The role of GlcNAc-PI-de-N-acetylase gene by gene knockout through homologous recombination and its consequences on survival, growth and infectivity of Leishmania major in in vitro and in vivo conditions

At present, there are no efficacious vaccines or effective drugs against leishmaniasis; therefore new and innovative control methods are urgently required. One way to achieve this important goal is through using reverse genetic engineering to evaluate important enzymes, proteins and macromolecules. One of the most important enzymes for Glycosylphosphatidylinositol (GPI) biosynthetic pathways is GlcNAc-PI-deN-acetylase (GPI12). The molecular constructs were cloned in Escherichia coli strain Top 10 and confirmed by molecular methods and were transfected by electroporation into Leishmania major. We demonstrated that two alleles of the GPI12 gene in L. major were successfully removed and enabling the generation of a null mutant, which supports the idea that GPI12 is not an essential gene for the growth and survival of Leishmania and the homozygous knockouts of Leishmania are able to survive. We were able to produce a mutant parasite that caused no damaged to the host. Further investigations are essential to check the safety profile in laboratory animal

Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms

Parts of the natural life cycle of Echinococcus granulosus can be retraced in vitro such as the development of protoscoleces into semiadult worms with three or more proglottids, or the redifferentiation of in vitro cultured protoscoleces into metacestode-like cystic structures. Most in vitro generated samples share-at the microscopical level-high similarities with those naturally grown, but developmental differences have also been documented, such as missing egg production in in vitro grown adults or unusual bladder/vesicle formation in protoscoleces cultured into the metacestode direction. The aim of the present study was to explore how far different in vitro generated stage-specific materials/structures match the natural situation on the transcriptome level, based on testing five exemplarily chosen different genes: the frizzled receptor eg-fz4 (posterior marker), the FGF receptor-like factor eg-fgfrl (anterior association), the cell differentiation protein eg-rcd1 (part of the CCR4-NOT complex, a key regulator of eukaryotic gene expression), the rapidly accelerated fibrosarcoma serin/threonin kinase eg-braf (part of the MAPK pathway involved, e.g., in EGF signaling) and the co-smad eg-smadD (downstream factor of TGFβ/BMP2/activin signaling). These genes-tested via qPCR-were selected such as to allow a discussion on their potential role in the development of E. granulosus into the adult stage. Thus, testing took place with three ex vivo isolated samples, namely (i) egg-containing adult worms, (ii) invaginated protoscoleces, and (iii) protoscolex-free germinal layer tissue. Respective data were compared (a) with in vitro generated metacestode-like microcysts developed from protoscolices, and (b) different development stages of protoscoleces in vitro cultured toward adult maturation. As a finding, only eg-smadD and partially eg-fz4 showed high expression similarities between ex vivo harvested and in vitro cultured E. granulosus, thus suggesting a putative role in adult maturation. Conclusively, the fact of using "only" five genes did not allow answering the question if ex vivo and in vitro materials are similar on the transcriptome level. Another experimental restriction arises from different growth conditions of the in vitro cultured materials, and comparing these to the ex vivo harvested ones. Future experiments may solve the problems by using fully standardized E. granulosus sample collection and fully standardized culture conditions

Serological Evidence and Associated Risk Factors for Toxoplasma gondii in Gorgan and a Review of Recent Studies in Golestan Province

Background and purpose: Toxoplasma gondii is one of the most common parasitic infections in humans and other vertebrates worldwide. This study aimed at determining the seroprevalence of Toxoplasma infection and associated risk factors in individuals attending a Reference Laboratory in Gorgan, Iran. Moreover, recent studies in Golestan province were reviewed. Materials and methods: A descriptive cross-sectional survey was conducted in 551 blood samples obtained from February-June 2017. Serum samples were isolated and examined for IgG and IgM antibodies against T. gondii using ELISA. Data were analyzed in SPSS applying logistic regression. Results: Total prevalence of T. gondii was 63.5% (350 out of 551). Among the samples, 306 (55.5%), 12 (2.17%), and 32 (5.8%) were found to be positive for IgG, IgM and both IgG and IgM against T. gondii, respectively. There were significant associations between T. gondii infection and age and soil contact (P0.05). Conclusion: About two thirds of individuals were infected with T. gondii. Accordingly, a high number of subjects have been exposed to T. gondii in this area, which their seropositivity was seen to increase by age as a risk factor. High seropositivity indicates appropriate conditions for the life cycle of the parasite in this province. Therefore, preventive measurements at the community level, particularly in high-risk groups, are warranted

Transient knockdown of Nucleoside transporter 4 gene expression as a therapeutic target in Leishmania major by antisense RNA: In vitro and in vivo studies

Background & objectives: Leishmania parasites cause various clinical symptoms in humans such as cutaneous ulcers and fatal visceral diseases. These parasites cannot synthesize purine rings de novo and must uptake purines from their hosts via salvage. Salvage is regulated by permeases in the cell membrane. There are hundreds of membrane transporter proteins to receive nutrients in Leishmania. Nucleoside transporter 4 (NT4) is one of the purine transporters that is involved in enhancing the uptake of adenine in Leishmania major. They are important new drug targets for the treatment of leishmaniasis because they can be used to transport toxic purine analogs to kill parasitic cells, thus preventing the progression of the infection. The present study was conducted to silence the NT4 nucleobase involved in the salvage pathway to interrupt purine nucleotide membrane transport in the cells of L. major. Methods: In this study, a 502 bp segment of NT4 gene sequence was selected and designed as antisense transcripts after insertion in the parasite. The NT4 construct was transfected into L. major promastigotes for in vitro study of gene expression. Then, BALB/c mice infected with transgenic Leishmania and wild-type strain along with the number and size of lesions were studied in vivo. Results: The study showed that relative expression of NT4 gene in mutant Leishmania was lower than in the control on Day 3 to 20. The percentages and the number of amastigotes in infected macrophages with wild-type strain L. major were more than infected macrophages with mutant parasites. Infected BALB/c mice with transgenic Leishmania showed a lower number and size of lesions than the BALB/c mice infected with wild-type strain. Interpretation & conclusion: The results of the study indicated that the use of antisense RNA reduced NT4 gene expression in L. major. Further, studies are needed to ascertain that the use of antisense can be considered as a new treatment for leishmaniasis. Keywords Author Keywords:Antisense RNA; gene expression; Leishmania major; Nucleoside transporter 4 gene; transient knockdown KeyWords Plus:NUCLEOBASE TRANSPORTER; VIRULENCE; DONOVAN

Efficiency of Horizontal Roughing Filter in Removing Suspended Solids from Effluent of Waste Stabilization Ponds in 2013

Background & Aims of the Study: The Horizontal roughing filters (HRF) are generally formed with three layers. The size of the course at the beginning of the flow path is large and at the end is small. This kind of layering increases absorption capacity while the solids entering the bed&ndash;depth gradually separate because of the decline in the holes&rsquo; diameters. This study aimed to examine the efficiency of HRFs in removing total suspended solids (TSS) from effluent of a waste stabilization pond. Materials & Methods: This experimental study was conducted in 2013. The pilot project was transferred to the Karaj wastewater treatment plant (waste stabilization pond), Alborz province of Iran. Then, the installation, equipping and start-up of the system began, using an effluent treatment plant. Sampling was done from March to August in 3 rates of 0.5, 1 and 1.5 m/h, which was included simultaneous sampling from inlet and outlet HRF to determine the concentrations of TSS. Results: The results showed that at filtration rates of 0.5, 1, and 1.5 m/h, the average of TSS removal was equaled to 42.46, 56.65 and 33.22%, respectively. The removal efficiency of TSS at the rate of 1m/h was more than 0.5 and 1.5 m/h rate (p<0.05). The Outlet value of TSS was lower than the standards which are set by the Environmental Standards Organization (ESO) (P < 0.05). Conclusion: According to the results of this study, the HRF function was approximately adequate in TSS removal and can be a suitable option for improving the quality of stabilization ponds effluent

Efficiency of horizontal roughing filter in removing nitrate, phosphate and chemical oxygen demand from effluent of waste stabilization pond

Background: The effective size of the end grain of horizontal roughing filters (HRFs) is larger than 2 mm. This study aimed to examine the efficiency of HRFs in removing nitrate, phosphate, and chemical oxygen demand (COD) from effluent of a wastewater stabilization pond. Methods: This experimental study was conducted in 2013. The pilot project was transferred to the Karaj wastewater treatment plant (stabilization pond), and the installation, equipping, and start-up of the system began using an effluent treatment plant. Sampling was done from March to August in 3 rates, 0.5, 1 and 1.5 m/h, and included simultaneous sampling from inlet and outlet filtering to determine the concentrations of nitrate, phosphate, and COD. Results: At filtration rates of 0.5, 1, and 1.5 m/h, the average nitrate removal equaled 25%, 32%, and 34%, respectively, average phosphate removal equaled 29%, 26%, and 28%, respectively, and the average COD removal at filtration rates of 0.5, 1, and 1.5 m/h equaled 62%, 66%, and 68%, respectively. Outlet values of phosphate and nitrate were lower than the standards set by the Environmental Standards Organization (ESO) (P < 0.05). Conclusion: According to the results of this study, the HRF function was approximately adequate in COD removal, but its efficiency in nitrate and phosphate removal was lower

Differential Expression of Hox and Notch Genes in Larval and Adult Stages of Echinococcus granulosus

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus