Dual Assay Research Articles

The miR-216/miR-217 Cluster Regulates Lipid Metabolism in Laying Hens With Fatty Liver Syndrome via PPAR/SREBP Signaling Pathway.

Fatty liver syndrome (FLS), a common metabolic disease in laying hens, caused by excessive hepatic fat deposition is a bottleneck in the poultry industry. However, no specific therapeutic methods have been developed. Evidence suggests that microRNAs (miRNAs) are essential for liver lipid metabolism and homeostasis, providing strong evidence for targeting miRNAs as a potential treatment option for liver diseases. However, the roles of miRNAs in the pathogenesis of FLS remain unclear. In present study, RNA-sequencing was performed to discern the expression patterns of miRNAs in normal and fatty livers of laying hens. In total, 12 dysregulated miRNAs (2 down-regulated and 10 up-regulated) were detected between the normal and fatty livers. Functional enrichment analysis showed the potential impacts of the dysregulated miRNAs on lipid metabolism. Notably, miR-216a/b and miR-217-5p, which belong to the miR-216/miR-217 cluster, were up-regulated in the sera and livers of FLS chickens, as well as free fatty acid (FFA)-induced LMH cells. Oil-red O staining revealed that up-regulation of the miR-216/miR-217 cluster induced lipid accumulation in FFA-induced LMH cells. Furthermore, the dual luciferase gene reporter assay and RT-qPCR analysis demonstrated that 3-hydroxyacyl-CoA dehydratase 2, F-box protein 8, and transmembrane 9 superfamily member 3 (TM9SF3) were directly targeted by miR-216a/b and miR-217-5p, respectively, and suppressed in the fatty livers of laying hens. Moreover, overexpression of the miR-216/miR-217 cluster or reduction in TM9SF3 levels led to activation of the proliferator-activated receptor/sterol regulatory-element binding protein (PPAR/SREBP) pathway. Overall, these results demonstrate that the miR-216/miR-217 cluster regulates lipid metabolism in laying hens with FLS, which should prove helpful in the development of new interventional strategies.

Novel in vitro methods for simultaneous screening of two antagonistic bacteria against multiple fungal phytopathogens in a single agar plate.

Two novel and rapid in vitro agar plate methods for simultaneous screening of two bacterial isolates for antagonism against multiple fungal phytopathogens in a single agar plate are described. In the modified plus streak method (MPS), in a 10cm diameter PDA agar plate, two different bacterial isolates were screened against four fungal pathogens. The second method used 20cm diameter PDA agar plate surface and was termed as the parallel multiple streak (PMS) method. Here, two bacterial isolates were screened for antagonism against ten fungal phytopathogens on a single agar plate surface. Our new methods are rapid and more economical, and could reduce the number of agar plates and the medium required for in vitro screening by several fold, compared to that used in the conventional dual culture plate assay.

Transcriptome Analysis Reveals Key miRNA–mRNA Pathways in Ovarian Tissues of Yunshang Black Goats With Different Kidding Numbers

The granulosa cell (GC) is the basic functional unit of follicles, and it is important for promoting follicle growth and sex hormones, as well as growth factor secretion in the process of reproduction. A variety of factors influence granulocyte proliferation, yet there are still many gaps to be filled in target and non-coding RNA regulation. In our study, the differentially expressed (DE) mRNAs and miRNAs were detected by using RNA-seq, and we constructed a mRNA–miRNA network related to goat prolificacy. Then, the goat primary GCs were isolated from the follicle for the function validation of candidate genes and their regulator miRNAs. A total of 2,968 DE mRNAs and 99 DE miRNAs were identified in the high- and low-prolificacy goat by RNA-seq, of which there were 1,553 upregulated and 1,415 downregulated mRNAs, and 80 upregulated and 19 downregulated miRNAs, respectively. JAK3 was identified as highly expressed in the low-prolificacy goats (3 times higher than high-prolificacy goats), and the integrated analysis showed that chi-miR-493-3p was a potential regulator of JAK3. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that JAK3 was involved in the PI3K-Akt signaling pathway, the Jak-STAT signaling pathway, and signaling pathways regulating pluripotency of stem cells. In particular, the PI3K-Akt signaling pathway was a typical pathway for cell proliferation, differentiation, apoptosis, and migration. We found that the chi-miR-493-3p targets JAK3 directly via RT-qPCR, dual fluorescence assays, and Western blot. Furthermore, the expression of JAK3 was significantly decreased by the chi-miR-493-3p mimic and increased by the chi-miR-493-3p inhibitor. The CCK-8 assay showed that overexpression of JAK3 promoted cell proliferation, while inhibiting JAK3 had the opposite effect. The expression of cell proliferation markers CDK4 and cyclin D2 also showed the same results. Moreover, the enzyme-linked immunosorbent assay showed that steroid hormones E2 and PROG were increased by overexpressing JAK3 and decreased by inhibiting JAK3. Therefore, our results identified a chi-miR-439-3p-JAK3 regulatory pathway, which provided a new insight into the GC proliferation and prolificacy of goat.

Enzyme-free ratiometric fluorescence and colorimetric dual read-out assay for glyphosate with ultrathin g-C3N4 nanosheets

Herein, a sensing platform for the rapid quantitative detection of glyphosate with high sensitivity and selectivity was constructed by enzyme-free ratiometric fluorescence and colorimetric dual read-out assay based on ultrathin g-C3N4 nanosheets (UCN NS).Fluorescent diaminophenylazine (DAP) with fluorescence was from the oxidation of o-phenylenediamine (OPD) in the presence of Cu2+. DAP could inspire a significant fluorescence quenching of nanosheets due to photo-induced electron transfer (PET), therefore exhibiting a ratiometric fluorescence response. In the presence of glyphosate, the strong chelation of Cu2+ by glyphosate inhibited the oxidation of OPD, which reduced the fluorescence intensity of DAP, while the fluorescence of the ultrathin nanosheets was restored. Moreover, the oxidation of OPD (colorless) to DAP (yellow) was a distinctly visible color change process, which provides well-suited conditions for colorimetric analysis. At the optimum experimental conditions, the detection concentration range of glyphosate was 0.001–0.48 μg/mL, and the limit of fluorometric detection was 0.49 ng/mL (S/N = 3). This assay was simple and easy to use for the detection of glyphosate in food safety and the environment with satisfactory results.

Cytotoxic Activity of Aquilaria malaccensis Ethanolic Leaves Extract on Human Chronic Myeloid Leukaemia K-562 Cell Line

Aquilaria malaccensis, also known as “Pokok Karas” in Malaysia, has various benefits and widely used in Chinese herbal medicines. Natural products such as medicinal plant have been found to be effective anti-cancer agents, killing cancer cells with fewer side effects than chemotherapy. Therefore, the purpose of this study was to evaluate the cytotoxic activities of ethanolic extract of A. malaccensis on the human chronic myeloid leukaemia K-562 cell line. In this study, cytotoxic activities of ethanolic extract of A. malaccensis leaves on K-562 cells was determined by using (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) assay. K-562 cells were treated with 70% ethanolic extracts and incubated for 24, 48 and 72 hours. The half-maximal inhibitory concentration (IC50) of ethanolic extract of A. malaccensis leaves was observed at 72 hours of incubation; 90 µg/mL for 70% ethanolic extract. Moreover, the morphological changes of K-562 cells were observed by using acridine orange/ propidium iodide (AO/PI) dual staining assay. The results showed that the ethanolic extract of A. malaccensis leaves induced K-562 cells into apoptotic cell death mode manner. This finding suggested that the ethanolic extract of A. malaccensis has the potential to become an anti-cancer agent for chronic myeloid leukaemia K-562 cell line.

The transcription factor TaMYB31 regulates the benzoxazinoid biosynthetic pathway in wheat.

Benzoxazinoids are specialized metabolites that are highly abundant in staple crops, such as maize and wheat. Although their biosynthesis has been studied for several decades, the regulatory mechanisms of the benzoxazinoid pathway remain unknown. Here, we report that the wheat transcription factor MYB31 functions as a regulator of benzoxazinoid biosynthesis genes. A transcriptomic analysis of tetraploid wheat (Triticum turgidum) tissue revealed the up-regulation of two TtMYB31 homoeologous genes upon aphid and caterpillar feeding. TaMYB31 gene silencing in the hexaploid wheat Triticum aestivum significantly reduced benzoxazinoid metabolite levels and led to susceptibility to herbivores. Thus, aphid progeny production, caterpillar body weight gain, and spider mite oviposition significantly increased in TaMYB31-silenced plants. A comprehensive transcriptomic analysis of hexaploid wheat revealed that the TaMYB31 gene is co-expressed with the target benzoxazinoid-encoded Bx genes under several biotic and environmental conditions. Therefore, we analyzed the effect of abiotic stresses on benzoxazinoid levels and discovered a strong accumulation of these compounds in the leaves. The results of a dual fluorescence assay indicated that TaMYB31 binds to the Bx1 and Bx4 gene promoters, thereby activating the transcription of genes involved in the benzoxazinoid pathway. Our finding is the first report of the transcriptional regulation mechanism of the benzoxazinoid pathway in wheat.

YTHDF1 alleviates sepsis by upregulating WWP1 to induce NLRP3 ubiquitination and inhibit caspase-1-dependent pyroptosis

Pyroptosis is inflammation-associated caspase-1-dependent programmed cell death, which confers a crucial role in sepsis. The present study intends to investigate the regulatory network and function of the microarray-predicted YTHDF1 in caspase-1-dependent pyroptosis of sepsis. Peripheral blood of patients with sepsis was collected to determine WWP1 and YTHDF1 expression. An in vitro sepsis cell model was induced in RAW264.7 cells using lipopolysaccharide (LPS) and ATP and an in vivo septic mouse model by cecal ligation and perforation (CLP). After gain- and loss-of-function assays in vitro and in vivo, TNF-α and IL-1β levels and the cleavage of gasdermin-D (GSDMD) were detected by ELISA and Western blot assay, followed by determination of lactate dehydrogenase (LDH) activity. Immunoprecipitation and meRIP assay were performed to detect the ubiquitination of NLRP3 and the m6A modification of WWP1 mRNA. The binding of WWP1 to YTHDF1 was explored using RIP-RT-qPCR and dual luciferase gene reporter assay. It was noted that WWP1 and YTHDF1 were downregulated in clinical sepsis samples, LPS + ATP-treated RAW264.7 cells, and CLP-induced mice. The ubiquitination of NLRP3 was promoted after overexpression of WWP1. WWP1 translation could be promoted by YTHDF1. Then, WWP1 or YTHDF1 overexpression diminished LDH activity, NLRP3 inflammasomes and caspase-1-mediated cleavage of GSDMD in LPS + ATP-induced RAW264.7 cells. Overexpressed YTHDF1 restrained inflammatory response in CLP-induced mice. Collectively, the alleviatory effect of m6A reader protein YTHDF1 may be achieved through promotion of NLRP3 ubiquitination and inhibition of caspase-1-dependent pyroptosis by upregulating WWP1.

Combined detection of molecular and serological signatures of viral infections: The dual assay concept

The recent worldwide spread of viral infections has highlighted the need for accurate, fast, and inexpensive disease diagnosis and monitorization methods. Current diagnostics tend to focus either on molecular or serological testing. In this work we propose a dual detection assay approach for viral diseases, where both serological and molecular assays are combined in a single analysis performed on a magnetoresistive system. This type of assay guarantees an accurate assessment of the infection phase, saving time and costs associated with multiple independent tests. Zika and dengue viruses were used as model diseases for the validation of the system. Human IgG anti-zika and anti-dengue antibodies were successfully detected in infected patients’ serum, using a novel approach combining competitive and sandwich strategies in a magnetoresistive portable platform. Specificity and sensitivity values of 100% were obtained. Calibration curves with dynamic ranges between 10 ng/mL and 1 μg/mL were established achieving LODs of 1.26 and 1.38 nM for IgG anti-ZIKV and anti-DENV antibodies, respectively. Viral RNA detection down to a few hundreds of pM was also successfully carried out after the design of specific oligo probes and primers for RT-PCR amplification. Dual assays were performed for both viruses, where viral RNA and anti-virus antibodies in serum samples were simultaneously detected. The results obtained for the detection of the molecular and serological targets in the dual assay format show no significant difference between the ones obtained individually, proving the feasibility and accuracy of the dual detection assay. This assay format represents a new paradigm in viral infections diagnostics.

Functional and Structural Insights into Human PPARα/δ/γ Subtype Selectivity of Bezafibrate, Fenofibric Acid, and Pemafibrate.

Among the agonists against three peroxisome proliferator-activated receptor (PPAR) subtypes, those against PPARα (fibrates) and PPARγ (glitazones) are currently used to treat dyslipidemia and type 2 diabetes, respectively, whereas PPARδ agonists are expected to be the next-generation metabolic disease drug. In addition, some dual/pan PPAR agonists are currently being investigated via clinical trials as one of the first curative drugs against nonalcoholic fatty liver disease (NAFLD). Because PPARα/δ/γ share considerable amino acid identity and three-dimensional structures, especially in ligand-binding domains (LBDs), clinically approved fibrates, such as bezafibrate, fenofibric acid, and pemafibrate, could also act on PPARδ/γ when used as anti-NAFLD drugs. Therefore, this study examined their PPARα/δ/γ selectivity using three independent assays—a dual luciferase-based GAL4 transactivation assay for COS-7 cells, time-resolved fluorescence resonance energy transfer-based coactivator recruitment assay, and circular dichroism spectroscopy-based thermostability assay. Although the efficacy and efficiency highly varied between agonists, assay types, and PPAR subtypes, the three fibrates, except fenofibric acid that did not affect PPARδ-mediated transactivation and coactivator recruitment, activated all PPAR subtypes in those assays. Furthermore, we aimed to obtain cocrystal structures of PPARδ/γ-LBD and the three fibrates via X-ray diffraction and versatile crystallization methods, which we recently used to obtain 34 structures of PPARα-LBD cocrystallized with 17 ligands, including the fibrates. We herein reveal five novel high-resolution structures of PPARδ/γ–bezafibrate, PPARγ–fenofibric acid, and PPARδ/γ–pemafibrate, thereby providing the molecular basis for their application beyond dyslipidemia treatment.

Bacillus subtilis suppresses the charcoal rot disease by inducing defence responses and physiological attributes in soybean.

Endophytes can induce the defence responses and modulates physiological attributes in host plants during pathogen attacks. In the present study, 127 bacterial endophytes (BEs) were isolated from different parts of healthy soybean plant. Among them, two BEs (M-2 and M-4) resulted a significant antagonistic property against Macrophomina phaseolina, causes charcoal rot disease in soybean. The antagonistic potential was evaluated through dual culture plate assay, where M-4 expressed higher antifungal activity than M-2 against M. phaseolina. The M-4 produces cell wall degrading enzymes viz. cellulase (145.71 ± 1.34 μgmL-1), chitinase (0.168 ± 0.0009 unitmL-1) and β,1-3 endoglucanase (162.14 ± 2.5 μgmL-1), which helps in cell wall disintegration of pathogens. Additionally, M-4 also can produce siderophores, indole-3-acetic acid (IAA) (17.03 ± 1.10 μgmL-1) and had a phosphate solubilization potential (19.89 ± 0.26 μgmL-1). Further, GC-MS profiling of M-4 has been carried out to demonstrate the production of lipophilic secondary metabolites which efficiently suppress the M. phaseolina defensive compounds under co-culture conditions. Bio-efficacy study of M-4 strain shown a significant reduction in disease incidence around 60 and 80% in resistant and susceptible varieties of soybean, respectively. The inoculation of M-4 potentially enhances the physiological attributes and triggers various defence responsive enzymes viz. superoxide dismutase (SOD), phenol peroxidase (PPO), peroxidase (PO) and catalase (CAT). The histopathological study also confirmed that M-4 can reduce the persistence of microsclerotia in root and shoot tissue. Conclusively, M-4 revealed as an efficient biocontrol agent that can uses multifaceted measures for charcoal rot disease management, by suppress the M. phaseolina infection and enhance the physiological attributes of soybean.

Biocontrol Activity of Trichoderma Species Isolated from Grapevines in British Columbia against Botryosphaeria Dieback Fungal Pathogens.

Botryosphaeria dieback (BD) is a grapevine trunk disease (GTD) causing significant yield losses and limiting the lifespan of vineyards worldwide. Fungi responsible for BD infect grapevines primarily through pruning wounds, and thus pruning wound protection, using either synthetic chemicals or biological control agents (BCAs), is the main available management strategy. However, no products to control GTDs are currently registered in Canada. With a focus on more sustainable grapevine production, there is an increasing demand for alternatives to chemical products to manage GTDs. Accordingly, the objective of this study was to identify Trichoderma species from grapevines in British Columbia (BC) and evaluate their potential biocontrol activity against BD fungi Diplodia seriata and Neofusicoccum parvum. Phylogenetic analyses identified seven species, including T. asperelloides, T. atroviride, T. harzianum, T. koningii, T. tomentosum, and two novel species, T. canadense and T. viticola. In vitro dual culture antagonistic assays showed several isolates to inhibit fungal pathogen mycelial growth by up to 75%. In planta detached cane assays under controlled greenhouse conditions identified T. asperelloides, T. atroviride and T. canadense isolates from BC as providing 70% to 100% pruning wound protection against BD fungi for up to 21 days after treatment. In addition, these isolates were shown to provide similar or better control when compared against commercial chemical and biocontrol products. This study demonstrates the potential that locally sourced Trichoderma species can have for pruning wound protection against BD fungi, and further supports the evaluation of these isolates under natural field conditions.

Interaction of Bdellovibrio bacteriovorus with Gram-Negative and Gram-Positive Bacteria in Dual Species and Polymicrobial Communities.

The interaction of Bdellovibrio bacteriovorus PF13 with mixed bacterial communities, consisting of Gram-negative (Pseudomonas fluorescens and Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus and Enterococcus faecium) bacteria, was investigated to determine if this wild-type predator preferentially preys on certain bacteria and whether the presence of Gram-positive organisms influences its predation efficiency. In co-culture with P. fluorescens and K. pneumoniae, the cell counts (PFU/mL) of PF13 increased by 5.79 and 5.17 logs (48 h), respectively, while in the dual species assay (P. fluorescens, K. pneumoniae and PF13), the cell counts of PF13 increased by 1.95 logs (24 h). Using ethidium monoazide bromide quantitative polymerase chain reaction (EMA-qPCR), the concentration of PF13 increased by 1.25 to 3.62 logs in the co-culture experiments, by 1.41 to 5.05 logs in dual species cultures and by 2.65 logs in a polymicrobial culture. However, PF13 preferentially preyed on K. pneumoniae in the dual species and polymicrobial cultures, highlighting that the presence of Gram-positive bacteria did not affect the predation efficiency of PF13. This is significant as it implies that the predator can be applied in mixed microbial communities to target Gram-negative pathogens which may pose a health risk to patients, consumers or for the treatment of contaminated water.

Fungal endophytes in Fraxinus excelsior petioles and their in vitro antagonistic potential against the ash dieback pathogen Hymenoscyphus fraxineus.

Fungal endophytes were isolated from 250 asymptomatic leaf petioles of Fraxinus excelsior collected from trees showing symptoms of ash dieback in five forest sites in southern Poland. Fungal isolations yielded 1646 colonies representing 97 taxa, including 92 Ascomycota and 5 Basidiomycota species. The most common Ascomycota comprised Nemania serpens (38.0 % of colonized petioles), Diaporthe eres (33.6 %), Venturia fraxini (26.4 %), Diaporthe sp. 1 (20.4 %), Alternaria sp. 1 (14.8 %), Colletotrichum acutatum (14.8 %), Nemania diffusa (14.0 %), Colletotrichum gloeosporioides (12.4 %) and Colletotrichum sp. (12.4 %). The occurrence of all these taxa except Alternaria sp. 1 was significantly different between the studied forest sites. Two yeast species, Vishniacozyma foliicola (4.8 %) and Cystobasidium pinicola (2.8 %), dominated among the Basidiomycota endophytes detected. All the fungal endophytes were tested in dual culture antagonistic assays against two strains of Hymenoscyphus fraxineus, resulting in the development of four interaction types. The interactions included the physical contact of co-partners' mycelia (41.8 %), development of an inhibition zone (47.4 %), growth of endophyte mycelia over H. fraxineus colonies (9.3 %) and growth of H. fraxineus mycelia over endophyte colonies (1.5 %). The strongest antibiotic activity against H. fraxineus, measured by the width of the inhibition zone, was observed for Cytospora pruinosa, Fusarium lateritium, Phoma sp. 2, Pleosporales sp. 2 and Thielavia basicola. A variety of morphophysiological deformations of H. fraxineus hyphae were observed under endophyte pressure: spiral twist of the hyphae, formation of cytoplasmic extrusions, development of torulose hyphae and excessive lateral branching of the hyphae. The strongest antagonistic effects, coupled with the potential to overgrow H. fraxineus colonies, was shown by Clonostachys rosea, Nemania diffusa, N. serpens, Peniophora cinerea, Rosellinia corticium and Xylaria polymorpha. Some of these species were able to attack H. fraxineus hyphae in a mycoparasitic manner. The antagonistic activities included the physical penetration of H. fraxineus hyphae, dissolution of hyphal cell walls, disappearance of pigmentation, disintegration of hyphae and degradation of other fungal structures. In contrast, one of the most commonly detected endophytes in ash leaves, Venturia fraxini, did not show in vitro antagonistic potential against H. fraxineus. Finally, we discuss the potential of the detected fungal endophytes to combat H. fraxineus invasion, the cause of ash decline in Europe.

Novel probe-based melting curve assays for the characterization of fluoroquinolone resistance in Mycoplasma genitalium.

Background Mycoplasma genitalium infection is a sexually transmitted infection that has rapidly become resistant to mainstay treatments. While individualized treatment approaches have been recommended and adopted for macrolides, individualized therapy for fluoroquinolones has not yet been explored, due to a lack of commercial molecular assays and a lack of confidence in specific mutations associated with resistance. In another recent study, we defined a clear role and diagnostic utility in focusing on the absence of resistance mutations to inform microbial cure with fluoroquinolone antimicrobials.MethodsWe developed two proof-of-concept molecular tests that focus on detection of M. genitalium and characterization of WT parC sequences that are strongly linked to fluoroquinolone susceptibility.ResultsWe screened a total of 227 M. genitalium-positive samples using novel molecular beacon and dual hybridization probe assays. These assays were able to detect M. genitalium and characterize fluoroquinolone susceptibility in 143/227 (63%) samples, based on clear differences in melting peak temperatures. The results of these molecular assays were in 100% agreement with ‘gold standard’ Sanger sequencing. Additionally, WT parC sequences were readily distinguished from M. genitalium samples harbouring parC mutations of known or suspected clinical significance. The ability of the assays to successfully characterize fluoroquinolone susceptibility and resistance was reduced in low M. genitalium load samples.ConclusionsThese proof-of-concept assays have considerable potential to improve individualized treatment approaches and rationalize tests of cure for M. genitalium infection. The ability to initiate individualized treatment in up to two-thirds of cases will enhance antimicrobial stewardship for this challenging pathogen.

One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.

Thiazolidinedione-Based Structure Modification of Celastrol Provides Thiazolidinedione-Conjugated Derivatives as Potent Agents against Non-Small-Cell Lung Cancer Cells through a Mitochondria-Mediated Apoptotic Pathway.

In order to improve the potential of celastrol against non-small-cell lung cancer cells, the privileged structure, thiazolidinedione, was introduced into its C-20 carboxylic group with acetylpiperazine as a linker, and the thiazolidinedione-conjugated compounds 10a-10t were prepared. The target compounds were evaluated for their cytotoxic activities against the A549 cell line, and the results showed that most of the compounds 10a-10t displayed improved potency over celastrol, and compound 10b exhibited significant activity against the A549 cell line, with an IC50 value of 0.08 μM, which was 13.8-fold more potent than celastrol (IC50 = 1.10 μM). The mechanistic studies suggested that 10b could induce A549 cell apoptosis, as evidenced by Hoechst 33342 staining and annexin V-FITC/propidium iodide dual staining assays. Western blot analysis suggested that compound 10b could upregulate Bax expression, downregulate Bcl-2 expression, and activate the mitochondria-mediated apoptotic pathway. Furthermore, compound 10b could effectively inhibit tumor growth when tested in an A549 cell xenograft mouse model. Collectively, compound 10b is worthy of further investigation to support the discovery of effective agents against non-small-cell lung cancer.

In Vitro Screening of New Biological Limiters against Some of the Main Soil-Borne Phytopathogens

This study explored the role of Aphanocladium album (strain MX95), Pleurotus ostreatus (strain ALPO) and Pleurotus eryngii (strain AL142PE) as potential biological limiters. MX95, ALPO and AL142PE were screened under laboratory studies against Phytophthora nicotianae (PN), Fusarium oxysporum f. sp. radicis-lycopersici (FORL), Fusarium oxysporum f. sp. lycopersici (FOL), Fusarium solani (FS), Sclerotinia minor (SM), Sclerotinia sclerotiorum (SS), Athelia (Sclerotium) rolfsii (AR) and Verticillium dahliae (VD). The radial growth inhibition and the over-growth of potential antagonists on the target organisms were used to assess the interactions in the in vitro dual culture plate assay. The antagonistic ability of each challenge isolate was evaluated by calculating an index of the antagonism (AI) based on the interaction type in the dual cultures. MX95, reducing the growth of SS (20%) and FS (40%), displayed deadlock at mycelial contact against FOL and FORL, deadlock at distance versus VD and completely over-grew PN and SM. ALPO reduced (43 to 88%) the mycelial growth of tested pathogens except FORL and replaced PN and VD. AL142PE reducing (53 to 67%) SS, VD, FS and FOL mycelial growth and completely over-grew PN. AR showed combative ability against all the experienced biological limiters. Based on the results of the AI values, MX95 (AI = 16.5) was considered an active antagonist, while ALPO (AI = 11.5) and AL142PE (AI = 12.0) were moderately active antagonists. Strains MX95, ALPO and AL142PE were suitable as environment-friendly potential biocontrol agents to manage some of the main soil-borne agents of foot, root, soft rot and wilt diseases. These results are the first step in the assessment of the potential capacity of these organisms as biological limiters. Nevertheless, additional experiments should be performed for the translation to the field conditions in plant protection against soil-borne plant pathogens. In particular, the optimisation of dose and application time validation should be performed for a solid conclusion about the competitive ability of MX95, ALPO and AL142PE and the usefulness of potential biological limiters.

Antagonistic activities of bioagent fungi Trichoderma harzianum and Pleurotus ostreatus against three species of Fusarium in cucumber plants

Many bioagent fungi have promising potential as eco-friendly alternatives to fungicides, with considerable antagonistic activity against various phytopathogenic fungi. The present study aimed to investigate the antagonistic activity of Trichoderma harzianum and Pleurotus ostreatus isolates against Fusarium spp., the causative agents of wilt disease in cucumber plants, through a dual plate assay of volatile and nonvolatile compounds from these bioagent fungi. The results showed significant (P < 0.05) antagonistic activities of T. harzianum against the growth of F. solani AJA2 (62.3%), followed by F. oxysporum AJA (55.2%), F. incarnatum AJA (53.2%), and F. solani AJA1 (50.8%). The effectiveness of P. ostreatus against the four Fusarium species was notably less than that of T. harzianum. In contrast, in the dual culture assay, the bioagent fungal filtrate exhibited inhibitory effects on the growth of all pathogens at 25% concentration. The highest inhibition rate (85%) was shown by T. harzianum against F. incarnatum. The percent of inhibition caused by P. ostreatus was substantially lower than that caused by T. harzianum, which reached 35% in F. incarnatum followed by other pathogens. The volatile compounds of T. harzianum led to a high percentage of inhibition of all the three Fusarium species, while the highest percentage of inhibition due to the compounds of P. ostreatus was observed only for F. solani AJA1 (41.5%). From these results, we concluded that despite the diverse inhibitory effects of both bioagent fungi against Fusarium species, they exhibited successful antagonistic activity and the ability to compete against these species.

Carbon dots derived from cellobiose for temperature and phosalone detection

Cellobiose, the hydrolysis product of cellulose and the dimer of glucose, was employed to synthesize carbon dots (CDs) via a simple hydrothermal method. The inducing of nitrogen can act as a “molecular glue”, more and more carbon clusters were “glued” together, which would push the hydrothermal reaction in the positive direction. Besides, the linear response of the fluorescence of N-CDs to temperature ensured its potential application in temperature sensing. In the temperature range of 10∼80 °C, the fluorescence of as-prepared N-CDs was reversible and recoverable. Moreover, the dual fluorescence centers and dual-absorption bands of the as-prepared N-CDs permitted the novel dual-mode ratiometric assays for phosalone detection. The linear range and limit of detection (LOD) for dual fluorescence center-based ratiometric assay were 0.12∼5.45 μg/mL and 42.90 ng/mL, respectively. While that for the dual absorption-based ratiometric assay were 0.02∼1.40 μg/mL, and 6.67 ng/mL, respectively.

Anticancer effect of selenium/chitosan/polyethylene glycol/allyl isothiocyanate nanocomposites against diethylnitrosamine-induced liver cancer in rats

BackgroundNano-based drug delivery systems have shown several advantages in cancer treatment like specific targeting of cancer cells, good pharmacokinetics, and lesser adverse effects. Liver cancer is a fifth most common cancer and third leading cause of cancer-related mortalities worldwide. ObjectiveThe present study focusses to formulate the selenium (S)/chitosan (C)/polyethylene glycol (Pg)/allyl isothiocyanate (AI) nanocomposites (SCPg-AI-NCs) and assess its therapeutic properties against the diethylnitrosamine (DEN)-induced liver cancer in rats via inhibition of oxidative stress and tumor markers. MethodologyThe SCPg-AI-NCs were synthesized by ionic gelation technique and characterized by various characterization techniques. The liver cancer was induced to the rats by injecting a DEN (200 mg/kg) on the 8th day of experiment. Then DEN-induced rats treated with 10 mg/kg of formulated SCPg-AI-NCs an hour before DEN administration for 16 weeks. The 8-hydroxy-2′ -deoxyguanosine (8-OHdG) content, albumin, globulin, and total protein were examined by standard methods. The level of glutathione (GSH), vitamin-C & -E, and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities were examined using assay kits. The liver marker enzymes i.e., alanine transaminase (ALT), aspartate tansaminase (AST), γ-glutamyl transaminase (GGT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activities, alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA), Bax, and Bcl-2 levels, and caspase-3&9 activities was examined using assay kits and the liver histopathology was assessed microscopically by hematoxylin and eosin staining method. The effect of formulated SCPg-AI-NCs on the viability and apoptotic cell death on the HepG2 cells were examined using MTT and dual staining assays, respectively. ResultsThe results of different characterization studies demonstrated the formation of SCPg-AI-NCs with tetragonal shape, narrowed distribution, and size ranging from 390 to 450 nm. The formulated SCPg-AI-NCs treated liver cancer rats indicated the reduced levels of 8-OHdG, albumin, globulin, and total protein. The SCPg-AI-NCs treatment appreciably improved the GSH, vitamin-C & -E contents, and SOD, CAT, GPx, and GR activities in the serum of liver cancer rats. The SCPg-AI-NCs treatment remarkably reduced the liver marker enzyme activities in the DEN-induced rats. The SCPg-AI-NCs treatment decreased the AFP and CEA contents and enhanced the Bax and caspase 3&9 activities in the DEN-induced rats. The SCPg-AI-NCs effectively decreased the cell viability and induced apoptosis in the HepG2 cells. ConclusionThe present findings suggested that the formulated SCPg-AI-NCs remarkably inhibited the DEN-induced liver carcinogenesis in rats. These findings provide an evidence that SCPg-AI-NCs can be a promising anticancer nano-drug in the future to treat the liver carcinogenesis.