Comparison and Correlation of Corynespora cassiicola Populations from Kiwifruit and Other Hosts Based on Morphology, Phylogeny, and Pathogenicity.

Corynespora leaf spot, which is caused by Corynespora cassiicola (Berk. & M. A. Curtis) C.T. Wei (C. cassiicola), has been globally reported in many plant species. 'Hongyang' was reported as highly sensitive kiwifruit cultivar to C. cassiicola. This cultivar is an important germplasm resource in the Actinidiaceae family and is widely cultivated throughout China. Even though C. cassiicola has been identified as the pathogen associated with kiwifruits in China, the C. cassiicola population from kiwifruit has not been characterized based on morphology, phylogeny, and pathogenicity. In this study, 133 and 48 representative C. cassiicola isolates from kiwifruit and 11 other hosts, respectively, recovered from symptomatic leaves were classified into eight morphological subgroups based on host origins. Using three loci (rDNA ITS, caa5, and act1), a phylogenetic tree showed that C. cassiicola isolates in Sichuan Province were grouped into three clades. All kiwifruit isolates were genetically identical to the rubber isolates from different countries. However, most isolates from other hosts in this study were genetically identical to the cucumber, soybean, and cowpea isolates in China, Brazil, and the United States, and two strawberry isolates clustered with isolates from tomato and other hosts in China, Brazil, and the United States. Furthermore, we confirmed host shift of C. cassiicola among different plant species in this study. Although 51 isolates from kiwifruit and different hosts were pathogenic to kiwifruit, blueberry, cucumber, and soybean, virulence levels of the pathogen were diverse for four hosts. Kiwifruit isolates exhibited host specificity with regards to the original host in degree. In addition, those isolates revealed a correlation between morphology and pathogenicity. The results suggest that C. cassiicola in Sichuan Province were derived from three different phylogenetic lineages. Promotion of the susceptible 'Hongyang' cultivar led to the emergence of a regnant C. cassiicola population from kiwifruit. In conclusion, rapid development of the C. cassiicola-sensitive crop in agricultural systems led to the emergence of a regnant C. cassiicola population. In some dominant populations (e.g., the C. cassiicola population from kiwifruit in this study), host origin was found to be a key factor influencing the morphologic, genetic, and pathogenic characterization of C. cassiicola.

Role of rhizobia in promoting non-enzymatic antioxidants to mitigate nitrogen-deficiency and nickel stresses in Pongamia pinnata.

The contribution of rhizobia in the mitigation of non-enzymatic antioxidants against nitrogen deficiency and heavy metal toxicity for legume plant is not clear. Therefore, it is hypothesized that the inoculation of rhizobia could mitigate nitrogen deficiency and nickel (Ni) stresses in P. pinnata tissues by enhancing the formation of certain non-enzymatic antioxidants. The effect of symbiotic nitrogen-fixing rhizobia on the mitigation of nitrogen-deficiency and Ni stresses in P. pinnata was evaluated by inoculating two different rhizobia, i.e., Rhizobium pisi PZHK2 and Ochrobacterium pseudogrignonense PZHK4, around the rhizosphere of P. pinnata grown in soil containing 40 mg kg-1 Ni2+ and without nitrogen addition. The inoculation with both rhizobial strains promoted the growth of P. pinnata under nickel stress or nitrogen-deficiency condition, increased nitrogen content in all plant tissues and nickel content in shoots and leaves, but reduced nickel accumulation in roots. The four non-enzymatic antioxidants including glutathione (GSH), proanthocyanidin (OPC), ascorbic acid (ASA) and flavonoids (FLA) distributed in roots, shoots and leaves were followed in descending order: GSH > OPC > ASA > FLA. The four non-enzymatic antioxidants showed different levels of change under the nitrogen-deficiency and nickel stresses and in the non-stress control. The inoculation of PZHK2 and PZHK4 significantly (p < 0.05) increased the four non-enzymatic antioxidants in P. pinnata tissues, especially in roots. Some non-enzymatic antioxidants showed correlations with nickel or nitrogen in P. pinnata tissues, and the four non-enzymatic antioxidants also had correlations among each other. Therefore, this research revealed an excellent role of rhizobia in promoting non-enzymatic antioxidants to mitigate nitrogen-deficiency or nickel stress for P. pinnata.

Synergistic anti-oxidative effects of Pongamia pinnata against nickel mediated by Rhizobium pisi and Ochrobacterium pseudogrignonense.

Nickel is widely spread by different anthropogenic activities and shows toxicity for plant growth and development. Whether rhizobia symbiotically fix nitrogen can eliminate or reduce nickel toxic effect on plant or not is still unknown. This study was aimed to investigate the effect of different rhizobia genus inoculation on growth, nitrogen fixing ability, metal accumulation and enzymatic antioxidative balance of Pongamia pinnnaa. Inoculation with Rhizobium pisi and Ochrobacterium pseudogrignonense increased the all the growth parameters both in 0 and 40 mg/kg nickel as comparison with control. Only shoot length increased in presence of nitrogen as compared with no supply of nitrogen. Nitrogen content also increased both in rhizobia inoculation as compared to no nitrogen supply and non-inoculation control, respectively. Nickel uptake was higher in shoots and leaves but lower in roots in case of inoculation as compared to non-inoculation control. Rhizobia inoculation improved the plant antioxidant capacity by increasing the activity of enzymatic scavengers catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and ascorbate (GR). However, 40 mg/kg of nickel adding showed mostly effect on the activity CAT, SOD, POD in leaves. All the enzymatic activity showed a significant increase in absence of nitrogen supply as compared nitrogen supply. Our results suggested that rhizobia inoculation effectively mediated nickel stress for legume plants by increasing nitrogen supplement and inducing antioxidant capacity.

Development of a Highly Sensitive Enzyme-Linked Immunosorbent Assay for Mouse Soluble Epoxide Hydrolase Detection by Combining a Polyclonal Capture Antibody with a Nanobody Tracer.

Enzyme-linked immunosorbent assays (ELISA) for the detection of soluble epoxide hydrolase (sEH), a key enzyme in the metabolism of fatty acids and a biomarker, may increasingly represent an important diagnostic tool. However, there is a lack of ELISAs for mouse sEH quantification, thus resulting in a bottleneck in understanding the pathogenesis of many diseases related to sEH based on mouse models. In this work, nanobodies recognizing mouse sEH were obtained through rebiopanning against mouse sEH in the previous phage display library of human sEH. Later, we developed four ELISAs involving a combination of anti-mouse sEH polyclonal antibodies (pAbs) and nanobodies. It was found that the double antibodies worked as dual filters and had a huge impact on both the sensitivity and selectivity of sandwich immunoassays. The switch from anti-human sEH pAbs to anti-mouse sEH pAbs led to over a 100-fold increase in the sensitivity and a dramatic decrease of the limit of detection to a picogram per milliliter range in format B (pAb/biotin-VHH/streptavidin-poly-horseradish peroxidase). Moreover, we found that the four sandwich ELISAs might demonstrate excellent selectivities to mouse sEH, despite the antibodies alone showing significant cross-reactivity to the matrix, indicating the enhanced selectivity of double antibodies as dual filters. Eventually, for the first time, the ELISA (format B) was successfully used to measure the mouse sEH level in cancer cells with ultralow abundances. The ELISAs proposed here represent a sensitive tool for tracking sEH in various biological processes and also provide deep insights into developing sandwich immunoassays against various targets in terms of both the sensitivity and selectivity.

Dissipation and residue determination of penicillin G and its two metabolites in citrus under field conditions by DSPE/UPLC-MS/MS.

A rapid determination method of residual penicillin G and its two metabolites in citrus was developed and validated by dispersive solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (DSPE/UPLC-MS/MS). The samples were extracted with 80% acetonitrile and purified with octadecylsilane. High linearity was obtained with correlation coefficients (r2 ) >0.9981. The limits of quantification were 0.005-0.01 mg/kg. The recoveries of penicillin G and its metabolites spiked in blank citrus were within 76.7-107%, with relative standard deviations of 1.3-9.6%. The dissipation dynamics and distribution of penicillin G in citrus followed first-order kinetics, with half-life of 1.7-2.7 days. Penicillin G degraded easily in citrus and the metabolite was mainly penilloic acid, which can exist stably for long time. The terminal residues of penicillin G in pulp, whole citrus and peels were 0.015-0.701, 0.047-7.653 and 0.162-13.376 mg/kg, respectively. The hazard indexes for risk assessment of citrus were significantly <1, suggesting that the health risks to humans after consumption of citrus were insignificant and negligible. These results could provide necessary data for evaluating the safe and proper use of penicillin G in citrus.

Changes of root microbial populations of natively grown plants during natural attenuation of V-Ti magnetite tailings.

Mine tailings contain dangerously high levels of toxic metals which pose a constant threat to local ecosystems. Few naturally grown native plants can colonize tailings site and the existence of their root-associated microbial populations is poorly understood. The objective of this study was to give further insights into the interactions between native plants and their microbiota during natural attenuation of abandoned V-Ti magnetite mine tailings. In the present work, we first examined the native plants' potential for phytoremediation using plant/soil analytical methods and then investigated the root microbial communities and their inferred functions using 16 S rRNA-based metagenomics. It was found that in V-Ti magnetite mine tailings the two dominant plants Bothriochloa ischaemum and Typha angustifolia were able to increase available nitrogen in the rhizosphere soil by 23.3% and 53.7% respectively. The translocation factors (TF) for both plants indicated that B. ischaemum was able to accumulate Pb (TF = 1.212), while T. angustifolia was an accumulator of Mn (TF = 2.502). The microbial community structure was more complex in the soil associated with T. angustifolia than with B. ischaemum. The presence of both plants significantly reduced the population of Acinetobacter. Specifically, B. ischaemum enriched Massilia, Opitutus and Hydrogenophaga species while T. angustifolia significantly increased rhizobia species. Multivariate analyses revealed that among all tested soil variables Fe and total organic carbon (TOC) could be the key factors in shaping the microbial structure. The putative functional analysis indicated that soil sample of B. ischaemum was abundant with nitrate/nitrite reduction-related functions while that of T. angustifolia was rich in nitrogen fixing functions. The results indicate that these native plants host a diverse range of soil microbes, whose community structure can be shaped by plant types and soil variables. It is also possible that these plants can be used to improve soil nitrogen content and serve as bioaccumulators for Pb or Mn for phytoremediation purposes.

A new sensitive LC/MS/MS analysis of vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine.

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.

Pongamia pinnata inoculated with Bradyrhizobium liaoningense PZHK1 shows potential for phytoremediation of mine tailings.

Mine tailings contain high concentrations of metal contaminants and only little nutrients, making the tailings barren for decades after the mining has been terminated. Effective phytoremediation of mine tailings calls for deep-rooted, metal accumulating, and soil fertility increasing plants with tolerance against harsh environmental conditions. We assessed the potential of the biofuel leguminous tree Pongamia pinnata inoculated with plant growth promoting rhizobia to remediate iron-vanadium-titanium oxide (V-Ti magnetite) mine tailing soil by pot experiment and in situ remediation test. A metal tolerant rhizobia strain PZHK1 was isolated from the tailing soil and identified as Bradyrhizobium liaoningense by phylogenetic analysis. Inoculation with PZHK1 increased the growth of P. pinnata both in V-Ti magnetite mine tailings and in Ni-contaminated soil. Furthermore, inoculation increased the metal accumulation capacity and superoxide dismutase activity of P. pinnata. The concentrations of Ni accumulated by inoculated plants were higher than the hyperaccumulator threshold. Inoculated P. pinnata accumulated high concentration of Fe, far exceeding the upper limit (1000 mg kg-1) of Fe in plant tissue. In summary, P. pinnata-B. liaoningense PZHK1 symbiosis showed potential to be applied as an effective phytoremediation technology for mine tailings and to produce biofuel feedstock on the marginal land.

Development of a Specific Monoclonal Antibody for the Quantification of Artemisinin in Artemisia annua and Rat Serum.

Artemisinin, extracted from Artemisia annua, and its derivatives are important frontline antimalarials. To produce specific antibodies for the detection and quantification of artemisinin, artemisinin was transformed to 9-hydroxyartemisinin by microbial fermentation, which was used to prepare a 9-succinate artemisinin hapten for conjugation with ovalbumin. A monoclonal antibody (mAb), designated as 3H7A10, was selected from hybridoma cell lines which showed high specificity to artemisinin. No competitive inhibition was observed with artesunate, dihydroartemisinin, and artemether for up to 20,000 ng mL(-1). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed, which showed a concentration causing 50% of inhibition (IC50) for artemisinin as 2.6 ng mL(-1) and a working range of 0.6-11.5 ng mL(-1). The icELISA was applied for the quantification of artemisinin in crude extracts of wild A. annua and the study of pharmacokinetics of artemisinin in rat serum after intraperitoneal injection. The results were highly correlated with those determined by HPLC-UV analysis (R(2) = 0.9919). In comparison with reported antiartemisinin mAbs which have broad cross-reactivity with other artemisinin derivatives, the high specificity of 3H7A10 for artemisinin will enable development of methods for quantification of artemisinin in Artemisia plants and antimalarial drugs such as Arco and for pharmacokinetic studies.

VHH antibodies: emerging reagents for the analysis of environmental chemicals.

A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Discovered nearly 25 years ago, they have been investigated for their use in clinical therapeutics and immunodiagnostics, and more recently for environmental monitoring applications. A new and valuable immunoreagent for the analysis of small molecular weight environmental chemicals, VHH will overcome many pitfalls encountered with conventional reagents. In the work so far, VHH antibodies often perform comparably to conventional antibodies for small molecule analysis, are amenable to numerous genetic engineering techniques, and show ease of adaption to other immunodiagnostic platforms for use in environmental monitoring. Recent reviews cover the structure and production of VHH antibodies as well as their use in clinical settings. However, no report focuses on the use of these VHH antibodies to detect small environmental chemicals (MW < 1500 Da). This review article summarizes the efforts made to produce VHHs to various environmental targets, compares the VHH-based assays with conventional antibody assays, and discusses the advantages and limitations in developing these new antibody reagents particularly to small molecule targets. Graphical Abstract Overview of the production of VHHs to small environmental chemicals and highlights of the utility of these new emerging reagents.

Tunable Nanosupramolecular Aggregates Mediated by Host-Guest Complexation.

Nanosupramolecular assemblies with controlled topological features have inventive applications in fundamental studies and material manufacturing. Herein, a variety of morphologically interesting aggregates have been constructed using the supramolecular modulation with bipyridinium-modified diphenylalanine derivative (BP-FF). Benefiting from the high binding affinity of bipyridinium group with four different macrocyclic receptors, namely cucurbit[7]uril, cucurbit[8]uril, pillar[5]arene, and tetrasulfonated crown ether, we have succeeded in tuning the topological aggregates of BP-FF from fine nanofibers to nanorods, octahedron-like nanostructure, helical nanowires, and rectangular nanosheets without any tedious chemical modification. This supramolecular approach may provide us a powerful method to construct well-defined nanostructures with different morphologies that can be conveniently controlled by facile host-guest interactions.

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays.

Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase.

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

Identification of Botryosphaeriaceae Species Causing Kiwifruit Rot in Sichuan Province, China.

Species of Botryosphaeriaceae fungi are important plant pathogens causing cankers, blight, and fruit rot in an extremely wide range of host. In recent years, kiwifruit rot has been a serious problem in Sichuan Province, one of the important kiwifruit production areas of China. Botryosphaeria dothidea has previously been associated with kiwifruit rot but little is known regarding whether other Botryosphaeriaceae genera also constitute kiwifruit rot pathogens in China. Accordingly, diseased fruit were collected from six different areas of Sichuan Province. Based on morphological characteristics, pathogenicity testing, and comparisons of DNA sequences of the internal transcribed spacer, transcription elongation factor 1-α, and ß-tubulin genes, 135 isolates of Botryosphaeriaceae were identified as B. dothidea, Lasiodiplodia theobromae, and Neofusicoccum parvum. All of these species were found to cause kiwifruit rot. To understand the infection cycle of kiwifruit rot pathogens, these three species were used to inoculate leaves and shoots of kiwifruit. The results showed that these species could cause spots on leaves and lesions on shoots, producing abundant pycnidia on leaves and shoots surfaces. Moreover, B. dothidea conidia and ascospores from overwintered pycnidia and pseudothecia in kiwifruit orchards in April and August could cause fruit rot and spots on leaves of kiwifruit. Therefore, we concluded that overwintered pycnidia and pseudothecia of B. dothidea in kiwifruit orchards are the primary inoculum for kiwifruit rot, with new pycnidia that develop during the growing season serving as a secondary inoculum. This is the first report of N. parvum and L. theobromae causing kiwifruit rot in China and is also the first report that B. dothidea is able to overwinter as pycnidia and pseudothecia in kiwifruit orchards and serve as the primary inoculum for kiwifruit rot.

[Effects of Rapamycin and Rapamycin-loaded Poly(lactic-co-glycolic)Acid Nanoparticles on Apoptosis and Expression of bcl-2 and p27(kip1) Proteins of Human Umbilical Arterial Vascular Smooth Muscle Cell].

OBJECTIVE: To investgate the effects of rapamycin(RPM)and RPM-loaded poly(lactic-co-glycolic)acid(PLGA)nanoparticles(NPs)on the apoptosis of human umbilical arterial vascular smooth muscle cells(HUASMCs)in vitro and expression of bcl-2 and p27(kip1) protein. METHODS: HUASMCs were cultured in vitro and divided to RPM and RPM-PLGA-NPs groups treated at 3 different concentration by 12 and 24 hours,with M231-smooth muscle growth supplements medium and null-PLGA-NPs treated groups as controlled. The apoptosis of HUASMCs was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining and flow cytometry. The expressions of bcl-2 and p27(kip1) were detected by streptacidin/peroxidase immunohistochemical method. The effect on cellular proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidecolorimetry. RESULTS: The proliferation of HUASMCs was inhibited by RPM and RPM-PLGA-NPs in a dose-dependent manner. DNA electrophoresis showed DNA ladder in RPM and RPM-PLGA-NPs groups and classical scalar strips in control groups. The apoptotic indexes of RPM 100 ng/ml group and RPM-PLGA-NPs 500 ng/ml group detected by flow cytometry were(45.45<2.36)% and(35.04<5.64)%,respectively,which were significantly higher than that of M231-smooth muscle growth supplements control group [(2.60<0.95)%,all P<0.01]. The apoptotic indexes of groups incubated with RPM and RPM-PLGA-NPs for 24 hours were significantly higher than those of groups which incubated for 12 hours(P<0.05,P<0.01). The positive expression indexes(PEI)of p27(kip1) and bcl-2 protein were higher in RPM and RPM-PLGA-NPs groups than that of control groups. The Spearman's rank correlation coefficient test showed that there was no significant correlation between the PEI of p27(kip1) and the apoptotic indexes in the RPM group and RPM-PLGA-NPs group(P>0.05). CONCLUSIONS: Rapamycin-loaded PLGA nanoparticles and rapamycin have similar effects in inhibiting proliferation and inducing apoptosis;meanwhile,they upregulate the expression of p27(kip1) protein without downregulating the expression of bcl-2 protein in HUASMCs in vitro. RPM-PLGA-NPs has more potent pro-apoptotic effect than equivalent dose of RPM but is not linearly correlated with the p27(kip1) expression level.

Development of a colloidal gold-based lateral flow dipstick immunoassay for rapid qualitative and semi-quantitative analysis of artesunate and dihydroartemisinin.

BACKGROUND: Artemisinin-based combination therapy (ACT) plays an indispensable role in malaria control and elimination. However, the circulation of counterfeit, substandard drugs has greatly threatened malaria elimination campaigns. Most methods for the analysis of artemisinin and its derivatives require expensive equipment and sophisticated instrumentation. A convenient, easy-to-use diagnostic device for rapid evaluation of the quality of artemisinin drugs at the point-of-care is still lacking. In this study a lateral flow dipstick immunoassay was developed for qualitative and semi-quantitative analysis of artesunate (ATS) and dihydroartemisinin (DHA) in anti-malarial drugs. METHODS: This assay was based on a monoclonal antibody (mAb) raised against ATS. ATS-bovine serum albumin and goat anti-mouse IgG, used as the test capture reagent and the control capture reagent, were coated on the nitrocellulose membrane to form the test line and control line, respectively. The conjugate pad was saturated with the gold-labelled anti-ATS mAb. RESULTS: The indicator range of the dipsticks, defined as lowest concentration of the target analytes between which the test line was not visible, were 100-200 and 200-500 ng mL(-1) for ATS and DHA, respectively. No competitive inhibition was observed up to 5,000 ng mL(-1) of quinine, chloroquine diphosphate salt, primaquine phosphate, pyrimethamine, lumefantrine, amodiaquine, piperaquine tetraphosphate tetrahydrate or pyronaridine tetraphosphate. Semi-quantitative analysis of ATS and DHA in commercial drugs and raw drug materials with the dipsticks produced result agreeable with those determined by high performance liquid chromatography (HPLC). Storage test showed that the indicator range for artemisinins remained unchanged after a week at 37 °C and increased four-folds after six months of storage at 4 °C or ambient temperature. CONCLUSIONS: The new selected mAb 3D82G7 with high avidity and broad cross reactivity for artemisinins was used to develop and optimize a dipstick immunoassay for qualitative and semi-quantitative analysis of ATS and DHA in anti-malarial drugs. The semi-quantitative analysis of ATS and DHA in commercial drugs and raw drug materials, and the specificity test of the artemisinin-related drugs both proved the accurate performance of the developed dipsticks for semi-quantitation of ACT samples. The dipstick may be used as a point-of-care device for identifying substandard and counterfeit ATS- and DHA-containing anti-malarial drugs.

Chalcomoracin promotes apoptosis and endoplasmic reticulum stress in hepatocellular carcinoma cells.

Chalcomoracin (CMR), a Diels-Alder adduct obtained from mulberry leaves, demonstrated wide-spectrum anti-cancer activity. Herein, we aimed to explore the function of CMR and how it works in hepatocellular carcinoma (HCC). Human HCC cell lines Hep3B and SNU-387 were cultured and treated with various concentrations of CMR (1.5, 3, and 6 µM). Subsequently, the effects of CMR on cell viability, colony formation, apoptosis, migration, and invasion abilities were studied in vitro. Furthermore, the levels of endoplasmic reticulum (ER) stress-related proteins and mitogen-activated protein kinase (MAPK) pathway-related proteins in cells under CMR exposure were detected using western blot. Experiments in vivo were conducted to examine the effects of CMR on tumor growth in HCC. CMR administration inhibited the viability and clonogenic, migration, and invasion abilities, as well as promoted cell apoptosis and ER stress in Hep3B and SNU-387 cells. In addition, CMR treatment reduced the phosphorylation levels of ERK, P38, and JNK in the MAPK pathway. Moreover, an in vivo study showed that CMR administration could inhibit tumorigenesis and MAPK pathway activity in HCC. Our data indicate that CMR has the potential to inhibit the development of HCC, potentially through the inhibition of the MAPK pathway. These findings suggest that CMR may have promising applications as an anticancer agent in future therapeutics for HCC.

Pan-cancer analyses of immunogenic cell death-derived gene signatures: Potential biomarkers for prognosis and immunotherapy.

BACKGROUND: Immunogenic cell death (ICD) is a type of regulated cell death that is capable of initiating an adaptive immune response. Induction of ICD may be a potential treatment strategy, as it has been demonstrated to activate the tumor-specific immune response. AIMS: The biomarkers of ICD and their relationships with the tumor microenvironment, clinical features, and immunotherapy response are not fully understood in a clinical context. Therefore, we conducted pan-cancer analyses of ICD gene signatures across 33 cancer types from The Cancer Genome Atlas database. METHODS AND RESULTS: We identified key genes that had strong relationships with survival and the tumor microenvironment, contributing to a better understanding of the role of ICD genes in cancer therapy. In addition, we predicted therapeutic agents that target ICD genes and explored the potential mechanisms by which gemcitabine induce ICD. Moreover, we developed an ICD score based on the ICD genes and found it to be associated with patient prognosis, clinical features, tumor microenvironment, radiotherapy access, and immunotherapy response. A high ICD score was linked to the immune-hot phenotype, while a low ICD score was linked to the immune-cold phenotype. CONCLUSION: We uncovered the potential of ICD gene signatures as comprehensive biomarkers for ICD in pan-cancer. Our research provides novel insights into immuno-phenotypic assessment and cancer therapeutic strategies, which could help to broaden the application of immunotherapy to benefit more patients.

Hydrogel: a new material for intravesical drug delivery after bladder cancer surgery.

The standard treatment for non-muscle invasive bladder cancer (NMIBC) is transurethral resection of bladder tumor (TURBT). However, this procedure may miss small lesions or incompletely remove them, resulting in cancer recurrence or progression. As a result, intravesical instillation of chemotherapy or immunotherapy drugs is often used as an adjunctive treatment after TURBT to prevent cancer recurrence. In the traditional method, drugs are instilled into the patient's bladder through a urinary catheter under sterile conditions. However, this treatment exposes the bladder mucosa to the drug directly, leading to potential side effects like chemical cystitis. Furthermore, this treatment has several limitations, including a short drug retention period, susceptibility to urine dilution, low drug permeability, lack of targeted effect, and limited long-term clinical efficacy. Hydrogel, a polymer material with a high-water content, possesses solid elasticity and liquid fluidity, making it compatible with tissues and environmentally friendly. It exhibits great potential in various applications. One emerging use of hydrogels is in intravesical instillation. By employing hydrogels, drug dilution is minimized, and drug absorption, retention, and persistence in the bladder are enhanced due to the mucus-adhesive and flotation properties of hydrogel materials. Furthermore, hydrogels can improve drug permeability and offer targeting capabilities. This article critically examines the current applications and future prospects of hydrogels in the treatment of bladder cancer.