Molecular typing of a collection of Iranian clinical Trichophyton tonsurans isolates based on the non-transcribed spacer region of rDNA and antifungal susceptibility testing of the species.

INTRODUCTION: Wrestling, considered the national sport of Iran, has gained immense popularity among Iranians. Wrestlers frequently encounter skin conditions, with dermatophyte fungal infections, particularly tinea gladiatorum (TG), being a common issue. TG, caused by the Trichophyton genus, has emerged as a major health concern for wrestlers and other contact sport athletes worldwide. This study aimed to assess the genotypic diversity and antifungal susceptibility of Trichophyton tonsurans isolates responsible for TG in Iranian wrestlers from Mazandaran province, northern Iran. MATERIALS AND METHODS: A total of 60 clinical T. tonsurans isolates collected from various cities in Mazandaran, were included in the study. The isolates were identified through PCR-restriction fragment length polymorphism and sequencing methods. Genomic DNA was extracted from these isolates, and the non-transcribed spacer (NTS) region of ribosomal RNA (rRNA) was targeted for genotyping using newly designed primers. Haplotype analysis was performed to explore genetic diversity, and antifungal susceptibility to terbinafine (TRB) and itraconazole (ITC) was assessed. RESULTS: The results revealed five distinct NTS types: NTS-I, NTS-II, NTS-III, NTS-IV and NTS-V, with NTS-IV being the most prevalent. The distribution of NTS types varied across different cities, suggesting potential transmission patterns among wrestlers. Antifungal susceptibility testing showed that all isolates were susceptible to TRB, while one isolate demonstrated resistance to ITC. Genotypic diversity was not correlated with antifungal susceptibility, emphasising the importance of monitoring susceptibility to ensure effective treatment. Haplotype analysis highlighted significant genetic diversity among the T. tonsurans isolates. This diversity may be attributed to factors such as human-to-human transmission, geographic location and lifestyle changes. The study's findings underscore the need for comprehensive genotypic analysis to understand the epidemiology and evolution of T. tonsurans infections in athletes. CONCLUSION: In conclusion, this study provides valuable insights into the genotypic diversity and antifungal susceptibility of T. tonsurans isolates causing TG in Iranian wrestlers. The presence of multiple NTS types and varying susceptibility patterns highlights the complexity of T. tonsurans infections in this population. Further research is warranted to track the transmission routes and genetic evolution of T. tonsurans strains among wrestlers and develop effective control measures.

High Prevalence of Terbinafine Resistance Among Trichophyton mentagrophytes/T. interdigitale Species Complex, a Cross-Sectional Study from 2021 to 2022 in Northern Parts of Iran.

Treatment-resistant dermatophytosis caused by the members of the Trichophyton mentagrophytes/Trichophyton interdigitale species group (TMTISG) is increasing worldwide. We aimed to determine the prevalence of TMTISG in patients with dermatophytosis in two centers from north of Iran and detect the possible mutations in the squalene epoxidase (SQLE) gene in relevant terbinafine (TRB) resistant pathogenic isolates. From November 2021 to December 2022, 1960 patients suspected to dermatophytosis and referred to two mycology referral laboratories in the north of Iran were included in the study. Identification of all dermatophyte isolates was confirmed by RFLP of rDNA internal transcribed spacer (ITS) regions. Antifungal susceptibility testing against five common antifungals using the CLSI-M38-A3 protocol was performed. The TMTISG isolates resistant to TRB, were further analyzed to determine the possible mutations in the SQLE gene. Totally, 647 cases (33%) were positive for dermatophytosis of which 280 cases (43.3%) were identified as members of TMTISG. These were more frequently isolated from tinea corporis 131 (44.56%) and tinea cruris 116 (39.46%). Of 280 TMTISG isolates, 40 (14.3%) were resistant to TRB (MIC ≥ 4 µg/mL), all found to be T. indotineae in ITS sequencing. In SQLE sequencing 34 (85%) of TRB-resistant isolates had coincident mutations of Phe397Leu and Ala448Thr whereas four and two isolates had single mutations of Phe397Leu and Leu393Ser, respectively. Overall, the resistance of Iranian TMTISG isolates to TRB greatly occurred by a mutation of Phe397Leu in the SQLE gene as alone or in combination with Ala448Thr. Nevertheless, for the occurrence of in vitro resistance, only the presence of Phe397Leu mutation seems to be decisive.

Enhancement of immune responses by vaccine potential of three antigens, including ROP18, MIC4, and SAG1 against acute toxoplasmosis in mice.

Toxoplasma gondii (T. gondii) causes considerable financial losses in the livestock industry and can present serious threats to pregnant women, as well as immunocompromised patients. Therefore, it is required to design and produce an efficient vaccine for controlling toxoplasmosis. The present study aimed to evaluate the protective immunity induced by RMS protein (ROP18, MIC4, and SAG1) with Freund adjuvant, calcium phosphate nanoparticles (CaPNs), and chitosan nanoparticles (CNs) in BALB/c mice. The RMS protein was expressed in Escherichia coli (E. coli) and purified using a HisTrap HP column. Thereafter, cellular and humoral immunity was assessed by injecting RMS protein on days 0, 21, and 35 into four groups [RMS, RMS-chitosan nanoparticles (RMS-CNs), RMS-calcium phosphate nanoparticles (RMS-CaPNs), and RMS-Freund]. Phosphate buffered saline (PBS), CNs, CaPNs, and Freund served as the four control groups. The results displayed that vaccination with RMS protein and adjuvants significantly elicited the levels of specific IgG antibodies and cytokines against toxoplasmosis. There were high levels of total IgG, IgG2a, and IFN-γ in vaccinated mice, compared to those in the control groups, especially in the RMS-Freund, indicating a Th-1 type response. The vaccinated and control mice were challenged intraperitoneally with 1 × 103 tachyzoites of the T. gondii RH strain four weeks after the last injection, and in RMS-Freund and RMS-CaPNs groups, the highest increase in survival time was observed (15 days). The RMS can significantly increase Th1 and Th2 responses; moreover, multi-epitope vaccines with adjuvants can be a promising strategy for the production of a vaccine against toxoplasmosis.

Five-year surveillance study of clinical and environmental Triazole-Resistant Aspergillus fumigatus isolates in Iran.

BACKGROUND: Invasive aspergillosis is one of the most common fungal infections and azole resistance in Aspergillus fumigatus (ARAf) is a growing medical concern in high-risk patients. To our knowledge, there is no comprehensive epidemiological surveillance study on the prevalence and incidence of ARAf isolates available in Iran. OBJECTIVES: The study aimed to report a five-year survey of triazole phenotypes and genotype patterns concerning the resistance in clinical and environmental A. fumigatus in Iran. METHODS: During the study time frame (2016-2021), a total of 1208 clinical and environmental Aspergillus species were collected. Isolates were examined and characterised by in vitro antifungal susceptibility testing (CLSI M38 broth microdilution) and cyp51A sequencing. RESULTS: In total, 485 Aspergillus section Fumigati strains were recovered (clinical, n = 23; 4.74% and environment, n = 462; 95.26%). Of which A. fumigatus isolates were the most prevalent species (n = 483; 99.59%). Amphotericin B and the echinocandins demonstrated good in vitro activity against the majority of isolates in comparison to triazole. Overall, 16.15% (n = 78) of isolates were phenotypically resistant to at least one of the azoles. However, 9.73% of A. fumigatus isolates for voriconazole were classified as resistant, 89.03% were susceptible, and 1.24% were intermediate. While, for itraconazole and posaconazole, using the epidemiological cut-off value 16.15% and 6.83% of isolates were non-wild types, respectively. Remarkably, in 21.79% (n = 17) phenotypically resistant isolates, no mutations were detected within the cyp51A gene. CONCLUSION: Although the incidence of ARAf varies from country to country, in Iran the rate has ranged from 3.3% to 18%, significantly increasing from 2013 to 2021. Strikingly, a quarter of the phenotypically resistant isolates harboured no mutations in the cyp51A gene. It seems that other mechanisms of resistance are importantly increasing. To fill a gap in our understanding of the mechanism for azole resistance in the non-cyp51A strains, we highly recommend further and more extensive monitoring of the soil with or without exposure to fungicides in agricultural and hospital areas.

Iranian National Survey on Tinea Capitis: Antifungal Susceptibility Profile, Epidemiological Characteristics, and Report of Two Strains with a Novel Mutation in SQLE Gene with Homology Modeling.

BACKGROUND: The data on the epidemiological and antifungal susceptibility profile of tinea capitis (TC) in Iran has not been updated in recent decades. This report presents the Iranian epidemiological and drug susceptibility data regarding the distribution of dermatophytes species isolated by six national mycology centers for a period of one year (2020-2021). MATERIAL AND METHODS: A total of 2100 clinical samples from individuals suspeted to TC were subjected to mycological analysis of direct microscopy and culture. For definite species identification, the culture isolates were additionally subjected to PCR-RFLP and PCR-sequencing of the ITS ribosomal DNA (ITS-rDNA) region. Antifungal susceptibility profiles for eight common antifungal drugs were determined by CLSI M38-A3 guidelines. The SQLE gene was partially amplified and sequenced in two terbinafine-resistant and two susceptible T. mentagrophytes isolates to elucidate probable substitutions involved in resistance. RESULTS: TC (n = 94) was diagnosed in 75 children (79.8%) and 19 adults (20.2%) by direct microscopy and culture. Frequency of TC was significantly more among males (66 males = 70.2% vs 28 females = 29.8%). The prevalent age group affected was 5-9 years (39.36%). Thirty-two (34.04%) T. mentagrophytes, 27 (28.7%) T. tonsurans, 14 (14.9%) M. canis, 13 (13.8%) T. violaceum, 5 (5.32%) T. indotineae, 2 (2.1%) T. benhamiae, and 1 (1.1%) T. schoenleinii were identified as the causative agents. MIC values of isolates showed susceptibility to all antifungal agents, except for fluconazole and griseofulvin with GM MIC of 11.91 µg/ml and 2.01 µg/ml, respectively. Terbinafine exhibited more activity against isolates, with GM MIC 0.084 µg/ml followed by ketoconazole (0.100 µg/ml), econazole (0.107 µg/ml), itraconazole (0.133 µg/ml), butenafine (0.142 µg/ml), and miconazole (0.325 µg/ml). Two resistant T. mentagrophytes isolates harbored missense mutations in SQLE gene, corresponding to amino acid substitution F397L. Remarkably, one unique mutation, C1255T, in the SQLE sequence of two terbinafine-susceptible T. mentagrophytes strains leading to a change of leucine at the 419th position to phenylalanine (L419F) was detected. CONCLUSIONS: T. mentagrophytes, T. tonsurans, and M. canis remained the main agents of TC in Iran, however less known species such as T. indotinea and T. benhamiae are emerging as new ones. Terbinafine could still be the appropriate choice for the treatment of diverse forms of TC.

Investigation of vaginal and rectal swabs of women infected with COVID-19 in two hospitals covered by Mazandaran University of Medical Sciences, 2020.

Due to the emergence of COVID-19 virus worldwide and need to identify ways of transmitting the virus, we conducted a cross-sectional study from July to November 2020 on 80 women with COVID-19 infection was confirmed by nasopharyngeal proper time polymerase chain reaction (RT-PCR). We investigated SARS-CoV-2 in their vaginal and rectal swabs. The results showed that (n = 6, 7.5%) patients had positive rectal PCR and (n = 10, 12.5%) had positive vaginal PCR. There was a statistically significant relationship between positive rectal test and positive vaginal test (p=.001). Positive rectal PCR was significantly higher in women over 60 years old than in other age groups (p=.004).Impact StatementWhat is already known on this subject? In the past studies, the presence of the virus in the vagina and rectum was less or not confirmed.What do the results of this study add? The results of our study showed that the COVID-19 virus can infect the vagina and rectum of women.What are the implications of these findings for clinical practice and/or further research? This finding should be considered in sexual transmission and mother to child transmission and also vaginal colonisation, especially at the time of delivery.

Protective efficacy by a novel multi-epitope vaccine, including MIC3, ROP8, and SAG1, against acute Toxoplasma gondii infection in BALB/c mice.

Toxoplasma gondii is an intracellular apicomplexan parasite, which can cause a serious infectious disease in pregnant women and immunocompromised individuals. Therefore, the development of a polyvalent vaccine consisting of all stages of the parasite life cycle using the epitopes from tachyzoites, bradyzoites, and sporozoites is likely to be required for complete protective immunity. In this study, we designed protein vaccine candidate based on the prediction of specific epitopes (i.e., B cell and T cell) from three Toxoplasma gondii antigens. The MRS protein (MIC3: 30-180, ROP8: 85-185, and SAG1: 85-235) was expressed in Escherichia coli, and purification was performed using a HisTrap HP column and then we evaluated immunogenicity and protective property in BALB/c mice. Seventy-two mice were randomly divided into six groups, including three vaccinations (i.e., MRS, MRS-Freund, and MRS-Calcium Phosphate Nanoparticles (MRS-CaPNs)) and three control (i.e., Phosphate-buffered saline, Freund, and CaPNs) groups. All groups were immunized three times via subcutaneous injection within three-week intervals. In the vaccination groups, the BALB/c mice were injected with 20 µg of MRS protein for the first time and 10 µg of MRS for the next two times. Antibodies, cytokines, and splenocytes proliferation in the immunized mice were assayed using the enzyme-linked immunosorbent assay. Protective efficacy was analyzed by challenging the immunized mice with T. gondii of RH strain. Antibody, cytokine, and lymphocyte proliferation assays showed that the mice immunized with MRS induced stronger humoral and T helper type 1 cell-mediated immune responses, compared to the control mice. However, co-immunization with adjuvants (i.e., Freund and CaNPs) resulted in impaired immune responses. Effective protection against the parasite achieved an increase in survival time in the immunized mice, especially in the MRS-CaNPs group. The obtained results of the present study demonstrated that multi-epitope protein vaccination, MRS, is a potential strategy against toxoplasmosis infection. In addition, the vaccine co-delivered with CaPNs could provide an important key for vaccine candidate to control T. gondii infection.

Genetic Polymorphisms of Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) and clinical outcomes post-allogeneic hematopoietic stem cell transplantation: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVE: Although HLA matching is considered as a key genetic predictor of allo-HSCT outcomes, genetic polymorphisms in non-HLA genes, especially in genes encoding immunoregulatory proteins, have also been proposed as additional risk factors linked to the occurrence of transplant complications. This study aimed to carry out a systematic review and meta-analysis from all eligible cohort studies to determine the effect of CTLA-4 gene polymorphisms, including rs231775, rs3087243, rs4553808, rs5742909, and rs733618, on clinical outcomes in patients receiving an allo-HSCT. METHODS: A systematic literature search in PubMed, Web of Science, and Scopus was performed to identify the relevant studies, and related information was extracted. The effect size (ES) and corresponding 95% confidence intervals (CIs) were calculated to estimate the association. RESULTS: 16 studies were eligible and included in the meta-analysis. The pooled results showed that only the dominant models of rs3087243 were significantly associated with chronic GVHD (cGVHD), while other SNPs were not significantly associated with overall survival, disease-free survival, relapse, and GVHD. CONCLUSIONS: Our study represents, for the first time, a comprehensive meta-analysis on the role of CTLA-4 polymorphisms on outcomes after allo-HSCT. The results indicate that the CT60 CTLA-4 polymorphism could be a significant risk factor for cGVHD.

A novel recombinant protein vaccine containing the different E7 proteins of the HPV16, 18, 6, 11 E7 linked to the HIV-1 Tat (47-57) improve cytotoxic immune responses.

OBJECTIVES: Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. RESULTS: In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E. coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD + 8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8 + T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. CONCLUSION: Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8 + T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.

Different types of adjuvants in prophylactic and therapeutic human papillomavirus vaccines in laboratory animals: a systematic review.

Human papillomavirus (HPV) causes cervical carcinoma, which and is the third most common cancer, accounting for 275,000 deaths annually worldwide. Adjuvants have a key role in promotion of vaccine efficacy; therefore, using prophylactic and therapeutic vaccines combined with adjuvant could be of great benefit in prevention and treatment of cervical cancer. There are different types of adjuvants, including MF59TM adjuvants, RNA-based, JY (interleukin2/chitosan), cholera toxin (CT), heat-labile enterotoxin (LT), Freund's adjuvant, alum, SA-4-1BBL, λ-carrageenan (λ-CGN), heat shock proteins (HSPs), juzen-taiho-to (JTT) and hochu-ekki-to (HET), ISCOM and ISCOMATRIX™, very small size proteoliposomes (VSSPs), granulocyte macrophage colony-stimulating factor (GM-CSF), and Toll-like receptors (TLRs). Adjuvants have various functions, especially in therapeutic vaccines, and they lead to an increase in cytotoxic T lymphocytes (CTLs), so they are important in the design of vaccines. Here, we review the currently used adjuvants and their combinations with HPV protein vaccines in order to introduce an appropriate adjuvant for HPV vaccines.

Tropolone alkaloids from Colchicum kurdicum (Bornm.) Stef. (Colchicaceae) as the potent novel antileishmanial compounds; purification, structure elucidation, antileishmanial activities and molecular docking studies.

Natural compounds played an important role for prevention and treatment of the disease as well as are the important compounds for the design of the new bioactive compounds. In this study, eight tropolone alkaloids were isolated from Colchicum kurdicum including colchicoside, 2-demethyl colchicine, 3-demethyl colchicine, demecolcine, colchifoline, N-deacetyl-N-formyl colchicine, colchicine and cornigerine by column and preparative thin layer chromatography. The chemical structures were identified by 1H NMR and 13C NMR spectroscopy. Moreover, the antileishmanial activity on Leishmania major, anti-inflammatory activity, iron chelating activity and toxicity studies including hemolytic activity, brine shrimp toxicity, cytotoxicity and acute toxicity and docking study of all isolated bioactive compounds were evaluated. As result, colchicoside and colchicine had potent leishmanicidal effects and N-deacetyl-N-formyl colchicine and cornigerine had the highest anti-inflammatory effects. All compounds had the significant iron chelating activity. According to toxicity studies, isolated compounds showed the low hemolytic activity and cytotoxicity, high LC50, LC90 and LD50. In the molecular docking study, colchicoside had the high dockscore. According to the study, with future studies all isolated compounds could be used for design the novel antileishmanial drugs.

Genetic diversity and antifungal susceptibility patterns of Aspergillus nidulans complex obtained from clinical and environmental sources.

The molecular epidemiology and antifungal susceptibility of Aspergillus nidulans species complex has not been well studied. To evaluate the genetic diversity and antifungal susceptibility patterns of clinical and environmental isolates of A. nidulans complex. Sixty clinical and environmental isolates of Aspergillus section Nidulantes were collected from five countries (Iran, The Netherlands, Spain, Portugal and Greece). The species were molecularly identified by sequencing of ß-tubulin gene. The genetic diversity of A nidulans complex isolates (n = 54) was determined with a microsatellite genotyping assay. Antifungal susceptibility profile was determined using EUCAST method. The isolates were classified as A nidulans (46.7%), A spinulosporus (26.6%), A quadrilineatus (10%), A pachycristatus (3.3%), A rugulosus (3.3%), A unguis (5%), A creber, (1.7%), A olivicola (1.7%) and A sydowii (1.7%). Thirty-four sequence types (STs) were identified among the 54 A nidulans complex isolates. A high level of genetic diversity was found among A nidulans sensu stricto strains but low diversity was found among A spinulosporus strains. Amphotericin B showed high MICs to all species. The most active azole was posaconazole (GM = 0.64 mg/L), while itraconazole showed the highest MICs among azoles (GM = 2.95 mg/L). A spinulosporus showed higher MICs than A nidulans sensu stricto for all antifungals except for micafungin and anidulafungin. Interspecies variations may result in differences in antifungal susceptibility patterns and challenge antifungal therapy in infections caused by A nidulans. Differences in the distribution of STs or persistence of multiple STs might be related to the sources of isolation and niche specialisation.

In silico analysis and expression of a novel chimeric antigen as a vaccine candidate against Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that causes one of the most common parasitic infections in humans and other warm-blooded animals. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the development of a vaccine to protect against toxoplasmosis is an attractive option for avoiding infection. The aim of this study was to design an epitope-based vaccine for T. gondii. In the present study, an in silico approach was used to predict and analyze B-cell and T-cell epitopes and the transmembrane domain of proteins SAG1, MIC3, and ROP8. We also predicted the antigenicity, allergenicity, secondary and tertiary structures, and physicochemical characteristics of a chimeric protein. Next, codon optimization and mRNA structure prediction were conducted using bioinformatics tools, and the designed construct was chemically synthesized and cloned into the pET28a vector. SAG1 (amino acid positions 85-235), MIC3 (30-180), and ROP8 (85-185) were found to have several strong immunodominant epitopes that were joined with a rigid linker A(EAAAK)2A. Although the resultant protein called MRS (MIC3, ROP8, and SAG1) did not turn out to be an allergen, its antigenicity was estimated to be 0.7983. Additionally, MRS was selected as the best vaccine candidate on the basis of its secondary and tertiary structures. The number of amino acids, molecular weight, and numbers of negatively and positively charged residues of MRS were 427 and 45,661.31 Da, 45, and 50, respectively. ΔG of the best-predicted structure was -413.0 kcal/mol, and the first nucleotides at the 5' end did not form a stable hairpin or pseudoknot. Finally, successful expression and verification of the expressed MRS protein showed that in silico analysis was almost accurate. This vaccine candidate selected by in silico tools should be validated in experimental studies.

Protective efficacy induced by DNA prime and recombinant protein boost vaccination with Toxoplasma gondii GRA14 in mice.

Toxoplasma gondii, the etiological agent of toxoplasmosis, can cause severe or lethal damages in both animals and man. So, tends to develop a more effective vaccine to prevent this disease is extremely needed and would be so prominent. The novel dense granule antigen 14 (GRA14) has been identified as a potential vaccine candidate against T. gondii infection. The aim of this study was evaluation of protective immunity induced by prime/boost vaccination strategy of GRA14 antigen with calcium phosphate (CaPNs) or Aluminum hydroxide (Alum) nano-adjuvants in BALB/c mice. The finding showed that immunization with the prime-boost strategy using plasmid DNA (pcGRA14) and recombinant protein (rGRA14) with nano-adjuvants significantly elicited levels of specific IgG antibodies and cytokines against T. gondii infection. Given that, there were the high levels of total IgG, IgG2a, IFN-γ in mice of rGRA14-CaPNs and pcGRA14 + rGRA14-CaPNs groups, which indicating a Th-1 type response. While immunization of mice with Alum based rGRA14 and pcGRA14 + rGRA14 elicited specific IgG1 and IL-4 levels, which was confirmed a Th-2 type response. Mice immunized with DNA prime-protein boost vaccine with nano-adjuvants produce more vigorous specific lymphoproliferative responses than mice immunized with other antigen formulations. In addition, the CaPNs-based prime-boost vaccine of pcGRA14 + rGRA14 showed the longest survival time in mice and the lowest parasitic load in their brain tissue compared to the other groups. The results obtained in this study show that the use of GRA14 based DNA prime-protein boost vaccination regime with CaPNs can dramatically enhanced both humoral and cellular immune responses. Therefore, this strategy can provide a promising approach to the development of an effective vaccine against T. gondii infection in the future.

Status of insecticide resistance and its biochemical and molecular mechanisms in Anopheles stephensi (Diptera: Culicidae) from Afghanistan.

BACKGROUND: Insecticide resistance of Anopheles stephensi, the main malaria vector in eastern Afghanistan, has been reported previously. This study describes the biochemical and molecular mechanisms of resistance to facilitate effective vector control and insecticide resistance management. METHODS: Mosquito larvae were collected from the provinces of Kunar, Laghman and Nangarhar from 2014 to 2017. The susceptibility of the reared 3-4 days old adults was tested with deltamethrin 0.05%, bendiocarb 0.1%, malathion 5%, permethrin 0.75% and DDT 4%. Cytochrome P450 content and general esterase, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were measured in the three field populations and the results were compared with those of the laboratory susceptible An. stephensi Beech strain. Two separate allele-specific PCR assays were used to identify L1014, L1014F and L1014S mutations in the voltage gated sodium channel gene of An. stephensi. Probit analysis, ANOVA and Hardy-Weinberg equilibrium were used to analyse bioassay, biochemical assay and gene frequency data respectively. RESULTS: The population of An. stephensi from Kunar was susceptible to bendiocarb, apart from this, all populations were resistant to all the other insecticides tested. The differences between all values for cytochrome P450s, general esterases, GSTs and AChE inhibition rates in the Kunar, Laghman and Nangarhar populations were statistically significant when compared to the Beech strain, excluding GST activities between Kunar and Beech due to the high standard deviation in Kunar. The three different sodium channel alleles [L1014 (wild type), L1014F (kdr west) and L1014S (kdr east)] were all segregated in the Afghan populations. The frequencies of kdr east mutation were 22.9%, 32.7% and 35% in Kunar, Laghman and Nangarhar populations respectively. Kdr west was at the lowest frequency of 4.44%. CONCLUSIONS: Resistance to different groups of insecticides in the field populations of An. stephensi from Kunar, Laghman and Nangarhar Provinces of Afghanistan is caused by a range of metabolic and site insensitivity mechanisms, including esterases, cytochrome P450s and GSTs combined with AChE and sodium channel target site insensitivity. The intensity and frequency of these mechanisms are increasing in these populations, calling for urgent reorientation of vector control programmes and implementation of insecticide resistance management strategies.

Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain.

Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. Leishmania major is the most common cause of cutaneous leishmaniasis. In the Leishmania parasites, sterol 14α-demethylase (CYP51), which is involved in the biosynthesis of sterols, has been identified as an attractive target for development of new therapeutic agents. In this study, the sequence and structure of CYP51 in a laboratory strain (MRHO/IR/75/ER) of L. major were determined and compared to the wild-type strain. The results showed 19 mutations including seven non-synonymous and 12 synonymous ones in the CYP51 sequence of strain MRHO/IR/75/ER. Importantly, an arginine to lysine substitution at position of 474 resulted in destabilization of CYP51 (ΔΔG = 1.17 kcal/mol) in the laboratory strain; however, when the overall effects of all substitutions were evaluated by 100 ns molecular dynamics simulation, the final structure did not show any significant changes (p-value < 0.05) in stability parameter of the strain MRHO/IR/75/ER compared to the wild-type protein. The energy level for the CYP51 of wild-type and MRHO/IR/75/ER strain were -40,027.1 and -39,706.48 Kcal/mol respectively. The overall Root-mean-square deviation (RMSD) deviation between two proteins was less than 1 Å throughout the simulation and Root-mean-square fluctuation (RMSF) plot also showed no substantial differences between amino acids fluctuation of the both protein. The results also showed that, these mutations were located on the protein periphery that neither interferes with protein folding nor with substrate/inhibitor binding. Therefore, L. major strain MRHO/IR/75/ER is suggested as a suitable laboratory model for studying biological role of CYP51 and inhibitory effects of sterol 14α-demethylase inhibitors.

Effects of moderate treadmill exercise and fluoxetine on behavioural and cognitive deficits, hypothalamic-pituitary-adrenal axis dysfunction and alternations in hippocampal BDNF and mRNA expression of apoptosis - related proteins in a rat model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a condition that develops after an individual has experienced a major trauma. Currently, selective serotonin reuptake inhibitors (SSRIs) like fluoxetine are the first-line choice in PTSD drug treatment but their moderate response rates and side effects indicate an urgent need for the development of new treatment. Physical activity is known to improve symptoms of certain neuropsychiatric disorders. The present study investigated the effects of moderate treadmill exercise, the antidepressant fluoxetine and the combined treatment on behavioural deficits, and hypothalamic-pituitary-adrenal (HPA) axis dysfunction. We also examined alternations in hippocampal brain-derived neurotrophic factor (BDNF) and mRNA expression of apoptosis - related proteins in a rat model of PTSD: the single prolonged stress (SPS) model. Rats were exposed to SPS (restraint for 2h, forced swimming for 20min and ether anaesthesia) and were then kept undisturbed for 14days. After that, SPS rats were subjected to chronic treatment with fluoxetine (10mg/kg/day, for 4weeks), moderate treadmill running (4weeks, 5day per week) and the combined treatment (fluoxetine plus treadmill exercise), followed by behavioural, biochemical and apoptosis markers assessments. SPS rats exhibited increased anxiety levels in the elevated plus maze and light/dark box, impaired fear conditioning and extinction in inhibitory avoidance (IA) task, impaired spatial memory in a recognition location memory task and enhanced negative feedback on the HPA axis following a dexamethasone suppression test. SPS rats also showed reduced hippocampal BDNF and enhanced apoptosis. Moderate treadmill exercise, fluoxetine and the combined treatment alleviated the SPS-induced alterations in terms of anxiety levels, HPA axis inhibition, IA conditioning and extinction, hippocampal BDNF and apoptosis markers. Furthermore, the combined treatment was more effective than fluoxetine alone, but in most tests, the effects of the combined treatment were similar to those of exercise alone, suggesting that exercise is the main factor in the beneficial effects of the combined therapy in PTSD patients.

Immunological evaluation of a DNA cocktail vaccine with co-delivery of calcium phosphate nanoparticles (CaPNs) against the Toxoplasma gondii RH strain in BALB/c mice.

Many recent studies have been conducted to evaluate protective immunity mediated by DNA vaccines against toxoplasmosis. Cocktail DNA vaccines showed better immune responses compared to single vaccines. The objective of the current study was to evaluate the protective efficacy of rhomboid 4 (ROM4) and cocktail DNA vaccines (ROM4 + GRA14) of the Toxoplasma gondii RH strain with or without coated calcium phosphate nanoparticles (CaPNs) as the adjuvant to improve the immunogenicity against the T. gondii RH strain in BALB/c mice. Cocktail DNA vaccines of pcROM4 + pcGRA14 of the T. gondii RH strain were constructed. CaPNs were synthesized and the cocktail DNA vaccine was coated with the adjuvant of CaPNs. Immunogenicity and the protective effects of cocktail DNA vaccines with or without CaPNs against lethal challenge were evaluated in BALB/c mice. pcROM4 and cocktail DNA vaccine coated with CaPNs significantly enhanced cellular and humoral immune responses against Toxoplasma compared to pcROM4 and cocktail DNA vaccine without CaPNs (p < 0.05). These findings indicate that the survival time of immunized mice after challenge with the RH strain of T. gondii was increased compared to that of controls and the DNA vaccine provided significant protection in mice (p < 0.05). The CaPN-based cocktail DNA vaccine of pcROM4 + pcGRA14 showed the longest survival time compared to the other groups. Co-immunization with CaPN-based cocktail DNA vaccine (pcROM4 + pcGRA14) boosted immune responses and increased the protective efficacy against acute toxoplasmosis in BALB/c mice compared to both single gene and bivalent DNA vaccine without nano-adjuvants.

Avian gametologs as molecular tags for sex identification in birds of prey of Iran.

Global environmental change and rapid destruction of natural habitats necessitate the conservation of endangered and threatened birds of prey. Recently, molecular sex identification methods based on amplification of introns of chromodomain-helicase DNA binding protein1 (CHD1) have provided valuable tools for ecological study and conservation breeding programs of birds. These methods employ a primer pair flanking an intron which varies considerably in length between the avian gametologs CHD1Z and CHD1W. Herein, we test the applicability of CHD1Z and CHD1W as universal tags for molecular sex identification in birds of prey of Iran. We showed successful sex identification in 22 species of birds of prey using feathers as the source of DNA. The results suggest that the regions of CHD1W and CHD1Z amplified in this study are conserved among most of Falconiformes, enabling accurate sex identification in birds of prey.

Pseudogene-free amplification of HPRT1 in quantitative reverse transcriptase polymerase chain reaction.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a powerful tool for precise gene expression analysis. The accuracy of the results highly depends on careful selection of a reference gene for data normalization. HPRT1 (hypoxanthine phosphoribosyl transferase 1) is a frequently used housekeeping gene for normalizing relative expression values. However, the existence of processed pseudogenes for HPRT1 might interfere with reliable results obtained in qRT-PCR due to amplification of unintended products. Here, we designed a primer pair for pseudogene-free amplification of HPRT1 in qRT-PCR. We demonstrate that this primer pair specifically amplified HPRT1 messenger RNA (mRNA) sequence while avoiding coamplification of the pseudogenes.