Discovering single cannabidiol or synergistic antitumor effects of cannabidiol and cytokine-induced killer cells on non-small cell lung cancer cells.

Introduction: A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern. Methods: Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes. Results: Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-g) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L_CD45RA+terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. Conclusions: Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients.

Profiling antibody epitopes induced by mRNA-1273 vaccination and boosters.

Background: Characterizing the antibody epitope profiles of messenger RNA (mRNA)-based vaccines against SARS-CoV-2 can aid in elucidating the mechanisms underlying the antibody-mediated immune responses elicited by these vaccines. Methods: This study investigated the distinct antibody epitopes toward the SARS-CoV-2 spike (S) protein targeted after a two-dose primary series of mRNA-1273 followed by a booster dose of mRNA-1273 or a variant-updated vaccine among serum samples from clinical trial adult participants. Results: Multiple S-specific epitopes were targeted after primary vaccination; while signal decreased over time, a booster dose after >6 months largely revived waning antibody signals. Epitope identity also changed after booster vaccination in some subjects, with four new S-specific epitopes detected with stronger signals after boosting than with primary vaccination. Notably, the strength of antibody responses after booster vaccination differed by the exact vaccine formulation, with variant-updated mRNA-1273.211 and mRNA-1273.617.2 booster formulations inducing significantly stronger S-specific signals than a mRNA-1273 booster. Conclusion: Overall, these results identify key S-specific epitopes targeted by antibodies induced by mRNA-1273 primary and variant-updated booster vaccination.

Expression profiles and gene set enrichment analysis of the transcriptomes from the cancer tissue, white adipose tissue and paracancer tissue with colorectal cancer.

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide and is related to diet and obesity. Currently, crosstalk between lipid metabolism and CRC has been reported; however, the specific mechanism is not yet understood. In this study, we screened differentially expressed long non-coding RNAs (lncRNAs) and mRNAs from primary cancer, paracancer, and white adipose tissue of CRC patients. We screened and analyzed the genes differentially expressed between primary and paracancer tissue and between paracancer and white adipose tissue but not between primary and white adipose tissue. According to the results of the biological analysis, we speculated a lncRNA (MIR503HG) that may be involved in the crosstalk between CRC and lipid metabolism through exosome delivery. Methods: We screened differentially expressed long non-coding RNAs (lncRNAs) and mRNAs from primary cancer, paracancer, and white adipose tissue of CRC patients. We screened and analyzed the genes differentially expressed between primary and paracancer tissue and between paracancer and white adipose tissue but not between primary and white adipose tissue. Results: We speculated a lncRNA (MIR503HG) that may be involved in the crosstalk between CRC and lipid metabolism through exosome delivery. Conclusions: In this study, the findings raise the possibility of crosstalk between lipid metabolism and CRC through the exosomal delivery of lncRNAs.

6-Gingerol attenuates hepatic ischemia/reperfusion injury through regulating MKP5-mediated P38/JNK pathway.

6-Gingerol, the main bioactive compound of ginger, has antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. However, it is unclear whether 6-Gingerol has protective effects against hepatic ischemia/reperfusion (I/R) injury. In this study, the mouse liver I/R injury model and the mouse AML12 cell hypoxia/reoxygenation (H/R) model were established by pretreatment with 6-Gingerol at different concentrations to explore the potential effects of 6-Gingerol. Serum transaminase levels, liver necrotic area, cell viability, inflammatory response, and cell apoptosis were used to assess the effect of 6-Gingerol on hepatic I/R or cell H/R injury. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the mRNA and protein expression. The results show that 6-Gingerol decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, liver necrosis, inflammatory cytokines IL-1ß, IL-6, MCP-1, TNF-α expression, Ly6g+ inflammatory cell infiltration, protein phosphorylation of NF-κB signaling pathway, Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positive cells, cell apoptosis rate, the protein expression of pro-apoptotic protein BAX and C-Caspase3, increased cell viability, and expression of anti-apoptotic protein BCL-2. Moreover, 6-Gingerol could increase the mRNA and protein expression of mitogen activated protein kinase phosphatase 5 (MKP5) and inhibit the activation of P38/JNK signaling pathway. In MKP5 knockout (KO) mice, the protective effect of 6-gingerol and the inhibition of P38/JNK pathway were significantly weakened. Therefore, our results suggest that 6-Gingerol exerts anti-inflammatory and anti-apoptotic effects to attenuate hepatic I/R injury by regulating the MKP5-mediated P38/JNK signaling pathway.

FAM120A deficiency improves resistance to cisplatin in gastric cancer by promoting ferroptosis.

The occurrence of chemoresistance is an inescapable obstacle affecting the clinical efficacy of cisplatin in gastric cancer (GC). Exploring the regulatory mechanism of cisplatin resistance will help to provide potential effective targets for improving the prognosis of gastric cancer patients. Here, we find that FAM120A is upregulated in GC tissues and higher in cisplatin-resistant GC tissues, and its high expression is positively correlated with the poor outcome of GC patients. Functional studies indicate that FAM120A confers chemoresistance to GC cells by inhibiting ferroptosis. Mechanically, METTL3-induced m6A modification and YTHDC1-induced stability of FAM120A mRNA enhance FAM120A expression. FAM120A inhibits ferroptosis by binding SLC7A11 mRNA and enhancing its stability. FAM120A deficiency enhances cisplatin sensitivity by promoting ferroptosis in vivo. These results reveal the function of FAM120A in chemotherapy tolerance and targeting FAM120A is an effective strategy to alleviate cisplatin resistance in GC.

Desmin gene expression is not ubiquitous in all upper airway myofibers and the pattern differs between healthy and sleep apnea subjects.

BACKGROUND: Desmin is a major cytoskeletal protein considered ubiquitous in mature muscle fibers. However, we earlier reported that a subgroup of muscle fibers in the soft palate of healthy subjects and obstructive sleep apnea patients (OSA) lacked immunoexpression for desmin. This raised the question of whether these fibers also lack messenger ribonucleic acid (mRNA) for desmin and can be considered a novel fiber phenotype. Moreover, some fibers in the OSA patients had an abnormal distribution and aggregates of desmin. Thus, the aim of the study was to investigate if these desmin protein abnormalities are also reflected in the expression of desmin mRNA in an upper airway muscle of healthy subjects and OSA patients. METHODS: Muscle biopsies from the musculus uvulae in the soft palate were obtained from ten healthy male subjects and six male patients with OSA. Overnight sleep apnea registrations were done for all participants. Immunohistochemistry, in-situ hybridization, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) techniques were used to evaluate the presence of desmin protein and its mRNA. RESULTS: Our findings demonstrated that a group of muscle fibers lacked expression for desmin mRNA and desmin protein in healthy individuals and OSA patients (12.0 ± 5.6% vs. 23.1 ± 10.8%, p = 0.03). A subpopulation of these fibers displayed a weak subsarcolemmal rim of desmin accompanied by a few scattered mRNA dots in the cytoplasm. The muscles of OSA patients also differed from healthy subjects by exhibiting muscle fibers with reorganized or accumulated aggregates of desmin protein (14.5 ± 6.5%). In these abnormal fibers, the density of mRNA was generally low or concentrated in specific regions. The overall quantification of desmin mRNA by RT-qPCR was significantly upregulated in OSA patients compared to healthy subjects (p = 0.01). CONCLUSIONS: Our study shows evidence that muscle fibers in the human soft palate lack both mRNA and protein for desmin. This indicates a novel cytoskeletal structure and challenges the ubiquity of desmin in muscle fibers. Moreover, the observation of reorganized or accumulated aggregates of desmin mRNA and desmin protein in OSA patients suggests a disturbance in the transcription and translation process in the fibers of the patients.

TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. METHODS: We cultured sexed hippocampal neurons from ERα+/+ and ERα-/- mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. RESULTS: Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. CONCLUSIONS: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Honokiol induces apoptosis and autophagy in dexamethasone-resistant T-acute lymphoblastic leukemia CEM-C1 cells.

Objective: To study whether and, if so, how honokiol overcome dexamethasone resistance in DEX-resistant CEM-C1 cells. Methods: We investigated the effect of honokiol (0-20 µM) on cell proliferation, cell cycle, cell apoptosis and autophagy in DEX-resistant CEM-C1 cells and DEX-sensitive CEM-C7 cells. We also determined the role of c-Myc protein and mRNA in the occurrence of T-ALL associated dexamethasone resistance western blot and reverse transcription-qPCR (RT-qPCR) analysis. Results: Cell Counting Kit (CCK)-8 assay shows that DEX-resistant CEM-C1 cell lines were highly resistant to dexamethasone with IC50 of 364.1 ± 29.5 µM for 48 h treatment. However, upon treatment with dexamethasone in combination with 1.5 µM of honokiol for 48 h, the IC50 of CEM-C1 cells significantly decreased to 126.2 ± 12.3 µM, and the reversal fold was 2.88. Conversely, the IC50 of CEM-C7 cells was not changed combination of dexamethasone and honokiol as compared to that of CEM-C7 cells treated with dexamethasone alone. It has been shown that honokiol induced T-ALL cell growth inhibition by apoptosis and autophagy via downregulating cell cycle-regulated proteins (Cyclin E, CDK4, and Cyclin D1) and anti-apoptotic proteins BCL-2 and upregulating pro-apoptotic proteins Bax and led to PARP cleavage. Honokiol may overcome dexamethasone resistance in DEX-resistant CEM-C1 cell lines via the suppression of c-Myc mRNA expression. Conclusion: The combination of honokiol and DEX were better than DEX alone in DEX-resistant CEM-C1 cell lines. Honokiol may regulate T-ALL-related dexamethasone resistance by affecting c-Myc.

A long non-coding RNA functions as a competitive endogenous RNA to modulate TaNAC018 by acting as a decoy for tae-miR6206.

Increasing evidence indicates a strong correlation between the deposition of cuticular waxes and drought tolerance. However, the precise regulatory mechanism remains elusive. Here, we conducted a comprehensive transcriptome analysis of two wheat (Triticum aestivum) near-isogenic lines, the glaucous line G-JM38 rich in cuticular waxes and the non-glaucous line NG-JM31. We identified 85,143 protein-coding mRNAs, 4,485 lncRNAs, and 1,130 miRNAs. Using the lncRNA-miRNA-mRNA network and endogenous target mimic (eTM) prediction, we discovered that lncRNA35557 acted as an eTM for the miRNA tae-miR6206, effectively preventing tae-miR6206 from cleaving the NAC transcription factor gene TaNAC018. This lncRNA-miRNA interaction led to higher transcript abundance for TaNAC018 and enhanced drought-stress tolerance. Additionally, treatment with mannitol and abscisic acid (ABA) each influenced the levels of tae-miR6206, lncRNA35557, and TaNAC018 transcript. The ectopic expression of TaNAC018 in Arabidopsis also improved tolerance toward mannitol and ABA treatment, whereas knocking down TaNAC018 transcript levels via virus-induced gene silencing in wheat rendered seedlings more sensitive to mannitol stress. Our results indicate that lncRNA35557 functions as a competing endogenous RNA to modulate TaNAC018 expression by acting as a decoy target for tae-miR6206 in glaucous wheat, suggesting that non-coding RNA has important roles in the regulatory mechanisms responsible for wheat stress tolerance.

Diminished neutralizing activity against the XBB1.5 strain in 55.9% of individuals post 6 months COVID-19 mRNA booster vaccination: insights from a pseudovirus assay on 1,353 participants in the Fukushima vaccination community survey, Japan.

This study investigates the neutralizing activity against the XBB1.5 variant and the ancestral strain in a population post-bivalent vaccination using a pseudo virus assay validated with authentic virus assay. While bivalent booster vaccination and past infections enhanced neutralization against the XBB 1.5 strain, individuals with comorbidities showed reduced responses. The study suggests the need for continuous vaccine updates to address emerging SARS-CoV-2 variants and highlights the importance of monitoring real-world immune responses.

Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.

Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.

[Inhibition of connexin 43-mediated hemichannel activity promotes odontoblast differentiation of human dental pulp cells induced by lipopolysaccharide].

PURPOSE: To investigate the role and mechanism of connexin 43（Cx43）in odontoblast differentiation of human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). METHODS: The maxillary first molar injury model of SD rats was established. The expression pattern of Cx43 in dental pulp repair after injury was detected by immunofluorescence(IF) staining. hDPCs was respectively stimulated with 0, 1, 10, 100 and 1 000 ng/mL LPS for 6 h to screen the optimal concentration, and then the expression of Cx43 was inhibited and overexpressed in hDPCs. Quantitative real-time PCR(qRT-PCR) and Western blot(WB) were used to detect the expression of Cx43 and dentin sialophosphoprotein (DSPP), dental matrix protein-1 (DMP-1), osterix (Osx) and extracellular signal-regulated kinase (ERK) activity. Furthermore, hDPCs were treated with specific Cx43 channel inhibitors to investigate the effect of Cx43-mediated channel activity in odontoblast differentiation of hDPCs, and to explore the role and mechanism of Cx43 in regulating odontoblast differentiation of hDPCs induced by LPS. Statistical analysis was performed with SPSS 26.0 software package. RESULTS: IF results showed that Cx43 was mainly expressed in the odontoblast layer in healthy dental pulp tissues. At 3-24 h after tooth injury, the expression of Cx43 decreased and then gradually increased to the normal level; from 3 days to 2 weeks after injury, the expression of Cx43 tended to be down-regulated which was in the odontoblast layer and pulp proper. The expression of DSPP mRNA was significantly up-regulated in the hDPCs stimulated with 10 ng/mL LPS for 6 h(P＜0.01). Inhibition of Cx43 significantly up-regulated the expression of DSPP, DMP-1 and Osx mRNA induced by LPS in hDPCs(P＜0.05), while overexpression of Cx43 obviously inhibited the expression of factors related to LPS-induced odontoblast differentiation(P＜0.01) and the fluorescence intensity of DSPP. 10 ng/mL LPS activated ERK signal in hDPCs, and overexpression of Cx43 significantly attenuated the activity of ERK signal induced by LPS(P＜0.01). Inhibition of Cx43-mediated hemichannel (HC) promoted mRNA expression of factors related to odontoblast differentiation in hDPCs and the activity of ERK signal induced by LPS(P＜0.05), while blocking Cx43-mediated gap junction channel (GJC) inhibited odontoblast differentiation. CONCLUSIONS: Cx43 participates in the regulation of dental pulp repair after injury, and its expression shows a downward trend as a whole. Inhibition of Cx43 or blocking of HC promotes LPS-induced ERK signal activity and odontoblast differentiation of hDPCs.

[Effects of M2-type macrophages and GKT137831 on oxidative stress in hepatic stellate cells].

Objective: To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4 (NOX4) inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the rat hepatic stellate cell line (HSC-T6). Methods: Rat bone marrow macrophages were extracted and induced using interleukin (IL)-4 to differentiate them into M2 phenotype macrophages. HSC-T6 activation was performed with 5 µg/L transforming growth factor ß1 (TGF-ß1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 µmol/L after 48 hours was detected using the Cell Counting Kit-8 (CCK-8) assay. The optimal drug concentration was chosen and divided into an HSC co-culture group (the control group) and five experimental groups: the TGF-ß1 stimulation group, the TGF-ß1 +GKT137831 stimulation group, the M2-type macrophage + HSC co-culture group, the M2-type macrophage +TGF-ß1 stimulation group, and the M2-type + TGF-ß1 + GKT137831 stimulation group. Reactive oxygen species (ROS) production level was detected in each cell using the DCFH-DA probe method. NOX4, α-smooth muscle actin (α-SMA), Nrf2, and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method. The t-test was used to compare the two groups. The one-way ANOVA method was used to compare multiple groups. Results: Intracellular ROS increased significantly following TGF-ß1 stimulation. ROS relative levels in each cell group were 1.03±0.11, 3.88±0.07, 2.90±0.08, 0.99±0.06, 3.30±0.05, 2.21±0.11, F = 686.1, P = 0.001, respectively. The mRNA and protein expressions of NOX4, α-SMA, Nrf2, and HO-1 were significantly increased (P < 0.05). After the addition of GKT137831, ROS, and NOX4, α-SMA mRNA and protein expression were comparatively decreased in the TGF-ß1 stimulation group (P < 0.05), while mRNA and protein expressions of Nrf2 and HO-1 were increased (P < 0.05). The expression of ROS and NOX4, as well as α-SMA mRNA and protein, produced by HSC were significantly decreased in the co-culture group compared to the single culture group after TGF-ß1 stimulation (P < 0.05). After the addition of GKT137831, ROS, NOX4, α-SMA mRNA, and protein expression were further reduced in the co-culture group compared with the single culture group (P < 0.05), while the mRNA and protein expression of Nrf2 and HO-1 were further increased (P < 0.05). Conclusion: NOX4 inhibitor GKT137831 can reduce RO, NOX4, and α-SMA levels while increasing Nrf2 and HO-1 levels in hepatic stellate cells. After M2-type macrophage co-culture, GKT137831 assists in lowering ROS, NOX4, and α-SMA levels while accelerating Nrf2 and HO-1 levels in hepatic stellate cells, which regulates the balance between oxidative stress and anti-oxidative stress systems, thereby antagonizing the fibrosis process.

Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system.

We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5'-untranslated regions (5'UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5'UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5'UTRs.

Role of kidney function on Nrf2 mRNA levels in type 2 diabetes.

INTRODUCTION: Diabetic kidney disease (DKD) is a major complication in patients with diabetes and the main contributor to the chronic kidney disease (CKD) global burden. Oxidative stress is a crucial factor in DKD pathogenesis but the role of the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) and its molecular regulators has been poorly investigated in man. RESEARCH DESIGN AND METHODS: In this case-control study, we analyzed the roles of Nrf2, a transcription factor shielding cells from oxidative stress, its repressor Kelch-like ECH-associated protein 1 (Keap1) and six microRNAs (miRNAs) that potentially suppress Nrf2. We categorized 99 participants into 3 groups: 33 non-dialysis patients with type 2 diabetes with DKD, 33 patients with type 2 diabetes without DKD and 33 control subjects and quantified the gene expression (messenger RNA (mRNA)) levels of Nrf2, Keap1 and 6 miRNAs. Moreover, we studied the correlation between gene expression levels and clinical indicators of kidney health. RESULTS: In patients with diabetes with DKD, Nrf2 mRNA levels were significantly lower than in patients without DKD (p=0.01) and controls (p=0.02), whereas no difference in Nrf2 expression levels existed between patients without DKD and controls. Conversely, in patients with and without DKD, Keap1 expression levels were significantly higher than in controls. Of the six miRNAs studied, miRNA 30e-5p showed differential expression, being markedly reduced in patients with DKD (p=0.007). Nrf2 mRNA levels directly correlated with estimated glomerular filtration rate (eGFR) in patients with DKD (r=0.34, p=0.05) and in a formal mediation analysis the eGFR emerged as the first factor in rank for explaining the difference in Nrf2 mRNA levels between patients with and without DKD. CONCLUSIONS: The observed dysregulation in the Nrf2-Keap1 axis and the unique expression pattern of miRNA30e-5p in DKD underscore the need for more focused research in this domain that can help identify novel intervention strategies for DKD in patients with type 2 diabetes.

Heat shock factor 1 inhibition enhances the effects of modulated electro hyperthermia in a triple negative breast cancer mouse model.

Female breast cancer is the most diagnosed cancer worldwide. Triple negative breast cancer (TNBC) is the most aggressive type and there is no existing endocrine or targeted therapy. Modulated electro-hyperthermia (mEHT) is a non-invasive complementary cancer therapy using an electromagnetic field generated by amplitude modulated 13.56 MHz frequency that induces tumor cell destruction. However, we have demonstrated a strong induction of the heat shock response (HSR) by mEHT, which can result in thermotolerance. We hypothesized that inhibition of the heat shock factor 1 (HSF1) can synergize with mEHT and enhance tumor cell-killing. Thus, we either knocked down the HSF1 gene with a CRISPR/Cas9 lentiviral construct or inhibited HSF1 with a specific small molecule inhibitor: KRIBB11 in vivo. Wild type or HSF1-knockdown 4T1 TNBC cells were inoculated into the mammary gland's fat pad of BALB/c mice. Four mEHT treatments were performed every second day and the tumor growth was followed by ultrasound and caliper. KRIBB11 was administrated intraperitoneally at 50 mg/kg daily for 8 days. HSF1 and Hsp70 expression were assessed. HSF1 knockdown sensitized transduced cancer cells to mEHT and reduced tumor growth. HSF1 mRNA expression was significantly reduced in the KO group when compared to the empty vector group, and consequently mEHT-induced Hsp70 mRNA upregulation diminished in the KO group. Immunohistochemistry (IHC) confirmed the inhibition of Hsp70 upregulation in mEHT HSF1-KO group. Demonstrating the translational potential of HSF1 inhibition, combined therapy of mEHT with KRIBB11 significantly reduced tumor mass compared to either monotherapy. Inhibition of Hsp70 upregulation by mEHT was also supported by qPCR and IHC. In conclusion, we suggest that mEHT-therapy combined with HSF1 inhibition can be a possible new strategy of TNBC treatment with great translational potential.

[Effect of CircCCND1 on the Malignant Biological Behaviors of H446 Lung Cancer Cells by Regulating the MiR-340-5p/TGIF1 Axis].

BACKGROUND: Lung cancer is a common malignant tumor of the lung. To explore the molecular mechanism of the occurrence and development of lung cancer is a hot topic in current research. Cyclic RNA D1 (CircCCND1) is highly expressed in lung cancer and may be a potential target for the treatment of lung cancer. The aim of this study was to investigate the effect of CircCCND1 on the malignant biological behaviors of lung cancer cells by regulating the miR-340-5p/transforming growth factor ß-induced factor homeobox 1 (TGIF1) axis. METHODS: The expression of CircCCND1, miR-340-5p, and TGIF1 mRNA in human normal lung epithelial cells BEAS-2B and human lung cancer H446 cells were detected. H446 cells cultured in vitro were randomly divided into control group, CircCCND1 siRNA group, miR-340-5p mimics group, negative control group, and CircCCND1 siRNA+miR-340-5p inhibitor group. Cell proliferation, mitochondrial membrane potential, apoptosis, migration, and invasion were detected, and the expressions of CircCCND1, miR-340-5p, TGIF1 mRNA, BCL2-associated X protein (Bax), cleaved Caspase-3, N-cadherin, E-cadherin, and TGIF1 proteins in each group were detected. The targeting relationship of miR-340-5p with CircCCND1 and TGIF1 was verified. RESULTS: Compared with BEAS-2B cells, CircCCND1 and TGIF1 mRNA were increased in H446 cells, and miR-340-5p expression was decreased (P<0.05). Knocking down CircCCND1 or up-regulating the expression of miR-340-5p inhibited the proliferation, migration and invasion of H446 cells, decreased the expression of TGIF1 mRNA and TGIF1 protein, and promoted cell apoptosis. Down-regulation of miR-340-5p could antagonize the inhibitory effect of CircCCND1 knockdown on the malignant biological behavior of H446 lung cancer cells. CircCCND1 may target the down-regulation of miR-340-5p, and miR-340-5p may target the down-regulation of TGIF1. CONCLUSIONS: Knocking down CircCCND1 can inhibit the malignant behaviors of lung cancer H446 cells, which may be achieved through the regulation of miR-340-5p/TGIF1 axis.

Oral post-exercise garlic extract supplementation enhances glycogen replenishment but does not up-regulate mitochondria biogenesis mRNA expression in human-exercised skeletal muscle.

PURPOSE: Garlic extract (GA) is purported to enhance antioxidant and anti-inflammatory activity and glucose regulation in humans. The present study investigated the effects of post-exercise GA supplementation on GLUT4 expression, glycogen replenishment, and the transcript factors involved with mitochondrial biosynthesis in exercised human skeletal muscle. METHODS: The single-blinded crossover counterbalanced study was completed by 12 participants. Participants were randomly divided into either GA (2000 mg of GA) or placebo trials immediately after completing a single bout of cycling exercise at 75% Maximal oxygen uptake (VO2max) for 60 minutes. Participants consumed either GA (2000 mg) or placebo capsules with a high glycemic index carbohydrate meal (2 g carb/body weight) immediately after exercise. Muscle samples were collected at 0-h and 3-h post-exercise. Muscle samples were used to measure glycogen levels, GLUT4 protein expression, as well as transcription factors for glucose uptake, and mitochondria biogenesis. Plasma glucose, insulin, glycerol, non-esterified fatty acid (NEFA) concentrations, and respiratory exchange ratio (RER) were also analyzed during the post-exercise recovery periods. RESULTS: Skeletal muscle glycogen replenishment was significantly elevated during the 3-h recovery period for GA concurrent with no difference in GLUT4 protein expression between the garlic and placebo trials. PGC1-α gene expression was up-regulated for both GA and placebo after exercise (p < 0.05). Transcript factors corresponding to muscle mitochondrial biosynthesis were significantly enhanced under acute garlic supplementation as demonstrated by TFAM and FIS1. However, the gene expression of SIRT1, ERRα, NFR1, NFR2, MFN1, MFN2, OPA1, Beclin-1, DRP1 were not enhanced, nor were there any improvements in GLUT4 expression, following post-exercise garlic supplementation. CONCLUSION: Acute post-exercise garlic supplementation may improve the replenishment of muscle glycogen, but this appears to be unrelated to the gene expression for glucose uptake and mitochondrial biosynthesis in exercised human skeletal muscle.

Bioinformatics-based dynamics of cuproptosis -related indicators in experimental silicosis.

Pneumoconiosis is one of the most serious occupational diseases worldwide. Silicosis due to prolonged inhalation of free silica dust during occupational activities is one of the main types. Cuproptosis is a newly discovered mode of programmed cell death characterized by the accumulation of free copper in the cell, which ultimately leads to cell death. Increased copper in the serum of silicosis patients, suggests that the development of silicosis is accompanied by changes in copper metabolism, but whether cuproptosis is involved in the progression of silicosis is actually to be determined. To test this hypothesis, we screened the genetic changes in patients with idiopathic fibrosis by bioinformatics methods and predicted and functionally annotated the cuproptosis-related genes among them. Subsequently, we established a mouse silicosis model and detected the concentration of copper ions and the activity of ceruloplasmin (CP) in serum, as well as changes of the concentration of copper and cuproptosis related genes in mouse lung tissues. We identified 9 cuproptosis-related genes among the differential genes in patients with IPF at different times and the tissue-specific expression levels of ferredoxin 1 (FDX1) and Lipoyl synthase (LIAS) proteins. Furthermore, serum CP activity and copper ion levels in silicosis mice were elevated on days 7th and 56th after silica exposure. The expression of CP in mouse lung tissue elevated at all stages after silica exposure. The mRNA level of FDX1 decreased on days 7th and 56th, and the protein level remained in accordance with the mRNA level on day 56th. LIAS and Dihydrolipoamide dehydrogenase (DLD) levels were downregulated at all times after silica exposure. In addition, Heatshockprotein70 (HSP70) expression was increased on day 56. In brief, our results demonstrate that there may be cellular cuproptosis during the development of experimental silicosis in mice and show synchronization with enhanced copper loading in mice.