Differential effects of HIF2α antagonist and HIF2α silencing in renal cancer and sensitivity to repurposed drugs.

BACKGROUND: In clear cell renal cell carcinoma, 80% of cases have biallelic inactivation of the VHL gene, leading to constitutive activation of both HIF1α and HIF2α. As HIF2α is the driver of the disease promoting tumour growth and metastasis, drugs targeting HIF2α have been developed. However, resistance is common, therefore new therapies are needed. METHODS: We assessed the effect of the HIF2α antagonist PT2385 in several steps of tumour development and performed RNAseq to identify genes differentially expressed upon treatment. A drug screening was used to identify drugs with antiproliferative effects on VHL-mutated HIF2α-expressing cells and could increase effectiveness of PT2385. RESULTS: PT2385 did not reduce cell proliferation or clonogenicity but, in contrast to the genetic silencing of HIF2α, it reduced in vitro cell invasion. Many HIF-inducible genes were down-regulated upon PT2385 treatment, whereas some genes involved in cell migration or extracellular matrix were up-regulated. HIF2α was associated with resistance to statins, addition to PT2385 did not increase the sensitivity. CONCLUSIONS: this study shows key differences between inhibiting a target versus knockdown, which are potentially targetable.

The circulating transcriptome as a source of cancer liquid biopsy biomarkers.

Non-invasive biomarkers or liquid biopsies have the potential to revolutionise cancer patient management as repeated sampling allows real-time monitoring of disease progression and response to treatment. This allows for earlier intervention and dynamic treatment management; both cornerstones of personalised medicine. The circulating transcriptome represents a rich source of potential cancer biomarkers that includes many classes of RNA, both coding and non-coding, that are only now beginning to be explored. In particular the increasing power and availability of RNAseq techniques have pushed studies beyond circulating miRNAs, to other classes of RNA including mRNA, snRNA, snoRNA, piRNA, YRNA, lncRNA and circRNA. In this review we focus on the emerging potential for these different classes of RNA as cancer biomarkers, and in particular the barriers and limitations that remain to be overcome if these molecules are to become part of routine clinical practice.

CircRNAs and cancer: Biomarkers and master regulators.

Circular RNAs (circRNAs) are a novel class of regulatory RNAs that despite being relatively abundant have only recently begun to be explored. There are many thousands of genes that appear capable of producing circRNAs, however the function of all but a handful remain to be determined. What is emerging about these highly conserved molecules is that they play important roles in biology and cancer biology in particular. The most explored function of circRNAs is as master regulators of gene expression that act to sequester or ´sponge´ other gene expression regulators, in particular miRNAs. They have also been demonstrated to function via direct modulation of transcription, and by interfering with splicing mechanisms. Although generally expressed in low abundance when compared to their linear counterparts, they are often expressed in a tissue- and developmental stage- specific manner. Coupled with their remarkable resistance to RNAse activity due to a covalent closed cyclic structure, circRNAs show great promise as novel biomarkers of cancer and other diseases. In this review we consider the current state of knowledge regarding these molecules, their synthesis, function, and association with cancer. We will also review some of the challenges that remain to be resolved if this emerging class of RNAs are really to become useful in the clinic.

Nitrous Oxide Abatement Coupled with Biopolymer Production As a Model GHG Biorefinery for Cost-Effective Climate Change Mitigation.

N2O represents ∼6% of the global greenhouse gas emission inventory and the most important O3-depleting substance emitted in this 21st century. Despite its environmental relevance, little attention has been given to cost-effective and environmentally friendly N2O abatement methods. Here we examined, the potential of a bubble column (BCR) and an internal loop airlift (ALR) bioreactors of 2.3 L for the abatement of N2O from a nitric acid plant emission. The process was based on the biological reduction of N2O by Paracoccus denitrificans using methanol as a carbon/electron source. Two nitrogen limiting strategies were also tested for the coproduction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) coupled with N2O reduction. High N2O removal efficiencies (REs) (≈87%) together with a low PHBV cell accumulation were observed in both bioreactors in excess of nitrogen. However, PHBV contents of 38-64% were recorded under N limiting conditions along with N2O-REs of ≈57% and ≈84% in the ALR and BCR, respectively. Fluorescence in situ hybridization analyses showed that P. denitrificans was dominant (>50%) after 6 months of experimentation. The successful abatement of N2O concomitant with PHBV accumulation confirmed the potential of integrating biorefinery concepts into biological gas treatment for a cost-effective GHG mitigation.

A HIF independent oxygen-sensitive pathway for controlling cholesterol synthesis.

Cholesterol biosynthesis is a highly regulated, oxygen-dependent pathway, vital for cell membrane integrity and growth. In fungi, the dependency on oxygen for sterol production has resulted in a shared transcriptional response, resembling prolyl hydroxylation of Hypoxia Inducible Factors (HIFs) in metazoans. Whether an analogous metazoan pathway exists is unknown. Here, we identify Sterol Regulatory Element Binding Protein 2 (SREBP2), the key transcription factor driving sterol production in mammals, as an oxygen-sensitive regulator of cholesterol synthesis. SREBP2 degradation in hypoxia overrides the normal sterol-sensing response, and is HIF independent. We identify MARCHF6, through its NADPH-mediated activation in hypoxia, as the main ubiquitin ligase controlling SREBP2 stability. Hypoxia-mediated degradation of SREBP2 protects cells from statin-induced cell death by forcing cells to rely on exogenous cholesterol uptake, explaining why many solid organ tumours become auxotrophic for cholesterol. Our findings therefore uncover an oxygen-sensitive pathway for governing cholesterol synthesis through regulated SREBP2-dependent protein degradation.

Role of Hypoxia in the Interferon Response.

In solid tumors, as the tumor grows and the disease progresses, hypoxic regions are often generated, but in contrast to most normal cells which cannot survive under these conditions, tumour cells adapt to hypoxia by HIF-driven mechanisms. Hypoxia can further promote cancer development by generating an immunosuppressive environment within the tumour mass, which allows tumour cells to escape the immune system recognition. This is achieved by recruiting immunosuppressive cells and by upregulating molecules which block immune cell activation. Hypoxia can also confer resistance to antitumor therapies by inducing the expression of membrane proteins that increase drug efflux or by inhibiting the apoptosis of treated cells. In addition, tumor cells require an active interferon (IFN) signalling pathway for the success of many anticancer therapies, such as radiotherapy or chemotherapy. Therefore, hypoxic effects on this pathway needs to be addressed for a successful treatment.

Biogas-based production of glycogen by Nostoc muscorum: Assessing the potential of transforming CO2 into value added products.

The potential of the filamentous N2-fixing cyanobacterium Nostoc muscorum for CO2 capture from high-loaded streams (i.e. flue gas or biogas) combined with the accumulation of glycogen (GL) and polyhydroxybutyrate (PHB), was evaluated under nutrient-sufficient and nutrient-limited conditions. N. muscorum was able to grow under CO2 contents from 0.03 up to 30% v/v, thus tolerating CO2 concentrations similar to those found in raw biogas or flue-gas, with maximum CO2-fixation rates of 191.9 ± 46 g m-3 d-1 at a biomass concentration of 733.3 ± 207.4 mg TSS L-1. Despite N. muscorum was inhibited by the presence of H2S, the co-inoculation with activated sludge resulted in both CO2 and H2S depletion. Moreover, N. muscorum accumulated GL up to ∼54% dcw under N and P-deprivation, almost 36 times higher than that recorded under nutrients sufficient condition. The addition of 10% extra carbon in the form of valeric acid not only did not hamper the growth of N. muscorum (336.0 ± 113.1 mg TSS L-1) but also increased the GL content to ∼58% dcw. On the contrary, a negligible PHB accumulation was found under the tested conditions, likely due to the high CO2 concentration of 30% v/v in the headspace and therefore the high availability of inorganic carbon for the cultures. N. muscorum cultures achieved VFAs degradations up to ∼78% under controlled pH. These results supported N. muscorum as a sustainable alternative for CO2-capture and greenhouse gas mitigation or for photosynthetic biogas upgrading coupled with value added biomass production.

Hypoxia Regulates Endogenous Double-Stranded RNA Production via Reduced Mitochondrial DNA Transcription.

Hypoxia is a common phenomenon in solid tumours strongly linked to the hallmarks of cancer. Hypoxia promotes local immunosuppression and downregulates type I interferon (IFN) expression and signalling, which contribute to the success of many cancer therapies. Double-stranded RNA (dsRNA), transiently generated during mitochondrial transcription, endogenously activates the type I IFN pathway. We report the effects of hypoxia on the generation of mitochondrial dsRNA (mtdsRNA) in breast cancer. We found a significant decrease in dsRNA production in different cell lines under hypoxia. This effect was HIF1α/2α-independent. mtdsRNA was responsible for induction of type I IFN and significantly decreased after hypoxia. Mitochondrially encoded gene expression was downregulated and mtdsRNA bound by the dsRNA-specific J2 antibody was decreased during hypoxia. These findings reveal a new mechanism of hypoxia-induced immunosuppression that could be targeted by hypoxia-activated therapies.

The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes.

Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

Trimethylamine abatement in algal-bacterial photobioreactors.

Trimethylamine (TMA) is an odorous volatile organic compound emitted by industries. Algal-based biotechnologies have been proven as a feasible alternative for wastewater treatment, although their application to abate polluted air emissions is still scarce. This work comparatively assessed the removal of TMA in a conventional bacterial bubble column bioreactor (BC) and a novel algal-bacterial bubble column photobioreactor (PBC). The PBC exhibited a superior TMA abatement performance compared to the conventional BC. In this sense, the BC reached a removal efficiency (RE) and an elimination capacity (EC) of 78% and 12.1 g TMA m-3 h-1, respectively, while the PBC achieved a RE of 97% and a EC of 16.0 g TMA m-3·h-1 at an empty bed residence time (EBRT) of 2 min and a TMA concentration ~500 mg m-3. The outstanding performance of the PBC allowed to reduce the operating EBRT to 1.5 and 1 min while maintaining high REs of 98 and 94% and ECs of 21.2 and 28.1 g m-3·h-1, respectively. Moreover, the PBC improved the quality of the gas and liquid effluents discharged, showing a net CO2 consumption and decreasing by ~ 30% the total nitrogen concentration in the liquid effluent via biomass assimilation. A high specialization of the bacterial community was observed in the PBC, Mumia and Aquamicrobium sp. being the most abundant genus within the main phyla identified. GraphicalAbstract.

Elucidating the influence of environmental factors on biogas-based polyhydroxybutyrate production by Methylocystis hirsuta CSC1.

The valorization of biogas as a feedstock for the generation of added-value bioproducts will play a key role on the sustainability of anaerobic digestion. The present work assessed the influence of key environmental parameters (O2:CH4 ratio, temperature and nitrogen source) on the growth and polyhydroxybutyrate (PHB) synthesis under nitrogen limiting conditions of the type II methanotroph Methylocystis hirsuta CSC1 using biogas as a feedstock. The O2:CH4 ratios tested (1:1, 1.5:1 and 2:1) did not affect significantly M. hirsuta CSC1 growth yields (~5 g TSS mol-1 CH4), although lower CH4 removal rates were reached under O2-limiting conditions (ratio 1:1). The highest PHB content (45 wt%) was achieved at a ratio 2:1 and was threefold higher than those obtained at lower ratios (~15 wt%). The increase in temperature from 15 to 25 °C resulted in increases in the growth yield (from 5 to 6 g TSS mol-1 CH4) and PHB content (from 32 to 40 wt%). Conversely, the lowest PHB content (30 wt%) was reached at 37 °C, together with a negligible growth under nutrient sufficient conditions. The nitrogen source also played a key role on both M. hirsuta CSC1 growth and PHB synthesis. Thus, ammonium resulted in the highest growth yield (7 g TSS mol-1 CH4), although the maximum PHB content was achieved when biomass was previously grown in nitrate as the nitrogen source (41 wt%). Nitrite exerted an inhibitory effect on M. hirsuta CSC1 growth.

Hypoxia Induces Transcriptional and Translational Downregulation of the Type I IFN Pathway in Multiple Cancer Cell Types.

Hypoxia is a common phenomenon in solid tumors and is strongly linked to hallmarks of cancer. Recent evidence has shown that hypoxia promotes local immune suppression. Type I IFN supports cytotoxic T lymphocytes by stimulating the maturation of dendritic cells and enhancing their capacity to process and present antigens. However, little is known about the relationship between hypoxia and the type I IFN pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA) followed by IFNα/ß secretion and transcriptional activation of IFN-stimulated genes (ISG). In this study, we determined the effects of hypoxia on the type I IFN pathway in breast cancer and the mechanisms involved. In cancer cell lines and xenograft models, mRNA and protein expressions of the type I IFN pathway were downregulated under hypoxic conditions. This pathway was suppressed at each level of signaling, from the dsRNA sensors RIG-I and MDA5, the adaptor MAVS, transcription factors IRF3, IRF7, and STAT1, and several ISG including RIG-I, IRF7, STAT1, and ADAR-p150. Importantly, IFN secretion was reduced under hypoxic conditions. HIF1α- and HIF2α-mediated regulation of gene expression did not explain most of the effects. However, ATAC-seq data revealed in hypoxia that peaks with STAT1 and IRF3 motifs had decreased accessibility. Collectively, these results indicate that hypoxia leads to an overall downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in an HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumors. SIGNIFICANCE: These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine/paracrine effects on tumor growth.

MicroRNAs as Biomarkers of B-cell Lymphoma.

B-cell lymphomas represent a diverse group of neoplasms classified primarily by histopatholgy and are often challenging to accurately diagnose. Despite having been recognized less than 20 years ago, microRNAs (miRNAs) have emerged as one of the most promising class of cancer molecular biomarkers and are particularly attractive as they can be readily detected in formalin-fixed paraffin-embedded biopsy material and biological fluids such as blood. Many of the identified B-cell lymphoma miRNA biomarkers also play crucial regulatory roles in normal B-cell development. Below we consider the identity, function, and biomarker potential of miRNAs in B-cell lymphoma and most importantly the barriers that remain to be overcome if they are really to become part of routine clinical practice.

Comprehensive two-dimensional gas chromatography with capillary flow modulation to separate FAME isomers.

A method to separate FAME and the linoleic and linolenic acids isomers by GCxGC using an apparatus equipped with a capillary flow technology (CFT) based modulator and a FID detector has been developed. Four different column combinations (one conventional and three inverted phase sets) were used in these experiments. The conventional set first involved a DB5-MS non-polar column followed by a highly polar HP-INNOWax column in the second dimension. The inverted phase set comprised of a highly polar BPX-70 column in the first dimension and a non-polar ZB5-MS column for the second dimension. Furthermore, the influence of the length of the second dimension column on FAME isomer separation was studied in the inverted phase sets, along with other parameters like the modulation time and column flow. The best results in terms of the time required for the analysis and number of FAME identified with the inverted set were achieved with the shorter second dimension column. After supercritical fluid extraction, the method was applied to identify FAMEs in broccoli leaves from three different cultivars (Naxos, Nubia and Viola).