Biosynthesis of precious metabolites in callus cultures of <Emphasis Type="Italic">Eclipta alba</Emphasis>

Eclipta alba (False daisy) is an important medicinal plant with well-known antihepatotoxic activity. However, no previous in vitro studies are available for its callus culture for increased production of antioxidant secondary metabolites. Herein, we maintained a competent protocol for callus culture of E. alba using stem and leaf explants grown on MS medium containing various concentrations of thidiazuron, 6-benzylaminopurine (BAP) either alone or in association with α-naphthalene acetic acid (NAA). Among all the applied plant growth regulators, BAP along with NAA resulted in maximal dry biomass of 18.0 and 13.8 g/l for stem and leaf explants, respectively. Furthermore, the highest production of phenolics (375.7 mg/l for stem-associated callus and 298 mg/l for leaf-associated callus) and flavonoids (62.0 and 52.3 mg/l for stem- and leaf-associated callus, respectively) were found to be present in optimized callus culture. Antioxidant activity was also elucidated for both stem and leaf derived calli. The highest antioxidant activities (~?93.5%) were witnessed for stem and leaf associated calli at set concentrations of 3.0 mg/l BAP?+?1.0 mg/l NAA and 4.0 mg/l BAP, respectively. High-performance liquid chromatography analyses revealed optimum accumulation of coumarin (1.98 mg/g DW) and wedelolactone (49.63 mg/g DW) in leaf associated callus and desmethylwedelolactone (69.96 mg/g DW), β-amyrin (0.8179 mg/g DW) and eclalbatin (0.3202 mg/g DW) in stem associated callus at optimized concentration.     

Phenotype and frequency of cells secreting IL-2, IL-4, IL-6, IL-10, IFN and TNF-α in human peripheral blood

The phenotype and frequency of cells in normal human peripheral blood spontaneously secreting IL-2, IL-4, IL-6, IL-10, IFN and TNF-α ex vivo was determined using ELIspot assays. CD4⁺T cells were the dominant source of IL-2 and IL-4 while multiple cell types (primarily CD8⁺lymphocytes) produced IFN. Fewer than 0.05% of mononuclear cells were spontaneously secreting these T cell derived factors. By comparison, IL-6, IL-10 and TNF-α were produced by 0.7–20% of PBMC. The primary sources of the latter cytokines were CD14⁺macrophages/monocytes. A significant positive correlation was found in the frequency of cells secreting IL-6, IL-10 and TNF-α ex vivo, suggesting that the release of such factors was coordinately regulated. No such correlation was found among IL-2, IL-4 and IFN secreting cells, indicating that the production of predominantly T cell derived cytokines was regulated independently.     

Evaluation of the differentiation status of neural stem cells based on cell morphology and the expression of Notch and Sox2

Neural stem cells (NSCs) isolated from a variety of sources are being developed as cellular therapies aimed at treating neurodegenerative diseases. During NSC culture and expansion it is important the cells do not differentiate prematurely because this may have an unfavorable effect on product quality and yield. In our study, we evaluated the use of Notch and Sox2 as markers for undifferentiated human and mouse NSCs. The expression of Notch2 and Sox2 during extensive-passage, low-oxygen culture and differentiation conditions were analyzed to confirm that the presence of these signature proteins directly correlates with the ability of NSCs to form new neurospheres and differentiate into multiple cell types. Using expression of Notch1, Notch2 and Sox2 as a reference, we then used flow cytometry to identify a specific morphological profile for undifferentiated murine and human NSCs. Our studies show that Notch and Sox2 expression, along with flow cytometry analysis, can be used to monitor the differentiation status of NSCs grown in culture for use in cellular therapies.     

N-(5-Methyl-1,3-Thiazol-2-yl)-2-{[5-((Un)Substituted- Phenyl)1,3,4-Oxadiazol-2-yl]Sulfanyl}acetamides. Unique Biheterocycles as Promising Therapeutic Agents

An electrophile, 2-bromo-N-(5-methyl-1,3-thiazol-2-yl)acetamide, was synthesized by the reaction of 5-methyl-1,3-thiazol-2-amine and bromoacetyl bromide in an aqueous medium. In a parallel scheme, a series of (un)substituted benzoic acids was converted sequentially into respective esters, acid hydrazides, and then into 1,3,4-oxadiazole heterocyclic cores. The electrophile was coupled with the aforementioned 1,3,4-oxadiazoles to obtain the targeted bi-heterocyles. Structural analysis of the synthesized compounds was performed by IR, EI-MS, ¹H NMR, and ¹³C NMR. The enzyme inhibition study of these molecules was carried out against four enzymes, namely, acetylcholinesterase, butyrylcholinesterase, α-glucosidase, and urease. The interactions of these compounds with respective enzymes were recognized by their in silico study. Moreover, their cytotoxicity was also determined to find out their utility as possible therapeutic agents.

     

The antimicrobial potential of a new derivative of cathelicidin from <Emphasis Type="Italic">Bungarus fasciatus</Emphasis> against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32–128 μg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 μg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.     

Design of ultrasensitive bisphenol A–aptamer based on platinum nanoparticles loading to polyethyleneimine-functionalized carbon nanotubes

Here, a highly sensitive electrochemical aptasensor based on a novel signal amplification strategy for the determination of bisphenol A (BPA) was developed. Construction of the aptasensor began with the deposition of highly dispersed platinum nanoparticles (PtNPs)/acid-oxidized carbon nanotubes (CNTs–COOH) functionalized with polyethyleneimine (PEI) at the surface of glassy carbon (PtNPs/PEI/CNTs–COOH/GC) electrode. After immobilizing the amine-capped capture probe (ssDNA1) through the covalent amide bonds formed by the carboxyl groups on the nanotubes and the amino groups on the oligonucleotides, we employed a designed complementary BPA–aptamer (ssDNA2) as a detection probe to hybridize with the ssDNA1. By adding BPA as a target, the aptamer specifically bound to BPA and its end folded into a BPA-binding junction. Because of steric/conformational restrictions caused by aptamer–BPA complex formation at the surface of modified electrode, the interfacial electron transfer of [Fe(CN)₆]^3−/4− as a probe was blocked. Sensitive quantitative detection of BPA was carried out by monitoring the decrease of differential pulse voltammetric responses of [Fe(CN)₆]^3−/4− peak current with increasing BPA concentrations. The newly developed aptasensor embraced a number of attractive features such as ease of fabrication, low detection limit, excellent selectivity, good stability and a wide linear range with respect to BPA.