A Novel G Protein-Coupled Receptor of Schistosoma mansoni (SmGPR-3) Is Activated by Dopamine and Is Widely Expressed in the Nervous System

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of “orphan” amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs

Liposomal formulations loaded with a eugenol derivative for application as insecticides: encapsulation studies and In silico identification of protein targets

Supplementary Materials can be downloaded at: https://www.mdpi.com/article/10.3390/nano12203583/s1,A recently synthesized new eugenol derivative, ethyl 4-(2-methoxy-4-(oxiran-2-ylmethyl)phenoxy)butanoate, with a high insecticidal activity against Sf9 (Spodoptera frugiperda) insect cells, was encapsulated in the liposomal formulations of egg-phosphatidylcholine/cholesterol (Egg-PC:Ch) 70:30 and 100% dioleoylphosphatidylglycerol (DOPG), aiming at the future application as insecticides. Compound-loaded DOPG liposomes have sizes of 274 ± 12 nm, while Egg-PC:Ch liposomes exhibit smaller hydrodynamic diameters (69.5 ± 7 nm), high encapsulation efficiency (88.8% ± 2.7%), higher stability, and a more efficient compound release, thus, they were chosen for assays in Sf9 insect cells. The compound elicited a loss of cell viability up to 80% after 72 h of incubation. Relevantly, nanoencapsulation maintained the toxicity of the compound toward insect cells while lowering the toxicity toward human cells, thus showing the selectivity of the system. Structure-based inverted virtual screening was used to predict the most likely targets and molecular dynamics simulations and free energy calculations were used to demonstrate that this molecule can form a stable complex with insect odorant binding proteins and/or acetylcholinesterase. The results are promising for the future application of compound-loaded nanoliposome formulations as crop insecticides.This research was funded by project PTDC/ASP-AGR/30154/2017 (POCI-01-0145-FEDER 030154) of the COMPETE2020 program, co-financed by the FEDER and the European Union. The authors also acknowledge the Foundation for Science and Technology (FCT, Portugal) and FEDERCOMPETE QREN-EU for financial support to the research centers CQUM (UID/QUI/00686/2021), CF-UM-UP (UIDB/04650/2020) and REQUIMTE (UIDB/50006/2020). Renato B. Pereira acknowledges PRIMA Foundation (H2020-PRIMA 2018—Section 2, Project MILKQUA) and FCT (PTDC/QUI-QFI/2870/2020) for additional funding. The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network and was purchased within the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005, with funds from POCI 2010 (FEDER) and FCT

Yeast Biological Networks Unfold the Interplay of Antioxidants, Genome and Phenotype, and Reveal a Novel Regulator of the Oxidative Stress Response

Background Identifying causative biological networks associated with relevant phenotypes is essential in the field of systems biology. We used ferulic acid (FA) as a model antioxidant to characterize the global expression programs triggered by this small molecule and decipher the transcriptional network controlling the phenotypic adaptation of the yeast Saccharomyces cerevisiae. Methodology/Principal Findings By employing a strict cut off value during gene expression data analysis, 106 genes were found to be involved in the cell response to FA, independent of aerobic or anaerobic conditions. Network analysis of the system guided us to a key target node, the FMP43 protein, that when deleted resulted in marked acceleration of cellular growth (~15% in both minimal and rich media). To extend our findings to human cells and identify proteins that could serve as drug targets, we replaced the yeast FMP43 protein with its human ortholog BRP44 in the genetic background of the yeast strain Δfmp43. The conservation of the two proteins was phenotypically evident, with BRP44 restoring the normal specific growth rate of the wild type. We also applied homology modeling to predict the 3D structure of the FMP43 and BRP44 proteins. The binding sites in the homology models of FMP43 and BRP44 were computationally predicted, and further docking studies were performed using FA as the ligand. The docking studies demonstrated the affinity of FA towards both FMP43 and BRP44. Conclusions This study proposes a hypothesis on the mechanisms yeast employs to respond to antioxidant molecules, while demonstrating how phenome and metabolome yeast data can serve as biomarkers for nutraceutical discovery and development. Additionally, we provide evidence for a putative therapeutic target, revealed by replacing the FMP43 protein with its human ortholog BRP44, a brain protein, and functionally characterizing the relevant mutant strain

MODULATORY ACTIONS OF SEROTONERGIC SYSTEM IN CARDIAC FUNCTION, BEHAVIOR, AND SENSORIMOTOR CIRCUIT ACTIVITY IN DROSOPHILA MELANOGASTER

In this dissertation, I have focused on the role of serotonin (5-HT) as a modulator in heart rate, feeding and locomotion behaviors as well as sensorimotor circuit activity in Drosophila melanogaster. A general overview in the actions of the serotonergic (5-HTergic) system on the larval heart and nervous system in larvae and adults is reviewed in Chapter One. I sought to further study the actions of serotonergic system to provide additional insights into cellular and molecular underpinnings in the actions of 5-HT.In Chapter two, I present studies on mechanisms of action by 5-HT in larvae cardiac system. For this purpose, genetic and pharmacological approaches were used. The transgenic flies used expressed hM4Di receptors (designer receptors exclusively activated by designer drugs (DREADDs)) which were employed to manipulate the activity of Gαi heterotrimeric protein through activation of engineered G-protein coupled receptors hM4Di DREADD. The activation of hM4Di DREADD receptors by clozapine-N-oxide (CNO) arrested the heart beat; however, pharmacological manipulation of adenylyl cyclase activity and cAMP levels had no significant effect on heart rate. In Chapter Three the role of various 5-HT receptor subtypes that mediate 5-HT action in larval cardiac tissue is addressed. In this study, various 5-HT agonists and antagonists were employed. The pharmacological results demonstrate that a 5-HT2 agonist significantly increases the heart rate. Furthermore, 5-HT2 antagonist, markedly reduces the effect of 5-HT. In addition, I employed genetic approaches to corroborate the pharmacological results. In addition, I investigated the role of the 5-HTergic system in locomotion and feeding behaviors as well as in modulation of sensorimotor circuits. This study is delineated in Chapter Four. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae various pharmacological agents. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or induced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotor activity and feeding behavior in larvae. In addition, acute activation of 5-HT neurons disrupts normal locomotor activity in adult flies. In Chapter Five, I addressed direct actions of fluoxetine on synaptic transmission at neuromuscular junctions (NMJs), neural properties, and cardiac function unrelated to fluoxetine’s action as a selective 5-HT reuptake inhibitor using Drosophila, crayfish and primary neurons in mouse model system. Fluoxetine application blocked action potentials in crayfish axons, enhanced occurrences of spontaneous synaptic vesicle fusion events at NMJs of both Drosophila and crayfish. In rodent primary neurons, fluoxetine application resulted in increase of cytoplasmic Ca2+. I also developed teaching modules, which are presented in Chapter Seven, to guide students how to exploit a vast array of genetic tools, such as optogenetics in Drosophila to manipulate various neural circuits and to observe their effects on behavior and sensorimotor circuit activity. I also developed a module to teach college level students a hands-on experiment regarding proprioception and tension receptors in crab limb, which is detailed in Chapter Eight

Case Record

UNDIFFERENTIATED SCHIZOPHRENIA: Mr. R was reported to be normal 4 years back, he was found to be preoccupied and not communicating well with his family members and preferred to stay alone. He was talking and laughing to self. When asked he said that he heard voices speaking to him and he were replying to it. Gradually his sleep decreased and he would sleep only for 3 to 4 hours at night. He did not attend classes. He was started washing his room many times. He was taking bath frequently. He was started worshiping god Anjanaya many times a day and also avoiding to speak with female family members. This continued for 2 months after which he started to be abusive and assaultive others for no reason. So he was taken to a psychiatrist, was admitted for 10 days and treated with ECT. After discharge he would discontinue medications on and off during which his symptoms would get exacerbated. He continued his studies but performance was poor. He also exhibited suicidal gestures on three occasions in the form of cutting his harms and carrying kerosene and matchbox inside the bathroom, saved by family members. Again the mother took him to psychiatrist and continued medication the symptoms were under the control. He completed his course but did not complete his studies. Then he got employment into Ford Company, his performance was average, he continued his job for 5 months with medication. After discontinue of treatment he became self-withdrawn, slowly he was neglecting self-care, attempted suicide, coworkers informed to his parents. Hence he was brought to IMH and admitted. No H/O sad mood, crying spells. No H/O tall claims, spending spree. No H/O thoughts being known to others or withdrawn. No H/O substance use. No H/O head injury, LOC, seizures. No H/O fever or any prolonged drug intake. SUBSTANCE INDUCED MOOD DISORDER: The patient was introduced to using betelnut along with his friends around 18 years of age. Later be continued to use tobacco in the form of Panparag, Hans, Shanthi betelnut 4-6 packets per day. Later after 3 years of tobacco intake, he started consuming alcohol for the first time along with his friends on some occasion, he consumed beer around 200ml. As he enjoyed the high produced by the drink, he continued to take alcohol at regular interval. After 1 year of beginning alcohol intake, he got married, after 6months of marriage life he started consuming alcohol in the form of brandy almost every night. He would become intoxicated, come home, abuse and assault his wife frequently. Due to frequent marrital dishormony his wife left and living with her grand parants for past 8 months. Now according to his be continued to drink alcohol, but the past two months he was engaged in some temple work, where he is supposedto have been introduced to cannabis.After consuming cannabis, his behavior became unmanageble. He frequently keep standing in the middle of the road and appear to make gesturesas if regulating the traffic. He would keep talking excessively and laugh for unprovoked reasons. His sleep patterns also worsened. All through out the night he would keep wandering in the street. And also he was started talking irrelevantly and would not be able to brought back home. He would also talk high about himself. He would claim himself to be God and capable of doing lot of things and able to grant wishes to all people. His selfcare also deteriorated, he started picking up quarrles and assaulted others. Nighbours made complaint against him. So, the family members brought him in the confused state to IMH. He was treated with Ing. Lorazepam 4 mg IM stat and referred to GGH for favour ofadmission and rule out other causes of delirium. He was treated at GGH for one week with Inj. Haloperidal 5 mg, Inj. Lorazepam 4 mg im, Inj.Thiamine and has been referred to IMH for further management. No h/o head injury/LOC/seizures. No h/o low mood/crying spells/suicidal attempts. No h/o hearing voices No h/o repititive washing/cheeking etc., DELUSIONAL DISORDER-MIXED: The patient was reported to be normal till one year eight months back. She claimed hat her co-tenant Mr. V called her for sexual relationship and she refused after that she started telling that he is setting people against her to harm her and also setup prostitutes as a co-tenants, to move her away from the place She also says that he tried to kill her with ambulance 108 and milk van by using his political influence. Patient gave complaint in nearby police station about Mr. Enquiry done. But the police Also turned against her by his political influence. So, she used to go to SP office, collector office daily and shout to arrest Mr. V. Mean while she vacated that house and shifted to Mr. S house who is friend of her brother. He is a widower, living alone. After 2 months of shifting to new house she started believing that the house owner was deeply loved with her whom she understands by his Gestures. And he did not admit his love for her has he did not want others to know. She was fought with the co-tenant once for silly reason. They were assaulted her with an aluminum mug and broken house hold article. And she assumed that Mr. V. only arranged them to fight with her. And also without any reason the patient was fought with the female Co-tenants whoever talking to Mr. S. She uses to tell everybody that the Mr. S. loves with her. The house owner warned them to vacate the house. But she did not vacate the house. The house owner slowly cut power supply, water supply to her portion. After 4 months she vacated the house to Next Street. Even after vacating, patient goes to Mr. S. house and starts quarrel with the new tenants that they should vacate and only because of them he is avoiding her. Every day she was going to her old house and tells everybody that the house owner loves with her. The husband told her not to go there, but she poured kerosene on him and try to kill him. So, the house owner filed a case against her, she was arrested and kept in observation at IMH. During observation she was continuously blaming the old co-tenant that all because him only it happen. Still the house owner his loves with her, she also loves him deeply. DEMENTIA IN ALZHEIMER’S DISEASE: The patient was reported to be normal till one year back. Then, her daughter noticed that the patient repeatedly searched for certain things in the house. She would forget simple things in the house like the way for going to toilet. At times she also found it difficult to return to her house after going for a walk. In course of time, she was not able to identify her close relatives. She was not able to remember whether she had taken her food or not. Her personal hygiene decreased gradually. She did not take bath and did not dress properly. She would pass urine inside the house itself at times. She slept for very little time and would wake up in the middle of the night and keep pacing inside the house.Slowly she was not able to identify her own family members. MENTAL RETARDATION-MILD: Patient was born out of non consanguineous marriage, full term normal delivery. Mother was 22 yrs and fathers age was 298 yrs. No history of any drug intake, fever or exanthematous eruptions in the ante natal period. No ante natal checkup was done. No history of radiation, injury, malnutrition, or vaginal bleeding. Delivery was conducted by local dhai; h/o prolonged 2nd stage of labor, the baby cried soon after birth and was breast fed after a short while. No h/o neonatal seizures or difficulty in feeding. No h/o of jaundice, breast fed up to 10 months, and there were no weaning difficulties