Advancing hydrogen generation: Kinetic insights and process refinement for sorption-enhanced steam gasification of biomass utilizing waste carbide slag.

Sorption enhanced steam gasification of biomass (SESGB) presents a promising approach for producing high-purity H2 with potential for zero or negative carbon emissions. This study investigated the effects of gasification temperature, CaO to carbon in biomass molar ratio [CaO/C], and steam flow on the SESGB process, employing carbide slag (CS) and its modifications, CSSi2 (mass ratio of CS to SiO2 is 98:2) and CSCG5 (mass ratio of CS to coal gangue (CG) is 95:5), as CaO-based sorbents. The investigation included non-isothermal and isothermal gasification experiments and kinetic analyses using corn cob (CC) in a macro-weight thermogravimetric setup, alongside a fixed-bed pyrolysis-gasification system to assess operational parameter effects on gas product. The results suggested that CO2 capture by CaO reduced the mass loss during the main gasification as the [CaO/C] increased. The appropriate temperature for SESGB process should be selected between 550 and 700 °C at atmospheric pressure. The appropriate amount of sorbent or steam could facilitate the gasification reaction, but excessive addition led to adverse effects. Operational parameters influenced the apparent activation energy (Ea) by affecting various gasification reactions. For each test, Ea at the char gasification stage was significantly higher than that at the rapid pyrolysis stage. The addition of CS notably increased H2 concentration and yield, while sharply reducing CO2 levels. H2 concentration initially rose and then fell with greater steam flow, peaking at 76.11 vol% for a steam flow of 1.0 g/min. H2 yield peaked at 298 mL/g biomass with a steam flow of 1.5 g/min, a gasification temperature of 600 °C and a [CaO/C] of 1.0. Increasing gasification temperature remarkably boosted the H2 and CO2 yields. Optimal conditions for the SESGB using CS as a sorbent, determined via response surface methodology (RSM), include a gasification temperature of 666 °C, a [CaO/C] of 1.99, and a steam flow of 0.5 g/min, under which H2 and CO2 yields were 464 and 48 mL/g biomass, respectively. CSSi2 and CSCG5 demonstrated excellent cyclic H2 production stability, maintaining H2 yields around 440 mL/g biomass and low CO2 yields (∼60 mL/g biomass) across five cycles. The study results offer new insights for the high-value utilization of agroforestry biomass and the reduction and resource utilization of industrial waste.

Enhanced Gasdermin-E-mediated Pyroptosis in Alzheimer's Disease.

Amyloid ß protein (Aß) is a critical factor in the pathogenesis of Alzheimer's disease (AD). Aß induces apoptosis, and gasdermin-E (GSDME) expression can switch apoptosis to pyroptosis. In this study, we demonstrated that GSDME was highly expressed in the hippocampus of APP23/PS45 mouse models compared to that in age-matched wild-type mice. Aß treatment induced pyroptosis by active caspase-3/GSDME in SH-SY5Y cells. Furthermore, the knockdown of GSDME improved the cognitive impairments of APP23/PS45 mice by alleviating inflammatory response. Our findings reveal that GSDME, as a modulator of Aß and pyroptosis, plays a potential role in Alzheimer's disease pathogenesis and shows that GSDME is a therapeutic target for AD.

Transcriptional activation of CSTB gene expression by transcription factor Sp3.

CSTB has been reported to be associated with the pathogenesis of many malignant tumors, especially hepatocellular carcinoma (HCC). However, how the expression of this gene is regulated is largely unknown. We initially cloned and analyzed the promoter region of the CSTB gene by luciferase assay and the Sp3 binding site (CCCCGCCCCGCG) was found in it. The results of electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments verified that the transcription factor, Sp3 could bind to the " CCCCGCCCCGCG ″ site of the CSTB gene promoter. We showed that the overexpression of Sp3 significantly increased the endogenous mRNA and protein expression levels of CSTB, whereas knockdown of Sp3 decreased the mRNA and protein expression levels according to quantitative real-time PCR (qRT‒PCR) and western blotting. In conclusion, CSTB gene expression is closely regulated by transcription factor Sp3, which may be a potential mechanism for the dysregulation of CSTB expression in HCC.

Sp4 Regulates PTTG1IP Gene Transcription and Expression.

Pituitary tumor-transforming gene 1 protein (PTTG)-interacting protein, also known as PTTG-binding factor (PBF), is encoded by a proto-oncogene PTTG1IP. PBF has been identified through its interaction with PTTG. Similar to PTTG, PBF has been implicated in the etiology of several tumors, including pituitary, thyroid, and breast cancer. PBF can induce the translocation of PTTG into the nucleus, and then lead to tumorigenesis. Studies have shown that PBF plays a vital and complex role in increasing tumor development. However, the transcriptional regulation of PTTG1IP gene remains undefined. In this study, we have cloned a 467-bp fragment of the 5' flanking region of the human PTTG1IP gene and identified the region (-212 to +7 bp) necessary for PTTG1IP gene promoter activity by luciferase assay. Electrophoretic mobility shift assay revealed PTTG1IP gene promoter containing Sp4 response elements. Overexpression of Sp4 increased PTTG1IP gene transcription and expression in HeLa cells. Our study demonstrates that Sp4 regulates PTTG1IP gene transcription and expression.

JAM2 predicts a good prognosis and inhibits invasion and migration by suppressing EMT pathway in breast cancer.

OBJECTIVES: Large-scale epidemiological surveys have shown that patients with Down syndrome, which is caused by a chromosomal abnormality (an extra chromosome 21), are significantly less likely to develop solid tumors, including breast cancer, than those without. This feature has prompted the search for oncogenes located on chromosome 21. Junctional adhesion molecule 2 (JAM2), which is located on chromosome 21, is expressed at low levels in breast cancer and is associated with a good prognosis. These findings strongly suggest that JAM2 may be a potential oncogene suppressor in breast cancer. However, the role and function of JAM2 in breast cancer are not yet clear. Therefore, this study aimed to explore the biological functions and mechanisms of JAM2 in breast cancer. METHODS: Several databases were used to explore JAM2 expression in breast cancer and to analyze its diagnostic and prognostic value in breast cancer. Changes in relevant markers were examined at the gene and protein levels using RT-qPCR and Western blot techniques, in addition, cell migration and invasion abilities were identified by scratch assays and transwell assays. Untargeted metabolomics, transcriptome sequencing and Luminex liquid suspension chip detection were performed in combination to study the mechanisms. RESULTS: JAM2 is expressed at low levels in breast cancer, and patients with high JAM2 expression have a good prognosis, indicating that JAM2 has good clinical diagnostic and prognostic value. Overexpression of JAM2 can block the invasion and migration of breast cancer cells, and the mechanism may be that JAM2 inhibits the EMT pathway. Finally, combined multiomics analysis revealed that JAM2 may affect the immune microenvironment of breast cancer by influencing the secretion of CXCL9/10 from tumor cells.

[Corrigendum] HORMAD1 promotes docetaxel resistance in triple negative breast cancer by enhancing DNA damage tolerance.

Following the publication of this article, the authors realized that the published version of Fig. 4A contained an erroneous label; essentially, the information purported to relate to experiments having been performed with docetaxel should not have been included in this figure. The correctly labelled version of Fig. 4 is shown with the remainder of Fig. 4 on the next page. This change does not affect the data shown in the paper, and the text in the published article did accurately describe the information shown in this figure. The authors sincerely apologize for the error that was introduced during the preparation of this figure, and thank the Editor for allowing them the opportunity to publish a Corrigendum. Furthermore, they regret any inconvenience caused. [the original article was published in Oncology Reports 46: Article no. 138, 2021; DOI: 10.3892/or.2021.8089].

HORMAD1 promotes docetaxel resistance in triple negative breast cancer by enhancing DNA damage tolerance.

HORMA domain­containing protein 1 (HORMAD1), is normally expressed only in the germline, but is frequently re­activated in human triple­negative breast cancer (TNBC); however, its function in TNBC is largely unknown. In the present study, the expression and biological significance of HORMAD1 in human TNBC was evaluated. Bioinformatics analysis and reverse transcription­quantitative PCR were used to evaluate HORMAD1 expression in datasets and cell lines. HORMAD1 protein expression was detected in TNBC samples using immunohistochemical assays, and the effect of HORMAD1 on cell proliferation was determined using Cell Counting Kit­8, plate colony formation and standard growth curve assays. Cell cycle, reactive oxygen species (ROS) and apoptosis analyses were conducted using flow cytometry. The activity of caspases was measured using caspase activity assay kit. The levels of key apoptosis regulators and autophagy markers were detected by western blot analysis. TNBC cell survival and apoptosis were not influenced by small interfering RNA targeting HORMAD1 alone; however, HORMAD1 knockdown enhanced autophagy and docetaxel (Doc)­induced apoptosis, compared with the control group. Furthermore, higher ROS levels and caspase­3, ­8 and ­9 activity were detected in MDA­MB­436 TNBC cells with HORMAD1 knockdown upon exposure to Doc. The levels of the induced DNA damage marker γH2AX were also higher, while those of the DNA repair protein RAD51 were lower in TNBC cells with HORMAD1 knockdown compared with the controls. Furthermore, the expression of the autophagy marker P62 was enhanced in MDA­MB­231 cells in response to HORMAD1 overexpression. Notably, Doc­induced apoptosis was similarly increased by both HORMAD1 overexpression and treatment with the autophagy inhibitor, 3­methyladenine (3MA); however, the Doc­induced increase in autophagy was not inhibited by 3MA. The present data indicated that HORMAD1 was involved in autophagy and that the inhibition of autophagy can partially enhance the induction of apoptosis by Doc. The role of HORMAD1 in the DNA damage tolerance of tumor cells may be the main reason for Doc resistance; hence, HORMAD1 could be an important therapeutic target in TNBC.

AGR3 promotes estrogen receptor-positive breast cancer cell proliferation in an estrogen-dependent manner.

Breast cancer is one of the most common malignancies and the leading cause of cancer-associated death among women. Anterior gradient 3 (AGR3) is a cancer-associated gene and is similar to its homologous oncogene AGR2. However, whether AGR3 participates in breast cancer progression remains unclear. The present study aimed to investigate the function of AGR3 in ER-positive breast cancer. In the present study, reverse transcription-quantitative PCR was used to detect AGR3 mRNA expression in breast cancer tissues and cell lines; linear correlation analysis was used to investigate the correlation between AGR3 and estrogen receptor 1 (ESR1) expression in breast cancer via GEO dataset analysis; western blotting was used to assess the levels of AGR3, ER and GAPDH; small interfering (si)RNA transfection was used to knock down AGR3 and ESR1 expression; and finally the Cell Counting Kit-8 assay was used to evaluate cell viability. In the present study, AGR3 expression was markedly increased in estrogen receptor (ER)-positive breast cancer tissues and cell lines compared with that in ER-negative breast cancer. AGR3 expression was upregulated in estrogen-treated T47D cells, whereas 4-hydroxytamoxifen, an inhibitor of estrogen-ER activity in breast cancer cells, downregulated AGR3 expression in T47D cells. Functional assays demonstrated that knockdown of AGR3 using siRNAs inhibited T47D cell proliferation compared with that of the negative control group. Additionally, AGR3 expression was decreased after knocking down ESR1. The present results suggested that AGR3 may serve an important role in estrogen-mediated cell proliferation in breast cancer and that AGR3 knockdown may be a potential therapeutic strategy for ER-positive breast cancer.

Although c­MYC contributes to tamoxifen resistance, it improves cisplatin sensitivity in ER­positive breast cancer.

Tamoxifen (TAM) resistance is a major challenge in the treatment of estrogen receptor­positive (ER+) breast cancer. To date, to the best of our knowledge, there are only a few studies available examining the response of patients with TAM­resistant breast cancer to chemotherapy, and the guidelines do not specify recommended drugs for these patients. In the present study, TAM­resistant cells were shown to exhibit increased proliferation and invasion compared with the parent cells, and the increased expression of c­MYC was demonstrated to play an important role in TAM resistance. Furthermore, the TAM­resistant cells were significantly more sensitive to cisplatin compared with the parent cells, and the silencing of c­MYC expression desensitized the cells to cisplatin through the inhibition of the cell cycle. An increased c­MYC expression was observed in 28 pairs of primary and metastatic tumors from patients treated with TAM, and the clinical remission rate of cisplatin­based chemotherapy was significantly higher compared with other chemotherapy­based regimens in 122 patients with TAM resistant breast cancer. Taken together, the data of the present study demonstrated that although c­MYC was involved in TAM resistance, it increased the sensitivity of ER+ breast cancer to cisplatin. Thus, cisplatin may be a preferred chemotherapeutic agent for the treatment of patients with TAM­resistant breast cancer, particularly in patients where the rapid control of disease progression is required.

Sulfasalazine­induced ferroptosis in breast cancer cells is reduced by the inhibitory effect of estrogen receptor on the transferrin receptor.

The aim of the present study was to clarify the activation of ferroptosis in different breast cancer cells by sulfasalazine (SAS) and to explore the relationship between the estrogen receptor (ER) and the transferrin receptor (TFRC). MDA­MB­231 and T47D cells were treated with SAS for 24 h. Changes in cell morphology were observed under a microscope. CCK­8 was used to detect the proliferation inhibition rate and determine the IC50 values. Western blotting was used to detect the expression of glutathione peroxidase 4 (GPX4) and xCT. Flow cytometry was used to identify changes in the production of reactive oxygen species (ROS). Mitochondrial morphological changes in T47D were observed using transmission electron microscopy. Changes in the mitochondrial membrane potential (MMP) were observed using confocal fluorescence microscopy. RT­PCR was used to detect the mRNA expression levels of TFRC and divalent metal transporter 1 (DMT1). Bioinformatics analysis was performed on TFRC expression in 1,208 breast cancer samples and its relationship with ER. TFRC expression was detected in various breast cancer tissues using immunohistochemistry and in various breast cancer cells using western blotting. Small interfering RNA (siRNA) knocked down ER expression in T47D cells, and changes in the TFRC mRNA and protein levels were observed. RT­PCR was used to detect TFRC expression in 87 clinical specimens. The results of the present study revealed that SAS could inhibit breast cancer cell viability, which was accompanied by an abnormal increase in ROS and a depletion of GPX4 and system xc­. Liproxstatin­1 reversed the SAS­induced increase in ROS. The cells treated with SAS had shrunken mitochondria and decreased MMP. SAS upregulated TFRC and DMT1. Knockdown of the ER increased TFRC expression in breast cancer cells. Immunohistochemistry indicated that TFRC expression was lower in ER+ tissues than in ER­ tissues. After confirmation with RT­PCR in 87 clinical specimens, TFRC expression in ER­ tissue was revealed to be significantly higher than that of ER+ tissue. In conclusion SAS could trigger ferroptosis in breast cancer cells, especially in cells with low ER expression. Therefore, SAS is a potential agent for breast cancer treatment.

Lysophosphatidic Acid Receptor 6 (LPAR6) Expression and Prospective Signaling Pathway Analysis in Breast Cancer.

BACKGROUND AND OBJECTIVE: Lysophosphatidic acid (LPA) has widely been reported to participate in the numerous biological behaviors of tumors through its receptors. LPA receptor 6 (LPAR6) is a newly identified G protein-coupled receptor of LPA, and few studies have explored the role of LPAR6 in cancer. In breast cancer (BC), LPAR6 has not, as yet, been studied. This study aimed to evaluate LPAR6 expression in BC patients and to explore its possible role in BC. METHODS: A total of 98 pairs of clinical BC and para-cancer tissues were collected, and LPAR6 expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Kaplan-Meier plots were employed for survival analysis. Human BC cell lines were cultured to study decitabine (5-aza-2'-deoxycytidine [5-Aza]) intervention. Bioinformatic analyses were carried out to support the study conclusions and predictions. RESULTS: LPAR6 expression was significantly reduced in BC tissues (p < 0.001). In the analysis of clinical parameters, LPAR6 expression was related to BC molecular classification (p < 0.05). Furthermore, patients with higher LPAR6 expression had better prognoses (p < 0.001). The CpG islands of LPAR6 were hypermethylated in BC tissues relative to those in para-cancer tissues (p < 0.01). 5-Aza significantly upregulated LPAR6 expression in BC cell lines. Additionally, LPAR6 knockdown significantly promoted cell migration and proliferation in the ZR-75-1 cell line (p < 0.001). Finally, through Gene Set Enrichment Analysis (GSEA), LPAR6 was found to be negatively correlated with cancer-promoting factors and positively correlated with tumor-suppressing factors. CONCLUSION: LPAR6 was downregulated in BC, and low LPAR6 expression was related to poor prognosis. The anti-tumor drug 5-Aza significantly upregulated LPAR6 expression in vitro, and LPAR6 might act as a tumor suppressor in BC.

Novel Breast-Specific Long Non-coding RNA LINC00993 Acts as a Tumor Suppressor in Triple-Negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) was characterized by breast cancers that do not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor (HER)-2 genes. TNBC patients are associated with a shorter median time to relapse and death for the lack of available treatment targets. Long non-coding RNAs (LncRNAs) have been reported to play an important role in the development of TNBC. We identified a novel breast-specific long non-coding RNA LINC00993, but less was known about its expression pattern and functional role in TNBC. Methods: LINC00993 RNA expression was detected across different types of clinical breast cancer samples by using qRT-PCR. Bioinformatic methods "guilt by association" and gene set enrichment analysis (GSEA) were used to predict LINC00993 functions. Subcellular localization of LINC00993 in cells was detected by RNA fluorescence in situ hybridization (FISH). Effect of LINC00993 on cell growth was measured by plate colony formation assays, typical growth curve, and an in vivo tumor model. Cell cycle analysis was done by flow cytometry analysis. Key cell cycle regulators were detected by Western blot. Results: LINC00993 was largely downregulated in TNBC, and higher expression indicated better outcome. LINC00993 located mainly in the nucleus. LINC00993 suppressed TNBC growth both in vitro and in vivo. LINC00993 was predicted to be involved in cell cycle pathways by using "guilt by association" and GSEA methods. Key cell cycle regulators like p16INK4A, p14ARF, p53, and p21 were affected by LINC00993 overexpression. Conclusions: A new breast-specific lincRNA LINC00993 was identified with a tumor-suppressive feature and with prognostic value. This is the first research on LINC00993 function. Our results suggest that controlling LINC00993 level may be beneficial for breast cancer treatment.

Estrogen receptor α (ERα) status evaluation using RNAscope in situ hybridization: a reliable and complementary method for IHC in breast cancer tissues.

Estrogen receptor α (ERα) plays a significant role in the development of breast cancer and has been used clinically as an endocrine therapeutic target. Currently, clinical laboratories use immunohistochemistry (IHC) to determine the ERα status of patients in order to distinguish those who would benefit from endocrine therapy. This method is highly subjective, requires a large amount of tumor tissue, and may generate false-negative results. To improve the detection of ERα, we used a new RNA in situ hybridization technique (RNAscope) and compared its use with IHC in 72 breast cancer tissues (47 ERα positive and 25 ERα negative). Then we evaluated ERα mRNA by RT-qPCR with RNAscope. An unobvious difference was found between reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and IHC, but a positive correlation was found between RNAscope and IHC. In addition, breast cancer is a highly heterogeneous cancer, and RNAscope could easily reveal the heterogeneity in breast cancer. Moreover, we found that some ERα IHC-based negative and RNAscope-based positive test results were detected as positive after testing with IHC again. Our findings suggest that RNAscope may be a complementary method for improving the detection of patient ERα status and has potential clinical utility.

Large-pore mesoporous silica nanospheres as vehicles for delivering TRAF3-shRNA plasmids to Kupffer cells.

The currently available techniques for transferring exogenous genes into macrophages, especially the targeted import of exogenous genes into Kupffer cells (KCs) in vivo, are inefficient and achieve only low targeting. Novel Large-Pore Mesoporous Silica Nanospheres (LPMSNs) may be a promising gene transfection agent for KCs because of their superior biodegradation and hypotoxic characteristics, as well as their ability to retain the biological function of KCs and the high loading-rate of exogenous plasmid. LPMSNs were able to completely adsorb shRNA-TRAF3 (tumor necrosis factor receptor-associated factor-3) plasmid at a mass ratio as low as 30:1, and exhibited a low cytotoxicity for KCs. LPMSNs were detected in KC cytoplasm in vitro, and transmission electron microscopy (TEM) revealed that they were present only in KCs in liver tissue in vivo. The max KC transfection efficiency with LPMSNs was 34.8± 0.07%, as evaluated using flow cytometry, and the protein and mRNA levels of TRAF3 were significantly inhibited (P < 0.05) by shRNA-TRAF3 plasmid transfection after 24 h in vitro and 48 h in vivo. In conclusion, KC targeted transfection was achieved successfully by LPMSNs carrying shRNA-TRAF3 plasmids in vitro and vivo. The protein and mRNA levels of TRAF3 were suppressed significantly. These results suggest that LPMSNs are a promising vehicle for delivering exogenous genes into KCs in vitro and vivo.

[Inhibition of nuclear factor kappa B mediated by miRNA plasmid carried by novel polyethyleneimine nanogel particles in Kupffer cells of rats].

OBJECTIVE: To test a novel gene carrier, named polyethyleneimine nanogel particles (PEI-NP), for delivering the mircroRNA (miRNA) into Kupffer cells (KCs) for silencing the expression of nuclear transcription factor κB (NF-κB) P65. METHODS: The capacity of PEI-NPs to carry miRNA was tested by gel electrophoresis. RAW264.7 cell line was transfected with the cationic polymers which mixed together PEI-NP and miRNA. Then, the cytotoxicity of the RAW264.7 cells was detected by cell counting kit-8 (CCK-8); the cell apoptosis and transfection efficiency were analyzed by flow cytometry combined with annexin V-FITC/PI staining; the gene and protein levels of NF-κB P65 were determined respectively by real-time quantitative PCR (qRT-PCR) and Western blotting. The distribution of the cationic polymers in RAW264.7 cells and liver KCs was observed by transmission electron microscope (TEM). The protein level of NF-κB P65 in KCs was detected by immunohistochemistry. RESULTS: PEI-NPs were able to completely combine with miRNA at the least mass ratio of 20:1. The highest transfection efficiency of 48 hours was (33.63±1.94)% in vitro. The protein level of NF-κB P65 at 48 hours of PEI-NP/miRNA transfection into RAW264.7 cells was significantly inhibited. TEM showed the cationic polymers in RAW264.7 cytoplasm and only in KCs of liver tissue after transfection. Immunohistochemistry indicated that the protein expression of NF-κB P65 in KCs was significantly lower than that in control group. CONCLUSION: Macrophages and KCs were successfully transfected with the cationic polymers in vitro and in vivo, respectively. The expression of NF-κB P65 was knocked down significantly after transfection.