Quantitative Polymerase Chain Research Articles

Synthesis, Biological and Molecular Docking Studies of New Polysubstituted 2‐Amino‐3‐Cyano‐4H‐Chromene Derivatives

AbstractA series of new substituted 2‐amino‐3‐cyano‐4H‐chromene derivatives have been synthesised by one pot multicomponent reaction of substituted benzaldehyde, malononitrile and 4‐chloro resorcinol. All the synthesised compounds have been characterised using FT‐IR, 1H NMR, 13C NMR and HR‐MS spectra and screened for bioactivities such as swarming behaviour, biofilm formation, biofilm disruption, urease production in Proteus mirabilis. Whereas swarming inhibition was shown by all synthesized chromene derivatives, biofilm inhibition was shown only by nitrophenyl and fluorophenyl substituted chromene derivatives. Interestingly, urease activity was shown by meta substituted nitrophenyl and fluorophenyl derivatives. Quantitative polymerase chain (qPCR) reaction used to investigate the molecular mechanism of most active chromene derivatives showed prominent down regulations of urease enzyme genes. Molecular docking study against urease supported the observed bioactive effect and provide valuable insights into the binding modes and affinities of these new chromene derivatives. The per‐residue interaction analysis proposed the involvement of non‐bonded (steric and electrostatic) and bonded (hydrogen bonding and pi‐pi stacking) interactions with the active site.

Achatina fulica Mucus Ameliorates UVB-induced Human Dermal Fibroblast Photoaging via the TGF-β/Smad Pathway

BACKGROUND: Ultraviolet B (UVB) induces skin photoaging by reducing collagen deposition via impairment of the TGF-β/Smad signaling pathway. Achatina fulica mucus (AFM) is a native medicine acting as vehicle of anti-aging ingredients. The present investigation examined the effect of AFM on UVB-induced fibroblast photoaging by assessing TGF-β, Smad3, and Smad7 mRNA expressions.METHODS: AFM was extracted from A. fulica using electrical shock and freeze-dried into a powder. Normal human dermal fibroblast (NHDF) cultures were irradiated with/without 100 mJ/cm2 UVB and treated with/without 10% platelet-rich plasma or different concentrations of AFM: 3.9 μg/mL in AF3 group; 15.625 μg/mL in AF15 group, and 62.5 μg/mL in AF62 group. The mRNA expressions of TGF-β, Smad3, and Smad7 in NHDF were evaluated by quantitative polymerase chain reaction.RESULTS: TGF-β mRNA expressions in the AF3 (0.85±0.01), AF15 (0.94±0.02) and AF62 (1.64±0.03) groups were significantly higher (p<0.05) compared with that in the UVB group (0.55±0.04). Moreover, Smad3 expressions in the AF3 (1.42±0.25), AF15 (1.89±0.13), and AF62 (2.50±0.31) groups were significantly higher (p<0.05) compared with that in the UVB group (0.57±0.08). Furthermore, Smad7 expressions in the AF3 (1.57±0.18), AF15 (0.87±0.03), and AF62 (0.25±0.09) groups were significantly lower (p<0.05) than that in the UVB group (2.57±0.06).CONCLUSION: AFM ameliorates UVB-induced fibroblast photoaging by upregulating the TGF-β/Smad3 expressions and downregulating Smad7 expression.KEYWORDS: Achatina fulica, TGF-β, Smad, collagen, UVB, fibroblast, photoaging

Resolution of Cerebral Inflammation Following Subarachnoid Hemorrhage

BackgroundAneurismal subarachnoid hemorrhage (SAH) is a type of hemorrhagic stroke that, despite improvement through therapeutic interventions, remains a devastating cerebrovascular disorder that has a high mortality rate and causes long-term disability. Cerebral inflammation after SAH is promoted through microglial accumulation and phagocytosis. Furthermore, proinflammatory cytokine release and neuronal cell death play key roles in the development of brain injury. The termination of these inflammation processes and restoration of tissue homeostasis are of utmost importance regarding the possible chronicity of cerebral inflammation and the improvement of the clinical outcome for affected patients post SAH. Thus, we evaluated the inflammatory resolution phase post SAH and considered indications for potential tertiary brain damage in cases of incomplete resolution.MethodsSubarachnoid hemorrhage was induced through endovascular filament perforation in mice. Animals were killed 1, 7 and 14 days and 1, 2 and 3 months after SAH. Brain cryosections were immunolabeled for ionized calcium-binding adaptor molecule-1 to detect microglia/macrophages. Neuronal nuclei and terminal deoxyuridine triphosphate-nick end labeling staining was used to visualize secondary cell death of neurons. The gene expression of various proinflammatory mediators in brain samples was analyzed by quantitative polymerase chain reaction.ResultsWe observed restored tissue homeostasis due to decreased microglial/macrophage accumulation and neuronal cell death 1 month after insult. However, the messenger RNA expression levels of interleukin 6 and tumor necrosis factor α were still elevated at 1 and 2 months post SAH, respectively. The gene expression of interleukin 1β reached its maximum on day 1, whereas at later time points, no significant differences between the groups were detected.ConclusionsBy the herein presented molecular and histological data we provide an important indication for an incomplete resolution of inflammation within the brain parenchyma after SAH. Inflammatory resolution and the return to tissue homeostasis represent an important contribution to the disease’s pathology influencing the impact on brain damage and outcome after SAH. Therefore, we consider a novel complementary or even superior therapeutic approach that should be carefully rethought in the management of cerebral inflammation after SAH. An acceleration of the resolution phase at the cellular and molecular levels could be a potential aim in this context.

Preliminary Study of the Cancer Stem Cells' Biomarker CD147 in Leukoplakia: Dysplasia and Squamous Cell Carcinoma of Oral Epithelial Origin.

Objectives Cancer stem cells (CSCs) are responsible for initiating the process of carcinogenesis de novo, as well as through the transformation of oral potential malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of our study was to detect the expression of stemness-type CSC marker CD147 in oral leukoplakias (OLs), the most common OPMD, and OSCCs as well. Materials and methods This study focuses on the semiquantitative immunohistochemical pattern of the expression of the CSCprotein biomarker CD147 in paraffin-embedded samples of 20 OSCCs of different grades of differentiation and 30 cases of OLs without or with different grades of dysplasia, compared to the normal oral epithelium in terms of cells' stain positivity. Statistical analysis wasperformed through Statistical Package for Social Sciences (SPSS) version 25.0(IBM SPSS Statistics, Armonk, NY) with Pearson chi-square test, and the significance level was set at 0.05 (p=0.05). In addition, the study clarified the expression of the respective gene of CD147 through quantitative polymerase chain (qPCR), in paraffin-embedded samples of the two extreme graduations: OLs of mildly dysplastic or non-dysplastic cases (n=10 cases) andOSCCs of moderately/poorly differentiated cases(n=17). Statistical analysis wasthen performed through SPSS version 25.0 with an independent paired t-test, and the significance level was set at 0.05 (p=0.05). Results The gene CD147 was expressed in all cases, although no statistically significant correlations were established. Regarding its protein products, the characteristic membranous staining of CD147 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. CD147 was upregulated significantly in the moderately and severely dysplastic OLs than in the mildlydysplastic and non-dysplastic OLs (p=0.008). Also, CD147 was upregulated significantly in the mildly dysplastic and non-dysplastic OLs than in the normal oral epithelium (p=0.012). Discussion The characteristic expression of CD147 in OLs and OSCCs' lesions suggests the presence of stemlike cancer cells, illustrating an underlying effect on the early stages of oral dysplasia, in the OL stage. The clinical application of CD147 as prognostic factor requires the experimental evaluation in larger number of samples. Conclusion Stem cells play an important role in the process of carcinogenesis. A major goal in cancer researchis the identification of specific biomarkers for the detection of cancer stem cells. CD147 is considered as an innovative stem cell marker. Our findings in oral mucosal potentially malignant disorders showed that CD147 is expressed more intenselyin parallel with the progression of the grade of dysplasia in OL.On the other hand, in oral squamous cell carcinoma, CD147 expression remains stable regardless of the degree of differentiation.

Early activation of Toll-like receptor-3 reduces the pathological progression of Alzheimer’s disease in APP/PS1 mouse

BackgroundToll-like receptor 3 (TLR3) plays an important role in the immune/inflammatory response in the nervous system and is a main pathological feature of Alzheimer’s disease (AD). This study investigates the role of early activation of TLR3 in the pathophysiological process of AD.MethodsIn the experiment, the agonist of TLR3, Poly(I:C), was intraperitoneally injected into the APP/PS1 mouse model of AD and wild-type control mice starting from the age of 4 to 9 months. At the age of 14 months, behavioral tests were conducted. Western blot and immunohistochemistry staining were used to evaluate the level of amyloid β-protein (Aβ), the activation of inflammatory cells, and neuron loss. In addition, the levels of inflammatory cytokines were measured using a quantitative polymerase chain reaction.ResultsThe results demonstrated that the early activation of TLR3 attenuated neuronal loss and neurobehavioral dysfunction. Moreover, the early activation of TLR3 reduced Aβ deposition, inhibited the activation of microglia and astrocytes, and decreased the transcription of pro-inflammatory factors in the hippocampus.ConclusionsThe results indicated that the activation of TLR3 by Poly (I:C) in the early stage of development of AD in a mouse model attenuated neuron loss and improved neurobehavioral functions. The underlying mechanisms could be attributed to its role in Aβ clearance, the inhibition of glial cells, and the regulation of neuroinflammation in the hippocampus.

EXPRESSION OF miRNA AS PROGNOSTIC MARKERS OF RENAL CELL CARCINOMA COURSE

To investigate the expression and clinical significance of miR-99b, -144, -155, -210, -222, -302а, -377in RCC ex vivo. Retrospective analysis of the expression levels of miRNAs -99b, -144, -155, -210, -222, -302а, -377in tumor tissue of 126patients with clear cell RCC was performed using quantitative polymerase chain reaction. Overexpression of miR-99b, -222, -302a and low levels of miR-155in RCC tissues were significantly associated with advanced tumor stage (p < 0.05). In majority of patients with distant metastases (>60%), high levels of miR-99b and 377were detected (p < 0.05). High/low miR-99b, -144, -155, -210, -222, and -302a ratio was significantly associated with Fuhrman grade of tumors. Aberrant expression of miR-99b, -144, -155, -210, -222, -302а, -377is strongly associated with advanced RCC, and panel of these miRNAs may be a useful prognostic marker for RCC course.

Myofibroblasts Are Not Characteristic Features of Keloid Lesions.

Myofibroblasts in tissue sections from keloids, hypertrophic scars (HTSs), and normal skin were localized by α-SMA immunostaining. Expression of α-SMA mRNA (ACTA2 gene) in normal skin and keloid tissue, and in fibroblasts from normal skin, keloid, and HTSs, was measured using quantitative polymerase chain reaction. Normal skin did not exhibit α-SMA-expressing myofibroblasts, but myofibroblasts were identified in 50% of keloids and 60% of HTSs. No significant differences in ACTA2 expression between keloid and normal skin tissue were observed. Mean ACTA2 expression was higher in HTS (2.54-fold, P = 0.005) and keloid fibroblasts (1.75-fold, P = 0.046) versus normal fibroblasts in vitro. However, α-SMA expression in keloids in vivo was not associated with elevated ACTA2 in keloid fibroblasts in vitro. Despite elevated ACTA2 in cultured keloid fibroblasts, myofibroblast presence is not a consistent feature of keloids. Therefore, therapies that target myofibroblasts may not be effective for all keloids. Further research is required to define the mechanisms driving keloid formation for development of more effective therapies.

Conjunctiva Resident γδ T Cells Expressed High Level of IL-17A and Promoted the Severity of Dry Eye.

Conjunctival inflammation promotes ocular surface disorders in dry eye disease (DED). Here we identified γδ T cells as the predominant source of IL-17A in the murine conjunctiva and assessed their contribution to the pathogenesis of DED. We enrolled 22 patients with DED, and analyzed the proportion of γδ T cells in the conjunctival epithelial samples by flow cytometry. Adult C57Bl/6 wild-type and TCRδ-/- mice were used to induce DED models to investigate the role of γδ T cells. The characteristics of immune cell infiltration and the expression of immune-related cytokines or markers in mouse conjunctiva were analyzed by flow cytometry, Western blot, and quantitative polymerase chain reaction. The proportion of γδ T cells in the human DED conjunctiva is significantly higher in patients with severe corneal epithelial defects than in mild ones, which is consistently observed in the murine DED model. Further, a high level of IL-17A but not IFN-γ is detected in the conjunctiva of mice. The increased murine IL-17A-producing cells on the conjunctiva are identified as γδ T cells predominantly and Th17 cells to a lesser extent. Ablation of γδ T cells by antibody depletion or genetic deletion of TCRδ alleviates ocular surface damage in the murine DED model. Our studies evaluate human and experimental murine DED for evidence of γδ T-cell-mediated inflammation and highlight a potential therapeutic synergy by targeting IL-17 and γδ T cells in DED treatment.

The nonopioid cholinergic agonist GTS-21 mitigates morphine-induced aggravation of burn injury pain together with inhibition of spinal microglia activation in young rats

BackgroundRepetitive opioid use does not always alleviate basal pain, procedural pain, or both after burn injury. Mitigation of burn injury-site pain can be achieved by GTS-21 stimulation of α7-acetylcholine nicotinic receptors (α7AChRs) and reduced microglia activation in rat. We tested the hypothesis that morphine exaggerates burn injury-site pain and GTS-21 alleviates both morphine-induced aggravated burn injury pain and microglia activation. MethodsYoung rats with dorsal paw burn injury or sham-burn received intraperitoneal saline, morphine, GTS-21, or a combination twice daily for 14 days. Ipsilateral plantar pain thresholds were tested every other day before morning drugs from days 0–20. Spinal microglia activation, evidenced as pain-transducer (tumour necrosis factor-α [TNF-α], interleukin [IL]-6, IL-1β, nuclear factor kappa B [NF-κB], Toll-like receptor 4 [TLR4]) expression, was examined using immunohistochemistry and immunoblot. In cultured microglia, morphine-induced cytokine expression was measured (quantitative polymerase chain reaction/enzyme-linked immunosorbent assay [qPCR/ELISA]). ResultsMorphine aggravated allodynia at day 5 in sham-burn (P=0.039, n=8–11) but significantly aggravated burn injury site allodynia by day 3 (P=0.010, n=8–11). Microgliosis paralleled nociceptive behaviour changes where burn injury with morphine had highest microgliosis compared with burn injury, morphine alone, or controls (number of cells per field [SD]: 33.8 [2.4], 18.0 [4.1], 8.2 [1.9], and 4.8 [2.0], respectively; P<0.001, n=4–5]. GTS-21 reversed the morphine-induced pain component in sham-burn and burn injury rats together with reduced microgliosis and spinal pain-transducer expression (TNF-α, IL-6, IL-1β, NF-κB, and TLR4). Morphine-exposed microglial cells showed increased cytokine expression, which was mitigated by GTS-21. ConclusionsMorphine or burn injury alone increases pain together with microgliosis and pain-transducer expression. Morphine administration augments burn injury-site nociception sooner and aggravated spinal microgliosis and inflammatory pain-transducer expression. GTS-21 has the potential to treat morphine-induced pain in burn injury.

An environmental DNA approach to informing restoration of the functionally extinct oyster, Ostrea angasi

Abstract The success of oyster reef restoration can be enhanced by data on the distribution of remnant populations to inform the selection of suitable restoration locations. A quantitative polymerase chain reaction‐based environmental DNA (eDNA) assay was designed to provide distribution data for the oyster, Ostrea angasi, whose reefs are functionally extinct in Port Phillip Bay, Australia. Ostrea angasi eDNA accumulation and decay was measured in aquaria containing oysters in low and high densities, prior to testing the efficacy of the eDNA approach for detection of oysters at 15 field sites. Ostrea angasi eDNA accumulated significantly faster in aquaria where more individuals were present, while eDNA became undetectable 2–6 days after oysters were removed in low‐density treatments. The eDNA samples were successful at detecting O. angasi in the field when taken in close proximity of an oyster population. Increasing the sample number and volume could maximize oyster detection, demonstrating the potential of eDNA to identify suitable sites for the restoration of functionally extinct marine ecosystems.

Melatonin attenuates acute and chronic itch in mice: the antioxidant and anti-inflammatory effects of melatonin receptors.

BackgroundItch is a common symptom of skin diseases and significantly reduces patients’ quality of life. Melatonin has anti-inflammatory and antioxidant effects. Our study examined the potential anti-itch effects of melatonin (N-acetyl-5-methoxytryptamine) in mice.MethodsWe detected the effects of melatonin and its receptors on acute and chronic itch by conducting itching behavioral experiments in male C57 mice. Reactive oxygen species (ROS) levels and calcium ion (Ca2+) mobilization during acute itching production were explored using flow cytometry and calcium imaging techniques. Melatonin expression in the serum of the chronic itch model mice was determined by enzyme-linked immunoassays. Hematoxylin and eosin staining show the effects of melatonin on skin thickness in a chronic itch model. Cytokine and chemokine levels were determined by quantitative polymerase chain reaction.ResultsWe discovered that compound 48/80 (C48/80)- and chloroquine (CQ)-induced scratching were significantly decreased by intraperitoneal (i.p), intradermal, and intrathecal administration of melatonin in a dose-dependent manner in mice, and the co-administration of melatonin receptor antagonists abolished the anti-itch effects of i.d melatonin. The incubation of melatonin significantly decreased the intracellular ROS levels induced by C48/80 and CQ in cultured ND7/23 cells from a mouse x rat hybridoma nerve as neuron. Melatonin inhibited intracellular Ca2+ increases induced by CQ (but not C48/80) in cultured dorsal root ganglia (DRG) neurons. Melatonin (50 mg/kg i.p) attenuated imiquimod (IMQ)- or acetone and diethyl ether followed by acetone-ether-water (AEW)-induced chronic itch and epidermal hyperplasia in mice. Finally, melatonin treatment reduced the IMQ-induced expression of ST2 and interleukin-33 (IL-33) or the AEW-induced expression of interleukin 31 (IL-31) and interleukin 31 receptor A (IL-31 RA) in the mice.ConclusionsCollectively, our results indicate that melatonin attenuates acute and chronic itch, possibly via melatonin receptors, and its antioxidant, and anti-inflammatory effects in mice.

C-reactive protein perturbs alveolar bone homeostasis: An experimental study of periodontitis and diabetes in the rat.

To explore the role of C-reactive protein (CRP) in periodontitis and diabetes and its mechanism in alveolar bone homeostasis. In vivo, normal, and Crp knockout (KO) rats were randomly divided into control, diabetes, periodontitis, and diabetes and periodontitis groups, respectively. The diabetes model was established using a high-fat diet combined with streptozotocin injection. The periodontitis model was established by ligature combined with lipopolysaccharide (LPS) injection. Alveolar bones were analysed using micro-computed tomography, histology, and immunohistochemistry. In vitro, human periodontal ligament cells (hPDLCs) were treated with LPS and high glucose. CRP knockdown lentivirus or CRP overexpression adenovirus combined with a PI3K/AKT signalling inhibitor or agonist were used to explore the regulatory mechanism of CRP in osteogenesis and osteoclastogenesis of hPDLCs, as evidenced by alkaline phosphatase staining, Western blot, and quantitative polymerase chain reaction. In periodontitis and diabetes, CRP KO decreased the alveolar bone loss and the expression levels of osteoclastogenic markers, while increasing the expression levels of osteogenic markers. CRP constrained osteogenesis while promoting the osteoclastogenesis of hPDLCs via PI3K/AKT signalling under high glucose and pro-inflammatory conditions. CRP inhibits osteogenesis and promotes osteoclastogenesis via PI3K/AKT signalling under diabetic and pro-inflammatory conditions, thus perturbing alveolar bone homeostasis.

A decellularized flowable placental connective tissue matrix supports cellular functions of human tenocytes in vitro

PurposeInjectable connective tissue matrices (CTMs) may promote tendon healing, given their minimally invasive properties, structural and biochemical extracellular matrix components, and capacity to fill irregular spaces. The purpose of this study is to evaluate the effects of placental CTMs on the cellular activities of human tenocytes. Decellularization, the removal of cells, cell fragments, and DNA from CTMs, has been shown to reduce the host’s inflammatory response. Therefore, the authors hypothesize that a decellularized CTM will provide a more cell-friendly matrix to support tenocyte functions.MethodsThree human placental CTMs were selected for comparison: AmnioFill® (A-CTM), a minimally manipulated, non-viable cellular particulate, BioRenew™ (B-CTM), a liquid matrix, and Interfyl® (I-CTM), a decellularized flowable particulate. Adhesion and proliferation were evaluated using cell viability assays and tenocyte migration using a transwell migration assay. Gene expression of tenocyte markers, cytokines, growth factors, and matrix metalloprotease (MMP) in tenocytes were assessed using quantitative polymerase chain reaction.ResultsAlthough A-CTM supported more tenocyte adhesion, I-CTM promoted significantly more tenocyte proliferation compared with A-CTM and B-CTM. Unlike A-CTM, tenocyte migration was higher in I-CTM than the control. The presence of I-CTM also prevented the loss of tenocyte phenotype, attenuated the expression of pro-inflammatory cytokines, growth factors, and MMP, and promoted the expression of antifibrotic growth factor, TGFβ3.ConclusionCompared with A-CTM and B-CTM, I-CTM interacted more favorably with human tenocytes in vitro. I-CTM supported tenocyte proliferation with reduced de-differentiation and attenuation of the inflammatory response, suggesting that I-CTM may support tendon healing and regeneration in vivo.

Feasibility of community at-home dried blood spot collection combined with pooled reverse transcription PCR as a viable and convenient method for malaria epidemiology studies

BackgroundMany Plasmodium infections in endemic regions exist at densities below the limit of detection of standard diagnostic tools. These infections threaten control efforts and may impact vaccine and therapeutic drug studies. Simple, cost-effective methods are needed to study the natural history of asymptomatic submicroscopic parasitaemia. Self-collected dried blood spots (DBS) analysed using pooled and individual quantitative reverse transcription polymerase chain reaction (qRT-PCR) provide such a solution. Here, the feasibility and acceptability of daily at-home DBS collections for qRT-PCR was studied to better understand low-density infections.MethodsRapid diagnostic test (RDT)-negative individuals in Katakwi District, northeastern Uganda, were recruited between April and May 2021. Venous blood samples and clinic-collected DBS were taken at enrollment and at four weekly clinic visits. Participants were trained in DBS collection and asked to collect six DBS weekly between clinic visits. Opinions about the collection process were solicited using daily Diary Cards and a Likert scale survey at the final study visit. Venous blood and DBS were analysed by Plasmodium 18S rRNA qRT-PCR. The number of participants completing the study, total DBS collected, and opinions of the process were analysed to determine compliance and acceptability. The human internal control mRNA and Plasmodium 18S rRNA were evaluated for at-home vs. clinic-collected DBS and venous blood to assess quality and accuracy of at-home collected samples.ResultsOne-hundred two adults and 29 children were enrolled, and 95 and 26 completed the study, respectively. Three individuals withdrew due to pain or inconvenience of procedures. Overall, 96% of participants collected ≥ 16 of 24 at-home DBS, and 87% of DBS contained ≥ 40 µL of blood. The procedure was well tolerated and viewed favourably by participants. At-home collected DBS were acceptable for qRT-PCR and showed less than a one qRT-PCR cycle threshold shift in the human control mRNA compared to clinic-collected DBS. Correlation between Plasmodium falciparum 18S rRNA from paired whole blood and DBS was high (R = 0.93).ConclusionsAt-home DBS collection is a feasible, acceptable, and robust method to obtain blood to evaluate the natural history of low-density Plasmodium infections by qRT-PCR.

Neuroprotective Effects of Curcumin against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury in Cultured Primary Rat Astrocyte by Improving Mitochondrial Function and Regulating the ERK Signaling Pathway.

Objectives Curcumin (Cur) is a natural polyphenol isolated from turmeric and has potent anti-inflammatory and antioxidant activities. This study aimed to explore the effects and possible mechanisms of curcumin on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in cultured rat astrocyte primary cells. Methods After screening for effective doses, the cultured rat astrocyte primary cells were divided into three groups: control, OGD/R, and OGD/R + curcumin (10 μM, 20 μM, and 40 μM). Cell viability was detected using CCK8 assays. The level of malondialdehyde and superoxide dismutase activity was determined using commercial kits. The endothelial nitric oxide synthase and adenosine triphosphate concentrations were determined by enzyme-linked immunosorbent assay. The mRNA levels of the inflammatory indexes interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1β were evaluated by quantitative reverse-transcription polymerase chain reaction. Annexin V-fluorescein isothiocyanate/propidium iodide was used to detect apoptosis. JC-1 was used to assess the mitochondrial membrane potential. The protein expression of apoptosis-related proteins (B-cell lymphoma-2 (Bcl-2), BCL-2-associated X (Bax), and cleaved caspase 3), mitochondria-related proteins (dynamin-related protein 1 (DRP1), phosphorylated DRP1 (p-DRP1), and mitofusin 2), and essential proteins of the extracellular signal-regulated kinase (ERK) signaling pathway (ERK1/2, p-ERK1/2) were analyzed by western blot. Results Our data indicated that curcumin reversed OGD/R-induced cell viability loss, oxidative stress, inflammatory cytokine production, and cell apoptosis in a dose-dependent manner. Furthermore, curcumin attenuated OGD/R-induced mitochondrial dysfunction and ERK1/2 phosphorylation in a dose-dependent manner. Conclusions Curcumin protected against OGD/R-induced injury in rat astrocyte primary cells through improving mitochondrial function and regulating the ERK signaling pathway.

Genome-wide identification and expression analysis of the ftsH protein family and its response to abiotic stress in Nicotiana tabacum L

BackgroundThe filamentous temperature-sensitive H protease (ftsH) gene family plays an important role in plant growth and development. FtsH proteins belong to the AAA protease family. Studies have shown that it is a key gene for plant chloroplast development and photosynthesis regulation. In addition, the ftsH gene is also involved in plant response to stress. At present, the research and analysis of the ftsH gene family are conducted in microorganisms such as Escherichia coli and Oenococcus and various plants such as Arabidopsis, pear, rice, and corn. However, analysis reports on ftsH genes from tobacco (Nicotiana tabacum L.), an important model plant, are still lacking. Since ftsH genes regulate plant growth and development, it has become necessary to systematically study this gene in an economically important plant like tobacco.ResultsThis is the first study to analyze the ftsH gene from Nicotiana tabacum L. K326 (NtftsH). We identified 20 ftsH genes from the whole genome sequence, renamed them according to their chromosomal locations, and divided them into eight subfamilies. These 20 NtftsH genes were unevenly distributed across the 24 chromosomes. We found four pairs of fragment duplications. We further investigated the collinearity between these genes and related genes in five other species. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis identified differential expression patterns of NtftsH in different tissues and under various abiotic stress conditions.ConclusionsThis study provides a comprehensive analysis of the NtftsH gene family. The exon–intron structure and motif composition are highly similar in NtftsH genes that belong to the same evolutionary tree branch. Homology analysis and phylogenetic comparison of ftsH genes from several different plants provide valuable clues for studying the evolutionary characteristics of NtftsH genes. The NtftsH genes play important roles in plant growth and development, revealed by their expression levels in different tissues as well as under different stress conditions. Gene expression and phylogenetic analyses will provide the basis for the functional analysis of NtftsH genes. These results provide a valuable resource for a better understanding of the biological role of the ftsH genes in the tobacco plant.

CircCEP85 upregulates IGF1 expression to promote breast cancer progression via sponging miR-1193

BackgroundIncreasing evidence has suggested that circular RNAs (circRNAs) play critical roles in breast cancer (BC) progression. However, the expression level and potential functional role of circRNA centrosomal protein 85 (circCEP85) in BC remains largely unknown. Here, we aimed to explore the role of circCEP85 in BC.MethodsThe levels of circCEP85, insuline-like growth factor I (IGF1) mRNA and microRNA-1193 (miR-1193) were examined by quantitative real-time polymerase chain reaction. The protein level was measured by Western blot. Cell proliferation, migration, apoptosis, angiogenesis and stemness were assessed by cell counting kit-8, 5-ethynyl-2’-deoxyuridine assay, transwell assay, flow cytometry, tube formation and sphere formation assays. Xenograft mouse models were conducted to evaluate the effect of circCEP85 in BC in vivo. Moreover, dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays were preformed to confirm the interaction between miR-1193 and circCEP85 or IGF1.ResultsCircCEP85 was upregulated in BC tissues and cells. Silencing of circCEP85 inhibited proliferation, invasion, angiogenesis and stemness, but promoted apoptosis in BC cells in vitro. In addition, circCEP85 silencing inhibited tumor growth in vivo. Mechanistically, circCEP85 elevated IGF1 expression via sponging miR-1193 to promote breast cancer progression.ConclusionThe circCEP85-miR-1193-IGF1 axis regulated BC progression via the competitive endogenous RNA (ceRNA) mechanism. CircCEP85 might be a prognostic biomarker and therapeutic target for BC.

Exosomal circWDR62 promotes temozolomide resistance and malignant progression through regulation of the miR-370-3p/MGMT axis in glioma

Exosome-mediated delivery of circular RNAs (circRNAs) is implicated in cancer progression. However, the role of exosomal circRNAs in the chemotherapy resistance of tumours remains poorly understood. Here we identified a novel circRNA, circWDR62. It was found that circWDR62 expression was upregulated in TMZ-resistant glioma cells and TMZ-resistant glioma cell-derived exosomes compared with their controls by using high-throughput microarray analysis and quantitative real-time polymerase chain reaction, and high circWDR62 expression was associated with poor prognosis of glioma. Functionally, downregulation of circWDR62 expression could significantly inhibit the TMZ resistance and malignant progression of glioma. Further mechanistic studies showed that circWDR62 plays a role by sponging miR-370-3p as a competing endogenous RNA. Rescue experiments confirmed that MGMT is the downstream target of the circWDR62/miR-370-3p axis in glioma. In addition, circWDR62 could be transported between TMZ-resistant and TMZ-sensitive glioma cells via exosomes. Exosomal circWDR62 from TMZ-resistant cells conferred TMZ resistance in recipient sensitive cells while also enhancing the proliferation, migration and invasion of these cells. A series of clinical and in vivo trials corroborated that exosomal circWDR62 could promote TMZ chemoresistance and malignant progression of glioma. Our results demonstrate for the first time that exosome-mediated delivery of circWDR62 can promote TMZ resistance and malignant progression via targeting of the miR-370-3p/MGMT axis in vitro and in vivo in glioma, providing a new therapeutic strategy. Moreover, exosomal circWDR62 in human serum may serve as a promising therapeutic target and prognostic marker for glioma therapy.

Near-infrared light reduces glia activation and modulates neuroinflammation in the brains of diet-induced obese mice

Neuroinflammation is a key event in neurodegenerative conditions such as Alzheimer’s disease (AD) and characterizes metabolic pathologies like obesity and type 2 diabetes (T2D). Growing evidence in humans shows that obesity increases the risk of developing AD by threefold. Hippocampal neuroinflammation in rodents correlates with poor memory performance, suggesting that it contributes to cognitive decline. Here we propose that reducing obesity/T2D-driven neuroinflammation may prevent the progression of cognitive decline associated with AD-like neurodegenerative states. Near-infrared light (NIR) has attracted increasing attention as it was shown to improve learning and memory in both humans and animal models. We previously reported that transcranial NIR delivery reduced amyloid beta and Tau pathology and improved memory function in mouse models of AD. Here, we report the effects of NIR in preventing obesity-induced neuroinflammation in a diet-induced obese mouse model. Five-week-old wild-type mice were fed a high-fat diet (HFD) for 13 weeks to induce obesity prior to transcranial delivery of NIR for 4 weeks during 90-s sessions given 5 days a week. After sacrifice, brain slices were subjected to free-floating immunofluorescence for microglia and astrocyte markers to evaluate glial activation and quantitative real-time polymerase chain reaction (PCR) to evaluate expression levels of inflammatory cytokines and brain-derived neurotrophic factor (BDNF). The hippocampal and cortical regions of the HFD group had increased expression of the activated microglial marker CD68 and the astrocytic marker glial fibrillary acidic protein. NIR-treated HFD groups showed decreased levels of these markers. PCR revealed that hippocampal tissue from the HFD group had increased levels of pro-inflammatory interleukin (IL)-1β and tumor necrosis factor-α. Interestingly, the same samples showed increased levels of the anti-inflammatory IL-10. All these changes were attenuated by NIR treatment. Lastly, hippocampal levels of the neurotrophic factor BDNF were increased in NIR-treated HFD mice, compared to untreated HFD mice. The marked reductions in glial activation and pro-inflammatory cytokines along with elevated BDNF provide insights into how NIR could reduce neuroinflammation. These results support the use of NIR as a potential non-invasive and preventive therapeutic approach against chronic obesity-induced deficits that are known to occur with AD neuropathology.

Differential Expression of Long Noncoding RNAs in Murine Myoblasts After Short Hairpin RNA-Mediated Dysferlin Silencing In Vitro: Microarray Profiling

Background Long noncoding RNAs (lncRNAs) are noncoding RNA transcripts greater than 200 nucleotides in length and are known to play a role in regulating the transcription of genes involved in vital cellular functions. We hypothesized the disease process in dysferlinopathy is linked to an aberrant expression of lncRNAs and messenger RNAs (mRNAs). Objective In this study, we compared the lncRNA and mRNA expression profiles between wild-type and dysferlin-deficient murine myoblasts (C2C12 cells). Methods LncRNA and mRNA expression profiling were performed using a microarray. Several lncRNAs with differential expression were validated using quantitative real-time polymerase chain reaction. Gene Ontology (GO) analysis was performed to understand the functional role of the differentially expressed mRNAs. Further bioinformatics analysis was used to explore the potential function, lncRNA-mRNA correlation, and potential targets of the differentially expressed lncRNAs. Results We found 3195 lncRNAs and 1966 mRNAs that were differentially expressed. The chromosomal distribution of the differentially expressed lncRNAs and mRNAs was unequal, with chromosome 2 having the highest number of lncRNAs and chromosome 7 having the highest number of mRNAs that were differentially expressed. Pathway analysis of the differentially expressed genes indicated the involvement of several signaling pathways including PI3K-Akt, Hippo, and pathways regulating the pluripotency of stem cells. The differentially expressed genes were also enriched for the GO terms, developmental process and muscle system process. Network analysis identified 8 statistically significant (P&lt;.05) network objects from the upregulated lncRNAs and 3 statistically significant network objects from the downregulated lncRNAs. Conclusions Our results thus far imply that dysferlinopathy is associated with an aberrant expression of multiple lncRNAs, many of which may have a specific function in the disease process. GO terms and network analysis suggest a muscle-specific role for these lncRNAs. To elucidate the specific roles of these abnormally expressed noncoding RNAs, further studies engineering their expression are required.