Real-time Quantitative Reverse Transcription-polymerase Research Articles

Inpp5e Regulated the Cilium-Related Genes Contributing to the Neural Tube Defects Under 5-Fluorouracil Exposure.

Primary cilia are crucial for neurogenesis, and cilium-related genes are involved in the closure of neural tubes. Inositol polyphosphate-5-phosphatase (Inpp5e) was enriched in primary cilia and closely related to the occurrence of neural tube defects (NTDs). However, the role of Inpp5e in the development of NTDs is not well-known. To investigate whether Inpp5e gene is associated with the neural tube closure, we established a mouse model of NTDs by 5-fluorouracil (5-FU) exposure at gestational day 7.5 (GD7.5). The Inpp5e knockdown (Inpp5e-/-) mouse embryonic stem cells (mESCs) were produced by CRISPR/Cas9 system. The expressions of Inpp5e and other cilium-related genes including intraflagellar transport 80 (Ift80), McKusick-Kaufman syndrome (Mkks), and Kirsten rat sarcoma viral oncogene homolog (Kras) were determined, utilizing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), western blot, PCR array, and immunofluorescence staining. The result showed that the incidence of NTDs was 37.10% (23 NTDs/62 total embryos) and significantly higher than that in the control group (P < 0.001). The neuroepithelial cells of neural tubes were obviously disarranged in NTD embryos. The mRNA and protein levels of Inpp5e, Ift80, Mkks, and Kras were significantly decreased in NTD embryonic brain tissues, compared to the control (P < 0.05). Knockdown of the Inpp5e (Inpp5e-/-) reduced the expressions of Ift80, Mkks, and Kras in mESCs. Furthermore, the levels of α-tubulin were significantly reduced in NTD embryonic neural tissue and Inpp5e-/- mESCs. These results suggested that maternal 5-FU exposure inhibited the expression of Inpp5e, which resulted in the downregulation of cilium-related genes (Ift80, Mkks, and Kras), leading to the impairment of primary cilium development, and ultimately disrupted the neural tube closure.

Inactivation of Human Norovirus GII.4's Infectivity in Fresh Oysters (Crassostrea gigas) through Thermal Treatment in Association with Propidium Monoazide.

The current study investigated the effects of heat treatment (85 °C or 100 °C for 5-20 min) on human norovirus (HuNoV) GII.4's capsid stability in fresh oysters. In addition, propidium monoazide (PMA) was used in viral samples to distinguish infectious viruses and evaluated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Further, we explored the effect of the heat treatment on oyster quality (Hunter color and hardness). The titer of HuNoV for oysters significantly (p < 0.05) decreased to 0.39-1.32 and 0.93-2.27 log10 copy number/μL in the non-PMA and PMA-treated groups, respectively, after heat treatment. HuNoV in oysters not treated with PMA showed a decrease of <1.5 - log10, whereas in PMA-treated oysters, a decrease of >1 - log10 was observed after treatment at 85 °C for 10 min. Treatments for both 15 min and 20 min at 100 °C showed a >99% log10 reduction using PMA/RT-qPCR. In the Hunter color, an increase in heat temperature and duration was associated with a significant decrease in 'L' (brightness+, darkness-) and an increase in 'a' (redness+, greenness-) and 'b' (yellowness+, blueness-) (p < 0.05). Our findings confirmed that the hardness of oyster meat significantly increased with increasing temperature and time (p < 0.05). This study demonstrated that PMA/RT-qPCR was effective in distinguishing HuNoV viability in heat-treated oysters. The optimal heat treatment for oysters was 10 min at 85 °C and 5 min at 100 °C.

Synthesis and evaluation of febrifugine derivatives as anticoccidial agents.

A series of new febrifugine derivatives with a 4(3H)-quinazolinone scaffold were synthesized and evaluated for their anticoccidial activity both in vitro and in vivo. The targets' in vitro activity against Eimeria tenella was studied using quantitative real-time reverse transcription polymerase chain reaction and Madin-Darby bovine kidney cells. Most of these compounds demonstrated anticoccidial efficacy, with inhibition ratios ranging from 3.3% to 85.7%. Specifically, compounds 33 and 34 showed significant inhibitory effects on the proliferation of E. tenella and exhibited lower cytotoxicity compared to febrifugine. The IC50 values of compounds 33 and 34 were 3.48 and 1.79 μM, respectively, while the CC50 values were >100 μM for both compounds. Furthermore, in a study involving 14-day-old chickens infected with 5 × 104 sporulated oocysts, treatment with five selected compounds (22, 24, 28, 33, and 34), which exhibited in vitro inhibition rate of over 50% at 100 μM, at a dose of 40 mg/kg in daily feed for 8 consecutive days showed that compound 34 possessed moderate in vivo activity against coccidiosis, with an anticoccidial index of 164. Structure-activity relationship studies suggested that spirocyclic piperidine may be a preferable substructure to maintain high effectiveness in inhibiting Eimeria spp., when the side chain 1-(3-hydroxypiperidin-2-yl)propan-2-one was replaced.

Changes in the expression of mexB, mexY, and oprD in clinical Pseudomonas aeruginosa isolates.

Changes in expression levels of drug efflux pump genes, mexB and mexY, and porin gene oprD in Pseudomonas aeruginosa were investigated in this study. Fifty-five multidrug-resistant P. aeruginosa (MDRP) strains were compared with 26 drug-sensitive strains and 21 strains resistant to a single antibiotic. The effect of the efflux inhibitor Phe-Arg-β-naphthylamide on drug susceptibility was determined, and gene expression was quantified using real-time quantitative real-time reverse transcription polymerase chain reaction. In addition, the levels of metallo-β-lactamase (MBL) and 6'-N-aminoglycoside acetyltransferase [AAC(6')-Iae] were investigated. Efflux pump inhibitor treatment increased the sensitivity to ciprofloxacin, aztreonam, and imipenem in 71%, 73%, and 29% of MDRPs, respectively. MBL and AAC(6')-Iae were detected in 38 (69%) and 34 (62%) MDRP strains, respectively. Meanwhile, 76% of MDRP strains exhibited more than 8-fold higher mexY expression than the reference strain PAO1. Furthermore, 69% of MDRP strains expressed oprD at levels less than 0.01-fold of those in PAO1. These findings indicated that efflux pump inhibitors in combination with ciprofloxacin or aztreonam might aid in treating MDRP infections.

DWN12088, A Prolyl-tRNA Synthetase Inhibitor, Alleviates Hepatic Injury in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a liver disease caused by obesity that leads to hepatic lipoapoptosis, resulting in fibrosis and cirrhosis. However, the mechanism underlying NASH is largely unknown, and there is currently no effective therapeutic agent against it. DWN12088, an agent used for treating idiopathic pulmonary fibrosis, is a selective prolyl-tRNA synthetase (PRS) inhibitor that suppresses the synthesis of collagen. However, the mechanism underlying the hepatoprotective effect of DWN12088 is not clear. Therefore, we investigated the role of DWN12088 in NASH progression. Mice were fed a chow diet or methionine-choline deficient (MCD)-diet, which was administered with DWN12088 or saline by oral gavage for 6 weeks. The effects of DWN12088 on NASH were evaluated by pathophysiological examinations, such as real-time quantitative reverse transcription polymerase chain reaction, immunoblotting, biochemical analysis, and immunohistochemistry. Molecular and cellular mechanisms of hepatic injury were assessed by in vitro cell culture. DWN12088 attenuated palmitic acid (PA)-induced lipid accumulation and lipoapoptosis by downregulating the Rho-kinase (ROCK)/AMP-activated protein kinase (AMPK)/sterol regulatory element-binding protein-1c (SREBP-1c) and protein kinase R-like endoplasmic reticulum kinase (PERK)/α subunit of eukaryotic initiation factor 2 (eIF2α)/activating transcription factor 4 (ATF4)/C/EBP-homologous protein (CHOP) signaling cascades. PA increased but DWN12088 inhibited the phosphorylation of nuclear factor-κB (NF-κB) p65 (Ser536, Ser276) and the expression of proinflammatory genes. Moreover, the DWN12088 inhibited transforming growth factor β (TGFβ)-induced pro-fibrotic gene expression by suppressing TGFβ receptor 1 (TGFβR1)/Smad2/3 and TGFβR1/glutamyl-prolyl-tRNA synthetase (EPRS)/signal transducer and activator of transcription 6 (STAT6) axis signaling. In the case of MCD-diet-induced NASH, DWN12088 reduced hepatic steatosis, inflammation, and lipoapoptosis and prevented the progression of fibrosis. Our findings provide new insights about DWN12088, namely that it plays an important role in the overall improvement of NASH. Hence, DWN12088 shows great potential to be developed as a new integrated therapeutic agent for NASH.

Histone Deacetylase 2 Stabilizes SPARC-related Modular Calcium Binding 2 to Promote Metastasis and Stemness in Gallbladder Cancer.

We aimed to investigate the relationship between histone deacetylase 2 (HDAC2) and SPARC-related modular calcium binding 2 (SMOC2) and the role of SMOC2 in gallbladder cancer (GBC). The expression of HDAC2 and SMOC2 in GBC and normal cells was detected by quantitative real-time reverse transcription polymerase chain reaction (qRTPCR), which was also used to detect the mRNA stability of SMOC2. The combination between HDAC2 and SMOC2 was detected by Chromatin immunoprecipitation (ChIP) assay. After silencing and/or overexpressing HDAC2 and SMOC2, cell viability, migration, invasion, and stemness were respectively tested by the Cell Counting Kit-8 (CCK-8), cell scratch, transwell, and sphere-formation assay. In GBC cells, HDAC2 and SMOC2 were highly expressed. HDAC2 combined with SMOC2 promoted mRNA stability of SMOC2. HDAC2 or SMOC2 overexpression promoted GBC cell metastasis and stemness. SMOC2 overexpression rescued the negative effects of silencing HDAC2 in GBC. HDAC2 stabilizes SMOC2 to promote metastasis and stemness in gallbladder cancer.

Anti-ulcerative colitis effects of chemically characterized extracts from Calliandra haematocephala in acetic acid-induced ulcerative colitis.

Background: Ulcerative colitis is a chronic immune-mediated inflammatory bowel disease that involves inflammation and ulcers of the colon and rectum. To date, no definite cure for this disease is available. Objective: The objective of the current study was to assess the effect of Calliandra haematocephala on inflammatory mediators and oxidative stress markers for the exploration of its anti-ulcerative colitis activity in rat models of acetic acid-induced ulcerative colitis. Methods: Methanolic and n-hexane extracts of areal parts of the plant were prepared by cold extraction method. Phytochemical analysis of both extracts was performed by qualitative analysis, quantitative methods, and high-performance liquid chromatography (HPLC). Prednisone at 2mg/kg dose and plant extracts at 250, 500, and 750mg/kg doses were given to Wistar rats for 11days, which were given acetic acid on 8th day through the trans-rectal route for the induction of ulcerative colitis. A comparison of treatment groups was done with a normal control group and a colitis control group. To evaluate the anti-ulcerative colitis activity of Calliandra haematocephala, different parameters such as colon macroscopic damage, ulcer index, oxidative stress markers, histopathological examination, and mRNA expression of pro and anti-inflammatory mediators were evaluated. mRNA expression analysis was carried out by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Results: The phytochemical evaluation revealed polyphenols, flavonoids, tannins, alkaloids, and sterols in both extracts of the plant. Results of the present study exhibited that both extracts attenuated the large bowel inflammation and prevented colon ulceration at all tested doses. Macroscopic damage and ulcer scoreswere significantly decreased by both extracts. Malondialdehyde (MDA) levels and nitrite/nitrate concentrations in colon tissues were returned to normal levels while superoxide dismutase (SOD) activity was significantly improved by all doses. Histopathological examination exhibited that both extracts prevented the inflammatory changes, cellular infiltration, and colon thickening. Gene expression analysis by RT-qPCR revealed the downregulation of pro-inflammatory markers such as tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) whereas the anti-inflammatory cytokines including Interleukin-4 (IL-4) and Interleukin-10 (IL-10) were found to be upregulated in treated rats. Conclusion: It was concluded based on study outcomes that methanolic and n-hexane extracts of Calliandra haematocephala exhibited anti-ulcerative colitis activity through modulation of antioxidant defense mechanisms and the immune system. In this context, C. haematocephala can be considered as a potential therapeutic approach for cure of ulcerative colitis after bioassay-directed isolation of bioactive phytochemicals and clinical evaluation.

Bladder Cancer Patients with Elevated SPRR1B Expression Experiencing a Poor Prognosis.

SPRR1B, a member of the small proline-rich protein family, is implicated in various epithelial cancers as a potential oncogene linked to tumour growth and poor survival outcomes. However, its role in urothelial bladder carcinoma (UBC) remains to be fully elucidated. Transcriptional profiling data from The Cancer Genome Atlas grouped UBC samples in accordance with SPRR1B expression. Bioinformatic analysis was conducted to evaluate whether SPRR1B is a prognostic factor and a survival factor in UBC. Gene set enrichment analysis (GSEA) was performed to study immune cells and pathways. Reverse transcription quantitative real-time polymerase chain reaction detected gene expression. Immunohistochemistry assessed protein expression. Spearman correlation test analysed the correlation between SPRR1B and the protein p53. The bioinformatics results indicated that the expression level of SPRR1B in UBC tissues was significantly increased compared with that in normal bladder tissues, correlating with clinical characteristics. A high expression predicted poor prognosis and survival. Univariate Cox statistics showed that a high expression level of SPRR1B was correlated with UBC patients having poor overall survival (OS) (p < 0.05). In addition, on the basis of the multivariate Cox analysis, SPRR1B expression was independently correlated with OS (p = 0.005). GSEA analysis revealed enrichment in the p53, apoptosis, and cell cycle signalling pathways, and an association with B cells, lymphocytes, and natural killer cells. In addition, SPRR1B was found to be associated with immune infiltration based on the analysis of immune cell infiltration. Performing corresponding verification on a small number of tissues collected from bladder cancer patients revealed that the expression of this protein was negatively correlated with the expression of p53. SPRR1B overexpression predicts poor UBC outcomes, suggesting its role as a prognostic marker and therapeutic target. Further research is necessary to elucidate its role in UBC progression.

Curcuma longa extract inhibits migration by reducing MMP-9 and Rac-1 expression in highly metastatic breast cancer cells.

Highly metastatic breast cancer is a population of cancer cells that has metastasized to other organs in the body leading to apoptosis resistance. It was reported that MDAMB-231 cells contain lower levels of reactive oxygen species associated with metastatic capability. Curcuma longa (CL) possesses cytotoxic effects in several cancer cells including metastatic breast cancer cells. This study aimed to investigate the effect of CL-inhibited cell migration in highly metastatic breast cancer MDAMB-231 cells. CL was extracted under maceration with methanol. The cytotoxic effect on single and combination treatment of CL was assessed through the MTT assay. Migration analysis was evaluated using scratch wound healing assay, MMP-9 expression by gelatine zymography, Rac-1, and MMP-9 gene expression using Real-Time Quantitative Reverse transcription polymerase chain reaction (qRT-PCR). The apoptosis induction was analyzed through Bax gene expression and Bcl-2 protein expression. We found that CL inhibits the growth of MDAMB-231 cells, induces Bax gene expression, and suppresses Bcl-2 expression in a dose-dependent manner. Moreover, cancer cell migration was suppressed by the presence of CL. qRT-PCR and gelatine zymography assay showed that CL downregulates Rac-1 and MMP-9 gene expression. CL could inhibit the growth and migration of highly metastatic breast cancer cells by reducing the Rac-1 gene expression and regulating apoptosis protein expression.

A novel SERS-lateral flow assay (LFA) tray for monitoring of miR-155-5p during pyroptosis in breast cancer cells.

In the study, a novel surface-enhanced Raman scattering (SERS)-lateral flow assay (LFA) tray for the real-time detection of pyroptosis-associated miR-155-5p in breast cancer cells was established and validated. The SERS probe modified with monoclonal antibodies and functionalized HP1@5-FAM was first synthesized. When miR-155-5p was present, HP1@5-FAM on the SERS probe specifically recognized target miRNAs and hybridized with them, resulting in HP2 on the T line only capturing some SERS probes that were not bound to miR-155-5p. The T line appeared as a light orange band or there was no color change, and the corresponding Raman detection result showed a weak or insignificant Raman signal. The SERS probe showed high selectivity, satisfactory stability, and excellent reproducibility, and the limit of detection (LOD) for miR-155-5p was 7.26 aM. Finally, the proposed SERS-LFA tray was applied to detect miR-155-5p in MBA-MD-468 cells that underwent varying degrees of pyroptosis, and the detection results of SERS were consistent with those of the conventional real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. The study demonstrated that the SERS-LFA tray was a convenient and ultrasensitive method for miR-155-5p real-time detection, which could provide more detailed information for pyroptosis and be of potential value in guiding the treatment of breast cancer.

Linc00662 sponges miR-15b-5p to promote hypopharyngeal squamous cell carcinoma progression by facilitating cancer stem cell-like phenotypes.

Background: Long non-coding RNAs (lncRNAs) are associated with multiple head and neck tumors and play important roles in cancer. This study explored the molecular mechanism of Linc00662 in hypopharyngeal squamous cell carcinoma (HSCC). Methods: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect gene expression in HSCC tissues. The viability and proliferation of tumor cells were measured using CCK-8 assays. HSCC cell apoptosis was measured using flow cytometry and western blotting. Cell stemness was examined using the sphere formation assay. A xenograft tumor model was established to investigate the role of Linc00662 in vivo. Results: The expression level of Linc00662 in HSCC tissues was significantly higher than that in adjacent normal tissues. The expression of Linc00662 had no significant relationship with the tumor stage. Patients with high Linc00662 expression were found to have shorter overall survival than those with low Linc00662 expression. Linc00662 over-expression promoted cell viability and inhibited apoptosis. Using online databases and a dual luciferase reporter, miR-15b-5p was confirmed as a potential downstream sponge of Linc00662. Moreover, Linc00662 was negatively associated with miR-15b-5p in HSCC cells. Depletion of miR-15b-5p can reverse the function of Linc00662 in vivo and in vitro. Furthermore, Linc00662 promotes tumor growth, which was abolished by miR-15b-5p mimics. Importantly, the stemness of cancer stem cells was mediated by the Linc00662/miR-15b-5p axis. Conclusion: Patients with HSCC with high Linc00662 showed poor prognosis and high Linc00662 induced stemness of tumor cells by targeting miR-15b-5p. Linc00662 may serve as a novel diagnostic and target marker for head and neck squamous cell carcinoma.

Formononetin Restrains Tumorigenesis of Breast Tumor by Restraining STING-NF-κB and Interfering with the Activation of PD-L1.

Breast cancer (BC), a common tumor in women, has high morbidity and mortality. Formononetin, an active ingredient in red clover and Astragalus membranaceus, has a wide range of pharmacological applications, including as an anticancer agent. Since immunotherapy is a hot topic in the treatment strategy of BC, it was dedicated to appraising the specific mechanism of formononetin in BC immunotherapy in this research. Different formononetin concentrations (0, 20, 40, 60, 80, 100 μM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot. Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone. Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.

MicroRNA-15a-5p suppresses hypoxia-induced tumor growth and chemoresistance in bladder cancer by binding to eIF5A2.

In various malignant tumors (including bladder cancer) poor prognosis is associated with hypoxia and therapeutic resistance. Evidence indicates that in bladder cancer, microRNAs (miRNAs) have vital functions in acquired drug resistance. However, the involvement of miRNAs in hypoxia-mediated bladder cancer doxorubicin (Dox) resistance is unknown. Herein, we showed that hypoxia and Dox treatment downregulated miR-15a-5p expression. Using UM-UC-3 and J82 bladder cancer cell lines and in vivo mouse models of bladder cancer, we confirmed that miR-15a-5p arrests tumor cell growth and Dox resistance in vitro and in vivo. Furthermore, we determined the interaction between miR-15a-5p and eukaryotic translation initiation factor 5A-2 (eIF5A2) using dual luciferase reporters and quantitative real-time reverse transcription polymerase chain reaction assays. We also showed that a miR-15a-5p agomir repressed EIF5A2 expression in bladder cancer cells, thereby inhibiting the epithelial-mesenchymal transition (EMT) induced by Dox or hypoxia. Moreover, ectopic expression of miR-15a-5p abrogated eIF5A2-mediated Dox resistance in bladder cancer cells. Collectively, these data indicated that hypoxia promotes tumor growth and chemoresistance through the HIF-1α/miR-15a-5p/eIFTA2/EMT pathway. This new finding not only has implications for improving our understanding of the Dox resistance process during bladder cancer progression but also indicates that the miR-15a-5p agomir is a promising tool to prevent Dox resistance in patients with bladder cancer.

Tranexamic Acid Ameliorates Skin Hyperpigmentation by Downregulating Endothelin-1 Expression in Dermal Microvascular Endothelial Cells.

Although reports suggest that tranexamic acid (TXA) has clinical benefits for melasma patients by oral, intralesional and topical treatment, the optimal route of TXA therapy and the underlying mechanism involved remain poorly defined. To compare the skin lightening effect between oral TXA and topical TXA and to dissect the molecular mechanisms using ultraviolet B (UVB)-induced hyperpigmentation mouse model, ex vivo cultured human skin explant, and cultured melanocytes (MCs) and endothelial cells. Melanin content and cluster of differentiation 31 (CD31)-positive cell numbers were measured in tail skins from UVB-irradiated mice treated by intragastral or topical TXA using immunofluorescent and Fontana-Masson staining. The conditioned medium (CM) was harvested from human umbilical vein endothelial cells treated with or without 3 mM TXA and was used to treat MCs for 48 hours. mRNA and protein levels of tyrosinase and microphthalmia-associated transcription factor were measured using quantitative real-time reverse transcription polymerase chain reaction and western blotting assays. HMB45- and CD31-positive cell numbers as well as melanin content were also examined in ex vivo cultured human skin explants. The hyperpigmented phenotype were significantly mitigated in UVB-irradiated tail skin plus intragastral TXA-treated mice compared with mice treated with UVB only or with UVB plus topical TXA. CD31-positive cell numbers correlated with the anti-melanogenic activity of TXA therapy. The data from cultured cells and skin tissues showed that suppression of endothelin-1 (ET-1) in vascular endothelial cells by TXA reduced melanogenesis and MC proliferation. Oral TXA outperforms topical TXA treatment in skin lightening, which contributes to suppression of ET-1 in dermal microvascular endothelial cells by TXA.

Effect of m6A Methylation Modification on IncRNA ENST00000425005 in Doxorubicin Resistance and Epithelial-Mesenchymal Transition Progression in Lung Cancer Cells

Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for 18.4% of all cancer deaths. This study aims to investigate the underlying mechanism by which long non-coding RNA (lncRNA) ENST00000425005 mediates doxorubicin resistance and epithelial-mesenchymal transition (EMT) in lung cancer cells. The expression levels of ENST00000425005 in non-small cell lung cancer (NSCLC) cells were determined using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The protein levels of alkB homolog 5 (ALKBH5) and EMT markers (including Snail1, E-cadherin, N-cadherin, and Vimentin) were assessed using Western Blot analysis. RNA binding protein immunoprecipitation assays were conducted to detect the interaction between ENST00000425005 and ALKBH5. Cell viability was evaluated using cell counting kits assay, and cell invasion was determined by transwell assay. It was found that ENST00000425005 expression was downregulated, while ALKBH5 expression was upregulated in NSCLC cells. Additionally, ALKBH5 bound to ENST00000425005 and downregulated its expression. Overexpression of ALKBH5 reduced m6A modification and RNA levels of ENST00000425005. Moreover, co-overexpression of ENST00000425005 and ALKBH5 rescued loss of NSCLC cell viability, invasion, and doxorubicin resistance caused by overexpression of ENST00000425005. Furthermore, this co-overexpression rescued ENST00000425005-induced changes in expression of E-cadherin, Snail1, N-cadherin, and Vimentin. The reduction of m6A methylation modification on lncRNA ENST00000425005 caused by binding to ALKBH5 promoted doxorubicin resistance and EMT progression in NSCLC cells. In summary, targeting lncRNA ENST00000425005 holds promise as a therapeutic strategy for NSCLC.

PLIN5 Suppresses Lipotoxicity and Ferroptosis in Cardiomyocyte via Modulating PIR/NF-κB Axis.

Cardiomyocyte lipotoxicity and ferroptosis are the key to the development of diabetic cardiomyopathy (DCM). Perilipin 5 (PLIN5) is perceived as a significant target of DCM. This study aimed to focus on the role and mechanism of PLIN5 on lipotoxicity and ferroptosis in DCM.Following transfection, mouse cardiomyocytes HL-1 were induced by 0.1 mM palmitic acid (PA) to set up lipotoxic cardiomyocyte models. The cell viability and lipid accumulation were evaluated by cell counting kit-8 assay and Oil red O staining, respectively. Ferrous ion (Fe2+), glutathione (GSH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels were determined to verify the effects of PLIN5 or Pirin (PIR) on ferroptosis. Quantitative real-time reverse transcription polymerase chain reaction or Western blot was performed for quantitative analysis.PLIN5 overexpression promoted the viability, GSH level, and expression of GPX4/PIR/intracellular P65, yet suppressed lipid accumulation, level of Fe2+/MDA/ROS, and expression of interleukin (IL)-1β/IL-18/intranuclear P65 in PA-stimulated HL-1 cells. PIR silencing counteracted the roles of PLIN5 overexpression in PA-stimulated HL-1 cells.PLIN5 suppresses lipotoxicity and ferroptosis in cardiomyocyte via modulating PIR/NF-κB axis, hinting its potential as a therapeutic target in DCM.

Flowering-associated gene expression and metabolic characteristics in adzuki bean (Vigna angularis L.) with different short-day induction periods.

The adzuki bean is a typical short-day plant and an important grain crop that is widely used due to its high nutritional and medicinal value. The adzuki bean flowering time is affected by multiple environmental factors, particularly the photoperiod. Adjusting the day length can induce flower synchronization in adzuki bean and accelerate the breeding process. In this study, we used RNA sequencing analysis to determine the effects of different day lengths on gene expression and metabolic characteristics related to adzuki bean flowering time. 'Tangshan hong xiao dou' was used as the experimental material in this study and field experiments were conducted in 2022 using a randomized block design with three treatments: short-day induction periods of 5 d (SD-5d), 10 d (SD-10d), and 15 d (SD-15d). A total of 5,939 differentially expressed genes (DEGs) were identified, of which 38.09% were up-regulated and 23.81% were down-regulated. Gene ontology enrichment analysis was performed on the target genes to identify common functions related to photosystems I and II. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified two pathways involved in the antenna protein and circadian rhythm. Furthermore, florescence was promoted by down-regulating genes in the circadian rhythm pathway through the blue light metabolic pathway; whereas, antenna proteins promoted flowering by enhancing the reception of light signals and accelerating electron transport. In these two metabolic pathways, the number of DEGs was the greatest between the SD-5d VS SD-15d groups. Real-time reverse transcription‒quantitative polymerase chain reaction analysis results of eight DEGs were consistent with the sequencing results. Thus, the sequencing results were accurate and reliable and eight genes were identified as candidates for the regulation of short-day induction at the adzuki bean seedling stage. Short-day induction was able to down-regulate the expression of genes related to flowering according to the circadian rhythm and up-regulate the expression of certain genes in the antenna protein pathway. The results provide a theoretical reference for the molecular mechanism of short-day induction and multi-level information for future functional studies to verify the key genes regulating adzuki bean flowering.

Age-related dynamics in acute myeloid leukemia: Implications for prognosis, risk stratification, and treatment response

Abstract: BACKGROUND: Acute myeloid leukemia (AML) is a complex, heterogeneous disease driven by acquired somatic mutations. The presence of specific mutations advances stratification, treatment, and prognosis. Linear accumulation of mutations over time is a crucial factor in cancer development, particularly among elderly patients. Our recent study on gene rearrangement in AML revealed a significant association between age and adverse risk cases. AIM: The aim of this study was to examine the distribution of age, molecular characteristics, risk stratification, and treatment response based on age among patients with de novo AML in Iraq. PATIENTS AND METHODS: A prospective cohort study enrolled 115 Iraqi adult patients diagnosed with de novo AML using morphology and flow cytometry from December 2020 to May 2022. The Leukemia Q-Fusion Screening Kit, employing multiplex reverse transcription–real-time quantitative polymerase chain reaction with 30 gene rearrangements, was employed for the identification of gene rearrangement. The patients received care and follow-up at the Hematology Unit of Baghdad Teaching Hospital in Medical City. Ethical approval from the College of Medicine’s Ethical Committee at the University of Baghdad was secured before commencing the research, ensuring adherence to ethical standards throughout the study. RESULTS: The age distribution exhibited a bimodal pattern, with a mean of 45.1 ± 17.5 years, ranging from 18 to 84 years, and a median of 46 years. A total of 39.1% of patients were diagnosed with AML before the age of 35 years, while 43% were diagnosed after the age of 51 years. AML patients with RARA mutations, RUNX1::RUNX1T1 alterations, and NPM1 mutations were predominantly observed in younger individuals, as well as those diagnosed with AML defined by differentiation. Conversely, KMT2A rearrangements were more prevalent among older age groups, with a statistically significant difference in the distribution of AML classifications according to the World Health Organization (WHO) by age categories (P = 0.001). The risk stratification based on age and response assessment showed a notable higher risk profile observed among elderly patients that was associated with adverse risk and poorer response and mortality (P &lt; 0.05). The prediction of treatment response accuracy rate was improved by adding age to the WHO classification and ELN 2022 risk stratification (73.5%–87.9%). CONCLUSION: Age significantly influences AML prognosis and treatment response. Incorporating age into risk stratification improves accuracy. Tailored approaches considering age are vital for optimizing AML management and outcomes.

Can Multiplex SYBR Green Real-Time PCR Assay Serve as a Detection and Quantification Method Comparable to the TaqMan Method for SARS-CoV-2 Diagnosis?

The reopening of schools, business, and social sectors during the COVID-19 pandemic has caused a current increase in the number of COVID-19 cases and clusters all over the globe. While the COVID-19 pandemic is far from over, the reopening and resumption of all economic sectors are essential to recovering the world economy. Health experts all over the world have determined that the real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) method is the gold standard for diagnosing COVID-19 infections due to the test’s high sensitivity and specificity. During the past 3 years when WHO declared the COVID-19 pandemic, the cost of laboratory diagnosis of COVID-19 using a robust RT-qPCR assay is still considerably expensive, especially for low and middle-income countries. Therefore, numerous studies have reported optimized SYBR green methods which are more economical than the qPCR probe assay. Continuous diagnostic testing is vital to mitigate the spread of COVID-19. However, there is a question as to whether SYBR Green may serve as an excellent detection and quantification method for molecular diagnosis to perform SARS-CoV-2 screening. This review summarizes the numerous studies using SYBR Green RT-PCR to detect SARS-CoV-2. The reliability of SYBR Green qPCR assays for determining gene expression based on their performance is justified and the quality is comparable to the TaqMan method.

MicroRNA-21 and MicroRNA-125b expression in skin tissue and serum as predictive biomarkers for psoriasis.

Psoriasis is a chronic, inflammatory, and hyperproliferative skin disease. We have investigated the role of miR-21 and miR-125b in the development of psoriasis and atopic eczema and their relation with the severity of the diseases. Participants included 40 psoriasis patients, 40 healthy controls, and 40 atopic eczema patients as a positive control group. In addition, analysis of mRNA expression of miR-125b and miR-21 was carried out utilizing quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) in serum samples and skin tissue. Our results have demonstrated that miR-21 was significantly overexpressed in the psoriatic and atopic eczema skin tissue and serum samples compared to controls, whereas miR-125b was significantly down-expressed in psoriatic and atopic eczema skin tissues and serum samples. There was a statistically significant positive correlation between the psoriasis area and severity index (PASI) score and miR-21 among the studied groups in both serum and tissue samples. In contrast, there was a statistically significant negative association between the miR-125b and PASI score. On the other hand, there was no significant relation between the extent of body surface area (BSA), intensity, and subjective symptoms using visual analog scale (VAS) of atopic eczema disease and miRNA-21 and miRNA-125b in both tissue and serum. In conclusion, miR-21 gene expression was significantly increased in psoriatic and atopic eczema skin samples and serum samples, whereas miR-125b was statistically lowered in psoriatic and atopic eczema patient samples. The miR-21 and miR-125b expression level has a possible predictive value as a marker for psoriasis severity but not for atopic eczema severity.