Hypoxia research, where to now?

Investigating how cells and organisms sense and respond to O2 levels is essential to our understanding of physiology and pathology. This field has advanced considerably since the discovery of the major transcription factor family, hypoxia-inducible factor (HIF), and the enzymes that control its levels: prolyl hydroxylases (PHDs). However, with its expansion, new complexities have emerged. Herein we highlight three main areas where, in our opinion, the research community could direct some of their attention. These include non-transcriptional roles of HIFs, specificity and O2 sensitivity of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), and new tools and methods to detect O2 concentrations in cells and organs. A greater understanding of these areas would answer big questions and help drive our knowledge of cellular responses to hypoxia forward.

Oxygen-regulated post-translation modifications as master signalling pathway in cells.

Oxygen is essential for viability in mammalian organisms. However, cells are often exposed to changes in oxygen availability, due to either increased demand or reduced oxygen supply, herein called hypoxia. To be able to survive and/or adapt to hypoxia, cells activate a variety of signalling cascades resulting in changes to chromatin, gene expression, metabolism and viability. Cellular signalling is often mediated via post-translational modifications (PTMs), and this is no different in response to hypoxia. Many enzymes require oxygen for their activity and oxygen can directly influence several PTMS. Here, we review the direct impact of changes in oxygen availability on PTMs such as proline, asparagine, histidine and lysine hydroxylation, lysine and arginine methylation and cysteine dioxygenation, with a focus on mammalian systems. In addition, indirect hypoxia-dependent effects on phosphorylation, ubiquitination and sumoylation will also be discussed. Direct and indirect oxygen-regulated changes to PTMs are coordinated to achieve the cell's ultimate response to hypoxia. However, specific oxygen sensitivity and the functional relevance of some of the identified PTMs still require significant research.

Global acetylome profiling indicates EPA impedes but OA promotes prostate cancer motility through altered acetylation of PFN1 and FLNA.

Prostate cancer (PCa) is one of the leading causes of cancer morbidity and mortality in men. Metastasis is the main cause of PCa-associated death. Recent evidence indicated a significant reduction in PCa mortality associated with higher ω-3 polyunsaturated fatty acids (PUFAs) consumption. However, the underlying mechanisms remained elusive. In this study, we applied global acetylome profiling to study the effect of fatty acids treatment. Results indicated that oleic acid (OA, monounsaturated fatty acid, MUFA, 100 µM) elevates while EPA (eicosapentaenoic acid, 100 µM) reduces the acetyl-CoA level, which alters the global acetylome. After treatment, two crucial cell motility regulators, PFN1 and FLNA, were found with altered acetylation levels. OA increased the acetylation of PFN1 and FLNA, whereas EPA decreased PFN1 acetylation level. Furthermore, OA promotes while EPA inhibits PCa migration and invasion. Immunofluorescence assay indicated that EPA impedes the formation of lamellipodia or filopodia through reduced localization of PFN1 and FLNA to the leading edge of cells. Therefore, perturbed acetylome may be one critical step in fatty acid-affected cancer cell motility. This study provides some new insights into the response of ω-3 PUFAs treatment and a better understanding of cancer cell migration and invasion modulation.

Autophagy is the main driver of radioresistance of HNSCC cells in mild hypoxia.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1ß leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

Characterization of novel aurantiactinomyxon types (Cnidaria, Myxosporea) from the oligochaete Ilyodrilus templetoni (Southern, 1909), with a comprehensive phylogeny of the collective group.

Three new aurantiactinomyxon types are described from the oligochaete Ilyodrilus templetoni (Southern, 1909) (Naididae) collected from a northern Portuguese estuary, based on light microscopy and sequencing of the 18S rDNA. The addition of I. templetoni to the group of freshwater annelids known to be permissive for aurantiactinomyxon development reinforces the crucial role of naidids in the evolution and settlement of myxozoans in estuarine environments. Maximum likelihood and Bayesian inference analyses of a comprehensive 18S rDNA dataset placed the novel types within the Paramyxidium clade. This positioning suggests them as probable life cycle counterparts to Paramyxidium spp. that most likely infect the European eel Anguilla anguilla, as the sole representative of Elopomorpha in Portuguese rivers. Although distance estimation revealed a genetic difference of only 0.4 % between Aurantiactinomyxon types 1 and 3, this value was determined to be representative of interspecific variability based on the consistent matching of both genotypes with distinct actinospore morphologies, and potential richness of closely related species of Paramyxidium infecting the European eel in Portuguese waters. The clustering of aurantiactinomyxon types within distinct myxosporean lineages, representative of the suborders Variisporina and Platysporina, demonstrates that the aurantiactinomyxon morphotype is highly functional in promoting myxozoan infections in estuarine environments.

Synopsis of the species of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae).

A synopsis of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae) is presented and identifies 26 nominal species presently allocated within this genus. Species morphological and morphometric features, tissue tropism, type-host, and type-locality are provided from original descriptions. Data from subsequent redescriptions and reports is also given. Accession numbers to sequences deposited in GenBank are indicated when available, and the myxospores were redrawn based on original descriptions. The information gathered shows that Ortholinea infect a wide taxonomic variety of freshwater and marine fish. Nonetheless, the broad host specificity reported for several species is not fully supported by morphological descriptions and requires molecular corroboration. The members of this genus are coelozoic and mainly parasitize the urinary system, with few species occurring in the gallbladder. Ortholinea visakhapatnamensis is the only exception, being histozoic in the visceral peritoneum. Molecular data of the small subunit ribosomal RNA gene (SSU rDNA) is available for about one third of Ortholinea species, with genetic interspecific variation ranging between 1.65% and 29.1%. Phylogenetic analyses reveal Ortholinea to be polyphyletic, with available SSU rDNA sequences clustering within the subclades of the highly heterogenous freshwater urinary clade of the oligochaete-infecting lineage. The life cycles of two Ortholinea species have been clarified based on molecular inferences and identify triactinomyxon actinospores as counterparts, and marine oligochaetes of the family Naididae as permissive hosts to this genus.

Morphological, histopathological, ultrastructural and phylogenetic analysis of Henneguya archosargus n. sp. (Cnidaria, Myxosporea) infecting the sparid fish Archosargus probatocephalus in Brazilian waters.

The introduction of new fish species to the aquaculture industry is essential to halt the progressive decline of natural fish stocks. The sheepshead Archosargus probatocephalus is a commercially valuable sparid fish with potential for breeding in captivity, but with limited information regarding parasitic infections that could pose a significant threat for its sustainable production. Thus, the present study aimed to study the myxozoan diversity infecting A. probatocephalus. A novel Henneguya sp. was detected forming plasmodia in the gill lamellae of specimens inhabiting the Brazilian coast, and is characterized based on morphological, histopathological, ultrastructural, molecular, and phylogenetic data. Myxospore total length was 21.3 ± 0.8 µm, with myxospore body 10.0 ± 0.5 µm long, 6.2 ± 0.3 µm wide, and 4.8 ± 0.5 µm thick. Caudal appendages were 10.3 ± 0.5 µm long and did not present any type of coating. Two pyriform polar capsules, 3.4 ± 0.3 µm long and 1.5 ± 0.2 µm wide, each containing an isofilar polar tubule with 4-5 coils. Histopathological analyses showed large intralamellar polysporic plasmodia associated with vascular congestion of the gill filament and gill lamellae, as well as epithelial hyperplasia causing partial or total fusion of gill lamellae. Maximum likelihood and Baysesian inference SSU rDNA-based phylogenetic analyses showed the novel sequence grouped within the marine clade of Henneguya spp. that mostly parasitize fishes belonging to Eupercaria incertae sedis.

Systems approaches to understand oxygen sensing: how multi-omics has driven advances in understanding oxygen-based signalling.

Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insight into how cells detect, and respond to low oxygen is crucial to understanding the role of hypoxia in disease. Central to the hypoxic response is rapid changes in the expression of genes essential to carry out a wide range of functions to adapt the cell/tissue to decreased oxygen availability. These changes in gene expression are co-ordinated by specialised transcription factors, changes to chromatin architecture and intricate balances between protein synthesis and destruction that together establish changes to the cellular proteome. In this article, we will discuss the advances of our understanding of the cellular oxygen sensing machinery achieved through the application of 'omics-based experimental approaches.

Regulation of chromatin accessibility by hypoxia and HIF.

Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) co-ordinate a transcriptional programme required for survival and adaptation to hypoxia. However, the effects of HIF on chromatin accessibility are currently unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility are enriched at a subset hypoxia transcriptionally responsive genes, agreeing with previous data using other models. We show for the first time that hypoxia inducible changes in chromatin accessibility across the genome are predominantly HIF dependent, rapidly reversible upon reoxygenation and partially mimicked by HIF-α stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that HIF is central to chromatin accessibility alterations in hypoxia, and has implications for our understanding of gene expression regulation by hypoxia and HIF.

Synopsis of the aurantiactinomyxon collective group (Cnidaria, Myxozoa), with a discussion on the validity of morphotype definition and demise of guyenotia.

Aurantiactinomyxon is one of the most diverse myxozoan collective groups, comprising types that mostly infect freshwater and marine oligochaetes belonging to the family Naididae Ehrenberg, 1828, but also Lumbriculidae Claus, 1872. In this study, a comprehensive revision of all known aurantiactinomyxon types is performed and highlights the fallibility of using the form and length of the valvular processes as main criterion for differentiating among style-less actinospore morphotypes. The demise of the guyenotia collective group is proposed based on the ambiguous features of several types that allow conformity with both the aurantiactinomyxon and guyenotia definitions. Nonetheless, the information presently available clearly shows that a general shift is needed in our approach to actinospore grouping, which should probably be based on actinospore functionality relative to environment and host ecology, rather than on morphology. Life cycle studies based on experimental transmission and molecular inferences of the 18S rDNA have linked aurantiactinomyxon (including former guyenotia) to myxozoans belonging to a diverse array of genera, including Chloromyxum, Henneguya, Hoferellus, Myxobolus, Paramyxidium, Thelohanellus and Zschokkella. This undoubtedly shows a high capacity of the aurantiactinomyxon morphotype to promote infection in intrinsically distinct vertebrate hosts and environmental habitats, consequently increasing interest in its study for attaining a better understanding of myxozoan-host interactions. The identification of novel and known types, however, is impeded by the lack of concise information allowing a comprehensive analysis of biological, morphological, and molecular criteria. In this sense, the compilation of data presented in this study will ultimately help researchers seeking to perform reliable identifications.

Synopsis of the species of Henneguya Thélohan, 1892 (Cnidaria: Myxosporea: Myxobolidae) described since 2012.

The genus Henneguya Thélohan, 1892 (Cnidaria: Myxosporea: Myxobolidae) encompasses a large number of species that mostly infect freshwater fish belonging to 71 families of Actinopterygii. A synopsis of Henneguya species described between 2012 and 2022 is herein presented. It includes 57 species described during the last decade, and one species missing from the previous synopses, adding to a total of 254 species that have been formally described within this genus. Biological characters and myxospore morphometry are presented for each species record.

Von Hippel-Lindau (VHL) small-molecule inhibitor binding increases stability and intracellular levels of VHL protein.

Von Hippel-Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought.

Antiphospholipid Antibodies and Lipids in Hematological Malignancies.

One of the main groups of lipids is phospholipids, which are mainly involved in forming cell membranes. Neoplastic processes such as cell replication have increased lipid synthesis, making tumor cells dependent on this synthesis to maintain their requirements. Antiphospholipid antibodies attack phospholipids in the cell membranes. Three main types of antiphospholipid antibodies are recognized: anti-ß2 glycoprotein I (anti-ß2GP-I), anticardiolipin (aCL), and lupus anticoagulant (LA). These types of antibodies have been proven to be present in hematological neoplasms, particularly in LH and NHL. This review on antiphospholipid antibodies in hematological neoplasms describes their clinical relationship as future implications at the prognostic level for survival and even treatment.

Biogas and calcite handling in calcium fluoride precipitation from aqueous solution guided by thermodynamic simulation with PHREEQC.

In this innovative study, biogas has been associated with calcium carbonate [CaCO3] to promote the precipitation of fluorite, aiming at the treatment of wastewater with high content of fluoride. The work associates distinct sources of calcium and CO2 for the precipitation of fluorite according to previous simulation with the free software PHREEQC. Considering the reaction at equilibrium, the minimal predicted Ca dosage was 215 mg/L, lower than the 430 mg/L that was experimentally determined, independent of Ca source. The simultaneous use of CaCO3 and CO2 from distinct gas sources (pure CO2, 1:1 CO2:N2, and biogas) exhibited high performance permitting the reduction of fluoride content from 134 to 10 mg/L, with low gas consumption. The biogas consumption of 66.0 mmol/L, equivalent to 33.4 mmol/L of CO2 (1.47 kgCO2/m3treated wastewater) was predicted, indicating that the biogas storage bag of 700 L would be able to treat 469 L of wastewater. Furthermore, the inert fraction of biogas (CH4) did not impact the reaction and it may be used after the reaction as an alternative source of power, equivalent to 8.25 kWh/m3treated wastewater. Final solids were composed by fluorite and non-dissolved calcite, confirming the predictions obtained by PHREEQC.

Nano-based drug delivery systems used as vehicles to enhance polyphenols therapeutic effect for diabetes mellitus treatment.

Diabetes mellitus is one of the biggest health emergencies of the 21st century worldwide, characterized by deficiency in insulin secretion and/or action, leading to hyperglycemia. Despite the currently available antidiabetic therapeutic options, 4.2 million people died in 2019 due to diabetes. Thus, new effective interventions are required. Polyphenols are plant secondary metabolites and have been recognized for their vast number of biological activities, including potential antidiabetic effects. However, the poor bioavailability and high metabolization of polyphenols restrict their biological effects in vivo. Nanotechnology is a promising area of research to improve the therapeutic effect of several compounds. Therefore, this review provides an overview of the literature about the utility of nano-based drug delivery systems as vehicles of polyphenols in diabetes treatment. It was possible to conclude that, in general, nano-based drug delivery systems can potentiate the beneficial antidiabetic properties of polyphenols, when compared with the free compounds, opening a new field of research in diabetology.

Identification and functional characterization of FMN2, a regulator of the cyclin-dependent kinase inhibitor p21.

The ARF tumor suppressor is a central component of the cellular defense against oncogene activation in mammals. p14ARF activates p53 by binding and inhibiting HDM2, resulting, inter alia, in increased transcription and expression of the cyclin-dependent kinase inhibitor p21 and consequent cell-cycle arrest. We analyzed the effect of p14ARF induction on nucleolar protein dynamics using SILAC mass spectrometry and have identified the human Formin-2 (FMN2) protein as a component of the p14ARF tumor suppressor pathway. We show that FMN2 is increased upon p14ARF induction at both the mRNA and the protein level via a NF-κB-dependent mechanism that is independent of p53. FMN2 enhances expression of the cell-cycle inhibitor p21 by preventing its degradation. FMN2 is also induced by activation of other oncogenes, hypoxia, and DNA damage. These results identify FMN2 as a crucial component in the regulation of p21 and consequent oncogene/stress-induced cell-cycle arrest in human cells.

Role of Hypoxia in the Control of the Cell Cycle.

The cell cycle is an important cellular process whereby the cell attempts to replicate its genome in an error-free manner. As such, mechanisms must exist for the cell cycle to respond to stress signals such as those elicited by hypoxia or reduced oxygen availability. This review focuses on the role of transcriptional and post-transcriptional mechanisms initiated in hypoxia that interface with cell cycle control. In addition, we discuss how the cell cycle can alter the hypoxia response. Overall, the cellular response to hypoxia and the cell cycle are linked through a variety of mechanisms, allowing cells to respond to hypoxia in a manner that ensures survival and minimal errors throughout cell division.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Gene transcription and chromatin regulation in hypoxia.

Oxygen sensing is an essential feature of metazoan biology and reductions in oxygen availability (hypoxia) have both physiological and pathophysiological implications. Co-ordinated mechanisms have evolved for sensing and responding to hypoxia, which involve diverse biological outputs, with the main aim of restoring oxygen homeostasis. This includes a dynamic gene transcriptional response, the central drivers of which are the hypoxia-inducible factor (HIF) family of transcription factors. HIFs are regulated in an oxygen-dependent manner and while their role in hypoxia is well established, it is apparent that other key players are required for gene expression control in hypoxia. In this review, we highlight the current understanding of the known and potential molecular mechanisms underpinning gene transcriptional responses to hypoxia in mammals, with a focus on oxygen-dependent effects on chromatin structure.

Chronic hepatitis C treatment in HIV co-infection in Portugal: Results from a cohort OF 2133 patients presented by GEPCOI (Portuguese Coinfection Study Group).

Direct-acting antiviral drugs (DAAs) have recently changed the paradigm of hepatitis C therapy, significantly improving treatment response rates, patient life expectancy and quality of life. In Portugal, sofosbuvir (SOF) and SOF/ledipasvir (SOF/LDV) were fully reimbursed by the National Health System since early 2015 and generalized use of interferon-free DAA based regimens became current practice. During 2016, the remaining DAAs were sequentially added and covered by the same health access policy. The Portuguese Study Group of Hepatitis and HIV Co-infection (GEPCOI) collected data from 15 clinical centres in Portugal, pertaining to the HCV treatment experience with DAA regimens. A cohort of 2133 patients was analysed, representing one of the largest DAA treated HCV/HIV co-infected individuals. The global sustained virologic response (SVR) achieved was 95% in this real-life cohort setting. Linear regression analysis showed significant differences in treatment response rates when using SOF plus ribavirin (RBV) combination in genotype 2 or 3 infected individuals (P < .002) and in those with liver cirrhosis (P < .002). These findings corroborate that early treatment is mandatory in HIV/HCV co-infected patients, as response rates may be negatively influenced by higher fibrosis stages and suboptimal DAA regimens. The current national Portuguese health policy should continue to promote wider treatment access and individualized therapy strategies, aiming at the elimination of HCV infection in this high-risk co-infected population.