[Study on mechanism of Chinese Yam polysaccharide synergizing nucleoside analogues against hepatitis B virus through p38 MAPK signaling pathway].

This study explore the molecular mechanism of the synergistic effect of Chinese Yam polysaccharides and nucleoside analogues(NAs) on hepatitis B virus(HBV) resistance. Different concentrations of Chinese Yam polysaccharide and entecavir were ad-ded to HepG2.2.15 cells. After the cytotoxicity was detected by cell counting kit-8(CCK-8), the optimal concentration and time of the two drugs to inhibit HepG2.2.15 cells were screened out. They were divided into control group, Chinese Yam polysaccharide group, entecavir group and combination drug group(Chinese Yam polysaccharide + entecavir). The drugs were added to HepG2.2.15 cells, ELISA was used to detect the effects of each group of drugs on the secretion of hepatitis B virus surface antigen(HBsAg) and hepatitis B virus e antigen(HBeAg) in cell supernatant, probe quantitative real-time PCR(probe qRT-PCR) was used to detect the effects of drugs on HBV-DNA in HepG2.2.15 cells, and Western blot was used to detect the effects of each group of drugs on the expression of p38 MAPK, p-p38 MAPK, NTCP proteins in HepG2.2.15 cells. The qRT-PCR was used to detect the effect of drugs on the expression of p38 MAPK and NTCP mRNA in HepG2.2.15 cells. The results showed that compared with control group, the concentrations of HBeAg and HBsAg in Chinese Yam polysaccharide group, entecavir group and combination group decreased(P<0.01 or P<0.001), and both of them inhibited HBV-DNA in HepG2.2.15 cells(P<0.01), and the HBV-DNA inhibition of HepG2.2.15 cells in the combination group was more obvious(P<0.001), and the protein expression levels of p-p38 MAPK and NTCP were significantly decreased(P<0.05 or P<0.01), the mRNA expression level of p38 MAPK increased, and the mRNA expression level of NTCP decreased(P<0.05 or P<0.01). To sum up, Chinese Yam polysaccharide can reduce the expression of NTCP protein and mRNA through p38 MAPK signaling pathway and cooperate with entecavir in anti-HBV.

Improving thermo-tolerance of Saccharomyces cerevisiae by precise regulation of the expression of small HSP.

The level of heat resistance in microbial cells is an important factor in determining the energy consumption and product synthesis efficiency of fermentation processes. Current research generally believes that heat shock proteins (HSPs) are the most closely related functional molecules to heat resistance inside cells. They can stabilize cell structures and allow cells to perform their normal physiological functions. Based on our previous transcriptome data, this study applies synthetic biology methods to validate the functionality of heat-resistant elements. The researchers introduced gene circuits expressing small HSPs (sHSP-HB8, HSP12, HSP26, HSP30, HSP42, and ibpa-MB4) with different promoter strengths (TDH3p, YNL247wp) into Saccharomyces cerevisiae strains for functional verification. All engineered strains, with the exception of No. 3 and No. 8, demonstrated a significantly higher growth rate and cell viability at 42 °C. Among them, No. 7 (YNL247wp-HSP12-SLM5t) and No. 11 (YNL247wp-sHSP-HB8-SLM5t), the two best performing engineered strains, exhibited a 19.8% and 17.2% increase in cell density, respectively, compared to the control strain. Additionally, the analysis of pyruvate kinase (PK) and malate dehydrogenase (MDH) enzyme activities indicated that the engineered strains enhanced protein quality at higher temperatures. The research methods and ideas presented in this paper have significant scientific reference value for exploring and applying other stress-resistant gene circuits.

NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1.

In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.

Analyze the SUMOylation of IKK γ/NEMO During Genotoxic Stress.

SUMOylation is an important posttranslational modification of substrate proteins that regulates their functions in a variety of cellular processes including epigenetic and transcriptional regulation of gene expression, genomic stability, DNA repair, subcellular translocation, and protein turnover. The critical roles of SUMOylation in regulating NF-κB signaling is exemplified by the findings that it regulates IκBα stability, transactivity of RelA and RelB, as well as initiating the export of nuclear DNA damage signal to cytoplasmic IKK complex through NEMO SUMOylation. Detection of SUMOylated protein is technically challenging due to only a small fraction of substrate proteins is SUMOylated and this process is also reversible by highly active SUMO-deconjugating enzymes. In this protocol, we outline a method for detecting SUMOylation of NEMO in mammalian cells treated by genotoxic agents.

Immune checkpoint molecules: "new" kids on the block of skin photoimmunology.

Ultraviolet radiation (UVR) is a prominent etiological factor of the pathogenesis of skin diseases such as squamous cell carcinoma and melanoma. Excessive exposure to the natural sources of UVR such as sunlight or artificially from tanning lamps has been linked to the increasing incidence of skin cancers in the United States. Besides the skin inflammation, DNA damage and oncogenic mutation caused by UVR, UV exposure also plays a critical role in suppressing local and systemic immune responses which enable premalignant and cancer cells to escape immune surveillance. A variety of mechanisms have been reported to regulate the immune-suppressive effects of UVR. Here we discuss the current understanding of how UV modulates the local and systemic immunity, the recent progress in roles of immune checkpoint molecules in UVR-induced immune suppression, and how the crosstalk between the immune cells may shape the immune landscape of the skin upon UVR.

OTULIN couples WNT signaling to resistance in triple-negative breast cancer.

Oncogenic Wnt/ß-catenin activation promotes cancer development and drug resistance to cancer treatments. We recently revealed an underlying mechanism linking linear ubiquitination with Wnt/ß-catenin activation upon genotoxic treatments. We showed that ABL1 (ABL proto-oncogene 1)-dependent phosphorylation of OTULIN (OTU deubiquitinase with linear linkage specificity) upon DNA damage drives ß-catenin activation which promotes drug resistance in triple-negative breast cancer.

ABL1-dependent OTULIN phosphorylation promotes genotoxic Wnt/ß-catenin activation to enhance drug resistance in breast cancers.

Dysregulated Wnt/ß-catenin activation plays a critical role in cancer progression, metastasis, and drug resistance. Genotoxic agents such as radiation and chemotherapeutics have been shown to activate the Wnt/ß-catenin signaling although the underlying mechanism remains incompletely understood. Here, we show that genotoxic agent-activated Wnt/ß-catenin signaling is independent of the FZD/LRP heterodimeric receptors and Wnt ligands. OTULIN, a linear linkage-specific deubiquitinase, is essential for the DNA damage-induced ß-catenin activation. OTULIN inhibits linear ubiquitination of ß-catenin, which attenuates its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association with ß-catenin is enhanced by OTULIN Tyr56 phosphorylation, which depends on genotoxic stress-activated ABL1/c-Abl. Inhibiting OTULIN or Wnt/ß-catenin sensitizes triple-negative breast cancer xenograft tumors to chemotherapeutics and reduces metastasis. Increased OTULIN levels are associated with aggressive molecular subtypes and poor survival in breast cancer patients. Thus, OTULIN-mediated Wnt/ß-catenin activation upon genotoxic treatments promotes drug resistance and metastasis in breast cancers.

Fully-Differential TPoS Resonators Based on Dual Interdigital Electrodes for Feedthrough Suppression.

As one of the core components of MEMS (i.e., micro-electro-mechanical systems), thin-film piezoelectric-on-silicon (TPoS) resonators experienced a blooming development in the past decades due to unique features such as a remarkable capability of integration for attractive applications of system-on-chip integrated timing references. However, the parasitic capacitive feedthrough poses a great challenge to electrical detection of resonance in a microscale silicon-based mechanical resonator. Herein, a fully-differential configuration of a TPoS MEMS resonator based on a novel structural design of dual interdigital electrodes is proposed to eliminate the negative effect of feedthrough. The fundamental principle of feedthrough suppression was comprehensively investigated by using FEA (i.e., finite-element analysis) modeling and electrical measurements of fabricated devices. It was shown that with the help of fully-differential configuration, the key parameter of SBR (i.e., signal-to-background ratio) was significantly enhanced by greatly suppressing the in-phase signal. The S-parameter measurement results further verified the effectiveness of this novel feedthrough suppression strategy, and the insertion loss and SBR of proposed TPoS resonators were improved to 4.27 dB and 42.47 dB, respectively.

Progress of Individualized Chemotherapy for Gastric Carcinoma Under the Guidance of Genetic Testing.

BACKGROUND: Gastric cancer is a major malignancy that has high incidence rates worldwide. Approximately 30% of patients with gastric cancer have progressed into advanced stages at the time of diagnosis. Chemotherapy is the standard-of-care for most advanced gastric cancer and elicits variable responses among patients. Personalized chemotherapy based on genetic information of individual patients with gastric cancer has gained increasing attention among oncologists for guiding chemotherapeutic regimens. METHODS: This review summarizes recent progress of individualized chemotherapy in gastric cancer guided by pharmacogenomics. Variable medical research search engines, such as PubMed, Google Scholar, SpringerLink and ScienceDirect, were used to retrieve related literature. Only peerreviewed journal articles were selected for further analyses. RESULTS AND CONCLUSION: The efficiency of chemotherapy in patients with gastric cancer is not only determined by chemotherapeutic drugs but is also directly and indirectly influenced by functionally correlative genes. Individual gene alteration or polymorphism remarkably affects patients' responses to particular chemotherapy. Most studies have focused on the influence of single-gene alteration on a selected drug, and only a few works explored the interaction between therapeutics and a panel of genes. Individualized chemotherapy regimens guided by a genetic survey of a multiple-gene panel are expected to remarkably improve the treatment efficacy in patients with advanced gastric cancer and may become the new standard for personalizing chemotherapy for gastric cancer in the near future.

LINC02273 drives breast cancer metastasis by epigenetically increasing AGR2 transcription.

BACKGROUND: The majority of breast cancer patients die of metastasis rather than primary tumors, whereas the molecular mechanisms orchestrating cancer metastasis remains poorly understood. Long noncoding RNAs (lncRNA) have been shown to regulate cancer occurrence and progression. However, the lncRNAs that drive metastasis in cancer patients and their underlying mechanisms are still largely unknown. METHODS: lncRNAs highly expressed in metastatic lymph nodes were identified by microarray. Survival analysis were made by Kaplan-Meier method. Cell proliferation, migration, and invasion assay was performed to confirm the phenotype of LINC02273. Tail vein model and mammary fat pad model were used for in vivo study. RNA pull-down and RIP assay were used to confirm the interaction of hnRNPL and LINC02273. Chromatin isolation by RNA purification followed by sequencing (ChIRP-seq), RNA-seq, ChIP-seq, and luciferase reporter assay reveal hnRNPL-LINC02273 regulates AGR2. Antisense oligonucleotides were used for in vivo treatment. RESULTS: We identified a novel long noncoding RNA LINC02273, whose expression was significantly elevated in metastatic lesions compared to the primary tumors, by genetic screen of matched tumor samples. Increased LINC02273 promoted breast cancer metastasis in vitro and in vivo. We further showed that LINC02273 was stabilized by hnRNPL, a protein increased in metastatic lesions, in breast cancer cells. Mechanistically, hnRNPL-LINC02273 formed a complex which activated AGR2 transcription and promoted cancer metastasis. The recruitment of hnRNPL-LINC02273 complex to AGR2 promoter region epigenetically upregulated AGR2 by augmenting local H3K4me3 and H3K27ac levels. Combination of AGR2 and LINC02273 was an independent prognostic factor for predicting breast cancer patient survival. Moreover, our data revealed that LINC02273-targeting antisense oligonucleotides (ASO) substantially inhibited breast cancer metastasis in vivo. CONCLUSIONS: Our findings uncover a key role of LINC02273-hnRNPL-AGR2 axis in breast cancer metastasis and provide potential novel therapeutic targets for metastatic breast cancer intervention.

Upregulation of PD-L1 via HMGB1-Activated IRF3 and NF-κB Contributes to UV Radiation-Induced Immune Suppression.

Solar ultraviolet radiation (UVR) suppresses skin immunity, which facilitates initiation of skin lesions and establishment of tumors by promoting immune evasion. It is unclear whether immune checkpoints are involved in the modulation of skin immunity by UVR. Here, we report that UVR exposure significantly increased expression of immune checkpoint molecule PD-L1 in melanoma cells. The damage-associated molecular patterns molecule HMGB1 was secreted by melanocytes and keratinocytes upon UVR, which subsequently activated the receptor for advanced glycation endproducts (RAGE) receptor to promote NF-κB- and IRF3-dependent transcription of PD-L1 in melanocytes. UVR exposure significantly reduced the susceptibility of melanoma cells to CD8+ T-cell-dependent cytotoxicity, which was mitigated by inhibiting the HMGB1/TBK1/IRF3/NF-κB cascade or by blocking the PD-1/PD-L1 checkpoint. Taken together, our findings demonstrate that UVR-induced upregulation of PD-L1 contributes to immune suppression in the skin microenvironment, which may promote immune evasion of oncogenic cells and drive melanoma initiation and progression. SIGNIFICANCE: These findings identify PD-L1 as a critical component of UV-induced immune suppression in the skin, which facilitates immunoevasion of oncogenic melanocytes and development of melanoma.See related commentary by Sahu, p. 2805.

BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion.

BACKGROUND: Metastasis is responsible for a significant number of breast cancer-related deaths. Hypoxia, a primary driving force of cancer metastasis, induces the expression of BHLHE40, a transcription regulator. This study aimed to elucidate the function of BHLHE40 in the metastatic process of breast cancer cells. METHODS: To define the role of BHLHE40 in breast cancer, BHLHE40 expression was knocked down by a lentiviral construct expressing a short hairpin RNA against BHLHE40 or knocked out by the CRISPR/Cas9 editing system. Orthotopic xenograft and experimental metastasis (tail vein injection) mouse models were used to analyze the role of BHLHE40 in lung metastasis of breast cancer. Global gene expression analysis and public database mining were performed to identify signaling pathways regulated by BHLHE40 in breast cancer. The action mechanism of BHLHE40 was examined by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (CoIP), exosome analysis, and cell-based assays for metastatic potential. RESULTS: BHLHE40 knockdown significantly reduced primary tumor growth and lung metastasis in orthotopic xenograft and experimental metastasis models of breast cancer. Gene expression analysis implicated a role of BHLHE40 in transcriptional activation of heparin-binding epidermal growth factor (HBEGF). ChIP and CoIP assays revealed that BHLHE40 induces HBEGF transcription by blocking DNA binding of histone deacetylases (HDAC)1 and HDAC2. Cell-based assays showed that HBEGF is secreted through exosomes and acts to promote cell survival and migration. Public databases provided evidence linking high expression of BHLHE40 and HBEGF to poor prognosis of triple-negative breast cancer. CONCLUSION: This study reveals a novel role of BHLHE40 in promoting tumor cell survival and migration by regulating HBEGF secretion.

The conserved RNA recognition motif and C3H1 domain of the Not4 ubiquitin ligase regulate in vivo ligase function.

The Ccr4-Not complex controls RNA polymerase II (Pol II) dependent gene expression and proteasome function. The Not4 ubiquitin ligase is a Ccr4-Not subunit that has both a RING domain and a conserved RNA recognition motif and C3H1 domain (referred to as the RRM-C domain) with unknown function. We demonstrate that while individual Not4 RING or RRM-C mutants fail to replicate the proteasomal defects found in Not4 deficient cells, mutation of both exhibits a Not4 loss of function phenotype. Transcriptome analysis revealed that the Not4 RRM-C affects a specific subset of Pol II-regulated genes, including those involved in transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. The Not4 RING, RRM-C, or RING/RRM-C mutations cause a generalized increase in Pol II binding at a subset of these genes, yet their impact on gene expression does not always correlate with Pol II recruitment which suggests Not4 regulates their expression through additional mechanisms. Intriguingly, we find that while the Not4 RRM-C is dispensable for Ccr4-Not association with RNA Pol II, the Not4 RING domain is required for these interactions. Collectively, these data elucidate previously unknown roles for the conserved Not4 RRM-C and RING domains in regulating Ccr4-Not dependent functions in vivo.

Survivin Inhibitors Mitigate Chemotherapeutic Resistance in Breast Cancer Cells by Suppressing Genotoxic Nuclear Factor-κB Activation.

Therapeutic resistance developed after chemotherapy and aggressive metastasis are the major causes of cancer-related death in patients with triple-negative breast cancer (TNBC). Survivin is the smallest member of the inhibitor-of-apoptosis proteins (IAPs) family, which plays critical roles in cell division and cell survival. High expression levels of survivin have been associated with therapeutic resistance in various cancers. We recently developed a novel small-molecule survivin inhibitor mimicking the IAP-binding motif of second mitochondria-derived activator of caspase, which showed high potency in promoting survivin degradation. Here, we show that survivin inhibitor MX106/MX107 suppresses TNBC cell proliferation. Moreover, MX106/MX107 synergized with chemotherapeutic drugs or radiation and significantly enhanced tumoricidal efficacy of genotoxic treatments. Mechanistically, MX106/MX107 induced degradation of XIAP and/or cIAP1, which inhibited nuclear factor κB (NF-κB) activation by genotoxic agents. Treatment with MX106/MX107 alone did not activate alternative NF-κB signaling in breast cancer cells, which is likely attributable to their selective potency in degrading survivin in these cells. In addition, survivin degradation by MX106/MX107 dramatically increased abnormal mitotic spindle formation and cell division failure, which led to cell cycle arrest in breast cancer cells. Overall, our study suggests that combination treatment of TNBC using survivin inhibitors MX106/MX107 with cytotoxic chemotherapeutic drugs can achieve significantly improved therapeutic efficacy, which depends on MX106/MX107-mediated inhibition of genotoxic NF-κB activation.

The Electrochemical Performance of Silicon Nanoparticles in Concentrated Electrolyte.

Silicon is a promising material for anodes in energy-storage devices. However, excessive growth of a solid-electrolyte interphase (SEI) caused by the severe volume change during the (de)lithiation processes leads to dramatic capacity fading. Here, we report a super-concentrated electrolyte composed of lithium bis(fluorosulfonyl)imide (LiFSI) and propylene carbonate (PC) with a molar ratio of 1:2 to improve the cycling performance of silicon nanoparticles (SiNPs). The SiNP electrode shows a remarkably improved cycling performance with an initial delithiation capacity of approximately 3000 mAh g-1 and a capacity of approximately 2000 mAh g-1 after 100 cycles, exhibiting about 6.8 times higher capacity than the cells with dilute electrolyte LiFSI-(PC)8 . Raman spectra reveal that most of the PC solvent and FSI anions are complexed by Li+ to form a specific solution structure like a fluid polymeric network. The reduction of FSI anions starts to play an important role owing to the increased concentration of contact ion pairs (CIPs) or aggregates (AGGs), which contribute to the formation of a more mechanically robust and chemically stable complex SEI layer. The complex SEI layer can effectively suppress the morphology evolution of silicon particles and self-limit the excessive growth, which mitigates the crack propagation of the silicon electrode and the deterioration of the kinetics. This study will provide a new direction for screening cycling-stable electrolytes for silicon-based electrodes.

MiR-181 family-specific behavior in different cancers: a meta-analysis view.

The involvement of microRNAs in malignant transformation and cancer progression was previously grounded. The observations made by multiple published studies led to the conclusion that some of these small sequences could be eventually used as biomarkers for diagnosis/prognosis. This meta-analysis investigated whether microRNA-181 family members could predict the outcome of patients carrying different types of cancer. We searched the PubMed and Embase databases for studies evaluating the expression levels of miR-181a/b/c/d in patients with cancer, selecting the publications that assessed the relation between low and high levels of one of these four microRNAs and patients' outcome. Hazard ratios (HRs) or risk ratios (RRs) were extracted from the studies, and random-effect model was performed to investigate the role of miR-181 in the outcome of these patients. The meta-analysis comprised 26 studies including 2653 cancer patients from 6 countries. The results showed significant association between the expression of miR-181 family members and colorectal cancer. Considering the heterogeneity of the pathologies, the analysis, including all types of cancer and the expression of all the miR-181 family members together, showed no association with distinct outcome (HR = 1.099, p = 0.435). When the analysis was performed on each microRNA separately, the expression of miR-181c was significantly associated with the outcome of patients with cancer (HR = 2.356, p = 0.011) and miR-181a expression levels significantly correlated with survival in patients with non-small-cell lung cancer (HR = 0.177, p < 0.05). This meta-analysis revealed evidence regarding the involvement of miR-181 family members in the outcome of patients with some types of cancer, according to their expression level.

DNA damage-induced nuclear factor-kappa B activation and its roles in cancer progression.

DNA damage is a vital challenge to cell homeostasis. Cellular responses to DNA damage (DDR) play essential roles in maintaining genomic stability and survival, whose failure could lead to detrimental consequences such as cancer development and aging. Nuclear factor-kappa B (NF-κB) is a family of transcription factors that plays critical roles in cellular stress response. Along with p53, NF-κB modulates transactivation of a large number of genes which participate in various cellular processes involved in DDR. Here the authors summarize the recent progress in understanding DNA damage response and NF-κB signaling pathways. This study particularly focuses on DNA damage-induced NF-κB signaling cascade and its physiological and pathological significance in B cell development and cancer therapeutic resistance. The authors also discuss promising strategies for selectively targeting this genotoxic NF-κB signaling aiming to antagonize acquired resistance and resensitize refractory cancer cells to cytotoxic treatments.

SMARCE1 regulates metastatic potential of breast cancer cells through the HIF1A/PTK2 pathway.

BACKGROUND: While aberrant activation of the chromatin-remodeling SWI/SNF complexes has been associated with cancer development and progression, the role of each subunit in tumor cells is poorly defined. This study is aimed to characterize the role of SMARCE1/BAF57 in regulating metastasis of breast cancer cells. METHODS: Genetic approaches and chemical inhibitors were used to manipulate the activities of SMARCE1 and its downstream targets in multiple breast cancer cell lines. Xenograft mouse models were used to analyze the role of SMARCE1 in lung metastasis in vivo. Nonadherent culture conditions were used to elucidate the role of SMARCE1 in regulating anoikis. Chromatin immunoprecipitation (ChIP), immunoprecipitation, and immunoblotting assays were designed to dissect the mechanism of action of SMARCE1. Public databases were used to investigate the relationship between SMARCE1 deregulation and breast cancer prognosis. RESULTS: SMARCE1 knockdown reduced lung metastasis of breast cancer cells and sensitized tumor cells to anoikis. In response to loss of attachment, SMARCE1 interacted with and potentiated transcriptional activity of HIF1A, resulting in rapid PTK2 activation. Both HIF1A and PTK2 were indispensable for SMARCE1-mediated protection against anoikis by promoting activation of ERK and AKT pathways while suppressing the expression of pro-apoptotic BIM protein. Expression data analysis of a large cohort of human breast tumors revealed that high expression of SMARCE1 or PTK2 is associated with poor prognosis and tumor relapse, and PTK2 expression is positively correlated with SMARCE1 expression in basal-like and luminal B subtypes of breast tumors. CONCLUSIONS: SMARCE1 plays an essential role in breast cancer metastasis by protecting cells against anoikis through the HIF1A/PTK2 pathway. SMARCE1-mediated PTK2 activation likely plays a key role in promoting metastasis of basal-like and luminal B subtype of breast tumors.