Comparison of different methods for erythroid differentiation in the K562 cell line

Objective

To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis.

Results

Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001).

Conclusions

Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

Organic two-photon nanoparticles modulate reactive oxygen species,intracellular calcium concentration,and mitochondrial membrane potential during apoptosis of human gastric carcinoma SGC-7901 cells

Objectives

To investigate the biocompatibility of human gastric carcinoma cells (SGC-7901) with organic two-photon nanoparticles (NPs).

Results

Different concentrations of NPs were incubated with SGC-7901 cells for different times. The levels of cell apoptosis, reactive oxygen species (ROS), intracellular calcium, and mitochondrial membrane potential (MMP) were measured by staining the SGC-7901 cells with Annexin V-FITC/PI, 2′,7′-dichlorofluorescin diacetate, Fluo-3 AM, and Rhodamine 123, followed by the flow cytometry assay. NPs at <4 µg/ml, did not have any significant effect on apoptosis, necrosis, generation of ROS, increase of intracellular Ca²⁺ concentration or decrease of MMP in SGC-7901 cells, but >4 µg/ml had a major effects on all the above mentioned parameters.

Conclusion

2,5,2′,5′-Tetra(4-N,N-diphenylamine styryl) biphenyl NPs can be used at an appropriate concentration as a safe drug carrier or imaging marker and may serve as an effective tool for developing a photodynamic cancer therapy.

     

Molecular Characterization and Antifungal Susceptibility Testing of Sequentially Obtained Clinical <Emphasis Type="Italic">Cryptococcus deneoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Isolates from Ljubljana,Slovenia

Aim

To retrospectively investigate the epidemiology of cryptococcosis in Ljubljana, Slovenia.

Methodology

Forty-six sequentially obtained isolates from 19 patients were subjected to amplified fragment length polymorphism (AFLP) genotyping, microsatellite typing, mating- and serotype PCRs and antifungal susceptibility testing.

Results

Majority of the isolates were Cryptococcus deneoformans (n = 29/46; 63%) followed by Cryptococcus neoformans (n = 16/46; 34.8%) and their interspecies hybrid (n = 1/46; 2.2%). Mating-type α was predominant, two mating-type a C. deneoformans isolates and one mating-type a/α isolate were observed. Several mixed infections were found by microsatellite typing; one patient had a persisting C. deneoformans infection for > 2.5 years. For C. deneoformans, the in vitro antifungal MIC₉₀ and susceptibility ranges were for amphotericin B 0.25 µg/ml (0.031–0.25 µg/ml), 5-fluorocytosine 0.25 µg/ml (0.063–4 µg/ml), fluconazole 8 µg/ml (0.5–16 µg/ml), voriconazole 0.063 µg/ml (0.008–0.125 µg/ml), posaconazole 0.063 µg/ml (0.008–0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031–0.125 µg/ml). For C. neoformans, these values were for amphotericin B 0.25 µg/ml (0.063–0.5 µg/ml), 5-fluorocytosine 1 µg/ml (0.063–1 µg/ml), fluconazole 16 µg/ml (0.5–64 µg/ml), voriconazole 0.125 µg/ml (0.008–0.25 µg/ml), posaconazole 0.063 µg/ml (0.008–0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031–0.125 µg/ml).

Conclusions

Majority of the cases were caused by C. deneoformans; mating-type α was predominant. Several mixed infections were identified by AFLP genotyping and microsatellite typing. Despite antifungal therapy, a cryptococcal isolate could persist for years. Voriconazole, itraconazole and posaconazole were the most potent antifungal drugs.

     

A metabolomic,geographic, and seasonal analysis of the contribution of pollen-derived adenosine to allergic sensitization

Background

Studies on ragweed and birch pollen extracts suggested that the adenosine content is an important factor in allergic sensitization. However, exposure levels from other pollens and considerations of geographic and seasonal factors have not been evaluated.

Objective

This study compared the metabolite profile of pollen species important for allergic disease, specifically measured the adenosine content, and evaluated exposure to pollen-derived adenosine.

Methods

An NMR metabolomics approach was used to measure metabolite concentrations in 26 pollen extracts. Pollen count data was analyzed from five cities to model exposure.

Results

A principal component analysis of the various metabolites identified by NMR showed that pollen extracts could be differentiated primarily by sugar content: glucose, fructose, sucrose, and myo-inositol. In extracts of 10 mg of pollen/ml, the adenosine was highest for grasses (45 µM) followed by trees (23 µM) and weeds (19 µM). Pollen count data showed that tree pollen was typically 5–10 times the amount of other pollens. At the daily peaks of tree, grass, and weed season the pollen-derived adenosine exposure per day is likely to be only 1.1, 0.11, and 0.12 µg, respectively. Seasonal models of pollen exposure and respiration suggest that it would be a rare event limited to tree pollen season for concentrations of pollen-derived adenosine to approach physiological levels.

Conclusion

Sugar content and other metabolites may be useful in classifying pollens. Unless other factors create localized exposures that are very different from these models, pollen-derived adenosine is unlikely to be a major factor in allergic sensitization.

     

Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells

Background

Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.?

Methods

Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the E?xpi293 cells with PEI (which has a negligible cost).

Results

The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aβ) aggregates in vivo.

Conclusions

This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

     

Triterpenoid-rich loquat leaf extract induces growth inhibition and apoptosis of pancreatic cancer cells through altering key flux ratios of glucose metabolism

Introduction

Loquat leaf extract (LLE) is a mixture rich in terpenoids, and has broad biological activities including the inhibition of cancer cell growth. The exact metabolic mechanism of this growth inhibiting effect is not known.

Objectives

We investigated the cellular metabolic effect of LLE, and ursolic acid (UA) on pancreatic cancer cells using a ¹³C carbon tracing technology.

Methods

MIA PaCa-2 cells were cultured in medium containing [1, 2 ¹³C₂]-glucose in the presence of either LLE (50 µg/ml), UA (50 µM), or metformin (1 mM). The mass isotopomer distribution of glucose, lactate, ribose, glutamate and palmitate in medium was determined. Based on the mass isotopomer distribution in metabolites we were able to determine individual ¹³C enrichment (∑M?×?n) and the minimum fraction of new synthesis?(1-M0) in each metabolite. Several flux ratios of energy metabolic pathways were calculated from the mass isotopomer ratios of these metabolites.

Results

We found that tumor viability was suppressed by LLE and UA in a dose dependent manner, and the tumor-inhibiting effect was associated with the changes in oxidative/non-oxidative pentose (Ox/Non-ox) and pyruvate dehydrogenase/isocitrate dehydrogenase (PDH/ICDH) flux ratios resulting in decreased new syntheses of ribose and fatty acids.

Conclusion

Metabolic homeostasis (balance of fluxes) in cancer cells is maintained through the regulation of metabolic fluxes by oncogenes and tumor-suppressor genes. Treatment of MIA PaCa-2 cells by LLE, UA and metformin likely altered key metabolic flux ratios affecting metabolic homeostasis required for energy and macromolecular production in tumor growth.

     

Pranlukast,a novel binding ligand of human Raf1 kinase inhibitory protein

Objectives

To study the binding of pranlukast to hRKIP and its regulatory role in the Raf1/MEK/ERK signal pathway.

Results

NMR and fluorescence experiments demonstrated hRKIP could bind pranlukast with a binding constant of 1016 mM^?1. Residues (Y81, S109 and Y181) on the conserved ligand-binding pocket of hRKIP played a crucial role in binding pranlukast, and their mutations reduced the binding affinity more than 85 %. Furthermore, 25 μM pranlukast could up-regulate the ERK phosphorylation by about 17 %.

Conclusion

Pranlukast may be used as a potential drug precursor for treating hRKIP involved diseases.

     

Differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in fibrin scaffold by a histone deacetylase inhibitor

Background

Human adipose-derived stem cells (hADSCs) are capable of differentiating into many cells such as cardiac cells. Different types of inducers are used for cardiac cell differentiation, but this question still remains to be investigated, which one is the best. The aim of this paper was to investigate the effect of combination of fibrin scaffold and trichostatin A (TSA), for differentiation of hADSCs into cardiomyocyte-like cells.

Methods

After approval of characteristics of hADSCs and fibrin scaffold, hADSCs were cultured in fibrin scaffold with 10 µM TSA for 72 h and kept in standard conditions for 4 weeks. QRT-PCR and immunostaining assay were performed for evaluating the expression pattern of special cardiac genes and proteins.

Results

In particular, our study showed that fibrin scaffold alongside TSA enhanced expression of the selected genes and proteins.

Conclusions

We concluded that the TSA alone or with fibrin scaffold can lead to the generation of cardiac like cells in a short period of time.

     

CDK inhibitor SU9516 induces tetraploid blastocyst formation from parthenogenetically activated porcine embryos

Objective

To examine the effect of SU9516, a cyclin-dependent kinase inhibitor, on the induction of tetraploid blastocyst formation in porcine embryos by parthenogenetic activation.

Results

Karyotype analysis of blastocysts showed that in the SU9516-treatment group 56% were tetraploid, whereas in the cytochalasin B (CB) group 67% were diploid. The level of maturation-promoting factor (MPF) in stimulated embryos treated with 10 µM SU9516 for 4 h was lower than in embryos treated with CB group (103 vs. 131 pg/ml). The mRNA expression levels of Nanog significantly increased in SU9516-treated embryos than CB group.

Conclusion

SU9516 can induce tetraploid blastocyst formation at high efficiency. SU9516 can significantly influence the in vitro developmental competence of porcine parthenogenetically activated embryos by influencing the level of MPF and the gene related apoptosis and pluripotency.

     

Inactivation of virginiamycin by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Objective

To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans.

Results

Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three previously identified strains from clade 5 were compared for inactivation of virginiamycin. Laccase-induced culture supernatants from clade 7 strains were more effective at inactivation of virginiamycin, particularly at 50 °C. Clade 7 strain NRRL Y-2567 inactivated 6 µg virginiamycin/ml within 24 h. HPLC analyses indicated that virginiamycin was degraded by A. pullulans.

Conclusions

A. pullulans has the potential for the bioremediation of virginiamycin-contaminated materials, such as distiller’s dry grains with solubles (DDGS) animal feed produced from corn-based fuel ethanol production.

     

Enhancement of production and activity of alkaline zinc metalloprotease from <Emphasis Type="Italic">Salinivibrio proteolyticus</Emphasis> using low intensity direct electric current and zinc nanoparticles

Objectives

To improve the production and activity of an alkaline zinc metalloprotease from Salinivibrio proteolyticus in response to ZnSO₄ (ionic and nanoparticle forms) and low intensity direct electric current (LIDC).

Results

A DC of 50 µA for 10 min increased enzyme production from 35 to 53 U ml^?1 when applied to the stationary phase bacterial cells. Zn²⁺ improved enzyme production better than zinc nanoparticles (52 vs. 43.5 U ml^?1). Zinc nanoparticles (0.5 mM) added to an enzyme reaction mixture containing casein (0.65 %) and 20 mM Tris/HCl buffer (pH 8) improved enzyme activity more than Zn²⁺ (42 vs. 36 U ml^?1).

Conclusion

LIDC exposure (50 µA, 10 min) to the stationary phase bacterial cells increases metalloprotease production in Salinivibrio. A low concentration of zinc nanoparticles (0.5 mM) increases maximum enzyme activity.

     

Gelatin microspheres containing calcitonin gene-related peptide or substance P repair bone defects in osteoporotic rabbits

Objectives

To investigate the therapeutic effect of gelatin microspheres containing different concentrations of calcitonin gene-related peptide (CGRP) or substance P on repairing bone defects in a rabbit osteoporosis model.

Results

Gelatin microspheres containing different concentrations of CGRP or substance P promoted osteogenesis after 3 months in a rabbit osteoporotic bone defective model. From micro-computed tomography imaging results, 10 nM CGRP was optimal for increasing the trabecular number and decreasing the trabecular bone separation degree; similar effects were observed with the microspheres containing 1 µM substance P. Histological analysis showed that the gelatin microspheres containing CGRP or substance P, regardless of the concentration, effectively promoted osteogenesis, and the highest effect was achieved in the groups containing 1 µM CGRP or 1 µM substance P.

Conclusions

Gelatin microspheres containing CGRP or substance P effectively promoted osteogenesis in a rabbit osteoporotic bone defect model dose-dependently, though their effects in repairing human alveolar ridge defects still need further investigation.

     

Transient expression of recombinant tissue plasminogen activator (rt-PA) gene in cucurbit plants using viral vector

Objective

To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants.

Results

The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml.

Conclusions

The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.

     

Heteroresistance to Fluconazol in Clinical and Environmental Brazilian Strains of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>/<Emphasis Type="Italic">C. gattii</Emphasis> Species Complex

Purpose of Study

To review the literature on heteroresistance to fluconazole (FLC) and investigate the level of heteroresistance to FLC (LHF), we analyzed 100 clinical and environmental Brazilian Cryptococcus strains.

Recent Findings

Heteroresistance is a phenomenon described as the emergence of resistant subpopulation cells within a single susceptible strain that can tolerate higher concentrations of fluconazole above the minimal inhibitory concentration (MIC) level.

Summary

We found lower FLC-MICs (0.12–64 mg/L) than LHF (8–128 mg/L). Highly heteroresistant adapted subpopulations (256 mg/L) was found in minority (9%) strains, but importantly, 33% showed low FLC-MIC (8 mg/L). We concluded for similar LHF in both species, but higher LHF in clinical strains in comparison to environmental ones. Our findings stressed that the LHF is not correlated to species and pretty is strain-dependent and alert about high heteroresistant subpopulations that hardly reverts to the original LHF even upon the removal of drug pressure.

     

Knockout of <Emphasis Type="Italic">CTNNB1</Emphasis> by CRISPR-Cas9 technology inhibits cell proliferation through the Wnt/β-catenin signaling pathway

Objective

To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/β-catenin signaling pathway.

Results

CTNNB1 gene of HEK 293T cells was knocked out by CRISPR-Cas9. This was confirmed by sequencing and western blotting. Methylthiazolyl-tetrazolium bromide assays indicated that deletion of β-catenin significantly weakened adhesion ability and inhibited proliferation rate (P < 0.01) of HEK 293T cells. Nevertheless, deletion of β-catenin did not affect apoptosis of HEK 293T cells, which was analyzed by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. In addition, expression level of GSK-3β, CCND1, and CCNE1 detected by qPCR and expression level of N-Cadherin and cyclin D1 detected by western blotting were significantly decreased (P < 0.01) while expression of γ-catenin detected by western blotting was significantly increased (P < 0.001).

Conclusions

Knockout of CTNNB1 disturbed Wnt/β-catenin signaling pathway and significantly inhibited adhesion and proliferation of HEK 293T cells.

     

PEGylation of cytochrome <Emphasis Type="Italic">c</Emphasis> at the level of lysine residues mediated by a microbial <Emphasis Type="Italic">trans</Emphasis>glutaminase

Objectives

To establish a method for microbial transglutaminase (mTG)-mediated PEGylation of proteins at the level of lysine (Lys) residues.

Results

Carboxybenzyl-glutaminyl–glycinyl-methoxypolyethylene glycol (CBZ-QG-mPEG) was prepared by introducing carboxybenzyl-glutaminyl-glycine (CBZ-QG) to mPEG amine. The analysis by Fourier transform infrared spectroscopy and SDS-PAGE showed that CBZ-QG-mPEG was successfully synthesized and can be recognized by mTG as an acyl donor to modify therapeutic protein, cytochrome c (cyt c). Finally, under an optimized condition (cyt c 0.5 mg/ml, CBZ-QG-mPEG 11.25 mg/ml, mTG 0.5 mg/ml, 37 °C, 2 h), the PEGylation yield reached 76.5 %.

Conclusions

This is the first study regarding the PEGylation of protein at the level of Lys residues catalyzed by mTG. The novel method could be employed to immobilize active proteins and modify therapeutic proteins.

     

Tryptase as a marker of severity of aortic valve stenosis

Background

Severe aortic valve stenosis is one of the most common cause of mortality in adult patients affected with metabolic syndrome, a condition associated with an active inflammatory process involving also mast cells and their mediators, in particular tryptase. The aim of this study was to characterize the possible long-term prognostic role of tryptase in severe aortic valve stenosis.

Case presentation

The baseline serum tryptase was measured in 5 consecutive patients admitted to our Hospital to undergo aortic valve replacement for severe acquired stenosis. Within 2 years after, the patients were evaluated for the occurrence of major cardiovascular events (MACE). The tryptase measurements were higher in patients experiencing MACE (10.9, 11.7 and 9.32 ng/ml) than in non-MACE ones (5.69 and 5.58 ng/ml).

Conclusions

In patients affected with severe aortic stenosis, baseline serum tryptase may predict occurence of MACE. Further studies are needed to demonstrate the long-term prognostic role of this biomarker.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.