Zakaria Vahabzadeh

Purpose: Immune checkpoint blockade enhances antitumor immunity, and combined CTLA-4/PD-1 inhibition has improved outcomes in several malignancies. However, its effect on NK cell-mediated cytotoxicity in acute lymphoblastic leukemia (ALL) remains unclear. Considering NK cell dysfunction and exhaustion in patients with ALL, this study aimed to evaluated whether dual PD-1 and CTLA-4 blockade enhances NK cell activity against primary ALL lymphoblasts.Methods: NK cells were isolated from peripheral blood of 25 healthy donors and 20 patients with ALL. Purity and viability were assessed by flow cytometry. Cells were expanded ex vivo for 14 days using IL-15 (10 ng/mL), nicotinamide (NAM), and CD3⁺CD56⁻CD16⁻ feeder cells. PD-1 and CTLA-4 expression was analyzed after expansion. NK cell cytotoxicity against primary lymphoblasts was evaluated following PD-1 blockade, CTLA-4 blockade, or dual blockade. IFN-γ and granzyme B secretion were also measured.Results: NK cell purity was 96.58 ± 2.57%. NAM significantly increased feeder-based NK cell expansion (22.53 ± 2.16- fold; P < 0.05). PD-1 and CTLA-4 expression increased after culture. Dual PD-1/CTLA-4 blockade significantly enhanced NK cell cytotoxicity against primary lymphoblasts (53.78%) compared with the control, PD-1 blockade, and CTLA-4 blockade groups (P < 0.001, P < 0.05, and P < 0.05, respectively). IFN-γ and granzyme B secretion also increased significantly following dual blockade.Conclusion: Combined PD-1 and CTLA-4 blockade enhances NK cell-mediated antitumor activity against primary lymphoblasts from patients with ALL. These findings support the integration of NK cell-based therapy with immune checkpoint inhibition as a promising immunotherapeutic strategy for ALL.

Colorectal cancer (CRC) is a malignancy with a significant global disease burden.Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been implicated in tumorigenesis. The oncogenic long non-coding RNA highly upregulated in liver cancer (HULC) plays a pivotal role in CRC progression. However, the exact molecular mechanism of HULC and its correlation with TMAO in CRC pathogenesis has remained unclear. This study tested whether TMAO regulates HULC and whether HULC mediates changes in selected miRNAs relevant to CRC.

Objectives Trimethylamine N-oxide (TMAO) is related to the pathogenesis of Metabolic dysfunction-associated fatty liver disease (NAFLD). However, the molecular mechanism of how TMAO causes MAFLD development is still unclear. The present study attempted to investigate whether TMAO contributes to MAFLD development through HULC in a cellular model of MAFLD. Methods HepG2 cells were cultured and induced in a fatty liver cell model. HULC knockdown was induced using the CRISPR/Cas13 system. Fatty liver cells were exposed to TMAO concentrations (75µM and 300µM) before and after HULC knockdown. RT-qPCR was used to evaluate the expression of the target genes. Apoptosis was assessed using Annexin V-FITC and PI staining. Statistical analyses included ANOVA and post-hoc tests. Results TMAO upregulated the expression of HULC, followed by P38MAPK overexpression (P value < 0.05). Upon HULC knockdown, TMAO could not change P3MAPK expression and its downstream targets, including TNFα, IL-6, and PNPPLA3 in fatty liver cells. Additionally, TMAO significantly induced apoptosis in the fatty acid cellular model (P value < 0.05). Conclusion In conclusion, the results of this study provide evidence of the TMAO/HULC/P38MAPK axis involvement in the pathogenesis of MAFLD by increasing the expression of genes involved in inflammation and fibrosis. Our data suggests that TMAO reduction could be a therapeutic target in MAFLD through gut microbiome modulation.

Memory impairment is one of the cognitive symptoms in Huntington’s disease (HD) which appears before motor dysfunction in patients. Various molecular mechanisms, including disruptions in neurotrophins levels, are involved in the occurrence of memory problems in HD. Alpha-pinene (APN), a member of the monoterpene family, exhibited beneficial effects in animal models of neurodegenerative disorders. As a result, this study assessed the impact of APN on memory in the 3-nitropropionic acid (3-NP) induced model of HD in rats. Male Wistar rats received saline, 3-NP to model HD, or APN (1, 5, or 10 mg/kg) plus 3-NP for 21 days to assess APN’s effects. Working and spatial memory were examined by the Y-maze and Morris-water-maze (MWM) tests. The mRNA levels of neurotrophins and their receptors in the brain cortex and hippocampus of the rats were quantitatively assessed through Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. The results showed that APN, at all three doses, significantly prevented the disease phenotype induced by 3-NP administration. In addition, APN treatment elevated the gene expression levels of BDNF, TrkA, TrkB, and CREB, while significantly decreasing P75 NTR showing a dose-dependent effect in the brain cortex and hippocampus, compared to the 3-NP group. These findings suggest that APN alleviates 3-NP-induced memory deficits by enhancing neurotrophins and their receptor levels in an animal model of HD.

Objectives Non-alcoholic fatty Liver Disease (NAFLD) poses a growing global health concern, yet its complex aetiology remains incompletely understood. Emerging evidence implicates the gut microbiome and choline metabolism in NAFLD pathogenesis. This study aims to elucidate the association of choline-consuming bacteria in gut microbiome with choline level. Methods A population comprising 85 NAFLD patients and 30 healthy controls was selected. DNA extraction from stool samples was conducted using the FavorPrep™ Stool DNA Isolation Mini Kit, followed by polymerase chain reaction (PCR) detection of choline-consuming bacterial strains and quantitative PCR (qPCR) for Cut C gene expression. Choline content measurement was performed using fluorescence high-performance liquid chromatography (FL-HPLC). Results Our findings revealed a significant reduction in choline levels among NAFLD patients compared to healthy controls. ROC curve analysis demonstrated choline levels and Cut C expression as a promising diagnostic tool for NAFLD, with high sensitivity and specificity. The microbial analysis identified specific choline-consuming bacteria enriched in NAFLD patients, notably Anarococcus Hydrogenalis and Clostridium asparagiforme. This was consistent with higher Cut C gene expression in patients compared to healthy individuals, which is responsible for encoding an enzyme to consume choline by these bacteria. Conclusion The current study gives a possible association between gut microbiota and the development of NAFLD, possibly due to an alteration in choline bioavailability. Further research is required to determine whether gut bacteria alter in the context of NAFLD or a change in their composition might lead to NAFLD progression, possibly via alternation in choline bioavailability.

Background: A549, a human lung adenocarcinoma cell line, is a KRAS mutant cell used for over 5 decades as a type II alveolar cell and non-small cell lung adenocarcinoma model. Cyclin-dependent kinase inhibitor 2 A (CDKN2A) and cyclin-dependent kinase inhibitor 2 B (CDKN2B) are protein-coding genes in the INK4-ARF locus and function as tumor suppressors and negative regulators of the cell cycle. These genes have been reported to be deleted in the A549 cell line. The Long non-coding RNAANRIL is located in the antisense direction of CDKN2B and shares a bidirectional promoter with CDKN2A. ANRIL is a negative regulator of the INK4-ARF locus genes and has an oncogenic role in cancers. ANRIL deletion in the A549 cell line has not been reported to date. Objectives: Herein, the presence of ANRIL was investigated in the A549 cell line. Methods: In this study, the A549 cell line from 2 different sources was tested for the presence of the INK4-ARF locus genes by polymerase chain reaction (PCR) using specific primers at both DNA and RNA levels. We compared our findings with Calu-6, MRC-5, and HepG2 cell lines. Results: Our analysis revealed that all protein-coding genes in the INK4-ARF locus, including CDKN2A and CDKN2B, were deleted in the A549 cell lines. Furthermore, we observed that ANRIL was entirely deleted in the A549 cells. The evaluated locus and all of its genes are present and expressed in other investigated cell lines. Conclusions: For the deletion of ANRIL in the A549 cell line, 2 scenarios are possible: First, from a structural point of view, the deletion of the protein-coding genes in the antisense of ANRIL in the INK4-ARF locus implies the possibility of a concurrent loss of ANRIL with the deletion of these genes in the A549 cell line. Second, as cancer cell lines are genetically unstable and are always susceptible to the acquisition of new mutations, ANRIL loss may have occurred later, following a novel genetic alteRNAtion in a population derived from a mutated cell.

Non-alcoholic fatty liver disease is a multifactorial disorder with complicated pathophysiology ranging from simple steatosis to steatohepatitis and liver fibrosis. Trimethylamine-N-oxide (TMAO) production is believed to be correlated with choline deficiency. This study investigated the expression of miRNA-34a, miRNA-122, and miRNA-192 in the fatty liver cell model treated with different concentrations of TMAO. A fatty liver cell model was developed by exposing HepG2 cells to a mixture of palmitate and oleate in a ratio of 1:2 at a final concentration of 1200 μM for 24 h. The confirmed fatty liver cells were treated with 37.5, 75, 150, and 300 μM of TMAO for 24 h. RT-qPCR was used to quantify the expression of microRNAs in a cellular model. The cellular expression of all microRNAs was significantly higher in treated fatty liver cells compared to normal HepG2 cells (P < 0.05). Only 75 and 150 µM of TMAO significantly increased the expression of miRNA-34a and miRNA-122 compared to both fatty and normal control cells (P < 0.05). Our results provided an experimental documentation for the potential effect of TMAO to change the expression of miR-34a and miR-22 as a mechanism for contributing to the pathogenesis of non-alcoholic fatty liver disease.