Dual-target one-step nested PCR for sensitive detection of SARS-CoV-2 nucleic acids.

The Corona Virus Disease reported in 2019 (COVID-19) poses a significant threat to human and public health. Its early and accurate detection can reduce the spread and recurrence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Real-time reverse transcription fluorescent quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for detecting the nucleic acid of SARS-CoV-2. This study developed and tested a dual-target (ORF1ab and N gene) one-step nested RT-qPCR (DTO-N-PCR) to detect SARS-CoV-2. Ten-fold serial dilutions of mixed synthetic DNA from SARS-CoV-2 ORF1ab and N gene were used as templates to test the sensitivity of DTO-N-PCR. Its specificity was subsequently tested using throat swab specimens from 10 COVID-19 patients and 35 healthy participants. DTO-N-PCR was more sensitive and specific than conventional RT-qPCR. It has unique features, including a dual-target (ORF1ab and N gene), rapid one-step operation of reverse transcription and PCR, four pairs of inner and outer primers, and specific probes. These features aid in its rapid, accurate, and efficient detection of SARS-CoV-2 RNA.

Effects of Alpha-Synuclein on Primary Spinal Cord Neurons Associated with Apoptosis and CNTF Expression.

Spinal cord injury (SCI) often causes neurological deficits with poor recovery; the treatment, however, is far from satisfaction, and the mechanisms remain unclear. Using immunohistochemistry and western blotting analysis, we found α-synuclein (SNCA) was significantly up-regulated in the spinal caudal segment of rats subjected to spinal cord transection at 3 days post-operation. Moreover, the role of SNCA on neuronal growth and apoptosis in vitro was determined by using overexpressing and interfering SNCA recombined plasmid vectors, and the underlying mechanism was detected by QRT-PCR and western blotting. Spinal neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival, while it results in cell apoptosis in SNCA-ORF group. In molecular level, SNCA silence induced the up-regulation of CNTF and down-regulation of Caspase7/9. Together, endogenous SNCA plays a crucial role in spinal neuronal survival, in which the underlying mechanism may be linked to the regulation both apoptotic genes (Caspase7/9) and CNTF. The present findings therefore provide novel insights into the role of SNCA in spinal cord and associated mechanism, which may provide novel cue for the treatment of SCI in future clinic trials.

Knockdown of α-synuclein in cerebral cortex improves neural behavior associated with apoptotic inhibition and neurotrophin expression in spinal cord transected rats.

Spinal cord injury (SCI) often causes severe functional impairment with poor recovery. The treatment, however, is far from satisfaction, and the mechanisms remain unclear. By using proteomics and western blot, we found spinal cord transection (SCT) resulted in a significant down-regulation of α-synuclein (SNCA) in the motor cortex of SCT rats at 3 days post-operation. In order to detect the role of SNCA, we used SNCA-ORF/shRNA lentivirus to upregulate or knockdown SNCA expression. In vivo, SNCA-shRNA lentivirus injection into the cerebral cortex motor area not only inhibited SNCA expression, but also significantly enhanced neurons' survival, and attenuated neuronal apoptosis, as well as promoted motor and sensory function recovery in hind limbs. While, overexpression SNCA exhibited the opposite effects. In vitro, cortical neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival and neurite outgrowth, while it was accompanied by reverse efficiency in SNCA-ORF group. In molecular level, SNCA silence induced the upregulation of Bcl-2 and the downregulation of Bax, and the expression of NGF, BDNF and NT3 was substantially upregulated in cortical neurons. Together, endogenous SNCA play a crucial role in motor and sensory function regulation, in which, the underlying mechanism may be linked to the regulation of apoptosis associated with apoptotic gene (Bax, Bcl2) and neurotrophic factors expression (NGF, BDNF and NT3). These finds provide novel insights to understand the role of SNCA in cerebral cortex after SCT, and it may be as a novel treatment target for SCI repair in future clinic trials.

Bone Marrow Stromal Cells Promote Neuronal Restoration in Rats with Traumatic Brain Injury: Involvement of GDNF Regulating BAD and BAX Signaling.

BACKGROUND/AIMS: To investigate the effects of bone marrow stromal cells (BMSCs) and underlying mechanisms in traumatic brain injury (TBI). METHODS: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43) and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. RESULTS: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF). The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. CONCLUSION: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) improves brain ischemia-induced pulmonary injury in rats associated to TNF-α expression.

BACKGROUND: Bone marrow mesenchymal stem cell (BMSCs)-based therapy seems to be a promising treatment for acute lung injury, but the therapeutic effects of BMSCs transplantation on acute lung injury induced by brain ischemia and the mechanisms have not been totally elucidated. This study explores the effects of transplantation of BMSCs on acute lung injury induced by focal cerebral ischemia and investigates the underlying mechanism. METHODS: Acute lung injury model was induced by middle cerebral artery occlusion (MCAO). BMSCs (with concentration of 1 × 10(6)/ml) were transplanted into host through tail vein 1 day after MCAO. Then, the survival, proliferation and migration of BMSCs in lung were observed at 4 days after transplantation, and histology observation and lung function were assessed for 7 days. Meanwhile, in situ hybridization (ISH), qRT-PCR and western blotting were employed to detect the expression of TNF-α in lung. RESULTS: Neurobehavioral deficits and acute lung injury could be seen in brain ischemia rats. Implanted BMSCs could survive in the lung, and relieve pulmonary edema, improve lung function, as well as down regulate TNF-α expression. CONCLUSIONS: The grafted BMSCs can survive and migrate widespread in lung and ameliorate lung injury induced by focal cerebral ischemia in the MCAO rat models. The underlying molecular mechanism, at least partially, is related to the suppression of TNF-α.

Intrathecal injection of lentivirus-mediated glial cell line-derived neurotrophic factor RNA interference relieves bone cancer-induced pain in rats.

Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.

Limb remote ischemic postconditioning protects cerebral ischemia from injury associated with expression of HIF-1α in rats.

BACKGROUND: Limb remote ischemic postconditioning (LRIP) can ameliorate cerebral ischemia-reperfusion injury (IRI), while the underlying mechanism remains elusive. Hypoxia-inducible factor 1α (HIF-1α) is an important transcription factor during cerebral ischemia damage. However, whether the neuroprotective effect of LRIP could be associated with HIF-1α is somewhat unclear. Here we tested the hypothesis that Limb remote ischemic postconditioning (LRIP) protecting brain from injury in middle cerebral artery occlusion (MCAO) rat model was associated with HIF-1α expression. RESULTS: LRIP was conducted with 3 cycles of 10 min occlusion/10 min reperfusion at the beginning of reperfusion. The analysis of neurobehavioral function and triphenyltetrazolium chloride (TTC) staining showed the neurological deficit, brain infarct and cerebral edema, caused by ischemia-reperfusion injury (IRI), were dramatically ameliorated in LRIP administrated animals. Meanwhile, the result of Q-PCR and western blot revealed that the overexpression of HIF-1α induced by IRI could be notably suppressed by LRIP treatment. CONCLUSIONS: LRIP exhibits a protective effect against cerebral ischemia/reperfusion and the possible mechanism is associated with the suppression of HIF-1α in stroke rats.

Co-grafting of neural stem cells with olfactory en sheathing cells promotes neuronal restoration in traumatic brain injury with an anti-inflammatory mechanism.

BACKGROUND: We sought to investigate the effects of co-grafting neural stem cells (NSCs) with olfactory ensheathing cells (OECs) on neurological behavior in rats subjected to traumatic brain injury (TBI) and explore underlying molecular mechanisms. METHODS: TBI was established by percussion device made through a weight drop (50 g) from a 30 cm height. Cultured NSCs and OECs isolated from rats were labeled by Hoechst 33342 (blue) and chloromethyl-benzamidodialkyl carbocyanine (CM-Dil) (red), respectively. Then, NSCs and/or OECs, separately or combined, were transplanted into the area surrounding the injury site. Fourteen days after transplantation, neurological severity score (NSS) were recorded. The brain tissue was harvested and processed for immunocytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Significant neurological function improvement was observed in the three transplant groups, compared to the TBI group, and co-transplantation gave rise to the best improvement. Morphological evaluation showed that the number of neurons in cortex from combination implantation was more than for other groups (P <0.05); conversely, the number of apoptotic cells showed a significant decrease by TUNEL staining. Transplanted NSCs and OECs could survive and migrate in the brain, and the number of neurons differentiating from NSCs in the co-transplantation group was significantly greater than in the NSCs group. At the molecular level, the expressions of IL-6 and BAD in the co-graft group were found to be down regulated significantly, when compared to either the NSC or OEC alone groups. CONCLUSION: The present study demonstrates for the first time the optimal effects of co-grafting NSCs and OECs as a new strategy for the treatment of TBI via an anti-inflammation mechanism.

Transplantation of olfactory ensheathing cells promotes the recovery of neurological functions in rats with traumatic brain injury associated with downregulation of Bad.

BACKGROUND AIMS: The neuroprotective effects of olfactory ensheathing cells (OECs) after transplantation have largely been known in the injured nervous system. However, the underlying mechanisms still must be further elucidated. We explored the effects of OEC transplantation on the recovery of neurophysiologic function and the related anti-apoptosis mechanism in acute traumatic brain injury. METHODS: The OECs from neonatal Sprague-Dawley rats were isolated, identified and labeled and then were immediately transplanted into the regions surrounding the injured brain site that is resulted from free-weight drop injury. RESULTS: Nerve growth factor and it's recepor, p75 was expressed in cultured OECs. Transplanted OECs survived, migrated around the injury site and significantly improved the neurological severe scores compared with the control group (P < 0.05). OEC transplantation significantly increased the number of GAP-43-immunopositive fibers and synaptophysin-positive vesicles (P < 0.05) but significantly decreased the number of apoptotic cells (P < 0.05). On the molecular level, the expression of Bad in the OEC transplantation group was significantly downregulated (P < 0.05). CONCLUSIONS: OEC transplantation could effectively improve neurological deficits in TBI rats; the underlying mechanism may be related with their effects on neuroprotection and regeneration induction, which is associated with the downregulation of the apoptotic molecule Bad.

Predictive potentials of glycosylation-related genes in glioma prognosis and their correlation with immune infiltration.

Glycosylation is currently considered to be an important hallmark of cancer. However, the characterization of glycosylation-related gene sets has not been comprehensively analyzed in glioma, and the relationship between glycosylation-related genes and glioma prognosis has not been elucidated. Here, we firstly found that the glycosylation-related differentially expressed genes in glioma patients were engaged in biological functions related to glioma progression revealed by enrichment analysis. Then seven glycosylation genes (BGN, C1GALT1C1L, GALNT13, SDC1, SERPINA1, SPTBN5 and TUBA1C) associated with glioma prognosis were screened out by consensus clustering, principal component analysis, Lasso regression, and univariate and multivariate Cox regression analysis using the TCGA-GTEx database. A glycosylation-related prognostic signature was developed and validated using CGGA database data with significantly accurate prediction on glioma prognosis, which showed better capacity to predict the prognosis of glioma patients than clinicopathological factors do. GSEA enrichment analysis based on the risk score further revealed that patients in the high-risk group were involved in immune-related pathways such as cytokine signaling, inflammatory responses, and immune regulation, as well as glycan synthesis and metabolic function. Immuno-correlation analysis revealed that a variety of immune cell infiltrations, such as Macrophage, activated dendritic cell, Regulatory T cell (Treg), and Natural killer cell, were increased in the high-risk group. Moreover, functional experiments were performed to evaluate the roles of risk genes in the cell viability and cell number of glioma U87 and U251 cells, which demonstrated that silencing BGN, SDC1, SERPINA1, TUBA1C, C1GALT1C1L and SPTBN5 could inhibit the growth and viability of glioma cells. These findings strengthened the prognostic potentials of our predictive signature in glioma. In conclusion, this prognostic model composed of 7 glycosylation-related genes distinguishes well the high-risk glioma patients, which might potentially serve as caner biomarkers for disease diagnosis and treatment.

Neural stem cells grafts decrease neural apoptosis associated with caspase-7 downregulation and BDNF upregulation in rats following spinal cord hemisection.

Transplantation of neural stem cells (NSCs) into lesioned spinal cord demonstrated a beneficial effect for neural repair, the underlying mechanism, however, remains to be elusive. Here, we showed that NSCs, possessing the capacity to differentiate toward into neurons and astrocytes, exhibit a neuroprotective effect by anti-apoptosis mechanism in spinal cord hemi-transected rats despite it did not improve behavior. Intravenous NSCs injection substantially upregulated the level of BDNF mRNA but not its receptor TrkB in hemisected spinal cord, while caspase-7, a downstream apoptosis gene of caspase-3, has been largely down-regulated. TUNEL staining showed that the number of apoptosis cells in injured spinal cord decreased significantly, compared with seen in rats with no NSCs administration. The present finding therefore provided crucial evidence to explain neuroprotective effect of NSCs grafts in hemisected spinal cord, which is associated with BDNF upregulation and caspase-7 downregulation.

Losartan supports liver regrowth via distinct boost of portal vein pressure in rodents with 90 % portal branch ligation.

OBJECTIVES: The study used a model of 90 % portal branch ligation (PBL) in rats to study the effect of losartan on portal vein pressure (PVP) and liver regeneration in rats after PBL. METHODS: A total of 144 male Sprague-Dawley rats were arbitrarily designated into three treatment method groups: a sham operation group (Sham), a PBL treatment group (PBL), and a PBL plus losartan treatment group (PBL + L). Losartan (2 mg/day) was intragastrically gavaged 3 days before the PBL or sham operation to time points of study. RESULTS: Both the PBL and PBL + L groups showed an intense surge in PVP after PBL treatment, peaking at 12 h postsurgery, then lessening progressively afterwards. PVP was substantially greater in these two groups compared with the Sham group at 6-72 h postsurgery (p < 0.01). Compared with the PBL group, the PBL + L group showed a noticeable reduction in PVP 6-48 h postsurgery (p < 0.05); the PBL group showed considerably raised levels of plasma ALT and AST 6-72 h postsurgery (p < 0.01). Compared to the PBL group, the PBL + L group showed drastically reduced plasma ALT and AST levels 12-72 h postsurgery (p < 0.05). CONCLUSIONS: Losartan supports liver regeneration in 90 % of rats that underwent PBL. The mechanism may be related to losartan's ability to regulate PVP and increase serum hepatocyte growth factor levels.

Molecular detection of trisomy 21 by bicolor competitive fluorescent PCR.

OBJECTIVE: To develop a reliable and specific method for rapid prenatal diagnosis of Trisomy 21 (Down syndrome). METHODS: We established a dual color competitive fluorescent Polymerase Chain Reaction (PCR) to measure the gene dosage of Down syndrome critical region (DSCR), a single copy sequence in chromosome 21. Another unique single copy sequence located on chromosome 2 (USC2) but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chose as reference gene. RESULTS: The DSCR3/USC2 ratio of peripheral blood in trisomy 21 syndrome patients to normal subjects was 1.41∼1.74 to 0.93∼1.15, respectively (p < 0.01). Dual color competitive fluorescent PCR technique effectively differentiates the normal subjects from the Down syndrome patients. Next, according to the dual color competitive fluorescence quantitative PCR, among the 46 pregnant women, 3 cases were Down syndrome and 43 cases were normal, and these were confirmed by cytogenetic karyotype analysis. CONCLUSION: This indicated that the new technique may be a reliable and specific method for the rapid prenatal diagnosis of Trisomy 21.

Identification of necroptosis-related long non-coding RNAs prognostic signature and the crucial lncRNA in bladder cancer.

BACKGROUND: Research on the relationships between long non-coding RNAs (lncRNAs) and cancer is attractive and has progressed very rapidly. Necroptosis-related biomarkers can potentially be used for predicting the prognosis of cancer patients. This study aimed to establish a necroptosis-related lncRNA (NPlncRNA) signature to predict the prognosis of patients with bladder cancer (BCa). METHODS: First, NPlncRNAs were identified using Pearson correlation analysis and machine learning algorithms, including SVM-RFE, least absolute shrinkage and selection operator (LASSO) regression, and random forest. The prognostic NPlncRNA signature was constructed using univariate and multivariate Cox regression analyses and the diagnostic efficacy and clinically predictive efficiency were evaluated and validated. The biological functions of the signature were analysed using gene set enrichment analysis (GSEA) and functional enrichment analysis. We further integrated the RNA-seq dataset (GSE133624) with our outcomes to reveal the crucial NPlncRNA that was functionally verified by assessing cell viability, proliferation, and apoptosis in BCa cells. RESULTS: The prognostic NPlncRNAs signature was composed of PTOV1-AS2, AC083862.2, MAFG-DT, AC074117.1, AL049840.3, and AC078778.1, and a risk score based on this signature was proven to be an independent prognostic factor for the BCa patients, indicated by poor overall survival (OS) of patients in the high-risk group. Additionally, the NPlncRNAs signature had a higher diagnostic validity than that of other clinicopathological variables, with a greater area under the receptor operating characteristic and concordance index curves. A nomogram established by integrating clinical variables and risk score confirmed that the signature can accurately predict the OS of patients and has high clinical practicability. Functional enrichment analysis and GSEA revealed that some cancer-related and necroptosis-related pathways were enriched in high-risk groups. The crucial NPlncRNA MAFG-DT was associated with poor prognosis and was highly expressed in BCa cells. MAFG-DT silencing notably inhibited proliferation and enhanced apoptosis of BCa cells. CONCLUSIONS: A novel prognostic NPlncRNAs signature was identified in BCa in this study, which provides potential therapeutic targets among which MAFG-DT plays critical roles in the tumorigenesis of BCa.

Effects of pulsed electromagnetic fields on the mRNA expression of CAII and RANK in ovariectomized rats.

The present study was designed to determine the effects of pulsed electromagnetic fields (PEMFs) on the mRNA expression of the carbonic anhydrase II (CAII) and receptor activator of NF-κB (RANK) in ovariectomized rats. A total of 48 SD rats were randomly divided into four groups [Sham, OVX, PEMFs, and E(2) (premarin)], 12 rats in each group. Rats in the Sham group received sham ovariectomy, while rats in OVX, PEMFs, and E(2) groups received ovariectomy. Twelve weeks following the surgery, rats (whole body) in the PEMFs group were exposed to PEMFs for 30 days with 3.8 mT, 8 Hz, and 40 min per day; rats in the E(2) group were administered premarin (0.0625 mg/kg/d; intragastric administration 1-2 ml/100 g). Rats in the Sham and OVX groups housed in the same conditions. At the end of intervention, the level of serum estradiol of rats was measured. The gene expression of CAII and RANK in the left ilium of rats was determined with real-time fluorescent-nested quantitative polymerase chain reaction. Compared with the Sham group, the level of serum estradiol in the ovariectomized group was significantly decreased (P < 0.05); compared with the OVX group, CAIImRNA expression was significantly decreased in the PEMFs group and E group (P < 0.05, 0.01, respectively). Compared with the E group, RANKmRNA expression was significantly higher in the PEMFs group (P < 0.05); although RANKmRNA expression decreased in PEMFs group, no statistically significant difference was found between PEMF group and OVX group (P = 0.82). These data suggest that PEMFs could regulate the expression of CAIImRNA in ovariectomized rats.

[Effect of dihydroartemisinin on the expression of BCR/ABL fusion gene in leukemia K562 cells].

OBJECTIVE: To investigate the effect of dihydroartemisinin (DHA) on the BCR/ABL fusion gene in leukemia K562 cell. METHODS: K562 cells were cultured in vitro. The rate of proliferation inhibition of cells treated with various concentrations of DHA were determined by using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) method. Expression of BCR/ABL fusion gene was analyzed by reverse transcription(RT-PCR) before and after DHA treatment. Apoptosis of K562 cells was detected by flow cytometry. RESULTS: The growth of K562 cells was inhibited when the concentrations of DHA were 10-160 umol/L. With the added dose of DHA, the growth inhibition was remarkable, with the rate of inhibition risen from 52.76% to 94.65%. The expression of BCR/ABL fusion gene, as detected by RT-PCR after incubating the K562 cells with 20 umol/L DHA, measured as ΔCt = 4.45 ± 0.25 after 12 h and ΔCt = 5.23 ± 0.21 after 24 h, which was significantly lower compared with that of the control ( ΔCt = 4.23 ± 0.21, P < 0.05). CONCLUSION: DHA can inhibit the proliferation of leukemia K562 cells and facilitate the induction of apoptosis by downregulating the expression of BCR/ABL fusion gene.

[Detecting Down syndrome with a novel dual-color competitive quantitative fluorescent polymerase chain reaction method].

OBJECTIVE: To develop a rapid method for the detection of Down syndrome (DS) using dual-color competitive quantitative fluorescent polymerase chain reaction (DCC-QF-PCR), and to assess its feasibility for the prenatal diagnosis of Down syndrome. METHODS: DNA was extracted from peripheral blood of 30 DS patients and 60 normal men, common primers for DSCR and USC2 genes and respective TaqMan probes were designed and synthesized. The results of DCC-QF-PCR were compared with those of QF-PCR which measured the ratio between DSCR and GAPDH. Forty-six amniotic fluid samples were assayed with DCC-QF-PCR. The results were compared with that of karyotyping. Monoclone fragments for DSCR and USC2 genes were obtained from direct cloning of PCR products. DCC-QF-PCR was carried out using different DNA ratios of DSCR and USC2 as the template. The dosage ratio between DSCR and USC2 was calculated. RESULTS: The gene dosage ratio of the DS patients was 1.41-1.74, which was significantly higher than that of normal men (0.93-1.15). The dosage ratio range of DSCR and GAPDH by QF-PCR was comparatively greater than that of DSCR and USC2. Three samples were diagnosed as DS, which was in good agreement with that of karyotyping analysis. There was no significant difference between the gene dosage ratio from DCC-QF-PCR and that of predetermined (P>0.05). CONCLUSION: DCC-QF-PCR is an accurate, rapid, and low cost method, which only requires tiny amount of sample and therefore has broad application in the genetic and prenatal diagnosis.

Dihydroartemisinin Inhibits Laser-Induced Choroidal Neovascularization in a Mouse Model of Neovascular AMD.

Purpose: Choroidal neovascularization (CNV) is the main pathogenic process and a leading cause of severe vision loss in neovascular age-related macular degeneration (AMD). We investigated the antiangiogenic efficacy of dihydroartemisinin (DHA) in an experimental laser-induced CNV mouse model. Methods: After fluorescein angiography confirmed that CNV was induced by laser photocoagulation in C57BL/6J mice, DHA or vehicle was given by intragastric administration once a day. On day 6 and day 12, fluorescein angiography, optic coherence tomography, and flat-mounting analysis were performed to grade CNV leakage, measure CNV thickness and evaluate CNV areas, respectively. Immunofluorescence staining and Western blot analysis were performed to evaluate the expression of NF-κB, VEGF, and VEGFR2. To confirm the safety of intragastric DHA application, changes in retinal morphology and neural cell apoptosis were tested by histopathological examination and TUNEL assay, and retinal function was determined by electroretinogram (ERG). Results: Intragastric administration of DHA significantly suppressed CNV leakage and CNV formation in both thickness and area. Immunofluorescence showed that DHA suppressed VEGFR2 and NF-κB p65 expression in laser-induced lesions. Compared to the normal group, the protein expression of VEGF, VGFER2, NF-κB p65, and NF-κB1 p50 increased significantly in the vehicle group after laser photocoagulation, while it was profoundly inhibited by DHA treatment. In addition, histopathological examination, TUNEL analysis, and ERG test showed no obvious evidence of retinal toxicity caused by DHA. Conclusion: Systemic administration of DHA can effectively inhibit laser-induced CNV formation in mice, which might be due to the suppression of the classic NF-κB signaling pathway and downregulation of VEGFR2 and VEGF expression. The current results suggest that DHA could be a natural potential alternative therapeutic strategy for neovascular AMD.

Corrigendum: Combined Scutellarin and C18H17NO6 Imperils the Survival of Glioma: Partly Associated With the Repression of PSEN1/PI3K-AKT Signaling Axis.

[This corrects the article DOI: 10.3389/fonc.2021.663262.].

Downregulation of TGF-beta receptor types II and III in oral squamous cell carcinoma and oral carcinoma-associated fibroblasts.

BACKGROUND: The purpose of this study was to assess the expression levels for TßRI, TßRII, and TßRIII in epithelial layers of oral premalignant lesions (oral leukoplakia, OLK) and oral squamous cell carcinoma (OSCC), as well as in oral carcinoma-associated fibroblasts (CAFs), with the final goal of exploring the roles of various types of TßRs in carcinogenesis of oral mucosa. METHODS: Normal oral tissues, OLK, and OSCC were obtained from 138 previously untreated patients. Seven primary human oral CAF lines and six primary normal fibroblast (NF) lines were established successfully via cell culture. The three receptors were detected using immunohistochemical (IHC), quantitative RT-PCR, and Western blot approaches. RESULTS: IHC signals for TßRII and TßRIII in the epithelial layer decreased in tissue samples with increasing disease aggressiveness (P < 0.05); no expression differences were observed for TßRI, in OLK and OSCC (P > 0.05); and TßRII and TßRIII were significantly downregulated in CAFs compared with NFs, at the mRNA and protein levels (P < 0.05). Exogenous expression of TGF-ß1 led to a remarkable decrease in the expression of TßRII and TßRIII in CAFs (P < 0.05). CONCLUSION: This study provides the first evidence that the loss of TßRII and TßRIII expression in oral epithelium and stroma is a common event in OSCC. The restoration of the expression of TßRII and TßRIII in oral cancerous tissues may represent a novel strategy for the treatment of oral carcinoma.