Initiation of a ZAKα-dependent ribotoxic stress response by the innate immunity endoribonuclease RNase L.

RNase L is an endoribonuclease of higher vertebrates that functions in antiviral innate immunity. Interferons induce oligoadenylate synthetase enzymes that sense double-stranded RNA of viral origin leading to the synthesis of 2',5'-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L remodels the host cell transcriptome. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A is directly introduced into cells. Here, we report that RNase L activation by 2-5A causes a ribotoxic stress response involving the MAP kinase kinase kinase (MAP3K) ZAKα, MAP2Ks, and the stress-activated protein kinases JNK and p38α. RNase L activation profoundly alters the transcriptome by widespread depletion of mRNAs associated with different cellular functions but also by JNK/p38α-stimulated induction of inflammatory genes. These results show that the 2-5A/RNase L system triggers a protein kinase cascade leading to proinflammatory signaling and apoptosis.

Initiation of a ZAKα-dependent Ribotoxic Stress Response by the Innate Immunity Endoribonuclease RNase L.

RNase L is a regulated endoribonuclease in higher vertebrates that functions in antiviral innate immunity. Interferons induce OAS enzymes that sense double-stranded RNA of viral origin leading to synthesis of 2',5'-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L inhibits viral infections. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A was directly introduced into cells. Here we report that RNase L activation by 2-5A causes a ribotoxic stress response that requires the ribosome-associated MAP3K, ZAKα. Subsequently, the stress-activated protein kinases (SAPK) JNK and p38α are phosphorylated. RNase L activation profoundly altered the transcriptome by widespread depletion of mRNAs associated with different cellular functions, but also by SAPK-dependent induction of inflammatory genes. Our findings show that 2-5A is a ribotoxic stressor that causes RNA damage through RNase L triggering a ZAKα kinase cascade leading to proinflammatory signaling and apoptosis.

Single cell multiomic analysis reveals diabetes-associated ß-cell heterogeneity driven by HNF1A.

Broad heterogeneity in pancreatic ß-cell function and morphology has been widely reported. However, determining which components of this cellular heterogeneity serve a diabetes-relevant function remains challenging. Here, we integrate single-cell transcriptome, single-nuclei chromatin accessibility, and cell-type specific 3D genome profiles from human islets and identify Type II Diabetes (T2D)-associated ß-cell heterogeneity at both transcriptomic and epigenomic levels. We develop a computational method to explicitly dissect the intra-donor and inter-donor heterogeneity between single ß-cells, which reflect distinct mechanisms of T2D pathogenesis. Integrative transcriptomic and epigenomic analysis identifies HNF1A as a principal driver of intra-donor heterogeneity between ß-cells from the same donors; HNF1A expression is also reduced in ß-cells from T2D donors. Interestingly, HNF1A activity in single ß-cells is significantly associated with lower Na+ currents and we nominate a HNF1A target, FXYD2, as the primary mitigator. Our study demonstrates the value of investigating disease-associated single-cell heterogeneity and provides new insights into the pathogenesis of T2D.

Single-cell lineage analysis reveals extensive multimodal transcriptional control during directed beta-cell differentiation.

The in vitro differentiation of insulin-producing beta-like cells can model aspects of human pancreatic development. Here, we generate 95,308 single-cell transcriptomes and reconstruct a lineage tree of the entire differentiation process from human embryonic stem cells to beta-like cells to study temporally regulated genes during differentiation. We identify so-called 'switch genes' at the branch point of endocrine/non-endocrine cell fate choice, revealing insights into the mechanisms of differentiation-promoting reagents, such as NOTCH and ROCKII inhibitors, and providing improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identify a stage-specific enhancer at the TCF7L2 locus for diabetes, uncovered by genome-wide association studies, that drives a transient wave of gene expression in pancreatic progenitors. Finally, we develop a web app to visualize gene expression on the lineage tree, providing a comprehensive single-cell data resource for researchers studying islet biology and diabetes.

Novel Bi2+xWO6 p-n Homojunction Nanostructure: Preparation, Characterization, and Application for a Self-Powered Cathodic Photoelectrochemical Immunosensor.

Synthesizing novel cathodic photoactive materials with high photoelectrochemical (PEC) performance is urgently important for the development of photocathodic sensors. Herein, a novel photocathode material, Bi self-doped Bi2WO6 (i.e., Bi2+xWO6) p-n homojunction, is prepared via a simple ethylene glycol-assisted solvothermal reduction for the first time. Compared with pristine Bi2WO6, Bi2+xWO6 possesses a narrower band gap and higher light harvesting ability. Among the synthesized materials, Bi2.1WO6 exhibits the highest photocurrent response, which is 23 times that of pure Bi2WO6 because of the synergistic effect of doped Bi and the p-n homojunction. The open circuit potential, "V-shaped" Mott-Schottky plot, linear sweep voltammetry curve, and transient photocurrent demonstrate the p-n homojunction characteristics of the material well. By using the Bi2+xWO6 p-n homojunction as the photocathode for sensing and the plasmonic WO3/Au composite as the photoanode for signal amplification, a new self-powered membraneless PEC immunosensor is established for a highly sensitive detection of human epididymal protein 4. This study offers a new idea for designing novel photocatalysts with satisfactory performance, and the Bi2+xWO6 p-n homojunction is expected to act as a promising PEC platform for developing various self-powered biosensors.

Space-confined synthesis of ordered mesoporous carbon doped with single-layer MoS2-boron for the voltammetric determination of theophylline.

A space-confined synthesis method is employed for the preparation of a single-layer MoS2-boron doped ordered mesoporous carbon nanocomposite. A phenol-formaldehyde resin is used as carbon source to create a confined space for the formation of single-layer MoS2. The addition of pluronic F127, as a soft template, suppresses the stacking of MoS2 layers and makes the composite porous. The nanocomposite is characterized by scanning electron and transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy. The single-layer MoS2 sheets have a lateral size of about 5 nm and are uniformly embedded in the composite. They possess numerous active edge sites and display a strong synergistic effect with other components. The composite is modified on a glassy carbon electrode, followed by the electrochemical imprinting of theophylline, and the resulting electrode exhibits good electrochemical response to theophylline. The linear response range is 0.01-250 µM by differential pulse voltammetry, and the lower detection limit is 5 nM. It has been successfully applied to the determination of theophylline in spiked tea drink samples. Graphical abstract Single-layer MoS2-boron doped ordered mesoporous carbon nanocomposite has large surface area and high catalysis, when coupling with molecularly imprinted polymer the resulting electrode shows highly sensitive and selective response to theophylline.

Z-scheme I-BiOCl/CdS with abundant oxygen vacancies as highly effective cathodic material for photocathodic immunoassay.

A novel label-free photocathodic immunosensor was constructed by introducing a direct Z-scheme I-BiOCl/CdS cathodic material as highly effective photocatalyst for the selective detection of carcino embryonic antigen (CEA). The Z-scheme photocatalyst could promote the separation of photogenerated carriers and showed a more negative conduction band potential. In addition, the I-BiOCl with abundant oxygen vacancies could activate electron acceptors (i.e. O2 and H2O2) and made them reduce more completely, thus the sensitivity of the photocathodic immunosensor was significantly improved. Afterward, CEA antibody (Ab) was employed for the selective recognition of CEA target, which was covalently bonded to the substrate material. The formation of immune complexes hindered the diffusion of electron acceptors, thus the photocurrent decreased. Under the optimized conditions, the photocathodic immunosensor displayed abroad linear range (0.01-40.0 ng/mL) and a low detection limit (0.002 ng/mL) for CEA detection. Furthermore, acceptable reproducibility, excellent selectivity and high anti-interference ability were achieved. This work provides a new horizon for the design and development of Z-scheme cathodic materials as photoactive material for photocathodic biosensing.

Correction to: miR-153 suppresses IDO1 expression and enhances CAR T cell immunotherapy.

The original article [1] contains a spelling error in the authorship; the authors would like to note the correct spelling of the second author, Jiajia Xi.

miR-21 depletion in macrophages promotes tumoricidal polarization and enhances PD-1 immunotherapy.

MicroRNA-21 (miR-21) is one of the most abundant microRNAs in mammalian cells. It has been intensively studied for its role in regulating apoptosis and oncogenic transformation. However, the impact of miR-21 on host anti-tumor immunity remains unknown. Tumor-associated macrophages are a major leukocyte type that infiltrates tumors and predominantly develops into immunosuppressive, tumor-promoting M2-like macrophages. In contrast, the pro-inflammatory M1-like macrophages have tumoricidal activity. In this study, we show that genetic deficiency of miR-21 promotes the polarization of macrophages toward an M1-like phenotype in vivo and in vitro in the presence of tumor cells; thus it confers host mice with enhanced anti-tumor immunity. By downregulating JAK2 and STAT1, miR-21 inhibits the IFN-γ-induced STAT1 signaling pathway, which is required for macrophage M1 polarization. We also show that the expression of miR-21 in macrophages is regulated upon polarization stimuli as well as upon macrophages co-culturing with tumor cells. Thus, tumor cells may stimulate miR-21 expression in tumor-associated macrophages to prevent tumoricidal M1 polarization. However, augmented STAT1 signaling mediated by miR-21 deficiency upregulates PD-L1 expression in macrophages, which is known to inhibit phagocytic anti-tumor activity. This adverse effect can be alleviated by PD-1 blockade; indeed, miR-21 depletion in macrophages and PD-1 antibody treatment offer superior anti-tumor activity than either agent alone. These studies shed lights on potential application of the combination of miR-21 inhibition and immune checkpoint blockade to target the tumor microenvironment.

TAK1, more than just innate immunity.

Transforming growth factor ß-activated kinase 1 (TAK1) is a key regulator of the innate immunity and the proinflammatory signaling pathway. In response to interleukin-1, tumor necrosis factor-α, and toll-like receptor agonists, it mediates the activation of the nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), and p38 pathways. In addition, TAK1 plays a central role in adaptive immunity, in which it mediates signaling from T- and B-cell receptors. This review will focus on recent developments and also examine the regulation of TAK1 in response to a diverse range of other stimuli including DNA damage, transforming growth factor-ß, Wnt, osmotic stress, and hypoxia.

The role of TBK1 and IKKε in the expression and activation of Pellino 1.

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-ß]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB (inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKKε. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3)-/- BMDMs, and its induction is only reduced slightly in type 1 interferon receptor-/- BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKε/TBK1 and is reversed by phosphatase treatment. Thus IKKε/TBK1 mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.

[Downregulation of HER2 by adenovirus-mediated RNA interference and its inhibitory effect on growth of SKBR3 breast cancer cell].

AIM: To explore the possibility of RNA interference (RNAi)-based gene therapy against HER2-overexpressing tumors using adenovirus-mediated vector. METHODS: A plasmid named pHER2-GFP containing HER2 and green fluorescent protein (GFP) fusion was constructed and cotransfected into CHO-K1 cells respectively with nine small interference RNA (siRNA)-expressing plasmids targeting different regions of HER2. The siRNA-expressing plasmids with best interference effect were screened out and then used to identify the gene silence effect in HER2-overexpressing SKBR3 breast cancer cells. Subsequently, the siRNA-expressing cassettes were subcloned into adenoviral vectors. Downregulation of HER2 by adenovirus-mediated RNAi and its effect on SKBR3 cell proliferation were identified again. RESULTS: Two siRNA-expressing plasmids with best interference effect were screened out and HER2 was also efficiently downregulated in SKBR3 cells infected with the adenovirus containing these siRNA-expressing cassettes. Downregulation of HER2 resulted in the increase of cells in G1 phase and the induction of apoptosis. Furthermore, infection of adenovirus inhibited SKBR3 cell growth, which was confirmed by MTT and cell long-term proliferation assays. CONCLUSION: The adenovirus-mediated RNAi could downregulate the HER2 expression efficiently and exert an inhibitory effect on growth of HER2-overexpressing breast cancer cell.