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Background: Wnt signaling has been linked with P-glycoprotein (P-gp) overexpression and which was mainly mediated by β-catenin nuclear translocation. Flavonoids have already been reported as modulators of the Wnt/β-catenin pathway and hence they may serve as promising agents in the reversal of P-gp mediated cancer multi drug resistance (MDR).Methods: In this study, we screened selected flavonoids against Wnt/β-catenin signaling molecules. The binding interaction of flavonoids (theaflavin, quercetin, rutin, epicatechin 3 gallate and tamarixetin) with GSK 3β was determined by molecular docking. Flavonoids on P-gp expression and the components of Wnt signaling in drug-resistant KBCHR8-5 cells were analyzed by western blotting and qRT-PCR. The MDR reversal potential of these selected flavonoids against P-gp mediated drug resistance was analyzed by cytotoxicity assay in KBCHR8-5 and MCF7/ADR cell lines. The chemosensitizing potential of flavonoids was further analyzed by observing cell cycle arrest in KBCHR8-5 cells.Results: In this study, we observed that the components of Wnt/β-catenin pathway such as Wnt and GSK 3β were activated in multidrug resistant KBCHR8-5 cell lines. All the flavonoids selected in this study significantly decreased the expression of Wnt and GSK 3β in KBCHR8-5 cells and subsequently modulates P-gp overexpression in this drug-resistant cell line. Further, we observed that these flavonoids considerably decreased the doxorubicin resistance in KBCHR8-5 and MCF7/ADR cell lines. The MDR reversal potential of flavonoids were found to be in the order of theaflavin > quercetin > rutin > epicatechin 3 gallate > tamarixetin. Moreover, we observed that flavonoids pretreatment significantly induced the doxorubicin-mediated arrest at the phase of G2/M. Further, the combinations of doxorubicin with flavonoids significantly modulate the expression of drug response genes in KBCHR8-5 cells.Conclusion: The present findings illustrate that the studied flavonoids significantly enhances doxorubicin-mediated cell death through modulating P-gp expression pattern by targeting Wnt/β-catenin signaling in drug-resistant KBCHR8-5 cells. [ABSTRACT FROM AUTHOR]

Background: Mosquitoes are responsible for transmission of viruses, including dengue, West Nile and chikungunya viruses. Female mosquitoes are infected when they blood-feed on vertebrates, a required step for oogenesis. During this process, mosquitoes encounter high iron loads. Since iron is an essential nutrient for most organisms, including pathogens, one of the defense mechanisms for the host includes sequestration of iron away from the invading pathogen. Here, we determine whether iron availability affects viral replication in mosquitoes. Methods: To elucidate effect of iron availability on mosquito cells during infection, Culex cells were treated with either ferric ammonium citrate (FAC) or the iron chelator, deferoxamine (DFX). Real time RT-PCR was performed using ferritin (heavy chain) and NRAMP as a measure of iron homeostasis in cells. To determine iron requirement for viral replication, Culex cells were knocked down for NRAMP using dsRNA. Finally, the results were validated in Culex mosquito-infection model, by treating infected mosquitoes with DFX to reduce iron levels. Results: Our results show that infection of Culex cells led to induction in levels of ferritin (heavy chain) and NRAMP mRNAs in time-dependent manner. Results also showed that treatment of cells with FAC, reduced expression of NRAMP (iron transporter) and increase levels of ferritin (heavy chain). Interestingly, increasing iron levels increased viral titers; while reducing intracellular iron levels, either by NRAMP knock-down or using DFX, reduced viral titers. The results from Culex mosquito infection showed that mosquitoes treated with DFX had reduced viral titers compared with untreated controls in midgut as well as carcass 8 days pi. Saliva from mosquitoes treated with DFX also showed reduced viral titers compared with untreated controls, indicating low viral transmission capacity. Conclusions: Our results indicate that iron is required for viral replication in mosquito cells. Mosquitoes respond to viral infection, by inducing expression of heavy chain ferritin, which sequesters available iron, reducing its availability to virus infected cells. The data indicates that heavy chain ferritin may be part of an immune mechanism of mosquitoes in response to viral infections. [ABSTRACT FROM AUTHOR]

An abstract of the article "The effect of a standardized massage application on spinal stiffness in asymptomatic subjects," by G. N. Kawchuk, N. Prasad, R. Chamberlain, A. Klymkiv, and L. Peter is presented.

Background: Indacaterol is a novel, once-daily (o.d.) inhaled, long-acting beta2-agonist in development for chronic obstructive pulmonary disease (COPD). This 12-week, double-blind study compared the efficacy, safety, and tolerability of indacaterol to that of placebo in patients with moderate-to-severe COPD.Methods: Efficacy variables included 24-h trough FEV1 (mean of 23 h 10 min and 23 h 45 min post-dose) at Week 12 (primary endpoint) and after Day 1, and the percentage of COPD days with poor control (i.e., worsening symptoms). Safety was assessed by adverse events (AEs), mean serum potassium and blood glucose, QTc (Fridericia), and vital signs.Results: Patients were randomised (n = 416, mean age 63 years) to receive either indacaterol 150 microg o.d. (n = 211) or placebo (n = 205) via a single-dose dry-powder inhaler; 87.5% completed the study. Trough FEV1 (LSM +/- SEM) at Week 12 was 1.48 +/- 0.018 L for indacaterol and 1.35 +/- 0.019 L for placebo, a clinically relevant difference of 130 +/- 24 mL (p < 0.001). Trough FEV1 after one dose was significantly higher with indacaterol than placebo (p < 0.001). Indacaterol demonstrated significantly higher peak FEV1 than placebo, both on Day 1 and at Week 12, with indacaterol-placebo differences (LSM +/- SEM) of 190 +/- 28 (p < 0.001) and 160 +/- 28 mL (p < 0.001), respectively. Standardised AUC measurements for FEV1 (between 5 min and 4 h, 5 min and 1 h, and 1 and 4 h post-dose) at Week 12 were all significantly greater with indacaterol than placebo (p < 0.001), with LSM (+/- SEM) differences of 170 +/- 24, 180 +/- 24, and 170 +/- 24 mL, respectively. Indacaterol significantly reduced the percentage of days of poor control versus placebo by 22.5% (p < 0.001) and was also associated with significantly reduced use of rescue medication (p < 0.001). The overall rates of AEs were comparable between the groups (indacaterol 49.3%, placebo 46.8%), with the most common AEs being COPD worsening (indacaterol 8.5%, placebo 12.2%) and cough (indacaterol 6.2%, placebo 7.3%). One patient died in the placebo group. Serum potassium and blood glucose levels did not differ significantly between the two groups, and no patient had QTc >500 ms.Conclusions: Indacaterol 150 microg o.d. provided clinically significant and sustained bronchodilation, reduced rescue medication use, and had a safety and tolerability profile similar to placebo.Trial Registration: NCT00624286. [ABSTRACT FROM AUTHOR]

Background: Early initiation of physical therapy (PT) has been associated with lower healthcare costs and utilization; however, these studies have been limited to single institutions or healthcare systems. Our goal was to assess healthcare utilization and spending among patients who present for the first time with low back pain (LBP), according to whether they received early physical therapy (PT), using a large, nationwide sample; and geographic variation in rates of early PT and 30-day LBP-related spending., Methods: Using the Truven MarketScan database, we identified nearly 980,000 US adults ages 18-64 years who initially presented with acute LBP from 2010 through 2014 and did not have nonmusculoskeletal causes of LBP. Approximately 110,000 patients (11%) received early PT (≤2 weeks after presentation). We compared healthcare utilization and spending at 30 days and 1 year after presentation between patients who received early PT and those who did not. Alpha = 0.05., Results: At 30 days, early PT was associated with lower odds of chiropractor visits (odds ratio [OR] = 0.41, 95% confidence interval [CI] = 0.40-0.42), pain specialist visits (OR = 0.49, 95% CI = 0.47-0.51), emergency department visits (OR = 0.51, 95% CI = 0.49-0.54), advanced imaging (OR = 0.57, 95% CI = 0.56-0.58), orthopaedist visits (OR = 0.67, 95% CI = 0.66-0.69), and epidural steroid injections (OR = 0.68, 95% CI = 0.65-0.70). At 1 year, early PT was associated with less healthcare utilization. At 30 days, patients with early PT had lower mean LBP-related spending ($1180 ± $1500) compared with those without early PT ($1250 ± $2560) (P < 0.001). At 1 year, LBP-related spending was significantly less among patients who did not receive early PT ($2510 ± $3826) versus those who did ($2588 ± $3704). Early PT rates (range, 4-25%; P < 0.001) and 30-day LBP-related spending differed by state (range, $421 to -$410; P < 0.001)., Conclusion: Early PT for acute LBP was associated with less 30-day and 1-year healthcare utilization and less 30-day LBP-related spending. Early PT rates and 30-day spending differed by US state., Level of Evidence: IV., (© 2022. The Author(s).)

Background: In clear cell renal cell carcinoma, 80% of cases have biallelic inactivation of the VHL gene, leading to constitutive activation of both HIF1α and HIF2α. As HIF2α is the driver of the disease promoting tumour growth and metastasis, drugs targeting HIF2α have been developed. However, resistance is common, therefore new therapies are needed., Methods: We assessed the effect of the HIF2α antagonist PT2385 in several steps of tumour development and performed RNAseq to identify genes differentially expressed upon treatment. A drug screening was used to identify drugs with antiproliferative effects on VHL-mutated HIF2α-expressing cells and could increase effectiveness of PT2385., Results: PT2385 did not reduce cell proliferation or clonogenicity but, in contrast to the genetic silencing of HIF2α, it reduced in vitro cell invasion. Many HIF-inducible genes were down-regulated upon PT2385 treatment, whereas some genes involved in cell migration or extracellular matrix were up-regulated. HIF2α was associated with resistance to statins, addition to PT2385 did not increase the sensitivity., Conclusions: this study shows key differences between inhibiting a target versus knockdown, which are potentially targetable., (© 2021. The Author(s).)

Background: Breast (mammary) cancers in human (BC) and canine (CMT) patients share clinical, pathological, and molecular similarities that suggest dogs may be a useful translational model. Many cancers, including BC, shed exosomes that contain microRNAs (miRs) into the microenvironment and circulation, and these may represent biomarkers of metastasis and tumor phenotype., Methods: Three normal canine mammary epithelial cell (CMEC) cultures and 5 CMT cell lines were grown in serum-free media. Exosomes were isolated from culture media by ultracentrifugation then profiled by transmission electron microscopy, dynamic light scattering, and Western blot. Exosomal small RNA was deep-sequenced on an Illumina HiSeq2500 sequencer and validated by qRT-PCR. In silico bioinformatic analysis was carried out to determine microRNA gene and pathway targets., Results: CMEC and CMT cell lines shed round, "cup-shaped" exosomes approximately 150-200 nm, and were immunopositive for exosomal marker CD9. Deep-sequencing averaged ~ 15 million reads/sample. Three hundred thirty-eight unique miRs were detected, with 145 having > ±1.5-fold difference between one or more CMT and CMEC samples. Gene ontology analysis revealed that the upregulated miRs in this exosomal population regulate a number of relevant oncogenic networks. Several miRNAs including miR-18a, miR-19a and miR-181a were predicted in silico to target the canine estrogen receptor (ESR1α)., Conclusions: CMEC and CMT cells shed exosomes in vitro that contain differentially expressed miRs. CMT exosomal RNA expresses a limited number of miRs that are up-regulated relative to CMEC, and these are predicted to target biologically relevant hormone receptors and oncogenic pathways. These results may inform future studies of circulating exosomes and the utility of miRs as biomarkers of breast cancer in women and dogs.

Background: Splenic masses are common in older dogs; yet diagnosis preceding splenectomy and histopathology remains elusive. MicroRNAs (miRNAs) are short, non-coding RNAs that play a role in post-transcriptional regulation, and differential expression of miRNAs between normal and tumor tissue has been used to diagnose neoplastic diseases. The objective of this study was to determine differential expression of miRNAs by use of RNA-sequencing in canine spleens that were histologically confirmed as hemangiosarcoma, nodular hyperplasia, or normal., Results: Twenty-two miRNAs were found to be differentially expressed in hemangiosarcoma samples (4 between hemangiosarcoma and both nodular hyperplasia and normal spleen and 18 between hemangiosarcoma and normal spleen only). In particular, mir-26a, mir-126, mir-139, mir-140, mir-150, mir-203, mir-424, mir-503, mir-505, mir-542, mir-30e, mir-33b, mir-365, mir-758, mir-22, and mir-452 are of interest in the pathogenesis of hemangiosarcoma., Conclusions: Findings of this study confirm the hypothesis that miRNA expression profiles are different between canine splenic hemangiosarcoma, nodular hyperplasia, and normal spleens. A large portion of the differentially expressed miRNAs have roles in angiogenesis, with an additional group of miRNAs being dysregulated in vascular disease processes. Two other miRNAs have been implicated in cancer pathways such as PTEN and cell cycle checkpoints. The finding of multiple miRNAs with roles in angiogenesis and vascular disease is important, as hemangiosarcoma is a tumor of endothelial cells, which are driven by angiogenic stimuli. This study shows that miRNA dysregulation is a potential player in the pathogenesis of canine splenic hemangiosarcoma.

Background: Widespread in the tropics, the mosquito Aedes aegypti is an important vector of many viruses, posing a significant threat to human health. Vector monitoring often requires fecundity estimation by counting eggs laid by female mosquitoes. Traditionally, manual data analyses have been used but this requires a lot of effort and is the methods are prone to errors. An easy tool to assess the number of eggs laid would facilitate experimentation and vector control operations., Results: This study introduces a built-in software called ICount allowing automatic egg counting of the mosquito vector, Aedes aegypti. ICount egg estimation compared to manual counting is statistically equivalent, making the software effective for automatic and semi-automatic data analysis. This technique also allows rapid analysis compared to manual methods. Finally, the software has been used to assess p-cresol oviposition choices under laboratory conditions in order to test the system with different egg densities., Conclusions: ICount is a powerful tool for fast and precise egg count analysis, freeing experimenters from manual data processing. Software access is free and its user-friendly interface allows easy use by non-experts. Its efficiency has been tested in our laboratory with oviposition dual choices of Aedes aegypti females. The next step will be the development of a mobile application, based on the ICount platform, for vector monitoring surveys in the field.

Background: Malignant glioma is one of the most devastating tumors in adults with poor patient prognosis. Notably, glioma often exhibits resistance to conventional chemotherapeutic approaches, complicating patient treatments. However, the molecular mediators involved in tumor chemoresistance remain poorly defined, creating a barrier to the successful management of glioma. In the present study, we hypothesized that the antioxidant transcription factor, Nrf2 (nuclear factor erythroid-derived 2 like 2), attenuates glioma cytotoxicity to Carmustine (BCNU), a widely used chemotherapeutic agent known to modulate cellular oxidative balance., Methods: To test the hypothesis, we employed human malignant glioma cell line, U87MG and overexpression of Nrf2 in glioma cells was achieved using both pharmacological and genetic approaches., Results: Notably, induction of Nrf2 was associated with increased expression of heme oxygenase-1 (HO-1), a stress inducible enzyme involved in anti-oxidant defense. In addition, over expression of Nrf2 in U87MG cells significantly attenuated the cytotoxicity of Carmustine as evidenced by both cellular viability assay and flow cytometry analysis. Consistent with this, antioxidants such as glutathione and N-acetyl cysteine significantly reduced Carmustine mediated glioma cytotoxicity., Conclusions: Taken together, these data strongly implicate an unexplored role of Nrf2 in glioma resistance to Carmustine and raise the possible use of Nrf2 inhibitors as adjunct to Carmustine for the treatment of malignant glioma.

Background: As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors., Results: Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number., Conclusions: The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.

Background: Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol., Results and Conclusion: Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

Background: The main cause of relapse in smokers attempting to quit is inability to resist urges to smoke. Pharmacotherapy ameliorates but does not entirely prevent urges to smoke when abstinent, so other methods to resist urges to smoke might be helpful. Exercise is effective, but aerobic exercise is often impractical when urges strike. Two techniques, body scan and isometric exercise, have been shown to reduce urge intensity and nicotine withdrawal symptoms in temporarily abstinent smokers. It is unclear whether they would be used or effective in typical smokers attempting to quit., Methods: In a pilot trial set in a UK smoking cessation clinic, 20 smokers were randomised to receive emails containing .mp3 files and .pdf illustrations of the instructions for doing the body scan and isometric exercises. Twenty smokers received no other intervention, although all 40 were receiving weekly behavioural support and nicotine replacement therapy. Carbon monoxide confirmed abstinence, nicotine withdrawal symptoms, urges to smoke, and use of the techniques to resist urges were recorded weekly for four weeks after quit day., Results: 60-80% of quitters reported using the isometric exercises each week and 40-70% reported using the body scan to deal with urges. On average, these techniques were rated as 'slightly helpful' for controlling the urges. There were no large or significant differences in withdrawal symptoms or urge intensity between the two groups. The risk ratio and 95% confidence interval for exercises compared with controls for prolonged confirmed abstinence at four weeks was 0.82 (0.44-1.53). 81% of quitters intended to continue using isometric exercises and 25% body scan, while 81% and 50% respectively would recommend using these techniques to others trying to stop., Conclusion: Isometric exercises, and to a lesser extent body scan, were popular and perceived as somewhat helpful by quitters. The trial showed that these techniques were used and a larger trial could now be developed to examine the influence of the methods on reducing urges to smoke and increasing abstinence.