Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr‐activated sepharose‐4B affinity chromatography

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA‐aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (k_d = 2.3 × 10^?11). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.     

Umbelliprenin shows antitumor,antiangiogenesis, antimetastatic,anti‐inflammatory,and immunostimulatory activities in 4T1 tumor‐bearing Balb/c mice

Umbelliprenin (UMB) has shown various pharmacological properties in vitro. We investigated the antineoplastic and immunostimulatory effects of UMB in 4T1 mammary‐tumor‐bearing mice. Two‐hundred microliter of UMB (12.5 mg/ml) was intraperitoneally administrated to healthy and tumor‐bearing female Balb/c mice for a period of 18 days. Data was analyzed using GraphPad Prism 5 software for Windows (version 5, La Jolla, CA). UMB caused a significant decrease in tumor size (P < 0.01). Serum interferon gamma (IFNγ) was augmented in both healthy and tumor‐bearing animals (P < 0.01), and IL‐4 declined in healthy animals (P < 0.01) treated with UMB. Expressions of Ki‐67, VEGF, CD31, MMP2, MMP9, VCAM1, and NF‐κB were significantly decreased in tumors from UMB‐treated animals (P < 0.001), whereas E‐Cadherin and TNFR1 expressions were markedly increased (P < 0.001). The rates of liver and lung metastases in UMB‐administrated animals were smaller compared to the control. UMB can potently inhibit tumor growth, angiogenesis, metastasis, and inflammation and potentiate an antitumor immune response in vivo. However, further investigations are required to evaluate the UMB mechanisms of action in cancerous cells.     

Improvement of Mercuric Chloride-Induced Testis Injuries and Sperm Quality Deteriorations by Spirulina platensis in Rats

The present study was undertaken to investigate the protective effect of the filamentous cyanobacterium Spirulina platensis (S. platensis) on mercury (II) chloride (HgCl₂)-induced oxidative damages and histopathological alterations in the testis of Wistar albino rats. The animals were divided into four equal groups, i ) control, ii ) HgCl₂, iii ) S. platensis and iv ) combination of HgCl₂+S. platensis. Oxidative stress, induced by a single dose of HgCl₂ (5 mg/kg, bw; subcutaneously, s.c.), substantially decreased (P<0.01) the activity level of testicular key enzymatic antioxidant biomarkers (superoxide dismutase, SOD; catalase, CAT and glutathione peroxidase, GPx), oxidative stress makers (blood hydroperoxide; testicular reduced glutathione, GSH and malondialdehyde, MDA), and testicular mercury levels. Moreover, HgCl₂ administration resulted in a significant (P<0.01) increase in the number of sperms with abnormal morphology and decrease in epididymal sperm count, motility, plasma testosterone level and testicular cholesterol. Furthermore, HgCl₂ exposure induced histopathological changes to the testis including morphological alterations of the seminiferous tubules, and degeneration and dissociation of spermatogenic cells. Notably, oral pretreatment of animals with Spirulina (300 mg/kg, bw) lowered the extent of the observed HgCl₂-mediated toxicity, whereby significantly reducing the resulting lipid peroxidation products, mercury accumulation in the testis, histopathological changes of the testes and spermatozoal abnormalities. In parallel, the pretreatment with Spirulina also completely reverted the observed Hg-Cl₂-induced inhibition in enzymatic activities of antioxidant biomarkers (SOD, CAT and GPx) back to control levels. The pretreatment of rats with S. platensis significantly recovered the observed HgCl₂-mediated decrease in the weight of accessory sex organs. Taken together, our findings clearly highlight the role of S. platensis as a protective modulator of HgCl₂-induced testicular injuries and suggest some therapeutic potential in mammals. Further investigation of therapeutic strategies employing Spirulina against heavy metals toxicity in humans is therefore warranted.     

The diagnostic impact of implementing a molecular-based algorithm to standard mycobacterial screening at a reference laboratory with an intermediate prevalence for non-respiratory samples

Rapid, reliable results can be given by molecular, direct detection and identification of the Mycobacterium tuberculosis (MTB/Mtb) complex from clinical samples. The Xpert MTB/RIF assay is an assay that has been availablefor more than a decade for identification of Mycobacterium tuberculosis and resistance to rifampicin. However, there is minimal evidence on its clinical usefulness in paucibacillary, non-respiratory samples. The Xpert MTB/RIF assay clinical utility index, its diagnostic characteristics and the number required to diagnose 2935 non-respiratory specimens submitted for routine mycobacterial work-up in a reference laboratory in an intermediate prevalence setting per specimen form were evaluated. The Xpert MTB/RIF assay showed a variable clinical utility index and number required to diagnose (NND) depending on the type of specimen, which was moderate in tissue biopsies (NND = 1.8) and excellent in pus and urine samples, compared to acid-fast microscopy and culture as a gold standard technique (NND = 1.1 and 1.2). Microscopy, on the other hand, consistently showed a weak to fair index of clinical usefulness in all specimen forms, with in NND of 2.3–12.5. The NND for detecting tuberculous infection in the cerebrospinal fluid by the Xpert MTB/RIF assay was noted to be 1.2, with a moderate clinical utility index of 0.8. The evidence presented indicates that the overall appropriate diagnostic utility of the Xpert MTB/RIF assay is clinically successful in most non-respiratory samples. To check the cost-effectiveness and prognostic effect of integrating this completely automated molecular-based assay into the routine testing algorithm for non-respiratory mycobacterial specimens, further data must be collected.     

Cloning,high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain and a heavy chain (HC) linked through a disulfide bond. In the present study we explored the immunogenicity and protective capability of the most effective part corresponding to 1163-1256 residues of botulinum type E neurotoxin HC gene. DNA encoding the 93 C-terminal amino acid of HC residues was synthesized with optimal codon usage for expression. These DNA fragments were ligated into a pLivSelect vector and subcloned into expression vector pET32a. Recombinant plasmids were then transformed into Escherichia coli strain BL21 DE3. The recombinant protein was purified by nickel affinity gel column chromatography. The HC1163-1256 was identified by antibodies raised against BoNT/E. HC1163-1256 was shown to bind with synaptotagmin and gangliosides, indicating that the expressed and purified HC1163-1256 protein retains a functionally active conformation. The immunization with recombinant protein induced a protection level in mice. The immunization with 2 μg of the recombinant protein induced a significant protection level in mice. In conclusion, availability of the recombinant protein provides an effective system to study the biochemical and physical interactions involved during BoNT binding to nerve cells and protection against its toxicity.     

Hepatoprotective activity of Schouwia thebica webb

Oral administration of alcoholic extracts of Schouwia thebica Webb showed that extracts are safe for human use. The studied extracts are considered safe, since they failed to induce death of mice in doses up to 4000 mg/kg body weight. Hepatoprotective activity was studied for the total alcoholic extracts. The total extract was fractionated in turn with diethyl ether, chloroform, ethyl acetate, and n-butanol, respectively. These extracts were tested for possible hepatoprotective activity. It was found that the ethyl acetate and n-butanol extracts of S. thebica Webb showed hepatoprotective activity. These extracts significantly reduced the increase in activities of ALT, AST, and GGT, and levels of glucose, triglycerides, and cholesterol in serum of CCl(4)-treated rats. The extracts showing activity were found to contain flavonoids; one new compound, chrysoeriol-7-O-xylosoide- (1,2)-arabinofuranoside (2), in addition to another known four compound chrysoeriol (1), quercetin (3), quercetin-7-O-rhamnoside (4), and kaempferol-3-O-beta-D-glucoside (5). The isolated new compound was mainly found to be responsible for this activity when tested on animals in the laboratory. The structures were established by melting point, UV spectroscopy, EI-Mass, Fab-Mass, and 1D and 2D NMR spectroscopic techniques on a 600MHz instrument.     

Cytotoxic isoferulic acidamide from Myricaria germanica (Tamaricaceae)

Tamgermanitin, a unique N-trans-Isoferuloyltyramine, together with the hitherto unknown polyphenolics, 2,4-di-O-galloyl-(α/β)-glucopyranose and kaempferide 3,7-disulphate have been isolated from the leaf aqueous ethanol extract of the false tamarisk, Myricaria germanica DESV. In addition, 18 known phenolics were also separated and characterized. All structures were elucidated on the basis of detailed analysis of 1D- ¹H and ¹³C NMR, COSY, HSQC, HMBC and HRFTESIMS spectral data. The extract, its chromatographic column fractions and the isolated isoferuloyltyramine, tamgermanetin demonstrated potential cytotoxic effect against three different tumor cell lines, namely liver (Huh-7), breast (MCF-7) and prostate (PC-3). The IC₅₀''s were found to be substantially low with low-resistance possibility. DNA flow-cytometic analysis indicated that column fractions and tamgermanetin enhanced pre-G apoptotic fraction. Both materials showed inhibiting activity against PARP enzyme activity. In conclusion, we report the isolation and identification of a novel compound, tamgermanitin, from the aqueous ethanol extract of Myricaria germanica leaves. Further, different fractions of the extract and tamgermanitin exhibit potent cytotoxic activities which warrant further investigations.     

Synthesis and characterization of transition metal dithiocarbamate derivatives of 1-aminoadamantane: Crystal structure of (N-adamantyldithiocarbamato)nickel(II)

Four bis(N-adamantyldithiocarbamato)metal(II) complexes [M(S₂CNHC₁₀H₁₅)₂] (M = Ni, Zn, Cd, and Hg) were prepared by metathesis of the corresponding potassium salt, K[S₂CNHC₁₀H₁₅]. Characterization of the prepared complexes shown that the presence of only one band in the region 1000 cm⁻¹ in IR spectra can be attributed to a completely symmetrical bonding of the dithiocarbamate ligand, acting in a symmetrical bidentate mode. Also the short thioureide C-N distance in X-ray analysis of Ni complex confirm the delocalization of the π electrons over the S₂CN moiety and strong double bond character.