic echography (same band as 4G technology), Medical Imaging / localization of tumors (same bands as 2G, 3G, 4G and 5G technologies) and Medical Monitoring / Measurement of vital function as respiration and heart rate (same band as the forthcoming mmWave 5G). By utilizing new technologies that are involved in 5G communication (IoT and mmWave frequencies) healthcare systems can improve the quality of care, provide more personalized and preventive care and reduce the cost of care. © 2021 Zerbinis Publications. All rights reserved