High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the ...number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.
(1) Background: Many transporters of the SLC22 family (e.g., OAT1, OAT3, OCT2, URAT1, and OCTN2) are highly expressed in the kidney. They transport drugs, metabolites, signaling molecules, ...antioxidants, nutrients, and gut microbiome products. According to the Remote Sensing and Signaling Theory, SLC22 transporters play a critical role in small molecule communication between organelles, cells and organs as well as between the body and the gut microbiome. This raises the question about the potential role of SLC22 transporters in cancer biology and treatment. (2) Results: In two renal cell carcinoma RNA-seq datasets found in TCGA, KIRC and KIRP, there were multiple differentially expressed (DE) SLC22 transporter genes compared to normal kidney. These included SLC22A6, SLC22A7, SLC22A8, SLC22A12, and SLC22A13. The patients with disease had an association between overall survival and expression for most of these DE genes. In KIRC, the stratification of patient data by pathological tumor characteristics revealed the importance of SLC22A2, SLC22A6, and SLC22A12 in disease progression. Interaction networks combining the SLC22 with ADME genes supported the centrality of SLC22 transporters and other transporters (ABCG2, SLC47A1) in disease progression. (3) Implications: The fact that many of these genes are uric acid transporters is interesting because altered uric acid levels have been associated with kidney cancer. Moreover, these genes play key roles in processing metabolites and chemotherapeutic compounds, thus making them potential therapeutic targets. Finally, our analyses raise the possibility that current approaches may undertreat certain kidney cancer patients with low SLC22 expression and only localized disease while possibly overtreating more advanced disease in patients with higher SLC22 expression. Clinical studies are needed to investigate these possibilities.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Noninvasive biomarkers are needed to monitor stable patients after kidney transplant (KT), because subclinical acute rejection (subAR), currently detectable only with surveillance biopsies, can lead ...to chronic rejection and graft loss. We conducted a multicenter study to develop a blood‐based molecular biomarker for subAR using peripheral blood paired with surveillance biopsies and strict clinical phenotyping algorithms for discovery and validation. At a predefined threshold, 72% to 75% of KT recipients achieved a negative biomarker test correlating with the absence of subAR (negative predictive value: 78%‐88%), while a positive test was obtained in 25% to 28% correlating with the presence of subAR (positive predictive value: 47%‐61%). The clinical phenotype and biomarker independently and statistically correlated with a composite clinical endpoint (renal function, biopsy‐proved acute rejection, ≥grade 2 interstitial fibrosis, and tubular atrophy), as well as with de novo donor‐specific antibodies. We also found that <50% showed histologic improvement of subAR on follow‐up biopsies despite treatment and that the biomarker could predict this outcome. Our data suggest that a blood‐based biomarker that reduces the need for the indiscriminate use of invasive surveillance biopsies and that correlates with transplant outcomes could be used to monitor KT recipients with stable renal function, including after treatment for subAR, potentially improving KT outcomes.
The authors present data for a blood‐based gene expression profile that can be used to detect and monitor the treatment of subclinical rejection in patients following kidney transplantation. See Naesens's editorial on page 5.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mutations at position K171 in the kinase activation loop of Inhibitor of κB kinase beta (IKKβ) occur in multiple myeloma, spleen marginal zone lymphoma and mantle cell lymphoma. Previously, we ...demonstrated that these result in constitutive kinase activation and stimulate Signal Transducer and Activator of Transcription 3 (STAT3). This work also identified K147 as a site of K63-linked regulatory ubiquitination required for activation of signaling pathways. We now present a more detailed analysis of ubiquitination sites together with a comprehensive examination of the signaling pathways activated by IKKβ K171E mutants. Downstream activation of STAT3 is dependent upon the activity of: UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; TAK1 (MAP3K7), or TGFβ Activated Kinase, which forms a complex required for NFκB activation; JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; and gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKβ K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKKβ K171E mutants trigger an autocrine loop in which IL-6 is secreted and binds to the IL-6 receptor complex gp130, resulting in JAK activation. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKβ K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKKβ are likely to also exhibit activated STAT3 and p44/42 MAPK (Erk1/2), this suggests the possibility of using MAPK (Erk1/2) and JAK inhibitors, or specific ubiquitination inhibitors. K63-linked ubiquitination occurs in other kinases at sites homologous to K147 in IKKβ, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Irreversible electroporation (IRE) is a nonthermal ablation technique that is used clinically in selected patients with locally advanced pancreatic cancer, but most patients develop recurrent distant ...metastatic disease. We hypothesize that IRE can induce an
vaccination effect by releasing tumor neoantigens in an inflammatory context. Using an immunocompetent mouse model, we demonstrated that IRE alone produced complete regression of subcutaneous tumors in approximately 20% to 30% of mice. IRE was not effective in immunodeficient mice. Mice with complete response to IRE demonstrated prophylactic immunity and remained tumor free when rechallenged with secondary tumors on the contralateral flank. CD8
T cells from IRE-responsive mice were reactive against peptides representing model-inherent alloantigens and conferred protection against tumor challenge when adoptively transferred into immunocompromised, tumor-naïve mice. Combining IRE with intratumoral Toll-like receptor-7 (TLR7) agonist (1V270) and systemic anti-programmed death-1 receptor (PD)-1 checkpoint blockade resulted in improved treatment responses. This combination also resulted in elimination of untreated concomitant distant tumors (abscopal effects), an effect not seen with IRE alone. These results suggest that the systemic antitumor immune response triggered by IRE can be enhanced by stimulating the innate immune system with a TLR7 agonist and the adaptive immune system with anti-PD-1 checkpoint blockade simultaneously. Combinatorial approaches such as this may help overcome the immunosuppressive pancreatic cancer microenvironment.
In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a ...hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new 'D-site' class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is ...largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.
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•Neuronal activity drives dynamic O-GlcNAcylation in neuronal processes•Mitochondria are the loci of O-GlcNAc modification in neurons•O-GlcNAcylation boosts the respiratory capacity of neuronal mitochondria•Mitochondrial O-GlcNAcome supports neuronal activity-driven, on-demand ATP synthesis
Despite their high metabolic demands, neurons rely on ATP synthesis as needed. Yu et al. demonstrate that O-GlcNAcylation, a post-translational modification responsive to glucose flux, rapidly adjusts to neuronal activity, boosting mitochondrial plasticity and bioenergetics, thus coupling ATP synthesis rates to fuel availability for meeting energy demands.
Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular ...signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gene expression of BAL cells, which samples the cellular milieu within the lower respiratory tract, has not been well studied in severe asthma.
To identify new biomolecular mechanisms underlying ...severe asthma by an unbiased, detailed interrogation of global gene expression.
BAL cell expression was profiled in 154 asthma and control subjects. Of these participants, 100 had accompanying airway epithelial cell gene expression. BAL cell expression profiles were related to participant (age, sex, race, and medication) and sample traits (cell proportions), and then severity-related gene expression determined by correlating transcripts and coexpression networks to lung function, emergency department visits or hospitalizations in the last year, medication use, and quality-of-life scores.
Age, sex, race, cell proportions, and medications strongly influenced BAL cell gene expression, but leading severity-related genes could be determined by carefully identifying and accounting for these influences. A BAL cell expression network enriched for cAMP signaling components most differentiated subjects with severe asthma from other subjects. Subsequently, an
cellular model showed this phenomenon was likely caused by a robust upregulation in cAMP-related expression in nonsevere and β-agonist-naive subjects given a β-agonist before cell collection. Interestingly, ELISAs performed on BAL lysates showed protein levels may partly disagree with expression changes.
Gene expression in BAL cells is influenced by factors seldomly considered. Notably, β-agonist exposure likely had a strong and immediate impact on cellular gene expression, which may not translate to important disease mechanisms or necessarily match protein levels. Leading severity-related genes were discovered in an unbiased, system-wide analysis, revealing new targets that map to asthma susceptibility loci.
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