In silico designing of multiepitope-based-peptide (MBP) vaccine against MAPK protein express for Alzheimer's disease in Zebrafish.

Understanding the role of the mitogen-activated protein kinases (MAPKs) signalling pathway is essential in advancing treatments for neurodegenerative disorders like Alzheimer's. In this study, we investigate in-silico techniques involving computer-based methods to extract the MAPK1 sequence. Our applied methods enable us to analyze the protein's structure, evaluate its properties, establish its evolutionary relationships, and assess its prevalence in populations. We also predict epitopes, assess their ability to trigger immune responses, and check for allergenicity using advanced computational tools to understand their immunological properties comprehensively. We apply virtual screening, docking, and structure modelling to identify promising drug candidates, analyze their interactions, and enhance drug design processes. We identified a total of 30 cell-targeting molecules against the MAPK1 protein, where we selected top 10 CTL epitopes (PAGGGPNPG, GGGPNPGSG, SAPAGGGPN, AVSAPAGGG, AGGGPNPGS, ATAAVSAPA, TAAVSAPAG, ENIIGINDI, INDIIRTPT, and NDIIRTPTI) for further evaluation to determine their potential efficacy, safety, and suitability for vaccine design based on strong binding potential. The potential to cover a large portion of the world's population with these vaccines is substantial-88.5 % for one type and 99.99 % for another. In exploring the molecular docking analyses, we examined a library of compounds from the ZINC database. Among them, we identified twelve compounds with the lowest binding energy. Critical residues in the MAPK1 protein, such as VAL48, LYS63, CYS175, ASP176, LYS160, ALA61, LEU165, TYR45, SER162, ARG33, PRO365, PHE363, ILE40, ASN163, and GLU42, are pivotal for interactions with these compounds. Our result suggests that these compounds could influence the protein's behaviour. Moreover, our docking analyses revealed that the predicted peptides have a strong affinity for the MAPK1 protein. These peptides form stable complexes, indicating their potential as potent inhibitors. This study contributes to the identification of new drug compounds and the screening of their desired properties. These compounds could potentially help reduce the excessive activity of MAPK1, which is linked to Alzheimer's disease.

Comparative characterization of microRNA-71 of Echinococcus granulosus exosomes.

Cystic echinococcosis (CE) is a global zoonotic disease caused by Echinococcus granulosus, posing a great threat to human and animal health. MiRNAs are small regulatory noncoding RNA involved in the pathogenesis of parasitic diseases, possibly via exosomes. Egr-miR-71 has been identified as one of the miRNAs in the blood of CE patients, but its secretory characteristics and functions remains unclear. Herein, we studied the secretory and biological activity of exosomal egr-miR-71 and its immunoregulatory functions in sheep peripheral blood mononuclear cells (PBMCs). Our results showed that egr-miR-71 was enriched in the exosome secreted by protoscoleces with biological activity. These egr-miR-71-containing exosomes were easily internalized and then induced the dysregulation of cytokines (IL-10 and TNF-α), nitric oxide (NO) and key components (CD14 and IRF5) in the LPS/TLR4 pathway in the coincubated sheep PBMCs. Similarly, egr-miR-71 overexpression also altered the immune functions but exhibited obvious differences in regulation of the cytokines and key components, preferably inhibiting proinflammatory cytokines (IL-1α, IL-1ß and TNF-α). These results demonstrate that exosomal egr-miR-71 is bioactive and capacity of immunomodulation of PBMCs, potentially being involved in immune responses during E. granulosus infection.

Title: Caractérisation comparative du microARN-71 des exosomes d'Echinococcus granulosus. Abstract: L'échinococcose kystique (EK) est une maladie zoonotique mondiale causée par Echinococcus granulosus, représentant une grande menace pour la santé humaine et animale. Les miARN sont des petits ARN régulateurs non codants impliqués dans la pathogenèse des maladies parasitaires, éventuellement via les exosomes. Egr-miR-71 a été identifié comme l'un des miARN présents dans le sang des patients atteints d'EK, mais ses caractéristiques et fonctions sécrétoires restent floues. Ici, nous avons étudié l'activité sécrétoire et biologique du egr-miR-71 exosomal et ses fonctions immunorégulatrices dans les cellules mononucléées du sang périphérique (CMSP) de mouton. Nos résultats ont montré qu'egr-miR-71 était enrichi dans l'exosome sécrété par les protoscolex ayant une activité biologique. Ces exosomes contenant egr-miR-71 ont été facilement internalisés et ont ensuite induit la dérégulation des cytokines (IL-10 et TNF-α), de l'oxyde nitrique (NO) et des composants clés (CD14 et IRF5) de la voie LPS/TLR4 dans les CMSP de mouton co-incubées. De même, la surexpression d'egr-miR-71 a également modifié les fonctions immunitaires mais a montré des différences évidentes dans la régulation des cytokines et des composants clés, inhibant de préférence les cytokines pro-inflammatoires (IL-1α, IL-1ß et TNF-α). Ces résultats démontrent que l'egr-miR-71 exosomal est bioactif et possède une capacité d'immunomodulation des CMSP, potentiellement impliquée dans les réponses immunitaires lors d'une infection à E. granulosus.

Analysis of sheep peripheral blood mononuclear cells in response to Echinococcus granulosus microRNA-71 overexpression.

Cyst echinococcosis, caused by Echinococcus granulosus, remains a zoonotic disease posing a great threat to public health and meat production industry. Sheep infected with E. granulosus show relatively high abundance of egr-miR-71 in the sera, but its role is unknown. Using bioinformatics and cell migration and Transwell assays, we comparatively analyzed the proteomes and cell invasion of sheep PBMCs in response to egr-miR-71 overexpression. The results showed that the egr-miR-71 induced a total of 157 proteins being differentially expressed and mainly involved in immune responses. In sheep PBMCs, egr-miRNA-71 overexpression induced significant downregulation of macrophage migration inhibitory factor (MIF) and accordingly promoted cell migration and invasion compared with the control. The results will provide a clue for further investigation of a role of circulating egr-miR-71 in immune responses during E. granulosus infection.

Characterization of Fructose-1,6-Bisphosphate Aldolase 1 of Echinococcus multilocularis.

Glycolysis is one of the important ways by which Echinococcus multilocularis acquires energy. Fructose-1, 6-bisphosphate aldolase (FBA) plays an important role in this process, but it is not fully characterized in E. multilocularis yet. The results of genome-wide analysis showed that the Echinococcus species contained four fba genes (FBA1-4), all of which had the domain of FBA I and multiple conserved active sites. EmFBA1 was mainly located in the germinal layer and the posterior of the protoscolex. The enzyme activity of EmFBA1 was 67.42 U/mg with Km and Vmax of 1.75 mM and 0.5 mmol/min, respectively. EmFBA1 was only susceptible to Fe3+ but not to the other four ions (Na+, Ca2+, K+, Mg2+), and its enzyme activity was remarkably lost in the presence of 0.5 mM Fe3+. The current study reveals the biochemical characters of EmFBA1 and is informative for further investigation of its role in the glycolysis in E. multilocularis.

Comparative analysis of different extracellular vesicles secreted by Echinococcus granulosus protoscoleces.

Extracellular vesicles (EVs) are heterogeneous populations of different membrane-wrapped vesicles in size and encapsulated cargo and have recently emerged as a crucial carrier with the functions in intercellular communication, being involved in host-parasite interactions. However, Echinococcus granulosus EVs are not fully described. To separate EVs with a different size, the culture supernatant of E. granulosus protoscoleces (PSCs) was sequentially centrifuged at 2,000g, 10,000g and 110,000g, and the resulting precipitates were accordingly named as 2K, 10K and 110K EVs, respectively. The size and morphology of three different EVs were identified using ZETASIZER NANO and transmission electron microscopy (TEM), respectively. Then mass spectrometry was applied to define protein cargo of EVs and EV internalization was assessed using fluorescent microscopy and flow cytometry. The results showed that 2K EVs mainly ranged from 450 to 950 nm in diameter, 10K EVs ranged from 220 to 390 nm and 110K EVs from 60 to 150 nm. A total of 901 EV proteins were identified, 328 of which were commonly found in the three types of EVs. GO analysis revealed that these proteins were mainly involved in binding (44%) and catalytic activity (44%). Three types of EVs were different in biomarkers (Enolase and 14-3-3) and in reactivity with anti-echinococcosis positive serum. Moreover, 110K EVs were more easily internalized by hepatic cells than 10K EVs as well as 2K EVs (p < 0.0001). These results reveal the physical and biological discrepancy among 2K, 10K and 110K EVs, suggesting a distinct role in host-parasite interactions.

miRNA-seq of Echinococcus multilocularis Extracellular Vesicles and Immunomodulatory Effects of miR-4989.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease. In the infected mice, emu-miR-4989-3p is present in sera, but its role remains unknown. Using high-throughput sequencing and qPCR, emu-miR-4989-3p was herein confirmed to be encapsulated into E. multilocularis extracellular vesicles. In the transfected macrophages, emu-miR-4989-3p was demonstrated to significantly inhibit NO production compared to the control (p < 0.05). Moreover, transfection of emu-miR-4989-3p also gave rise to the increased expression of TNF-α (p < 0.01). Furthermore, emu-miR-4989-3p induced the dysregulation of several key components in the LPS/TLR4 signaling pathway compared with the control, especially TLR4 and NF-κB that both were upregulated. Conversely, the NO production and the expression of TNF-α, TLR4 and NF-κB tended to be increased and decreased in the mimics-transfected cells upon emu-miR-4989-3p low expression, respectively. These results suggest that emu-miR-4989-3p is one of 'virulence' factors encapsulated into the extracellular vesicles, potentially playing a role in the pathogenesis of E. multilocularis.

Determination of antimicrobial effect of protamine by transmission electron microscopy and SDS PAGE on Pseudomonas aeruginosa isolates from diabetic foot infection.

OBJECTIVES: Diabetic foot infection is one of the major complications of diabetes leading to lower limb amputations. Isolation and identification of bacteria causing diabetic foot infection, determination of antibiotic resistance, antimicrobial potential of protamine by electron microscopy and SDS-PAGE analysis, arethe aims of this study. MATERIALS AND METHODS: 285 pus samples from diabetic foot infection patients were collected from different hospitals of Karachi and Capital Health Hospital, Halifax, Canada. Clinical history of each patient was recorded. Bacterial isolates were cultured on appropriate media; identification was done by morphology, cultural and biochemical tests. Effect of protamine against multi drug resistant strains of Pseudomona aeruginosa was checked by minimum inhibitory concentration in 96 well micro-titer plates. The isolates were grown in bactericidal concentration of protamine on plates to isolate mutants. Effect of protamine on protein expression was checked by SDS- PAGE and ultra-structural morphological changes by transmission electron microscopy. RESULTS: Results indicated prevalence of foot infection as 92% in diabetic patients. Major bacterial isolates were Staphylococcus aureus 65 (23%), P. aeruginosa 80 (28.1%), Klebsiella spp. 37 (13%), Proteus mirabilis 79 (27.7%), and Escherichia coli 24 (12%). These isolates were highly resistant to different antibiotics. MIC value of protamine was 500 µg/ml against P. aeruginosa. SDS-PAGE analysis revealed that protamine can suppress expression of various virulence proteins and electron micrographs indicated condensation of cytoplasm and accumulation of protamine in cytoplasm without damaging the cell membrane. CONCLUSION: P. aeruginosa and S. aureus were the major isolates expressing multi-drug resistance and protamine sulfate represented good antimicrobial potential.

Effect of glycine: Studying memory and behavioral changes in mice.

Glycine is an important chemical mediator of nervous system that plays a vital role in memory and other neurological functions. Therefore, the effect of glycine on these traits must be studied to understand biological mechanisms of intricate neurological system. We investigated the effect of different doses of glycine on memory and behavior using 30 albino mice models (treated and control). After two weeks of glycine dosing, we performed light and dark activity and novel-object recognition (NOR) tests to assess the cognitive traits. Brain and blood samples were taken and kept at -70°C using ultra-low temperature freezer. Neurochemical estimation of blood glycine level was estimated by high-performance liquid chromatography with electrochemical detectors (HPLC-ECD). Concentration of glycine (100, 300 and 500 mg/kg) is significantly observed (p<0.01) and it changes due to physiological variations in N-methyl-Daspartate (NMDA) an important neurotransmitter for memory. We observed significant increase in serotonin metabolites including 5-hydroxy tryptophan (5-HT, p<0.05) and 5-hydroxy indole acetic acid (5-HIAA, p<0.001) levels. Similarly,effects were found in case of dopamine (DA, p<0.05) and its metabolites: 3, 4-Dihydroxyphenylacetic acid (DOPAC,p<0.001) and homovanillic acid (HVA, p<0.001). Histopathological investigation of brain tissues showed cellular clumps at cortical junctions at higher doses of glycine as compared to control. These findings revealed that dose dependent concentration of glycine can be useful for memory loss and behavior deficits.

Molecular detection of Ehrlichia canis in dogs from three districts in Punjab (Pakistan).

Canine monocytic ehrlichiosis is a tick-borne disease caused by an intracellular alpha-proteobacterium, Ehrlichia canis, which replicates within mononuclear cells in the host. This study was designed to use a polymerase chain reaction (PCR) protocol for the molecular detection of E. canis by the amplification of a portion of its 16S rRNA gene, as well as the effects of this alpha-proteobacterium on the haematological parameters of the sampled dogs and the risk factors associated with E. canis infection. A total of 151 blood samples were collected from dogs of various breeds at three sampling sites (Lahore, Rawalpindi/Islamabad and Multan) in Punjab, Pakistan. Data regarding the epidemiological factors (including age, gender, breed, body temperature, deworming, vaccination, mucous membrane status, hydration status, the presence of haematuria and tick infestation) were collected through a questionnaire at the time of sample collection. A 400 bp DNA fragment of the 16S rRNA gene of E. canis was amplified from 42 dog blood samples (28% of the total), [Lahore (N = 24), Rawalpindi/Islamabad (N = 13) and Multan (N = 05)] through PCR. Data analysis revealed that the character of the animals (age, sex and breed) had no significant association (P > 0.05) with the presence of E. canis. Various haematological parameters were also compared, and the results revealed that all of the parameters remained unaffected, except significantly lower white blood cell counts (P = 0.004) in E. canis-positive blood samples, as compared with the control group. We concluded that this is the first molecular confirmation of canine infection by E. canis using PCR. Moreover, no specific epidemiological parameter was found associated with the prevalence of E. canis in dogs.

Identification of emu-TegP11, an EF-hand domain-containing tegumental protein of Echinococcus multilocularis.

Tegumental proteins (TegPs) are a group of proteins that coat on the surface of worms, mainly being involved in ion uptake and immune evasion. Echinococcus species have many TegPs, but none of them have been characterized and their role remains unclear. The genome-wide analysis revealed that there were at least 14 tegp genes (tegp1 - 14) in Echinococcus species, the majority of which were found to contain an EF-hand domain or a dynein light chain-like domain or both. Despite low identity, all TegP11 proteins from 25 flatworms were conserved in structure. Echinococcus multilocularis TegP11 (emu-TegP11) was verified to be secreted by extracellular vesicles and to be localized in different spatiotemporal patterns in protoscoleces. Moreover, emu-TegP11 was also shown to have weak or no Ca2+-binding capacity. In treated macrophages, emu-TegP11 interfered with the small RNA-induced silencing pathway via inducing ectopic expression of some key component genes. Additionally, emu-TegP11 remarkably promoted NO secretion possibly by upregulation of inos gene expression (p < 0.05). It was further shown that emu-TegP11 acted as a suppressor of inflammation, with il-12B and il-1ß being significantly down-regulated (p < 0.01), and il-10 and il-4 being significantly upregulated (p < 0.05). The study demonstrates a regulatory role of emu-TegP11, likely acting as a immunomodulator to be involved in regulation of host immune system during Echinococcus infection.

Correlates of myopia in students of Bahauddin Zakariya University, Multan.

The present study was conducted to determine the prevalence of myopia in young students of Bahaudin Zakariya University, Multan. A total sample of 620 students of both gender (male=295; female=325) was collected during 2014. The data was divided in two breeding pattern groups, five groups on account of age at myopia onset and different family size. Out of 620 subjects, 150 had myopia (male=85; female=65). The overall prevalence of myopia was 24.19%. The myopia prevalence was apparently higher in males 28.8% as compared to females 20%. It was observed that myopia was more in age group 21 (37.33%) and less in age group 18 (2.67%). Myopia was found to be higher in inbreeding group (cousin marriage) 56.67% when compared with out-breeding group 43.33% and was found significantly (P< 0.05) more 69.33% in family size of 6-9 as compared 5 (5.33%) respectively.

Suppression of nemo-like kinase by miR-71 in Echinococcus multilocularis.

Echinococcus multilocularis metacestodes are a causative pathogen for alveolar echinococcosis in human beings, and have been found to express miRNAs including emu-miR-71. miR-71 is evolutionarily conserved and highly expressed across platyhelminths, but little is known about its role. Here it was shown that emu-miR-71 was differentially expressed in protoscoleces and was unlikely to be expressed in neoblasts. The results of the luciferase assay indicated that emu-miR-71 was able to bind in vitro to the 3'-UTR of emu-nlk, encoding a key regulator of cell division, causing significant downregulation of luciferase activity (p < 0.01) compared to the negative control and the construct with mutations in the binding site. Consistent with the decreased luciferase activity, transfection of emu-miR-71 mimics into protoscoleces notably repressed emu-NLK (p < 0.05). These results demonstrate the suppression of emu-nlk by emu-miR-71, potentially involved in the protoscolex development.

Regulatory effects of Echinococcus multilocularis extracellular vesicles on RAW264.7 macrophages.

Extracellular vesicles (EVs) play a role in intercellular communications via exchanging biological molecules, being involved in host-parasite interplay. Little is to date known about E. multilocularis EVs and their biological activities. Here spherical EVs secreted by E. multilocularis metacestodes were shown to range predominately from 34nm to 95nm in diameter. A total of 433 proteins were identified in the EVs, and the proteins involved in binding (42%) and catalytic activity (41%) were most frequently represented. Moreover, the proteins associated with EV biogenesis and trafficking, including annexin, 14-3-3, tetraspanin and heat shock protein 70kDa, were highly enriched. It was shown that the EVs remarkably suppressed NO produced by activated RAW macrophages via downregulation of inducible nitric oxide synthase expression (p <0.01). Suppression of pro-inflammatory cytokines, especially IL-1α and IL-1ß, was also observed post treatment with the EVs. Conversely, increased expression of the majority (10/11) of key components involved in the LPS/TLR4 pathway was induced by the EVs. These results demonstrate a regulatory effect of E. multilocularis EVs on macrophages, suggesting a role in parasite-host interactions.

miRNA profiling in the mice in response to Echinococcus multilocularis infection.

miRNAs are small non-coding regulatory RNAs and actively contribute to the pathogenesis of parasitic diseases in multiple ways. The influence of Echinococcus multilocularis infection on host miRNAs remains unclear. Herein, it was shown that E. multilocularis infection disturbed the expression of 4 of 10 genes essential to miRNA biogenesis in the mouse liver, including ago1, ago4, tarbp2 and xrn2. Comparative analysis of deep sequencing data identified 46 differentially expressed miRNAs with 93.5% (43/46) being down-regulated, some of which are associated with modulation of liver cell death and fibrosis, and GO analysis revealed that these miRNAs were mainly enriched in signal transduction (p<0.008). Moreover, 57 miRNAs were commonly found to be edited in complex patterns in both control and E. multilocularis-infected samples. In some miRNAs, editing of nucleotides at the same or/and distinct positions in a given miRNA occurred in different frequencies. Correlation analysis showed that the mutation and editing rates of 57 commonly edited miRNAs were significantly correlated between both samples (r=0.9974, p<0.0001), suggesting little effect of E. multilocularis infection on miRNA mutation and editing. These results provide a rich and informative data for further studies of a role of host miRNAs during E. multilocularis infection.

Neospora caninum prevalence in dogs raised under different living conditions.

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of N. caninum because they act as definitive hosts shedding oocysts in the environment. Vertical transmission of the parasite is well recognized as an important aspect of the epidemiology of the parasite but the importance of horizontal transmission has been less studied. A N. caninum competitive ELISA was used to examine serum samples from 600 dogs that were raised under 4 different living conditions. Samples from 138 dogs living on 24 dairies with a prevalence (0-70%) of anti-N. caninum antibodies in the cattle, 294 pet dogs without neurological signs, 76 from pet dogs exhibiting neurological signs, and 92 stray dogs were examined. The overall seroprevalence of N. caninum was 23.5% (95% CI = ± 2.99) in the 600 dogs. Significant (P < 0.05) differences were observed between the 4 different populations of dogs. The number of N. caninum positive samples were: 51 (36.9%, 95% CI = ± 3.09) of 138 dogs from dairies, 31 (10.5%, 95% CI = ± 6.38) of 294 pet dogs without neurological signs, disorders, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with neurological signs, and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs. Seropositivity to N. caninum in dogs from dairies was associated with a high prevalence of N. caninum antibodies in the cattle. At the 3 dairies where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P < 0.05) than in the 21 dairies where dogs were present. Seroprevalence was significantly higher (P < 0.05) in male dogs (97 of 366; 26.5%, 95% CI = ± 3.40) than in female dogs (44 of 234; 18.8%, 95% CI = ± 5.65). Seroprevalence in dogs increased with age suggesting postnatal exposure to N. caninum infection however, this increase was not significant (P > 0.05). The prevalence of N. caninum antibodies was not significantly (P>0.05) different in dogs based on breed. These findings suggest a relationship between N. caninum infection of dogs from dairies and cattle on these dairies. However, further research is required to determine what is the most important way dogs acquire infection and how to prevent dogs from shedding oocysts.