Variational Mode Decomposition for Raman Spectral Denoising.

As a fast and non-destructive spectroscopic analysis technique, Raman spectroscopy has been widely applied in chemistry. However, noise is usually unavoidable in Raman spectra. Hence, denoising is an important step before Raman spectral analysis. A novel spectral denoising method based on variational mode decomposition (VMD) was introduced to solve the above problem. The spectrum is decomposed into a series of modes (uk) by VMD. Then, the high-frequency noise modes are removed and the remaining modes are reconstructed to obtain the denoised spectrum. The proposed method was verified by two artificial noised signals and two Raman spectra of inorganic materials, i.e., MnCo ISAs/CN and Fe-NCNT. For comparison, empirical mode decomposition (EMD), Savitzky-Golay (SG) smoothing, and discrete wavelet transformation (DWT) are also investigated. At the same time, signal-to-noise ratio (SNR) was introduced as evaluation indicators to verify the performance of the proposed method. The results show that compared with EMD, VMD can significantly improve mode mixing and the endpoint effect. Moreover, the Raman spectrum by VMD denoising is more excellent than that of EMD, SG smoothing and DWT in terms of visualization and SNR. For the small sharp peaks, some information is lost after denoising by EMD, SG smoothing, DWT and VMD while VMD loses fewest information. Therefore, VMD may be an alternative method for Raman spectral denoising.

A facile method to synthesize 3D nanosheets of Fe/S dopedα-Ni(OH)2 as an electrocatalyst for improved oxygen evolution reaction.

S-doped Fe/Ni oxide and Fe/Ni hydride oxide catalysts exhibit good oxygen evolution reaction (OER) performance. Nevertheless, the over-doping of S and the agglomeration of active sites still hinder the improvement of the performance of these catalysts. The S/O ratio regulation can optimize the electronic structure effectively so as to improve the OER performance of the catalysts, but few studies have focused on this study. Here, we find a facile room-temperature method to synthesize the unique 3D ultra-thin FeNiOS nanosheets with an adjustable S/O ratio for OER. The FeNiOS-NS catalysts exhibit excellent OER performance with an overpotential of 235 mV at 10 mA cm-2and a small Tafel slope of 64.2 mV dec-1in 0.1 M KOH, which originated from the sufficient exposure of the active Fe-Ni component and the optimized electronic structure due to the tunable S/O ratio. This study demonstrates a novel strategy to optimize the OER performance of Ni-based catalysts.

Ubiquitin C-Terminal Hydrolase L1 regulates autophagy by inhibiting autophagosome formation through its deubiquitinating enzyme activity.

Ubiquitination modification has been shown to play a key role in autophagy. Increasing studies reported the involvement of de-ubiquitinating enzymes (DUBs) in autophagy pathway. To systematically search how DUBs manipulate autophagy, we utilized a double fluorescence tagged LC3 stable HeLa cell line, and did a genome wide screen of 55 human DUBs which is about 60% coverage of the DUB family. We found a bunch of DUBs have impact on autophagy by either changing the LC3 puncta formation or the autophagy flux. One of them, Ubiquitin C-Terminal Hydrolase L1 (UCHL1) correlated to Parkinson's disease, strongly affects autophagy by inhibiting autophagosome formation. We found UCHL1 overexpression inhibits LC3 puncta formation and is dependent on its DUB activity. Knockdown of UCHL1 significantly promotes LC3 puncta formation. Further study revealed that UCHL1 may affect autophagy by interacting with LC3 but not other autophagy related proteins. Interestingly, a Parkinson's disease related mutant UCHL1 I93 M defects its DUB activity and can no longer inhibit autophagosome formation. We further screened 22 commercially available DUB inhibitors and found two potent UCHL1 inhibitors LDN-57444 (LDN) and NSC632839 (NSC), when treating cells, both strongly induce LC3 puncta formation. Taken together, our results indicated a new insight into the manner in which DUB regulates autophagy and provided potential drugs for the Parkinson's disease.

Lupeol alters ER stress-signaling pathway by downregulating ABCG2 expression to induce Oxaliplatin-resistant LoVo colorectal cancer cell apoptosis.

Colorectal cancer (CRC) is one of the most common cancers and causes of cancer-related death. There are several first-line chemotherapeutic drugs used to treat CRC. Oxaliplatin (OXA) is an alkylating cytotoxic agent that is usually combined with other chemotherapeutic drugs to treat stage II and stage III CRC. However, cancer cells commonly acquire multidrug resistance (MDR), which is a major obstruction to cancer treatment. Recent studies have shown that natural components from traditional Chinese medicine or foods that have many biological functions may be new adjuvant therapies in clinical trials. We challenged LoVo CRC cell lines with OXA in a dose-dependent manner to create an OXA-resistant model. The expression of ABCG2 was significantly higher, and levels of endoplasmic reticulum (ER) stress markers were lower than those Parental cells. However, Lupeol, which is found in fruits and vegetables, has been shown to have bioactive properties, including anti-tumor properties that are relevant to many diseases. In our study, Lupeol downregulated cell viability and activated cell apoptosis. Moreover, Lupeol decreased the expression of ABCG2 and activated ER stress to induce OXA-resistant cell death. Importantly, the anti-tumor effect of Lupeol in OXA-resistant cells was higher than that of LoVo Parental cells. In addition, we also confirmed our results with a xenograft animal model, and the tumor size significantly decreased after Lupeol injections. Our findings show that Lupeol served as a strong chemoresistant sensitizer and could be a new adjuvant therapy method for chemoresistant patients.

Evaluation of a consulting training course for international development assistance for health.

BACKGROUND: Development assistance for health (DAH) is an important component of foreign assistance. International health consultants usually play a key role in the international DAH field. However, there is still a shortage of consulting training in China. To address this issue and develop new backup force of DAH for China, the Global Health Institute of Wuhan University (GHIWHU) launched a training program called the "Consulting Training Course for International Development Assistance for Health". The purpose of this article is to evaluate the impact of the training on participants. METHODS: We conducted the analysis using Kirkpatrick's model. An evaluation survey examining participants' reaction (level 1) and learning (level 2) was carried out among trainees following the training, and a follow-up telephone interview of application (level 3) was made in three months after the training. RESULTS: A total of 25 participants from Chinese Consortium of Universities for Global Health (CCUGH) attended the training program. Results of satisfaction evaluation indicated that the training program was well received, with more than 85% of participants felt satisfied or relatively satisfied with the training. Trainees' self-ratings of the consulting knowledge and skills showed a significant increase (p < 0.001) from pre- to post-training. The follow-up interview revealed that the majority of participants applied the acquired knowledge and skills under various circumstances such as consulting program, teaching processes, writing reports, and et al. Meanwhile, participants considered that the lack of opportunities was one of the major application barriers. In addition, they expressed the willingness to participate in more relevant training and the need for more practice opportunities. CONCLUSIONS: This is the first study evaluating a consulting training program in China. The results show that the training course has been successfully implemented and participants have been given consulting knowledge and skills. Future research should use better-designed training methods based on demand surveys and consider providing participants with practice or practicum opportunities. Also, it is necessary to conduct both primary and advanced training courses and evaluate participants' long-term behavior changes resulting from the training.

beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation.

ß-Naphthoflavone (ß-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of ß-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with ß-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, ß-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by ß-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by ß-NF treatment. Our findings show that ß-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that ß-NF might prevent TNF-α-induced inflammation.

A20 and CYLD do not share significant overlapping functions during B cell development and activation.

The ubiquitin-editing enzyme A20 (TNFAIP3) and the deubiquitinase CYLD are central negative regulators of NF-κB signaling. Both can act by removing nonproteolytic K63-linked polyubiquitin chains from an overlapping set of signaling molecules. In B cells, A20 deficiency results in hyperactivity, loss of immune homeostasis, inflammation, and autoimmunity. The reported consequences of CYLD deficiency are controversial, ranging from an absence of effects to dramatic B cell hyperplasia. These differences could be due to varying compensation for the loss of CYLD function by A20. Therefore, to explore potential overlapping physiological functions between A20 and CYLD, we generated and characterized A20/CYLD double-deficient B cells. Interestingly, the lack of both A20 and CYLD did not exacerbate the developmental defects and hyperresponsive activity of A20-deficient B cells. In addition, the extent of B cell activation after in vitro stimulation with anti-CD40, LPS, and CpG was comparable in B cells lacking A20/CYLD and A20 alone. However, in response to BCR cross-linking, we observed small but reproducible additive effects of the lack of A20 and CYLD. Taken together, our results demonstrate that A20 and CYLD do not share significant functions during B cell development and activation.

Preparation and catalytic performance of temperature-responsive cell-like particles.

A novel kind of cell-like particles as temperature-responsive catalysts was presented in this paper. First, uniform α-Fe2O3shuttle-like nanoparticles were prepared by homogeneous hydrolysis. Then, these α-Fe2O3particles were coated by Au nanoparticles (AuNPs), SiO2and poly (N-isopropylacrylamide) (PNIPAM), respectively. After the removal of SiO2layer by etching with HF solution, the cell-like particles were prepared when the α-Fe2O3, AuNPs, and PNIPAM were as cell nucleus, catalysts, and cell membranes, respectively. These cell-like particles showed a novel temperature-responsively catalytic performance because the PNIPAM shell could change its hydrophilicity and swelling capacity under different temperature. When the temperature was 25°C, the yield of 4-aminophenol (4-AP) from 4-nitrophenol (4-NP) by reduction of NaBH4was about 100% in 15 min, while the yield of 4-AP was about 90.5% in 40 min. when the temperature was 40°C.

CpG adjuvant enhances humoral and cellular immunity against OVA in different degrees in BALB/c, C57BL/6J, and C57BL/6N mice.

In lab settings, inbred mouse strains like BALB/c, C57BL/6J, and C57BL/6N are commonly used. Research in immunology and infectious diseases indicates that their Th1 and Th2 immune responses differ. However, the specific differences in the immune response to the vaccination still require investigation. In this study, ovalbumin (OVA) was used as an antigen and CpG-enriched recombinant plasmid (pUC18-CpG) as an adjuvant for immunisation. The level of serum-specific antibody IgG was detected by indirect ELISA. At 35dpi, serum cytokine levels were measured using MILLIPLEX®. T lymphocyte clusters from mouse spleen were examined using flow cytometry to investigate the immunological effects of the CPG-OVA vaccine on three different types of mice. The results showed that pUC18-CpG as an adjuvant could successfully enhance the immune response. BALB/c had the highest level of IgG antibody. In the OVA-only group, the CD4+/CD8+ ratio of the three types of mice was generally increased, and the BALB/c group had the highest ratio. After inoculation with CpG-OVA, the CD4+/CD8+ ratio of the three types of mice was lower than that of the OVA-only group, and C57BL/6J was the lowest. Compared with the CpG-OVA group of the three kinds of mice, the levels of Th2 cytokines IL-6 and IL-10 in BALB/c were increased compared with C57BL/6J and C57BL/6N. After OVA, the six cytokines secreted in C57BL/6J were higher than those in the C57BL/6N OVA group. Therefore, C57 is a better model for examining the function of the vaccine in cellular immunity, whereas BALB/c mice are more prone to humoral immunity. In addition to highlighting the CpG plasmid's ability to successfully activate the immune response of Th1 and Th2, as well as the expression of IgG in vivo and promote T cell immune typing, this study provides valuable insights into immunology and the selection of mouse models for infectious diseases, providing a valuable resource for designing more effective vaccines in the future.

Immunogenicity Analysis and Identification of Potential T-Cell Epitopes in C129R Protein of African Swine Fever Virus.

The highly conserved C129R protein of AFSV was utilized in the development of an ASFV recombinant adenovirus vaccine, demonstrating strong immunogenicity. In this study, we immunized 6-week-old female C57BL/6J mice via subcutaneous injection with 10 µg of purified C129R protein. Humoral and cellular immune effects were assessed using ELISA, flow cytometry, and ELISpot assays. Additionally, 19 peptides of the C129R protein were synthesized and screened for the use of bioinformatics. Positive T-cell epitopes were screened using ELISpot. The results indicated a higher proportion of CD4+ and CD8+ T lymphocytes in immunized mice compared to control mice. ELISA analysis revealed a serum titer of approximately 1:1, 638, 400 in the experimental group of mice. Additionally, peptides C11(53-61aa), C14(81-89aa), C16(97-105aa), and C18(116-124aa) from the C129R protein were able to activate mice spleen lymphocytes to produce IFN-γ. These findings suggest that the C129R protein significantly enhances both humoral and cellular immunity in immunized mice. Moreover, peptides C11, C14, C16, and C18 may serve as potential T-cell epitopes for the C129R protein. These results lay the groundwork for the further exploration of ASFV C129R protein and the identification of novel ASF vaccine antigens.

Analysis of the Immunogenicity of African Swine Fever F317L Protein and Screening of T Cell Epitopes.

The African swine fever virus (ASFV) encodes numerous proteins characterized by complex immune escape mechanisms. At present, the structure and function of these proteins, including the F317L protein, have yet to be fully elucidated. In this study, we examined the immunogenicity of the F317L protein. Mice were subcutaneously immunized with the F317L protein using initial and subsequent booster doses, and, at the 28th day post-treatment, we assessed the humoral and cellular immune responses of mice. The F317L protein stimulated production of specific antibodies and activated humoral immune responses. In addition, F317L stimulated the production of large amounts of IFN-γ by splenic lymphocytes, thereby activating cellular immune responses. Using informatics technology, we predicted and synthesized 29 F317L protein T cell epitopes, which were screened using IFN-γ ELISpot. Among these, the F25 (246SRRSLVNPWT255) peptide was identified as having a stronger stimulatory effect than the full-length protein. Collectively, our findings revealed that the ASFV F317L protein can stimulate both strong humoral and cellular immunity in mice, and that the F25 (246SRRSLVNPWT255) peptide may be a potential active T cell epitope. These findings will provide a reference for further in-depth studies of the F317L protein and screening of antigenic epitopes.

Actomyosin-II protects axons from degeneration induced by mild mechanical stress.

Whether, to what extent, and how the axons in the central nervous system (CNS) can withstand sudden mechanical impacts remain unclear. By using a microfluidic device to apply controlled transverse mechanical stress to axons, we determined the stress levels that most axons can withstand and explored their instant responses at nanoscale resolution. We found mild stress triggers a highly reversible, rapid axon beading response, driven by actomyosin-II-dependent dynamic diameter modulations. This mechanism contributes to hindering the long-range spread of stress-induced Ca2+ elevations into non-stressed neuronal regions. Through pharmacological and molecular manipulations in vitro, we found that actomyosin-II inactivation diminishes the reversible beading process, fostering progressive Ca2+ spreading and thereby increasing acute axonal degeneration in stressed axons. Conversely, upregulating actomyosin-II activity prevents the progression of initial injury, protecting stressed axons from acute degeneration both in vitro and in vivo. Our study unveils the periodic actomyosin-II in axon shafts cortex as a novel protective mechanism, shielding neurons from detrimental effects caused by mechanical stress.

B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice.

The ubiquitin-editing enzyme A20/TNFAIP3 is essential for controlling signals inducing the activation of nuclear factor-κB transcription factors. Polymorphisms and mutations in the TNFAIP3 gene are linked to various human autoimmune conditions, and inactivation of A20 is a frequent event in human B-cell lymphomas characterized by constitutive nuclear factor-κB activity. Through B cell-specific ablation in the mouse, we show here that A20 is required for the normal differentiation of the marginal zone B and B1 cell subsets. However, loss of A20 in B cells lowers their activation threshold and enhances proliferation and survival in a gene-dose-dependent fashion. Through the expression of proinflammatory cytokines, most notably interleukin-6, A20-deficient B cells trigger a progressive inflammatory reaction in naive mice characterized by the expansion of myeloid cells, effector-type T cells, and regulatory T cells. This culminates in old mice in an autoimmune syndrome characterized by splenomegaly, plasma cell hyperplasia, and the presence of class-switched, tissue-specific autoantibodies.

Salmonella enterica serovar Typhi plasmid impairs dendritic cell responses to infection.

Salmonella enterica serovar Typhi (S. typhi) evades from innate immunity by expression of a variety of pathogenic factors. The "pR(ST98)" plasmid of S. typhi is involved in multidrug-resistant and virulence of S. typhi. However, its exact effect on host cell function remains elusive. Dendritic cells (DCs) play an important role in shaping immune response against Salmonella. For the purpose of investigation whether pR(ST98) might target DCs involved in adaptive immune response, murine DCs were infected with S. typhi wild type and mutant strains. S. typhi stimulation resulted in up-regulation of costimulatory molecules on DCs. S. typhi wild type resulted in decreased up-regulation of CD40, CD80, and CD86 expression. Experiments with S. typhi pR(ST98) mutant (S. typhi-Δ-pR(ST98)) and S. typhi-Δ-pR(ST98) with a complemented plasmid encoding pR(ST98) (S. typhi-c-pR(ST98)) revealed that pR(ST98) accounts for inhibition of surface molecule expression and functional maturity. S. typhi-Δ-pR(ST98) gave maximal levels of IL-12 and IFN-γ release compared with wild type S. typhi or the complemented strains. In contrast to IL-12 and IFN-γ, IL-10 secretion by S. typhi-Δ-pR(ST98)-infected DCs was significantly lower than induction by S. typhi wild type. This indicates that immunity in response to pR(ST98) is skewed away from a protective Th1 response. Moreover, infection with S. typhi-Δ-pR(ST98) induced autophagy in DCs. We herein demonstrate S. typhi pR(ST98) plays essential roles in modulating DCs maturation, activation, inflammatory responses, and autophagy. Together, these data prove that pR(ST98) targets functions of DCs that are required for T-cell activation. This might contribute to evasion of adaptive immune responses by S. typhi.

Spectral denoising based on Hilbert-Huang transform combined with F-test.

Due to the influence of uncontrollable factors such as the environment and instruments, noise is unavoidable in a spectral signal, which may affect the spectral resolution and analysis result. In the present work, a novel spectral denoising method is developed based on the Hilbert-Huang transform (HHT) and F-test. In this approach, the original spectral signal is first decomposed by empirical mode decomposition (EMD). A series of intrinsic mode functions (IMFs) and a residual (r) are obtained. Then, the Hilbert transform (HT) is performed on each IMF and r to calculate their instantaneous frequencies. The mean and standard deviation of instantaneous frequencies are calculated to further illustrate the IMF frequency information. Third, the F-test is used to determine the cut-off point between noise frequency components and non-noise ones. Finally, the denoising signal is reconstructed by adding the IMF components after the cut-off point. Artificially chemical noised signal, X-ray diffraction (XRD) spectrum, and X-ray photoelectron spectrum (XPS) are used to validate the performance of the method in terms of the signal-to-noise ratio (SNR). The results show that the method provides superior denoising capabilities compared with Savitzky-Golay (SG) smoothing.

Radiomics for differentiating minimally invasive adenocarcinoma from precursor lesions in pure ground-glass opacities on chest computed tomography.

OBJECTIVE: To explore the correlation between radiomic features and the pathology of pure ground-glass opacities (pGGOs), we established a radiomics model for predicting the pathological subtypes of minimally invasive adenocarcinoma (MIA) and precursor lesions. METHODS: CT images of 1521 patients with lung adenocarcinoma or precursor lesions appearing as pGGOs on CT in our hospital (The Third Affiliated Hospital of Sun Yat-sen University) from January 2015 to March 2021 were analyzed retrospectively and selected based on inclusion and exclusion criteria. pGGOs were divided into an atypical adenomatous hyperplasia (AAH)/adenocarcinoma in situ (AIS) group and an MIA group. Radiomic features were extracted from the original and preprocessed images of the region of interest. ANOVA and least absolute shrinkage and selection operator feature selection algorithm were used for feature selection. Logistic regression algorithm was used to construct radiomics prediction model. Receiver operating characteristic curves were used to evaluate the classification efficiency. RESULTS: 129 pGGOs were included. 2107 radiomic features were extracted from each region of interest. 18 radiomic features were eventually selected for model construction. The area under the curve of the radiomics model was 0.884 [95% confidence interval (CI), 0.818-0.949] in the training set and 0.872 (95% CI, 0.756-0.988) in the test set, with a sensitivity of 72.73%, specificity of 88.24% and accuracy of 79.47%. The decision curve indicated that the model had a high net benefit rate. CONCLUSION: The prediction model for pathological subtypes of MIA and precursor lesions in pGGOs demonstrated a high diagnostic accuracy. ADVANCES IN KNOWLEDGE: We focused on lesions appearing as pGGOs on CT and revealed the differences in radiomic features between MIA and precursor lesions. We constructed a radiomics prediction model and improved the diagnostic accuracy for the pathology of MIA and precursor lesions.

Metabolic Effects of Breastfeeding in Women with Previous Gestational Diabetes Mellitus: A Meta-Analysis.

Objective: To investigate the effects of breastfeeding (BF) on metabolic-related outcomes in women with previous gestational diabetes mellitus. Methods: Databases, including PubMed, Embase, Cochrane Library, and Web of Science, were searched until March 5, 2020. Finally, 14 high-quality articles were included. Relative risk (RR) and weighted mean difference (WMD) with 95% confidence interval (CI) were pooled using Stata14.0 Software. Results: Subjects in the BF group had a lower incidence of diabetes (RR: 0.611, 95% CI: 0.452-0.826, p < 0.001) and lower fasting plasma glucose level (WMD: -4.762, 95% CI: -5.552 to -3.973, p < 0.001), fasting insulin level (WMD: -21.513, 95% CI: -37.594 to -5.431, p = 0.009), homeostasis model assessment of insulin resistance (HOMA-IR) (WMD: -1.107, 95% CI: -1.683 to -0.532, p < 0.001), and triglyceride level (WMD: -33.951, 95% CI: -50.714 to -17.189, p < 0.001) than those in the non-BF group. The high-density lipoprotein level (WMD: 3.855, 95% CI: 2.629-5.081, p < 0.001), low-density lipoprotein level (WMD: 4.223, 95% CI: 0.6712-7.774, p = 0.020), and insulin sensitivity index (WMD: 1.503, 95% CI: 0.857-2.160, p < 0.001) in the BF group were higher than that in the non-BF group. No difference was found in the 2-hour postprandial blood glucose (WMD: -3.804, 95% CI: -8.237 to 0.630, p = 0.093), incidence of prediabetes mellitus (RR: 0.870, 95% CI: 0.750-1.009, p = 0.065), or cholesterol level (WMD: 1.377, 95% CI: -8.178 to 10.931, p = 0.778) between the two groups. Conclusion: BF may improve several metabolic markers and decrease the risk of developing diabetes.

LUBAC and OTULIN regulate autophagy initiation and maturation by mediating the linear ubiquitination and the stabilization of ATG13.

Macroautophagy/autophagy is a membrane-mediated intracellular degradation pathway, through which bulky cytoplasmic content is digested in lysosomes. How the autophagy initiation and maturation steps are regulated is not clear. In this study, we found an E3 ubiquitin ligase complex, linear ubiquitin chain assembly complex (LUBAC) and a deubiquitinating enzyme (DUB) OTULIN localize to the phagophore area to control autophagy initiation and maturation. LUBAC key component RNF31/HOIP translocates to the LC3 puncta area when autophagy is induced. RNF31 knockdown inhibits autophagy initiation, and cells are more sensitive to bacterial infection. OTULIN knockdown, however, promotes autophagy initiation but blocks autophagy maturation. In OTULIN knockdown cells, excessive ubiquitinated ATG13 protein was recruited to the phagophore for prolonged expansion, and therefore inhibits autophagosome maturation. Together, our study provides evidence that LUBAC and OTULIN cooperatively regulate autophagy initiation and autophagosome maturation by mediating the linear ubiquitination and the stabilization of ATG13.Abbreviations: ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; CUL1-FBXL20: cullin 1-F-box and leucine rich repeat protein 20; CUL3-KLHL20: cullin 3-kelch like family member 20; CUL4-AMBRA1: cullin 4-autophagy and beclin 1 regulator 1; CYLD: CYLD lysine 63 deubiquitinase; DAPI: 4',6-diamidino-2-phenylindole; DUB: deubiquitinating enzyme; EBSS: Earle's Balanced Salt Solution; GFP: green fluorescent protein; GST: glutathione S-transferase; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; LUBAC: linear ubiquitin chain assembly complex; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3B; MIM: MIT-interacting motif; mRFP: monomeric red fluorescent protein; NEDD4: NEDD4 E3 ubiquitin protein ligase; NFKB: NF-kappaB complex; OPTN: optineurin; OTULIN: OTU deubiquitinase with linear linkage specificity; PIK3C3/Vps34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns: phosphatidylinositol; PtdIns3K: class III phosphatidylinositol 3-kinase complex; PtdIns3P: phosphatidylinositol 3-phosphate; RBCK1/HOIL1: RANBP2-type and C3HC4-type zinc finger containing 1; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RIPK1: receptor interacting serine/threonine kinase 1; RNF216: ring finger protein 216; RNF31/HOIP: ring finger protein 31; RT-PCR: reverse transcriptase polymerase chain reaction; S. Typhimurium: Salmonella enterica serovar Typhimurium; SHARPIN: SHANK associated RH domain interactor; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING: stimulator of interferon response cGAMP interactor 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TNF/TNF-alpha: tumor necrosis factor; TNFAIP3/A20: TNF alpha induced protein 3; TRAF6: TNF receptor associated factor 6; TRIM32: tripartite motif containing 32; UBAN: ubiquitin binding in TNIP/ABIN and IKBKG/NEMO proteins; ULK1/2: unc-51 like autophagy activating kinase 1/2; USP: ubiquitin specific peptidase; UVRAG: UV radiation resistance associated; VCPIP1: valosin containing protein interacting protein 1; WIPI2: WD repeat domain, phosphoinositide interacting protein 2; ZBTB16-CUL3-RBX1: zinc finger and BTB domain containing protein 16-cullin 3-ring-box 1; ZRANB1: zinc finger RANBP2-type containing 1.

Runt-related transcription factor 1 promotes apoptosis and inhibits neuroblastoma progression in vitro and in vivo.

BACKGROUND: Runt-related transcription factor 1 (RUNX1) is a heterodimeric transcription factor that binds to the core element of many enhancers and promoters and can accelerate apoptosis in various tumors. However, the regulatory mechanisms underlying RUNX1 expression in neuroblastoma (NB), a highly malignant tumor in childhood, remain largely unclear. In this study, we aimed to assess the role of RUNX1 in NB and to reveal the underlying mechanisms that may contribute to finding a potential therapeutics strategy against NB. METHODS: Growth, invasion, metastasis and angiogenesis were assessed using Cell Counting Kit-8 (CCK-8) immunocytochemistry, and studies involving soft agar, cell invasion, tube formation and whole animals. The levels of expression were measured using real-time quantitative PCR for RNA, Western blot and immunostaining analyses for proteins. Luciferase reporter and chromatin immunoprecipitation assays indicated that RUNX1 directly binds within the BIRC5, CSF2RB and NFKBIA promoter regions to facilitate transcription. The level of apoptosis was assessed by determining mitochondrial membrane potential and flow cytometry. RESULTS: RUNX1 was highly expressed in ganglioneuroma (GN) and well-differentiated (WD) tissues relative to the poorly differentiated (PD) and undifferentiated (UD) ones. Moreover, RUNX1 effectively reduced cell viability, invasion, metastasis, angiogenesis, and promoted apoptosis in vitro and in vivo. RUNX1 reduced BIRC5 transcription and increased CSF2RB and NFKBIA transcription by directly binding BIRC5, CSF2RB and NFKBIA promoters. In addition, cytotoxic drugs, especially cisplatin, significantly increased RUNX1 expression in NB cells and promoted apoptosis. CONCLUSIONS: These data show that RUNX1 is an independent surrogate marker for the progression of NB and it can be used for monitoring NB prognosis during therapy.

The Chaperone BAG6 Regulates Cellular Homeostasis between Autophagy and Apoptosis by Holding LC3B.

AMFR/gp78 and USP13 are a pair of ubiquitin ligase and deubiquitinase that ensure the accuracy of endoplasmic reticulum-associated degradation (ERAD). Depletion of USP13 leads to caspase activation and cleavage of the ERAD chaperone BAG6, which is reversed by knockdown of AMFR. However, the mechanism and physiological relevance of this regulation are still unclear. Here, by using the NEDDylator system, we screened out TXN as a substrate of AMFR and USP13 and showed its involvement in regulating CASP3 activation and BAG6 cleavage. Furthermore, we showed that the cleaved N-terminal BAG6 is located in the cytosol and interacts with both LC3B-I and unprocessed form of LC3B (Pro-LC3B) through the LIR1 motif to suppress autophagy. An NMR approach verified the direct interaction between BAG6 LIR1 and LC3B-I or Pro-LC3B. Collectively, our findings uncover a mechanism that converts BAG6 from an ERAD regulator to an autophagy tuner and apoptosis inducer during ER stress.