qPCR Studies Research Articles

Comprehensive in silico, characterization and expression profiling of MST genes in soybean (Glycine max) reveal their roles in development and metal ion responses

Monosaccharide transporters (MSTs) are important plant glucose transporters that play roles in carbon allocation, growth, development, and stress regulation. However, research on MST regulatory genes in soybeans remains unexplored. In this study, we identified 110 MST-like candidate genes in the Glycine max L. (soybean) genome. The MST genes were distributed throughout 20 chromosomes, with many undergoing segmental duplication. The majority of MST group proteins exhibit significant preservation in Arabidopsis and soybean, and phylogenetic analysis reveals seven main categories. The promoters of the GmMST genes comprise cis-acting elements associated with plant responses to abscisic acid, auxin signaling, methyl jasmonate, low temperatures, and abiotic stresses. The RNA-seq data demonstrated diverse expression levels of MST genes across various tissues or organs, categorizing them into 11 primary clusters. The co-expression study of the network complex indicated that the GmMST genes encode proteins that interact with many essential genes associated with gibberellin, which are pivotal in seed and stamen development. A qPCR study validated the expression patterns of seven MST genes with exposure to copper (Cu) and cadmium (Cd) stress. Certain MST genes were notably stimulated by both Cu and Cd stress treatments, suggesting their involvement in defense responsiveness. Therefore, we provide a comprehensive analysis of the MST genes in soybeans and emphasize their substantial role in plant development and tolerance to metal ion stressors.

Evaluation of the toxicity of perfluorooctanoic acid toward human colorectal cancer cells using multi-dimensional approaches

While human exposure to perfluorooctanoic acid (PFOA) can lead to ulcerative colitis, the molecular mechanisms responsible for PFOA-induced intestinal toxicity are unclear. Herein, we examined the toxicity of PFOA toward human colorectal cancer cells (HCT116) from three dimensions: the cytotoxic phenotype, cell respiration, and transcription levels of metabolism-related genes. Formazan was used to assess how PFOA exposure affects HCT116-cell relative viability, after which the mitochondrial respiratory activities of these cells were determined by analyzing extracellular flux. The quantitative real-time polymerase chain reaction (qPCR) method was used to detect metabolism-related gene expression levels. The cytotoxicity assay revealed that the HCT116 showed significantly inhibited relative activities compared to those of the control when exposed to 300 μmol/L PFOA for 48 h (p<0.01), with most cells retained at the G0/G1 stage. In contrast, the mitochondrial respiratory activities of the HCT116 were promoted by concentrations of PFOA as low as 50 μmol/L. Two genes related to cellular metabolism (dipeptidase 1 (DPEP1) and sphingosine kinase 1 (SPHK1)) were found to be related to the PFOA-promoted formation of ulcerative colitis using our self-developed Metabolic Gene and Pathway Query software and Comparative Toxicogenomics Database (CTD). The qPCR studies revealed that DPEP1 and SPHK1 expression levels were enhanced by 8-10 times in HCT116 exposed to 300 μmol/L PFOA relative to the control, whereas this trend was not observed for HCT116 exposed to 50 μmol/L PFOA. Collectively, these results suggest that the respiratory activity of cellular mitochondria may serve as an index for determining the interference effects associated with PFOA and that metabolic pathways mediated by DPEP1 and SPHK1 may be involved in the development of PFOA-induced ulcerative colitis. Future studies should investigate the relationships between changes in metabolism-related genes (DPEP1 and SPHK1) and the mitochondrial respiratory activities of intestinal cells, and verify the roles played by the DPEP1 and SPHK1 genes in PFOA-induced intestinal inflammation using in-vivo models.

The emerging role of S100A4 and S100A14 proteins in colorectal cancer progression.

Colorectal cancer (CRC) is the third most frequent type of cancer and the second leading cause of cancer-related deaths globally. Despite a thorough understanding of its biology, etiology, and epidemiology, an estimated 1.8 million new cases are diagnosed each year, and 900000 people die as a result of malignancy. The current study aims to investigate the expression pattern of S100A4 and S100A14 proteins in CRC tissue specimens and a panel of cell lines. Furthermore, to explore the metastatic potential of the aforementioned proteins in relation to the epithelial-mesenchymal transition and their possible association with the clinical features of CRC. The present study involved 80 patients diagnosed with CRC. Upon identification of the sociodemographic and clinicopathological features of the participants, immunohistochemical studies were conducted to measure the expression pattern of the S100 proteins in CRC tissues. In addition to qPCR and western blot studies, a series of in vitro experiments were conducted in a panel of CRC cell lines to assess the effects of S100 protein expression in cell migration, invasion, and proliferation. The number of CRC patients with high S100A4 expression levels was considerably higher than those with low expression (p < 0.0001). S100A4 is positively correlated with TNM staging, nodal metastasis, distant metastasis, and perineural invasion and was statistically significant (p = 0.02, 0.01, 0.0001, and 0.02, respectively). In vitro studies demonstrated that S100A14 knockdown induced EMT and resulted in a substantial increase in cell proliferation, migration, and invasion in HT29 cells. Moreover, S100A4 knockdown substantially inhibited migration, invasion, and proliferation in LoVo cells. The findings collectively suggest that both S100A4 and S100A14 play a pivotal role in colorectal cancer progression. Overexpression of S100A4 consistently with S100A14 downregulation is associated with the activation of epithelial-mesenchymal transition, which in turn enhances cell proliferation, migration, and invasion.

Chemical Synthesis and Antifibrotic Properties of (±)-Cochlearol T, (±)-Ganocochlearin A, and (±)-Cochlearol Y.

The cochlearols and ganocochlearins are natural products with unique antifibrotic and renoprotective activities in models of kidney disease. They represent compelling lead compounds for pharmacological intervention against kidney disease, often characterized by renal fibrosis. We report a four-step synthesis of (±)-cochlearol T (1) and the first reported syntheses of (±)-ganocochlearin A (2) and (±)-cochlearol Y (3) through a strategy that includes a Robinson annulation and unexpected oxidative aromatization. We also access tricyclic intermediate 12 that represents a formal synthesis of ganocins A-C and ganocochlearins C-D. We investigated the activity of these synthesized compounds in vitro by inducing fibrosis in a human kidney cell line with TGF-β1. The effect on fibrosis was assessed by qPCR and Western blot studies. We detected significantly lower mRNA gene and protein expression of fibrosis markers for all three natural products.

Investigating the possible mechanism of Cornus officinalis in the therapy of ischemic stroke by UHPLC-Q-TOF-MS, network pharmacology, molecular docking, and experimental verification

Ethnopharmacological relevanceCornus officinalis is a conventional Chinese medicine for tonifying liver and kidney in ancient China. The active ingredients from Cornus officinalis can delay the progression of cerebral aneurysms, alleviate experimental autoimmune encephalomyelitis, and show a good intervention effect on brain diseases. Loganin, the active ingredient of Cornus officinalis, has a neuroprotective effect on cerebral ischemia-reperfusion injury in mice. It is yet unknown, nevertheless, how Cornus officinalis works to treat ischemic stroke. Aim of the studyBased on ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), network pharmacology and molecular docking, Cornus officinalis's mechanism of intervention in ischemic stroke is explored and verified by experiments. Materials and methodsTo examine the chemical components of Cornus officinalis, UHPLC-Q-TOF-MS was used. The network pharmacology was used to construct the “active ingredient-core target-main pathway” network of Cornus officinalis. Then, the link between the main active components and the key protein targets, as determined by network pharmacology, was verified through the application of molecular docking. The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model used in this study was created using the suture technique. The pharmacological effects of Cornus officinalis were explored by neurological function score, behavior, TTC staining, ultrasound and flow cytometry. Western blot and qPCR were used to confirm the core target. ResultsThe outcomes of the investigation demonstrated that Cornus officinalis had a potent anti-ischemic stroke effect. UHPLC-Q-TOF-MS method was used to determine 24 chemical constituents in Cornus officinalis, of which 22 components had a close relationship with protein targets relevant to ischemic stroke. The 27 protein targets screened by “active ingredient-core target-main pathway” may be the possible targets of Cornus officinalis in the therapy of ischemic stroke. Most of the 27 protein targets had to do with the inflammatory response, apoptosis and energy metabolism. KEGG enrichment analysis showed that AGE/RAGE ranked high and was closely related to inflammatory response. Molecular docking predicted that the top 10 components in the network diagram had good binding with inflammatory factors IL6, IL-1β and TNF-α protein targets. Western blot research outcomes stated that Cornus officinalis could firmly impede the production of AGE, RAGE, and P-NFκB P65. Cornus officinalis had the potential to prevent ischemic stroke by drastically inhibiting the production of TNF-α, IL-1β, and IL-6, according to the results of qPCR study. ConclusionThis study found that Cornus officinalis can improve the brain injury, motor ability and blood flow velocity of MCAO/R rats and suppress the inflammatory reaction through the AGE/RAGE/NFκB pathway to exert the therapeutic effect on ischemic stroke.

Gene expression & biochemical analysis in alkaptonuria caused by a founder pathogenic variant across different age groups from India.

Background & objectives Alkaptonuria (AKU) is an autosomal recessive disease wherein biallelic pathogenic variants in the homogentisate 1,2- dioxygenase (HGD) gene encoding the enzyme homogentisate 1,2 dioxygenase cause high levels of homogentisic acid (HGA) to circulate within the body leading to its deposition in connective tissues and excretion in urine. A homozygous splice donor variant (c.87+1G>A) has been identified to be the founder variant causing alkaptonuria among Narikuravars, a group of gypsies settled in Tamil Nadu. Methods Blood and urine samples of 30 homozygous splice site donor variant individuals (2 groups aged 7-20 and 21-83 yr, with 9 and 21 individuals, respectively), carriers and 30 wild-type individuals from the Narikuravars were collected during field visits after obtaining informed consent. Clinical evaluation and genetic counselling were done. The plasma and urine HGA levels were estimated by high-performance liquid chromatography. RNA was extracted from the peripheral blood and reverse transcribed. Sanger sequencing was done to check the consequence of the splice donor variant. Relative quantification of the cDNA in the three groups was done by real-time qPCR (RT-qPCR) studies using reference genes followed by Pearson's correlation analysis. Results In our cohort, among the affected alkaptonuria individuals, the minimum age for eye pigmentation detected was 23 yr. Similarly, the minimum age for back pain and any joint pain was 30 yr and 38 yr, respectively. Sequencing of the cDNA confirmed exon 2 skipping in affected individuals. In comparison to the normal individuals, the affected individuals showed reduced HGD expression. HGD relative expression showed a significant correlation (P<0.05) with mean plasma HGA levels in the younger (≤22 yr) age group but not in the older one. There was also a significant correlation (P<0.05) of reduced HGD expression with back pain in the 21-37 yr age group. Increasing age showed a positive correlation with circulating mean plasma HGA levels and a negative correlation with excreted HGA. Interpretation & conclusions As per the authors' knowledge, this is the first study to confirm the functional effect by RT-PCR of this highly prevalent founder HGD variant causing alkaptonuria in the Narikuravar community. Both plasma and urinary HGA levels correlated well with the gene expression of this variant and could serve as potential markers of AKU severity for those with this variant.

Abstract A051: Innate immune cell dynamics and pancreatic tumor progression in alcoholic pancreatitis

Abstract Introduction: Persistent inflammation due to alcoholic chronic pancreatitis (ACP) is associated with pancreatic ductal adenocarcinoma (PDAC). Myeloid cell infiltration within the pancreas, particularly with oncogenic mutant KrasG12D/+ (*Kras), is crucial for driving PDAC carcinogenesis. Our previous work demonstrated that hyperactivation of cyclic AMP response element binding protein (CREB) accelerates ADM/PanIN progression with heightened fibroinflammation. However, the association between CREB in precursor neoplastic lesions and innate myeloid cells remains unclear. Methods: We established an ACP mouse model using an alcohol diet along with prolonged caerulein administration in Ptf1aCreERTM/+; LSL-KrasG12D/+ (KC), and CREB deleted [Crebfl/fl in KC (KCC-/-)] mice. Single-cell RNA sequencing (scRNA-seq) of the mouse pancreas, multiparametric immunophenotyping of tumor-infiltrating myeloid cells and chromatin immunoprecipitation (ChIP-seq) with CREB pulldown were performed. Cytokine arrays and qPCR studies followed co-culture assays using mouse neutrophils (J774M) and macrophages (RAW 264.7) with epithelial cell lines harboring *Kras with in vitro ACP induction were conducted. Results: ACP induction in KC mice led to increased infiltration of neutrophils and alternatively activated macrophages with the pancreas. Conversely, Creb-deleted mice exhibited significantly reduced myeloid cell infiltration despite ACP induction. ChIP-Seq and qPCR identified CREB-regulated chemokines and immunomodulators as mediators of myeloid cell trafficking. Co-culture of mouse neutrophils and macrophages with KC-ACP conditioned media significantly increased IL-1 receptor antagonist (Il-1ra) expression and secretion. Single-cell transcriptomic analysis revealed heightened Il1rn mRNA levels in neutrophils and macrophages in KC-ACP when compared with KC pancreatic tumors. Creb-deleted PDAC models showed strong downregulation of Il1rn in macrophages, confirming CREB’s role in modulating myeloid cell interactions via epithelial cell signaling. Conclusions: Our findings highlight that CREB activation in epithelial cells under ACP conditions facilitates crosstalk with myeloid cells, promoting IL-1ra upregulation in neutrophils and macrophages. This CREB-driven regulation of IL-1ra in myeloid cells contributes to inflammation-associated tumorigenesis. Targeting this axis may offer a novel therapeutic approach to curb pancreatic cancer progression. Citation Format: Siddharth Mehra, Sudhakar Jinka, Varun Krishnamoorthy, Supriya Srinivasan, Anna Bianchi, Andrew Adams, Haleh Amirian, Manan Patel, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. Innate immune cell dynamics and pancreatic tumor progression in alcoholic pancreatitis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A051.

Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses.

Holotrichia parallela is among the world's most destructive pests. For accurate qPCR and gene expression studies, the selection of stable and appropriate reference genes is crucial. However, a thorough evaluation of potential reference genes for use in H. parallela research is lacking. In this study, 11 reference genes (GAPDH, RPL32, RPL7A, RPS18, RPL13a, RPL18, Actin, RPS7, RPS3, VATB,and EF1A) were evaluated under different biological conditions and environmental stresses. The stability of 11 potential reference gene transcripts was evaluated through various computational tools, including geNorm, BestKeeper, NormFinder, theΔCt method, and the RefFinder program. Under various developmental stages and RNAi conditions, RPL18 and RPL13a exhibited the greatest stability. RPL13a, RPL18, and RPL32 were the most stable genes in both male and female adults. Under differing tissue conditions, RPL13a and RPS3 stood out as the most reliable. Moreover, under varying photoperiod conditions, RPL13a, RPS3 and RPL32 were the most stable genes. Lastly, Actin and RPL13a were the most stable genes across different temperatures. These findings offer essential criteria for selecting suitable reference genes across diverse experimental settings, thereby establishing a solid basis for accurate gene expression studies in H. parallela using RT-qPCR.

CO2 protects cells from iron-Fenton oxidative DNA damage in E. coli and humans.

Whereas hydroxyl radical is commonly named as the Fenton product responsible for DNA and RNA damage in cells, here we demonstrate that the cellular reaction generates carbonate radical anion due to physiological levels of bicarbonate. Analysis of the metabolome, transcriptome and, in human cells, the nuclear genome shows a consistent buffering of H2O2-induced oxidative stress leading to one common pathway, namely guanine oxidation. Particularly revealing are nanopore-based studies of direct RNA sequencing of cytosolic and mitochondrial ribosomal RNA along with glycosylase-dependent qPCR studies of oxidative DNA damage in telomeres. The focusing of oxidative modification on one pathway is consistent with the highly evolved base excision repair suite of enzymes and their involvement in gene regulation in response to oxidative stress.

Characterization of the impact of dietary immunostimulant CpG on the expression of mRNA biomarkers involved in the immune responses in Atlantic salmon (Salmo salar)

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.

Antioxidants Hydroxytyrosol and Thioredoxin-Mimetic Peptide CB3 Protect Irradiated Normal Tissue Cells.

Reducing side effects in non-cancerous tissue is a key aim of modern radiotherapy. Here, we assessed whether the use of the antioxidants hydroxytyrosol (HT) and thioredoxin-mimetic peptide CB3 (TMP) attenuated radiation-induced normal tissue toxicity in vitro. We used primary human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (HaCaT) as normal tissue models. Cells were treated with HT and TMP 24 h or immediately prior to irradiation. Reactive oxygen species (ROS) were assessed via luminescent- and fluorescence-based assays, migration was investigated using digital holographic microscopy, and clonogenic survival was quantified by colony formation assays. Angiogenesis and wound healing were evaluated via time-dependent microscopy. Secreted cytokines were validated in quantitative polymerase chain reaction (qPCR) studies. Treatment with HT or TMP was well tolerated by cells. The application of either antioxidant before irradiation resulted in reduced ROS formation and a distinct decrease in cytokines compared to similarly irradiated, but otherwise untreated, controls. Antioxidant treatment also increased post-radiogenic migration and angiogenesis while accelerating wound healing. HT or TMP treatment immediately before radiotherapy increased clonogenic survival after radiotherapy, while treatment 24 h before radiotherapy enhanced baseline proliferation. Both antioxidants may decrease radiation-induced normal tissue toxicity and deserve further pre-clinical investigation.

Abstract We003: Identification of CircRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathways Related to Cardiomyocyte Proliferation in Neonatal Mice

Introduction: Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis. Methods: Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining. Results: Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle. Conclusion: Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.

Exploring the Common Pathogenic Mechanisms of Psoriasis and Atopic Dermatitis: The Interaction between SGK1 and TIGIT Signaling Pathways.

This study aims to explore the common pathogenic mechanisms of psoriasis and atopic dermatitis, two T-cell-mediated autoimmune diseases. Utilizing single-cell transcriptomic sequencing data, we revealed that Treg cells primarily express TIGIT in both psoriasis and atopic dermatitis, and identified a subset of macrophages that highly express SGK1. These cells can interact with T cells via the NECTIN2-TIGIT signaling pathway, inhibiting the differentiation of T cells into a pro-inflammatory phenotype, thereby uncovering a common immunoregulatory mechanism in both diseases. Furthermore, we discovered that inhibition of SGK1 exacerbates the inflammatory response in disease models of both conditions. These findings not only provide a new perspective for a common therapeutic strategy for psoriasis and atopic dermatitis but also highlight the importance of considering these molecular interactions in future treatments. Validation of these observations through further qPCR, immunofluorescence, and animal studies has identified potential new targets for the treatment of psoriasis and atopic dermatitis.

The SP1/SNHG16/GLUT1 axis promotes prostate cancer proliferation and invasion by regulating glucose metabolism

IntroductionThe SP1/SNHG16/GLUT1 is involved in diverse cancer-related process. This study was designed to study the role of SP1/SNHG16/GLUT1 axis in prostate cancer (PCa) via modulation of glucose metabolism.Material and methodsThe expression profile of SNHG16 in PCa tissues was obtained from the online public database GEPIA (http://gepia.cancer-pku.cn/). Real-time qPCR was used to ascertain the mRNA expression. To find cellular proliferation and invasion, respectively, CCK-8, transwell, and glycolysis were used to monitor proliferation, invasion, and glycolysis.ResultsAnalysis of GEPIA2 data revealed upregulation of SNHG16 in PCa tissues and a positive association between GLUT1 (SLC2A1) and SP1/SNHG16 expression in the correlation study. Consistently, SPI and SNHG16 were either overexpressed or knocked down in PCa cells to unveil the role of the SP1/SNHG16/GLUT1 axis. Results demonstrated that PC-3 and DU145 cell proliferation were promoted by the overexpression of either SPI or SNHG16. On the other hand, PC-3 and DU145 cell proliferation were reduced upon knockdown of SP1 or SNHG16. A real-time qPCR study revealed that GLUT1 mRNA was upregulated by SP1/SNHG16 overexpression and downregulated by SP1/SNHG16 knockdown. In PCa cells, overexpression of SNHG16/SP1 resulted in enhanced utilization of glucose, lactose, and ATP production, whereas SNHG16/SP1 knockdown had the reverse effect. Lastly, transwell assay results showed that overexpression of SNHG16/SP1 promoted, while knockdown of SNHG16/SP1 inhibited the invasiveness of PC-3 and DU145 PCa cells.ConclusionsCollectively, the SP1/SNHG16/GLUT1 axis regulates the proliferation of PCa cells via the glycolytic route and thus may act as a therapeutic target for PCa treatment.

Reproducible and high sample throughput isomiR next-generation sequencing for cancer diagnosis.

e15013 Background: Next-generation sequencing (NGS) can produce up to 6 Tb of data per run with single-nucleotide accuracy, making it ideal for quantifying isomiRs, which encompass both canonical miRNAs and their variants, for clinical applications. However, NGS has poor reproducibility and low sample throughput in quantifying circulating isomiRs due to significant technical variations and the limitations of the multiplex strategy, as evidenced by the fact that no isomiR NGS technique has been successfully used to diagnose cancer. Methods: To address these challenges, a library construction method including a dual unique-dual-index (DUDI) technology was developed. DUDI uses a pair of Inner UDI (IUDI) and outer UDI (OUDI) to label a sample. Twelve independent batches of isomiR NGS were carried out, including three repeated batches. Each batch included 100 gastric cancer and 100 control plasma samples. Batch effect, correlation coefficient (R), and principal component (PCA) analyses were used to evaluate technical reproducibility. Machine learning binary classification was used to assess biological reproducibility, with each pair of batch data serving interchangeably as both training and testing data. Results: In this multicenter study, over 700G of isomiR data were generated from 402 gastric cancer and 498 control samples, with a maximum error rate of 1 in 7 million isomiRs being assigned to wrong samples. The PCA plot indicates high technical reproducibility across the three repeated batches, shown by the extensive intermingling of data points from each batch and the lack of distinct batch-wise clustering. This observation is reinforced by that the R value for each of 239 isomiRs between the repeated batches are close to 1. While the mutual machine learning validations between the repeated batches yielded ~95% accuracy, indicating high biological reproducibility. The accuracies of the validations between the different batches of different samples range from 70% to 82%. The lower accuracy is as expected, given the high genetic heterogeneity of cancer and the small sample size. Furthermore, the IsomiR differentiated expression profiles from the current NGS study closely match those from prior qPCR studies. Conclusions: The DUDI library construction method can produce reproducible high sample throughput NGS data, yet it is cost-effective and straightforward. The maximum number of samples that can be multiplexed in an NGS project is almost one million, i.e., 976 * 976, as IUDI and OUDI can be any of the 976 designed DUDIs. This number far exceeds the high sample throughput requirements of any NGS application. While the capability to distinguish true biological variations of IsomiRs from technical noise, demonstrated by the high technical and biological reproducibility and concordance with the qPCR data, enables the development of robust machine learning algorithms for cancer diagnostics.

Exercise role on inflammatory and MCH-1 plasticity markers in severely contused spinal cord injured rodents

Approximately 17,500 new cases of Spinal cord Injury (SCI) occur annually in the United States. Growing evidence suggests that mobilizing the body following SCI through physical therapy regimens can help patients regain locomotor function. The immune response and the role of inflammation following SCI represents an important factor contributing to plasticity and recovery of function post-injury. The immune response uses both pro and anti-inflammatory factors to facilitate healing at the primary and secondary injury. In the severe case of SCI, inflammation can lead to maladaptive plasticity and that can hinder locomotor recovery. Pro-inflammatory cytokines such as Tumor necrosis factor alpha (TNF-a) can enhance the C-fiber activation in the dorsal horn resulting in sensitization and maladaptive plasticity. Exercise can also be pro-inflammatory. Exercise has been shown to decrease TNF-a levels and increases Interleukin-10 (IL-10) in human skeletal muscle. Bodyweight treadmill training in SCI animal has shown improvements in locomotor function. Interleukin-10 (IL-10), demonstrates potent anti-inflammatory effects in which it down-regulates TNF-alpha and its secondary effects. In addition, major histocompatibility complex class I proteins (MHC1), play a vital role in maintaining the structural integrity of neurons which is critical for fostering an environment for spinal plasticity to occur. In this study, we focus on the role of training paradigms to be used to facilitate locomotor recovery in spinally contused rats. The Body-Weight Supported Treadmill Training (BWSTT) and circular Bodyweight-Supported Ambulatory Rodent Trainer (cBART) both represent locomotor training for SCI animals. Mid-thoracic spinal cord contusion was performed, and the animals were allowed to recover for two weeks. The contused BWSTT alone, or cBART alone or combined BWSTT and cBART training groups were initiated at beginning of the third week for a total of 8-weeks of training. Controls include contused rats that received no training, and intact group (SHAM). At 10 weeks, the spinal cord was dissected for qPCR study. We measure the levels of TNF-alpha, MHC1, and IL-10 in the thoracic segment above the injury site, the injury site, Lumbar 1-3 and Lumbar 3-5 segment. Results show that in the cBART trained group, the MHC-1 expression in the L3-L5 segment was significantly greater when compared to the contused injury with no training intervention. No training effect was realized in MHC-1 in the L1-L3 trained groups. The training paradigms did not modulate TNF-a levels in the lumbar spinal cord segments. Lastly, IL-10 expression was undetectable in all the spinal cord segments. In this severe spinal cord contused model, we were only able to show modulation in the MHC-1 expression in the trained lumbar segment as function of exercise. Additional work will be required to consider the intensity of locomotor training and the severity of spinal cord contusion impact on these plasticity markers. Future understanding of these mechanisms may be useful in developing better locomotor training methods and drug targets for facilitation of recovery following SCI. CSULA RSCA 2020 MINI Grant- Dr. Joseph CSULA MORE Programs Offce, MBRS-URISE (1734GM145503) Bridges to Doctorate 5T23GM146700 NIH R25HD097701-04 - Dr. Ray De Leon. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PARP1 promotes EGFR-TKI drug-resistance via PI3K/AKT pathway in non-small-cell lung cancer.

Tyrosine kinase inhibitor (TKI) resistance is the main type of drug resistance in lung cancer patients with epidermal growth factor receptor (EGFR) mutations, but its underlying mechanism remains unclear. The purpose of this work was to investigate the mechanism by which PARP1 regulates EGFR-TKI resistance to identify potential targets for combating drug resistance. The GEO databases, TCGA databases, western blot and qPCR studies were used to investigate the expression of PARP1 in lung cancer cells and tissues and its correlation with the prognosis of lung cancer. The expression of PARP1 in lung cancer TKI resistant cell PC9-ER and TKI sensitive cell PC9 was analyzed by qPCR and western blot. After knocking down of PARP1, CCK-8 assays, colony formation, flow cytometry were used to investigate its impact on erlotinib sensitivity, cell survival, cell cycle, and apoptosis. RNA-seq was used to investigate the mechanism by which PARP1 participates in EGFR-TKI resistance, and the results were validated in vitro and in vivo studies. PARP1 was highly expressed in both lung cancer tissues and cells. Subsequently, increased PARP1 expression was observed in PC9-ER compared with its parental cell line. Knockdown of PARP1 increased erlotinib sensitivity, promoted cell apoptosis, and suppressed cell growth. RNA-seq and previous studies have shown that the PI3K/AKT/mTOR/P70S6K pathway is involved in PARP1-mediated TKI resistance, and these results were confirmed by Western blot in vitro and in vivo. PARP1 may serve as a potential therapeutic target for reversing EGFR-TKI resistance in NSCLC via the PI3K/AKT/mTOR/P70S6K pathway.

Simultaneous treatment with cells and rosemary extract ameliorates 6-OHDA-induced toxicity in the hippocampus of mice.

In this study, we delved into the hippocampal region to understand the effects of adipose stem cells (ADSCs) and rosemary extract (RE). Our main objective was to explore how these substances influence spatial memory, neurotrophins, and changes in antioxidant enzymes. Moreover, we meticulously investigated the impact of dopamine deficiency, a notable characteristic linked with Parkinson's disease (PD), on memory impairment. This study comprised five groups of Wistar rats - all male, all selected randomly. We labeled two of these gatherings "lesion" (L) and "sham" (SH). Each got injections in the bilateral form with 6 μg - one group getting saline, while another got 6-OHDA. From couple weeks before the neurotoxin injection to 8 weeks later on, our lesion cohort was treated with rosemary at a dosage rate of 50 mg/kg body weight - let's call it RE for simplicity sake. Moreover, there is also this other lot, designated as cell-transplanted lesion group or catchy exercise (CE) as we prefer to interpret them; they had cell transplants conducted exactly 7 days after receiving their respective injections. Bringing up the rear, we got a group treated with both cell transplant and rosemary (CE+R). We performed spatial memory tests at 4 weeks, then again at 8. At the end of eighth week, the brains were extracted for q-PCR, enzymatic and immunohistochemical studies. Turning our gaze toward a comparison between the CE+R and CE groups versus the L group, we spot an intriguing drop in escape latency time. There is also more time spent in quadrants. Digging deeper into this matter, the CE+R bunch unveiled a clear surge when it comes to the expression of four genes, namely NGF, BDNF, NT3, and NT4! This was notable especially while comparing with both R and even other fellows from its very own broader group - CE. In a bit complex bit related to enzyme activity now, there is some good news as well for those in favor of potent antioxidants such as GPx or SOD. CE + R group, showed a significant increase of GPX and SOD enzymes, compared to the SH and L groups, and a significant decrease of MDA activity as compared to other treated groups. A significant decrease of escape latency and increase of time in quadrant were observed in the CE+R and CE groups compared to L group. What's more, the levels of MDA in the CE+R group plummeted significantly when set up against the SH group. Wrapping things up, a definite downscale was observed in the density of GFAP-positive cells throughout different regions located within the hippocampus; this decline presented itself not solely in treatment groups but gripped onto those falling under SH as well, especially when compared to its comrade - the L group. Using ADSCs and taking RE orally have shown promising results in improving memory issues linked with PD.

Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues.

Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues.

K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3

K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.