Chemiluminescence-Based Assay to Monitor Early Oxidative Bursts in Soybean (Glycine max) Lateral Roots.

The reactive oxygen species (ROS) burst assay is a valuable tool for studying pattern-triggered immunity (PTI) in plants. During PTI, the interaction between pathogen recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) leads to the rapid production of ROS in the apoplastic space. The resultant ROS can be measured using a chemiluminescent approach that involves the usage of horseradish peroxidase and luminol. Although several methods and protocols are available to detect early ROS bursts in leaf tissues, no dedicated method is available for root tissues. Here, we have established a reliable method to measure the PAMP-triggered ROS burst response in soybean lateral roots. In plants, lateral roots are the potential entry and colonization sites for pathogens in the rhizosphere. We have used important PAMPs such as chitohexaose, flagellin 22 peptide fragment, and laminarin to validate our method. In addition, we provide a detailed methodology for the isolation and application of fungal cell wall components to monitor the oxidative burst in soybean lateral roots. Furthermore, we provide methodology for performing ROS burst assays in soybean leaf discs with laminarin and fungal cell walls. This approach could also be applied to leaf and root tissues of other plant species to study the PTI response upon elicitor treatment. © 2023 Wiley Periodicals LLC. Basic Protocol: Reactive oxygen species (ROS) burst assay in soybean lateral root tissues Alternate Protocol: ROS burst assay in soybean leaf discs Support Protocol: Isolating fungal cell wall fractions.

Epigenetic reprogramming during prostate cancer progression: A perspective from development.

Conrad Waddington's theory of epigenetic landscape epitomize the process of cell fate and cellular decision-making during development. Wherein the epigenetic code maintains patterns of gene expression in pluripotent and differentiated cellular states during embryonic development and differentiation. Over the years disruption or reprogramming of the epigenetic landscape has been extensively studied in the course of cancer progression. Cellular dedifferentiation being a key hallmark of cancer allow us to take cues from the biological processes involving epigenetic reprogramming in development such as the cellular differentiation and morphogenesis. Here, we discuss the role of epigenetic landscape and its modifiers in cell-fate determination, differentiation and prostate cancer progression. Lately, the emergence of RNA-modifications has also furthered our understanding of epigenetics in cancer. The overview of the epigenetic code regulating androgen signalling, and progression to aggressive neuroendocrine stage of PCa reinforces its gene regulatory functions during the development of prostate gland as well as cancer progression. Additionally, we also highlight the clinical implications of cancer cell epigenome, and discuss the recent advancements in the therapeutic strategies targeting the advanced stage disease.

Transcriptional network involving ERG and AR orchestrates Distal-less homeobox-1 mediated prostate cancer progression.

Distal-less homeobox-1 (DLX1) is a well-established non-invasive biomarker for prostate cancer (PCa) diagnosis, however, its mechanistic underpinnings in disease pathobiology are not known. Here, we reveal the oncogenic role of DLX1 and show that abrogating its function leads to reduced tumorigenesis and metastases. We observed that ~60% of advanced-stage and metastatic patients display higher DLX1 levels. Moreover, ~96% of TMPRSS2-ERG fusion-positive and ~70% of androgen receptor (AR)-positive patients show elevated DLX1, associated with aggressive disease and poor survival. Mechanistically, ERG coordinates with enhancer-bound AR and FOXA1 to drive transcriptional upregulation of DLX1 in ERG-positive background. However, in ERG-negative context, AR/AR-V7 and FOXA1 suffice to upregulate DLX1. Notably, inhibiting ERG/AR-mediated DLX1 transcription using BET inhibitor (BETi) or/and anti-androgen drugs reduce its expression and downstream oncogenic effects. Conclusively, this study establishes DLX1 as a direct-target of ERG/AR with an oncogenic role and demonstrates the clinical significance of BETi and anti-androgens for DLX1-positive patients.

Epigenetic Silencing of miRNA-338-5p and miRNA-421 Drives SPINK1-Positive Prostate Cancer.

PURPOSE: Serine peptidase inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in prostate cancer remains largely unknown. EXPERIMENTAL DESIGN: miRNA prediction tools were employed to examine the SPINK1-3'UTR for miRNA binding. Luciferase reporter assays were performed to confirm the SPINK1-3'UTR binding of shortlisted miR-338-5p/miR-421. Furthermore, miR-338-5p/-421-overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and a mouse xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. IHC and RNA in situ hybridization were carried out on prostate cancer patients' tissue microarray for SPINK1 and EZH2 expression, respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421-regulatory regions. Bisulfite sequencing and methylated DNA immunoprecipitation were performed on prostate cancer cell lines and patients' specimens. RESULTS: We established a critical role of miRNA-338-5p/-421 in posttranscriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive cells abrogates oncogenic properties including cell-cycle progression, stemness, and drug resistance, and shows reduced tumor burden and distant metastases in a mouse model. Importantly, we show that patients with SPINK1-positive prostate cancer exhibit increased EZH2 expression, suggesting its role in epigenetic silencing of miRNA-338-5p/-421. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive prostate cancer cells and patients' specimens confirms epigenetic silencing. CONCLUSIONS: Our findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive prostate cancer, although restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.See related commentary by Bjartell, p. 2679.

Targeting NF-kappa B Signaling by Artesunate Restores Sensitivity of Castrate-Resistant Prostate Cancer Cells to Antiandrogens.

Androgen deprivation therapy (ADT) is the most preferred treatment for men with metastatic prostate cancer (PCa). However, the disease eventually progresses and develops resistance to ADT in majority of the patients, leading to the emergence of metastatic castration-resistant prostate cancer (mCRPC). Here, we assessed artesunate (AS), an artemisinin derivative, for its anticancer properties and ability to alleviate resistance to androgen receptor (AR) antagonists. We have shown AS in combination with bicalutamide (Bic) attenuates the oncogenic properties of the castrate-resistant (PC3, 22RV1) and androgen-responsive (LNCaP) PCa cells. Mechanistically, AS and Bic combination inhibits nuclear factor (NF)-κB signaling and decreases AR and/or AR-variant 7 expression via ubiquitin-mediated proteasomal degradation. The combination induces oxidative stress and apoptosis via survivin downregulation and caspase-3 activation, resulting in poly-ADP-ribose polymerase (PARP) cleavage. Moreover, preclinical castrate-resistant PC3 xenograft studies in NOD/SCID mice (n =28, seven per group) show remarkable tumor regression and significant reduction in lungs and bone metastases upon administering AS (50 mg/kg per day in two divided doses) and Bic (50 mg/kg per day) via oral gavage. Taken together, we for the first time provide a compelling preclinical rationale that AS could disrupt AR antagonist-mediated resistance observed in mCRPC. The current study also indicates that the therapeutic combination of Food and Drug Administration-approved AS or NF-κB inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC patients.

Molecular Discriminators of Racial Disparities in Prostate Cancer.

Recent molecular characterization of prostate cancer (PCa) identified novel genetic aberrations and disease subtypes. The frequencies of molecular aberrations show racial disparity. Clinical strategies and targeted therapies embracing these racial differences are required. Here we discuss ethnic differences in genetic alterations and their impact on the susceptibility, progression, and treatment of prostate cancer.

Impact of injectable progestogen contraception in early puerperium on lactation and infant health.

INTRODUCTION: Unmet need for family planning approaches 40% or higher and many women are at risk of unintended pregnancy. Progesterone contraception after birth is frequently recommended, but concern remain of inhibition of lactation or effects on infant health. In present study we aimed to evaluate impact of short term use of injectable depot medroxy progesterone acetate (DMPA) if given in early post partum period on infant health and breast feeding performance. MATERIAL AND METHODS: A prospective case control study was conducted and 250 women immediately after delivery were recruited in the study. 150 women who were recruited as study group received Inj. DMPA 150 mg intra muscularly after initiation of lactation before discharge from hospital (Day 2-Day 10 of their delivery). 100 post partum women, not using hormonal contraception were taken as controls. All the subjects were followed to complete a full 6 months follow up. Duration and frequency of lactation, gain in weight, gain in height and any illness spells in the infant were noted at 6 weeks, 3 months and 6 months follow up. In the study group 100 subjects completed 6 months follow-up. The statistical analysis was carried out by using SPSS software version 7. The statistical technique used was z score(significance shown by z score> 1.96) and repeated measure analysis (two way Anova technique). RESULTS: Hundred percent of primigravidas in the study group and 95% in the control group were satisfied with their lactation amount. The average gain in height was comparable in both study and control group {4.36±0.56 vs 4.33±0.54 (z score 0.38) at 6 weeks, 12.44±0.73 vs 12.40±0.71 (z score 0.39) at 3 months 17.30±0.91 vs 17.28±0.83 (z score 0.16) at 6 months}. Similarly average gain in weight was also not significantly different in two groups {0.89±0.11 vs 0.93±0.10 (z score 0.71) at 6 weeks, 1.77±0.17 vs 1.78±0.16 (z score 1.07) at 3 months and 3.53±0.30 vs 3.46±0.33 (z score 1.19) at 6 months}. The results were comparable with other studies. CONCLUSION: Injectable DMPA use as a contraceptive in the immediate post partum period was found to be a safe and effective alternate method with no deleterious effect on mother's milk and infant's growth.