Live attenuated-nonpathogenic Leishmania and DNA structures as promising vaccine platforms against leishmaniasis: innovations can make waves.

Leishmaniasis is a vector-borne disease caused by the protozoan parasite of Leishmania genus and is a complex disease affecting mostly tropical regions of the world. Unfortunately, despite the extensive effort made, there is no vaccine available for human use. Undoubtedly, a comprehensive understanding of the host-vector-parasite interaction is substantial for developing an effective prophylactic vaccine. Recently the role of sandfly saliva on disease progression has been uncovered which can make a substantial contribution in vaccine design. In this review we try to focus on the strategies that most probably meet the prerequisites of vaccine development (based on the current understandings) including live attenuated/non-pathogenic and subunit DNA vaccines. Innovative approaches such as reverse genetics, CRISP/R-Cas9 and antibiotic-free selection are now available to promisingly compensate for intrinsic drawbacks associated with these platforms. Our main goal is to call more attention toward the prerequisites of effective vaccine development while controlling the disease outspread is a substantial need.

Structural analysis of PpSP15 and PsSP9 sand fly salivary proteins designed with a self-cleavable linker as a live vaccine candidate against cutaneous leishmaniasis.

BACKGROUND: Leishmania parasites are deposited in the host through sand fly bites along with sand fly saliva. Therefore, salivary proteins are promising vaccine candidates for controlling leishmaniasis. Herein, two immunogenic salivary proteins, PpSP15 from Phlebotomus papatasi and PsSP9 from Phlebotomus sergenti, were selected as vaccine candidates to be delivered by live Leishmania tarentolae as vector. The stepwise in silico protocol advantaged in this study for multi-protein design in L. tarentolae is then described in detail. METHODS: All possible combinations of two salivary proteins, PpSP15 and PsSP9, with or without T2A peptide were designed at the mRNA and protein levels. Then, the best combination for the vaccine candidate was selected based on mRNA and protein stability along with peptide analysis. RESULTS: At the mRNA level, the most favored secondary structure was PpSP15-T2A-PsSP9. At the protein level, the refined three-dimensional models of all combinations were structurally valid; however, local quality estimation showed that the PpSp15-T2A-PsSP9 fusion had higher stability for each amino acid position, with low root-mean-square deviation (RMSD), compared with the original proteins. In silico evaluation confirmed the PpSP15-T2A-PsSP9 combination as a good Th1-polarizing candidate in terms of high IFN-γ production and low IL-10/TGF-ß ratio in response to three consecutive immunizations. Potential protein expression was then confirmed by Western blotting. CONCLUSIONS: The approach presented herein is among the first studies to have privileged protein homology modeling along with mRNA analysis for logical live vaccine design-coding multi-proteins.

Leishmania tarentolae as Potential Live Vaccine Co-Expressing Distinct Salivary Gland Proteins Against Experimental Cutaneous Leishmaniasis in BALB/c Mice Model.

Leishmaniasis is a neglected vector-borne disease caused by Leishmania parasites transmitted through the infected sand flies bite. Current treatments are limited, partly due to their high cost and significant adverse effects, and no human vaccine is yet available. Sand flies saliva has been examined for their potential application as an anti-Leishmania vaccine. The salivary protein, PpSP15, was the first protective vaccine candidate against L. major. Additionally, PsSP9 was already introduced as a highly immunogenic salivary protein against L. tropica. Herein, we aimed to develop an effective multivalent live vaccine to control Cutaneous Leishmaniasis induced by two main species, L. major and L. tropica. Hence, the two above-mentioned salivary proteins using T2A linker were incorporated inside the L. tarentolae genome as a safe live vector. Then, the immunogenicity and protective effects of recombinant L. tarentolae co-expressing PpSP15 and PsSP9 were evaluated in pre-treated BALB/c mice with CpG against L. major and L. tropica. Following the cytokine assays, parasite burden and antibody assessment at different time-points at pre and post-infection, promising protective Th1 immunity was obtained in vaccinated mice with recombinant L. tarentolae co-expressing PpSP15 and PsSP9. This is the first study demonstrating the potency of a safe live vaccine based on the combination of different salivary proteins against the infectious challenge with two different species of Leishmania.

Significant co-expression of putative cancer stem cell markers, EpCAM and CD166, correlates with tumor stage and invasive behavior in colorectal cancer.

BACKGROUND: The crucial oncogenic role of cancer stem cells (CSCs) in tumor maintenance, progression, drug resistance, and relapse has been clarified in different cancers, particularly in colorectal cancer (CRC). The current study was conducted to evaluate the co-expression pattern and clinical significance of epithelial cell adhesion molecules (EpCAM) and activated leukocyte cell adhesion (CD166 or ALCAM) in CRC patients. METHODS: This study was carried out on 458 paraffin-embedded CRC specimens by immunohistochemistry on tissue microarray (TMA) slides. RESULTS: Elevated expression of EpCAM and CD166 was observed in 61.5% (246/427) and 40.5% (164/405) of CRC cases. Our analysis showed a significant positive association of EpCAM expression with tumor size (P = 0.02), tumor stage (P = 0.007), tumor differentiate (P = 0.005), vascular (P = 0.01), neural (P = 0.01), and lymph node (P = 0.001) invasion. There were no significant differences between CD166 expression and clinicopathological parameters. Moreover, the combined analysis demonstrated a reciprocal significant correlation between EpCAM and CD166 expression (P = 0.02). Interestingly, there was a significant positive correlation between EpCAM/CD166 phenotypes expression and tumor stage (P = 0.03), tumor differentiation (P = 0.05), neural, and lymph node invasion (P =0.01). CONCLUSIONS: The significant correlation of EpCAM and CD166 expression and their association with tumor progression and aggressive behavior is the reason for the suggestion of these two CSC markers as promising targets to promote novel effective targeted-therapy strategies for cancer treatment in the present study.

Leishmania Parasite: the Impact of New Serum-Free Medium as an Alternative for Fetal Bovine Serum

Background: Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes. Methods: Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (complete serum-free medium [CSFM]) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared. Results: Diff-Quick staining and epi-fluores¬cence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice. Conclusion: This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes.

Evaluation of protection induced by in vitro maturated BMDCs presenting CD8+ T cell stimulating peptides after a heterologous vaccination regimen in BALB/c model against Leishmania major.

Leishmaniasis is a complex vector-borne disease mediated by Leishmania parasite and a strong and long-lasting CD4+ Th1 and CD8+-T cell immunity is required to control the infection. Thus far multivalent subunit vaccines have met this requirement more promisingly. However several full protein sequences cannot be easily arranged in one construct. Instead, new emerging immune-informatics based epitope formulations surpass this restriction. Herein, we aimed to examine the protective potential of a dendritic cell based vaccine presenting epitopes to CD8+ and CD4+-T cells in combination with DNA vaccine encoding the same epitopes against murine cutaneous leishmaniasis. Immature DCs were loaded with epitopes (selected from parasite proteome) in vitro with or without CpG oligonucleotides and were used to immunize BALB/c mice. Peptide coding DNA was used to boost the system and immunological responses were evaluated after Leishmania (L.) major infectious challenge. The pre-challenge response to included epitopes was Th1 polarized which potentially lowered the infection at early time points post-challenge but not at later weeks. Collectively, DC prime-DNA boost was found to be a promising approach for Th1 polarization however the constituent epitopes undoubtedly make a significant contribution in the protection outcome of the vaccine.

Comparison of Protective Potency of DNA and Live Vaccines Expressing A2-CPA-CPB-CTE Antigens against Visceral Leishmaniasis in Syrian Hamster as Preliminary Study.

BACKGROUND: Visceral leishmaniasis is the most severe form of leishmaniasis caused by Leishmania (L.) donovani complex. Drug-resistant strains have been developed as a consequence of the current chemotherapeutic interventions, which has increased the need for advanced preventive and therapeutic strategies. A2-CPA-CPB-CTE-recombinant strain of L. tarentolae, which is non-pathogenic to humans, was shown protective in live vaccine as well as its DNA vaccine counterpart in both murine and canine models. METHODS: We evaluated the effectiveness of these DNA and live vaccination harboring A2-CPA-CPB-CTE in protecting hamsters against L. infantum infection using prime-boost regimens, namely DNA/DNA and Live/Live (n=9 hamsters per group). Cationic solid lipid nanoparticles (cSLN) were utilized as an adjuvant for DNA priming and electroporation for boosting DNA. At different time points post-challenge, parasite burden and body weight as well as humoral immune responses were measured. RESULTS: Both immunization strategies partially protect hamsters against L. infantum challenge. This protective immunity is associated with remarkable decrease in parasite load in liver and spleen of vaccinated hamsters eight weeks after challenge compared to control group. CONCLUSION: Both test groups (DNA/DNA and Live/Live) elicited high levels of IgG2 and total IgG as humoral immune responses and lower level of parasite propagation in both liver and spleen.

Visualization of Leishmania tropica Infection in BALB/c Mice by Bioluminescence Imaging

Background: Leishmania tropica is the cause of more than one form of leishmaniasis and lacks a known reservoir animal. This study compares the potential infectivity of recombinant and wild-type L. tropica in BALB/c mice. Methods: The potential infectivity of recombinant L. tropicaEGFP or L. tropicaEGFP-LUC by two different, the subcutaneous and intradermal, routes was compared using a range of classical detection methods and bioluminescence imaging (BLI). Results: In addition to the results obtained from classical diagnostic approaches, the BLI signals were detected in footpads and ears of L. tropica-infected animals. The BLI revealed that a bioluminescence signal can be observed at the inoculation site. The stability of the BLI remained constant in the footpad, but the signal was detectable for only three months in the pinna due to the decline in infection over time. Conclusion: The presented data are a precise verification of the assumption that BALB/c mice could be used as an experimental model for L. tropica infectivity.

Lactococcus lactis expressing sand fly PpSP15 salivary protein confers long-term protection against Leishmania major in BALB/c mice.

Cutaneous leishmaniasisis a vector-borne disease transmitted by Leishmania infected sand flies. PpSP15 is an immunogenic salivary protein from the sand fly Phlebotomus papatasi. Immunization with PpSP15 was shown to protect against Leishmania major infection. Lactococcus lactis is a safe non-pathogenic delivery system that can be used to express antigens in situ. Here, the codon-optimized Ppsp15-egfp gene was cloned in pNZ8121 vector downstream of the PrtP signal peptide that is responsible for expression and secretion of the protein on the cell wall. Expression of PpSP15-EGFP recombinant protein was monitored by immunofluorescence, flow cytometry and Western blot. Also, expression of protein in cell wall compartment was verified using whole cell ELISA, Western blot and TEM microscopy. BALB/c mice were immunized three times with recombinant L. lactis-PpSP15-EGFPcwa, and the immune responses were followed up, at short-term (ST, 2 weeks) and long-term (LT, 6 months) periods. BALB/c mice were challenged with L. major plus P. papatasi Salivary Gland Homogenate. Evaluation of footpad thickness and parasite burden showed a delay in the development of the disease and significantly decreased parasite numbers in PpSP15 vaccinated animals as compared to control group. In addition, immunized mice showed Th1 type immune responses. Importantly, immunization with L. lactis-PpSP15-EGFPcwa stimulated the long-term memory in mice which lasted for at least 6 months.

The outcome of arginase activity inhibition in BALB/c mice hosting Leishmania tropica.

Two species of Leishmania (L), L. tropica and L. major, are among the main causative agents of cutaneous leishmaniasis. Arginase (ARG) is an essential enzyme for cell growth, thus an attractive drug target. In this study, we tried to survey the inhibitory impact of ARG by nor-NOHA (N-ω-hydroxy-L-nor-arginine) on in vivo infection caused by L. tropica. BALB/c mice were inoculated with L. tropicaEGFP-LUC (Ltrop) or L. majorEGFP-LUC (Lmj) and then were treated by nor-NOHA. ARG inhibitor only indicated a delay in generation of a cutaneous lesion in inoculated footpad with nor-NOHA-Ltrop and nor-NOHA-Lmj. ARG activity has been significantly reduced in nor-NOHA-Ltrop group. In this group, ARG activity inhibition correlated with increased levels of nitric oxide (NO). In both inoculated mice with Ltrop or Lmj, parasite load showed a significant decrease at later steps during the CL course post-treatment. In vivo bioluminescence intensity did not show any ARG's inhibitory effect on treated-Ltrop. The findings verified that the ARG activity may partially control the L. tropica infection in BALB/c mice through reduction of parasite proliferation and parasite killing through NO generation. This effect is dose-dependent.

Antileishmanial effect of rapamycin as an alternative approach to control Leishmania tropica infection.

Cutaneous leishmaniosis (CL) is a parasitic disease in animals and human with no satisfactory treatments and vaccination. Rapamycin is a potent inhibitor of mammalian target of rapamycin (mTOR) with various applications. Here, the effect of rapamycin alone or in combination with two other drugs, namely amphotericin B (AmB) and glucantime, was investigated against Leishmania tropica infection. In vitro viability and electron microscopy evaluation of the parasites showed detrimental changes in their appearance and viability. Treatment with clinically relevant dose of rapamycin (10.2 µg/dose) is able to control the parasite load in BALB/c mice infected with L. tropica. Furthermore, the cytokine profiles showed significant polarization towards Th1 immune response. Surprisingly, combination therapy with either AmB or glucantime was not efficient. Rapamycin is showed an effective alternative therapy against leishmaniosis caused by L. tropica.

Correction: DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica.

[This corrects the article DOI: 10.1371/journal.pntd.0007067.].

A non-pathogenic Leishmania tarentolae vector based- HCV polytope DNA vaccine elicits potent and long lasting Th1 and CTL responses in BALB/c mice model.

Despite successful anti-viral (DAAs) treatment of Hepatitis C virus (HCV) infection, recent data indicated the need for an effective vaccine. Preexisting anti-vector immunity is an obstacle for application of live vectors for antigen delivery and development of effective T-cell based HCV vaccines. Herein, we report construction of recombinant Leishmania tarentolae, a lizard (non-human) parasite, expressing an HCV polytope DNA, PT-NT(gp96), encoding for several immunogenic HCV epitopes and evaluation of its immunogenicity in three different prime/boost immunization groups (G) of BALB/c mice. Homologous prime/boost immunization by L.tarentolae-PT-NT(gp96) either with or without CpG (G1 and G2 respectively) and heterologous immunization with a PT-NT(gp96) encoding-pCDNA plasmid followed by L.tarentolae-PT-NT (G3) was undertaken. Immune responses were measured three and nine weeks (W) post immunization. Splenocytes (cultured with antigen-stimulant) of mice in G1 showed the highest percentage of specific CTL-cytolytic activity compared to G2 and G3 at both short (W3:70.98% versus 41.29% and 13.12%) and long (W9: 50% versus 24.5% and 20%) term periods, accompanied with high levels of secreted IFN-γ. Comparison of IFN-γ, IL-4, IL-17 and TNF-α cytokines levels obtained from the supernatant of antigen-stimulated splenocytes as well as antibodies level (as IgG1/IgG2a ratio; obtained from sera of immunized mice) indicated higher Th1 oriented responses for G1, G2 groups and balanced Th1-Th17 for G3. Results indicated the potential of L.tarentolae (+CpG), as a non-pathogenic live vaccine vector, for delivery and enhancement of immune responses against HCV-polytope antigens.

DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica.

BACKGROUND: The vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection. METHODS AND RESULTS: In this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls. CONCLUSION: This study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens.

Therapeutic control of leishmaniasis by inhibitors of the mammalian target of rapamycin.

Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert anti-pathogen properties. In this study, we tested the therapeutic efficacy of several mTOR inhibitors in controlling infection with Leishmania major. Rapamycin, GSK-2126458 and KU-0063794 were administered to BALB/c mice, which had received an intrafootpad injection of the parasite. Footpad swelling and parasite burden were assessed, and cytokine production by mouse splenocytes and phenotypic changes in draining lymph node cells were evaluated. Treatment with a clinically relevant dose of rapamycin or with GSK-2126458, but not with KU-0063794, dramatically lowered both the footpad swelling and the parasite load in the draining lymph node. Importantly, the employed dose of rapamycin did not kill the promastigotes in vitro as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and electron microscopy. Moreover, the IL-4 production capacity of splenocytes harvested from infected mice that were treated with rapamycin was significantly reduced. Consequently, the IFN-γ:IL-4 production ratio was elevated, suggesting a T helper-type 1 (Th1)-skewed cytokine profile. Finally, the expression level of CD69, an early activation marker, on splenic and lymph node CD4+ and CD8+ T cells was enhanced in rapamycin-treated mice. Taken together, our findings suggest that select mTOR inhibitors may be used in therapeutic settings for the management of leishmaniasis. We propose that the beneficial effects of such inhibitors stem from their immunomodulatory properties. Therefore, the adjuvanticity of mTOR inhibitors may also be considered in vaccination strategies against Leishmania species.

Human Neutrophil Peptide 1 as immunotherapeutic agent against Leishmania infected BALB/c mice.

Human Neutrophil Peptide 1 (HNP1) produced by neutrophils, is a well-known antimicrobial peptide which plays a role both in innate as well as in adaptive immunity and is under intensive investigation as a potential therapeutic agent. Previous in vitro experiments have indicated the leishmaniacidal effect of recombinant HNP1 on Leishmania major (L. major) promastigotes and amastigotes. In the current study, we further extended the idea to explore the remedial effect of HNP1 in the two modalities of peptide therapy (folded HNP1) and gene therapy in L. major infected BALB/c mice. To this end, mice in five different groups received synthetic folded HNP1 (G1), pcDNA-HNP1-EGFP (G2), pcDNA-EGFP (G3), Amphotericin B (G4) and PBS (G5), which was started three weeks after infection for three consecutive weeks. Footpad swelling was monitored weekly and a day after the therapy ended, IFN-γ, IL-4, IL-10, IL-6 and nitric oxide produced by splenocytes were analyzed together with the parasite load in draining lymph nodes. Arginase activity and dermal histopathological changes were also analyzed in the infected footpads. We demonstrated that both therapeutic approaches effectively induced Th1 polarization and restricted parasite burden. It can control disease progression in contrast to non-treated groups. However, pcDNA-HNP1-EGFP is more promising in respect to parasite control than folded HNP1, but less effective than AmB treatment. We concluded with the call for a future approach, that is, a DNA-based expression of HNP1 combined with AmB as it can improve the leishmaniacidal efficacy.

Live Leishmania tarentolae secreting HNP1 as an immunotherapeutic tool against Leishmania infection in BALB/c mice.

AIM: Several disadvantages about chemotherapy for leishmaniasis has reinforced discovery of novel therapeutic agents especially immunotherapeutics. HNP1, as a member of the mammalian antimicrobial peptides family, is an attractive molecule due to its broad functional spectrum. Here, the in vivo potency of HNP1 in transgenic Leishmania tarentolae as an immunotherapy tool against Leishmania major-infected BALB/c mice was examined. METHODS & RESULTS: 3 weeks after infection with L. major, the treatment effect of L. tarentolae-HNP1-EGFP was pursued. The results were promising in respect to parasite load control and Th1 immune response polarization compared with controls. CONCLUSION: Immunotherapy by live L. tarentolae secreting HNP1 can elicit cellular immune response in a susceptible mouse model in order to control L. major infection.

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity.

PUF Proteins: Cellular Functions and Potential Applications.

RNA-binding proteins play critical roles in the regulation of gene expression. Among several families of RNA-binding proteins, PUF (Pumilio and FBF) proteins have been the subject of extensive investigations, as they can bind RNA in a sequence-specific manner and they are evolutionarily conserved among a wide range of organisms. The outstanding feature of these proteins is a highly conserved RNA-binding domain, which is known as the Pumilio-homology domain (PUM-HD) that mostly consists of eight tandem repeats. Each repeat recognizes an RNA base with a simple three-letter code that can be programmed in order to change the sequence-specificity of the protein. Using this tailored architecture, researchers have been able to change the specificity of the PUM-HD and target desired transcripts in the cell, even in subcellular compartments. The potential applications of this versatile tool in molecular cell biology seem unbounded and the use of these factors in pharmaceutics might be an interesting field of study in near future.