Integrated dataset of screening hits against multiple neglected disease pathogens

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.     

Heterologous virus-induced gene silencing as a promising approach in plant functional genomics

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.     

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.     

Epstein-Barr virus induces an oxidative stress during the early stages of infection in B lymphocytes, epithelial, and lymphoblastoid cell lines

The study investigates the direct effect of Epstein-Barr virus infection on the oxidative profile of in vitro cultivated human cells. For this purpose, a panel of human EBV target cells presenting heterogeneity in their cellular and culture types (epithelial cells or lymphocytes; primary culture or continuous cell culture) was selected. These cells are purified human B lymphocytes, DG75, 293, and HepG2 cell lines. The oxidative stress was evaluated during the early stages of infection (2, 12, and 24 h) by measuring malondialdehyde, the end product of the lipid peroxidation, as well as the activities of two antioxidant enzymes: catalase and superoxide dismutase. The obtained results were compared with those of the untreated cells and the K562 cell line which has no interaction with EBV. The incubation of the different target cells with EBV induced an oxidative stress in the purified B lymphocytes, DG75, and 293, but not in HepG2 and K562. This oxidative stress was highlighted by an increase in MDA level (P < 0.05), which began 2 h after the addition of the virus and persisted after 12 and 24 h. Simultaneously, a decrease in catalase and superoxide dismutase activities was observed (P < 0.05), suggesting an alteration of the molecular mechanisms promoting cellular resistance to reactive oxygen species (ROS). The efficiency of EBV infection, assessed by viral DNA PCR amplification, was confirmed in 293 and DG75 but not in HepG2, which was in total concordance with their oxidative profiles. In conclusion, the EBV infection of B and epithelial cells leads to the establishment of an oxidative stress which can play a key role during the viral transformation.     

Semisynthesis of some novel thiourea and carbamimidothioic acid derivatives using natural alkaloid L-norephedrine and their anticancer activity

A novel series of thiourea and carbamimidothioic acid derivatives was synthesized using natural alkaloid L-norephedrine as a starting material. Structures of the newly synthesized compounds were confirmed by analytical and spectral data. The synthesized compounds were evaluated in vitro for anticancer activity against the human breast (MCF-7), human liver (HEPG2), and human colon (HCT116) cancer cell lines. Best activity of the synthesized compounds was expressed against HEPG2, however, none of the compounds exceeded the IC₅₀ of doxorubicin. The corresponding N-(1-(2-chloroacetoxy)-1-phenylpropan-2-yl)-N′-p-tolylcarbamimidothioic acid was the most potent compound and exhibited higher cytotoxic activity against the human colon cancer cell line (HCT116) when compared with the reference drug doxorubicin. Also, this compound was the most active against the MCF-7 cell line but less active than the positive control.     

Citrinin detection by intensified fluorescence signal of a FRET-based immunosensor using magnetic/silica core–shell

The specific immune-reaction between the anti-citrinin antibody immobilized on the surface of magnetic/silica core–shell (MSCS) and the citrinin–Rho123–BSA conjugate brings the Rho123 fluorophore as an acceptor and the QDs as a donor in close spatial proximity and causes FRET for occurring upon photo-excitation of the QDs. The novelties of this study include: (1) immobilization of the MSCS; (2) large amount of the immobilized QDs, and (3) immobilization of a large amount of Rho123 on the BSA macromolecule. Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and tellurium in the presence of sodium borohydride. Magnetic nanoparticles were synthesized using FeSO₄ and FeCl₃. The prepared magnetic nanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Transmission electron microscopy (TEM) analysis was used for investigating shape and monodispersity of the nanoparticles. EDC/NHS was used as a cross linking agent for immobilization of the QDs, conjugation of citrinin to amino groups of BSA, labeling of BSA with Rho123 and also for immobilization of the amino-functionalized MSCS on the immobilized QDs. Immobilization of the anti-citrinin antibody on the surface of the amino-functionalized MSCS was performed by Schiff-base mechanism. By using these three effective strategies, sensitivity of the designed nanobiosensor was incredibly enhanced as a very low limit of detection (up to 0.1 pM). The feasibility of this technique was tested by the detection of citrinin in the spiked human serum. Results showed that there was a linear correlation between the decreased fluorescence intensity of the Rho123 and increased fluorescence intensity of the QDs with increasing concentration of citrinin in the spiked samples in the range of 1–6 pM. According to obtained results, we conclude that this highly sensitive detection scheme is a easy, quick and impressive method that can be used in optical-based nanosensors.     

Anticonvulsant and analgesic activities of crude extract and its fractions of the defensive secretion from the Mediterranean sponge, Spongia officinalis

ABSTRACT: This study progresses in the direction of identifying component(s) from the Mediterranean sponge, Spongia officinalis with anticonvulsant and analgesic activities. We investigated the efficacy of crude extract and its semi-purified fractions (F1-F3) of the defensive secretion from Spongia officinalis for their in vivo anticonvulsant activity using the pentylenetetrazole (PTZ) seizure model and analgesic activity using the writhing test in mice. Among the series the crude extract exhibited interesting analgesic activity in a dose dependent manner. Similarly the fraction F2 showed a partial protection of mice from PTZ-induced seizure and interesting analgesic activity in a dose dependent manner. The purification and the determination of chemical structure(s) of compound(s) of this active fraction are under investigation.     

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.     

Design and synthesis of antimicrobial,anticoagulant, and anticholinesterase hybrid molecules from 4-methylumbelliferone

We designed and synthesized new series of diverse triazoles, isoxazoles, isoxazolines, and aziridines linked 4-methylumbelliferone 1 using intermolecular 1,3-dipolar cycloaddition reactions. Structures of these compounds were established on the basis of ¹H NMR, ¹³C NMR, and ESI-HRMS. All prepared compounds were evaluated for their antimicrobial, anticoagulant, and anticholinesterase activities. Interestingly, among the tested molecules, some of the analogs displayed better activities than the parent 4-methylumbelliferone 1 such as 6a and 6d for their antifungal properties. Moreover, compounds 4, 5, 6, and 7 showed the importance of the added fragments to 4-methylumbelliferone 1 via the linker methylene to have good activity.