Synthesis of benzologues of Nitazoxanide and Tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity

We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short synthetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Compound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing IC₅₀’s <5μM. This compound was five-times more active than NIT, and 18-times more potent than metronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a new broad spectrum antiprotozoal agent.     

Thioredoxin 80-activated-monocytes (TAMs) inhibit the replication of intracellular pathogens

Background

Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs).

Principal Findings

In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome.

Significance

Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria.     

Elimination of tumorigenic stem cells from differentiated progeny and selection of definitive endoderm reveals a Pdx1+ foregut endoderm stem cell lineage

Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM(+)SSEA1(-)SSEA3(-) cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity.     

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.     

The expression of a hunchback ortholog in the polychaete annelid Platynereis dumerilii suggests an ancestral role in mesoderm development and neurogenesis

Orthologs of the Drosophila gap gene hunchback have been isolated so far only in protostomes. Phylogenetic analysis of recently available genomic data allowed us to confirm that hunchback genes are widely found in protostomes (both lophotrochozoans and ecdysozoans). In contrast, no unequivocal hunchback gene can be found in the genomes of deuterostomes and non-bilaterians. We cloned hunchback in the marine polychaete annelid Platynereis dumerilii and analysed its expression during development. In this species, hunchback displays an expression pattern indicative of a role in mesoderm formation and neurogenesis, and similar to the expression found for hunchback genes in arthropods. These data suggest altogether that these functions are ancestral to protostomes.Pierre Kerner and Fabiola Zelada González contributed equally to this work.     

Elongating modified conserved peptides eliminates their immunogenicity and protective efficacy against P. falciparum malaria

Plasmodium falciparum malaria protein peptides were synthesised in the search for more effective routes for inducing a protective immune response against this deadly parasite and this information has been associated with such molecules' three-dimensional structure. These peptides had high red blood cell binding activity and their carboxy- and amino-terminal extremes were elongated for determining their immunogenic and protection-inducing activity against this disease in the Aotus monkey experimental model. 1H-NMR was used for analysing their three-dimensional structure; FAST ELISA, immunofluorescence antibody test, and Western blot were used for identifying their antibody inducing capacity and these previously immunised Aotus were inoculated with a highly infective P. falciparum strain to determine whether these elongated peptides were able to induce protection. This was aimed at establishing an association or correlation between long peptides' three-dimensional structure and their immunogenic and protection-inducing response in these monkeys. Peptides 20026 (25 residue), 20028 (30 residue), and 20030 (35 residues) were synthesised based on elongating the amino-terminal region of the 10022 highly immunogenic and protection-inducing modified peptide. 1H-NMR studies revealed that the first three had Classical type III beta-turn structures, different from the 20-amino acid long modified peptide 10022 which had a distorted type III beta-turn. Humoral immune response analysis showed that even when some antibodies could be generated against the parasite, none of the immunised Aotus could be protected with elongated peptides suggesting that elongating them eliminated modified peptide 10022 immunogenic and protection-inducing capacity.     

Two L1-peptides are excellent tools for serological detection of HPV-associated cervical carcinoma lesions

A persistent high risk human papillomavirus (HR-HPV) infection causes cervical intraepithelial lesions and cervical carcinoma. There is evidence that detecting anti-L1 antibodies could be successfully used for discriminating between cervical lesion patients and women having normal cytology. It was found that peptides 18283 (55PNNNKILVPKVSGLQYRVFR74) and 18294 (284LYIKGSGSTANLASSNYFPT300) from the L1-surface exposed regions were specifically recognised by antibodies from the cervical lesion patient sera. These peptides were tested against 165 womens' normal cytology sera and 148 cervical lesion or cervical cancer patients' sera. Less than 3.6% of women's normal cytology sera recognised peptides 18283 or 18294; on the contrary, 91% to 96% of the cervical lesion (CIN I to CIN III) or cervical cancer patient sera recognised peptides 18283 and 18294. These data show that anti-peptide 18283 and 18294 antibodies in the patients' sera are strongly associated with the presence of HR-HPV associated cervical lesions, showing 92-97% sensitivity and 89-95% specificity in recognising precancerous and cervical cancer patients. These two peptides could be excellent tools for use in large-scale serological screening of women populations at risk of developing cervical carcinoma.     

Protection against malaria induced by chirally modified Plasmodium falciparum's MSP-1 42 pseudopeptides

The C-terminal portion of the Plasmodium falciparum blood stage MSP-1 antigen plays a key role in invasion of human erythrocytes. The MSP-1(1282-1301) non-polymorphic 1585 peptide, from the processed MSP-1(42) fragment, is poorly immunogenic and highly alpha-helical [Angew. Chem. Int. Ed. 40 (2001) 4654]. Assessing the alpha-carbon asymmetry and its implication in the host immune response is proposed in this work to overcome the 1585 peptide's immunological properties. Accordingly, the effect of incorporating single D-amino acids and psi-[CH(2)-NH] isoster bonds into the 1585 peptide was examined both at the immunogenic and 3D-structure levels. Therefore, specific binding to RBCs is promoted by site-directed chiral modifications on the native peptide as well as by simultaneously combining specific D-substitutions with psi-[CH(2)-NH] isoster bonds transforming this molecule into a high specific HLAbeta1*1101 allele binder. D-analog pseudopeptide immunized animals induced antibodies selectively recognizing a recombinant as well as native MSP-1(42) and MSP-1(33) fragments. Protection and low parasitemia levels were induced in Aotus monkeys immunized with the EVLYL(dK)PLAGVYRSLKKQLE analog. Peptide alpha-carbon chiral transformation is therefore an important target for structural modulation and, consequently, represents a novel approach towards designing multi-component subunit-based malarial vaccines.     

A role for the P-body component GW182 in microRNA function

In animals, the majority of microRNAs regulate gene expression through the RNA interference (RNAi) machinery without inducing small-interfering RNA (siRNA)-directed mRNA cleavage. Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies. Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations that prevent Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a siRNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.     

The zebrafish mutant vps18 as a model for vesicle‐traffic related hypopigmentation diseases

Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky–Pudlak, Chediak–Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18^hi2499A, which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18^hi2499A the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18^hi2499A larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18^hi2499A can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.