Reverse Transcription-polymerase Chain Reaction Data Research Articles

Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response.

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.

ANKRD2 expression combined with TNFRSF19 expression for evaluating the prognosis of oral squamous cell carcinoma patients

ObjectiveAs an important chemotherapy drug, cisplatin has been widely used in the treatment of many cancers. However, many patients, including oral squamous cell carcinoma (OSCC) patients, experience unacceptable outcomes from cisplatin treatment. Thus, we devised a risk model for predicting the sensitivity of OSCC patients to cisplatin treatment, to provide a reference for clinical practice. MethodsCAL-27 and SCC-9 cell lines treated or not with cisplatin and data from The Cancer Genome Atlas (TCGA) were screened for simultaneously and significantly differentially expressed genes. Next, we built a risk model for predicting cisplatin sensitivity in OSCC patients. Reverse transcription-polymerase chain reaction (RT-PCR), pathological samples and clinical data were used to examine the reliability of the model. ResultsANKRD2 and TNFRSF19 were differentially expressed between the OSCC metastasis cell line HSC-3 treated and not treated with cisplatin, as well as between the OSCC cell line SCC-25 and the cell line SCC25-DDP, which has cisplatin chemoresistance. We found that the expression of ANKRD2 and TNFRSF19 had a significant influence on the prognosis of OSCC patients. The risk model that combined ANKRD2 and TNFRSF19 to predict sensitivity to cisplatin in OSCC patients was confirmed by analysing the pathological samples and follow-up information of clinical patients. ConclusionsThe expression of ANKRD2 and TNFRSF19 is associated with cisplatin sensitivity and prognosis in patients with OSCC. The survival outcome of patients with oral squamous cell carcinoma (OSCC) was found to be significantly worse in those with high expression of ANKRD2 combined with low expression of TNFRSF19. ANKRD2 and TNFRSF19 may be targets for cisplatin sensitivity prediction in OSCC patients. These findings may provide novel strategies for overcoming cisplatin resistance.

Evaluation of diagnostic performance of a rapid antigen test in diagnosing COVID-19

The coronavirus disease 2019 (COVID-19) pandemic is our time's major global health crisis and the greatest health challenge. Reverse transcription-polymerase chain reaction (RT-PCR) test for severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the gold standard technique for diagnosis of symptomatic cases and asymptomatic carriers. By 2020, antigen rapid tests have been approved for use in Covid-19 testing by regulatory bodies all over the world owing to their benefits as they are rapid and cost effective. This work aimed to determine the diagnostic sensitivity and accuracy of the SARS-CoV-2 rapid antigen test in the detection of SARS-CoV-2 infection compared to RT-PCR data. The study included 111 symptomatic COVID-19 patients and 20 control subjects. Of the 111 study patients, 91 patients (81.98%) were positive by RT-PCR and 20 patients negative. The BIOZEK antigen COVID-19 Ag rapid test device was evaluated using sera from the 111 symptomatic COVID-19 patients. Of the 91 RT-PCR positive patients, 81 (90.1%) were positive by the antigen rapid diagnostic test (Ag-RDT). The control subjects were negative by both tests. The overall sensitivity, specificity, PPV, NPV, and accuracy of the Ag-RDT were 91.11%, 100%, 100%, 68.9%, and 91.8%, respectively and these increased as the level of viremia increased. In conclusion, the used Ag-RDT showed high sensitivity and accuracy for detecting of a SARS-CoV-2 infection, especially when the viral load was high. However, the test lacks sensitivity particularly in those with low viral load.

Genome-Wide Identification and Expression Analysis of Cytokinin Response Regulator (RR) Genes in the Woody Plant Jatropha curcas and Functional Analysis of JcRR12 in Arabidopsis.

The cytokinin (CK) response regulator (RR) gene family plays a pivotal role in regulating the developmental and environmental responses of plants. Axillary bud outgrowth in the perennial woody plant Jatropha curcas is regulated by the crosstalk between CK and gibberellins (GA). In this study, we first analyzed the effects of gibberellin A3 (GA3), lovastatin (a CK synthesis inhibitor), decapitation, and their interaction, on the outgrowth of axillary buds. The results indicate that lovastatin completely inhibited GA-promoted axillary bud outgrowth and partially weakened the decapitation-promoted axillary bud outgrowth. To further characterize and understand the role of CK signaling in promoting the development of female flowers and branches, we performed bioinformatics and expression analyses to characterize the CK RR gene (JcRR) family in J. curcas. A total of 14 members of the JcRR family were identified; these genes were distributed on 10 chromosomes. Phylogenetic analysis indicated that the corresponding RR proteins are evolutionarily conserved across different plant species, and the Myb-like DNA-binding domain divides the 14 members of the JcRR family into type-A and type-B proteins. Further analysis of cis-acting elements in the promoter regions of JcRRs suggests that JcRRs are expressed in response to phytohormones, light, and abiotic stress factors; thus, JcRRs may be involved in some plant development processes. Genomic sequence comparison revealed that segmental duplication may have played crucial roles in the expansion of the JcRR gene family, and five pairs of duplicated genes were all subjected to purifying selection. By analyzing RNA sequencing (RNA-seq) and quantitative reverse transcription-polymerase chain reaction (qRT–PCR) data, we characterized that the temporospatial expression patterns of JcRRs during the development of various tissues and the response of these genes to phytohormones and abiotic stress. The JcRRs were mainly expressed in the roots, while they also exhibited differential expression patterns in other tissues. The expression levels of all six type-A and one type-B JcRRs increased in response to 6-benzylaminopurine (6-BA), while the four type-B JcRRs levels decreased. The expression levels of two type-B JcRRs increased in response to exogenous GA3 treatment, while those of three type-A and three type-B JcRRs decreased. We found that type-A JcRRs may play a positive role in the continuous growth of axillary buds, while the role of type-B JcRRs might be the opposite. In response to abiotic stress, the expression levels of two type-A and three type-B JcRRs strongly increased. The overexpression of JcRR12 in Arabidopsis thaliana slightly increased the numbers of rosette branches after decapitation, but not under normal conditions. In conclusion, our results provide detailed knowledge of JcRRs for further analysis of CK signaling and JcRR functions in J. curcas.

Inhibition of macrophage-derived foam cells by Adipsin attenuates progression of atherosclerosis

Phagocytosis of oxidized low-density lipoprotein (OxLDL) by macrophages yields “foam cells” and serves as a hallmark of atherosclerotic lesion. Adipsin is a critical component of the complement activation pathway. Recent evidence has indicated an obligatory role for Adipsin in pathological models including ischemia-reperfusion and sepsis. Adipsin levels are significantly decreased in patients with asymptomatic carotid atherosclerosis, implying the role for Adipsin as a potential marker of asymptomatic carotid atherosclerosis. This study was designed to evaluate the role for Adipsin in atherosclerosis and the mechanisms involved using both in vivo and in vitro experiments. ApoE−/−/AdipsinTg mice were constructed and were fed a high-fat diet for 12 weeks. Compared with ApoE−/− mice, area of the sclerotic plaques was reduced, along with lower macrophage deposition within the plaque in ApoE−/−/AdipsinTg mice. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were stimulated with oxLDL (50 μg/ml). Adenovirus vectors containing the Adipsin gene were transfected into macrophages. Lipid accumulation was observed by Oil red O staining. Western blot and reverse transcription-polymerase chain reaction data revealed that Adipsin overexpression inhibited oxLDL-induced lipid uptake and foam cell formation and upregulation of CD36 and PPARγ in Ad-Adipsin-transfected macrophages. In addition, the PPARγ-specific agonist GW1929 reversed Adipsin overexpression-evoked inhibitory effect on lipid uptake. These results demonstrate unequivocally that Adipsin inhibits lipid uptake in a PPARγ/CD36-dependent manner and prevents the formation of foam cells, implying that Adipsin may be a potential therapeutic target against atherosclerosis.

Serum miR-204 and miR-451 Expression and Diagnostic Value in Patients with Pulmonary Artery Hypertension Triggered by Congenital Heart Disease.

Objective To determine the level of expression and clinical importance of serum miR-204 and miR-451 in patients with pulmonary arterial hypertension caused by congenital heart disease (CHD-PAH). Methods From July 2019 to January 2021, 114 infants with congenital heart disease (CHD) were hospitalized at Qingdao's Fuwai Cardiovascular Hospital. They were grouped into categories: CHD (53 cases) and CHD-PAH (61 cases) based on whether they had pulmonary hypertension (PAH). In addition, 60 healthy children were selected as the control group. All children underwent routine biochemical examination, echocardiography, and pulmonary arterial pressure examination. By using an enzyme-linked immunosorbent assay (ELISA), the levels of brain natriuretic peptide (BNP) and asymmetric dimethylarginine (ADMA) in the blood were measured. Additionally, reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression levels of miR-204 and miR-451 in peripheral blood. The correlation between miR-204, miR-451, BNP, ADMA, and mPAP was investigated using Pearson correlation analysis. Results Consequently, the TC, BNP, and ADMA serum levels were considerably higher in the CHD and CHD-PAH groups than in the control group (P < 0.05), whereas BNP and ADMA serum levels were significantly higher in the CHD-PAH group than in the CHD group (P < 0.05). According to RT-PCR data, the expression levels of miR-204 and miR-451 in the peripheral blood of children in the CHD and CHD-PAH groups were significantly lower (P < 0.05) when compared to the control group. The expression levels of miR-204 and miR-451 in the peripheral blood of children in the CHD-PAH group were substantially lower (P < 0.05) than in the CHD group. Significantly, the findings of the ROC curve revealed that the area under the curve (AUC) of CHD-PAH diagnosed by miR-204 and miR-451 alone was 0.737 and 0.725, respectively, and the AUC of joint diagnosis was 0.840, which was greater than that of single diagnosis (P < 0.05). Patients with CHD-PAH had lower levels of miR-204 and miR-451 in their blood, and this was found to be associated with BNP, ADMA, and mPAP, analyzed by Pearson correlation. Conclusions Children with CHD-PAH can be diagnosed based on aberrant expressions of miR-204 and miR-451 in their peripheral blood serum. Moreover, CHD-PAH can be diagnosed if the combination of miR-204 and miR-451 is detected as a biomarker, which has a higher diagnostic value.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Automated Reverse Transcription Polymerase Chain Reaction Data Analysis for Sars-CoV-2 Detection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is a current public health concern. Rapid diagnosis is crucial, and reverse transcription polymerase chain reaction (RT-PCR) is presently the reference standard for SARS-CoV-2 detection. Automated RT-PCR analysis (ARPA) is a software designed to analyze RT-PCR data for SARSCoV-2 detection. ARPA loads the RT-PCR data, classifies each sample by assessing its amplification curve behavior, evaluates the experiment's quality, and generates reports. ARPA was implemented in the R language and deployed as a Shiny application. We evaluated the performance of ARPA in 140 samples. The samples were manually classified and automatically analyzed using ARPA. ARPA had a true-positive rate = 1, true-negative rate = 0.98, positive-predictive value = 0.95, and negative-predictive value = 1, with 36 samples correctly classified as positive, 100 samples correctly classified as negative, and two samples classified as positive even when labeled as negative by manual inspection. Two samples were labeled as invalid by ARPA and were not considered in the performance metrics calculation. ARPA is a sensitive and specific software that facilitates the analysis of RT-PCR data, and its implementation can reduce the time required in the diagnostic pipeline.

A novel gene expression signature-based on B-cell proportion to predict prognosis of patients with lung adenocarcinoma

BackgroundThis study aimed to develop a reliable immune signature based on B-cell proportion to predict the prognosis and benefit of immunotherapy in LUAD.MethodsThe proportion of immune cells in the TCGA-LUAD dataset was estimated using MCP-counter. The Least Absolute Shrinkage and Selector Operation was used to identify a prognostic signature and validated in an independent cohort. We used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) data and formalin-fixed paraffin-embedded (FFPE) specimens immunohistochemistry to illustrate the correlation between prognostic signature and leukocyte migration.ResultsWe found that the relative abundance of B lineage positively correlated with overall survival. Then, we identified a 13-gene risk-score prognostic signature based on B lineage abundance in the testing cohort and validated it in a cohort from the GEO dataset. This model remained strongly predictive of prognoses across clinical subgroups. Further analysis revealed that patients with a low-risk score were characterized by B-cell activation and leukocyte migration, which was also confirmed in FFPE specimens by qRT-PCR and immunohistochemistry. Finally, this immune signature was an independent prognostic factor in the composite nomogram of clinical characteristics.ConclusionsIn conclusion, the 13-gene immune signature based on B-cell proportion may serve as a powerful prognostic tool in LUAD.

Overview of Safety Measures at Selected Airports during the COVID-19 Pandemic

The year 2020 was very challenging for the whole world, given the outbreak of the ongoing coronavirus-related pandemic, and was marked in particular by overcoming new hitherto unknown obstacles. For air transport, in particular, airlines stopped flying altogether and were forced to ground hundreds of planes worldwide involuntarily. Airports had to close their terminals for a long time, wholly suspend operations, and its resumption required significant organizational changes. This article summarizes the measures related to the COVID-19 pandemic adopted by airports to minimize the risk of spreading the disease. The article focuses on countermeasures and their implementation at selected airports in a specific time frame and airports’ behavior during a pandemic which varies depending on country and time of the year. The results demonstrated that steps being taken at airports include the use of face coverings or masks, social distance, enhanced cleaning and disinfection, or temperature checks and/or symptoms (fever, loss of smell, chills, cough, shortness of breath), RT-PCR (reverse transcription-polymerase chain reaction) screening and data collection with health declaration. These measures have now become an essential standard for the operation of airports and can, therefore, be used to assess the level of airport safety achieved. In the final phase, the article evaluates the level of achieved airport safety based on the proposed scoring method.

Effect of total flavonoids from Drynaria rhizome on bone loss in ovariectomized rats

Purpose: To determine the potential effect of total flavonoids from Drynaria rhizome on bone loss in ovariectomized (OVX) rats.&#x0D; Methods: The rats were divided into four groups: normal control, ovariectomized (OVX) control, and two Drynaria rhizome (DR) flavonoids treatments. Post-operation, osteoporotic OVX rats were given Drynaria rhizome total flavonoids for 3 months. Thereafter, the expressions of bone-related genes and biochemical indices were investigated in samples taken from the serum and bone of the rats.&#x0D; Results: Treatment with total flavonoids from Drynaria rhizome prevented bone mineral loss and improved some related biochemical indices associated with osteoporosis: alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), bone gla protein (BGP) and estradiol (E2). Reverse transcription-polymerase chain reaction (RT-PCR) data showed that treatment with the total flavonoids significantly downregulated mRNA expression of Wnt10b, β-catenin, recombinant human bone morphogenetic protein-2 (BMP2) and BMP4 in OVX rats, but significantly reversed OVX-induced downregulation of dickkopf1 (Dkk1) mRNA expression.&#x0D; Conclusion: These results indicate that total flavonoids from Drynaria rhizome exert anti-osteoporotic effects in rats via WNT signaling and BMP-2 signaling pathways.

Effect of total flavonoids from Drynaria rhizome on bone loss in ovariectomized rats

Purpose: To determine the potential effect of total flavonoids from Drynaria rhizome on bone loss in ovariectomized (OVX) rats.&#x0D; Methods: The rats were divided into four groups: normal control, ovariectomized (OVX) control, and two Drynaria rhizome (DR) flavonoids treatments. Post-operation, osteoporotic OVX rats were given Drynaria rhizome total flavonoids for 3 months. Thereafter, the expressions of bone-related genes and biochemical indices were investigated in samples taken from rat serum and bone.&#x0D; Results: Treatment with total flavonoids from Drynaria rhizome prevented bone mineral loss and improved some related biochemical indices associated with osteoporosis, namely, alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), bone gla protein (BGP) and estradiol (E2). Reverse transcription-polymerase chain reaction (RT-PCR) data showed that treatment with the total flavonoids significantly downregulated mRNA expression of Wnt10b, β-catenin, recombinant human bone morphogenetic protein-2 (BMP2) and BMP4 in OVX rats, but significantly reversed OVX-induced downregulation of dickkopf1 (Dkk1) mRNA expression.&#x0D; Conclusion: These results indicate that total flavonoids from Drynaria rhizome exert anti-osteoporotic effects in rats through the WNT signaling and BMP-2 signaling pathways.

Evaluation of potential COVID-19 recurrence in patients with late repeat positive SARS-CoV-2 testing.

BackgroundSARS-CoV-2 reinfection and reactivation has mostly been described in case reports. We therefore investigated the epidemiology of recurrent COVID-19 SARS-CoV-2.MethodsAmong patients testing positive for SARS-CoV-2 between March 11 and July 31, 2020 within an integrated healthcare system, we identified patients with a recurrent positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) assay ≥60 days after an initial positive test. To assign an overall likelihood of COVID-19 recurrence, we combined quantitative data from initial and recurrent positive RT-PCR cycle thresholds—a value inversely correlated with viral RNA burden— with a clinical recurrence likelihood assigned based on independent, standardized case review by two physicians. “Probable” or “possible” recurrence by clinical assessment was confirmed as the final recurrence likelihood only if a cycle threshold value obtained ≥60 days after initial testing was lower than its preceding cycle threshold or if the patient had an interval negative RT-PCR.ResultsAmong 23,176 patients testing positive for SARS-CoV-2, 1,301 (5.6%) had at least one additional SARS-CoV-2 RT-PCRs assay ≥60 days later. Of 122 testing positive, 114 had sufficient data for evaluation. The median interval to the recurrent positive RT-PCR was 85.5 (IQR 74–107) days. After combining clinical and RT-PCR cycle threshold data, four patients (3.5%) met criteria for probable COVID-19 recurrence. All four exhibited symptoms at recurrence and three required a higher level of medical care compared to their initial diagnosis. After including six additional patients (5.3%) with possible recurrence, recurrence incidence was 4.3 (95% CI 2.1–7.9) cases per 10,000 COVID-19 patients.ConclusionsOnly 0.04% of all COVID-19 patients in our health system experienced probable or possible recurrence; 90% of repeat positive SARS-CoV-2 RT-PCRs were not consistent with true recurrence. Our pragmatic approach combining clinical and quantitative RT-PCR data could aid assessment of COVID-19 reinfection or reactivation by clinicians and public health personnel.

Natural-lipid nanoparticle-based therapeutic approach to deliver 6-shogaol and its metabolites M2 and M13 to the colon to treat ulcerative colitis

The anti-inflammatory drug candidate, 6-shogaol, has demonstrated excellent efficacies in various in vitro studies. However, its rapid metabolism after oral administration results in poor bioavailability and undetectable in vivo pharmacokinetics. Here, we constructed a natural-lipid (NL) nanoparticle drug delivery system (NP-DDS) to encapsulate 6-shogaol and undertake its controlled release to the proposed drug target (colon). Our in vitro drug-release assay revealed that NL-encapsulated 6-shogaol (6-S-NL) exhibits a delayed drug-release profile compared to free 6-shogaol (free-6-S). Consistent with our expectations, orally administrated 6-S-NL exhibits a superior anti-inflammatory efficacy likely due to the controlled release compared to free 6-S in a dextran sulfate sodium (DSS)-induced mouse model of colitis. Although 6-S-NL treatment yields an enhanced concentration of 6-shogaol at the target site (colon), this concentration is still far below the effective level. We hypothesize that the released 6-shogaol undergoes rapid metabolism and that the metabolites of 6-shogaol may contribute to the anti-inflammatory efficacy of 6-S-NL. We thus examined the in vitro anti-inflammatory efficacies of two highly abundant colonic metabolites, M2 (a cysteine-conjugated metabolite) and M13 (a glutathione-conjugated metabolite), against macrophage cells. Reverse transcription-polymerase chain reaction (RT-PCR) data showed that both M2 and M13 (at 1.0 μg/mL) could down-regulate pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and up-regulate an anti-inflammatory factor (IL-10) in inflamed Raw 264.7 cells. Subsequent in vitro wound-healing assays also confirmed that M2 and M13 accelerate the wound recovery process of Caco-2 cells at the concentrations seen in the colon (1.0 μg/mL). Further, in the DSS-induced mouse model of colitis, oral administration of M2- or M13-loaded NL nanoparticles (M2-NL, M13-NL) demonstrated excellent in vivo wound-healing effects, and these activities were better than those observed for 6-S-NL. Combined with the 6-S-NL's bio-distribution assay, our data show that: the 6-shogaol metabolites, M2 and M13, are more potent anti-inflammatory compounds than 6-shogaol itself; NL nanoparticles can effectively deliver 6-shogaol to the colon, with little accumulation seen in the kidney or liver; and the actions of M2 and M13 mostly confer the anti-inflammatory effect of 6-S-NL. Our results explained the discrepancy between the low tissue concentrations of NL delivered 6-shogaol and its effectiveness against ulcerative colitis (UC) in a mouse model. This study paved the way for further developing the NL-loaded active metabolites, M2 or M13, as novel targeted therapeutic approaches for curing UC.

MiR-25 overexpression promotes fracture healing by activating the Wnt signaling pathway.

To study the mechanism of micro-ribonucleic acid (miR)-25 in regulating the fracture healing in rats. A total of 45 male Sprague-Dawley (SD) rats were selected and randomly divided into group A [Phosphate Buffered Saline (PBS), n=15], group B (mimics NC, n=15) and group C (miR-25 mimics, n=15). The fracture model in rats was established via operation in all groups. From 1 d after the successful modeling, 50 μL (2 nmoL) of PBS was intraperitoneally injected into rats in group A, an equal amount of mimics NC was injected into rats in group B, and an equal amount of miR-25 mimics was injected into rats in group C. The above agents were injected once a week for consecutive 6 weeks. Fracture healing in rats was evaluated via X-ray imaging. At the same time, miR-25 expression in the three groups was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Protein expressions of β-catenin, proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) in the three groups were detected via Western blotting. The OCN-, PCNA- and BMP-2-positive osteoblasts in the three groups were detected via immunohistochemical staining and were further quantified. Moreover, the biomechanical properties of femoral fracture healing in the three groups were analyzed via the 4-point bending flexural test. The X-ray examination of the femoral fracture healing at postoperative 1 and 7 weeks revealed that the fracture line disappeared, and both callus formation and fracture healing were good in miR-25 mimics group. In PBS group and mimics NC group, a few fracture lines could be observed, and both callus formation and fracture healing were poor. RT-PCR data showed that the expression level of miR-25 significantly increased in the miR-25 mimics group compared with that in the other two groups, and the differences were statistically significant (p<0.01). Western blotting analyses showed upregulated levels of β-catenin, PCNA and BMP-2 in the miR-25 mimics group compared with those in the control group, and the differences were statistically significant (p<0.01). Immunohistochemical staining manifested that the numbers of OCN-, PCNA- and BMP-2-positive osteoblasts in miR-25 mimics group markedly increased compared with that in the other two groups (p<0.01), suggesting that osteoblast differentiation in miR-25 mimics group was affected. The above immunohistochemical results indicated that the osteoblast differentiation at the fracture end in miR-25 mimics group was markedly enhanced compared with that in control groups. The results of the biomechanical test of femur specimens at 7 weeks after operation showed that in miR-25 mimics group, the maximum load, fracture energy and stiffness increased by 188%, 333% and 90%, respectively, compared with those in the PBS group (p<0.01). It is indicated that miR-25 promoted the mechanical properties of fracture healing. The overexpression of miR-25 in the fracture in rats promotes fracture healing by activating the Wnt signaling pathway.

Extracellular calcium regulates the adhesion and migration of osteoclasts via integrin αv β 3 /Rho A/Cytoskeleton signaling.

Integrin αv β3 is a transmembrane integrin, which can initiate osteoclasts' attachment on bones, leading to downward signaling pathways and subsequent bone resorption. Different calcium concentrations have been reported to have an influence on the activation of integrin αv β3 . To elucidate the regulatory mechanism of extracellular calcium concentrations on osteoclasts, a controlled micro flow plate (M04S) was utilized in the ONIX flow control system to observe the osteoclasts' adhesion and migration in different calcium concentration media. Fluorescent staining is conducted to show the distribution of integrin αv β3 and cytoskeleton reorganization. In addition, western blots were performed to detect the expression of integrin αv β3 and its downstream signaling pathways related to bone resorption. Also, real-time reverse-transcription polymerase chain reaction data of transcription co-activator (YAP/TAZ) and hydrolytic enzymes (the matrix metalloproteinase 9 and cathepsin K) areevaluated. Our findings suggest that osteoclasts' migration and adhesion is better promoted at 0.5 mM than 1.2 mM, which can be partly explained by the induced cytoskeleton organization via integrin αv β3 /Rho GTPase. But the activation and nuclear localization of YAP/TAZ, and the secretion of hydrolytic enzymes were upregulated when the calcium concentration is at a higher level (1.2 mM). According to our study, there is a highpossibility that the migration and attachment of osteoclasts and subsequent osteoclastic bone resorption are regulated over a specific range of extracellular calcium concentration.

Effects of miR-26a/miR-146a/miR-31 on airway inflammation of asthma mice and asthma children.

This study detected the expressions of microRNA-26a (miR-26a), miR-146a and miR-31 in lung tissues and BALF (bronchoalveolar lavage fluid) of asthma mice and children. Besides, cytokine levels of interleukin-5 (IL-5), IL-8, IL-12 and tumor necrosis factor-α (TNF-α) were detected as well. We aim to provide an experimental basis for clinical treatment of asthma. Forty female BALB/c mice were randomly assigned into control group and asthma group, respectively. Mice in asthma group (n=20) were immunized by intraperitoneal injection of OVA (ovalbumin) and provoked by atomization inhalation of OVA from the 15th day for 10 days. Mice in control group (n=20) were immunized and provoked with isodose saline during the same period. At the 26th day, mice were sacrificed for collecting lung tissues and BALF. Besides, we enrolled 17 cases of asthma children and 13 cases of children with airway foreign body as controls. BALF of each subject was collected. Total cellular score and differential counting in BALF were recorded. Expression levels of miR-26a, miR-146a, and miR-31 were detected by reverse transcription-polymerase chain reaction (RT-PCR). Levels of IL-5, IL-8, IL-12, and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). The total cellular score in BALF of asthma mice and asthma children was higher than that of controls (p<0.05). Percentages of eosinophils, neutrophils, and lymphocytes in BALF of asthma mice and asthma children were higher than those of controls, whereas the percentage of macrophages was lower (p<0.05). Levels of IL-5, IL-8, IL-12, and TNF-α in lung tissues of asthma mice were markedly elevated compared with those of controls (p<0.05). Similarly, levels of IL-5, IL-8, IL-12, and TNF-α were higher in BALF of asthma children than controls (p<0.05). RT-PCR data showed higher mRNA levels of miR-26a, miR-146a, and miR-31 in lung tissues of asthma mice than controls (p<0.05). The mRNA levels of miR-26a, miR-146a, and miR-31 in BALF of asthma children were highly expressed compared with those of controls as well (p<0.05). MiR-26a, miR-146a, and miR-31 are involved in asthma progression mainly through regulating inflammatory factors and cells.

Multilocus Characterization, Gene Expression Analysis of Putative Immunodominant Protein Coding Regions, and Development of Recombinase Polymerase Amplification Assay for Detection of 'Candidatus Phytoplasma Pruni' in Prunus avium.

Western X (WX) disease, caused by 'Candidatus Phytoplasma pruni', is a devastating disease of sweet cherry resulting in the production of small, bitter-flavored fruits that are unmarketable. Escalation of WX disease in Washington State prompted the development of a rapid detection assay based on recombinase polymerase amplification (RPA) to facilitate timely removal and replacement of diseased trees. Here, we report on a reliable RPA assay targeting putative immunodominant protein coding regions that showed comparable sensitivity to polymerase chain reaction (PCR) in detecting 'Ca. Phytoplasma pruni' from crude sap of sweet cherry tissues. Apart from the predominant strain of 'Ca. Phytoplasma pruni', the RPA assay also detected a novel strain of phytoplasma from several WX-affected trees. Multilocus sequence analyses using the immunodominant protein A (idpA), imp, rpoE, secY, and 16S ribosomal RNA regions from several 'Ca. Phytoplasma pruni' isolates from WX-affected trees showed that this novel phytoplasma strain represents a new subgroup within the 16SrIII group. Examination of high-throughput sequencing data from total RNA of WX-affected trees revealed that the imp coding region is highly expressed, and as supported by quantitative reverse transcription PCR data, it showed higher RNA transcript levels than the previously proposed idpA coding region of 'Ca. Phytoplasma pruni'.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells.

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

Role of Annexin A2 isoform 2 on the aggregative growth of dermal papillae cells.

The dermal papilla is a major component of hair, which signals the follicular epithelial cells to prolong the hair growth process. Human Annexin A2 was preliminarily identified by two-dimensional gel electrophoresis (2-DE), MALDI-TOF-MS and database searching. The aim of the present study was to explore the role of Annexin A2 in the aggregative growth of dermal papillae cells (DPC). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were adopted to detect the expression of Annexin A2. And siRNA technique was used to suppress the expression of Annexin A2. Construction of over-expression vector was used to up-regulate the expression of Annexin A2. Cell Counting Kit 8 (CCK-8) and proliferating cell nuclear antigen (PCNA) were taken to detect the proliferation of DPC. The expression of Annexin A2 mRNA was up-regulated in passage 3 DPC compared with passage 10 DPC by RT-PCR. In line with the results at the mRNA level, Western blot analysis revealed that Annexin A2 isoform 2 was up-regulated significantly in passage 3 DPC compared with passage 10 DPC. The Annexin A2 isoform 2 siRNA was synthesized and transfected into passage 3 DPC. RT-PCR data showed the mRNA expression of Annexin A2 isoform 2 was suppressed in passage 3 DPC. Western blot results showed the expression level of Annexin A2 isoform 2 and PCNA were suppressed in passage 3 DPC. CCK-8 results showed that the proliferation of passage 3 DPC was suppressed (P < 0.05). Recombinant plasmid PLJM-Annexin A2 isoform 2-expression vector were constructed and were transfected into passage 10 DPC. RT-PCR data showed the mRNA expression of Annexin A2 isoform 2 was up-regulated in passage 10 DPC. Western blot results showed the expression level of annexin A2 isoform 2 and PCNA were up-regulated in passage 10 DPC. CCK-8 assay showed the proliferation of DPC was stimulated compared with control group (*P < 0.05). Our study proved that Annexin A2 isoform 2 may participate in regulating the proliferation of DPC and may be related to aggregative growth of dermal papilla cells. Therefore, our study suggests that Annexin A2 may be linked to hair follicle growth cycle.