Prediction of a competing endogenous RNA co‐expression network as a prognostic marker in glioblastoma

Due to its high proliferation capacity and rapid intracranial spread, glioblastoma (GBM) has become one of the least curable malignant cancers. Recently, the competing endogenous RNAs (ceRNAs) hypothesis has become a focus in the researches of molecular biological mechanisms of cancer occurrence and progression. However, there is a lack of correlation studies on GBM, as well as a lack of comprehensive analyses of GBM molecular mechanisms based on high‐throughput sequencing and large‐scale sample sizes. We obtained RNA‐seq data from The Cancer Genome Atlas (TCGA) and Genotype‐Tissue Expression (GTEx) databases. Further, differentially expressed mRNAs were identified from normal brain tissue and GBM tissue. The similarities between the mRNA modules with clinical traits were subjected to weighted correlation network analysis (WGCNA). With the mRNAs from clinical‐related modules, a survival model was constructed by univariate and multivariate Cox proportional hazard regression analyses. Thereafter, we carried out Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, we predicted interactions between lncRNAs, miRNAs and mRNAs by TargetScan, miRDB, miRTarBase and starBase. We identified 2 lncRNAs (NORAD, XIST), 5 miRNAs (hsa‐miR‐3613, hsa‐miR‐371, hsa‐miR‐373, hsa‐miR‐32, hsa‐miR‐92) and 2 mRNAs (LYZ, PIK3AP1) for the construction of a ceRNA network, which might act as a prognostic biomarker of GBM. Combined with previous studies and our enrichment analysis results, we hypothesized that this ceRNA network affects immune activities and tumour microenvironment variations. Our research provides novel aspects to study GBM development and treatment.     

Scaffold compound L971 exhibits anti‐inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways

JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune‐related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high‐throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli‐dependent activation of STAT1, STAT3 and IκBα and could significantly down‐regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high‐throughput RNA sequencing, and significant differentially up‐regulated and down‐regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti‐inflammatory effects of L971. Finally, L971 anti‐inflammatory character was further verified in LPS‐induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down‐regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.     

Acetate: Friend or foe against breast tumour growth in the context of obesity?

Acetate is reported as a regulator of fat mass but also as lipogenic source for cancer cells. Breast cancer is surrounded by adipose tissue and has been associated with obesity. However, whether acetate contributes to cancer cell metabolism as lipogenic substrate and/or by changing fat storage and eventually obesity‐induced breast cancer progression remains unknown. Therefore, we studied the contribution of acetate to breast cancer metabolism and progression. In vitro, we found that acetate is not a bioenergetic substrate under normoxia and did not result in a significant change of growth. However, by using lipidomic approaches, we discovered that acetate changes the lipid profiles of the cells under hypoxia. Moreover, while mice fed a high‐fat diet (HFD) developed bigger tumours than their lean counterparts, exogenous acetate supplementation leads to a complete abolishment of fat mass gain without reverting the HFD‐induced obesity‐driven tumour progression. In conclusion, although acetate protects against diet‐induced obesity, our data suggest that it is not affecting HFD‐driven tumour progression.     

A novel DNA damage and repair‐related gene signature to improve predictive capacity of overall survival for patients with gliomas

Gliomas, as the most lethal and malignant brain tumours in adults, remain a major challenge worldwide. DNA damage and repair‐related genes (DDRRGs) appear to play a significant role in gliomas, but the studies of DDRRGs are still insufficient. Herein, we systematically explored and analysed 1547 DDRRGs in 938 glioma samples from TCGA and CGGA datasets. Using least absolute shrinkage and selection operator (LASSO) Cox regression analysis, we identified a 16‐DDRRG signature, characterized by high‐risk and low‐risk patterns. This risk model harbours robust predictive capability for overall survival of glioma patients. We found the high‐risk score is strongly associated with well‐known malignant features of gliomas, such as the mesenchymal subtype, IDH‐wildtype, 1p/19q non‐codeletion and MGMT promoter unmethylated status. In addition, we found that the high‐risk score is also linked with multiple oncogenic pathways and therapeutic resistance. Significantly, we found the high‐risk group has higher enrichment of immunosuppressive cells (M2‐type macrophages, Tregs and MDSCs) and immune inhibition biomarkers (PD‐1, PD‐L1 and CTLA‐4). Lastly, we proved that SMC4, which has the highest positive regression coefficient in our risk model, is strongly linked with malignant progression and TMZ resistance of gliomas in a E2F1‐dependent manner.     

A genotyping‐in‐thousands by sequencing panel to inform invasive deer management using noninvasive fecal and hair samples

Studies in ecology, evolution, and conservation often rely on noninvasive samples, making it challenging to generate large amounts of high‐quality genetic data for many elusive and at‐risk species. We developed and optimized a Genotyping‐in‐Thousands by sequencing (GT‐seq) panel using noninvasive samples to inform the management of invasive Sitka black‐tailed deer (Odocoileus hemionus sitkensis) in Haida Gwaii (Canada). We validated our panel using paired high‐quality tissue and noninvasive fecal and hair samples to simultaneously distinguish individuals, identify sex, and reconstruct kinship among deer sampled across the archipelago, then provided a proof‐of‐concept application using field‐collected feces on SGang Gwaay, an island of high ecological and cultural value. Genotyping success across 244 loci was high (90.3%) and comparable to that of high‐quality tissue samples genotyped using restriction‐site associated DNA sequencing (92.4%), while genotyping discordance between paired high‐quality tissue and noninvasive samples was low (0.50%). The panel will be used to inform future invasive species operations in Haida Gwaii by providing individual and population information to inform management. More broadly, our GT‐seq workflow that includes quality control analyses for targeted SNP selection and a modified protocol may be of wider utility for other studies and systems where noninvasive genetic sampling is employed.     

Dopamine and glutamate in individuals at high risk for psychosis: a meta‐analysis of in vivo imaging findings and their variability compared to controls

Dopaminergic and glutamatergic dysfunction is believed to play a central role in the pathophysiology of schizophrenia. However, it is unclear if abnormalities predate the onset of schizophrenia in individuals at high clinical or genetic risk for the disorder. We systematically reviewed and meta‐analyzed studies that have used neuroimaging to investigate dopamine and glutamate function in individuals at increased clinical or genetic risk for psychosis. EMBASE, PsycINFO and Medline were searched form January 1, 1960 to November 26, 2020. Inclusion criteria were molecular imaging measures of striatal presynaptic dopaminergic function, striatal dopamine receptor availability, or glutamate function. Separate meta‐analyses were conducted for genetic high‐risk and clinical high‐risk individuals. We calculated standardized mean differences between high‐risk individuals and controls, and investigated whether the variability of these measures differed between the two groups. Forty‐eight eligible studies were identified, including 1,288 high‐risk individuals and 1,187 controls. Genetic high‐risk individuals showed evidence of increased thalamic glutamate + glutamine (Glx) concentrations (Hedges’ g=0.36, 95% CI: 0.12‐0.61, p=0.003). There were no significant differences between high‐risk individuals and controls in striatal presynaptic dopaminergic function, striatal D2/D3 receptor availability, prefrontal cortex glutamate or Glx, hippocampal glutamate or Glx, or basal ganglia Glx. In the meta‐analysis of variability, genetic high‐risk individuals showed reduced variability of striatal D2/D3 receptor availability compared to controls (log coefficient of variation ratio, CVR=–0.24, 95% CI: –0.46 to –0.02, p=0.03). Meta‐regressions of publication year against effect size demonstrated that the magnitude of differences between clinical high‐risk individuals and controls in presynaptic dopaminergic function has decreased over time (estimate=–0.06, 95% CI: –0.11 to –0.007, p=0.025). Thus, other than thalamic glutamate concentrations, no neurochemical measures were significantly different between individuals at risk for psychosis and controls. There was also no evidence of increased variability of dopamine or glutamate measures in high‐risk individuals compared to controls. Significant heterogeneity, however, exists between studies, which does not allow to rule out the existence of clinically meaningful differences.     

M2‐phenotype tumour‐associated macrophages upregulate the expression of prognostic predictors MMP14 and INHBA in pancreatic cancer

Pancreatic cancer is one of the most lethal gastrointestinal tumours, the most common pathological type is pancreatic adenocarcinoma (PAAD). In recent year, immune imbalanced in tumour microenvironment has been shown to play an important role in the evolution of tumours progression, and the efficacy of immunotherapy has been gradually demonstrated in clinical practice. In this study, we propose to construct an immune‐related prognostic risk model based on immune‐related genes MMP14 and INHBA expression that can assess the prognosis of pancreatic cancer patients and identify potential therapeutic targets for pancreatic cancer, to provide new ideas for the treatment of pancreatic cancer. We also investigate the correlation between macrophage infiltration and MMP14 and INHBA expression. First, the gene expression data of pancreatic cancer and normal pancreatic tissue were obtained from The Cancer Genome Atlas Program (TCGA) and The Genotype‐Tissue Expression public database (GTEx). The differentially expressed immune‐related genes between pancreatic cancer samples and normal sample were screened by R software. Secondly, univariate Cox regression analysis were used to evaluate the relationship between immune‐related genes and the prognosis of pancreatic cancer patients. A polygenic risk score model was constructed by Cox regression analysis. The prognostic nomogram was constructed, and its performance was evaluated comprehensively by internal calibration curve and C‐index. Using the risk model, each patient gets a risk score, and was divided into high‐ or low‐ risk groups. The proportion of 22 types of immune cells infiltration in pancreatic cancer samples was inferred by CIBERSOFT algorithm, correlation analysis (Pearson method) was used to analyse the correlation between the immune‐related genes and immunes cells. Then, we applied macrophage conditioned medium to culture pancreatic cancer cell line PANC1, detected the expression of MMP14 and INHBA by qRT‐PCR and Western blot methods. Knock‐down MMP14 and INHBA in PANC1 cells by transfected with shRNA lentiviruses. Detection of migration ability of pancreatic cells was done by trans‐well cell migration assay. A subcutaneous xenograft tumour model of human pancreatic cancer in nude mice was constructed. In conclusion, an immune‐related gene prognostic model was constructed, patients with high‐risk scores have poorer survival status, M2‐phenotype tumour‐associated macrophages (TAMs) up‐regulate two immune‐related genes, MMP14 and INHBA, which were used to establish the prognostic model. Knock‐down of MMP14 and INHBA inhibited invasion of pancreatic cancer.     

NGSpeciesID: DNA barcode and amplicon consensus generation from long‐read sequencing data

Third‐generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long‐read sequences. This is not only advantageous for genome sequencing projects, but also advantageous for amplicon‐based high‐throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high‐quality consensus sequences. Here, we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long‐read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines.     

Ancestral ecological regime shapes reaction to food limitation in the Least Killifish,Heterandria formosa

Populations with different densities often show genetically based differences in life histories. The divergent life histories could be driven by several agents of selection, one of which is variation in per‐capita food levels. Its relationship with population density is complex, as it depends on overall food availability, individual metabolic demand, and food‐independent factors potentially affecting density, such as predation intensity. Here, we present a case study of two populations of a small live‐bearing freshwater fish, one characterized by high density, low predation risk, low overall food availability, and presumably low per‐capita food levels, and the other by low density, high predation risk, high overall food availability, and presumably high per‐capita food levels. Using a laboratory experiment, we examined whether fish from these populations respond differently to food limitation, and whether size at birth, a key trait with respect to density variation in this species, is associated with any such differential responses. While at the lower food level growth was slower, body size smaller, maturation delayed, and survival reduced in both populations, these fitness costs were smaller in fish from the high‐density population. At low food, only 15% of high‐density fish died, compared to 75% of low‐density fish. This difference was much smaller at high food (0% vs. 15% mortality). The increased survival of high‐density fish may, at least partly, be due to their larger size at birth. Moreover, being larger at birth enabled fish to mature relatively early even at the lower food level. We demonstrate that sensitivities to food limitation differ between study populations, consistent with selection for a greater ability to tolerate low per‐capita food availability in the high‐density population. While we cannot preclude other agents of selection from operating in these populations simultaneously, our results suggest that variation in per‐capita food levels is one of those agents.     

Amplicon_sorter: A tool for reference‐free amplicon sorting based on sequence similarity and for building consensus sequences

Oxford Nanopore Technologies (ONT) is a third‐generation sequencing technology that is gaining popularity in ecological research for its portable and low‐cost sequencing possibilities. Although the technology excels at long‐read sequencing, it can also be applied to sequence amplicons. The downside of ONT is the low quality of the raw reads. Hence, generating a high‐quality consensus sequence is still a challenge. We present Amplicon_sorter, a tool for reference‐free sorting of ONT sequenced amplicons based on their similarity in sequence and length and for building solid consensus sequences.     

Dissecting molecular details and functional effects of the high‐affinity copper binding site in plasminogen activator Inhibitor‐1

Plasminogen activator inhibitor‐1 (PAI‐1) is the primary inhibitor for plasminogen activators, tissue‐type plasminogen activator (tPA) and urokinase‐type plasminogen activator (uPA). As a unique member in the serine protease inhibitor (serpin) family, PAI‐1 is metastable and converts to an inactive, latent structure with a half‐life of 1–2 hr under physiological conditions. Unusual effects of metals on the rate of the latency conversion are incompletely understood. Previous work has identified two residues near the N‐terminus, H2 and H3, which reside in a high‐affinity copper‐binding site in PAI‐1 [Bucci JC, McClintock CS, Chu Y, Ware GL, McConnell KD, Emerson JP, Peterson CB (2017) J Biol Inorg Chem 22:1123–1,135]. In this study, neighboring residues, H10, E81, and H364, were tested as possible sites that participate in Cu(II) coordination at the high‐affinity site. Kinetic methods, gel sensitivity assays, and isothermal titration calorimetry (ITC) revealed that E81 and H364 have different roles in coordinating metal and mediating the stability of PAI‐1. H364 provides a third histidine in the metal‐coordination sphere with H2 and H3. In contrast, E81 does not appear to be required for metal ligation along with histidines; contacts made by the side‐chain carboxylate upon metal binding are perturbed and, in turn, influence dynamic fluctuations within the region encompassing helices D, E, and F and the W86 loop that are important in the pathway for the PAI‐1 latency conversion. This investigation underscores a prominent role of protein dynamics, noncovalent bonding networks and ligand binding in controlling the stability of the active form of PAI‐1.     

Exosomal hsa_circ_0125310 promotes cell proliferation and fibrosis in diabetic nephropathy via sponging miR‐422a and targeting the IGF1R/p38 axis

Diabetic nephropathy (DN) is still on the rise worldwide, and millions of patients have to be treated through dialysis or transplant because of kidney failure caused by DN. Recent reports have highlighted circRNAs in the treatment of DN. Herein, we aimed to investigate the mechanism by which high glucose‐induced exo‐circ_0125310 promotes diabetic nephropathy progression. circ_0125310 is highly expressed in diabetic nephropathy and exosomes isolated from high glucose‐induced mesangial cells (MCs). High glucose‐induced exosomes promote the proliferation and fibrosis of MCs. However, results showed that the effects of exosomes on MCs can be reversed by the knockdown of circ_0125310. miR‐422a, which targets IGF1R, was the direct target of circ_0125310. circ_0125310 regulated IGF1R/p38 axis by sponging miR‐422a. Exo‐circ_0125310 increased the luciferase activity of the WT‐IGF1R reporter in the dual‐luciferase reporter gene assays and upregulated the expression level of IGF1R and p38. Finally, in vivo research indicated that the overexpression of circ_0125310 promoted the diabetic nephropathy progression. Above results demonstrated that the high glucose‐induced exo‐circ_0125310 promoted cell proliferation and fibrosis in diabetic nephropathy via sponging miR‐422a and targeting the IGF1R/p38 axis.     

Identification of novel bacterial biomarkers to detect bird scavenging by invasive rats

Rapid advances in genomic tools for use in ecological contexts and non‐model systems allow unprecedented insight into interactions that occur beyond direct observation. We developed an approach that couples microbial forensics with molecular dietary analysis to identify species interactions and scavenging by invasive rats on native and introduced birds in Hawaii. First, we characterized bacterial signatures of bird carcass decay by conducting 16S rRNA high‐throughput sequencing on chicken (Gallus gallus domesticus) tissues collected over an 11‐day decomposition study in natural Hawaiian habitats. Second, we determined if field‐collected invasive black rats (Rattus rattus; n = 51, stomach and fecal samples) had consumed birds using molecular diet analysis with two independent PCR assays (mitochondrial Cytochrome Oxidase I and Cytochrome b genes) and Sanger sequencing. Third, we characterized the gut microbiome of the same rats using 16S rRNA high‐throughput sequencing and identified 15 bacterial taxa that were (a) detected only in rats that consumed birds (n = 20/51) and (b) were indicative of decaying tissue in the chicken decomposition experiment. We found that 18% of rats (n = 9/51) likely consumed birds as carrion by the presence of bacterial biomarkers of decayed tissue in their gut microbiome. One species of native bird (Myadestes obscurus) and three introduced bird species (Lophura leucomelanos, Meleagris gallopavo, Zosterops japonicus) were detected in the rats’ diets, with individuals from these species (except L. nycthemera) likely consumed through scavenging. Bacterial biomarkers of bird carcass decay can persist through rat digestion and may serve as biomarkers of scavenging. Our approach can be used to reveal trophic interactions that are challenging to measure through direct observation.     

Novel DNA sequences at chromosome 10q26 are amplified in human gastric carcinoma cell lines: molecular cloning by competitive DNA reassociation.

Molecular cloning of genomic sequences altered in cancer cells is believed to lead to the identification of new genes involved in the initiation and progression of the malignant phenotype. DNA amplification is a frequent molecular alteration in tumor cells, and is a mode of proto-oncogene activation. The cytologic manifestation of this phenomenon is the appearance of chromosomal homogeneously staining regions (HSRs) or double minute bodies (DMs). The gastric carcinoma cell line KATO III is characterized by a large HSR on chromosome 11. In-gel renaturation analysis confirmed the amplification of DNA sequences in this cell line, yet none of 42 proto-oncogenes that we tested is amplified in KATO III DNA. We employed the phenol-enhanced reassociation technique (PERT) to isolate 21 random DNA fragments from the amplified domain, and used 6 of them to further clone some 150 kb from that genomic region. While in situ hybridization performed with some of these sequences indicated that in KATO III they are indeed amplified within the HSR on chromosome 11, somatic cell hybrid analysis and in situ hybridization to normal lymphocyte chromosomes showed that they are derived from chromosome 10, band q26. The same sequences were found to be amplified in another gastric carcinoma cell line, SNU-16, which contains DMs, but were not amplified in other 70 cell lines representing a wide variety of human neoplasms. One of these sequences was highly expressed in both KATO III and SNU-16. Thus, the cloned sequences supply a starting point for identification of novel genes which might be involved in the pathogenesis of gastric cancers, and are located in a relatively unexplored domain of the human genome.     

Circulating miR‐19a‐3p and miR‐19b‐3p characterize the human aging process and their isomiRs associate with healthy status at extreme ages

Blood circulating microRNAs (c‐miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti‐aging therapies. Based on a cross‐sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c‐miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR‐19a‐3p and miR‐19b‐3p, were found increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT‐qPCR, but only with a high‐resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR‐19a/b‐3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age‐related increase of plasma miR‐19a/b‐3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c‐miRs and isomiRs as additional parameter to track the onset of aging and age‐related diseases using new potential biomarkers.     

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization.     

Genotyping‐by‐sequencing analysis of Orobanche crenata populations in Algeria reveals genetic differentiation

Crenate broomrape (Orobanche crenata Forsk.) is a serious long‐standing parasitic weed problem in Algeria, mainly affecting legumes but also vegetable crops. Unresolved questions for parasitic weeds revolve around the extent to which these plants undergo local adaptation, especially with respect to host specialization, which would be expected to be a strong selective factor for obligate parasitic plants. In the present study, the genotyping‐by‐sequencing (GBS) approach was used to analyze genetic diversity and population structure of 10 Northern Algerian O. crenata populations with different geographical origins and host species (faba bean, pea, chickpea, carrot, and tomato). In total, 8004 high‐quality single‐nucleotide polymorphisms (5% missingness) were obtained and used across the study. Genetic diversity and relationships of 95 individuals from 10 populations were studied using model‐based ancestry analysis, principal components analysis, discriminant analysis of principal components, and phylogeny approaches. The genetic differentiation (F _ST) between pairs of populations was lower between adjacent populations and higher between geographically separated ones, but no support was found for isolation by distance. Further analyses identified four genetic clusters and revealed evidence of structuring among populations and, although confounded with location, among hosts. In the clearest example, O. crenata growing on pea had a SNP profile that was distinct from other host/location combinations. These results illustrate the importance and potential of GBS to reveal the dynamics of parasitic weed dispersal and population structure.