MicroRNA-186 suppresses cell proliferation and metastasis in bladder cancer.

Purpose: Bladder cancer (BCa) is a common malignancy in the urinary system. This study aims to explore the role of miR-186 in BCa tumorigenesis. Methods: The expression of miR-186 and ADAMTS12 in clinical BCa tissues and cell lines was detected. BCa cell lines T24, 5637 and EJ were used to transfect miR-186 mimics or inhibitors. Luciferase reporter gene detection confirmed the correlation between miR-186 and ADAMTS12. MTT method and flow cytometry were used to detect cell viability and apoptosis. Cell migration and invasion ability was detected by transwell assay. The protein level of ADAMTS12, ß-catenin, GSK-3ß and p-GSK-3ß was determined using western blot analysis. Results: MiR-186 was negatively correlated with the expression of ADAMTS12 in BCa tissues. Further research confirmed that ADAMTS12 is the direct target of miR-186. In addition, overexpression of miR-186 down-regulated the expression of ADAMTS12, inhibiting cell viability and apoptosis, while knockout of miR-186 led to the opposite result. miR-186 also inhibits the phosphorylation of GSK-3 ß and ß-catenin without changing the total GSK-3ß level. Our study shows that miR-186 has a negative regulatory effect on the expression of ADAMTS12 in clinical specimens and in vitro. miR-186 can inhibit the proliferation and invasion of BCa cells. Conclusions: miR-186 has the potential to be used as a biomarker in the early detection of BCa.

Bacterial Community Selection of Russula griseocarnosa Mycosphere Soil.

Russula griseocarnosa is a wild, ectomycorrhizal, edible, and medicinal fungus with high economic value in southern China. R. griseocarnosa fruiting bodies cannot be artificially cultivated. To better understand the effects of abiotic and biotic factors on R. griseocarnosa growth, the physicochemical properties of R. griseocarnosa and its associated bacterial communities were investigated in two soil types (mycosphere and bulk soil) from Fujian, Guangdong, and Guangxi Provinces. The results revealed that the diversity, community structure, and functional characteristics of the dominant mycosphere bacteria in all geographical locations were similar. Soil pH and available nitrogen (AN) are the major factors influencing the mycosphere-soil bacterial communities' structure. The diversity of soil bacteria is decreased in R. griseocarnosa mycosphere when compared with the bulk soil. Burkholderia-Paraburkholderia, Mycobacterium, Roseiarcus, Sorangium, Acidobacterium, and Singulisphaera may also be mycorrhiza helper bacteria (MHB) of R. griseocarnosa. The functional traits related to the two-component system, bacterial secretion system, tyrosine metabolism, biosynthesis of unsaturated fatty acids, and metabolism of cofactors and vitamins were more abundant in R. griseocarnosa mycosphere soil. The mycosphere soil bacteria of R. griseocarnosa play a key role in R. griseocarnosa growth. Application of management strategies, such as N fertilizer and microbial fertilizer containing MHB, may promote the conservation, propagation promotion, and sustainable utilization of R. griseocarnosa.

Adhesive Gold Nanoparticles for Easy and Controlled Surface Coating.

Gold nanoparticles (Au NPs) are one of the most important nanomaterials due to their unique properties and broad applications. Among these applications, decorating Au NPs on universal surfaces is highly desired. Herein, we report adhesive Au NPs functionalized by borated dopamine dithiocarbamate. Such Au NPs are nonreactive in colloidal solution but can be activated at an acidic pH to produce adhesive Au NPs and initiate spontaneous surface coating through deprotected catechol-mediated reactions. Easy and controllable surface coating was achieved on materials with distinguished chemical and physical properties because of the high reactivity of catechol. Adhesive Au NPs represent new surface coating method with wide application potentials.

Fungal Systematics and Evolution: FUSE 5.

Thirteen new species are formally described: Cortinarius brunneocarpus from Pakistan, C. lilacinoarmillatus from India, Curvularia khuzestanica on Atriplex lentiformis from Iran, Gloeocantharellus neoechinosporus from China, Laboulbenia bernaliana on species of Apenes, Apristus, and Philophuga (Coleoptera, Carabidae) from Nicaragua and Panama, L. oioveliicola on Oiovelia machadoi (Hemiptera, Veliidae) from Brazil, L. termiticola on Macrotermes subhyalinus (Blattodea, Termitidae) from the DR Congo, Pluteus cutefractus from Slovenia, Rhizoglomus variabile from Peru, Russula phloginea from China, Stagonosporopsis flacciduvarum on Vitis vinifera from Italy, Strobilomyces huangshanensis from China, Uromyces klotzschianus on Rumex dentatus subsp. klotzschianus from Pakistan. The following new records are reported: Alternaria calendulae on Calendula officinalis from India; A. tenuissima on apple and quince fruits from Iran; Candelariella oleaginescens from Turkey; Didymella americana and D. calidophila on Vitis vinifera from Italy; Lasiodiplodia theobromae causing tip blight of Dianella tasmanica 'variegata' from India; Marasmiellus subpruinosus from Madeira, Portugal, new for Macaronesia and Africa; Mycena albidolilacea, M. tenuispinosa, and M. xantholeuca from Russia; Neonectria neomacrospora on Madhuca longifolia from India; Nothophoma quercina on Vitis vinifera from Italy; Plagiosphaera immersa on Urtica dioica from Austria; Rinodina sicula from Turkey; Sphaerosporium lignatile from Wisconsin, USA; and Verrucaria murina from Turkey. Multi-locus analysis of ITS, LSU, rpb1, tef1 sequences revealed that P. immersa, commonly classified within Gnomoniaceae (Diaporthales) or as Sordariomycetes incertae sedis, belongs to Magnaporthaceae (Magnaporthales). Analysis of a six-locus Ascomycota-wide dataset including SSU and LSU sequences of S. lignatile revealed that this species, currently in Ascomycota incertae sedis, belongs to Pyronemataceae (Pezizomycetes, Pezizales).

The ATTEMPTS delivery systems for macromolecular drugs.

Aiming at successful targeted drug delivery - a system that possesses both targeting and prodrug features that can be activated once the system reaches the target site upon systemic administration - would be desired to reduce systemic toxicity. Previously we proposed a heparin/protamine-based system for delivery of protease drugs such as tissue-specific plasminogen activator (t-PA). This approach, termed 'antibody targeted, triggered, electrically modified prodrug-type strategy' (ATTEMPTS), would permit antibody-directed administration of inactive t-PA and allow a subsequent triggered release of the active t-PA at the target site. This system can be adapted to target tumor tissues when protein transduction domain (PTD) peptide such as TAT is incorporated in the ATTEMPTS construct. Both in vitro and preliminary in vivo studies using TAT-gelonin (TAT-Gel) and TAT-asparaginase (TAT-ASNase) conjugates have demonstrated that the on/off regulation of the membrane translocation activity of PTD at tumor target, followed by intracellular delivery of cytotoxic macromolecular drug, can be accomplished. Hence, the PTD-mediated delivery system derived from our previous ATTEMPTS approach is a system that incorporates all of the targeting function, prodrug feature, release mechanism and cell entry mechanism and could become a generic system for delivery of macromolecular drugs.

The minimal functional sequence of protamine.

Despite its nearly universal applications, protamine, a mixture of four major peptides with different sequences, is associated with clinically significant side effects. Through a well-designed enzyme digestion method, various low molecular weight protamine peptides were obtained. Among them, two low molecular weight protamine peptides with the same or even more potent heparin neutralization abilities as native protamine were identified through both in vitro and in vivo tests. In addition, in vivo experiments showed that compared to native protamine, these two low molecular weight protamine peptides were less toxic and would be safer for clinical use.

Nontoxic membrane translocation peptide from protamine, low molecular weight protamine (LMWP), for enhanced intracellular protein delivery: in vitro and in vivo study.

Naturally derived, nontoxic peptides from protamine by the authors, termed low molecular weight protamines (LMWPs), possess high arginine content and carry significant sequence similarity to that of TAT, by far the most potent protein transduction domain peptide. Therefore, it was hypothesized that these LMWPs would also inherit the similar translocation activity across the cell membrane, which enables any impermeable species to be transduced into the cells. LMWPs were prepared by enzymatic digestion of protamine, examined their capability of transducing an impermeable protein toxin into the tumor cells by chemical conjugation, and determined cytotoxicity of transduced protein toxin (e.g., gelonin) against cancer cell lines and a tumor-bearing mouse. In vitro results showed that LMWPs could indeed translocate themselves into several mammalian cell lines as efficiently as TAT, thereby transducing impermeable gelonin into the cells by chemical conjugation. In vivo studies further confirmed that LMWP could carry an impermeable gelonin across the tumor mass and subsequently inhibit the tumor growth. In conclusion, the presence of equivalent cell translocation potency, absence of toxicity of peptide itself, and the suitability for low-cost production by simple enzymatic digestion could expand the range of clinical applications of LMWPs, including medical imaging and gene/protein therapies.

Application of "ATTEMPTS" for drug delivery.

A novel enzyme drug delivery system, Antibody, Targeted, Triggered, Electrically, Modified, Prodrug, Type, Strategy ("ATTEMPTS"), was developed in our laboratory to attenuate the toxicity associated with drug activity at non-targeted tissues. Tissue plasminogen activator is a prime example of an enzyme drug that exhibits systemic toxicity due to its indiscriminate activation of both targeted (i.e., clot-bound) and non-targeted (i.e., systemic) plasminogen. In brief, tissue plasminogen activator (t-PA) was modified to contain positive surface charges and then rendered inactive upon electrostatic binding with a negatively charged heparin-antifibrin antibody conjugate. After targeting the complex to the clot site, t-PA activity was restored by administration of protamine, a clinical heparin antidote. Cation-modified t-PA (CM-tPA) was obtained by chemical conjugation of t-PA with a poly(Arg)7Cys peptide using the crosslinker N-succinimidyl 3-2-(pyridlydithio)propionate (SPDP). Anti-fibrin IgG was chemically conjugated with heparin via oxidation of the carbohydrate moiety on its Fc region. Both in vitro characterization and in vivo studies using a rat thrombosis model clearly demonstrated that heparin-IgG conjugate induced inhibition of CM-tPA could be effectively reversed upon addition of protamine. Overall, the ATTEMPTS system was proven to induce clot dissolution without causing t-PA associated systemic toxicity due to the degradation of critical plasma factors by systemic plasmin production.

[Experimental study on changes of sexual hormones in senior males living on high altitude].

OBJECTIVES: To investigate the characteristics of testosterone (T) and estradiol (E2) in senior males living on high altitude METHODS: According to the years of living on 3,100-meter high altitude. 90 senior males who were more than sixty years old were divided into three groups: group 1 (one year on the high altitude, n = 30), group 2 (two years on the high altitude, n = 30) and group 3 (over 10 years on the high altitude, n = 30). Additionally, there was a control group (living on the sea level). Radioimmunoassay technique was used to measure the level of T and E2 in their serum respectively. At the same time, the ratio of T/E2 was also examined. RESULTS: The levels of T, E2 and T/E2 of the three groups were 42.2 +/- 38.5, 70.0 +/- 31.5, 190.3 +/- 73.5 and 44.0 +/- 42.2, 60.6 +/- 28.3, 144.9 +/- 62.0 and 0.96 +/- 0.19, 1.16 +/- 0.11, 1.33 +/- 0.24, respectively. The levels of T, E2 and T/E2 of group 3 increased obviously than those of the other two groups (P < 0.01). CONCLUSIONS: The sexual hormone levels in senior males living on high altitude increase along with the living years.

Low molecular weight protamine as an efficient and nontoxic gene carrier: in vitro study.

BACKGROUND: The structural similarity between low molecular weight protamine (LMWP), prepared by enzymatic digestion of protamine, and HIV-TAT protein transduction peptide suggested the feasibility of LMWP as an efficient carrier for delivering therapeutic genes while alleviating the cytotoxicity of currently employed gene carriers. METHODS: LMWP was prepared by enzymatic digestion of protamine with thermolysine. The prepared LMWP peptide and TAT peptide, as well as their complexes with plasmid DNA (pDNA), were examined for cellular uptake behaviors by using confocal microscopy and flow cytometry. The complexation of pDNA and LMWP was monitored by gel retardation test as well as size and zeta potential measurements, and was then further assessed by DNase I protection assay. The transfection efficiency of pDNA/LMWP was examined by varying the pDNA content and charge ratio in the complex, and then compared with that of pDNA/PEI. Cytotoxicity induced by pDNA/LMWP and pDNA/PEI was also examined. RESULTS: Prepared LMWP showed similar transcellular localization behavior and kinetics to those of TAT, and efficiently transferred the pDNA into nucleus and cytoplasm in a short time period. The size and zeta potential of the pDNA/LMWP complex were 120 nm and 30 mV, respectively, which were adequately suitable for cellular uptake. After forming the complex, LMWP appeared to effectively protect pDNA against DNase I attack. The pDNA/LMWP complex showed significantly enhanced gene transfer than both naked pDNA and the pDNA/PEI complex, while exhibiting a markedly reduced cytotoxicity than that of the pDNA/PEI complex. CONCLUSIONS: The present study suggested that LMWP could be a useful and safe tool for enhancing delivery of bioactive molecules and therapeutic DNA products into cells when applied in gene therapy.

ATTEMPTS: a heparin/protamine-based triggered release system for the delivery of enzyme drugs without associated side-effects.

A prodrug type delivery system based on competitive ionic binding for the conversion of the prodrug to an active drug has been developed for delivery of enzyme drugs without their associated toxic side-effects. This approach, termed "ATTEMPTS" (antibody targeted, triggered, electrically modified prodrug-type strategy), would permit the administration of an inactive drug and then subsequently triggered release of the active drug at the target site. The underlying principle was to modify the enzyme with small cationic species so that it could bind a negatively charged heparin-linked antibody, and the latter would block the activity of the enzyme drug until it reached the target. To provide the enzyme drug with appropriate binding strength to heparin, a cationic poly(Arg)(7) peptide was incorporated onto the enzyme either by the chemical conjugation method using a bifunctional crosslinker or by the biological conjugation method using the recombinant methodology. Methods for drug modification, heparin-antibody conjugation, and the prodrug and triggered release features of the "ATTEMPTS" approach are described in detail in this review article.