Semiquantitative Real-time Polymerase Chain Reaction Research Articles

Reduced graphene oxide/gold nanoparticles based ultrasensitive resistive sensor for PCA3

Early prostate cancer detection can be improved by detecting more specific markers like PCA3 RNA. This study reports a novel and simple method for detecting PCA3 RNA using a sensor based on reduced graphene oxide (RGO), gold nanoparticles (AuNP), and a single-stranded DNA (ssDNA). The device was characterized at each fabrication step using atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM). The RGO/AuNP/ssDNA sensor exhibited a significant change in resistance (6.51–15.01%) when exposed to varying concentrations of PCA3 (0.1–100 ng/mL). The developed device showed selective response towards PCA3 (14.5% for 50 ng/mL) when compared with negative controls like phosphate buffer saline solution (0.28%), PCA3-negative control sample (1.03%), RNA extracted from lung cancer (0.88%), and breast cancer (1.17%) cell lines. The RGO/AuNP/ssDNA sensor could also be employed to quantify the PCA3 RNA present in the RNA mixture extracted from a prostate cancer cell line and the observed findings were in excellent agreement with semi-quantitative real-time polymerase chain reaction (RT-PCR) results.

Exploring the anti-angiogenic properties of diosgenin saponin from Trigonella graecum (fenugreek): A study on Zebrafish embryos

Background: Angiogenesis plays a role in both physiological and pathological processes such as wound healing, embryonic development, and cancer metastasis, and it is the process by which new blood vessels are formed from existing ones. Diosgenin is a naturally occurring steroidal sapogenin found in plants such as yam and fenugreek, which is a compound of interest because of its pharmacological properties, including anti-carcinogenic effects. Zebrafish embryos are used due to their translucent nature, which facilitates the direct observation of vascular development. Aims and Objectives: The purpose of this study is to utilize zebrafish embryos to examine the impact of diosgenin on angiogenesis. It specifically aims to determine whether diosgenin could inhibit the development of inter-segmental vessels (ISV) in zebrafish embryos and also to investigate its effect on vascular endothelial growth factor (VEGF-A), a key regulator of angiogenesis. Materials and Methods: To explore the anti-angiogenic properties of diosgenin, we performed in vivo experiments using the zebrafish angiogenesis model. This involved staining red blood cells with O-dianisidine and conducting semiquantitative real-time polymerase chain reaction analysis to assess VEGF-A mRNA expression levels. Results: Diosgenin was shown to hinder the growth and formation of ISV in dosage-dependent development using a red blood cell staining assay when compared to the positive control, SU5416. In addition, there was a significant decrease in VEGF-A expression. Conclusion: In the presence of diosgenin, there is an evidence of inhibition of ISV development and suppression of VEGF mRNA expression. These findings underscore the therapeutic potential of diosgenin for targeting angiogenesis in cancer. However, further research is needed to understand the mechanism underlying diosgenin’s effect and its clinical relevance.

Tumor Promoting Effect of Long Non-Coding RNA RP11-556E13.1 and its Clinical Significance in Hepatocellular Carcinoma.

The aim of this study was to reveal the function of the long non-coding RNA (lncRNA) RP11-556E13.1 (RP11) and its clinical significance in hepatocellular carcinoma (HCC). LncRNA and mRNA expression profiling was performed using lncRNA and mRNA microarrays in HCC and adjacent tissues. Human tissue samples were analyzed by semiquantitative real-time polymerase chain reaction (sqRT-PCR) to evaluate the expression of RP11. Smart silencer RNA (siRNA) was used to knockdown the expression of RP11 in HCC cells. The function of RP11 was determined by some cell function experiments in HCC cells. Western blotting (WB) was performed to detect proteins that were presumably associated with these function changes. An Affymetrix Human HTA2.0 microarray was used to detect the underlying mechanism of RP11 in HCC. lncRNA RP11 was the most significantly upregulated lncRNA in HCC tissues compared with the adjacent tissues (p < 0.05, fold change = 20.24). The expression of RP11 was significantly higher in HCC tissues compared to adjacent tissues in 112 tissue pairs (p < 0.05). The higher the expression of RP11 in HCC tissues, the bigger the tumor size, the poorer the histological differentiation, and the lower the overall survival rate of the patients (all p < 0.05). After the knockdown of RP11, HCC cells displayed inhibited proliferation, increased apoptosis rate, and G1/S arrest. Moreover, the expression of cleaved PARP1 and cleaved caspase-3 was increased. GO enrichment and KEGG pathway enrichment analysis showed some important pathways that might be related to the knockdown of RP11 in HCC cells. lncRNA RP11 is an HCC-promoting gene and a potential prognostic predictor of HCC.

Exploring the role of tryptophanyl-tRNA synthetase and associations with inflammatory markers and clinical outcomes in COVID-19 patients: A case-control study

Tryptophanyl-tRNA synthetase (WRS) is a critical enzyme involved in protein synthesis, responsible for charging tRNA with the essential amino acid tryptophan. Recent studies have highlighted its novel role in stimulating innate immunity against bacterial and viral infections. However, the significance of WRS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains elusive. In this study, we aimed to investigate the complex interplay between WRS, inflammatory markers, Toll-like receptor-4 (TLR-4), and clinical outcomes in coronavirus disease 19 (COVID-19) patients. A case-control investigation comprised 127 COVID-19 patients, carefully classified as severe or moderate upon admission, and 112 healthy individuals as a comparative group. Blood samples were meticulously collected before treatment initiation, and WRS, interleukin-6 (IL-6), and C-reactive protein (CRP) concentrations were quantified using a well-established commercial ELISA kit. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples, and RNA was extracted for cDNA synthesis. Semi-quantitative real-time polymerase chain reaction (PCR) was employed to assess the relative expression of TLR-4. COVID-19 patients exhibited elevated levels of WRS, IL-6, CRP, and TLR-4 expression compared to healthy individuals, with the severe group displaying significantly higher levels than the moderate group. Notably, severe patients demonstrated substantial fluctuations in CRP, IL-6, and WRS levels over time, a pattern not observed in their moderate counterparts. Although no significant distinctions were observed in the dynamic alterations of WRS, IL-6, CRP, and TLR-4 expression between deceased and surviving patients, a trend emerged indicating higher IL-6_1 levels in deceased patients and elevated lactate dehydrogenase (LDH) levels in severe patients who succumbed to the disease. This pioneering research highlights the dynamic alterations of WRS in COVID-19 patients, providing valuable insights into the correlation between WRS, inflammatory markers, and disease severity within this population. Understanding the role of WRS in SARS-CoV-2 infection may open new avenues for therapeutic interventions targeting innate immunity to combat COVID-19.

Evaluating the genotoxicity of salinity stress and secondary products gene manipulation in lime, Citrus aurantifolia, plants.

Salinity is a significant abiotic stress that has a profound effect on growth, the content of secondary products, and the genotoxicity of cells. Lime, Citrus aurantifolia, is a popular plant belonging to the family Rutaceae. The interest in cultivating this plant is due to the importance of its volatile oil, which is included in many pharmaceutical industries, but C. aurantifolia plants are affected by the NaCl salinity levels. In the present study, a comet assay test has been applied to evaluate the genotoxic impact of salinity at 0, 50, 100, and 200 mM of NaCl on C. aurantifolia tissue-cultured plants. Furthermore, terpene gene expression was investigated using a semi-quantitative real-time polymerase chain reaction. Results from the two analyses revealed that 200 mM of NaCl stress resulted in high levels of severe damage to the C. aurantifolia plants' DNA tail 21.8%, tail length 6.56 µm, and tail moment 3.19 Unit. The relative highest expression of RtHK and TAT genes was 2.08, and 1.693, respectively, when plants were exposed to 200 mM of NaCl, whereas pv4CL2RT expressed 1.50 in plants subjected to 100 mM of NaCl. The accumulation of transcripts for the RTMYB was 0.951 when plants were treated with NaCl at 50 mM, and RtGPPS gene was significantly decreased to 0.446 during saline exposure at 100 mM. We conclude that the comet assay test offers an appropriate tool to detect DNA damage as well as RtHK, TAT, and pv4CL2RT genes having post-transcriptional regulation in C. aurantifolia plant cells under salinity stress. Future studies are needed to assess the application of gene expression and comet assay technologies using another set of genes that show vulnerability to different stresses on lime and other plants.

Anti-Photoaging Effects of the Ethanol Extract from Eucommia ulmoides Oliv. Seed Meal on UVB-irradiated HaCaT Cells and Mice

Background Eucommia ulmoides Oliv. is a traditional Chinese medicine, and its flowers, leaves, and seeds are widely used as food. Currently, E. ulmoides seed oil is capable of improving glucose and lipid metabolism in type 2 diabetic mice, but E. ulmoides seed meal is not fully utilized, and the anti-photoaging effect of it is still unclear. Objectives We examined the anti-photoaging effect of the ethanol extracts of E. ulmoides seed meal (EUSM) in UVB-stimulated HaCaT cells and mice. Materials and Methods EUSM was analyzed using Q-Orbitrap-based high-resolution LC-MS/MS. In vivo and in vitro experiments were conducted using normal mice and HaCaT cells, respectively. Hematoxylin and eosin (H&amp;E) staining and Masson’s staining were used to evaluate epidermal thickness and degree of skin fibrosis. The production of hyaluronic acid (HA) and total protein using ELISA kits. The expression of levels for collagen type I α 1 (COL1A1), hyaluronidase-1 (HYAL-1), MMP-3, and MMP-9 were assessed using semi-quantitative real-time polymerase chain reaction (qRT-PCR). Results EUSM contains all-trans-retinoic acid, medicagenic acid, and other chemical constituents. EUSM increased the level of total collagen by reducing matrix metalloproteinase expression, and it enhanced the content of HA. In addition, EUSM reduced the production of wrinkles in UVB-induced mouse skin and showed that EUSM decreased the level of malondialdehyde (MDA) and increased the contents of superoxide dismutase (SOD) and catalase (CAT) in the mouse skin tissue. Conclusion EUSM has therapeutic potential for UVB-induced skin photoaging and provided a rationale for the application of EUSM in the cosmetic field.

Role of fungal burden in risk stratification of HIV-negative patients with Pneumocystis pneumonia: A 12-year, retrospective, observational, multicenter cohort

ObjectivesThis study aimed to explore the role of fungal burden in risk stratification of patients without HIV-negative patients with Pneumocystis pneumonia (PCP). MethodsThis was a retrospective analysis of the characteristics associated with 30-day mortality in patients who were positive for P. jirovecii using polymerase chain reaction in bronchoalveolar lavage fluid between 2006 and 2017 in a multicenter cohort from Central Norway. The fungal burden was indicated by the cycle threshold (CT) values from semiquantitative real-time polymerase chain reaction targeting the β-tubulin gene. ResultsWe included 170 patients with proven or probable PCP. The all-cause 30-day mortality was 18.2%. After adjusting for host characteristics and premorbid corticosteroid use, a higher fungal burden was associated with a higher risk of dying: adjusted odds ratio 1.42 (95% confidence interval 0.48-4.25) for a CT value 31-36, increasing to odds ratio 5.43 (95% confidence interval 1.48-19.9) for a CT value ≤30 compared with patients with a CT value ≥37. The Charlson comorbidity index (CCI) improved the risk stratification: patients with a CT value ≥37 and CCI ≤2 had a 9% mortality risk compared with 70% among those with a CT value ≤30 and CCI ≥6. Comorbid cardiovascular disease, solid tumors, immunological disorders, premorbid corticosteroids, hypoxemia, abnormal leukocyte counts, low serum albumin, and C-reactive protein ≥100 were also independently associated with 30-day mortality. The sensitivity analyses did not suggest selection bias. ConclusionFungal burden may improve the risk stratification of patients without HIV-negative patients with PCP.

Circ0005654 as a new biomarker of thyroid cancer interacting with SP1 to influence the prognosis: A case-control study.

Circular RNAs (CircRNAs) have been reported to play key roles in the progression of various cancers, including thyroid cancer (TC). Transcription factor 1 (SP1) promotes the development of thyroid cancer. This study aims at investigating the expression level of Circ0005654 in combination with Transcription factor1 (SP1) in patients with TC for diagnostic and therapeutic purposes. A total of 76 patients with thyroid cancer underwent radical surgery. Intraoperatively, thyroid cancer tissues and paired adjacent tissues and the corresponding clinicopathological data were collected. The expression of SP1 and β-catenin in thyroid cancer and adjacent tissues was determined by immunohistochemistry (IHC) while the Circ0005654 expression level was measured by semiquantitative real-time polymerase chain reaction (sqRT-PCR). Then, we compared the variability of Circ0005654, SP1, and Wnt/β-catenin expression in cancerous and adjacent tissues and determined the relationship between the correlation analysis and the clinicopathological features of the thyroid cancer patients. The diagnostic value of Circ0005654 in thyroid cancer tissues was analyzed with the help of the receiver operating characteristic (ROC) curve, counting the 3-year postoperative survival rate, and analyzing the effect of Circ0005654 and SP1 protein levels on the 3-year survival rate of the patients. sqRT-PCR showed that the expression level of Circ0005654 in thyroid cancer tissue was significantly higher than that of adjacent tissues. The area under the ROC of Circ0005654 was 0.9553, 95% confidence interval: (0.9211-0.9895) with a cutoff value of 0.7895, a sensitivity of 92.11%, and a specificity of 86.84%. The IHC results showed that the expression level of SP1, β-catenin, and Wnt was higher in cancer tissues than in adjacent tissues; Circ0005654, SP1, Wnt/β-catenin expression levels were associated with tumor diameter, lymph node metastasis, TNM stage, and envelope invasion (all P < .05). According to the Circ0005654 expression level in thyroid cancer tissue, the 3-year survival rate of the high expression group was 77.5% and 94.4% in the low expression group with a statistically significant difference; the 3-year survival rate of SP1 positive and negative patients was 78.6% and 100%, respectively, with the data being significantly different. Circ0005654 may serve as a potential biomarker for thyroid cancer diagnosis and may be involved in the development of thyroid cancer.

Antibiofilm Activity of 4-(Adamantyl-1)-1-(1-Aminobutyl) Benzol against Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is a widespread opportunistic pathogen, causing community-acquired and nosocomial infections with both acute and chronic recurrent courses. The process of chronicity of the disease is provided by biofilms. Features of the structure and functioning of biofilms, in particular the presence of matrix, quorum sensing systems, persistent cells, and efflux pumps, provide microbial communities with resistance to antimicrobial drugs under their action in therapeutic concentrations. The insufficient eff ectiveness of modern antimicrobial chemotherapy against biofi lm microorganisms indicates the urgency of the problem to search for compounds with antibiofilm activity that can affect various stages of the biofilm formation and the formed biofilm. The aim of the study is to establish the antibiofilm activity of 4-(adamantyl-1)-1-(1-aminobutyl) benzol against methicillin-resistant S. aureus (MRSA) and to determine the mechanism of its action. Methods. The ability of adamantane-containing compound 4-(adamantyl-1)-1-(1-aminobutyl) benzol (AM-166) to prevent biofilm formation and destroy the formed biofilm of S. aureus was investigated on polystyrene plates by the sorption of gentian violet on its structures followed with desorption of the dye into the organic solvent. The viability of S. aureus cells at the first stage of biofilm formation and in the composition of mature biofilms was evaluated using specific dyes for living (acridine orange) and non-viable (propidium iodide) cells. Detection of genes responsible for antibiotic resistance and biofi lm formation was performed by the polymerase chain reaction (PCR) with detection of PCR products in agarose gel. Evaluation of the effect of AM-166 on the expression of genes regulating the biofilm formation (ica, agrA, sarA, and sigB) was investigated by the real-time PCR and semi-quantitative PCR. Results. It was found that the compound AM-166 shows activity against S. aureus biofilm formation. The most pronounced effect was registered at a concentration of 5.0 minimum inhibitory concentration (MIC) (92.3%.) Under the action of AM-166 on the formed 2-day biofilms, their destruction was marked: the biomass decreases by 30.9% at 5.0 MIC. According to the results of fluorescence microscopy, the adamantane derivative at 5.0 MIC helps to reduce the number of viable cells at different stages of formation of the S. aureus biofilm. The results of molecular genetic studies indicate that the ica gene expression is significantly inhibited by the action of subinhibitory concentrations of the compound AM-116. No significant changes in the expression of sarA, agrA, and sigB genes were registered. Conclusions. Experiments on the effect of adamantane derivative on S. aureus biofilms showed that the most pronounced activity of AM-116 was observed at the stage of biofilm formation, as evidenced by the inhibition of transcriptional activity of the ica gene responsible for early stages of the biofilm formation, in particular the adhesion of planktonic cells to the substrate.

Reduced expression of CXCL16/CXCR6 is involved in the pathogenesis of hidradenitis suppurativa.

Mutations in the γ-secretase complex have been well-described in familial hidradenitis suppurativa (HS). No gene mutations have been identified in sporadic HS, which comprises 60%-70% of all HS cases. Obesity and smoking are risk factors for HS and are closely related to DNA methylation, an essential epigenetic phenomenon. Hence, we hypothesized that epigenetic modifications might be involved in sporadic HS. To investigate genes with aberrant methylation in sporadic HS cases and assess their expression in skin lesions and blood from patients with HS. Skin lesion samples and corresponding normal skin were obtained from three patients with HS and subjected to whole-genome DNA methylation sequencing. Blood samples were collected from 20 patients with HS and 20 healthy controls (HCs). The HS mouse model was established by applying tamoxifen to NcstnΔKC mice. Target gene expression was analysed by immunohistochemistry, immunofluorescence, western blotting, enzyme-linked immunosorbent assay (ELISA) and semiquantitative real-time polymerase chain reaction (RT-qPCR). Among 10807 differentially methylated genes, we filtered 2101 genes with hypermethylated promoter regions, and following bioinformatics analyses, we focused on CXC chemokine ligand 16 (CXCL16). Subsequent functional experiments confirmed the downregulation of CXCL16 and its receptor, CXC chemokine receptor (CXCR) 6, in skin tissue from HS patients and NcstnΔKC mice. Serum CXCL16 concentrations were also significantly decreased in patients with HS. Our data revealed the downregulation of CXCL16 and CXCR6 in HS.

Overexpression of GATA binding protein 4 and myocyte enhancer factor 2C induces differentiation of mesenchymal stem cells into cardiac-like cells.

BACKGROUNDHeart diseases are the primary cause of death all over the world. Following myocardial infarction, billions of cells die, resulting in a huge loss of cardiac function. Stem cell-based therapies have appeared as a new area to support heart regeneration. The transcription factors GATA binding protein 4 (GATA-4) and myocyte enhancer factor 2C (MEF2C) are considered prominent factors in the development of the cardiovascular system.AIMTo explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs).METHODShUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry, and by their potential to differentiate into osteocytes and adipocytes. hUC-MSCs were transfected with GATA-4, MEF2C, and their combination to direct the differentiation. Cardiac differentiation was confirmed by semiquantitative real-time polymerase chain reaction and immunocytochemistry.RESULTShUC-MSCs expressed specific cell surface markers CD105, CD90, CD44, and vimentin but lack the expression of CD45. The transcription factors GATA-4 and MEF2C, and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e., GATA-4, MEF2C, NK2 homeobox 5 (NKX2.5), MHC, and connexin-43, and cardiac proteins GATA-4, NKX2.5, cardiac troponin T, and connexin-43.CONCLUSIONTransfection with GATA-4, MEF2C, and their combination effectively induces cardiac differentiation in hUC-MSCs. These genetically modified MSCs could be a promising treatment option for heart diseases in the future.

Investigation of the Toll-like receptors expression profile in peripheral blood mononuclear cells in COVID-19 patients in Iran

Introduction: Coronavirus disease 2019 (COVID-19) spreads all around the world and leads to several new infection cases and mortality. A better understanding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis could lead to more efficient therapeutic approaches. Objectives: The current study aimed to investigate the Toll-like receptors expression profile in peripheral blood mononuclear cells in COVID-19 patients in Iran. Patients and Methods: In this cross-sectional study, we evaluated 32 COVID-19 patients. At the admission time based on the disease severity patients were divided into two groups of severe and mild COVID-19. A group of 16 normal people was evaluated as the healthy control. Blood samples were collected before any treatment from patients at the admission time. Peripheral blood mononuclear cell isolation preformed on blood samples. RNA is extracted from human peripheral blood mononuclear cells and use for the cDNA synthesis. The expression level of TLR-3, 4, and 7 were evaluated by using semi-quantitative real-time polymerase chain reaction (PCR). Results: The patient’s mean age was 57.12±3.08 years and 15 (47%) were male. The background disorders represent two patients in the severe group with respiratory disorders, four patients with cardiovascular disease, and seven patients with diabetes. The TLRs expression levels represent a higher expression of TLR-4 in COVID-19 in comparison with controls for TLR-4 (P=0.007), TLR-3 and 7 also represent up-regulation however there were no statistically significant differences in the relative fold changes (RFCs) between groups for all other evaluated TLRs (P&gt;0.05). Conclusion: The study highlights the importance of the TLR-4 in COVID-19 patients in expression level. Further studies for a clear conclusion about TLRs expression levels are recommended.

Dispersal of taeniid eggs: Experimental faecal contamination of forest environment followed by DNA detection in wild berries

To understand Taeniidae epidemiology, the principles of egg-dispersion dynamics under natural conditions must be known. In this study, non-zoonotic Taenia laticollis was used as a model parasite for the family Taeniidae (including Echinococcus spp.). An experiment to investigate dispersion from contaminated faeces to the surroundings was performed both with bilberries (Vaccinium myrtillus) and lingonberries (Vaccinium vitis-idaea), both of which are commercially harvested wild berries in Finland. For this experiment, 30 g of fox faeces was inoculated with 30,000 T. laticollis eggs for the bilberry experiment and 100,000 eggs for the lingonberry experiment. The faecal material was placed in the middle of good berry growth areas in four locations for bilberries and eight locations for lingonberries. After 41–42 days, berries at different distances (0–15 m) from the original contamination spot were collected and delivered to our laboratory. DNA was extracted from washed and sieved material and analysed using T. laticollis-specific semi-quantitative SYBR Green real-time polymerase chain reaction (qPCR). Taenia laticollis-specific DNA was recovered from 67% (8/12) of bilberry samples but not reliably from any of the lingonberry samples 0% (0/24), although the exposure dose was higher for those. The qPCR results suggest that under natural conditions, taeniid egg dispersion from the contamination spot is demonstrated but attachment is berry specific. The surface of bilberries may be more adhesive for taeniid eggs than the waxier and harder pericarp of the lingonberries or there might be a difference in the dispersal mechanism caused by different biotopes.

Effect of levetiracetam on the gene expression of placental transporters in a murine model.

Levetiracetam (LEV) is an antiseizure medication prescribed to women during childbearing age. The impact of LEV on placental transporters is poorly understood. This study aimed to assess the effect of LEV exposure on the messenger RNA (mRNA) expression of placental transporters for hormones and nutrients and to correlate their expression with the drug's serum concentration in pregnant mice. Studies were conducted on gestational days (GD) 13 and 18, following oral treatment with 100mg/kg LEV or the vehicle every 24h after weaning. Serum LEV measurements were performed by High-performance liquid chromatography with a UV detector (HPLC-UV). The weight, height, and width of the fetuses were also analyzed. In addition, the placental expression of transporters xCt, Lat1, Oatp4a1, Fr-α, Rfc, and Snat4 was evaluated through semi-quantitative real-time polymerase chain reaction (qPCR). The Kruskal-Wallis and the Mann-Whitney U tests were used to determine the statistical significance (p<.05). The correlation between serum LEV concentration and placental gene expression was evaluated using the Spearman test. The weight, height, and width were lower in the fetuses exposed to LEV compared with the control group (p<.05). The number of fetuses was lower in the LEV-exposed group than in the control GD 13group (p<.001). No significant differences were detected in the mRNA expression level at GD 13. At GD 18, the expression of Lat1, Oatp4a1, xCT, and Snat4 was higher in the group treated with LEV compared with the control group (p<.05), whereas the expression of Rfc was lower (p<.05). No correlation was identified between serum LEV concentrations and gene expression levels. The repression of the Rfc transcript by LEV at GD 18 suggests that the protein expression would be abolished contributing to the observed intrauterine growth restriction (IUGR). Furthermore, the significant increase in mRNA of xCt, Snat4, Oatp4a1, and Lat1 might be a compensatory mechanism for fetal survival at GD 18.

Mechanism of Myopic Defocus or Atropine for Myopia Control: Different or Similar Ways?

Introduction: Myopia is usually caused by excessive elongation of the eye during development. This condition is common worldwide. In clinical practice, the progression of myopia is commonly controlled through optical or drug measures, but the specific mechanisms underlying these two treatments remain unclear. To verify whether the effects of these two treatments on posterior-pole tissues are similar or different, we studied a set of common transcriptional changes in chicken models. Methods: Chicks were divided into four groups, and they were given the intervention measures of plus-lens induction, minus-lens induction, minus-lens induction with atropine injection, and minus-lens induction with saline injection. Then, the genetic changes in each tissue at the posterior pole were detected, and the results of different genes were compared. A semiquantitative real-time polymerase chain reaction method was used to further study the visually induced changes in the transcription of potential candidate genes. Results: Based on RNA sequencing (RNA-seq) analysis of the transcriptome, we identified variations between the differentially expressed transcripts in three tissues from the two treatment groups. Through Kyoto Encyclopedia of Genes and Genomes enrichment analyses, eukaryotic protein translation elongation factor 1α2 (EEF1A2) was enriched in the “leishmaniasis” pathway in the choroid and showed increased expression in both the plus-lens induction and injection atropine groups. The expression levels of selected genes verified by quantitative real-time PCR were concordant with the RNA-seq data. Conclusions: Overlapping differentially expressed mRNAs of only one-tenth could suggest a different mechanism of myopic defocus and intravitreal injection of atropine controlling myopia. EEF1A2 might play an important role in the choroid during the treatment of myopia.

Cutaneous Photorejuvenation of Light Emitting Diodes via the Melatonin Membrane Receptor Pathway.

Melatonin receptors are present in the human skin and retina. These receptors can be stimulated by light emitting diodes (LEDs) at specific wavelengths, thereby inducing cutaneous photorejuvenation. However, the underlying mechanism remains unclear. To evaluate the influence of LEDs at specific wavelengths on melatonin membrane receptor (MT1) and cutaneous photorejuvenation via the MT1 pathway in vitro. Normal human dermal fibroblasts (HDFs) were irradiated using LEDs at different wavelengths (410~940 nm) at a dose of 1 J/cm². MT1 activity was evaluated after melatonin stimulation and LED irradiation. Thereafter, the expressions of collagen (COL) and matrix metalloproteinases (MMPs), with and without luzindole (MT1/2 receptor antagonist), were investigated via semi-quantitative reverse transcription polymerase chain reaction (PCR), real-time PCR, western blotting, and enzyme-linked immunosorbent assay. In HDFs, the MT1 mRNA and protein levels increased significantly in response to melatonin (dose, 50 nM) (p<0.01) and LED irradiation at 595, 630, 850, and 940 nm (p<0.01). LED irradiation up-regulated COL type I and down-regulated MMP-1. Compared to LED irradiation without luzindole, LED irradiation with luzindole produced no significant increase in COL type I mRNA and protein levels (p<0.01). We found that LED irradiation induces collagen synthesis and MMP-1 inhibition in HDFs via MT1 activation. Additionally, multiple LED wavelengths (595, 630, 850, and 940 nm) stimulated MT1 in HDFs, unlike in the eyes, where only blue light induced plasma melatonin suppression. This suggests the possibility of the melatoninergic pathway in photorejuvenation.

NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib Via PTEN/PI3K/Akt Signaling Pathway

Background: Multiple myeloma (MM) is the second most common hematologic malignancy. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM.Methods: The level of NEDD4-1 and its substrates in MM cell lines (NCI-H929, ARP-1, RPMI8226, OPM2, CAG, MM.1S) and MM cells (CD138+ plasma cells of the bone marrow) from patients were determined by semiquantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. NEDD4-1 was over-expressed in MM cells by lentivirus vector-mediated NEDD4-1-cDNA transfection. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8(CCK8) and flow cytometry. To immunoprecipitate endogenous proteins, whole cell extracts were immunoprecipitated with antibodies against NEDD4-1, PTEN, PI3K or Akt, along with IgG control. Clinical significance of NEDD4-1 expression in MM was assessed with the publicly available gene expression profiling (Oncomine) datasets.Results: The Oncomine showed that expression of NEDD4-1 was higher in MM cells from patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) and MM, in comparison to those from patients with Plasma Cell Leukemia. Patients in recurrence showed a lower NEDD4-1 expression than in primary occurrence. Also, MM cell lines NCI-H929, ARP-1 and RPMI8226 highly expressed NEDD4-1 at mRNA and protein levels. CAG and OPM2 showed relatively low expression. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNA, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05), MM cells cycle arrested in G2/M phase (P<0.05) and NEDD4-1 over-expression sensitized MM cells to Bor (P<0.05). In the add back rescue experiment, NEDD4-1 overexpressed in NEDD4-1 KD cells re-sensitized the cells to Bor (P<0.05). In KD cells, Bcl-2, CDK4 and CDK6 expressions were markedly activated, while cleaved capase-3, PARP-1, P21 and P27 were repressed after Bor treatment; the results were opposite in NEDD4-1 over-expressed cells. NEDD4-1 KD promoted the activation of PI3K, pAkt (Ser473), but markedly diminished the expression of PTEN compared with the control group. On the other hand, NEDD4-1 overexpression inactivated the PTEN/PI3K/Akt pathway. Furthermore, we detected PTEN, PI3K and Akt in NEDD4-1 immunoprecipitates, but not in the IgG control. Reciprocally, NEDD4-1, PI3K and PTEN were detected in Akt immunoprecipitates.Conclusion: Our study reveals that NEDD4-1 can promote MM sensitivity to Bor via inhibiting PTEN/PI3K/Akt signaling pathway and NEDD4-1 can interact with PTEN/PI3K/Akt to form a putative complex. These results suggest that NEDD4-1 may serve as a novel drug target in the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.

Cell death of hippocampal CA1 astrocytes during early epileptogenesis.

Growing evidence suggests that dysfunctional astrocytes are crucial players in the development of mesial temporal lobe epilepsy (MTLE). Using a mouse model closely recapitulating key alterations of chronic human MTLE with hippocampal sclerosis, here we asked whether death of astrocytes contributes to the initiation of the disease and investigated potential underlying molecular mechanisms. Antibody staining was combined with confocal imaging and semiquantitative real-time polymerase chain reaction analysis to identify markers of different cellular death mechanisms between 4h and 3days after epilepsy induction. Four hours after kainate-mediated induction of status epilepticus (SE), we found a significant reduction in the density of astrocytes in the CA1 stratum radiatum (SR) of the ipsilateral hippocampus. This reduction was transient, as within the next 3days, astrocyte cell numbers recovered to the initial values, which was accompanied by enhanced proliferation. Four hours after SE induction, a small proportion of astrocytes in the ipsilateral CA1 SR expressed autophagy-related genes and proteins, whereas we did not find astrocytes positive for cleaved caspase 3 or terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling, ruling out apoptosis-related astrocytic death. Importantly, at the same early time point post-SE, many astrocytes in the ipsilateral CA1 SR showed strong expression of genes encoding pro-necroptosis factors, including receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Phosphorylation of MLKL (pMLKL), formation of necrosome complexes composed of RIPK3 and pMLKL, and translocation of pMLKL to the nucleus and to the plasma membrane were often observed in astrocytes of the ipsilateral hippocampus 4h post-SE. The present study revealed that astrocytes die shortly after induction of SE. Our expression data and immunohistochemistry suggest that necroptosis and autophagy contribute to astrocytic death. These findings help to better understand how dysfunctional and pathological remodeling of astrocytes contributes to the initiation of temporal lobe epilepsy.

Mesenchymal Stem Cell and Oxygen Modulate the Cocultured Endothelial Cells in the Presence of Magnesium Degradation Products.

The interaction between mesenchymal stem cells (MSCs) and endothelial cells (ECs) holds a promising potential for the revascularization of osteoconductive grafts in orthopedics regeneration. Magnesium (Mg), as a well-studied degradable biomaterial already used in current medical practice, possesses osteoinductive properties. We investigated whether the physiochemical microenvironment, that is, the Mg and oxygen contents, further influences the MSC-modulating EC activities. Hypoxia, normoxia, and Mg degradation were represented by 5 and 20% O2 and gradient Mg degradation products, respectively. The migration of ECs in both EC mono- and MSC-EC coculture was increased in Mg with normoxia. Tube formation of ECs was reduced by Mg, especially in coculture and under normoxia. Compared to the monoculture, MSC-EC coculture exhibited significantly decreased content of proangiogenic cytokines but an increased amount of chemotactic factors. Semiquantitative real-time polymerase chain reaction revealed significant different profiles of the gene regulation under hypoxia, normoxia, different cell populations, and cell status. Investigation of heterotypic MSC-EC interactions in a mixed coculture system exhibited significantly increased proliferation under hypoxia. Transdifferentiation between MSCs and ECs was found to be reciprocally regulated by Mg degradation products in the two different oxygen conditions, probably because of the variable regulating effects of Mg on hypoxia-inducible factors. These results indicated the modulatory roles of oxygen tension and MSCs in combination with Mg or Mg-based degradable materials.

RETRACTED ARTICLE: HnRNP A1 - mediated alternative splicing of CCDC50 contributes to cancer progression of clear cell renal cell carcinoma via ZNF395

BackgroundAberrant alternative splicing events play critical roles in carcinogenesis and progression of many cancers, while sparse studies regarding to alternative splicing are available for clear cell renal cell carcinoma (ccRCC). We identified that alternative splicing of coiled-coil domain containing 50 (CCDC50) was dysregulated in ccRCC, whereas the clinical significance of this splicing event and its splicing regulation mechanisms were still elusive.MethodsBioinformatic algorithm was utilized to identify significant exon skipping events in ccRCC via exon sequencing data from The Cancer Genome Atlas. Semi-quantitative real-time polymerase chain reaction and western blot were used to validate the aberrant expression of different transcripts in renal cancer tissues, cell lines and corresponding noncancerous controls. Short hairpin RNA targeting CCDC50 and overexpressing plasmids for each transcript were introduced into ccRCC cell lines, followed by a series of in vitro and in vivo functional experiments. Moreover, a panel of splicing factors were identified and their roles on splicing regulation of CCDC50 precursor mRNA (pre-mRNA) were studied. Furthermore, RNAseq data were analyzed to elucidate downstream molecules of CCDC50. Two-way analysis of variance and unpaired Student t test were used in statistical analysis.ResultsPre-mRNA of CCDC50 generated two transcripts, full-length transcript (CCDC50-FL) and truncated transcript (CCDC50-S) with exon 6 skipped. CCDC50-S was overexpressed in ccRCC tissues and cell lines compared to noncancerous counterparts, but CCDC50-FL was only detected in noncancerous tissues and normal renal epithelial cells. Higher percent spliced-in index was associated with better survival in ccRCC patients. In vitro and in vivo functional experiments indicated that CCDC50-S transcript promoted the proliferation, migration, invasion and tumorigenesis of ccRCC, while CCDC50-FL exerted opposite tumor suppressive functions. Besides, we identified that heterogeneous nuclear ribonucleoprotein A1 (HnRNP A1) could promote the skipping of exon 6, which resulted in higher portion of CCDC50-S and oncogenic transformation. Moreover, zinc finger protein 395 (ZNF395) was identified as a downstream protein of CCDC50-S, and the interaction initiated oncogenic pathways which were involved in ccRCC progression.ConclusionsAberrant alternative splicing of CCDC50 is regulated by HnRNP A1 in ccRCC. This splicing event contributes to cancer progression through the downstream pathway involving ZNF395.