Appropriateness, Acceptability, and Feasibility of a Neurodiversity-Based Self-determination Program for Autistic Adults.

Published self-determination programs do not adequately address the needs of autistic adults. We designed a multi-component self-determination program, grounded in the neurodiversity paradigm, to help autistic adults achieve goals to improve their quality of life. The first phase involved 5 days of psychoeducation, practice, and social events; the second phase included 3 months of telecoaching; and the third phase included follow-up. Thirty-four university students coached 31 autistic adults on three evolving goals. On average, participants completed one goal per week. Most participants were satisfied with the program. We found that the program was appropriate, acceptable, and feasible. This program is a promising approach to helping autistic adults gain self-determination skills and improve their quality of life.

Molecular Docking of SP40 Peptide towards Cellular Receptors for Enterovirus 71 (EV-A71).

Enterovirus 71 (EV-A71) is one of the predominant etiological agents of hand, foot and mouth disease (HMFD), which can cause severe central nervous system infections in young children. There is no clinically approved vaccine or antiviral agent against HFMD. The SP40 peptide, derived from the VP1 capsid of EV-A71, was reported to be a promising antiviral peptide that targeted the host receptor(s) involved in viral attachment or entry. So far, the mechanism of action of SP40 peptide is unknown. In this study, interactions between ten reported cell receptors of EV-A71 and the antiviral SP40 peptide were evaluated through molecular docking simulations, followed by in vitro receptor blocking with specific antibodies. The preferable binding region of each receptor to SP40 was predicted by global docking using HPEPDOCK and the cell receptor-SP40 peptide complexes were refined using FlexPepDock. Local molecular docking using GOLD (Genetic Optimization for Ligand Docking) showed that the SP40 peptide had the highest binding score to nucleolin followed by annexin A2, SCARB2 and human tryptophanyl-tRNA synthetase. The average GoldScore for 5 top-scoring models of human cyclophilin, fibronectin, human galectin, DC-SIGN and vimentin were almost similar. Analysis of the nucleolin-SP40 peptide complex showed that SP40 peptide binds to the RNA binding domains (RBDs) of nucleolin. Furthermore, receptor blocking by specific monoclonal antibody was performed for seven cell receptors of EV-A71 and the results showed that the blocking of nucleolin by anti-nucleolin alone conferred a 93% reduction in viral infectivity. Maximum viral inhibition (99.5%) occurred when SCARB2 was concurrently blocked with anti-SCARB2 and the SP40 peptide. This is the first report to reveal the mechanism of action of SP40 peptide in silico through molecular docking analysis. This study provides information on the possible binding site of SP40 peptide to EV-A71 cellular receptors. Such information could be useful to further validate the interaction of the SP40 peptide with nucleolin by site-directed mutagenesis of the nucleolin binding site.

Molecular mechanism of L-SP40 peptide and in vivo efficacy against EV-A71 in neonatal mice.

AIMS: Enterovirus A71 (EV-A71) is an etiological agent of hand foot and mouth disease (HFMD) and has the potential to cause severe neurological infections in children. L-SP40 peptide was previously known to inhibit EV-A71 by prophylactic action. This study aimed to identify the mechanism of inhibition in Rhabdomyosarcoma (RD) cells and in vivo therapeutic potential of L-SP40 peptide in a murine model. MAIN METHODS: A pull-down assay was performed to identify the binding partner of the L-SP40 peptide. Co-immunoprecipitation and co-localization assays with the L-SP40 peptide were employed to confirm the receptor partner in RD cells. The outcomes were validated using receptor knockdown and antibody blocking assays. The L-SP40 peptide was further evaluated for the protection of neonatal mice against lethal challenge by mouse-adapted EV-A71. KEY FINDINGS: The L-SP40 peptide was found to interact and co-localize with nucleolin, the key attachment receptor of Enteroviruses A species, as demonstrated in the pull-down, co-immunoprecipitation and co-localization assays. Knockdown of nucleolin from RD cells led to a significant reduction of 3.5 logs of viral titer of EV-A71. The L-SP40 peptide demonstrated 80% protection of neonatal mice against lethal challenge by the mouse-adapted virus with a drastic reduction in the viral loads in the blood (~4.5 logs), skeletal muscles (1.5 logs) and brain stem (1.5 logs). SIGNIFICANCE: L-SP40 peptide prevented severe hind limb paralysis and death in suckling mice and could serve as a potential broad-spectrum antiviral candidate to be further evaluated for safety and potency in future clinical trials against EV-A71.

Strategies to identify and develop antiviral peptides.

The emergence and re-emergence of viral pathogens capable of causing epidemics or pandemics pose a serious healthcare burden. Small molecule antivirals used in conventional therapy have given rise to the severe problem of viral resistance against them. Peptides are generally considered safe, effective and are less likely to induce viral resistance. Antiviral peptides can be identified from screening of phage display of combinational peptide libraries, peptide array libraries or designed against viral targets. Limitations of peptides such as bioavailability can be improved with chemical modifications. Nanotechnology can further improve the stability of peptides in systemic circulation and enhance the antiviral activity of peptides, making them an appealing therapeutic option.

Functional Insights into Silymarin as an Antiviral Agent against Enterovirus A71 (EV-A71).

Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.

'Targeting the feast of a sleeping beast': Nutrient and mineral dependencies of encysted Acanthamoeba castellanii.

Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta-oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully.

Antiviral peptides against Enterovirus A71 causing hand, foot and mouth disease.

The emergence of new and resistant viruses is a serious global burden. Conventional antiviral therapy with small molecules has led to the development of resistant mutants. In the case of hand, foot and mouth disease (HFMD), the absence of a US-FDA approved vaccine calls for urgent need to develop an antiviral that could serve as a safe, potent and robust therapy against the neurovirulent Enterovirus A71 (EV-A71). Natural peptides such as lactoferrin, melittin and synthetic peptides such as SP40, RGDS and LVLQTM have been studied against EV-A71 and have shown promising results as potent antivirals in pre-clinical studies. Peptides are considered safe, efficacious and pose fewer chances of resistance. Poor pharmacokinetic features of peptides can be overcome by the use of chemical modifications to improve in vivo delivery particularly by oral route. The use of nanotechnology can remarkably assist in the oral delivery of peptides and enhance stability in vivo. This can greatly increase patient compliance and make it more attractive as antiviral therapy.

Role of oxidative stress and altered thyroid hormones in unexplained infertility.

OBJECTIVE: To explore the link between altered thyroid profile and oxidative stress marker in females with unexplained infertility. METHODS: The cross-sectional case-control study was carried out at the Islamabad Clinic Serving Infertile Couples, Islamabad, Pakistan, from June 2016 to August 2017, and comprised women aged 18-40 years regardless of ethnic background who were divided into two groups; those with unexplained infertility were the cases, while fertile women acted as the controls. Serum was analysed for triiodothyronine, thyroxine and thyroid stimulating hormone as well as for oxidative stress markers including manganese superoxide dismutase, glutathione reductase and adrenaline using enzymelinked immunosorbent assay. Data was analysed using SPSS 19. RESULTS: Of the 88 subjects, there were 44(50%) in each of the two groups. There was no significant difference in terms of thyroids markers except thyroxine and thyroid stimulating hormone (p<0.05). There were significant differences in terms of oxidative stress markers between the groups (p<0.05). A significant positive correlation of thyroid stimulating hormone was observed with manganese superoxide dismutase and adrenaline (p<0.05) with a weak non significant association of glutathione reductase (p>0.05). CONCLUSIONS: Increased thyroxine levels in females with unexplained infertility was associated with decrease in the serum levels of antioxidants.

Correction: Lalani, S. and Poh, C.L. Flavonoids as Antiviral Agents for Enterovirus A71 (EV-A71). Viruses 2020, 12, 184.

We have recently been made aware by Mr. Saravanan (National University of Singapore) thatthe structure of prunin flavonoid used in their study was different to the one that we reported [...].

Antiviral activity of silymarin in comparison with baicalein against EV-A71.

BACKGROUND: The hand, foot and mouth disease (HFMD) is a febrile and exanthematous childhood disease mainly caused by Enterovirus 71 (EV-A71). In severe HFMD, virulent EV-A71 strains can cause acute flaccid paralysis and cardiopulmonary edema leading to death. Currently, no FDA approved antiviral treatment or vaccine is available for EV-A71. Flavonoids such as silymarin and baicalein are known to possess in vitro antiviral properties against viruses. In this study, the cytotoxicity and antiviral activity of silymarin, baicalein and baicalin were investigated. METHODS: The cytotoxic effects of three flavonoids towards rhabdomyosarcoma (RD) cells were first examined using cell proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Compounds found to be non-cytotoxic in RD cells were evaluated for their in vitro antiviral properties against the EV-A71 subgenotype B4 strain 41 (5865/SIN/000009) using antiviral assays. Viral infectivity was determined by reduction of the formation of plaques in RD cells. For the measurement of RNA copy number, the real time quantitative reverse transcription PCR (qRT-PCR) was used. The most potent compound was further evaluated to determine the mode of action of inhibition by time course, virus attachment and entry assays in Vero cells. RESULTS: Silymarin was shown to exert direct extracellular virucidal effects against EV-A71 at 50% inhibitory concentration (IC50) of 15.2 ± 3.53 µg/mL with SI of 10.53. Similarly, baicalein exhibited direct extracellular virucidal effects against EV-A71 at a higher IC50 value of 30.88 ± 5.50 µg/mL with SI of 13.64. Besides virucidal activity, silymarin was shown to block both viral attachment and entry of EV-A71 to inhibit infection in Vero cells. CONCLUSIONS: Silymarin has a stronger inhibition activity against EV-A71 in comparison to baicalein. It could serve as a promising antiviral drug to treat EV-A71 infections.

Flavonoids as Antiviral Agents for Enterovirus A71 (EV-A71).

Flavonoids are natural biomolecules that are known to be effective antivirals. These biomolecules can act at different stages of viral infection, particularly at the molecular level to inhibit viral growth. Enterovirus A71 (EV-A71), a non-enveloped RNA virus, is one of the causative agents of hand, foot and mouth disease (HFMD), which is prevalent in Asia. Despite much effort, no clinically approved antiviral treatment is available for children suffering from HFMD. Flavonoids from plants serve as a vast reservoir of therapeutically active constituents that have been explored as potential antiviral candidates against RNA and DNA viruses. Here, we reviewed flavonoids as evidence-based natural sources of antivirals against non-picornaviruses and picornaviruses. The detailed molecular mechanisms involved in the inhibition of EV-A71 infections are discussed.

Neuroleptic Drug Targets a Brain-Eating Amoeba: Effects of Promethazine on Neurotropic Acanthamoeba castellanii.

Acanthamoeba spp. has recently been reported to express diverse group of ion channels and receptors that are expressed by human cells which bind drugs that are used in noninfectious diseases. Bioinformatics computational tools, growth assays, and 3D structural modeling have enabled the discovery of primitive muscarinic receptors, voltage-gated calcium channels, and ion transport pumps such as Na-K ATPase in this protist pathogen. The significance of the reported receptors and ion channels in the biology of Acanthamoeba is yet to be determined. We selected promethazine, which is a known antagonist of proteins like dopaminergic, histaminergic, muscarinic receptors, and calmodulin, to determine its effects on the growth and proliferation of trophozoites and cysts of Acanthamoeba spp. In order to elucidate the receptors involved in the effects produced by promethazine, we also performed individual experiments on Acanthamoeba trophozoites and cysts in the presence of the agonist of the above-mentioned receptors. Our results show that promethazine in the range of 60-100 µg/mL proved to be amoebicidal for Acanthamoeba trophozoites and at slightly higher doses ranging around 125-250 µg/mL also showed partial cysticidal effects. We also show the evidence of homology between the human targets of promethazine and similar targets in Acanthamoeba by the use of bioinformatic computational tools and 3D modeling. Promethazine and its structural analogs, because of being FDA-approved, have a wider margin of safety that can be tested as potential anti- Acanthamoeba agents in diseases like keratitis and encephalitis caused by this protist pathogen.

Association Between Vitamin D, Reproductive Hormones and Sperm Parameters in Infertile Male Subjects.

Background: The prevalence of infertility and vitamin D deficiency is common in Pakistan. Therefore, our study aims were to assess and compare Vitamin D; 25-hydroxyvitamin (25OHD) and reproductive hormone levels in male fertile and infertile subjects with normal and abnormal sperm parameters. Furthermore, the study is aimed to explore the association of 25OHD levels with these sperm parameters in a selected population of Karachi, Pakistan. Methods: The cross-sectional study was carried out from August 2016 till December 2017, 313 study subjects were recruited from an Infertile Clinic from Islamabad, Pakistan, and the general population. First, we took the couples' history of parenting and then carried out a semen analysis and infertile and fertile male subjects were then subgrouped into "normal" and "altered sperm parameter/s." Forward linear regression was done for selection of 25OHD as a significant predictor of sperm parameters. Results: The median values of the total count, motility, morphology as well as serum 25OHD were significantly higher in the group with "normal" (186) as compared to subjects (127) in "abnormal sperm parameters" group. The 25OHD levels were significantly high in males with "normal sperm parameters"; 80.90 ± 23.33 nmol/L vs. "altered sperm parameter/s," 64.68 ± 24.21 nmol/L (mean ± SD) with p < 0.001. Serum testosterone level had a significant positive correlation with 25OHD while LH had a significant negative correlation with 25OHD (p < 0.001), and FSH level had a non-significant negative correlation with 25OHD. Results of regression model showed one unit increase of motility would give 0.15-unit positive significant impact on 25OHD; 20% variation in 25OHD was explained by the total count, motility, and morphology, while the model was adjusted for BMI. Conclusion: The impact of 25OHD levels on sperm parameters can be emphasized on the basis of detection of its high serum levels in "normal" subjects in both fertile as well as infertile males in comparison to subjects that had altered sperm parameters; total sperm count, motility, and normal morphology. The considerably positive association between 25OHD, testosterone, total count, motility, and morphology further accentuates its impact on normal spermatogenesis and the male reproductive functions required for acquiring fertility.

Traced on the Timeline: Discovery of Acetylcholine and the Components of the Human Cholinergic System in a Primitive Unicellular Eukaryote Acanthamoeba spp.

Acetylcholine (ACh) is the neurotransmitter of cholinergic signal transduction that affects the target cells via muscarinic (mAChR) and nicotinic (nAChR) cholinergic receptors embedded in the cell membrane. Of the cholinergic receptors that bind to ACh, the mAChRs execute several cognitive and metabolic functions in the human central nervous system (CNS). Very little is known about the origins and autocrine/paracrine roles of the ACh in primitive life forms. With the recent report of the evidence of an ACh binding mAChR1 like receptor in Acanthamoeba spp., it was tempting to investigate the origin and functional roles of cholinergic G-Protein coupled receptors (GPCRs) in the biology of eukaryotes. We inferred the presence of ACh, its synthetic, degradation system, and a signal transduction pathway in an approximately ∼2.0 billion year old primitive eukaryotic cell Acanthamoeba castellanii. Bioinformatics analysis, ligand binding prediction, and docking methods were used to establish the origins of enzymes involved in the synthesis and degradation of ACh. Notably, we provide evidence of the presence of ACh in A. castellanii by colorimetric analysis, which to date is the only report of its presence in this primitive unicellular eukaryote. We show the evidence for the presence of homology of evolutionary conserved key enzymes of the cholinergic system like choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in A. castellanii spp., which were found to be near identical to their human counterparts. Tracing the origin, functions of ACh, and primeval mAChRs in primitive eukaryotic cells has the potential of uncovering covert cholinergic pathways that can be extended to humans in order to understand the states of cholinergic deficiency in neurodegenerative diseases (ND).

Apoptosis in Acanthamoeba castellanii belonging to the T4 genotype.

Here we describe features of apoptosis in unicellular Acanthamoeba castellanii belonging to the T4 genotype. When exposed to apoptosis-inducing compounds such as doxorubicin, A. castellanii trophozoites exhibited cell shrinkage and membrane blebbing as observed microscopically, DNA fragmentation using agarose gel electrophoresis, and phosphatidylserine (PS) externalization using annexin V immunostaining. Overall, these findings suggest the existence of apoptosis in A. castellanii possibly mediated by intrinsic apoptotic cascade. Further research in this field could provide avenues to selectively induce apoptosis in A. castellanii by triggering intrinsic apoptotic cascade.

Association of Vitamin D with outcome after intra cytoplasmic sperm injection.

OBJECTIVE: To observe effects of vitamin D levels on pregnancy outcome after intra cytoplasmic sperm injection (ICSI). METHOD: It was a cross-sectional study conducted in Australian Concept Infertility Medical Center from July 2011 to August 2014. Estimation of 25-hydroxy cholecalciferol (25-OHD) of consented females (252) was done before treatment protocol for ICSI. Results of ß hCG performed 14 days after embryo transfer categorized groups; Pregnant with ß hCG more than 25 IU/mL and rest included in non-pregnant group. Both groups were compared by independent sample t-test and Pearson's Chi Square test. Binary Logistic Regression Analysis was used to estimate odds ratio of pregnancy outcome with its predictors including Vitamin D. RESULTS: The mean value of 25-OHD, number of oocytes, fertilized oocytes and endometrial thickness was significantly higher in pregnant women. A significant positive association of 25-OHD with clinical pregnancy and thickness of endometrium was observed. After adjustment with female age and BMI, positive association of vitamin D with endometrial thickness was observed. CONCLUSION: Deficiency of 25-OHD in females hinders the accomplishment of optimal endometrial thickness required for implantation of embryo after ICSI. The improvement in vitamin D status can thus improve success results in assisted reproductive clinics.

Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library.

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.

Synthesis of novel inhibitors of α-glucosidase based on the benzothiazole skeleton containing benzohydrazide moiety and their molecular docking studies.

In an effort to design and synthesize a new class of α-glucosidase inhibitor, we synthesized benzothiazole hybrid having benzohydrazide moiety (5). Compound 5 was reacted with various substituted aryl aldehyde to generate a small library of compounds 6-35. Synthesis of compounds was confirmed by the spectral information. These compounds were screened for their α-glucosidase activity. They showed a varying degree of α-glucosidase inhibition with IC50 values ranging between 5.31 and 53.34 µM. Compounds 6, 7, 9-16, 19, 21-30, 32-35 showed superior activity as compared to standard acarbose (IC50 = 906 ± 6.3 µM). This has identified a new class of α-glucosidase inhibitors. The predicted physico-chemical properties indicated the drug appropriateness for most of these compounds, as they obey Lipinski's rule of five (RO5). A hybrid B3LYP density functional theory (DFT) was employed for energy, minimization of 3D structures for all synthetic compounds using 6-311 + G(d,p) basis sets followed by molecular docking to explore their interactions with human intestinal C- and N-terminal domains of α-glucosidase. All compounds bind to the prospective allosteric site of the C- terminal domain, and consequently, may be considered as mixed inhibitors. It was hypothesized that both the dipole moment and H-bond interactions govern the biological activation of these compounds.

Synthesis and molecular docking studies of potent α-glucosidase inhibitors based on biscoumarin skeleton.

In our effort directed toward the discovery of new anti-diabetic agent for the treatment of diabetes, a library of biscoumarin derivative 1-18 was synthesized and evaluated for α-glucosidase inhibitory potential. All eighteen (18) compounds displayed assorted α-glucosidase activity with IC50 values 16.5-385.9 µM, if compared with the standard acarbose (IC50 = 906 ± 6.387 µM). In addition, molecular docking studies were carried out to explore the binding interactions of biscoumarin derivatives with the enzyme. This study has identified a new class of potent α-glucosidase inhibitors.