In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2 restricted CD8 T cell response

Background

As a potent CD8⁺ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population.

Methods and Findings

Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8⁺ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2⁺ individuals recovered from L. major. HLA-A2⁻ individuals recovered from L. major and HLA-A2⁺ healthy donors were included as control groups. Individual response of HLA-A2⁺ recovered volunteers as percent of CD8⁺/IFN-γ⁺ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2⁻ recovered individuals. Based on cutoff scores calculated from the response of HLA-A2⁻ recovered individuals, 31.6% and 13.3% of HLA-A2⁺ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2⁻ recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2⁺ recovered individuals.

Conclusion

Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis.     

Correlation of circulating omentin-1 with bone mineral density in multiple sclerosis: the crosstalk between bone and adipose tissue

Background

Patients with multiple sclerosis (MS) are at increased risk of osteoporosis and fractures. Adipose tissue-derived adipokines may play important roles in the osteoimmunology of MS. In order to determine whether omentin-1 and vaspin may be related to bone health in MS patients, we compared circulating levels of these recently identified adipokines, between MS patients and healthy controls.

Methods

A total of 35 ambulatory MS patients with relapsing-remitting courses were compared with 38 age- and sex-matched healthy controls. Bone mineral density (BMD) was determined for the lumbar spine (L2–L4) and the proximal femur using dual-energy x-ray absorptiometry. Circulating omentin-1, vaspin, osteocalcin, osteopontin, osteoprotegerin, the receptor activator of nuclear factor-κB ligand, matrix metalloproteinase 9, C-reactive protein and 25-hydroxy vitamin D levels were evaluated by highly specific enzyme-linked immunosorbent assay methods.

Results

There was no significant difference between the two groups regarding bone-related cytokines, adipocytokines, and the BMD measurements of patients with MS and the healthy controls. However, in multiple regression analysis, serum omentin-1 levels were positively correlated with BMD at the femoral neck (β = 0.49, p = 0.016), total hip (β = 0.42, p = 0.035), osteopontin (β = 0.42, p = 0.030) and osteocalcin (β = 0.53, p = 0.004) in MS patients. No correlations were found between vaspin, biochemical, and BMD measures in both groups.

Conclusions

Elevated omentin-1 serum levels are correlated with BMD at the femoral neck and the serum levels of osteocalcin and osteopontin in MS patients. Therefore, there is crosstalk between adipose tissue and bone in MS.     

Use of Mycological, nested PCR, and Real-time PCR Methods on BAL Fluids for Detection of Aspergillus fumigatus and A. flavus in Solid Organ Transplant Recipients

Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted β-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.     

First survey of the two polymorphisms (Arg194Trp and Arg399Gln) in XRCC1 gene in four Afghanistan populations and comparison with worldwide data

Genetic polymorphisms in gene encoding X-ray repair cross-complementation group 1 (MIM: 194360; XRCC1) have been defined. Previous studies have revealed that there was significant difference between populations for allelic frequency of Arg194Trp (rs. 1799782) and Arg399Gln (rs. 25487) polymorphisms of XRCC1. In order to get more insight into the genetic structure of Afghanistan populations the present study was carried out. Present study was done on 656 (257 Pashtuns, 217 Tajiks, 120 Hazaras, and 62 Uzbeks) unrelated healthy Afghanis refuges living in Fars province (southern Iran). Genotypes for Arg194Trp and Arg399Gln polymorphisms of the XRCC1 were detected by RFLP-PCR method. The prevalence of the 194Trp allele in Pashtuns, Tajiks, Hazaras, and Uzbeks was 0.072, 0.085, 0.108, and 0.145, respectively. The frequency of the 399Gln in Pashtuns, Tajiks, Hazaras, and Uzbeks was 0.362, 0.378, 0.296, and 0.234, respectively. There was significant difference between these ethnic groups for the genotypic distributions of the Arg194Trp (χ² = 16.70, df = 6, P = 0.010) and Arg399Gln (χ² = 12.67, df = 6, P = 0.049) polymorphisms. Based on the complete dataset, these polymorphisms showed significant linkage disequilibrium. There was significant difference between the ethnic groups for prevalence of the haplotypes (χ² = 16.67, df = 6, P = 0.011). Uzbeks showed significant difference with the other ethnic groups (χ² = 10.09, df = 2, P = 0.006). The allelic frequencies of 194Trp and 399Gln in Pashtuns and Tajiks seem to be more similar to the Caucasians than the Asian populations. However, Uzbeks seems to be intermediate between Afghanis’ Caucasian (Pashtuns and Tajiks) and Asians.     

Structure of the Sm Binding Site From Human U4 snRNA Derived From a 3 ns PME Molecular Dynamics Simulation

Abstract

A molecular dynamics simulation of the Sm binding site from human U4 snRNA was undertaken to determine the conformational flexibility of this region and to identify RNA conformations that were important for binding of the Sm proteins. The RNA was fully-solvated (>9,000 water molecules) and charge neutralized by inclusion of potassium ions. A three nanosecond MD simulation was conducted using AMBER with long-range electrostatic forces considered using the particle mesh Ewald summation method. The initial model of the Sm binding site region had the central and 3′ stem-loops that flanked the Sm site co-axial with one another, and with the single-stranded Sm binding site region ([I] conformation). During the course of the trajectory, the axes of the 3′ stem-loop, and later the central stem-loop, became roughly orthogonal from their original anti-parallel orientation. As these conformational changes occurred, the snRNA adopted first an [L] conformation, and finally a [U] conformation. The [U] conformation was more stable than either the [I] or [L] conformations, and persisted for the final 1 ns of the trajectory. Analysis of the structure resulting from the MD simulations revealed the bulged nucleotide, U₁₁₄, and the mismatched A₉₁-G₁₁₀ base pair provided distinctive structural features that may enhance Sm protein binding. Based on the results of the MD simulation and the available experimental data, we proposed a mechanism for the binding of the Sm protein sub-complexes to the snRNA. In this model, the D₁/D₂ and E/F/G Sm protein sub-complexes first bind the snRNA in the [U] conformation, followed by conformational re-arrangement to the [I] conformation and binding of the D₃/B Sm protein sub-complex.     

Dehydrogenation of 2-imidazolines with sodium periodate catalyzed by manganese(III) tetraphenylporphyrin

In the present work, dehydrogenation of 2-substituted imidazolines with sodium periodate in the presence of tetraphenylporphyrinatomanganese(III) chloride, [Mn(TPP)Cl], is reported. A wide variety of 2-imidazolines efficiently converted to their corresponding imidazoles by [Mn(TPP)Cl]/NaIO₄ catalytic system at room temperature in 1:2, CH₃CN/H₂O mixture. The effect of reaction parameters such as kind of solvent and catalyst amount was also investigated.     

Fifty years of struggle to control cutaneous leishmaniasis in the highest endemic county in Iran: A longitudinal observation inferred with interrupted time series model

Negligible data are available following major social activities and environmental changes on leishmaniasis. Therefore, how interactions between these events influence cutaneous leishmaniasis (CL) risk is not well-known. This longitudinal study was undertaken to explore the impact of interventions conducted between 1971 and 2020 in Bam county, which has had the highest disease burden in Iran. Only confirmed CL cases during this period were taken into account. Data were analyzed by SPSS 22 using the X² test to assess the significance of the difference between proportions. Moreover, we used interrupted time series (ITS) to assess the impact of three environmental events during this period. Overall, 40,164 cases of CL occurred in the past five decades. Multiple complex factors were among the leading causes that synergistically induced the emergence/re-emergence of CL outbreaks in Bam. The main factors attributed negatively to CL control were cessation of malaria spraying activity, expansion of the city spaces, and a massive earthquake creating new breeding potentials for the vectors. The highest impact on CL incidence during these years was related to the earthquake [coefficient = 17.8 (95% CI: 11.3, 22.7); p-value < 0.001]. Many factors can contribute to CL outbreaks in endemic foci. They also can cause new foci in new areas. Since humans are the single reservoir for CL in this area, early detection and effective management significantly contribute to controlling CL to reduce the disease burden. However, essential evidence gaps remain, and new tools are crucial before the disease can ultimately be controlled. Nevertheless, sustained funding and more trained task forces are essential to strengthen surveillance and case management and monitor the interventions’ impact.     

Lipid-protein cargo transfer: a mode of direct cell-to-cell communication for lipids and their associated proteins

Cells in tissues or in experimental cell colonies respond to stimuli in a co-ordinated manner when they are electrically and chemically coupled by gap junctions. These junctions permit the cell-to-cell passage of small molecules, such as inositol tris phosphate (IP(3)) within the colony and are important in co-ordinating tissue activity. This is the only recognised mechanism of direct chemical signalling that does not involve the release of an extracellular messenger between cells. However, the data in this article demonstrates a new mode of intercellular communication. Two potentially important signalling lipids, PIP(2) and ganglioside G-M1 were shown to move between cells in colonies by tracking (i) fluorescent lipids loaded into the plasma membranes of individual cells in a cell colony using a novel micropipette technique and (ii) movement of fluorescent lipids after localised photobleaching. Furthermore, a large protein molecule, cholera toxin B subunit bound to extracellularly facing ganglioside G-M1 was also shown to transfer between cells. The transfer was inhibited by pre-treatment with poly-L-lysine and polyethylenimine, suggesting a role for tight junctions, perhaps by permitting diffusion of lipids and their protein "cargo" across these cell-to-cell contact points. This is a hitherto unsuspected form of molecular signalling within cell colonies and tissues which may have implications for understanding co-ordinated cell colony behaviour.     

Sympatric morphotypes of the restricted-range Tashan Cave Garra: distinct species or a case of phenotypic plasticity?

Environmental Biology of Fishes - Among Tashan cave barb Garra tashanensis inhabiting a small cave in southwest Iran, two mental disc (sucking mouth disc) forms were observed. To assess their...     

3‐Aryl Coumarin Derivatives Bearing Aminoalkoxy Moiety as Multi‐Target‐Directed Ligands against Alzheimer's Disease

Two series of novel coumarin derivatives, substituted at 3 and 7 positions with aminoalkoxy groups, are synthesized, characterized, and screened. The effect of amine substituents and the length of cross‐linker are investigated in acetyl‐ and butyrylcholinesterase (AChE and BuChE) inhibition. Target compounds show moderate to potent inhibitory activities against AChE and BuChE. 3‐(3,4‐Dichlorophenyl)‐7‐[4‐(diethylamino)butoxy]‐2H‐chromen‐2‐one ( 4y ) is identified as the most potent compound against AChE (IC₅₀=0.27 μm ). Kinetic and molecular modeling studies affirmed that compound 4y works in a mixed‐type way and interacts simultaneously with the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. In addition, compound 4y blocks β‐amyloid (Aβ) self‐aggregation with a ratio of 44.11 % at 100 μm and significantly protects PC12 cells from H₂O₂‐damage in a dose‐dependent manner.