Antiprotozoal activity of dehydroabietic acid derivatives against Leishmania donovani and Trypanosoma cruzi - SPECIAL ISSUE "NEW TALENT: EUROPE"

Derivatives of dehydroabietic acid bearing different amino acids scaffolds have potent antiprotozoal activity against Leishmania donovani and Trypanosoma cruzi, with good to high selectivity, and can therefore be regarded as good models for further development into new drugs to fight leishmaniasis and Chagas disease. Several of the tested compounds were able to kill parasites residing inside cells, with IC50 values ranging from 2.3 to 9 mu M (L. donovani) and 1.4 to 5.8 mu M (T. cruzi), reflecting their ability to fight these infections at the relevant stage responsible for disease. One of the compounds, bearing a 3-pyridyl-Dalanine side chain, was 1.5-fold more potent against T. cruzi amastigotes residing in L6 cells than the reference compound benznidazole.Peer reviewe

Isolation and characterization of phenol degrading bacterium strain Bacillus thuringiensis J20 from olive waste in Palestine

This study aimed at isolation of phenol degrading bacteria from olive mill wastes in Palestine. The efficiency of phenol removal and factors affecting phenol degradation were investigated. A bacterial strain (J20) was isolated from solid olive mill waste and identified as Bacillus thuringiensis based on standard morphological, biochemical characteristics and 16SrRNA sequence analysis. The strain was able to grow in a phenol concentration of 700 mg/L as the sole carbon and energy source. The culture conditions showed a significant impact on the ability of these cells to remove phenol. This strain exhibited optimum phenol degradation performance at pH 6.57 and 30 C . Under the optimized conditions, this strain could degrade 88.6% of phenol (700 mg/L) within 96 h when the initial cell density was OD600 0.2. However, the degradation efficiency could be improved from about 88% to nearly 99% by increasing the cell density. Immobilization of J20 was carried out using 4% sodium alginate. Phenol degradation efficiency of the immobilized cells of J20 was higher than that of the free cells, 100% versus 88.6% of 700 mg/L of phenol in 120 h, indicating the improved tolerance of the immobilized cells toward phenol toxicity. The J20 was used in detoxifying crude OMWW, phenolic compounds levels were reduced by 61% compared to untreated OMWW after five days of treatment. Hence, B. thuringiensis-J20 can be effectively used for bioremediation of phenol-contaminated sites in Palestine. These findings may lead to new biotechnological applications for the degradation of phenol, related to olive oil production.The authors thank Prof. Charles Greenblatt from Department of Microbiology and Molecular Genetics, Hebrew University – Hadassah Medical School, Jerusalem, Israel for his help. Suheir I. Ereqat and Ahmad A. Abdelkader contributed equally to this manuscript

Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Protozoal parasites of the genus Leishmania are transmitted by sand fly bites to humans and animals. Three major forms of disease are caused by these parasites: cutaneous leishmaniasis, responsible for disfiguring skin wounds; mucocutaneous leishmaniasis, causing non-healing ulceration around the mouth and nose; and the potentially fatal visceral leishmaniasis, involving internal organs such as the spleen and liver. More than 2 million new human infections are caused annually by leishmaniasis globally, it is endemic in more than 88 countries and prevalent also as an imported disease in non-endemic regions due to travel and tourism. Most species of Leishmania that infect humans are zoonotic and transmitted from animal reservoir hosts. As various leishmanial parasites cause disease with similar symptoms, but require different therapeutic regimens and have dissimilar prognoses, reliable, sensitive and rapid diagnostic assays are needed. This study focuses on the five main species that cause leishmaniasis in the Old World. It presents a new assay for rapid detection, species identification and quantification of leishmanial parasites in clinical samples, reservoir hosts and sand flies. This technique could be especially valuable in regions where several leishmanial species exist, in non-endemic regions where infected patients require a rapid diagnosis, and for epidemiological host and vector studies leading to prevention programs

Pfsec13 is an unusual chromatin-associated nucleoporin of plasmodium falciparum that is essential for parasite proliferation in human erythrocytes

In Plasmodium falciparum, the deadliest form of human malaria, the nuclear periphery has drawn much attention due to its role as a subnuclear compartment involved in virulence gene expression. Recent data have implicated components of the nuclear envelope in regulating gene expression in several eukaryotes. Special attention has been given to nucleoporins that compose the nuclear pore complex (NPC). However, very little is known about components of the nuclear envelope in Plasmodium parasites. Here we characterize PfSec13, an unusual nucleoporin of P. falciparum, which shows unique structural similarities suggesting that it is a fusion between Sec13 and Nup145C of yeast. Using super resolution fluorescence microscopy (3D-SIM) and in vivo imaging, we show that the dynamiclocalization of PfSec13 during parasites' intra-erythrocytic development corresponds with that of the NPCs and that these dynamics are associated with microtubules rather than with F-actin. In addition, PfSec13 does not co-localize with the heterochormatin markers HP1 and H3K9me3, suggesting euchromatic location of the NPCs. The proteins associated with PfSec13 indicate that this unusual Nup is involved in several cellular processes. Indeed, ultrastructural and chromatin immunoprecipitation analyses revealed that, in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin. Finally, we used peptide nucleic acids (PNA) to downregulate PfSec13 and show that it is essential for parasite proliferation in human erythrocytes. © 2013. Published by The Company of Biologists Ltd

Antiplasmodial dihetarylthioethers target the coenzyme A synthesis pathway in Plasmodium falciparum erythrocytic stages

Background: Malaria is a widespread infectious disease that threatens a large proportion of the population in tropical and subtropical areas. Given the emerging resistance against the current standard anti-malaria chemotherapeutics, the development of alternative drugs is urgently needed. New anti-malarials representing chemotypes unrelated to currently used drugs have an increased potential for displaying novel mechanisms of action and thus exhibit low risk of cross-resistance against established drugs. Results: Phenotypic screening of a small library (32 kinase-inhibitor analogs) against Plasmodium falciparum NF54-luc asexual erythrocytic stage parasites identified a diarylthioether structurally unrelated to registered drugs. Hit expansion led to a series in which the most potent congener displayed nanomolar antiparasitic activity ( IC50 = 39 nM, 3D7 strain). Structure–activity relationship analysis revealed a thieno[2,3-d]pyrimidine on one side of the thioether linkage as a prerequisite for antiplasmodial activity. Within the series, the oxazole derivative KuWei173 showed high potency ( IC50 = 75 nM; 3D7 strain), good solubility in aqueous solvents (1.33 mM), and >100-fold selectivity toward human cell lines. Rescue experiments identified inhibition of the plasmodial coenzyme A synthesis as a possible mode of action for this compound class. Conclusions: The class of antiplasmodial bishetarylthioethers reported here has been shown to interfere with plasmodial coenzyme A synthesis, a mechanism of action not yet exploited for registered anti-malarial drugs. The oxazole congener KuWei173 displays double-digit nanomolar antiplasmodial activity, selectivity against human cell lines, high drug likeness, and thus represents a promising chemical starting point for further drug development

Development of a Simple Mechanical Screening Method for Predicting the Feedability of a Pharmaceutical FDM 3D Printing Filament

Purpose: The filament-based feeding mechanism employed by the majority of fused deposition modelling (FDM) 3D printers dictates that the materials must have very specific mechanical characteristics. Without a suitable mechanical profile, the filament can cause blockages in the printer. The purpose of this study was to develop a method to screen the mechanical properties of pharmaceutically-relevant, hot-melt extruded filaments to predetermine their suitability for FDM. Methods: A texture analyzer was used to simulate the forces a filament is subjected to inside the printer. The texture analyzer produced a force-distance curve referred to as the flexibility profile. Principal Component Analysis and Correlation Analysis statistical methods were then used to compare the flexibility profiles of commercial filaments to in-house made filaments. Results: Principal component analysis showed clearly separated clustering of filaments that suffer from mechanical defects versus filaments which are suitable for printing. Correlation scores likewise showed significantly greater values with feedable filaments than their mechanically deficient counterparts. Conclusion: The screening method developed in this study showed, with statistical significance and reproducibility, the ability to predetermine the feedability of extruded filaments into an FDM printer

Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples

Background: Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. Methods: The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. Results: The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3%, 58.6%, 40.5% and 10.8% by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97% for both methods. Conclusion: This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China

Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307

Background Many cases of cutaneous leishmaniasis (CL) have been recorded in the Jenin District based on their clinical appearance. Here, their parasites have been characterized in depth. Methods Leishmanial parasites isolated from 12 human cases of CL from the Jenin District were cultured as promastigotes, whose DNA was extracted. The ITS1 sequence and the 7SL RNA gene were analysed as was the kinetoplast minicircle DNA (kDNA) sequence. Excreted factor (EF) serotyping and multilocus enzyme electrophoresis (MLEE) were also applied. Results This extensive characterization identified the strains as Leishmania tropica of two very distinct sub-types that parallel the two sub-groups discerned by multilocus microsatellite typing (MLMT) done previously. A high degree of congruity was displayed among the results generated by the different analytical methods that had examined various cellular components and exposed intra-specific heterogeneity among the 12 strains. Three of the ten strains subjected to MLEE constituted a new zymodeme, zymodeme MON-307, and seven belonged to the known zymodeme MON-137. Ten of the 15 enzymes in the profile of zymodeme MON-307 displayed different electrophoretic mobilities compared with the enzyme profile of the zymodeme MON-137. The closest profile to that of zymodeme MON-307 was that of the zymodeme MON-76 known from Syria. Strains of the zymodeme MON-307 were EF sub-serotype A2 and those of the zymodeme MON-137 were either A9 or A9B4. The sub-serotype B4 component appears, so far, to be unique to some strains of L. tropica of zymodeme MON-137. Strains of the zymodeme MON-137 displayed a distinctive fragment of 417 bp that was absent in those of zymodeme MON-307 when their kDNA was digested with the endonuclease RsaI. kDNA-RFLP after digestion with the endonuclease MboI facilitated a further level of differentiation that partially coincided with the geographical distribution of the human cases from which the strains came. Conclusions The Palestinian strains that were assigned to different genetic groups differed in their MLEE profiles and their EF types. A new zymodeme, zymodeme MON-307 was discovered that seems to be unique to the northern part of the Palestinian West Bank. What seemed to be a straight forward classical situation of L. tropica causing anthroponotic CL in the Jenin District might be a more complex situation, owing to the presence of two separate sub-types of L. tropica that, possibly, indicates two separate transmission cycles involving two separate types of phlebotomine sand fly vector

Canine leishmaniosis and its relationship to human visceral leishmaniasis in Eastern Uzbekistan

<p>Abstract</p> <p>Background</p> <p>The Namangan Region in the Pap District, located in Eastern Uzbekistan is the main focus of visceral leishmaniasis (VL) in Uzbekistan. In total, 28 cases of human VL were registered during 2006-2008 in this region. A study on the epidemiology of VL in this area was carried out in 2007-2008 in the villages of Chodak, Oltinkan, Gulistan and Chorkesar located at elevations of 900-1200 above sea level.</p> <p>Results</p> <p>A total of 162 dogs were tested for <it>Leishmania </it>infection. Blood was drawn for serology and PCR. When clinical signs of the disease were present, aspirates from lymph nodes and the spleen were taken. Forty-two dogs (25.9%) had clinical signs suggestive of VL and 51 (31.5%) were sero-positive. ITS-1 PCR was performed for 135 dogs using blood and tissue samples and 40 (29.6%) of them were PCR-positive. Leishmanial parasites were cultured from lymph node or spleen aspirates from 10 dogs.</p> <p>Eight <it>Leishmania </it>strains isolated from dogs were typed by multi-locus microsatellite typing (MLMT) and by multilocus enzyme electrophoretic analysis (MLEE), using a 15 enzyme system. These analyses revealed that the strains belong to the most common zymodeme of <it>L. infantum</it>, i.e., MON-1, and form a unique group when compared to MON-1 strains from other geographical regions.</p> <p>Conclusions</p> <p>The data obtained through this study confirm the existence of an active focus of VL in the Namangan region of Uzbekistan. The fact that <it>L. infantum </it>was the causative agent of canine infection with typical clinical signs, and also of human infection affecting only infants, suggests that a zoonotic form of VL similar in epidemiology to Mediterranean VL is present in Uzbekistan.</p

High-Resolution Melting Analysis as a Powerful Tool to Discriminate and Genotype Pseudomonas savastanoi Pathovars and Strains

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants