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Purpose:Gene fusions are important oncogenic drivers and many are actionable. Whole-genome and transcriptome (WGS and RNA-seq, respectively) sequencing can discover novel clinically relevant fusions.Experimental Design:Using WGS and RNA-seq, we reviewed the prevalence of fusions in a cohort of 570 patients with cancer, and compared prevalence to that predicted with commercially available panels. Fusions were annotated using a consensus variant calling pipeline (MAVIS) and required that a contig of the breakpoint could be constructed and supported from ≥2 structural variant detection approaches.Results:In 570 patients with advanced cancer, MAVIS identified 81 recurrent fusions by WGS and 111 by RNA-seq, of which 18 fusions by WGS and 19 by RNA-seq were noted in at least 3 separate patients. The most common fusions were EML4-ALK in thoracic malignancies (9/69, 13%), and CMTM8-CMTM7 in colorectal cancer (4/73, 5.5%). Combined genomic and transcriptomic analysis identified novel fusion partners for clinically relevant genes, such as NTRK2 (novel partners: SHC3, DAPK1), and NTRK3 (novel partners: POLG, PIBF1).Conclusions:Utilizing WGS/RNA-seq facilitates identification of novel fusions in clinically relevant genes, and detected a greater proportion than commercially available panels are expected to find. A significant benefit of WGS and RNA-seq is the innate ability to retrospectively identify variants that becomes clinically relevant over time, without the need for additional testing, which is not possible with panel-based approaches.

The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, with a few notable exceptions. We have sequenced and assembled the complete 5.5-Mb mitochondrial genome of Sitka spruce (Picea sitchensis), to date, one of the largest mitochondrial genomes of a gymnosperm. We sequenced the whole genome using Oxford Nanopore MinION, and then identified contigs of mitochondrial origin assembled from these long reads based on sequence homology to the white spruce mitochondrial genome. The assembly graph shows a multipartite genome structure, composed of one smaller 168-kb circular segment of DNA, and a larger 5.4-Mb single component with a branching structure. The assembly graph gives insight into a putative complex physical genome structure, and its branching points may represent active sites of recombination.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic. Marra and colleagues describe POG570, a pan-cancer, whole-genome, transcriptome and clinical dataset stressing the molecular interactions in advanced and post-therapy cancer patients.

Saline stress has been identified as the primary factor inhibiting rice seedling growth, which represents a complex abiotic stress process. Most plant hormones have been shown to alleviate the plant damage caused by salt stress. The effects of synthetic strigolactone (GR24) on Jinongda 667 rice seedlings treated with 200 mM NaCl were studied. Photosynthesis and its related physiological characteristics were analyzed in salt-stressed rice seedlings treated with GR24. NaCL stress inhibited the growth of the rice, including plant height and root length, by approximately 14% and 40%, respectively. Compared to the control check group (CK), the adverse effects of salt stress on the growth status, leaf photosynthesis, and physiological/biochemical indices in the rice seedlings were alleviated in the GR24 treatment group. With increases in the GR24 concentration, the plant height and root length of the seedlings increased. The plant height in the groups treated with 1/2 Hoagland’s complete nutrient solution + 200 mM NaCl +1 μM GR24 (T4) and 1/2 Hoagland’s complete nutrient solution + 200 mM NaCl +5 μM GR24 (T5) were significantly different than the 1/2 Hoagland’s complete nutrient solution + 200 mM NaCl group (T1) (P 0.05). The transpiration rate, stomatal conductance, and intercellular CO2 concentrations showed the same trends as the net photosynthetic rate. The MAD, POD, and SOD activities were significantly increased by 68%, 60%, 14%, respectively, compared to the CK group (P

Three microalgae-based treatment technologies were applied for removing nutrients in piggery wastewater and CO2 in biogas simultaneously. Chlorella vulgaris (C. vulgaris), Scenedesmus obliquus (S. obliquus), and Neochloris oleoabundans (N. oleoabundans) were selected for mono-cultivation or co-cultivation with fungi or activated sludge. The effects of initial inoculum concentration (low of 62.06 ± 6.23 mg/L, medium of 121 ± 9.34 mg/L, and high of 180 ± 12.78 mg/L) of microalgae/algal symbiont on the pollutant removal efficiency were evaluated. The results showed that cultivation of S. obliquus with activated sludge had a relatively high CO2 and nutrients removal efficiency at a medium concentration of initial inoculum. The highest removal efficiency of total organic carbon, total nitrogen, and total phosphorus in piggery wastewater was 87.29%, 87.26%, and 90.17% by co-cultivation of S. obliquus with activated sludge. The highest CO2 removal of 64.28% could be achieved under co-cultivation of S. obliquus and fungi at medium initial inoculum concentrations. This work will be helpful to promote the study of microalgae-based biogas upgrading and piggery wastewater purification. PRACTITIONER POINTS: Nutrient and CO2 were efficiently removed in moderate microalgal initial inoculum. Scenedesmus obliquus-activated sludge cultivation achieved the highest nutrient removal. Co-cultivation of S. obliquus and fungi showed the highest CO2 removal. Zinc was removed efficiently in high microalgal initial inoculum. Chlorella vulgaris/S. obliquus-activated sludge performed economically.

PURPOSE This study investigated therapeutic potential of integrated genome and transcriptome profiling of metastatic sarcoma, a rare but extremely heterogeneous group of aggressive mesenchymal malignancies with few systemic therapeutic options. METHODS Forty-three adult patients with advanced or metastatic non-GI stromal tumor sarcomas of various histology subtypes who were enrolled in the Personalized OncoGenomics program at BC Cancer were included in this study. Fresh tumor tissues along with blood samples underwent whole-genome and transcriptome sequencing. RESULTS The most frequent genomic alterations in this cohort are large-scale structural variation and somatic copy number variation. Outlier RNA expression as well as somatic copy number variations, structural variations, and small mutations together suggest the presence of one or more potential therapeutic targets in the majority of patients in our cohort. Point mutations or deletions in known targetable cancer genes are rare; for example, tuberous sclerosis complex 2 provides a rationale for targeting the mammalian target of rapamycin pathway, resulting in a few patients with exceptional clinical benefit from everolimus. In addition, we observed recurrent 17p11-12 amplifications, which seem to be a sarcoma-specific event. This may suggest that this region harbors an oncogene(s) that is significant for sarcoma tumorigenesis. Furthermore, some sarcoma tumors carrying a distinct mutational signature suggestive of homologous recombination deficiency seem to demonstrate sensitivity to double-strand DNA–damaging agents. CONCLUSION Integrated large-scale genomic analysis may provide insights into potential therapeutic targets as well as novel biologic features of metastatic sarcomas that could fuel future experimental and clinical research and help design biomarker-driven basket clinical trials for novel therapeutic strategies.

The algae-based technology has a positive effect on the treatment of biogas slurry and the purification of biogas, while vitamin B12 (VB12) is one of the important regulatory substances in the algae-based cultivation system. In this study, different concentrations of VB12 were used in three microalgal treatment technologies to assess their effect on simultaneous removal of nutrients from biogas slurry and removal of CO2 from raw biogas. Results showed that Chlorella vulgaris exhibited higher growth rate, mean daily productivity, chlorophyll a content, carbonic anhydrase activity and better photosynthetic properties when co-cultivated with Ganoderma lucidum, rather than when co-cultivated with activated sludge or under mono-cultivation. Maximum mean chemical oxygen demand, total nitrogen, total phosphorus and CO2 removal efficiencies were found to be 84.29 ± 8.28%, 83.27 ± 8.14%, 85.27 ± 8.46% and 65.71 ± 6.35%, respectively when microalgae were co-cultivated with Ganoderma lucidum under 100 ng L-1 of VB12. This study shows the potential of microalgae and fungi co-cultivation supplemented with VB12 for the simultaneous upgradation of biogas production as well as for the purification of biogas slurry.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3′ or 5′ termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

The purification effect of two different microalgae-fungi-bacteria symbiosis technologies on biogas and biogas slurry was studied to determine the best symbiosis treatment technology and the suitable concentration of GR24. The results showed that the purification effect of biogas slurry in Chlorella vulgaris-Ganoderma lucidum-endophytic bacteria (S395–2) symbiont co-culture system was better than that of the biogas slurry in Scenedesmus obliquus-Pleurotus ostreatus-S395–2 symbionts. Following 10–9 M GR24 treatment, Chlorella vulgaris-Ganoderma lucidum-S395–2 symbionts had elevated mean daily production rate and growth rate by 1.92 and 1.46 folds in comparison with blank group. After adjusting the GR24 level within the range of 10–9 M-10–7 M, Ganoderma lucidum-assisted Chlorella vulgaris-S395–2 attained higher maximal removal rates for TN, COD, CO2, and TP by 10.78%, 14.62%, 3.86%, and 9.07%, respectively, compared to the rates when GR24 was not added.

Here, we present the chloroplast genome sequence of black spruce ( Picea mariana ), a conifer widely distributed throughout North American boreal forests. This complete and annotated chloroplast sequence is 123,961 bp long and will contribute to future studies on the genetic basis of evolutionary change in spruce and adaptation in conifers.

Different gradient concentrations of vitamin B12 (0, 10, 100, 1,000 ng L-1 ) were used in the symbiosis system (Chlorella vulgaris-Ganoderma lucidum or Chlorella vulgaris-Pleurotus ostreatus) to assess their effect on simultaneous purification of biogas and removal of nutrients in biogas slurry using co-culture of microalgae and fungi. When B12 was added to the symbiosis system, biomass growth, intracellular carbonic anhydrase activity (CA), chlorophyll a content (CHL-a), photosynthetic characteristics of the two cultivation system, and removal efficiency of nutrients in biogas slurry and CO2 in biogas were significantly higher than those in the control group. The optimal concentration of B12 was determined to be 100 ng L-1 considering the removal efficiency of nutrients and CO2 . Maximum mean chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP), and CO2 removal efficiencies were 75.98 ± 6.26%, 78.46 ± 6.21%, 80.21 ± 6.83% and 61.08 ± 5.21% in Chlorella vulgaris-Ganoderma lucidum, respectively. This study showed the potential of microalgae and fungi symbiosis system with B12 addition for nutrient removal and biogas upgrading. PRACTITIONER POINTS: Vitamin B12 had positive effects on algal-fungal pellets growth. The optimal vitamin B12 concentration was 100 ng L-1 . The highest CO2 remove rate was 61.08% by G. lucidum/C. vulgaris pellets. Vitamin B12 significantly improved photosynthetic performance of pellets.

Freshwater microalgae Chlorella vulgaris was cultured and induced with strigolactone (GR24) to simultaneously eliminate nutrients in biogas slurry and purify biogas. Treatment with 10−7 M GR24 yielded maximum growth rate and mean daily productivity for algae at 0.187 ± 0.06 d−1 and 0.097 ± 0.008 g L−1 d−1, respectively. Results from chlorophyll fluorescence transients method demonstrated that moderate concentration of GR24 could enhance the photosynthetic performance of microalgae. In addition, GR24 affected intracellular carbonic anhydrase activity and chlorophyll-a content. Maximum chemical oxygen demand, total nitrogen, and CO2 removal efficiencies were 78.62 ± 2.36%, 76.47 ± 1.53% and 64.05 ± 1.15% with 10−7 M GR24 induction, respectively. Further, highest total phosphorus removal efficiency (80.27 ± 1.93%) was observed at 10−9 M. The optimal GR24 concentration range was determined to be between 10−9 and 10−7 M in consideration with nutrient and CO2 removal efficiencies.

In this study, digestate and biogas were treated simultaneously based on fungi-microalgae symbionts and fungi-microalgae-bacteria symbionts, respectively. The effects of strigolactone (GR24) on the biomass of symbionts and photosynthesis of algae, and purification of digestate and biogas were explored. With the induction of GR24, the metabolism of microorganism-microalgae systems and photosynthesis of microalgae were enhanced causing the rapid growth of the symbionts, further strengthening the purification performance of the systems. Moreover, the GR24 enhanced the CO2 removal performance of the co-culture systems via increasing the carbonic anhydrase activity of microalgae cells. The fungi-microalgae-bacteria co-culture strategy with GR24 induction was found to obtain great photosynthetic and removal performance, possessing the 73.54 ± 6.83% CO2 removal rate and 88.61 ± 8.24% COD removal rate.

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.Article SummaryA scalable, non-proprietary, magnetic bead-based automated nucleic acid extraction protocol optimised for minimum cross-well contamination

In this study, effects of polystyrene microplastics (MPS) on Euglena gracilis were investigated via examination on its photosynthesis and motility, two typical properties of the protozoan. No adverse effects were observed after 4-d exposure except for decrease in motility at two high MPS concentrations (5 and 25 mg/L). After 8-d duration, MPS at 1 mg/L had no obvious effects on E. gracilis, but two higher concentrations (5 and 25 mg/L) of MPS inhibited protozoan growth, motility, and photosynthesis. The reduced protozoan photosynthetic activity was reflected by changes in Fv/Fm (the maximum photochemical yield of PSII), ΔFIP (difference between FP and FI) and PIABS (the performance index), indicative of reduced quantum yield of electron transport and enhanced energy dissipation. A dose-dependent effect of MPS on E. gracilis was found in protozoan growth, photosynthesis and motility, especially photosynthetic indices. MPS of small size (75 nm) seemed more toxic to the protozoa than large size (1000 nm). Internalization of MPS in the cells and chloroplasts was observed clearly for the first time, likely responsible for their toxicity. Analysis on photosynthetic process and motility of E. gracilis could provide more comprehensive understanding of MPS toxicity in the aquatic environment, and may potentially serve as a biomonitoring tool.

In order to purify biogas slurry and biogas simultaneously, three different fungi, Pleurotus geesteranus (P. geesteranus), Ganoderma lucidum (G. lucidum), and Pleurotus ostreatus (P. ostreatus) were pelletized with Chlorella vulgaris (C. vulgaris). The results showed that the optimal light wavelength ratio for red:blue was 5:5 for these three different fungi-assisted C. vulgaris, resulting in higher specific growth rate as well as nutrient and CO2 removal efficiency compared with other ratios. G. lucidum/C. vulgaris was screened as the best fungi-mialgae for biogas slurry purification and biogas upgrading with light/dark ratio of 14h:10h, which was also confirmed by the economic efficiency analysis of the energy consumptions. These results will provide a theoretical foundation for large-scale biogas slurry purifying and biogas upgrading using microalgae.

This work mainly aimed to compare the growth and tolerance of fungal–algal cultures in various dilutions of anaerobically digested swine wastewater (ASW) and to determine their nutrient removal efficiency and role in biogas upgrading. Three species of microalgae, namely, Chlorella vulgaris, Scenedesmus obliquus, and Pseudokirchneriella subcapitata, were selected for ASW purification and biogas upgrading in photobioreactors with four different concentrations of 1300, 1000, 700, and 300 mg L−1 COD (chemical oxygen demand). Co-cultivation of the fungus Ganoderma lucidum and C. vulgaris in 300 mg L−1 COD was the most efficient for reduction of COD, total nitrogen, and total phosphorus by 79.74 ± 4.87%, 74.28 ± 6.13% and 85.37 ± 6.84%, respectively. With respect to biogas upgrading, co-cultivation of G. lucidum and P. subcapitata in 700 mg L−1 COD showed the maximum CO2 removal efficiency of 84.77 ± 3.04%. These results show the feasibility of simultaneously removing nutrients in ASW and CO2 in biogas using fungi-microalgae pellets.

Background RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. Conclusions These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.

The microalgae-based technology has been developed to reduce biogas slurry nutrients and upgrade biogas simultaneously. In this work, five microalgal strains named Chlorella vulgaris, Scenedesmus obliquus, Selenastrum capricornutum, Nitzschia palea, and Anabaena spiroides under mono- and co-cultivation were used for biogas upgrading. Optimum biogas slurry nutrient reduction could be achieved by co-cultivating microalgae (Chlorella vulgaris, Scenedesmus obliquus, and Nitzschia palea) with fungi using the pelletization technology. In addition, the effects of different ratio of mixed LED light wavelengths applying mixed light-emitting diode during algae strains and fungi co-cultivation on CO2 and biogas slurry nutrient removal efficiency were also investigated. The results showed that the COD (chemical oxygen demand), TN (total nitrogen), and TP (total phosphorus) removal efficiency were 85.82 ± 5.37%, 83.31 ± 4.72%, and 84.26 ± 5.58%, respectively at red: blue = 5:5 under the co-cultivation of S. obliquus and fungi. In terms of biogas upgrading, CH4 contents were higher than 90% (v/v) for all strains, except the co-cultivation with S. obliquus and fungi at red: blue = 3:7. The results indicated that co-cultivation of microalgae with fungi under mixed light wavelengths treatments was most successful in nutrient removal from wastewater and biogas upgrading.

Bacterial communities play an important role in diffuse sediment pollution in aquaculture farms. Previous studies have revealed the short-term influence of wastewater drainage on the bacterial communities but the seasonal response of the sediment bacterial communities to wastewater drainage from aquaculture farms remains unclear. This study used the 16S rRNA approach to explore the profiles of bacterial communities over four seasons in a typical crab aquaculture farm that included a pond and an outlet ditch. Nineteen sediment samples and an equal number of water samples were collected and analysed during spring, summer, autumn, and winter during 2018-19. Our results showed that Proteobacteria, Chloroflexi, and Bacteroides were the predominant phyla in aquaculture pond sediment with the relative abundance of 28.95%, 17.32%, and 15.31%, respectively. The relative abundance of Proteobacteria and Bacteroides was higher in autumn and winter, and the relative abundance of Chloroflexi was highest in spring. The Shannon diversity index value ranged from 6.17 to 9.30 and showed significant positive correlation (P 0.01) with the concentrations of TN, NH

The Yi River, the second longest river in Shandong Province, China, flows through Linyi City and is fed by three tributary rivers, Beng River, Liuqing River, and Su River in the northeastern part of the city. In this study, we determined the concentrations of five heavy metals (Cr, Ni, Cu, Zn, and Pb) in water, sediment, and aquatic macrophyte samples collected from the junction of the four rivers and evaluated the potential ecological risk of heavy metal pollution. Most of the heavy metals in water were in low concentrations with the water quality index (WQI) below 1, suggesting low metal pollution. The sediments showed low heavy metal concentrations, suggesting a low ecological risk based on the potential ecological risk index (RI) and the geo-accumulation index (Igeo). The aquatic plant species Potamogeton crispus accumulated considerable amounts of heavy metals, which were closely related to the metal concentrations of the sediment. The plant species Salvinia natans also showed an excellent metal accumulation capability. Based on our results, the junction of the four rivers is only slightly polluted in terms of heavy metals, and the plant species P. crispus is a suitable bioindicator for sediment heavy metal pollution.

Engelmann spruce ( Picea engelmannii ) is a conifer found primarily on the west coast of North America. Here, we present the complete chloroplast genome sequence of Picea engelmannii genotype Se404-851. This chloroplast sequence will benefit future conifer genomic research and contribute resources to further species conservation efforts.

Here, we present the complete chloroplast genome sequence of white spruce (Picea glauca, genotype WS77111), a coniferous tree widespread in the boreal forests of North America. This sequence contributes to genomic and phylogenetic analyses of the Picea genus that are part of ongoing research to understand their adaptation to environmental stress.

The Steller sea lion is the largest member of the Otariidae family and is found in the coastal waters of the northern Pacific Rim. Here, we present the Steller sea lion genome, determined through DNA sequencing approaches that utilized microfluidic partitioning library construction, as well as nanopore technologies. These methods constructed a highly contiguous assembly with a scaffold N50 length of over 14 megabases, a contig N50 length of over 242 kilobases and a total length of 2.404 gigabases. As a measure of completeness, 95.1% of 4104 highly conserved mammalian genes were found to be complete within the assembly. Further annotation identified 19,668 protein coding genes. The assembled genome sequence and underlying sequence data can be found at the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA475770.

Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, with a few notable exceptions. We have sequenced and assembled the 5.5 Mbp mitochondrial genome of Sitka spruce (Picea sitchensis), the largest complete mitochondrial genome of a gymnosperm. We sequenced the whole genome using Oxford Nanopore MinION, and then identified contigs of mitochondrial origin assembled from these long reads. The assembly graph shows a multipartite genome structure, composed of one smaller 168 kbp circular segment of DNA, and a larger 5.4 Mbp component with a branching structure. The assembly graph gives insight into a putative complex physical genome structure, and its branching points may represent active sites of recombination.

The beluga whale is a cetacean that inhabits arctic and subarctic regions, and is the only living member of the genus Delphinapterus. The genome of the beluga whale was determined using DNA sequencing approaches that employed both microfluidic partitioning library and non-partitioned library construction. The former allowed for the construction of a highly contiguous assembly with a scaffold N50 length of over 19 Mbp and total reconstruction of 2.32 Gbp. To aid our understanding of the functional elements, transcriptome data was also derived from brain, duodenum, heart, lung, spleen, and liver tissue. Assembled sequence and all of the underlying sequence data are available at the National Center for Biotechnology Information (NCBI) under the Bioproject accession number PRJNA360851A.

Programmed death ligands (PDLs) are immune-regulatory molecules that are frequently affected by chromosomal alterations in B-cell lymphomas. Although PDL copy-number variations are well characterized, a detailed and comprehensive analysis of structural rearrangements (SRs) and associated phenotypic consequences is largely lacking. Here, we used oligonucleotide capture sequencing of 67 formalin-fixed paraffin-embedded tissues derived from primary B-cell lymphomas and 1 cell line to detect and characterize, at base-pair resolution, SRs of the PDL locus (9p24.1; harboring PDL1/CD274 and PDL2/PDCD1LG2). We describe 36 novel PDL SRs, including 17 intrachromosomal events (inversions, duplications, deletions) and 19 translocations involving BZRAP-AS1, CD44, GET4, IL4R, KIAA0226L, MID1, RCC1, PTPN1 and segments of the immunoglobulin loci. Moreover, analysis of the precise chromosomal breakpoints reveals 2 distinct cluster breakpoint regions (CBRs) within either CD274 (CBR1) or PDCD1LG2 (CBR2). To determine the phenotypic consequences of these SRs, we performed immunohistochemistry for CD274 and PDCD1LG2 on primary pretreatment biopsies and found that PDL SRs are significantly associated with PDL protein expression. Finally, stable ectopic expression of wild-type PDCD1LG2 and the PDCD1LG2-IGHV7-81 fusion showed, in coculture, significantly reduced T-cell activation. Taken together, our data demonstrate the complementary utility of fluorescence in situ hybridization and capture sequencing approaches and provide a classification scheme for PDL SRs with implications for future studies using PDL immune-checkpoint inhibitors in B-cell lymphomas.

In this work, a cooperative algal-bacterial system that efficiently upgrade biogas, simultaneously reduce the biogas slurry nutrient, and exhibits high biomass productivity, was developed. The method about removing H2S and CO2 from biogas by three microalgal strains (Chlorella vulgaris, Scenedesmus obliquus, and Neochloris oleoabundans) mixed with activated sludge using biogas slurry as nutrient medium. A CO2 concentration of 45% (v/v) was considered optimum to support CO2 and H2S removals of 74.11%–80.57% and 99.04%–99.42%, respectively. At the CO2 concentration of 45%–55%, the mixed culture containing S. obliquus and C. vulgaris efficiently removed COD (chemical oxygen demand) and TP (total phosphorus), respectively. The mixed culture containing S. obliquus demonstrated high N removal efficiency at CO2 concentration of 45%. Biomass productivity increased at increasing CO2 concentration in a certain range, with a maximum of 0.177 g L−1 d−1 at CO2 concentration of 45% (v/v) for the mixed culture containing S. obliquus, whereas the C, N, and P biomass contents remained constant at 46.73%–52.31%, 7.59%–9.08%, and 0.91%–1.08%, respectively. This study showed the potential of the combination of alga and bacteria to serve as a treatment for nutrient removal and biogas upgrading in algal-bacterial processes.

Endophytic bacteria were isolated from Chlorella vulgaris and co-cultured with its host microalgae to determine whether this symbiotic system is suitable for purifying biogas and biogas slurry. Results showed that endophytic bacteria S395-1 and S395-2 belonged to different genera. Both strains promoted microalgae growth while improving photosynthetic performance, carbonic anhydrase activity, nutrient removal efficiency, and CO2 fixation. The optimal bacteria (S395-2)-to-microalgae ratio and co-culture duration were 10:1 and 7 days. Under this condition, the growth rate and carbonic anhydrase activity were 0.196 ± 0.06 d-1 and 31.24 ± 0.28 EU/cell, respectively. The symbiotic system had removal efficiencies of 88.29 ± 5.03%, 88.31 ± 4.29%, 88.21 ± 4.51%, and 68.13 ± 1.69% for chemical oxygen demand, nitrogen, phosphorus, and CO2, respectively. These results will provide a framework for constructing a microalgal-bacterial consortium that can improve wastewater treatment and enhance biogas quality.

To verify the practicability of biogas production from liquid waste emanated from alkaline pretreatment of lignocellulose, the blended solid-liquid slurry of sugarcane rind (SR), which generated from Ca(OH)2 coupled microwave pretreatment and undergone decalcification via biogas bubbling, was anerobically co-digested with kitchen waste (KW) in batched mesophilic assays at five mixing ratios. Results showed that the pretreatment improved the cell-free enzyme activity (CFEA) on cellulose hydrolysis during the anaerobic co-digestion (AcoD). The strategy of AcoD of pretreated SR slurry (PSRS) or SR with KW not only increased methane production but also was benifical to the process stability. The synergistic effect was revealed based on methane production, alkalinity and enzyme activities in AcoD process. The 25% PSRS +75% KW demostrated the highest methane production of 430.4 N mL CH4/g volatile solids, which was 22% and 86% higher than 25% SR + 75% KW and 100% SR, respectively. The higher the proportion of PSRS or SR in feedstock, the higher the CFEA on cellulose hydrolysis revealed during fermentation. The maximum CFEA on cellulose hydrolysis of 0.293 U/g appeared on the 14th day in 100% PSR, which was 24.2% higher than that of 100% SR. The results will provide a reference for utilizing SR and KW in actual biogas plant.

Atrazine is a widely-applied herbicide used primarily to control weeds, which can persist in the ecosystem and exert potential toxicity to phytoplankton in the aquatic environment. In this study, acute toxicity of atrazine on microalgae Chlorella sp. was investigated with different initial cell densities (1 × 105 and 1 × 106 cells mL−1) and exposure periods (4 d and 8 d). Both growth rate and photosynthetic parameters of the microalgae in response of atrazine stress were determined to find out the sensitive indices and toxicological mechanisms. Because of the independence of initial cell density as well as the high sensitivity and reliability, the performance index PIABS was verified as the most convincing photosynthetic parameter for indicating IC50 of atrazine on Chlorella sp., being superior to the traditional parameters of growth rate and FV/FM. The IP amplitude (ΔFIP, fluorescence amplitude of the I-to-P-rise in the OJIP curve) was another sensitive biomarker to reflect atrazine stress. Results from chlorophyll fluorescence transient revealed that atrazine damaged the photosystem II (PS II) reaction center, suppressed the electron transport at the donor and receptor sides, and acted on the absorption, transfer, and utilization of light energy. Our results provide confirmatory references for understanding the toxicity and mechanisms of atrazine on freshwater microalgae.

Co-cultivation of microalgae and microbes for pollutant removal from sewage is considered as an effective wastewater treatment method. The aim of this study is to screen the optimal photoperiod, light intensity and microalgae co-cultivation method for simultaneously removing nutrients in biogas slurry and capturing CO₂ in biogas. The microalgae-fungi pellets are deemed to be a viable option because of their high specific growth rate and nutrient and CO2 removal efficiency under the photoperiod of 14 h light:10 h dark. The order of both the biogas slurry purification and biogas upgrading is ranked the same, that is Chlorella vulgaris-Ganodermalucidum > Chlorella vulgaris-activated sludge > Chlorella vulgaris under different light intensities. For all cultivation methods, the moderate light intensity of 450 μmol m-2 s-1 is regarded as the best choice. This research revealed that the control of photoperiod and light intensity can promote the biological treatment process of biogas slurry purification and biogas upgrading using microalgal-based technology.

The northern sea otter inhabits coastal waters of the northern Pacific Ocean and is the largest member of the Mustelidae family. DNA sequencing methods that utilize microfluidic partitioned and non-partitioned library construction were used to establish the sea otter genome. The final assembly provided 2.426 Gbp of highly contiguous assembled genomic sequences with a scaffold N50 length of over 38 Mbp. We generated transcriptome data derived from a lymphoma to aid in the determination of functional elements. The assembled genome sequence and underlying sequence data are available at the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA388419.

Frogs play important ecological roles, and several species are important model organisms for scientific research. The globally distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Unfortunately, there are currently no genomic resources for the former, important group of amphibians. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines. We report a 5.8 Gbp (NG50 = 69 kbp) genome assembly of a representative North American bullfrog (Rana [Lithobates] catesbeiana). The genome contains over 22,000 predicted protein-coding genes and 6,223 candidate long noncoding RNAs (lncRNAs). RNA-Seq experiments show thyroid hormone causes widespread transcriptional change among protein-coding and putative lncRNA genes. This initial bullfrog draft genome will serve as a key resource with broad utility including amphibian research, developmental biology, and environmental research., The globally-distributed Ranidae (true frogs) are the largest frog family. Here, Hammond et al. present a draft genome of the North American bullfrog, Rana (Lithobates) catesbeiana, as a foundation for future understanding of true frog genetics as amphibian species face difficult environmental challenges.

In this study, we investigated the ability of microalgae to remove biogas slurry nutrients and upgrade biogas simultaneously under various light-emitting diode light intensities. Three green algae, namely, Scenedesmus obliquus, Chlorella sp., and Selenastrum bibraianum, were selected and cultivated in photobioreactor bags. The capacity of each microalga to reduce the chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) of wastes was investigated. The growth rate and CO2 uptake of each microalga were also examined. Compared with S. bibraianum, S. obliquus and Chlorella sp. showed better combined results and were more suitable for simultaneous biogas upgrading and biogas effluent nutrient reduction. Results showed that the best results for biogas slurry nutrient reduction, CO2 removal, and upgrade effects were obtained from the treatment under moderate light intensity (150–170 μmol m−2 s−1) for S. obliquus. The highest reduction values of COD, TN, TP, dry weight, and CH4 concentration obtained were 93.08% ± 4.62%, 84.12% ± 4.37%, 86.76% ± 4.58%, 465.31 ± 19.44 mg L−1, and 94.41 ± 3.16 (v/v), respectively.

The three microalgal strains were cultivated, namely, Chlorella vulgaris, Scenedesmus obliquus, and Neochloris oleoabundans, by applying mixed light-emitting diode wavelength treatments to biogas slurry in a photobioreactor bag. This study aims to compare the growth and nutrient removal efficiency of the algae and determine their roles for biogas upgrading. At red:blue=5:5, S. obliquus and C. vulgaris efficiently removed COD and TP, respectively. S. obliquus demonstrated high N removal efficiency at red:blue=7:3. The same strain significantly improved removal capacity for all nutrients compared with C. vulgaris and N. oleoabundans, particularly at red:blue=5:5, 7:3, and 3:7. For biogas upgrade, CH4 contents were higher than 75% (v/v) for all strains. The algae exhibited particularly good CH4 enrichment at red:blue=7:3, 5:5. Results show that microalgal biomass production offers real opportunities for addressing issues, such as nutrient reduction, CO2 removal, and biogas enrichment.

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune-privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin-fixed, paraffin-embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base-pair resolution in a rare, aggressive lymphoma. Our data suggest immune-checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements.

An integrated approach that combined freshwater microalgae Scenedesmus obliquus (FACHB-31) cultivation with piggery anaerobic digestate liquid treatment was investigated in this study. The characteristics of algal growth, biogas production, and nutrient removal were examined using photobioreactor bags (PBRbs) to cultivate S. obliquus (FACHB-31) in digestate with various digestate dilutions (the concentration levels of 3200, 2200, 1600, 1200, 800, and 400 mg L(-1) chemical oxygen demand (COD)) during 7-day period. The effects of the level of pollutants on nutrient removal efficiency and CO2 removal process were investigated to select the optimum system for effectively upgrade biogas and simultaneously reduce the nutrient content in digestate. The treatment performance displayed that average removal rates of COD, total nitrogen (TN), total phosphorous (TP), and CO2 were 61.58-75.29, 58.39-74.63, 70.09-88.79, and 54.26-73.81 %, respectively. All the strains grew well under any the dilution treatments. With increased initial nutrient concentration to a certain range, the CO4 content (v/v) of raw biogas increased. Differences in the biogas enrichment of S. obliquus (FACHB-31) in all treatments mainly resulted from variations in biomass productivity and CO2 uptake. Notably, the diluted digestate sample of 1600 mg L(-1) COD provided an optimal nutrient concentration for S. obliquus (FACHB-31) cultivation, where the advantageous nutrient and CO2 removals, as well as the highest productivities of biomass and biogas upgrading, were revealed. Results showed that microalgal biomass production offered real opportunities to address issues such as CO2 sequestration, wastewater treatment, and biogas production.

Two floating treatment wetlands (FTWs) in experimental tanks were compared in terms of their effectiveness on removing nutrients. The results showed that the FTWs were dominated by emergent wetland plants and were constructed to remove nutrients from simulated urban stormwater. Iris pseudacorus and Thalia dealbata wetland systems were effective in reducing the nutrient. T. dealbata FTWs showed higher nutrient removal performance than I. pseudacorus FTWs. Nitrogen (N) and phosphorous (P) removal rates in water by T. dealbata FTWs were 3.95 ± 0.19 and 0.15 ± 0.01 g/m2/day, respectively. For I. pseudacorus FTWs, the TN and TP removal rates were 3.07 ± 0.15 and 0.14 ± 0.01 g/m2/day, respectively. The maximum absolute growth rate for T. dealbata corresponded directly with the maximum mean nutrient removal efficiency during the 5th stage. At harvest, N and P uptak of T. dealbata was 23.354 ± 1.366 g and 1.489 ± 0.077 g per plant, respectively, approximate twice as high as by I. pseudacorus.

The wide application and unique properties of graphene oxide (GO) make it to interact with other pollutants and subsequently alter their behavior and toxicity. We evaluated the influences of GO at different concentrations (1 and 5mg/L) on copper (Cu) stress in duckweed (Lemna minor L.) GO below a concentration of 5mg/L showed no adverse effects on L. minor. The addition of Cu above 10μM represented a stress condition, which was evidenced by various parameters such as frond number, percent inhibition of growth rate (Ir), total chlorophyll content, dry weight, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). When L. minor was simultaneously exposed to GO and Cu, especially at a GO concentration of 5mg/L and a Cu level above 10μM, the increase of Ir and decrease of chlorophyll content were inhibited, suggesting that the Cu stress was diminished in the presence of GO. The addition of Cu alone, ranging between 5 and 20μM, increased Cu, B, Mn, Fe, Co, and Zn uptake, but decreased P uptake. Our results suggest that GO can lessen Cu stress in L. minor via Cu adsorption, thereby protecting the plants from the damaging effects of high Cu concentrations.

We describe a woman with the known pathogenic germline variant CHEK2:c.1100delC and synchronous diagnoses of both pelvic genital type leiomyosarcoma (LMS) and metastatic invasive ductal breast carcinoma. CHEK2 (checkpoint kinase 2) is a tumor-suppressor gene encoding a serine/threonine-protein kinase (CHEK2) involved in double-strand DNA break repair and cell cycle arrest. The CHEK2:c.1100delC variant is a moderate penetrance allele resulting in an approximately twofold increase in breast cancer risk. Whole-genome and whole-transcriptome sequencing were performed on the leiomyosarcoma and matched blood-derived DNA. Despite the presence of several genomic hits within the double-strand DNA damage pathway (CHEK2 germline variant and multiple RAD51B somatic structural variants), tumor profiling did not show an obvious DNA repair deficiency signature. However, even though the LMS displayed clear malignant features, its genomic profiling revealed several characteristics classically associated with leiomyomas including a translocation, t(12;14), with one breakpoint disrupting RAD51B and the other breakpoint upstream of HMGA2 with very high expression of HMGA2 and PLAG1. This is the first report of LMS genomic profiling in a patient with the germline CHEK2:c.1100delC variant and an additional diagnosis of metastatic invasive ductal breast carcinoma. We also describe a possible mechanistic relationship between leiomyoma and LMS based on genomic and transcriptome data. Our findings suggest that RAD51B translocation and HMGA2 overexpression may play an important role in LMS oncogenesis.

Summary: Cell fate acquisition is heavily influenced by direct interactions between master regulators and tissue-specific enhancers. However, it remains unclear how lineage-specifying transcription factors, which are often expressed in both progenitor and mature cell populations, influence cell differentiation. Using in vivo mouse liver development as a model, we identified thousands of enhancers that are bound by the master regulators HNF4A and FOXA2 in a differentiation-dependent manner, subject to chromatin remodeling, and associated with differentially expressed target genes. Enhancers exclusively occupied in the embryo were found to be responsive to developmentally regulated TEAD2 and coactivator YAP1. Our data suggest that Hippo signaling may affect hepatocyte differentiation by influencing HNF4A and FOXA2 interactions with temporal enhancers. In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent, which is an important consideration for researchers studying cancer biology or mammalian development and/or using transformed cell lines. : It is unclear how key transcription factors are critical for both lineage specification during embryonic development and maintenance of a differentiated, adult phenotype. By profiling the enhancer occupancy of the key transcription factors HNF4A and FOXA2 during mouse liver development, Alder et al. have found that YAP1 can influence enhancer interactions and target gene expression levels. Enhancer switching enables HNF4A and FOXA2 to fulfill distinct roles during organ development.

SummaryCellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.

Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1-10 ng of intact total RNA and 100-500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues.

The grizzly bear (Ursus arctos ssp. horribilis) represents the largest population of brown bears in North America. Its genome was sequenced using a microfluidic partitioning library construction technique, and these data were supplemented with sequencing from a nanopore-based long read platform. The final assembly was 2.33 Gb with a scaffold N50 of 36.7 Mb, and the genome is of comparable size to that of its close relative the polar bear (2.30 Gb). An analysis using 4104 highly conserved mammalian genes indicated that 96.1% were found to be complete within the assembly. An automated annotation of the genome identified 19,848 protein coding genes. Our study shows that the combination of the two sequencing modalities that we used is sufficient for the construction of highly contiguous reference quality mammalian genomes. The assembled genome sequence and the supporting raw sequence reads are available from the NCBI (National Center for Biotechnology Information) under the bioproject identifier PRJNA493656, and the assembly described in this paper is version QXTK01000000.

Tissues used in pathology laboratories are typically stored in the form of formalin-fixed, paraffin-embedded (FFPE) samples. One important consideration in repurposing FFPE material for next generation sequencing (NGS) analysis is the sequencing artifacts that can arise from the significant damage to nucleic acids due to treatment with formalin, storage at room temperature and extraction. One such class of artifacts consists of chimeric reads that appear to be derived from non-contiguous portions of the genome. Here, we show that a major proportion of such chimeric reads align to both the ‘Watson’ and ‘Crick’ strands of the reference genome. We refer to these as strand-split artifact reads (SSARs). This study provides a conceptual framework for the mechanistic basis of the genesis of SSARs and other chimeric artifacts along with supporting experimental evidence, which have led to approaches to reduce the levels of such artifacts. We demonstrate that one of these approaches, involving S1 nuclease-mediated removal of single-stranded fragments and overhangs, also reduces sequence bias, base error rates, and false positive detection of copy number and single nucleotide variants. Finally, we describe an analytical approach for quantifying SSARs from NGS data.

In this study, we sought to identify influent carbon-to-nitrogen (C/N) ratios that yield relatively high nutrient removal efficiency with relatively low greenhouse gas (GHG) emissions. The earthworm eco-filter (EE) system, which is composed of earthworms and plants (EP group), was found to be optimal for maximizing nutrient removal while reducing GHG emissions. In this EE system, the optimal influent C/N ratio for nutrient removal and GHG emission under C2N treatment conditions. Nutrient removal efficiency under this condition was 85.19 ± 6.40% chemical oxygen demand, 71.99 ± 11.28% total nitrogen, and 77.91 ± 8.51% total phosphorus, while the CO2 emission rate was 678.89 ± 201.87 mg m(-2) h(-1). Moreover, the highest nutrient removal and GHG emission rates were both achieved in late summer (August). Thus, carbon variation, season, system variation, as well as synergistic interaction between system variations and seasons, significantly affect nutrient removal efficiencies and GHG emissions.

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment.