γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids.

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αß T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.

Color Stability of Different Denture Teeth Following Immersion in Staining Solutions.

Objectives: This study aimed to assess the color change (∆E00) of 7 brands of denture teeth (conventional acrylic and composite teeth) following immersion in staining solutions. Materials and Methods: Maxillary central incisor denture teeth made of 4 conventional acrylic resins (Vitapan, SR Vivodent PE, Beta Star and Crystal) and 3 composite resins (Finex, Emeral and Phonares II) were randomly divided into four groups (n=5). Denture teeth of different brands were immersed in tea, coffee, cola, and turmeric solutions. The solutions were incubated at 37°C. The baseline color of the teeth was measured using an intraoral spectrophotometer. The color of the teeth was measured after 24 h (∆E12), 1 week (∆E13), 2 weeks (∆E14), and 1 month (∆E15). ∆E00 was calculated and analyzed using one-way ANOVA. Pairwise comparisons were performed by the Tukey's post-hoc test (P<0.05). Results: The color stability of all teeth was significantly affected by the solutions (P<0.001). The type of tooth and coloring solution had significant interactions at all times (P<0.05). Turmeric caused the maximum color change in all teeth after 1 month. Repeated measures ANOVA showed that ∆E00 of all teeth was significantly affected by the duration of immersion in the solutions (P<0.001). Conclusion: within the limitations of this study, 1-month immersion of denture teeth in coffee, tea and cola solution altered the ∆E values; however, they were within the acceptable range, except for Beta Star. Turmeric solution caused unacceptable color change in all denture teeth even after 24 h of immersion.

The antimicrobial and antibiofilm effects of three herbal extracts on Streptococcus mutans compared with Chlorhexidine 0.2% (in vitro study).

There is a special focus on using natural materials and herbal plants to prevent dental caries. Previous studies showed that some herbal plants have antimicrobial effects on oral pathogens. Thus we investigated the antimicrobial effects of three herbal extracts (Carum copticum, Phlomis bruguieri, and Marrubium parviflorum) on the growth of Streptococcus mutans, as the most important bacteria causing dental caries. First, plant methanolic extracts were prepared. Then, to evaluate the antimicrobial activity of the three herbal extracts, the agar well diffusion method and MIC were performed. The biofilm formation was carried out using a broth dilution method with 2% glucose-supplemented BHIS in sterile 96-well microplates. Serial dilutions (50, 25, 12.5, 6.25, 3.12 mg/ml) of extracts were prepared. Next, a 0.5 McFarland Suspension of S. mutans was added to wells. The inhibitory effect on biofilm formation was measured by the ELISA reader apparatus. The assay was repeated three times, and the average was calculated as 3. The results were compared with those of Chlorhexidine 0.2%. Carum copticum showed a better effect in the agar well diffusion method than others. MIC of the extracts of Carum coptimum, Phlomis bruguieri, and Marrubium parviflorum were 3.12, 6.25, and 12.5 mg/ml, respectively. Overall, the highest activity belonged to Carum copticum extract. For the anti-biofilm effect, the OD values of Carum copticum and Marrubium parviflorum were significantly different from that of Phlomis bruguieri. Although all of the methanolic herbal extracts can inhibit S. mutans growth and remove the biofilm, the effect of Carum copticum was better than Phlomis bruguieri and Marrubium parviflorum. Further studies are recommended to indicate how these extracts perform against the bacteria.

The natural compound atraric acid suppresses androgen-regulated neo-angiogenesis of castration-resistant prostate cancer through angiopoietin 2.

Castration-resistant prostate cancer (CRPC) is an aggressive lethal form of prostate cancer (PCa). Atraric acid (AA) not only inhibits the wild-type androgen receptor (AR) but also those AR mutants that confer therapy resistance to other clinically used AR antagonists, indicating a different mode of AR antagonism. AA induces cellular senescence and inhibits CRPC tumour growth in in vivo xenograft mouse model associated with reduced neo-angiogenesis suggesting the repression of intratumoural neo-angiogenesis by AA. In line with this, the secretome of CRPC cells mediates neo-angiogenesis in an androgen-dependent manner, which is counteracted by AA. This was confirmed by two in vitro models using primary human endothelial cells. Transcriptome sequencing revealed upregulated angiogenic pathways by androgen, being however VEGF-independent, and pointing to the pro-angiogenic factor angiopoietin 2 (ANGPT2) as a key driver of neo-angiogenesis induced by androgens and repressed by AA. In agreement with this, AA treatment of native patient-derived PCa tumour samples ex vivo inhibits ANGPT2 expression. Mechanistically, in addition to AA, immune-depletion of ANGPT2 from secretome or blocking ANGPT2-receptors inhibits androgen-induced angiogenesis. Taken together, we reveal a VEGF-independent ANGPT2-mediated angiogenic pathway that is inhibited by AA leading to repression of androgen-regulated neo-angiogenesis.

The androgen receptor-lncRNASAT1-AKT-p15 axis mediates androgen-induced cellular senescence in prostate cancer cells.

The bipolar androgen therapy (BAT) to treat prostate cancer (PCa) includes cycles of supraphysiological androgen levels (SAL) under androgen-deprivation therapy (ADT). We showed previously that SAL induces cellular senescence in androgen-sensitive PCa cells and in ex vivo-treated patient PCa tumor samples. Here, we analyzed the underlying molecular pathway and reveal that SAL induces cellular senescence in both, castration-sensitive (CSPC) LNCaP and castration-resistant PCa (CRPC) C4-2 cells through the cell cycle inhibitor p15INK4b and increased phosphorylation of AKT. Treatment with the AKT inhibitor (AKTi) potently inhibited SAL-induced expression of p15INK4b and cellular senescence in both cell lines. Proximity-ligation assays (PLA) combined with high-resolution laser-scanning microscopy indicate that SAL promotes interaction of endogenous androgen receptor (AR) with AKT in the cytoplasm as well as in the nucleus detectable after three days. Transcriptome sequencing (RNA-seq) comparing the SAL-induced transcriptomes of LNCaP with C4-2 cells as well as with AKTi-treated cell transcriptomes revealed landscapes for cell senescence. Interestingly, one of the identified genes is the lncRNASAT1. SAL treatment of native patient tumor samples ex vivo upregulates lncRNASAT1. In PCa tumor tissues, lncRNASAT1 is downregulated compared with nontumor tissues of the same patients. Knockdown indicates that the lncRNASAT1 is crucial for SAL-induced cancer-cell senescence as an upstream factor for pAKT and for p15INK4b. Further, knockdown of lncRNASAT1 enhances cell proliferation by SAL, suggesting that lncRNASAT1 serves as a tumor suppressor at SAL. Interestingly, immunoprecipitation of AR detected lncRNASAT1 as an AR-interacting partner that regulates AR target-gene expression. Similarly, RNA-ChIP experiments revealed the interaction of AR with lncRNASAT1 on chromatin. Thus, we identified a novel AR-lncRNASAT1-AKT-p15INK4b signaling axis to mediate SAL-induced cellular senescence.

Assessing contribution of bottled water in nutrient absorption using the bottled water nutritional quality index (BWNQI) in Iran.

In this study, the contribution of bottled water in the absorption of nutritional minerals in Iran has been investigated. To calculate the nutritional quality index of bottled water (BWNQI) and evaluate the contribution of bottled water in nutrient absorption; the concentration of nutrient minerals, the standard level of these elements in bottled water, the recommended amount of nutrient mineral and the total consumption of drinking water in different age-sex groups were analyzed. The results showed that the average contribution of bottled water in absorbing the recommended amount of the nutrients of fluoride (F), magnesium (Mg), calcium (Ca), sodium (Na), copper (Cu), zinc (Zn) and manganese (Mn) was 12.16, 4.98, 4.85, 2.12, 0.49, 0.33 and 0.02%, respectively. According to the BWNQI index, the bottled water quality was as follows: 53.5% poor, 36.6% marginal, 7% fair, 2.81% good. Although most of the bottled water studied in this research were mineral water, a significant portion of them had poor nutritional quality, so the addition of minerals needed by the body through bottled water should be given more attention by the bottled water manufacturers and suppliers.

Antithetic hTERT Regulation by Androgens in Prostate Cancer Cells: hTERT Inhibition Is Mediated by the ING1 and ING2 Tumor Suppressors.

The human telomerase is a key factor during tumorigenesis in prostate cancer (PCa). The androgen receptor (AR) is a key drug target controlling PCa growth and regulates hTERT expression, but is described to either inhibit or to activate. Here, we reveal that androgens repress and activate hTERT expression in a concentration-dependent manner. Physiological low androgen levels activate, while, notably, supraphysiological androgen levels (SAL), used in bipolar androgen therapy (BAT), repress hTERT expression. We confirmed the SAL-mediated gene repression of hTERT in PCa cell lines, native human PCa samples derived from patients treated ex vivo, as well as in cancer spheroids derived from androgen-dependent or castration resistant PCa (CRPC) cells. Interestingly, chromatin immuno-precipitation (ChIP) combined with functional assays revealed a positive (pARE) and a negative androgen response element (nARE). The nARE was narrowed down to 63 bp in the hTERT core promoter region. AR and tumor suppressors, inhibitor of growth 1 and 2 (ING1 and ING2, respectively), are androgen-dependently recruited. Mechanistically, knockdown indicates that ING1 and ING2 mediate AR-regulated transrepression. Thus, our data suggest an oppositional, biphasic function of AR to control the hTERT expression, while the inhibition of hTERT by androgens is mediated by the AR co-repressors ING1 and ING2.

A Novel Splice Variant of the Inhibitor of Growth 3 Lacks the Plant Homeodomain and Regulates Epithelial-Mesenchymal Transition in Prostate Cancer Cells.

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial-mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.

Androgen Receptor-Dependent Mechanisms Mediating Drug Resistance in Prostate Cancer.

Androgen receptor (AR) is a main driver of prostate cancer (PCa) growth and progression as well as the key drug target. Appropriate PCa treatments differ depending on the stage of cancer at diagnosis. Although androgen deprivation therapy (ADT) of PCa is initially effective, eventually tumors develop resistance to the drug within 2-3 years of treatment onset leading to castration resistant PCa (CRPC). Castration resistance is usually mediated by reactivation of AR signaling. Eventually, PCa develops additional resistance towards treatment with AR antagonists that occur regularly, also mostly due to bypass mechanisms that activate AR signaling. This tumor evolution with selection upon therapy is presumably based on a high degree of tumor heterogenicity and plasticity that allows PCa cells to proliferate and develop adaptive signaling to the treatment and evolve pathways in therapy resistance, including resistance to chemotherapy. The therapy-resistant PCa phenotype is associated with more aggressiveness and increased metastatic ability. By far, drug resistance remains a major cause of PCa treatment failure and lethality. In this review, various acquired and intrinsic mechanisms that are AR­dependent and contribute to PCa drug resistance will be discussed.

LncRNAs associated with multiple sclerosis expressed in the Th1 cell lineage.

Multiple sclerosis (MS) is a type of inflammatory and demyelinating disorder of the central nervous system in which immune-mediated inflammatory processes are elicited by secreted cytokines from T helper (Th)-1 and Th17 cells. While some protein-coding genes expressed in T cell types have established involvement in MS disease progression, little is understood about the roles of long noncoding RNAs (lncRNAs) within the disease landscape. LncRNAs, noncoding RNAs longer than 200 nucleotides, likely control gene expression and function of Th1 cells, and offer the potential to act as therapeutic and biomarker candidates for MS. We identified lncRNAs in Th1 cells linked to MS. Expression levels of candidate lncRNAs and genes were evaluated in 50 MS patients and 25 healthy controls using quantitative real-time polymerase chain reaction, and their correlations were assessed. LncRNAs encoded by AC007278.2 and IFNG-AS1-001 showed significantly higher expression in relapsing Phase MS patients whereas IFNG-AS1-003 was elevated in patients in the remitting phase compared with relapsing patients. Collectively, these misregulated lncRNAs may provide valuable tools to understand the relationships between lncRNAs and MS, and possibly other related disorders.

Evaluation of fixation disparity curve parameters with the modified near mallett unit in symptomatic and asymptomatic university students.

BACKGROUND: Research suggests that fixation disparity data are extremely useful in the assessment of the binocular and accommodative systems. OBJECTIVES: The purpose of this study was to evaluate fixation disparity curve (FDC) parameters with a modified near Mallett unit in symptomatic and asymptomatic students of Paramedical Sciences School of Mashhad University of Medical Sciences in 2007. PATIENTS AND METHODS: In this analytical-descriptive study, 100 students were selected randomly and divided into symptomatic and asymptomatic groups. Fixation disparity curve parameters were determined for each subject and compared in symptomatic and asymptomatic groups. RESULTS: There were more subjects with exo fixation disparity than eso fixation disparity in the study sample. The means for fixation disparity, fixation disparity curve x-intercept, and slope with the modified Mallett unit were each significantly different by Mann-Whitney U test in the symptomatic and asymptomatic groups. Also there was a significant difference in the distributions of fixation disparity curve types in the two symptom groups by Chi-square test. CONCLUSIONS: The X-intercepts (point at which the FDC crosses the X-axis) were on average more in the base-in direction, Y-intercepts (point at which the FDC crosses the Y-axis) were shifted in the exo direction, and slopes were steeper in the symptomatic group.

Evaluation of fixation disparity curve with the modified near Mallett unit and the wesson fixation disparity card in symptomatic and asymptomatic subjects.

PURPOSE: The purpose of this study is the evaluation of fixation disparity curve (FDC) parameters with an instrument that includes a central fusion lock (the modified near Mallett unit) and another without one (the Wesson fixation disparity card) to determine which is useful for diagnosis of symptomatic from asymptomatic subjects. MATERIALS AND METHODS: In this analytical-descriptive study, 100 students were selected randomly and divided into symptomatic and asymptomatic groups. FDC parameters were determined with the Wesson card and the modified near Mallett unit for each subject and compared in symptomatic and asymptomatic groups. Data were analyzed by Wilcoxon, Mann-Whitney U, Spearman correlation coefficient and Chi-square tests. RESULTS: The mean slope, y-intercept, and x-intercept with the Mallett unit and the Wesson card were significantly different in the symptomatic and asymptomatic groups (p < 0.001). Significant correlations were obtained between the Mallett unit and the Wesson card in y-intercept, x-intercept and slope (p < 0.001). There was a significant difference in the distributions of fixation disparity curve types in the two symptom groups with the Mallett unit (p = 0.01) and the Wesson card (p = 0.002) by Chi-square test. CONCLUSIONS: Among symptomatic participants in this study, both Type I and Type III FD are more common than previously thought, but depend upon the method used to measure it. The x-intercepts were on average displaced in the base-in direction, y-intercepts were shifted in the exo direction, and the slopes were steeper with the Wesson FD card compared with the modified near Mallett unit. This may be related to the design of two devices. These differences were more significant in the symptomatic group.

Body mass index and binocular vision skills.

INTRODUCTION: Body Mass Index (BMI) is of increasing interest to eye care practitioners. Associations have recently been proven between high BMI and several diseases affecting the eyes, including AMD, intracranial hypertension, optic disc cupping, and glaucoma. The symptoms of dizziness and vertigo have also been associated with high BMI. However, to these authors' knowledge, there has been no study performed comparing BMI to binocular function. METHODS: In this analytical-descriptive study, 119 randomly selected young subjects had their BMI measured, along with refractive error, dissociated phoria, near point of convergence, vergence ranges and facility, and stereopsis. RESULTS: In most situations, the subjects classified as normal and overweight, based on their BMI had better performance than those classified as underweight or obese. The worst binocular performance was found in underweight subjects. The one-way ANOVA showed only statistically significant differences between mean of near point of convergence and vergence facility, in different states of BMI. CONCLUSION: Unlike most ocular diseases that are adversely affected by higher BMI values, most binocular vision skills are adversely affected by lower BMI values. The possible reasons for this are discussed.