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Tetrazoles are five-membered ring aromatic heterocyclic molecules that consist of one carbon and four nitrogen atoms. Several tetrazole-based drugs have shown promising activities against bacteria, fungi, asthma, cancer, hypertension etc. The overall aim of this study was to determine anti-Acanthamoebic properties of tetrazoles and tetrazole-conjugated silver nanoparticles. Tetrazole-conjugated silver nanoparticles were synthesized and confirmed using ultraviolet-visible spectrometry, Dynamic light scattering, and Fourier-transform infrared spectroscopy. Using amoebicidal, encystment, and excystment assays, the findings revealed that tetrazoles exhibited antiamoebic properties and these effects were enhanced when conjugated with silver nanoparticles. Importantly, conjugation with silver nanoparticles inhibited parasite-mediated human cell death in vitro, as measured by lactate dehydrogenase release, but it reduced toxic effects of drugs alone on human cells. Overall, these results showed clearly that tetrazoles exhibit potent antiamoebic properties which can be enhanced by conjugation with silver nanoparticles and these potential in the rational development of therapeutic interventions against parasitic infections such as keratitis and granulomatous amoebic encephalitis due to pathogenic Acanthamoeba., Competing Interests: Declaration of competing interest Authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cancer progression is a multifaceted process that entails several stages and demands the persistent expression or activation of transcription factors (TFs) to facilitate growth and survival. TFs are a cluster of proteins with DNA-binding domains that attach to promoter or enhancer DNA strands to start the transcription of genes by collaborating with RNA polymerase and other supporting proteins. They are generally acknowledged as the major regulatory molecules that coordinate biological homeostasis and the appropriate functioning of cellular components, subsequently contributing to human physiology. TFs proteins are crucial for controlling transcription during the embryonic stage and development, and the stability of different cell types depends on how they function in different cell types. The development and progression of cancer cells and tumors might be triggered by any anomaly in transcription factor function. It has long been acknowledged that cancer development is accompanied by the dysregulated activity of TF alterations which might result in faulty gene expression. Recent studies have suggested that dysregulated transcription factors play a major role in developing various human malignancies by altering and rewiring metabolic processes, modifying the immune response, and triggering oncogenic signaling cascades. This review emphasizes the interplay between TFs involved in metabolic and epigenetic reprogramming, evading immune attacks, cellular senescence, and the maintenance of cancer stemness in cancerous cells. The insights presented herein will facilitate the development of innovative therapeutic modalities to tackle the dysregulated transcription factors underlying cancer., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A comprehensive investigation utilized a column-type sequencing batch reactor (SBR) to efficiently remove nutrients throughout various phases of its operational cycle by forming granules. This study assessed the influence and mechanisms of a simultaneous nitrification and denitrification (SND) system employing a column-type sequential batch reactor (SBR). The primary focus was on elucidating the functional groups involved in nitrogen transformation and removal within the extracellular polymeric substances (EPS). The research findings demonstrate the superior performance of the SBR process compared to the control group. It achieved an impressive SND efficiency of 69%, resulting in a remarkable 66% total nitrogen removal. Furthermore, a detailed analysis unveiled that the SBR process had a beneficial impact on the composition and properties of EPS. This impact was observed through increased EPS content and enhanced capacity to transport, convert, and retain nitrogen effectively. Additionally, after initial acclimatization, the SBR process showed its effectiveness in removing nutrients (88-98%) and COD (93%) from the generated wastewater within a hydraulic retention time (HRT) of 6 h. A statistically significant difference between the treatments for the investigated mixing ratios was found by univariate analysis of variance (ANOVA). Machine learning (CatBoost model) was employed to understand each parameter's relationship and predict the outcomes in measurable quantity. The findings of the SBR trials showed that the concentration of generated wastewater and the HRT impacted the treatment efficiency. However, the effluent may still need other physicochemical processes, such as membrane filtering, coagulation, electrocoagulation, etc., as post-treatment options, even though COD, nutrients, and turbidity have been entirely or significantly effectively removed. Overall, this work offers insightful information on the critical function of the SBR bacterial community in promoting SND., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This investigation is centered on the effectiveness of methylene blue (MB), a cationic dye, adsorbed from an aqueous media by H 3 PO 4 activated papaya skin/peels (PS PAC ), with initial pH (2-10), contact time (30-180 min), MB dye concentration (varying from 10 to 50 mg/L), and MB dose (0.1-0.5 gm). The findings show that the best optimal conditions for MB dye removal occur at a 6 pH, 0.3 gm dose of PS PAC adsorbent for 10 mg/L MB dye concentration, with 90 min of contact time. To optimize and validate the extraction efficiency of MB dye, a response surface methodology (RSM) study was conducted using a central composite design (CCD) with a regression model showing R 2  = 0.9940. FT-IR spectroscopy shows, CO, and O-H stretching functional groups while FE-SEM is assessed to supervise morphological features of the PS PAC adsorbent. The peak adsorption capacity with 46.95 mg/g for the Langmuir isotherm model conveniently satisfies the adsorption process with R 2  = 0.9984 while with R 2  = 0.999, a kinetic model, pseudo-second-order, confirms MB dye adsorption by PS PAC adsorbent. Moreover, thermodynamic parameters including ΔGᵒ, ΔH°, and ΔS° were computed and found to be spontaneous and exothermic. Furthermore, regeneration studies employed with NaOH (0.1 M) and HCl (0.1 M) solution media show an acceptable MB removal efficiency consecutive up to three cycles. The study highlights that H 3 PO 4 papaya skin/peel (PS PAC ) is an effectual, sustainable, reasonably available biosorbent to remove industrial cationic dyes disposal., Competing Interests: Declaration of competing interest The authors confirm that they have no known financial conflicts or personal connection that could have perceived to influence the work reported in this manuscript., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Implementing unified municipal solid waste management (MSWM) is often difficult due to socio-economic variables. However, spatial GIS models and statistical analysis of solid waste characterized by the weekdays, weekends, and festivals can somewhat mitigate the variance and assist with selecting suitable waste management methods. This paper presents the example of Rajouri, India, to propose a suitable MSWM based on Inverse Distance Weighted (IDW) intensity maps and statistical findings. The considered region was divided into different sample sites based on the local population density, and Municipal Solid Waste (MSW) was collected from four locations in each site on weekdays, weekends, and festivals. Compositional analysis of the MSW was then used to generate spatial IDW models in QGIS 3.22.7 to interpolate MSW generation over the entire area. Finally, statistical analysis was conducted to gain insight into the waste generation and accumulation trends. The results show that Rajouri produces 245 tonnes of waste daily (per capita: 0.382 kg/day) with a large organic fraction compared to other waste categories. Besides, waste generation is observed to increase over weekends and festivals due to increased consumption of material goods. Composting could serve as a vector for municipal solid waste because of its increased organic component and cost constraints. However, further research on the potential segregation techniques for the organic fraction of solid waste is needed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Acanthamoeba castellanii causes granulomatous amoebic encephalitis, an uncommon but severe brain infection and sight-threatening Acanthamoeba keratitis. Most of the currently used anti-amoebic treatments are not always effective, due to persistence of the cyst stage, and recurrence can occur. Here in this study we synthesize cinnamic acid and lactobionic acid-based magnetic nanoparticles (MNPs) using co-precipitation technique. These nanoformulations were characterized by Fourier transform infrared spectroscopy and Atomic form microscopy. The drugs alone (Hesperidin, Curcumin and Amphotericin B), magnetic NPs alone, and drug-loaded nano-formulations were evaluated at a concentration of 100 μg/mL for antiamoebic activity against a clinical isolate of A. castellanii. Amoebicidal assays revealed that drugs and conjugation of drugs and NPs further enhanced amoebicidal effects of drug-loaded nanoformulations. Drugs and drug-loaded nanoformulations inhibited both encystation and excystation of amoebae. In addition, drugs and drug-loaded nanoformulations inhibited parasite binding capability to the host cells. Neither drugs nor drug-loaded nanoformulations showed cytotoxic effects against host cells and considerably reduced parasite-mediated host cell death. Overall, these findings imply that conjugation of medically approved drugs with MNPs produce potent anti-Acanthamoebic effects, which could eventually lead to the development of therapeutic medications., Competing Interests: Declarations of competing interest No conflict of interest exists and the manuscript was submitted with the authorization of all authors., (Copyright © 2023. Published by Elsevier Inc.)

The Tunisian population has experienced a nutrition transition with an increase in the incidence of obesity. As obesity has been associated with a poor orosensory detection of fat. We hypothesized that poor fat detection could be a driver of poor diet quality. This study examined the association between linoleic acid (LA) detection and adherence to a healthy diet among adult participants. A total of 104 LA taster participants were recruited for this study. Dietary assessment was conducted using the 24 h dietary recall method. Diet quality was assessed by determining the Mediterranean diet (MD) score and Health diet indicator (HDI). The relationship between diet quality and log LA detection threshold was done using adjusted linear regression for age, sex, and daily energy intake (only in the fully adjusted model). The predictive margins model (interaction: anthropometric status x LA threshold) was used to assess the difference between non-obese and subjects with obesity adherence to MD across LA detection values. We have observed that the increase in the concentration of linoleic acid detection by 1 log(mmol/L) is associated with an increase of HDI score by 0.12-point [95% CI: 0.02-0.21] and a decrease of the MD score by -0.14-point [-0.25 to -0.03] in the partially adjusted model. However, only the MD score remained negatively associated with LA detection threshold in the fully adjusted model. The subjects with obesity adherence to the Mediterranean diet was lower than subjects with normal weight for LA concentration less than 0 log(mmol/L). The present study suggests that poor orosensory detection of dietary lipids might be a driver for worsening diet quality. Hence, These subjects might be at risk for obesity and, consequently, exposed cumulatively to the harmful effects of excess adiposity and an unhealthy diet., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Acanthamoeba keratitis is a painful, sight-threatening infection. It is commonly associated with the use of contact lens. Several lines of evidence suggest inadequate contact lens solutions especially against the cyst forms of pathogenic Acanthamoeba, indicating the need to develop effective disinfectants., Objective: In this work, the application and assessment of montmorillonite clay (Mt-clay), cetylpyridinium chloride (CPC) and cetylpyridinium chloride-montmorillonite clay complex (CPC-Mt) against keratitis-causing A. castellanii belonging to the T4 genotype was studied., Methods: Adhesion to human cells and amoeba-mediated cytopathogenicity assays were conducted to determine the impact of Mt-clay, CPC and CPC-Mt complex on amoeba-mediated binding and host cell death. Furthermore, assays were also performed to determine inhibitory effects of Mt-clay, CPC and CPC-Mt complex on encystment and excystment. In addition, the cytotoxicity of Mt-clay, CPC and CPC-Mt complex against human cells was examined., Results: The results revealed that CPC and CPC-Mt complex presented significant antiamoebic effects against A. castellanii at microgram dose. Also, the CPC and CPC-Mt complex inhibited amoebae binding to host cells. Furthermore, CPC and CPC-Mt complex, were found to inhibit the encystment and excystment processes. Finally, CPC and CPC-Mt complex showed minimal host cell cytotoxicity. These results show that CPC and CPC-Mt complex exhibit potent anti-acanthamoebic properties., Conclusion: Given the ease of usage, safety, cost-effectiveness and long-term stability, CPC and CPC-Mt complex can prove to be an excellent choice in the rational development of contact-lens disinfectants to eradicate pathogenic Acanthamoeba effectively., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mycobacterium leprae is known to cause leprosy, a neurological and dermatological disease. In the past 20 years, 16 million leprosy cases have been recorded and more than 200,000 new cases were registered each year, indicating that the disease is still progressing without hindrance. M. leprae, an intracellular bacterium, infects the Schwann cells of the peripheral nervous system. Several types of leprosy have been described, including indeterminate, tuberculoid, borderline tuberculoid, mid-borderline, borderline lepromatous and lepromatous, and three different forms of leprosy reactions, namely type 1, 2 and 3, have been designated. Microscopic detection, serological diagnostic test, polymerase chain reaction and flow tests are employed in the diagnosis of leprosy. The recommended treatment for leprosy consists of rifampicin, dapsone, clofazimine, ofloxacin and minocycline and vaccines are also available. However, relapse may occur after treatment has been halted and hence patients must be educated on the signs of relapse to allow proper treatment and reduce severity. In this review, we depict the current understanding of M. leprae pathogenicity, clinical aspects and manifestations. Transmission of leprosy, diagnosis and treatment are also discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Balamuthia mandrillaris and Naegleria fowleri are free-living amoebae that can cause life-threatening infections involving the central nervous system. The high mortality rates of these infections demonstrate an urgent need for novel treatment options against the amoebae. Considering that indole and thiazole compounds possess wide range of antiparasitic properties, novel bisindole and thiazole derivatives were synthesized and evaluated against the amoebae. The antiamoebic properties of four synthetic compounds i.e., two new bisindoles (2-Bromo-4-(di (1H-indol-3-yl)methyl)phenol (denoted as A1) and 2-Bromo-4-(di (1H-indol-3-yl)methyl)-6-methoxyphenol (A2)) and two known thiazole (4-(3-Nitrophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (A3) and 4-(Biphenyl-4-yl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (A4)) were evaluated against B. mandrillaris and N. fowleri. The ability of silver nanoparticle (AgNPs) conjugation to enrich antiamoebic activities of the compounds was also investigated. The synthetic heterocyclic compounds demonstrated up to 53% and 69% antiamoebic activities against B. mandrillaris and N. fowleri respectively, while resulting in up to 57% and 68% amoebistatic activities, respectively. Antiamoebic activities of the compounds were enhanced by up to 71% and 51% against B. mandrillaris and N. fowleri respectively, after conjugation with AgNPs. These compounds exhibited potential antiamoebic effects against B. mandrillaris and N. fowleri and conjugation of synthetic heterocyclic compounds with AgNPs enhanced their activity against the amoebae., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hospital wastewater are a lurking threat to environment and human health security for any given moment of time owing to its complexity and high vulnerability to cause disease outbreak. Though there are a number of treatment process for wastewater., there is a high need for employing cost-efficient and sustainable method of treatment. Hence a pilot scale horizontal surface flow Constructed Wetland (HSFCW) coupled with Tubesettler was installed at New Delhi, India (February to may 2019). This study reports comparative pollutants removal from hospital wastewater using Constructed Wetlands and associated tubesettler dosed with Hospital wastewater. A pilot scale CW system was used for treating 10m 3 /day of hospital wastewater. The system was tested for 3 Months to evaluate its performance for removing pollutants from the wastewater. The HSFCW coupled with tubesettler achieved over all removal efficiency of 94% (COD), MLSS (97%), TSS (98%), BOD 5 (96%), Phosphate (79%). However, process of nitrification was not observed and accumulation of Nitrate up to 197% was observed. The study concluded that it may be due to the presence of pharmaceuticals and other elements present in hospital wastewater. This conclusion was based on the fact that Alkalinity increased by 52% in effluent and pH value also exhibited an average increase of 12%. Further research studies are required to investigate effect of pharmaceutical originating from hospital on treatment efficiency, to incorporate anaerobic setup to complete denitrification-nitrification process and also to determine efficiency of thermophilic, mesophilic, and psychrophilic bacteria with respect to climate and temperature., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Acanthamoeba castellanii is an opportunistic protozoan responsible for serious human infections including Acanthamoeba keratitis and granulomatous amoebic encephalitis. Despite advances in antimicrobial therapy and supportive care, infections due to Acanthamoeba are a major public concern. Current methods of treatment are not fully effective against both the trophozoite and cyst forms of A. castellanii and are often associated with severe adverse effects, host cell cytotoxicity and recurrence of infection. Therefore, there is an urgent need to develop new therapeutic approaches for the treatment and management of Acanthamoebic infections. Repurposing of clinically approved drugs is a viable avenue for exploration and is particularly useful for neglected and rare diseases where there is limited interest by pharmaceutical companies. Nanotechnology-based drug delivery systems offer promising approaches in the biomedical field, particularly in diagnosis and drug delivery. Herein, we conjugated an antihyperglycemic drug, metformin with silver nanoparticles and assessed its anti-acanthamoebic properties. Characterization by ultraviolet-visible spectrophotometry and atomic force microscopy showed successful formation of metformin-coated silver nanoparticles. Amoebicidal and amoebistatic assays revealed that metformin-coated silver nanoparticles reduced the viability and inhibited the growth of A. castellanii significantly more than metformin and silver nanoparticles alone at both 5 and 10 μM after 24 h incubation. Metformin-coated silver nanoparticles also blocked encystation and inhibited the excystation in Acanthamoeba after 72 h incubation. Overall, the conjugation of metformin with silver nanoparticles was found to enhance its antiamoebic effects against A. castellanii. Furthermore, the pretreatment of A. castellanii with metformin and metformin-coated silver nanoparticles for 2 h also reduced the amoebae-mediated host cell cytotoxicity after 24 h incubation from 73% to 10% at 10 μM, indicating that the drug-conjugated silver nanoparticles confer protection to human cells. These findings suggest that metformin-coated silver nanoparticles hold promise in the improved treatment and management of Acanthamoeba infections., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bacterial infections have remained significant despite our advances in the development of a plethora of disinfectants as well as antimicrobial chemotherapy. This is in part due to our incomplete understanding of the prevalence of bacterial pathogens in the environmental and clinical settings. Several lines of evidence suggest that Acanthamoeba is one of the most ubiquitous/resilient protists that also acts as a host/reservoir for pathogenic microbes. Thus targeting the hardy host, which harbour microbial pathogens, offer a potential avenue to counter infection transmission, particularly hospital/community-acquired infections. This will complement existing approach of applying disinfectants that are targeted against bacterial pathogens directly., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Crocodiles exist in unsanitary environments, feed on rotten meat, are often exposed to heavy metals such as arsenic, cadmium, cobalt, chromium, mercury, nickel, lead, selenium, tolerate high levels of radiation, and are amid the very few species to survive the catastrophic Cretaceous-Tertiary extinction event, nonetheless they can live for up to a 100 years. Moreover, as they live in unhygienic conditions, they regularly come across pathogens. Logically, we postulate that crocodiles possess mechanisms to defend themselves from noxious agents as well as protecting themselves from pathogens. To test this hypothesis, various organ lysates and serum of Crocodylus palustris were prepared. Amoebicidal assays were performed using Acanthamoeba castellanii belonging to the T4 genotype. Cytotoxicity assays were performed using Prostate cancer cells culture by measuring lactate dehydrogenase release as a marker for cell death. Growth inhibition assays were performed to determine the growth inhibitory effects of various organ lysates. Serum and heart lysates of Crocodylus palustris exhibited powerful anti-tumor activity exhibiting more than 70% Prostate cancer cell death (P < 0.05). Additionally, lysates from gall bladder and bile also showed significant host cell cytotoxicity, however intestine, lungs and brain showed partial cytotoxicity. Both sera and heart lysates of Crocodylus palustris abolished Prostate cells growth. Moreover, serum completely abolished A. castellanii viability. For the first time, these findings showed that the organ lysates of Crocodylus palustris exhibit potent anti-amoebic and anti-tumor activity. The discovery of antimicrobial and antitumor activity in crocodile will stimulate research in finding therapeutic molecules from unusual sources, and has potential for the development of novel antitumor/antimicrobial compound(s) that may also overcome drug resistance. Nevertheless, rigorous research in the next few years will be necessary to realize these expectations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of this study was (i) to assess the antimicrobial effects of contact lens disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact lens solutions including Oxysept ® , AO SEPT PLUS, OPTI-FREE ® pure moist ® , Renu ® fresh™, FreshKon ® CLEAR and COMPLETE RevitaLens™ were performed using Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we determined whether combination of both agents can enhance efficacy of marketed contact lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100% kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had potent effects against Acanthamoeba cysts viability. When tested against trophozoites, two disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents in contact lens disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy of contact lens disinfectants tested in this study, these findings present a significant concern to public health. These findings revealed that targeting cyst wall by using cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy against this devastating eye infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Among the genus Streptococcus, S. pyogenes and S. pneumoniae are the major causes of pharyngitis, impetigo, pneumonia and meningitis in humans. Streptococcus spp. are facultative anaerobes that are nutritionally fastidious, yet survive in the environment and target the predisposed population. Antibacterial disinfectants have been partially effective only, indicating the need for novel preventative measures and to understand mechanisms of bacterial resistance. Acanthamoeba is a free-living protist that is known to harbour microbial pathogens, provide shelter, and assist in their transmission to susceptible population. The overall aim of this study was to determine whether S. pyogenes and S. pneumoniae can interact with A. castellanii by associating, invading, and surviving inside trophozoites and cysts. It was observed that both S. pyogenes and S. pneumoniae were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. Notably, S. pyogenes and S. pneumoniae survived the encystation process, avoided phagocytosis, multiplied, and exhibited higher recovery from the mature cysts, compared with the trophozoite stage (approximately 2 bacteria per amoebae ratio for cyst stage versus 0.02 bacteria per amoeba ration for trophozoite stage). As Acanthamoeba cysts are resilient and can disperse through the air, A. castellanii can act as a vector in providing shelter, facilitating growth and possibly genetic exchanges. In addition, these interactions may contribute to S. pyogenes and S. pneumoniae survival in harsh environments, and transmission to susceptible population and possibly affecting their virulence. Future studies will determine the molecular mechanisms associated with Acanthamoeba interactions with Streptococcus and the evolution of pathogenic bacteria and in turn expedite the discovery of novel therapeutic and/or preventative measures., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Microbial keratitis is a sight-threatening ocular infection caused by bacteria, fungi, and protist pathogens. Epithelial defects and injuries are key predisposing factors making the eye susceptible to corneal pathogens. Among bacterial pathogens, the most common agents responsible for keratitis include Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia and Serratia species. Fungal agents of corneal infections include both filamentous as well as yeast, including Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia, Paecilomyces, Scedosporium and Candida species, while in protists, Acanthamoeba spp. are responsible for causing ocular disease. Clinical features include redness, pain, tearing, blur vision and inflammation but symptoms vary depending on the causative agent. The underlying molecular mechanisms associated with microbial pathogenesis include virulence factors as well as the host factors that aid in the progression of keratitis, resulting in damage to the ocular tissue. The treatment therefore should focus not only on the elimination of the culprit but also on the neutralization of virulence factors to minimize the damage, in addition to repairing the damaged tissue. A complete understanding of the pathogenesis of microbial keratitis will lead to the rational development of therapeutic interventions. This is a timely review of our current understanding of the advances made in this field in a comprehensible manner. Coupled with the recently available genome sequence information and high throughput genomics technology, and the availability of innovative approaches, this will stimulate interest in this field., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Non-steroidal anti-inflammatory drug, Diclofenac, targeting COX have shown promise in the treatment of Acanthamoeba keratitis, but the underlying mechanisms remain unknown. Using various NSAIDs, Diclofenac sodium, Indomethacin, and Acetaminophen, here we determined the effects of NSAIDs on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype. Using amoebicidal assays, the results revealed that Diclofenac sodium, and Indomethacin affected growth of A. castellanii. In contrast, none of the compounds tested had any effect on the viability of A. castellanii. Importantly, all NSAIDs tested abolished A. castellanii encystation. This is a significant finding as the ability of amoebae to transform into the dormant cyst form presents a significant challenge in the successful treatment of infection. The NSAIDs inhibit production of cyclo-oxegenase, which regulates the synthesis of prostaglandins suggesting that cyclooxygenases (COX-1 and COX-2) and prostaglandins play significant role(s) in Acanthamoeba biology. As NSAIDs are routinely used in the clinical practice, these findings may help design improved preventative strategies and/or of therapeutic value to improve prognosis, when used in combination with other anti-amoebic drugs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The completion of Escherichia coli K1 genome has identified several genomic islands that are present in meningitis-causing E. coli RS218 but absent in the non-pathogenic E. coli MG1655. In this study, the role of various genomic islands in E. coli K1 interactions with intestinal epithelial cells (Caco-2) and kidney epithelial cells (MA104) was determined. Using association assays, invasion assays, and intracellular survival assays, the findings revealed that the genomic island deletion mutants of RS218 related to P fimbriae, S fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, protein secretion system (T1SS for hemolysin; T2SS; T5SS for antigen 43), Iro system and hmu system), invasins (CNF1, IbeA), toxins (α-hemolysin), K1 capsule biosynthesis, metabolism (d-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism), prophage genes, showed reduced interactions with both cell types. Next, we determined the role of various genomic islands in E. coli K1 resistance to serum. When exposed to the normal human serum, the viability of the genomic island deletion mutants related to adhesins such as S fimbriae, P fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, antigen 43 and T5SS for antigen 43, T2SS, and T1SS for hemolysin, Iro system and hmu system, prophage genes, metabolism (sugar metabolism and d-serine catabolism), K1 capsule biosynthesis, and invasins such as CNF1 was affected, suggesting their role in bacteremia. The characterization of these genomic islands should reveal mechanisms of E. coli K1 pathogenicity that could be of value as therapeutic targets., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Granulomatous amoebic encephalitis due to Acanthamoeba is a chronic disease that almost always results in death. Hematogenous spread is a pre-requisite followed by amoebae invasion of the blood-brain barrier to enter the central nervous system. Given the systemic nature of this infection, a significant latent period of several months before the appearance of clinical manifestations is puzzling. Based on reported cases, here we propose pathogenetic mechanisms that explain the above described latency of the disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Identification of health behaviors and markers of physiological health associated with childhood cognitive function has important implications for public health policy targeted toward cognitive health throughout the life span. Although previous studies have shown that aerobic fitness and obesity exert contrasting effects on cognitive flexibility among prepubertal children, the extent to which diet plays a role in cognitive flexibility has received little attention. Accordingly, this study examined associations between saturated fats and cholesterol intake and cognitive flexibility, assessed using a task switching paradigm, among prepubertal children between 7 and 10 years (N = 150). Following adjustment of confounding variables (age, sex, socioeconomic status, IQ, VO2max, and BMI), children consuming diets higher in saturated fats exhibited longer reaction time during the task condition requiring greater amounts of cognitive flexibility. Further, increasing saturated fat intake and dietary cholesterol were correlated with greater switch costs, reflecting impaired ability to maintain multiple task sets in working memory and poorer efficiency of cognitive control processes involved in task switching. These data are among the first to indicate that children consuming diets higher in saturated fats and cholesterol exhibit compromised ability to flexibly modulate their cognitive operations, particularly when faced with greater cognitive challenge. Future longitudinal and intervention studies are necessary to comprehensively characterize the interrelationships between diet, aerobic fitness, obesity, and children's cognitive abilities., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Despite advances in antimicrobial chemotherapy and supportive care, the prognosis of Acanthamoeba infections remains poor, suggesting that new targets are needed that can affect parasite survival and host-pathogen interactions. G proteins and their coupled receptors are well known regulators of a variety of cellular functions. The overall aim of the present study was to study the role of G-protein coupled receptor, β adrenergic receptor on the biology and pathogenesis of keratitis isolate of Acanthamoeba castellanii of the T4 genotype. Inhibition of β adrenergic receptor using antagonist, propranolol had detrimental effects on the extracellular proteolytic activities A. castellanii as determined using zymographic assays. Conversely, β adrenergic receptor agonist, isoprenaline showed increased proteases. Interestingly, β adrenergic receptor inhibition affected A. castellanii growth (using amoebistatic assays), viability (using amoebicidal assays by measuring uptake of Trypan blue) and encystation as determined by trophozoite transformation into the cyst form. Pre-treatment of parasites with propranolol hampered A. castellanii-mediated human brain microvascular endothelial cell cytotoxicity, as measured by the lacatate dehydrogenase release. The aforementioned findings suggest that G-protein coupled receptor, β adrenergic receptor-mediated signaling in A. castellanii biology and pathogenesis may offer new pharmacological targets., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Granulomatous amoebic encephalitis is a rare but serious human disease leading almost always to death. The pathophysiology of amoebic encephalitis is better understood, while events leading to the constitution of brain infection are largely unknown. Traversal of the blood-brain barrier is a key step in amoebae invasion of the central nervous system and facilitated by amoebic extracellular proteases. By using specific inhibitors of protease-activated receptors 1, 2 and 4, here we studied the role of these host receptors in Acanthamoeba castellanii-mediated damage to human brain microvasculature endothelial cells (HBMEC), which constitute the blood-brain barrier. The primary HBMEC were incubated with A. castellanii-conditioned medium in the presence or absence of FR-171113 (selective inhibitor of protease-activated receptor 1), FSLLRY-NH2 (inhibitor of protease-activated receptor 2), and tcY-NH2 (inhibitor of protease-activated receptor 4). The HBMEC monolayer disruptions were assessed by microscopy using Eosin staining, while host cell cytotoxicity was determined by measuring the release of cytoplasmic lactate dehydrogenase. Zymographic assays were performed to determine the effects of inhibitors of protease-activated receptors on the extracellular proteolytic activities of A. castellanii. A. castellanii-conditioned medium produced severe HBMEC monolayer disruptions within 60 min. The selective inhibitors of protease-activated receptors tested did not affect HBMEC monolayer disruptions. On the contrary, pre-treatment of A. castellanii-conditioned medium with phenylmethylsulfonyl fluoride, a serine protease inhibitor, or heating for 10 min at 95°C abolished HBMEC monolayer disruptions. Additionally, inhibitors of protease-activated receptors tested, failed to block A. castellanii-mediated HBMEC cytotoxicity and did not affect extracellular proteolytic activities of A. castellanii. Protease-activated receptors 1, 2 and 4 do not appear to play a role in A. castellanii-mediated dysfunction of HBMEC, which constitute the blood-brain barrier. The role of additional protease-activated receptors in amoebic invasion of the central nervous system is discussed further., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Granulomatous amoebic encephalitis (GAE) is caused by two protist pathogens, Acanthamoeba spp., and Balamuthia mandrillaris. Although rare, it almost always results in death. In the present study, amoebae were treated with various combinations of clinically-approved drugs, targeting vital cellular receptors and biochemical pathways. The results revealed that among the seven different combinations tested, three proved highly effective against both Acanthamoeba castellanii as well as B. mandrillaris at a concentration of 100μM. These combinations included (i) prochlorperazine plus loperamide; (ii) prochlorperazine plus apomorphine; and (iii) procyclidine plus loperamide. In viability assays, none of the drug-treated amoebae emerged as viable trophozoites, suggesting irreversible amoebicidal effects. Four combinations of drugs tested showed varied potency against A. castellanii and B. mandrillaris at 100μM. The combination of haloperidol and loperamide was highly effective against A. castellanii at 100μM, but potent effects against B. mandrillaris were observed only at 250μM. Digoxin and amlodipine were effective against A. castellanii and B. mandrillaris at 100μM and 250μM, respectively. In contrast, the combination of apomorphine and haloperidol was effective against B. mandrillaris and A. castellanii at 100μM and 250μM, respectively. At 100μM, the combination of procyclidine and amiodarone was effective against neither A. castellanii nor B. mandrillaris. In this case, amoebicidal properties were observed at 750μM for A. castellanii, and 950μM for B. mandrillaris. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic encephalitis caused by A. castellanii and B. mandrillaris., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of this study was to assess the anti-amoebic effects of marketed contact lens disinfecting solutions. Using amoebistatic, amoebicidal, and cysticidal assays, nine different contact lens solutions were tested including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Replenish, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+, Kontex Soak. In vitro growth inhibition (amoebistatic) assays were performed by incubating Acanthamoeba castellanii with aforementioned contact lens disinfection solutions as per manufacturer's instructions in the growth medium for up to 48h at 30°C. To determine amoebicidal and cysticidal effects, amoebae were incubated with contact lens solutions in phosphate buffered saline for 24h and viability was determined by haemocytometer counting as well as re-inoculating them in the growth medium. For controls, solutions were tested against bacterial corneal pathogen, Pseudomonas aeruginosa, as well as amoebae were incubated with the solvent alone. Of the nine contact lens solutions tested, none of them showed potent amoebicidal effects. Only DuraPlus and OptiFree Replenish exhibited trophozoite lysis of 85.3% and 73.7% respectively. In contrast, all contact lens disinfection solutions except Kontex Clean, Kontex Normal, Kontex Multisol extra+, tested showed amoebistatic effects. Importantly, none of the contact lens disinfection solutions exhibited cysticidal effects using qualitative assays, i.e., cysts treated with aforementioned solutions re-emerged as viable amoebae upon inoculation in the growth medium. However, more than 3-log reduction was observed when ReNu MultiPlus, DuraPlus and OptiFree Express were tested against P. aeruginosa which is in accordance with the ISO Stand-Alone Primary acceptance criteria. These findings are of great concern for contact lens users., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this study, Acanthamoeba castellanii was cultivated under different stress conditions to induce possible encystation. The morphological and histological properties were analysed by light and electron microscopy as well as cyst-specific staining. The findings revealed that cysts prepared through liquid medium using higher osmolarity as a trigger (10% glucose with 50mM magnesium chloride for 72 h) are similar to cysts prepared using non-nutrient agar (nutrient deprivation as a trigger in plating assays for 14 days), as determined by SDS-resistance, cyst-specific Calcofluor white staining and transmission electron microscopy. Using liquid medium assay, A. castellanii encystation was studied by exposing trophozoites to media lacking growth ingredients (phosphate buffered saline or distilled water), inappropriate temperatures (4-45°C), pH (3-9), artificial light-dark cycles, 5% CO2, and microaerophilic conditions. Optimal encystation was observed when cells were incubated in PBS with 50mM MgCl2 and 10% glucose at 24-30°C at pH 7. Increasing temperature over 37°C or pH 9 adversely affected encystation, while light-dark cycles, 5% CO2 and microaerophilic conditions had no effect on encystation of A. castellanii. None of the aforementioned conditions had any effect on the viability of A. castellanii, as determined by Trypan blue exclusion assay. A complete knowledge of encystation in A. castellanii is crucial to our understanding of the biology of these ecologically and medically important organisms., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Acanthamoeba is an opportunist protist pathogen that is known to infect the cornea to produce eye keratitis and the central nervous system to produce fatal granulomatous encephalitis. Early diagnosis, followed by aggressive treatment using a combination of drugs is a prerequisite in successful treatment but even then, prognosis remains poor due to lack of effective drugs. The overall aim of the present study was to determine the anti-Acanthamoebic potential of natural compounds, resveratrol and curcuminoids. Adhesion and cytotoxicity assays were performed using primary human brain microvascular endothelial cells, which constitute the blood-brain barrier. Pre-exposure of organisms to 100 μg resveratrol and demethoxy curcumin prevented amoeba binding by 57% and 73%, respectively, while cytotoxicity of host cells was inhibited by 86%. In an assay for viability of amoebae in the absence of host cells, resveratrol and de-methoxy curcumin exhibited significant amoebicidal effects (23% and 25%, respectively) at 100 μg concentrations (P<0.01). Neither resveratrol nor demethoxycurcumin had any effect on the proteolytic activities of Acanthamoeba castellanii. Of both compounds, resveratrol is of most interest for further investigation, because of the selective toxicity of resveratrol on A. castellanii but not the human brain microvascular endothelial cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a leading cause of nosocomial infections. Haematogenous spread is a pre-requisite but it is not clear how S. aureus survive the onslaught of macrophages. Acanthamoeba is a protozoan pathogen that is remarkably similar to macrophages, particularly in their cellular structure (morphological and ultra-structural features), molecular motility, biochemical physiology, ability to capture prey by phagocytosis and interactions with microbial pathogens. Thus, we hypothesize that S. aureus exhibit similarities in their interactions with Acanthamoeba and ThP1 macrophage-like cells. Here, we studied interactions of methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) and Staphylococcus epidermidis (SE) with Acanthamoeba castellanii belonging to the T4 genotype and macrophage-like cells (ThP1). The findings revealed that both MRSA and MSSA exhibited similarities in their binding/association and invasion of A. castellanii and ThP1 cells. Long-term incubation showed that MRSA and MSSA can survive intracellularly of both Acanthamoeba and ThP1 cells. Overall, these findings suggest that Acanthamoeba exhibit similar characteristics with ThP1 macrophage-like cells in their interaction with MRSA and MSSA. Additionally it was shown that bacteria survive inside Acanthamoeba during the encystment process as evidenced by bacterial recovery from mature cysts. Given that Acanthamoeba cysts are airborne, these findings suggest that cysts may act as "Trojan horse" to help spread MRSA to susceptible hosts., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pathophysiology of Escherichia coli sepsis is complex involving circulating bacterial products, cytokine release, and sustained bacteremia resulting in the damage of vascular endothelium. Here, it is shown that E. coli K1 produced cytopathogenicity of human brain microvascular endothelial cells (HBMEC), that constitute the blood-brain barrier. Whole bacteria or their conditioned medium produced severe HBMEC damage suggesting E. coli K1-cytopathogenicity is a contact-independent process. Using lipopolysaccharide (LPS) inhibitor, polymyxin B, purified LPS extracted from E. coli K1 as well as LPS mutant derived from E. coli K1, we showed that LPS is not the sole determinant of E. coli K1-mediated HBMEC death. Bacterial product(s) for HBMEC cytopathogenicity was heat-labile suggesting LPS-associated proteins. Several isogenic gene-deletion mutants (ΔompA, ΔibeA, ΔibeB, Δcnf1) exhibited HBMEC cytopathogenicity similar to that produced by wild type E. coli K1. E. coli K1-mediated HBMEC death was independent of phosphatidylinositol 3-kinase (PI3K) but dependent partially on focal adhesion kinase (FAK) using HBMEC expressing dominant negative FAK and PI3K., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acanthamoeba granulomatous encephalitis (AGE), caused by Acanthamoeba castellanii, is a fatal infection of immunocompromised individuals. The pathogenesis of blood-brain barrier (BBB) breach remains unknown. Using a novel in vitro BBB infection model under flow conditions, demonstrates that increases in flow rates lead to decreased binding of A. castellanii to host cells. This is a distinct departure from previous findings under static conditions. However, similarly to static conditions binding of A. castellanii to host cells is host mannose dependent. Disruption of the host cell monolayer was independent of amoeba binding, but dependent on secreted serine proteases. For the first time we report the binding dynamics of A. castellanii under physiological conditions, showing that BBB disruption is not directly linked to binding, instead it is reliant on secreted proteases. Our results offer a platform on which therapies designed at modulating physiological parameters can improve the outcome of infection with A. castellanii., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Corticospinal motor neurons (CSMN) are the cortical component of motor neuron circuitry, which controls voluntary movement and degenerates in diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. By using dual labeling combined with molecular marker analysis, we identified AAV2-2 mediated retrograde transduction as an effective approach to selectively target CSMN without affecting other neuron populations both in wild-type and hSOD1(G93A) transgenic ALS mice. This approach reveals very precise details of cytoarchitectural defects within vulnerable neurons in vivo. We report that CSMN vulnerability is marked by selective degeneration of apical dendrites especially in layer II/III of the hSOD1(G93A) mouse motor cortex, where cortical input to CSMN function is vastly modulated. While our findings confirm the presence of astrogliosis and microglia activation, they do not lend support to their direct role for the initiation of CSMN vulnerability. This study enables development of targeted gene replacement strategies to CSMN in the cerebral cortex, and reveals CSMN cortical modulation defects as a potential cause of neuronal vulnerability in ALS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Acanthamoeba hosts diverse microbial organisms including viruses, bacteria, yeast and protists, some of which are potential human pathogens. The precise nature of this symbiosis is not clear, but it is suggested that such interactions enable pathogenic microbes to survive hostile conditions and lead to their transmission to susceptible hosts to establish infection. In particular, Acanthamoeba-bacteria interactions have gained significant attention by the scientific and the medical community and have led to speculations of employing anti-amoebic approaches in eradicating 'superbugs' from clinical settings. Here, we discuss the nature of these convoluted interactions and the benefit they represent for the symbionts., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Given the remarkable similarities in cellular structure (morphological and ultra-structural features), molecular motility, biochemical physiology, ability to capture prey by phagocytosis and interactions with microbial pathogens, here we pose the question whether Acanthamoeba and macrophages are evolutionary related. This is discussed in the light of evolution and functional aspects such as the astonishing resemblance of many bacteria to infect and multiply inside human macrophages and amoebae in analogous ways. Further debate and studies will determine if Acanthamoeba is an evolutionary ancestor of macrophages. Is this a myth or reality?, (Copyright © 2011 Elsevier Inc. All rights reserved.)

In addition to the possible role of Acanthamoeba as an evolutionary precursor of pathogenicity in microbial pathogens, it has been suggested that intracellular bacteria or other microbial endosymbionts may also enhance the pathogenicity of Acanthamoeba. Using transmission electron microscopy, polymerase chain reaction and simple culturing, our findings did not reveal any apparent evidence of microbial presence intracellularly of a recently recovered clinical isolate of Acanthamoeba. Based on these findings, it is tempting to speculate that the virulence of Acanthamoeba may not be attributed to the pathogenicity of the endosymbiont alone., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P<0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Acanthamoeba granulomatous encephalitis is a serious human infection with fatal consequences. The most distressing aspect of Acanthamoeba granulomatous encephalitis is the limited improvement in mortality. The underlying neurobiology is at present not well understood and treatment options are often of limited efficacy. There is therefore a real need to obtain more knowledge regarding the pathogenesis and pathophysiology of Acanthamoeba granulomatous encephalitis and to develop new chemotherapeutic approaches. However, the difficulties in using mammalian models to study this infection have hindered our search for therapeutic interventions. Recent availability of the blood-brain barrier, in vitro and use of locust as an in vivo model will undoubtedly allow us to investigate disease pathogenesis, mechanisms of parasite traversal across the blood-brain barrier and new drug therapies. It is argued that the models described here can offer several advantages in terms of speed, cost, technical convenience, and ethical acceptance. Furthermore, they are extremely valuable tools to discriminate molecules participating from both sides of the host-parasite interaction and will generate potentially useful leads in the identification of new potential drugs, as well as testing drug toxicity., (Copyright 2009 Elsevier Inc. All rights reserved.)

Balamuthia mandrillaris is a causative agent of granulomatous encephalitis that almost always proves fatal. A major concern during the course of therapy is that B. mandrillaris can transform into cysts. Cysts are highly resistant to physical and chemical conditions and present a problem in successful antimicrobial chemotherapy. However, the underlying mechanisms of B. mandrillaris transformation into cysts are not known. In this study, we examined the effects of exogenous sugars on B. mandrillaris encystment. The findings revealed that free exogenous galactose, but not other sugars, enhanced parasite differentiation into cysts, and apparently a galactose-binding protein is involved in B. mandrillaris encystment. Cytoskeletal re-arrangements and phosphatidylinositol 3-kinase (PI3K)-mediated pathways are involved in B. mandrillaris encystment based on inhibitor studies. Dual functionality of galactose-binding protein in B. mandrillaris pathogenesis and encystment is discussed further., (Copyright 2009. Published by Elsevier Inc.)

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting "cyst wall" material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.

Acanthamoeba is an opportunistic protozoan pathogen that can produce keratitis and rare but fatal encephalitis. In the present study, we examined secretory IgA antibody to Acanthamoeba castellanii of the T4 genotype in mucosal secretions from 114 individuals of 37 countries, inhabitants and/or visitors, aged 16-65 years in London, UK. Acanthamoeba antibody prevalence rate was more than 85%, without any significant differences between males (86.2%) and females (89.2%). Some epidemiological factors contributing to the high prevalence of antibody to Acanthamoeba in surveyed population are discussed further.

Balamuthia amoebic encephalitis (BAE), caused by the protozoan pathogen, Balamuthia mandrillaris, is a serious human disease with fatal consequences and a mortality rate of more than 95%. A key factor that contributes to the high mortality is the incomplete understanding of its pathogenesis and pathophysiology. The most distressing aspect is that the high level of mortality is due to lack of awareness combined with the lack of effective drugs. Early diagnosis followed by aggressive treatment may lead to cure. Several lines of evidence suggest that BAE develops as a result of haematogenous spread, but it is unclear how circulating amoebae enter the central nervous system and cause inflammation, blood-brain barrier disruption and neuronal injury. Recent studies have identified several parasite-host determinants for B. mandrillaris translocation of the blood-brain barrier, and host inflammatory markers that may be associated with neuronal injury. These determinants may provide important targets for the prevention and treatment of BAE. Here, we present a brief overview of the current understanding of the pathogenesis and pathophysiology of BAE, available diagnostic methods, possible therapeutic interventions and biology of the causative agent.

We assessed the implication of Th (helper)-cells and the modulation of the Th1/Th2 dichotomy by n-3 polyunsaturated fatty acids (PUFA) in type I diabetic pregnancy (DP) and macrosomia. Female gestant rats fed a standard diet or n-3 PUFA regimen were rendered diabetic by administration of five low doses of streptozotocin. The macrosomic (MAC) offspring were sacrificed at the age of 90 days. The mRNAs of IL-2 and IFN-gamma (Th1 cytokines) and IL-4 (Th2 cytokine) were downregulated in the pancreas and spleen of diabetic pregnant rats. The levels of IL-10 mRNA, another Th2 cytokine, were unchanged in the spleen or upregulated in the pancreas of these animals. Feeding an n-3 PUFA diet to rats with DP upregulated IL-10 mRNA in the pancreas and IL-4 and IL-10 mRNA in the spleen. In MAC offspring, high expression of IL-2 and IFN-gamma mRNA, but not of Th2 cytokines, was observed. The n-3 PUFA diet diminished Th1 mRNA quantities and increased the levels of IL-4, but not of IL-10, mRNA in MAC offspring. Our study shows that DP is associated with a decreased Th1 phenotype and IL-4 mRNA expression in the pancreas and spleen, and an n-3 PUFA diet upregulates Th2 profile. In MAC offspring, the Th1 phenotype is upregulated and an n-3 PUFA diet downregulates this phenomenon.

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis and eye keratitis, however the pathogenic mechanisms of Acanthamoeba remain unclear. In this study, we described the ability of live Acanthamoeba to hydrolyse extracellular ATP. Both clinical and non-clinical isolates belonging to genotypes, T1, T2, T3, T4 and T7 exhibited ecto-ATPase activities in vitro. Using non-denaturing polyacrylamide gel electrophoresis, ecto-ATPases were further characterized. All Acanthamoeba isolates tested, exhibited a single ecto-ATPase band (approximate molecular weight of 272 kDa). However, clinical isolates exhibited additional bands suggesting that ecto-ATPases may play a role in the pathogenesis of Acanthamoeba. This was supported using suramin (ecto-ATPase inhibitor), which inhibited Acanthamoeba-induced host cell cytotoxicity. Previously, we and others have shown that Acanthamoeba binds to host cells using their mannose-binding protein and binding can be blocked using exogenous alpha-mannose. In this study, we observed that alpha-mannose significantly increased ecto-ATPase activities of pathogenic Acanthamoeba belonging to T1, T2, T3 and T4 genotypes but had no effect on non-pathogenic Acanthamoeba (belonging to T7 genotype). Overall, we have shown, for the first time, that Acanthamoeba exhibit ecto-ATPase activities, which may play a role in the pathogenesis of Acanthamoeba as well as their potential role in the differentiation of pathogenic Acanthamoeba.

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis, however, the pathogenic mechanisms associated with this disease remain unclear. One of the primary factors in Acanthamoeba encephalitis is the haematogenous spread, followed by invasion of the blood-brain barrier resulting in the transmigration of Acanthamoeba into the central nervous system. In this study, we have used human brain microvascular endothelial cells, which constitute the blood-brain barrier and studied their interactions with Acanthamoeba. Using in vitro cultures, we showed that Acanthamoeba isolates belonging to genotypes T3, T4 and T11, exhibited increased cytotoxicity on human brain microvascular endothelial cells as well as exhibited higher binding and were considered potential pathogens. In contrast, Acanthamoeba isolates belonging to genotypes T2 and T7 exhibited minimal cytotoxicity and significantly less binding to human brain microvascular endothelial cells (P< 0.01). Furthermore, exogenous alpha-mannose inhibited binding but increased cytotoxicity of human brain microvascular endothelial cells. This is the first demonstration of Acanthamoeba interactions with primary human brain microvascular endothelial cells.

Escherichia coli K1 invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for penetration into the central nervous system. We previously have shown that outer membrane protein A (OmpA) and cytotoxic necrotizing factor-1 (CNF1) contribute to E. coli K1 invasion of BMEC. In this study we constructed a double-knockout mutant by deleting ompA and cnf1. We demonstrated that the double-knockout mutant was significantly less invasive in human BMEC as compared with its individual Delta ompA and Delta cnf1 mutants, suggesting that the contributions of OmpA and CNF1 to BMEC invasion are independent of each other. In addition, we showed that OmpA treatment of human BMEC resulted in phosphatidylinositol 3-kinase (PI3K) activation with no effect on RhoA, while CNF1 treatment resulted in RhoA activation with no effect on PI3K, supporting the concept that OmpA and CNF1 contribute to E. coli K1 invasion of BMEC using different mechanisms. This concept was further confirmed by using both PI3K inhibitor (LY294002) and Rho kinase inhibitor (Y27632), which exhibited additive effects on inhibiting E. coli K1 invasion of BMEC. We isolated a 96KD OmpA interacting human BMEC protein by affinity chromatography using purified OmpA, which was identified as gp96 protein, a member of the HSP90 family. This receptor differed from the CNF1 receptor (37LRP) identified from human BMEC. Taken together, these data indicate that OmpA and CNF1 contribute to E. coli K1 invasion of BMEC in an additive manner by interacting with different BMEC receptors and using diverse host cell signaling mechanisms.

Acanthamoeba are free-living, harmless organisms, however, given the opportunity and the appropriate conditions, they can cause painful, sight-threatening as well as fatal infections and, thus, are considered opportunistic pathogens. Acanthamoeba infections have become increasingly important in the past few years due to increasing populations of contact lens users and AIDS patients. The mechanisms associated with the pathogenesis of Acanthamoeba tend to be highly complex, depending on parasite, host and the environmental factors. Elucidation of the biochemical, cellular and molecular basis of the pathogenesis of diseases caused by Acanthamoeba may lead to the development of therapeutic interventions.