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Annexin B1 has been cloned in Escherichia coli ( E. coli) K802 in previous works. After temperature shift induction the protein was expressed as soluble form. Then a separation method for annexin B1 from E. coli cells by ion-exchange chromatography has been developed and validated. The method exhibited a good reproducibility and is easy to operate. It has shown that annexin B1 isolated by chromatography had high anticoagulant activity. [ABSTRACT FROM AUTHOR]

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time. [ABSTRACT FROM AUTHOR]

With the continuous growth of global energy demand, the artificial photosynthetic system of photocatalytic decomposition of hydrogen production has been widely concerned, CdZnS, a photocatalyst based on solid solution, has shown good performance. In order to avoid the rapid recombination of photogenerated electron and hole, metal boride is added as a catalyst to enhance its photocatalytic hydrogen production performance. We prepared CdZnS photocatalyst by hydrothermal method, and synthesized different proportions of NiB/CdZnS catalyst by programmed heating method and calcined it at 773K. Characterized by XRD, XPS, uv–vis diffuse reflection and other methods, CZS solid solution and NiB/CdZnS were synthesized, and the absorption edge of NiB/CZS was redshifted relative to the pure CdZnS. The addition of NiB can effectively inhibit the recombination of electrons and holes to improve the separation efficiency of photogenerated charge, and its content has an impact on photocatalytic activity. We found that under visible light irradiation, when the content is 15 wt%, the hydrogen production was the highest. The optimal quantum efficiency was 13.3%, and the hydrogen production reacheed 8137 μmol/g/h, which is 17 times that of pure CdZnS. The addition of NiB greatly improved the photocatalytic performance of CdZnS. • a new NiB/ZnCdS catalysts. • NiB/CdZnS shows 17 times activity of CdZnS. • NiB accelerates the migration rate of photoelectrons. [ABSTRACT FROM AUTHOR]

Background: Poor drug response of sorafenib is a major challenge which reduces clinical benefit of renal cell carcinoma (RCC) patients. It is therefore of great clinical significance to elucidate the underlying mechanism to restore the therapeutic response to sorafenib.Methods: Angiopoietin-like protein 3 (ANGPTL3) protein levels were measured by western blot and immunohistochemistry in two cohorts of RCC patients. Loss-of-function and gain-of-function experiments were performed to investigate the biological roles of ANGPTL3 in response to sorafenib treatment in RCC cells. Human proteome microarray and immunoprecipitation analysis were performed to explore the molecular mechanisms underlying the functions of ANGPTL3.Results: ANGPTL3 was upregulated in sorafenib-responsive RCC, which correlated with clinically good sorafenib response. Knockdown of ANGPTL3 conferred sorafenib-tolerance traits to RCC cells, whereas overexpression of ANGPTL3 restored sorafenib sensitivity in RCC cells. Mechanistically, ANGPTL3 bound to Focal Adhesion Kinase(FAK) and restained sorafenib induced nuclear translocation of FAK, leading to attenuate the ubiquitination of p53, which contributed to cellular apoptosis and enhanced sorafenib response.Conclusions: ANGPTL3 may be a novel predictor for the response of sorafenib therapy in RCC patients, and a potential target in improving its therapeutic effect. [ABSTRACT FROM AUTHOR]

Abstract: Store-operated Ca2+ entry (SOCE) is a main Ca2+ influx pathway controlling the intracellular Ca2+ concentration in normal hepatocytes and hepatocellular carcinoma (HCC) cells. Ca2+ influx has been demonstrated to be involved in liver oncogenesis. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca2+ stored in the endoplasmic reticulum, and Orai1 protein constitutes the pore-forming subunit of the store-operated channels. Recently, STIM1 and Orai1 were found critical for breast tumor cell migration and metastasis. However, the effects of Ca2+ influx pathway on migration and metastasis have not been studied in hepatocellular carcinoma. Here, we found that STIM1 had a higher expression in hepatoma tissues than in precancerous tissues of the same patients. In general, STIM expression is elevated in HCC cell lines compared to a normal hepatocyte cell line. HCC-LM3 cell, which has a higher migration ability, expresses five times higher level of STIM than other HCC cell lines. STIM1 could then be explored as a prognostic marker to screen liver cancer patients with high metastatic potential. Inhibition of SOCE and STIM1 enhance focal adhesions and decrease the focal adhesion turnover, suggesting the therapeutic potential of SOCE and STIM1 as new molecular targets for metastatic HCC. [Copyright &y& Elsevier]

Abstract: The present study aims at establishing a novel vaccine procedure based on HBc-VLP-pulsed DCs. Immature mice BMDCs could capture HBc-VLP or HBc-VLP packaging CpG efficiently and present the antigen to syngeneic mice spleen T cells in vitro. Immunization with DCs showed that compared to VLP-pulsed DCs, VLP packaging CpG-pulsed DCs elicit stronger T-cell responses in vivo, as measured by both intracellular production of IFN-γ and in vivo killing assays by Ag-specific T cells. In the B16-pIR-HH tumor therapy model, the growth of established tumors was significantly inhibited by single immunization of DCs pulsed with HBc-VLP packaged with CpG, resulting in significantly longer survival of immunized animals and strikingly, high frequencies (>10% of CD8+ cells) of protective CTL could be induced and maintained. The mice immunized with DCs treated with HBc-VLP, however, trigger an antitumor effect at the early phase of vaccination, after 20 days of tumor injection, the tumor growth inhibition of VLP-pulsed DCs vaccination was decreased gradually and the fact could be interpreted by the decreasing number of antigen-specific CD8+ T-cell and IFN-γ+-producing CD8+ T cell. This study therefore shows that the use of HBc-VLP packaging CpG-pulsed DCs could facilitate the development of effective T-cell-based vaccines. [Copyright &y& Elsevier]

Abstract: Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. HLA-A2-transgenic mouse is a useful tool for measuring the CTL responses in vitro. However, tumor vaccine development is required to address the variables that are not easily evaluated by in vitro assays. With the objective of extending the usage of A2-tansgenic mouse in vaccine efficacy assay, here, we established a B16 tumor cell line coexpressing HLA-A*0201/H-2Kb chimeric gene and a polyepitope construct based on the use of a mammalian expression vector pIRES. The value as a tool for evaluating the antitumor efficacy in vitro as well as in experimental tumor challenge model in vivo has been tested. We found that priming with the polyepitope construct and boosting with the mixture of peptide in A2-transgenic mice resulted in: (1) CTL responses not only against the peptide-sensitized T2 and SW480 cell lines but also the non-sensitized reconstructed B16 cell line; (2) expression of HLA-A*0201/H-2Kb chimeric gene and polyepitopes by B16 led to its rejection by immunized A2-transgenic mice. These data established that the reconstructed B16 cell line stably expressed and efficiently presented the HCC-derived CTL epitopes, making B16 based melanoma suitable for the evaluation of the antitumor efficacy of immune responses to these epitopes. Collectively, these data indicate that the use of this method allows for directly testing of HLA-A2 restricted epitope immunogenicity in the A2-transgenic mice. [Copyright &y& Elsevier]

Abstract: Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a β2-microglobulin (β2m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and β2m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 α1, HLA-A2 α2 and MHC-H2D α3) and β2m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18–27 FLPSDFFPSI, HBx52–60 HLSLRGLPV, and HBx92–100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice. [Copyright &y& Elsevier]

Rationale and Objectives: The aim of this study was to evaluate the capability of an ultrasound (US)-based deep learning (DL) nomogram for predicting axillary lymph node (ALN) status after neoadjuvant chemotherapy (NAC) in breast cancer patients and its potential to assist radiologists in diagnosis., Methods: Two medical centers retrospectively recruited 535 node-positive breast cancer patients who had undergone NAC. Center 1 included 288 patients in the training cohort and 123 patients in the internal validation cohort, while center 2 enrolled 124 patients for the external validation cohort. Five DL models (ResNet 34, ResNet 50, VGG19, GoogLeNet, and DenseNet 121) were trained on pre- and post-NAC US images, and the best model was chosen. A US-based DL nomogram was constructed using DL predictive probabilities and clinicopathological characteristics. Furthermore, the performances of radiologists were compared with and without the assistance of the nomogram., Result: ResNet 50 performed best among all DL models, achieving areas under the curve (AUCs) of 0.837 and 0.850 in the internal and external validation cohorts, respectively. The US-based DL nomogram demonstrated strong predictive ability for ALN status post-NAC, with AUCs of 0.890 and 0.870 in the internal and external validation cohorts, respectively, outperforming both the clinical model and the DL model (p all < 0.05, except p = 0.19 for DL model in external validation cohort). Moreover, the nomogram significantly improved radiologists' diagnostic ability., Conclusion: The US-based DL nomogram is promising for predicting ALN status post-NAC and could assist radiologists for better diagnostic performance., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

Background: Monoglycerides have emerged as a promising alternative to conventional practices due to their biological activities, including antimicrobial properties. However, few studies have assessed the efficacy of monoglyceride blend on weaned pigs and their impacts on performance, immune response, and gut health using a disease challenge model. Therefore, this study aimed to investigate the effects of dietary monoglycerides of short- and medium-chain fatty acids on the immunity and gut health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli F18., Results: Pigs supplemented with high-dose zinc oxide (ZNO) had greater (P < 0.05) growth performance than other treatments, but no difference was observed in average daily feed intake between ZNO and monoglycerides groups during the post-challenge period. Pigs in ZNO and antibiotic groups had lower (P < 0.05) severity of diarrhea than control, but the severity of diarrhea was not different between antibiotic and monoglycerides groups. Pigs fed with monoglycerides or ZNO had lower (P < 0.05) serum haptoglobin on d 2 or 5 post-inoculation than control. Pigs in ZNO had greater (P < 0.05) goblet cell numbers per villus, villus area and height, and villus height:crypt depth ratio (VH:CD) in duodenum on d 5 post-inoculation than pigs in other treatments. Pigs supplemented with monoglycerides, ZNO, or antibiotics had reduced (P < 0.05) ileal crypt depth compared with control on d 5 post-inoculation, contributing to the increase (P = 0.06) in VH:CD. Consistently, pigs in ZNO expressed the lowest (P < 0.05) TNFa, IL6, IL10, IL12, IL1A, IL1B, and PTGS2 in ileal mucosa on d 5 post-inoculation, and no difference was observed in the expression of those genes between ZNO and monoglycerides. Supplementation of ZNO and antibiotic had significant impacts on metabolic pathways in the serum compared with control, particularly on carbohydrate and amino acid metabolism, while limited impacts on serum metabolites were observed in monoglycerides group when compared with control., Conclusions: The results suggest that supplementation of monoglyceride blend may enhance disease resistance of weaned pigs by alleviating the severity of diarrhea and mitigating intestinal and systemic inflammation, although the effectiveness may not be comparable to high-dose zinc oxide., (© 2024. The Author(s).)

Bromophenols has been proven to synthesize hydroxylated polybrominated diphenyl ethers (OH-PBDEs), which may pose additional environmental and health risks in the process of bioremediation. In this study, the removal of 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP) and the biosynthetic of OH-PBDEs by Prorocentrum donghaiense were explored. The removal efficiencies of 2,4-DBP and 2,4,6-TBP ranged from 32.71% to 76.89% and 31.15%-78.12%, respectively. Low concentrations of 2,4-DBP stimulated algal growth, while high concentrations were inhibitory. Furthermore, exposure to 10.00 mg L -1 2,4-DBP resulted in the detection of 2'-hydroxy-2,3',4,5'-tetrabromodiphenyl ether (2'-OH-BDE-68) within P. donghaiense. In contrast, increasing concentrations of 2,4,6-TBP considerably inhibited P. donghaiense growth, with 4'-hydroxy-2,3',4,5',6-pentabromodiphenyl ether (4'-OH-BDE-121) detected within P. donghaiense under 5.00 mg L -1 2,4,6-TBP. Metabolomic analysis further revealed that the synthesized OH-PBDEs exhibited higher toxicity than their precursors and identified the oxidative coupling as a key biosynthetic mechanism. These findings confirm the capacity of P. donghaiense to remove bromophenols and biosynthesize OH-PBDEs from bromophenols, offering valuable insights into formulating algal bioremediation to mitigate bromophenol contamination., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Ferroptosis is associated with the pathological progression of hemorrhagic injury and ischemia-reperfusion injury. According to our previous study, exosomes formed through bone marrow mesenchymal stem cells modified with miR-340-3p (MB-exos) can restore damaged endometrium. However, the involvement of ferroptosis in endometrial injury and the effect of MB-exos on ferroptosis remain elusive., Methods: The endometrial injury rat model was developed. Exosomes were obtained from the supernatants of bone marrow mesenchymal stromal cells (BMSCs) and miR-340/BMSCs through differential centrifugation. We conducted RNA-seq analysis on endometrial tissues obtained from the PBS and MB-exos groups. Ferroptosis was induced in endometrial stromal cells (ESCs) by treating them with erastin or RSL3, followed by treatment with B-exos or MB-exos. We assessed the endometrial total m 6 A modification level after injury and subsequent treatment with B-exos or MB-exos by methylation quantification assay. We performed meRIP-qPCR to analyze m 6 A modification-regulated endogenous mRNAs., Results: We reveal that MB-exos facilitate the injured endometrium to recover by suppressing ferroptosis in endometrial stromal cells. The injured endometrium showed significantly upregulated N 6 -methyladenosine (m 6 A) modification levels; these levels were attenuated by MB-exos through downregulation of the methylase METTL3. Intriguingly, METTL3 downregulation appears to repress ferroptosis by stabilizing HMOX1 mRNA, thereby potentially elucidating the mechanism through which MB-exos inhibit ferroptosis in ESCs. We identified YTHDF2 as a critical m 6 A reader protein that contributes to HMOX1 mRNA degradation. YTHDF2 facilitates HMOX1 mRNA degradation by identifying the m 6 A binding site in the 3'-untranslated regions of HMOX1. In a rat model, treatment with MB-exos ameliorated endometrial injury-induced fibrosis by inhibiting ferroptosis in ESCs. Moreover, METTL3 short hairpin RNA-mediated inhibition of m 6 A modification enhanced the inhibitory effect of MB-exos on ferroptosis in endometrial injury., Conclusions: Thus, these observations provide new insights regarding the molecular mechanisms responsible for endometrial recovery promotion by MB-exos and highlight m 6 A modification-dependent ferroptosis inhibition as a prospective therapeutic target to attenuate endometrial injury., (© 2024. The Author(s).)

In the realm of prognosticating the remaining useful life (RUL) of pivotal components, such as aircraft engines, a prevalent challenge persists where the available historical life data often proves insufficient. This insufficiency engenders obstacles such as impediments in performance degradation feature extraction, inadequacies in capturing temporal relationships comprehensively, and diminished predictive accuracy. To address this issue, a 1D CNN-GRU prediction model for few-shot conditions is proposed in this paper. In pursuit of more comprehensive data feature extraction and enhanced RUL prognostication precision, the Convolutional Neural Network (CNN) is selected for its capacity to discern high-dimensional features amid the intricate dynamics of the data. Concurrently, the Gated Recurrent Unit (GRU) network is leveraged for its robust capability in extracting temporal features inherent within the data. We combine the two to construct a CNN-GRU hybrid network. Moreover, the integration of data distribution alongside correlation and monotonicity indices is employed to winnow the input of multi-sensor monitoring parameters into the CNN-GRU network. Finally, the engine RULs are predicted by the trained model. In this paper, experiments are conducted on a sub-dataset of the National Aeronautics and Space Administration (NASA) C-MAPSS multi-constraint dataset to validate the effectiveness of the method. Experimental results have demonstrated that this method has high accuracy in RUL prediction tasks, which can powerfully demonstrate its effectiveness., (© 2024. The Author(s).)

Hepatic lipid droplets (LDs) are implicated in ectopic lipid accumulation. The core of LDs, triacylglycerol (TAG), is synthesized from the esterification of fatty acids to a glycerol-3-phosphate (G-3-P) backbone. Albumin transports plasma free fatty acids, and previously albumin knockout (Alb -/- ) mice were shown to exhibit lower hepatic TAG levels than wildtype (WT). Exercise is a beneficial strategy to alter hepatic metabolism, but its impacts on reducing hepatic lipids are far from satisfactory. The aim of this study was to investigate the combined effect of albumin deficiency and acute exercise on hepatic LDs. Eight-week-old male Alb -/- and WT mice were divided into sedentary and exercise groups. Exercised mice performed a 30-min high-intensity exercise bout. Results showed that sedentary Alb -/- mice had smaller hepatic LDs (P < 0.0001), associated with mitochondria, while WT mice exhibited larger LDs, surrounded by glycogen granules. Following acute exercise, hepatic LDs in Alb -/- mice reduced by 40% in size, while in WT increased by 14% (P < 0.0001). The maintenance of WT hepatic LDs was associated with elevated G-3-P level (P < 0.05), potentially derived from glycogen (R = -0.32, %change in glycogen versus LD content, P < 0.05). The reduction in Alb -/- mice LDs after exercise was possibly due to their low glycogen level. In conclusion, Alb -/- mice exhibited an enhanced capacity for reducing hepatic LD size and content in response to exercise. These findings suggest that modulating albumin's functions combined with exercise could be a potential strategy to reduce ectopic lipid deposition in the liver., (© 2023. The Author(s).)

Antibiotic production wastewater usually contains high concentrations of antibiotic residues, which can cause instability and deterioration of biological wastewater treatment units and also domestication and proliferation of antibiotic-resistance bacteria. An effective pretreatment on antibiotics production wastewater is expected to selectively reduce the concentration of antibiotics and decrease the toxicity, rather than mitigate organic and other contaminants before further treatments. In this work, two polymer-based solid acids, PS-S and CPS-S bearing high concentrations of -SOH 3 groups (up to 4.57 mmol/g), were prepared and successfully used for hydrolytic mitigation of 100 mg/L tylosin within 20 min. The co-existence of high concentrations of COD and humic substances did not affect the mitigation of tylosin obviously, while more than 500 mg/L of nitrogenous compounds suppressed the hydrolytic efficiency. Recycle and reuse experiments showed that the solid acids performed well in five cycles after regeneration. Three transformation products (P1, P2 and P3) were identified using UPLC-QTOF-MS/MS. Sugar moieties including mycarse, mycaminose, and mycinose detached and released simultaneously or in order from the 16-member lactone ring through desugarization, which led to a dramatic decrease in antibacterial activity as revealed by cytotoxicity evaluations using S. aureus. Ecotoxicity estimation indicated the acute toxicities of the hydrolyzed products to model species (e.g., fish, daphnid and green algae) were classified as "not harmful". This work suggested an effective and selective method to pretreat tylosin-contained production wastewater by using polymer-based solid acids. These results will shed light on effective elimination of antibiotics pollution from pharmaceutical industries through strengthening the pretreatments., (Copyright © 2022. Published by Elsevier B.V.)

Bone marrow mesenchymal stem cells (BMSCs) have potential therapeutic benefits for the treatment of endometrial diseases and injury. BMSCs interact with uterus parenchymal cells by direct contact or indirect secretion of growth factors to promote functional recovery. In this study, we found that BMSC treatment in rats subjected to mechanical damage (MD) significantly increased microRNA-340 (miR-340) levels in the regenerated endometrium. Then we employed knockin and knockdown technologies to upregulate or downregulate the miR-340 level in BMSCs (miR-340 + BMSCs or miR-340 - BMSCs) and their corresponding exosomes, respectively, to test whether exosomes from BMSCs mediate miR-340 transfer. We found that the exosomes released from the primitive BMSCs or miR-340 + BMSCs but not miR-340 - BMSCs increased the miR-340 levels in primary cultured endometrial stromal cells (ESCs) compared with control. Further verification of this exosome-mediated intercellular communication was performed using exosomal inhibitor GW4869. Tagging exosomes with red fluorescent protein demonstrated that exosomes were released from BMSCs and transferred to adjacent ESCs. Compared with controls, rats receiving primitive BMSC treatment significantly improved functional recovery and downregulated collagen 1α1, α-SMA and transforming growth factor (TGF)-β1 at day 14 after MD. The outcomes were significantly enhanced by miR-340 + BMSC treatment, and were significantly weakened by miR-340 - BMSC treatment, compared with primitive BMSC treatment. In vitro studies reveal that miR-340 transferred from BMSCs suppresses the upregulated expression of fibrotic genes in ESCs induced by TGF-β1. These data suggest that the effective antifibrotic function of BMSCs is able to transfer miR-340 to ESCs by exosomes, and that enhancing the transfer of BMSC-derived miR-340 is an alternative modality in preventing intrauterine adhesion., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Non-coding RNAs (ncRNAs) have been shown to regulate gene expression involved in tumor progression of multiple malignancies. Numerous studies have indicated that N-acetylglucosaminyltransferase V (MGAT5), is an important tumorigenesis and metastasis-associated enzyme in breast cancer (BC). But, the underlying molecular mechanisms by which ncRNAs modulate MGAT5 expression in BC remain undetermined. In this study, we demonstrated that miR-124 expression at a low level in BC tissue was associated with poor prognosis of BC patients. Meanwhile, miR-124 reduced BC cell proliferation and metastasis. MGAT5 was confirmed as a direct target of miR-124. MGAT5 restoration attenuated the inhibitory effects of miR-124 on BC proliferation and metastasis in vitro and vivo . Overall, we provide new insight into the mechanisms by which miR-124 inhibits BC progression, suggesting the potential of miR-124 and MGAT5 as biomarkers for early diagnosis of breast cancer to provide innovative ideas and methods for the diagnosis and treatment of BC., Competing Interests: None.

Long noncoding RNAs (lncRNAs) are implicated in renal cell carcinoma (RCC), but remain largely unclear. Using publicly available transcriptome sequencing data from renal cancer (n = 703) and integrating bioinformatics analyses, we screened and identified a valuable lncRNA, EGFR-AS1. In our validation cohort (n = 204), EGFR-AS1 was significantly upregulated in RCC tissues (P < 0.001). Gain-of-function and loss-of-function studies showed that EGFR-AS1 promoted cell proliferation and invasion in vitro and in vivo. Based on previous studies and sequence complementarity of EGFR with EGFR-AS1, we demonstrated that EGFR-AS1 directly bound to EGFR mRNA and inhibited its degradation. Furthermore, RNA pull-down and mass spectrometry analyses showed that EGFR-AS1 interacted with HuR, which was responsible for the mRNA stability of EGFR. Multivariate analysis suggested that higher EGFR-AS1 expression predicted a poor prognosis in RCC patients (high vs low: P = 0.018, HR = 2.204, 95% CI: 1.145-4.241). In conclusion, EGFR-AS1 enhances the malignant phenotype of RCC cells by enhancing HuR-mediated mRNA stability of EGFR. Our data also provide biological rationales for EGFR-AS1 as a prognostic biomarker and a potential therapeutic target for RCC.

Chronic hepatitis B (CHB) appears to progress more rapidly in males than in females, and CHB-related hepatic cirrhosis and hepatocellular carcinoma are predominately diseases that tend to occur in men and postmenopausal women. To obtain more insight into the underlying mechanisms of gender disparity of CHB progress, two-dimensional difference gel electrophoresis was employed to compare liver proteome of C57BL/6 and HBV transgenic (HBV-Tg) mice both in male and female groups. We identified 8 differently expressed proteins in male HBV-Tg mice and 12 in female HBV-Tg mice. Apolipoprotein A-I (Apo A-I) was found to be down-regulated in male and female HBV-Tg mouse liver. It is more interesting that the pattern of liver Apo A-I isoforms was altered in male HBV-Tg mice but not in female HBV-Tg mice. Our further results indicated that the basic Apo A-I isoform, based on pI positions from serum 2-dimensional Western blotting, increased in male CHB patient sera but not in female CHB patient sera. Finally, we identified that the oxidative modification Apo A-I mainly reside in basic isoform. This pattern of selectively modified Apo A-I isoforms may be considered as a pathological hallmark that may extend our knowledge of the molecular pathogenesis of CHB progression.

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.