Rational design of novel allosteric EYA2 inhibitors as potential therapeutics for multiple brain cancers.

The Eyes Absent (EYA) family of developmental proteins, often in partnership with the sine oculis (SIX) homeobox proteins, promote cancer metastasis and recurrence in numerous tumor types. In addition to being a transcriptional coactivator, EYA2 is a Tyr phosphatase that dephosphorylates H2AX which leads to repair instead of apoptosis upon DNA damage and ERß which inhibits the anti-tumor transcriptional activity of ERß. The SIX members of the EYA-SIX complex are difficult to target, therefore, we targeted the EYA2 to promote cell death and prevent cancer progression. We conducted structural optimization of a previously discovered allosteric inhibitor of EYA2, 9987, using the combination of in silico modeling, biochemical and cell-based assays. A new series of compounds was developed with significantly improved cellular activity and physiochemical properties desirable for brain targets. Specifically, compound 2e showed >30 fold improvement in the medulloblastoma cell line D458, relative to 9987, while maintaining potent and selective inhibitory activity against EYA2 Tyr phosphatase activity and a good multiparameter optimization score for central nervous system drugs.

Biochemical characterization of the Eya and PP2A-B55α interaction.

The eyes absent (Eya) proteins were first identified as co-activators of the six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here, we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3's apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, MycP59A, a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α-mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability.

Saliva antibody profiles are associated with reaction threshold and severity of peanut allergic reactions.

BACKGROUND: Reaction threshold and severity in food allergy are difficult to predict, and noninvasive predictors are lacking. OBJECTIVE: We sought to determine the relationships between pre-challenge levels of peanut (PN)-specific antibodies in saliva and reaction threshold, severity, and organ-specific symptoms during PN allergic reactions. METHODS: We measured PN-specific antibody levels in saliva collected from 127 children with suspected PN allergy before double-blind, placebo-controlled PN challenges in which reaction threshold, severity, and symptoms were rigorously characterized. Low threshold (LT) PN allergy was defined as reaction to <300 mg of PN protein cumulatively consumed. A consensus severity grading system was used to grade severity. We analyzed associations between antibody levels and reaction threshold, severity, and organ-specific symptoms. RESULTS: Among the 127 children, those with high pre-challenge saliva PN IgE had higher odds of LT PN allergy (odds ratio [OR] 3.9, 95% CI 1.6-9.5), while those with high saliva PN IgA:PN IgE ratio or PN IgG4:PN IgE ratio had lower odds of LT PN allergy (OR 0.3, 95% CI 0.1-0.8; OR 0.4, 95% CI 0.2-0.9). Children with high pre-challenge saliva PN IgG4 had lower odds of severe PN reactions (OR 0.4, 95% CI 0.2-0.9). Children with high saliva PN IgE had higher odds of respiratory symptoms (OR 8.0, 95% CI 2.2-26.8). Saliva PN IgE modestly correlated with serum PN IgE levels (Pearson r = 0.31, P = .0004). High and low saliva PN IgE levels further distinguished reaction threshold and severity in participants stratified by serum PN IgE, suggesting endotypes. CONCLUSIONS: Saliva PN antibodies could aid in noninvasive risk stratification of PN allergy threshold, severity, and organ-specific symptoms.

Multi-omic integration reveals alterations in nasal mucosal biology that mediate air pollutant effects on allergic rhinitis.

BACKGROUND: Allergic rhinitis is a common inflammatory condition of the nasal mucosa that imposes a considerable health burden. Air pollution has been observed to increase the risk of developing allergic rhinitis. We addressed the hypotheses that early life exposure to air toxics is associated with developing allergic rhinitis, and that these effects are mediated by DNA methylation and gene expression in the nasal mucosa. METHODS: In a case-control cohort of 505 participants, we geocoded participants' early life exposure to air toxics using data from the US Environmental Protection Agency, assessed physician diagnosis of allergic rhinitis by questionnaire, and collected nasal brushings for whole-genome DNA methylation and transcriptome profiling. We then performed a series of analyses including differential expression, Mendelian randomization, and causal mediation analyses to characterize relationships between early life air toxics, nasal DNA methylation, nasal gene expression, and allergic rhinitis. RESULTS: Among the 505 participants, 275 had allergic rhinitis. The mean age of the participants was 16.4 years (standard deviation = 9.5 years). Early life exposure to air toxics such as acrylic acid, phosphine, antimony compounds, and benzyl chloride was associated with developing allergic rhinitis. These air toxics exerted their effects by altering the nasal DNA methylation and nasal gene expression levels of genes involved in respiratory ciliary function, mast cell activation, pro-inflammatory TGF-ß1 signaling, and the regulation of myeloid immune cell function. CONCLUSIONS: Our results expand the range of air pollutants implicated in allergic rhinitis and shed light on their underlying biological mechanisms in nasal mucosa.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yLuc7 protein (to mimic the lack of Luc7Ls in human U1 snRNP) and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

Joint transcriptomic and cytometric study of children with peanut allergy reveals molecular and cellular cross talk in reaction thresholds.

BACKGROUND: Reaction thresholds in peanut allergy are highly variable. Elucidating causal relationships between molecular and cellular processes associated with variable thresholds could point to therapeutic pathways for raising thresholds. OBJECTIVE: The aim of this study was to characterize molecular and cellular systemic processes associated with reaction threshold in peanut allergy and causal relationships between them. METHODS: A total of 105 children aged 4 to 14 years with suspected peanut allergy underwent double-blind, placebo-controlled food challenge to peanut. The cumulative peanut protein quantity eliciting allergic symptoms was considered the reaction threshold for each child. Peripheral blood samples collected at 0, 2, and 4 hours after challenge start were used for RNA sequencing, whole blood staining, and cytometry. Statistical and network analyses were performed to identify associations and causal mediation between the molecular and cellular profiles and peanut reaction threshold. RESULTS: Within the cohort (N = 105), 81 children (77%) experienced allergic reactions after ingesting varying quantities of peanut, ranging from 43 to 9043 mg of cumulative peanut protein. Peripheral blood expression of transcripts (eg, IGF1R [false discovery rate (FDR) = 5.4e-5] and PADI4 [FDR = 5.4e-5]) and neutrophil abundance (FDR = 9.5e-4) were associated with peanut threshold. Coexpression network analyses revealed that the threshold-associated transcripts were enriched in modules for FcγR-mediated phagocytosis (FDR = 3.2e-3) and Toll-like receptor (FDR = 1.4e-3) signaling. Bayesian network, key driver, and causal mediation analyses identified key drivers (AP5B1, KLHL21, VASP, TPD52L2, and IGF2R) within these modules that are involved in bidirectional causal mediation relationships with neutrophil abundance. CONCLUSION: Key driver transcripts in FcγR-mediated phagocytosis and Toll-like receptor signaling interact bidirectionally with neutrophils in peripheral blood and are associated with reaction threshold in peanut allergy.

Butyrate ameliorated ferroptosis in ulcerative colitis through modulating Nrf2/GPX4 signal pathway and improving intestinal barrier.

Oxidative stress and intestinal inflammation are main pathological features of ulcerative colitis (UC). Ferroptosis, characterized by iron accumulation and lipid peroxidation, is closely related to the pathologic process of UC. 16S rRNA sequencing for intestinal microbiota analysis and gas chromatography-mass spectrometry (GC-MS) for short-chain fatty acid (SCFA) contents clearly demonstrated lower amounts of butyrate-producing bacteria and butyrate in colitis mice. However, the precise mechanisms of sodium butyrate (NaB) in treating UC remain largely unclear. We found that ferroptosis occurred in colitis models, as evidenced by the inflammatory response, intracellular iron level, mitochondria ultrastructural observations and associated protein expression. NaB inhibited ferroptosis in colitis, significantly rescued weight loss and colon shortening in mice and reduced inflammatory lesions and mitochondrial damage. Furthermore, NaB improved intestinal barrier integrity and markedly suppressed the expression of pro-ferroptosis proteins. Conversely, the protein expression of anti-ferroptosis markers including nuclear factor erythroid-related Factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4), was significantly upregulated with NaB treatment. Moreover, the knockdown of Nrf2 reversed the anti-colitis effect of NaB. Taken together, NaB exhibited a protective effect by ameliorating ferroptosis in experimental colitis through Nrf2/GPX4 signaling and improving intestinal barrier integrity, which provides a novel mechanism for NaB prevention of UC.

Sclareol ameliorates liver injury by inhibiting nuclear factor-kappa B/NOD-like receptor protein 3-mediated inflammation and lipid metabolism disorder in diabetic mice.

Objectives: Sclareol (SCL) is a natural diterpene with anti-inflammation and antioxidant properties. This study aimed to assess the hepatoprotective effects of SCL in diabetic mice. Methods: SCL (10 mg/kg) was administered intragastrically to C57BL/6 mice with streptozotocin-induced diabetes daily for 5 weeks to evaluate its beneficial effects in liver injury. Body and liver weight and blood glucose levels were measured. Liver histopathology, fibrosis, and lipid accumulation were evaluated using hematoxylin and eosin, Masson's trichrome, and Oil Red O staining, respectively. Serum hepatic enzyme and lipid levels were measured using an automatic biochemical analyzer. Hepatocellular apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Oxidative stress markers and reactive oxygen species (ROS) were measured using appropriate assay kits. The effects of sclareol on inflammation and lipid metabolism was evaluated by enzyme-linked immunosorbent assay (ELISA), immunohistochemical analysis, and Western blot assays. Results: SCL significantly decreased serum liver enzymes and lipids levels, and alleviated adipogenesis and fibrosis. Moreover, the protein levels of acetyl-CoA carboxylase and sterol response element-binding protein 1 were downregulated, whereas the expression of carnitine palmitoyl transferase 1 was upregulated. SCL increased the antioxidant activity, and decreased ROS levels. SCL alleviated hepatic mitochondrial damage. Furthermore, SCL inhibited Kupffer cell infiltration and reduced serum inflammatory cytokine levels. SCL significantly downregulated the protein expression of nuclear factor-kappa B (NF-κB) P65, NOD-like receptor protein 3 (NLRP3), caspase 1, and interleukin-1ß. Conclusions: Our findings suggest that SCL improves diabetes-induced liver injury by alleviating the NF-κB/NLRP3-mediated inflammation and lipid metabolism disorder.

Integrated study of systemic and local airway transcriptomes in asthma reveals causal mediation of systemic effects by airway key drivers.

BACKGROUND: Systemic and local profiles have each been associated with asthma, but parsing causal relationships between system-wide and airway-specific processes can be challenging. We sought to investigate systemic and airway processes in asthma and their causal relationships. METHODS: Three hundred forty-one participants with persistent asthma and non-asthmatic controls were recruited and underwent peripheral blood mononuclear cell (PBMC) collection and nasal brushing. Transcriptome-wide RNA sequencing of the PBMC and nasal samples and a series of analyses were then performed using a discovery and independent test set approach at each step to ensure rigor. Analytic steps included differential expression analyses, coexpression and probabilistic causal (Bayesian) network constructions, key driver analyses, and causal mediation models. RESULTS: Among the 341 participants, the median age was 13 years (IQR = 10-16), 164 (48%) were female, and 200 (58.7%) had persistent asthma with mean Asthma Control Test (ACT) score 16.6 (SD = 4.2). PBMC genes associated with asthma were enriched in co-expression modules for NK cell-mediated cytotoxicity (fold enrichment = 4.5, FDR = 6.47 × 10-32) and interleukin production (fold enrichment = 2.0, FDR = 1.01 × 10-15). Probabilistic causal network and key driver analyses identified NK cell granule protein (NKG7, fold change = 22.7, FDR = 1.02 × 10-31) and perforin (PRF1, fold change = 14.9, FDR = 1.31 × 10-22) as key drivers predicted to causally regulate PBMC asthma modules. Nasal genes associated with asthma were enriched in the tricarboxylic acid (TCA) cycle module (fold enrichment = 7.5 FDR = 5.09 × 10-107), with network analyses identifying G3BP stress granule assembly factor 1 (G3BP1, fold change = 9.1 FDR = 2.77 × 10-5) and InaD-like protein (INADL, fold change = 5.3 FDR = 2.98 × 10-9) as nasal key drivers. Causal mediation analyses revealed that associations between PBMC key drivers and asthma are causally mediated by nasal key drivers (FDR = 0.0076 to 0.015). CONCLUSIONS: Integrated study of the systemic and airway transcriptomes in a well-phenotyped asthma cohort identified causal key drivers of asthma among PBMC and nasal transcripts. Associations between PBMC key drivers and asthma are causally mediated by nasal key drivers.

Longitudinal dynamics of the gut microbiome and metabolome in peanut allergy development.

BACKGROUND: Rising rates of peanut allergy (PA) motivate investigations of its development to inform prevention and therapy. Microbiota and the metabolites they produce shape food allergy risk. OBJECTIVE: We sought to gain insight into gut microbiome and metabolome dynamics in the development of PA. METHODS: We performed a longitudinal, integrative study of the gut microbiome and metabolome of infants with allergy risk factors but no PA from a multicenter cohort followed through mid-childhood. We performed 16S rRNA sequencing, short chain fatty acid measurements, and global metabolome profiling of fecal samples at infancy and at mid-childhood. RESULTS: In this longitudinal, multicenter sample (n = 122), 28.7% of infants developed PA by mid-childhood (mean age 9 years). Lower infant gut microbiome diversity was associated with PA development (P = .014). Temporal changes in the relative abundance of specific microbiota and gut metabolite levels significantly differed in children who developed PA. PA-bound children had different abundance trajectories of Clostridium sensu stricto 1 sp (false discovery rate (FDR) = 0.015) and Bifidobacterium sp (FDR = 0.033), with butyrate (FDR = 0.045) and isovalerate (FDR = 0.036) decreasing over time. Metabolites associated with PA development clustered within the histidine metabolism pathway. Positive correlations between microbiota, butyrate, and isovalerate and negative correlations with histamine marked the PA-free network. CONCLUSION: The temporal dynamics of the gut microbiome and metabolome in early childhood are distinct for children who develop PA. These findings inform our thinking on the mechanisms underlying and strategies for potentially preventing PA.

CRISPR arrays as high-resolution markers to track microbial transmission during influenza infection.

BACKGROUND: Disruption of the microbial community in the respiratory tract due to infections, like influenza, could impact transmission of bacterial pathogens. Using samples from a household study, we determined whether metagenomic-type analyses of the microbiome provide the resolution necessary to track transmission of airway bacteria. Microbiome studies have shown that the microbial community across various body sites tends to be more similar between individuals who cohabit in the same household than between individuals from different households. We tested whether there was increased sharing of bacteria from the airways within households with influenza infections as compared to control households with no influenza. RESULTS: We obtained 221 respiratory samples that were collected from 54 individuals at 4 to 5 time points across 10 households, with and without influenza infection, in Managua, Nicaragua. From these samples, we generated metagenomic (whole genome shotgun sequencing) datasets to profile microbial taxonomy. Overall, specific bacteria and phages were differentially abundant between influenza positive households and control (no influenza infection) households, with bacteria like Rothia, and phages like Staphylococcus P68virus that were significantly enriched in the influenza-positive households. We identified CRISPR spacers detected in the metagenomic sequence reads and used these to track bacteria transmission within and across households. We observed a clear sharing of bacterial commensals and pathobionts, such as Rothia, Neisseria, and Prevotella, within and between households. However, due to the relatively small number of households in our study, we could not determine if there was a correlation between increased bacterial transmission and influenza infection. CONCLUSION: We observed that airway microbial composition differences across households were associated with what appeared to be different susceptibility to influenza infection. We also demonstrate that CRISPR spacers from the whole microbial community can be used as markers to study bacterial transmission between individuals. Although additional evidence is needed to study transmission of specific bacterial strains, we observed sharing of respiratory commensals and pathobionts within and across households. Video Abstract.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe 2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.

Oxidative stress and Cx43-mediated apoptosis are involved in PFOS-induced nephrotoxicity.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that can cause nephrotoxicity. However, the underlying mechanisms are not fully understood and require further investigation. In the present study, we established a PFOS-exposed Sprague-Dawley (SD) rat kidney injury model by intraperitoneal injection of PFOS (1 mg/kg and 10 mg/kg body weight) every alternate day for 15 days and cytotoxicity models of normal rat kidney epithelial (NRK52E) and human renal proximal tubular (HK2) cells exposed to PFOS (20 µM and 60 µM) for 24 h to reveal the mechanisms underlying PFOS-induced nephrotoxicity. Data showed that PFOS increased the kidney index and induced nephrotoxicity in rats. Furthermore, PFOS significantly increased malondialdehyde (MDA) levels, decreased GSH peroxidase (GSH-PX) activity in kidney tissues, and increased intracellular reactive oxygen species (ROS) levels in NRK52E and HK2 cells. Following PFOS treatment, mitochondrial damage in the renal tubular epithelial cells of rats was observed and the mitochondrial membrane potential (ΔΨm) was decreased in NRK52E cells. PFOS upregulated apoptosis of tubular epithelial cells and expression of Connexin 43 (Cx43) in vitro and in vivo. The Cx43 inhibitor gap26 attenuated the apoptosis of tubular epithelial cells. In conclusion, our findings reveal that PFOS may trigger renal tubular cell apoptosis through oxidative stress and upregulation of Cx43, resulting in PFOS-induced nephrotoxicity.

The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'-O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

NAT10 and DDX21 Proteins Interact with RNase H1 and Affect the Performance of Phosphorothioate Oligonucleotides.

RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

Identification of immune-related and autophagy-related genes for the prediction of survival in bladder cancer.

BACKGROUND: Bladder cancer has the characteristics of high morbidity and mortality, and the prevalence of bladder cancer has been increasing in recent years. Immune and autophagy related genes play important roles in cancer, but there are few studies on their effects on the prognosis of bladder cancer patients. METHODS: Using gene expression data from the TCGA-BLCA database, we clustered bladder cancer samples into 6 immune-related and autophagy-related molecular subtypes with different prognostic outcomes based on 2208 immune-related and autophagy-related genes. Six subtypes were divided into two groups which had significantly different prognosis. Differential expression analysis was used to explore genes closely related to the progression of bladder cancer. Then we used Cox stepwise regression to define a combination of gene expression levels and immune infiltration indexes to construct the risk model. Finally, we built a Nomogram which consist of risk score and several other prognosis-related clinical indicators. RESULTS: The risk model suggested that high expression of C5AR2, CSF3R, FBXW10, FCAR, GHR, OLR1, PGLYRP3, RASGRP4, S100A12 was associated with poor prognosis, while high expression level of CD96, IL10, MEFV pointed to a better prognosis. Validation by internal and external dataset suggested that our risk model had a high ability to discriminate between the outcomes of patients with bladder cancer. The immunohistochemical results basically confirmed our results. The C-Index value and Calibration curves verified the robustness of Nomogram. CONCLUSIONS: Our study constructed a model that included a risk score for patients with bladder cancer, which provided a lot of helps to predict the prognosis of patients with bladder cancer.

RNA modifications can affect RNase H1-mediated PS-ASO activity.

Phosphorothioate modified antisense oligonucleotides (PS-ASOs) can reduce gene expression through hybridization to target RNAs and subsequent cleavage by RNase H1. Target reduction through this mechanism is influenced by numerous features of the RNA, which modulate PS-ASO binding affinities to the RNA target, and how the PS-ASO-RNA hybrid is recognized by RNase H1 for RNA cleavage. Endogenous RNAs are frequently chemically modified, which can regulate intra- and intermolecular interactions of the RNA. The effects of PS-ASO modifications on antisense activity have been well studied; however, much less is known regarding the effects of RNA modifications on PS-ASO hybridization and RNase H1 cleavage activity. Here, we determine the effects of three different RNA modifications on PS-ASO binding and antisense activity in recombinant and cell-based systems. Some RNA modifications can reduce PS-ASO hybridization, the cleavage activity of RNase H1, or both, while other modifications had minimal effects on PS-ASO function. In addition to these direct effects, RNA modifications can also change the RNA structure, which may affect PS-ASO accessibility in a cellular context. Our results elucidate the effects of three prevalent RNA modifications on PS-ASO-mediated RNase H1 cleavage activity, and such findings will help improve PS-ASO target site selection.

Multiscale study of the oral and gut environments in children with high- and low-threshold peanut allergy.

BACKGROUND: The oral and gut microbiomes have each been associated with food allergy status. Within food allergy, they may also influence reaction thresholds. OBJECTIVE: Our aim was to identify oral and gut microbiota associated with reaction thresholds in peanut allergy. METHODS: A total of 59 children aged 4 to 14 years with suspected peanut allergy underwent double-blind, placebo-controlled food challenge to peanut. Those children who reacted at the 300-mg or higher dose of peanut were classified as high-threshold (HT), those who reacted to lower doses were classified as low-threshold (LT), and those children who did not react were classified as not peanut allergic (NPA). Saliva and stool samples collected before challenge underwent DNA isolation followed by 16S rRNA sequencing and short-chain fatty acid measurement. RESULTS: The 59 participants included 38 HT children and 13 LT children. Saliva microbiome α-diversity (Shannon index) was higher in LT children (P = .017). We identified saliva and stool microbiota that distinguished HT children from LT children, including oral Veillonella nakazawae (amplicon sequence variant 1979), which was more abundant in the HT group than in the LT group (false discovery rate [FDR] = 0.025), and gut Bacteroides thetaiotaomicron (amplicon sequence variant 6829), which was less abundant in HT children than in LT children (FDR = 0.039). Comparison with NPA children revealed consistent ordinal trends between these discriminating species and reaction thresholds. Importantly, many of these threshold-associated species were also correlated with short-chain fatty acid levels at the respective body sites, including between oral V nakazawae and oral butyrate (r = 0.57; FDR = 0.049). CONCLUSION: Findings from this multiscale study raise the possibility of microbial therapeutics to increase reaction thresholds in children with food allergy.

Targeting EYA2 tyrosine phosphatase activity in glioblastoma stem cells induces mitotic catastrophe.

Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors.

Golgi-58K can re-localize to late endosomes upon cellular uptake of PS-ASOs and facilitates endosomal release of ASOs.

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs can trigger RNase H1 cleavage of cellular target RNAs to modulate gene expression. Internalized PS-ASOs must be released from membraned endosomal organelles, a rate limiting step that is not well understood. Recently we found that M6PR transport between Golgi and late endosomes facilitates productive release of PS-ASOs, raising the possibility that Golgi-mediated transport may play important roles in PS-ASO activity. Here we further evaluated the involvement of Golgi in PS-ASO activity by examining additional Golgi proteins. Reduction of certain Golgi proteins, including Golgi-58K, GCC1 and TGN46, decreased PS-ASO activity, without substantial effects on Golgi integrity. Upon PS-ASO cellular uptake, Golgi-58K was recruited to late endosomes where it colocalized with PS-ASOs. Reduction of Golgi-58K caused slower PS-ASO release from late endosomes, decreased GCC2 late endosome relocalization, and led to slower retrograde transport of M6PR from late endosomes to trans-Golgi. Late endosome relocalization of Golgi-58K requires Hsc70, and is most likely mediated by PS-ASO-protein interactions. Together, these results suggest a novel function of Golgi-58K in mediating Golgi-endosome transport and indicate that the Golgi apparatus plays an important role in endosomal release of PS-ASO, ensuring antisense activity.