Inhibitors of Alpha-Synuclein Aggregation

Alpha-Synuclein (αS) – a neuronal, disordered, presynaptic protein – aggregates into amyloid fibrils and accumulates in the substantia nigra pars compacta of Parkinson\u27s Disease (PD) patients. The aggregation and accumulation of αS amyloid fibrils leads to death of dopaminergic neurons; a hallmark of PD. Although it’s not clear why αS aggregates, prior studies have found that intrastriatal injection of fibril alone is sufficient to cause PD pathology in mouse and non-human primates models. These observations implicate αS as a therapeutic target against PD. Unfortunately, there are three caveats when attempting to target αS. First, αS is a neuronal protein expressed in the midbrain. A therapeutic agent should be able to cross the Blood Brain Barrier (BBB) and the cell membrane to target αS. Second, αS is intrinsically disordered and dynamic, and most drug design platforms target static tertiary protein structures. Third, it’s not clear which sequence domain initiates the aggregation process. αS has 140 residues with the first 90 N-terminal residues typically found in the amyloid core. The specific N-terminus sequence that nucleates aggregation isn’t characterized, thus there isn’t a known αS region to target. To address these problem, we prepared and screened a library of small molecule alpha-helical mimetics called ‘Foldamers’ against αS aggregation. Foldamers fold to a helical conformation and present their functional groups at similar spatial and angular locations to peptide alpha helix, making them great tools for targeting protein-protein interactions. Our screen identified SK129, an oligoquinoline foldamer that is a potent inhibitor of in vitro and in vivo αS aggregation as demonstrated in human cells (HEK293 Cells) and C. elegans (NL5901, PD model). Then, we tested the hypothesis that SK129 could be binding αS in the sequence that nucleates aggregation. We performed Heteronuclear Single Quantum Coherence NMR experiments and identified sequences 6- 12, 15-23, 36-45 and 47-53 as potential binding sites for SK129. We made αS mutants with deletions in the identified sequences – Δ6-12, Δ15-23, Δ36-45 and Δ47-53 – and performed aggregation experiments. Mutants Δ36-45 and Δ47-53 couldn’t form amyloid fibrils suggesting these sequences are critical for aggregation. Additionally, we developed an Oligopyridylamide (OP) based 2-Dimensional Fragment-Assisted Structure-based Technique (2D-FAST) and utilized it to develop a potent inhibitor of αS aggregation – NS163. This molecule was able to prevent cellular αS aggregation in body wall muscle cells (NL5901, C. elegans model) and dopaminergic neurons (UA196). NS163 was also able to halt disease progression in post-disease onset PD models. We also explored the effect polyamines have on αS aggregation. Since one of hallmarks of PD is degradation resistant αS aggregates, we tested if aggregating αS in the presence of different polyamines confers degradation resistance to resulting aggregates. We found αS aggregated with spermine is resistant to enzymatic degradation. Spermine also perturbs the aggregation equilibrium to favor the formation of higher amounts of insoluble protein

Antioxidant properties of Enterobacter cloacae C3 lipopeptides in vitro and in model food emulsion

The present work aims to investigate the in vitro antioxidant activities of Enterobacter cloacae C3 lipopeptides, as well as the stability of sunflower oil in water (o/w) emulsion and the conservation of raw beef patties. The C3 lipopeptides were assayed for their antioxidant activity through five different tests. The C3 lipopeptides showed good in vitro antioxidant activities. Lipopeptides C3 exhibited important antioxidant properties in 10% sunflower o/w emulsions during prolonged storage (28 days) at 30°C. The effect of C3 lipopeptides on the oxidative stability of raw beef patties showed effectiveness effect in preventing oxidative degradation of lipids via thiobarbituric acid reactive substance and peroxide methods. Cytotoxicity test using human kidney HEK293 cells showed that studied lipopeptides was nontoxic substances. The results of this study indicate that lipopeptides C3 could be appropriate antioxidant agent in food models as inhibitors of lipid oxidation.Peer ReviewedPostprint (author's final draft

Antioxidant properties, antimicrobial and anti-adhesive acivities of DCS1 lipopeptides from Bacillus methylotrophicus DCS1

Background: The present work aims to investigate the antioxidant and antimicrobial activities as well as the potential of DCS1 lipopeptides produced by Bacillus methylotrophicus DCS1 strain at inhibition and disruption of biofilm formation. Results: The produced biosurfactants were characterized as lipopeptides molecules by using thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR). The DCS1 lipopeptides were assayed for their antioxidant activity through five different tests. The scavenging effect on DPPH radicals at a concentration of 1 mg mL−1 was 80.6%. The reducing power reached a maximum value of 3.0 (OD700 nm) at 2 mg mL−1 . Moreover, the DCS1 lipopeptides exhibited a strong inhibition of β-carotene bleaching by linoleic acid assay with 80.8% at 1 mg mL−1 and showed good chelating ability and lipid peroxidation inhibition. The in vitro antimicrobial activity of DCS1 lipopeptides showed that they display significant antibacterial and antifungal activities. The anti-adhesive activity of DCS1 lipopeptides was evaluated against several pathogenic microorganisms. The lipopeptides showed excellent anti-adhesive activity, even at low concentrations, in a polystyrene surface pre-treatment against all the microorganisms tested. Further, they can disrupt performed biofilms. Conclusion: This study shows the potentiality of DCS1 lipopeptides as natural antioxidants, antimicrobial and/or anti-adhesive agent for several biomedical and industrial applications

Isolation and characterization of kurstakin and surfactin isoforms produced by Enterobacter cloacae C3 strain

In this work, the extraction, structural analysis, and identification as well as antimicrobial, anti‐adhesive, and antibiofilm activities of lipopeptides produced by Enterobacter cloacae C3 strain were studied. A combination of chromatographic and spectroscopic techniques offers opportunities for a better characterization of the biosurfactant structure. Thin layer chromatography (TLC) and HPLC for amino acid composition determination are used. Efficient spectroscopic techniques have been utilized for investigations on the biochemical structure of biosurfactants, such as Fourier transform infrared (FT‐IR) spectroscopy and mass spectrometry analysis. This is the first work describing the production of different isoforms belonging to kurstakin and surfactin families by E cloacae strain. Three kurstakin homologues differing by the fatty acid chain length from C10 to C12 were detected. The spectrum of lipopeptides belonging to surfactin family contains various isoforms differing by the fatty acid chain length as well as the amino acids at positions four and seven. Lipopeptide C3 extract exhibited important antibacterial activity against Gram‐positive and Gram‐negative bacteria, antifungal activity, and interesting anti‐adhesive and disruptive properties against biofilm formation by human pathogenic bacterial strains: Salmonella typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, and Candida albicans

Calidad de servicio y la fidelización del cliente en la Pizzería Tumi del cercado de Juliaca 2021

El objetivo de la presente investigación fue determinar la relación entre la calidad de servicio y la fidelización del cliente en la pizzería Tumi del cercado de Juliaca 2021. En este sentido, el tipo de investigación fue aplicado, descriptivo– correlacional y de diseño no experimental. La población estuvo conformada por 950 clientes de la pizzería y aplicando un muestreo probabilístico se obtuvo una muestra de 142 clientes. La técnica e instrumento aplicados fueron la encuesta y el cuestionario. La aplicación del instrumento proporciono resultados y respondió a la hipótesis de estudio, concluyendo que la calidad de servicio tiene relación directa y alta con la fidelización del cliente en la pizzería Tumi, puesto que se obtuvo un Rho=0.852 con un nivel de significancia 0.00=. Por tanto, frente a la existencia de alta (baja) calidad de servicio, se concentrará alta (baja) fidelización de clientes en la empresa

NIC 19: Beneficios a los empleados y su incidencia en la presentación de los Estados Financieros de empresas del sector textil del distrito de La Victoria, año 2022

La presente investigación tiene como propósito determinar cómo la “NIC 19: Beneficios a los Empleados incide en la presentación de los Estados Financieros de las empresas del sector textil del distrito de La Victoria, año 2022”. La NIC 19, de gran importancia y aplicabilidad en los diversos rubros empresariales, nos permite identificar y reflejar de manera confiable y oportuna los beneficios reconocidos a empleados. El trabajo de tesis se estructura en un total de cinco capítulos. El capítulo uno, aborda el marco teórico, antecedentes de la investigación, la descripción situacional del sector económico empresarial al que se dirige la investigación y las bases teóricas de las variables seleccionadas. En el capítulo 2, se desarrollan temas como la descripción de la problemática, la formulación del problema general y los problemas específicos, la justificación y relevancia, el objetivo general y los objetivos específicos, hipótesis y parámetros. El capítulo tres, se enfoca en la metodología de la investigación, el diseño metodológico, la investigación cualitativa y cuantitativa, y sus respectivas poblaciones junto con los instrumentos de recolección de datos respectivos. En el capítulo cuatro, se encuentran la formulación de entrevistas a profundidad a los expertos y la formulación de encuestas. Por último, en el capítulo cinco se encuentran las conclusiones de esta investigación, además conclusiones en base a el análisis de las hipótesis planteadas de las hipótesis planteadas, las cuales confirman que la NIC 19: Beneficios a los empleados incide en la presentación de los Estados Financieros en las empresas del sector textil del distrito de La Victoria, en el año 2022.The purpose of this investigation is to determine how "NIC 19: Employee Benefits affects the presentation of financial statements of companies in the textile sector of the district of La Victoria, year 2022". IAS 19, of great importance and applicability in various business areas, allows us to identify and reflect in a reliable and timely manner the benefits recognized for employees. The thesis work is structured in a total of five chapters. Chapter one addresses the theoretical framework, background of the research, the situational description of the business economic sector to which the research is directed and the theoretical bases of the selected variables. In chapter 2, topics such as the description of the problem, the formulation of the general problem and the specific problems, the justification and relevance, the general objective and the specific objectives, hypotheses and parameters are developed. Chapter three focuses on the research methodology, methodological design, qualitative and quantitative research, and their respective populations together with the respective data collection instruments. In chapter four, the formulation of in-depth interviews with experts and the formulation of surveys are found. Finally, in chapter five are the conclusions of this investigation, as well as conclusions based on the analysis of the hypotheses raised, which confirm that IAS 19: Employee benefits affects the presentation of the statements. financial statements in companies in the textile sector of the district of La Victoria, in the year 2022.Tesi

Evaluation of changes in liver function test in first, second and third trimester of normal pregnancy

Background: Understanding of physiological adaptations of normal pregnancy remains a major goal of obstetrics, and without such knowledge, it is almost impossible to understand disease processes that can threaten women during pregnancy. Aim of this study is to evaluate changes in serum liver function tests in normal pregnant women in first, second and third trimester.Methods: A hospital based cross sectional in vitro study conducted at Civil Hospital, Ahmedabad (India). A study consists of 150 pregnant women and 50 matched control. Among the 150 pregnant women, 50 were in first trimester, 50 were in second trimester and 50 were in third trimester. Serum sample was taken and assessed for routine liver function tests.Results: Serum total and direct bilirubin concentrations were significantly lower in second and third trimester. Serum ALT and AST activity was slightly but significantly increased in third trimester. Serum ALP activity was significantly higher in second and third trimester. ALP activity increases as pregnancy advances. Serum GGT values were significantly lowers in third trimester. No significant change in serum total proteins concentration, but serum albumin concentration was significantly lower and serum globulin concentration was significantly higher in all three trimester. Serum albumin/globulin ratio was significantly reduced in second and third trimester.Conclusion: Such changes in in liver function tests during normal pregnancy can be misinterpreted as pathological and can also unmask or worsen preexisting disease. So the identification and understanding of these physiological changes in pregnancy is important for the diagnosis of liver diseases during pregnancy

The conservative effects of lipopeptides from Bacillus methylotrophicus DCS1 on sunflower oil-in-water emulsion and raw beef patties quality

Lipid oxidation was considered as a problem in food conservation. The present study aims to investigate the effect of lipopeptides DCS1 on the conservation of food models against lipid oxidation by determining the primary and the secondary oxidation products. Lipopeptides DCS1 are able to preserve the nutritional properties of the emulsion during 23¿days of storage, at a concentration of 0.0125% (w/w of emulsion), by slowing down the formation of hydroperoxides and malondialdehyde (MDA) compounds. The direct incorporation of lipopeptides in ground beef patties at a concentration of 0.5% (w/w of meat) was found to be more effective than gelatin film enriched with lipopeptides (2.5%, w/w of gelatin) as a coating, in inhibiting lipid oxidation. Furthermore, lipopeptides DCS1 are not toxic to human kidney cells HEK293 up to a concentration of 250¿µg/ml. The results indicate that lipopeptides DCS1 are effective for the preservation of fatty foods against lipid oxidation.Peer ReviewedPostprint (author's final draft

Structural characterization and identification of cyclic lipopeptides produced by Bacillus methylotrophicus DCS1 strain

Bacillus methylotrophicus DCS1 strain was isolated from diesel contaminated soil and screened for its ability to produce biosurfactants; it was found effective for the production of surface active molecules. The structural characterization of the isolated lipopeptides was studied by a variety of analytical techniques. The organic extract of DCS1 'lipopeptides was fractionated by silica gel column chromatography (60 Mesh). Fractions containing lipopeptides were collected and identified by tandem mass spectrometry MALDI-TOF-MS and MALDI-TOF MS2. The crude biosurfactants contains a mixture of homologous lipopeptides with molecular weights between 1016 and 1556 Da. Mass spectrometry analysis of partially purified lipopeptides revealed that it contains different isoforms belonging to three families: surfactin, iturin and fengycin. To identify lipopeptides isoforms, MALDI-TOF MS2 was used and ions representing characteristic fragmentations were detected. The mass spectrometry characterization revealed the presence of four variants of surfactin lipopeptides, four variants of pumilacidin that differ according to the-beta-hydroxy fatty acid chain length as well as the type of amino acid at position 7, five variants of iturin A/mycosubtilin varying in the beta-amino fatty acid chain length from C12 to C16, C16 iturin Cl, five isoforms of bacillomycin D varying in the beta-amino fatty acid chain length from C14 to C18, and six fengycin isoforms that differ according to the length of the beta-hydroxy fatty acid side chain as well as the amino acid at position 6. The capacity of B. methylotrohicus DCS1 strain to produce many lipopeptides isoforms belonging to different families and having a structural diversity is a very interesting characteristic that allows them to be used in various fields of biotechnological applications