INVESTIGATION OF SITE-SPECIFIC CHANGES WITHIN THE N-TERMINAL DOMAIN OF CALMODULIN AND THE RELATIONSHIP TO STRUCTURAL AND THERMAL STABILITY

The goal of this dissertation was to develop a method to assess how solution conditions affect a protein's stability, and a well-characterized model protein, was used to accomplish this objective. Generally, stabilizing conditions for proteins are identified from screening a matrix of conditions using an array of biophysical methods that probe physical stability by determining melting temperatures (Tm) of secondary and tertiary structure. Although this approach has been invaluable for quickly obtaining stable formulations, these tools do not provide site-specific information, which is needed to gain mechanistic understanding of protein stability. Our methodology employs high-resolution solution NMR spectroscopy to acquire molecular information about how specific residues within the structure are impacted by solution conditions and how the conditions affect stability. The N-terminal domain of Calmodulin (N-CaM) was recombinantly expressed in 15N media along with supplemental calcium to enhance in vivo stability. Solution NMR spectroscopy was used to evaluate the role of site-specific changes in mobility and structure on the overall stability of this model system in different solution conditions. The 1H-15N HSQC experiment was used to investigate effects of pH and ionic strength on individual residues within or near the two homologous Ca2+-binding sites. Circular dichroism (CD) was also employed to assess the thermal stability of N-CaM and to help interpret the observed site-specific changes in 1H-15N HSQC spectra. To increase protein yield and produce a sufficient amount of protein for NMR analysis, varying amounts of calcium concentrations were screened in the expression media. Based on densitometric analysis of SDS-PAGE, 1.5 mM CaCl2 was determined to provide the optimal concentration for enhancing in vivo stability, leading to the highest yield of N-CaM expression. From 2D NMR experiments, quantitative information that reflects changes in dynamics was derived from changes in peak shape and chemical shift position to elucidate how the solution environment affects residues within N-CaM. 1H-15N HSQC experiments provided quantitative determination of specific residues near known degradation sites that undergo physical perturbations. Changes in the chemical shift position and peak shape observed during 1H-15N HSQC experiments confirmed that increasing pH and ionic strength affects a limited subset of residues within a localized region. Residues N60, G61, and D64 in the Ca2+-binding loop site I was affected, in increasing pH and high ionic strength, while residues in the corresponding position of Ca2+-binding loop site II remained unaffected. CD data confirmed that, at basic pH, increased ionic strength resulted in increased overall thermal stability. Our study used common methods of probing physical stability to evaluate the overall thermal stability of N-CaM in different solution conditions. Analysis by NMR spectroscopy provided more detailed, site-specific information to show that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. By utilizing NMR spectroscopy combined with CD analysis, we were able to show that a subset of individual residues play a role in overall stability. Employing 2D NMR experiments to provide molecular information can be utilized in evaluating solution conditions for formulation of proteins of therapeutic interest.

Evolution récente des conditions climatiques et des écoulements sur le bassin versant de la Macta (Nord-Ouest de l'Algérie)

24 pages, 5 tableaux, 24 figuresInternational audienceEn Algérie, les conditions climatiques qui prévalent depuis trois décennies ont une influence négative sur la ressource en eau. Ce travail a pour finalité de déterminer leur impact sur les cours d'eau du bassin versant de la Macta (Nord-Ouest de l'Algérie). L'étude des précipitations annuelles (de septembre à août) sur la période 1930-2002 montre une diminution très nette des pluies à partir de la première moitié des années 1970. Les débits des cours d'eau aux stations de Trois Rivières (sur l'oued El Hammam), de Sidi Ali Ben Youf et de Sidi Bel Abbès (sur l'oued Mekerra) en ont été affectés. Sur la période 1976-2002, les lames d'eau écoulées annuelles moyennes sont de 28 à 36 % plus faibles que sur la période 1949-1976. En valeurs moyennes, les précipitations se révèlent plus faibles pour toutes les saisons, mais les écoulements sont peu ou pas diminués en automne

Advanced Computing Systems for Scientific Research

An advanced computing system was constructed at Bridgewater State University to provide students access to computing machines tailored to the purpose of computational scientific research. This paper provides an overview of the construction, design, capability, and future potential of the computing system. Project Reasoning During the course of projects utilizing a numerical weather prediction (NWP) model, it became evident that the desktop computing machines provided to students in computer labs were not capable of yielding results in a reasonable amount of time for non-standard applications. The standard configuration of computers in the Conant Science and Mathematics Center at Bridgewater State University is sufficient for the majority of student purposes; however, these standard computers become insufficient for more comprehensive applications. Because of this insufficiency, it became necessary to seek better performing computer systems to support research applications. Project Execution To increase the computational power of computing systems on campus, two distinct steps were taken. The first step was the upgrade of a standard desktop computer. This upgrade provided a single desktop machine with superior computing capability to the standard machines available to students. The second step was the construction of a Linux computer cluster from several networked previous generation machines

Differing Alterations of Odor Volatiles among Pathogenic Stimuli

Alterations of the volatile metabolome (the collection of volatiles present in secretions and other emanations) that occur in response to inflammation can be detected by conspecifics and chemometric analyses. Using a model system where mouse urinary metabolites are altered by treatment with lipopolysaccharide (found in the outer cell membrane of gram-negative bacteria), we hypothesized that alteration of body odor volatiles will vary according to the pathogen responsible for inducing the inflammation. We tested this hypothesis by treating mice with different immunogens that engage different immune signaling pathways. Results suggest that alterations of body odor volatiles resulting from inflammation do contain detailed information about the type of pathogen that instigated the inflammation and these differences are not merely dependent on the severity of the inflammatory event. These results are encouraging for the future of differential medical diagnosis of febrile diseases by analysis of the volatile metabolome. In particular, our data support the possibility that bacterial infections can be differentiated from viral infections such that antibiotic drug stewardship could be drastically improved by reducing unneeded treatments with antibiotics

Cytokine contributions to alterations of the volatile metabolome induced by inflammation

Several studies demonstrate that inflammation affects body odor. Volatile signals associated with inflammation induced by pyrogens like LPS are detectable both by conspecifics and chemical analyses. However, little is known about the mechanisms which translate detection of a foreign molecule or pathogen into a unique body odor, or even how unique that odor may be. Here, we utilized C57BL/6J trained mice to identify the odor of LPS-treated conspecifics to investigate potential pathways between LPS-induced inflammation and changes in body odor, as represented by changes in urine odor. We hypothesized that the change in volatile metabolites could be caused directly by the pro-inflammatory cytokine response mediated by TNF or IL-1b, or by the compensatory anti-inflammatory response mediated by IL-10. We found that trained biosensors generalized learned LPS-associated odors to TNF-induced odors, but not to IL-1b or IL-10-induced odors. Analyses of urine volatiles using headspace gas chromatography revealed distinct profiles of volatile compounds for each treatment. Instrumental discrimination relied on a mixture of compounds, including 2-sec-butyl-4,5-dihydrothiazole, cedrol, nonanal, benzaldehyde, acetic acid, 2- ethyl-1-hexanol, and dehydro-exo-brevicomin. Although interpretation of LDA modeling differed from behavioral testing, it does suggest that treatment with TNF, IL-1b, and LPS can be distinguished by their resultant volatile profiles. These findings indicate there is information found in body odors on the presence of specific cytokines. This result is encouraging for the future of disease diagnosis via analysis of volatiles

Sharing an environment with sick conspecifics alters odors of healthy animals

Body odors change with health status and the odors of sick animals can induce avoidance behaviors in healthy conspecifics. Exposure to sickness odors might also alter the physiology of healthy conspecifics and modify the odors they produce. We hypothesized that exposure to odors of sick (but non-infectious) animals would alter the odors of healthy cagemates. To induce sickness, we injected mice with a bacterial endotoxin, lipopolysaccharide. We used behavioral odor discrimination assays and analytical chemistry techniques followed by predictive classification modeling to ask about differences in volatile odorants produced by two types of healthy mice: those cohoused with healthy conspecifics and those cohoused with sick conspecifics. Mice trained in Y-maze behavioral assays to discriminate between the odors of healthy versus sick mice also discriminated between the odors of healthy mice cohoused with sick conspecifics and odors of healthy mice cohoused with healthy conspecifics. Chemical analyses paired with statistical modeling revealed a parallel phenomenon. Urine volatiles of healthy mice cohoused with sick partners were more likely to be classified as those of sick rather than healthy mice based on discriminant model predictions. Sickness-related odors could have cascading effects on neuroendocrine or immune responses of healthy conspecifics, and could affect individual behaviors, social dynamics, and pathogen spread

Revealing cytotoxic substructures in molecules using deep learning

In drug development, late stage toxicity issues of a compound are the main cause of failure in clinical trials. In silico meth ods are therefore of high importance to guide the early design process to reduce time, costs and animal testing. Technical advances and the ever growing amount of available toxicity data enabled machine learning, especially neural networks, to impact the feld of predictive toxicology. In this study, cytotoxicity prediction, one of the earliest handles in drug discovery, is investigated using a deep learning approach trained on a highly consistent in-house data set of over 34,000 compounds with a share of less than 5% of cytotoxic molecules. The model reached a balanced accuracy of over 70%, similar to previ ously reported studies using Random Forest. Albeit yielding good results, neural networks are often described as a black box lacking deeper mechanistic understanding of the underlying model. To overcome this absence of interpretability, a Deep Taylor Decomposition method is investigated to identify substructures that may be responsible for the cytotoxic efects, the so-called toxicophores. Furthermore, this study introduces cytotoxicity maps which provide a visual structural interpretation of the relevance of these substructures. Using this approach could be helpful in drug development to predict the potential toxicity of a compound as well as to generate new insights into the toxic mechanism. Moreover, it could also help to de-risk and optimize compounds

Knowledge and ideas about the processes of empathy and environmental identity among students and teachers in a secondary school in Córdoba, Argentina

En este escrito se analizan los procesos de empatía e identidad ambiental en estudiantes y profesores de Biología de tres cursos de 4º año que asistieron durante 2021 a una institución de Educación Secundaria de la provincia de Córdoba, Argentina. Se administró una Escala de Identidad Ambiental adaptada a los estudiantes (N=73) y se realizaron entrevistas semiestructuradas a profesores encargados del espacio curricular Biología. Los resultados mostraron escasos abordajes de problemáticas ambientales en las aulas, lo que resultaría de utilidad para promover habilidades como la empatía ambiental. Respecto a este concepto, los docentes poseen nociones básicas. También se vislumbró la importancia de una identidad ambiental construida a partir de acercamientos a la naturaleza y de los contextos de actuación de las personas. Entre las reflexiones finales se demarca la necesidad de generar espacios de formación y aprendizaje permanente con énfasis en los aspectos sociales y emocionales del ambiente.This paper analyzes environmental empathy and identity processes among Biology students and teachers who attended three fourth-year courses in an institution of Secondary School in the province of Cordoba, Argentina, in 2021. The Environmental Identity Scale was administered an adapted to students (N=73), and semi-structured interviews were conducted with Biology teachers. The results showed few attempts to deal with environmental problems in the classrooms, which would otherwise have been useful to promote skills such as environmental empathy. Regarding this concept, teachers have basic notions. The importance of an environmental identity built from engagement with nature and the contexts with people. Among final reflections, the need should be highlighted to generate contexts for training and lifelong learning empathizing on social and emotional aspects of the environment.Fil: Caceres, Pilmaiquen Malen. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Enseñanza de la Ciencia y la Tecnología; ArgentinaFil: Pelli, Romina Talia. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Enseñanza de la Ciencia y la Tecnología; ArgentinaFil: Martinenco, Rebeca Mariel. Universidad Nacional de Villa María. Instituto Académico Pedagógico de Ciencias Sociales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Martin, Rocío Belén. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Enseñanza de la Ciencia y la Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Risk factors for antibiotic-resistant bacteria colonisation in children with chronic complex conditions

To assess drug-resistant bacterial colonisation rates and associated risk factors in children with complex chronic conditions admitted to a national reference unit in Spain. Cross-sectional study that included all children admitted to our unit from September 2018 to July 2019. Rectal swabs were obtained to determine multidrug-resistant Gram-negative bacilli (MR-GNB) colonisation, and nasal swab to determine S. aureus and methicillin-resistant S. aureus (MRSA) colonisation. Medical records were reviewed. 100 children were included, with a median of four complex chronic conditions. Sixteen percent had S. aureus colonisation, including two MRSA. S. aureus colonisation was associated with technology-dependent children, while being on antibiotic prophylaxis or having undergone antibiotic therapy in the previous month were protective factors. The prevalence of MR-GNB colonisation was 27%, which was associated with immunosuppressive therapy (aOR 31; 2.02-47]; p = 0.01), antibiotic prophylaxis (aOR 4.56; 1.4-14.86; p = 0.012), previously treated skin-infections (aOR 2.9; 1.07-8.14; p = 0.03), surgery in the previous year (aOR 1.4; 1.06-1.8; p = 0.014), and hospital admission in the previous year (aOR 1.79; [1.26-2.56]; p = 0.001). The rate of S. aureus nasal colonisation in this series was not high despite the presence of chronic conditions, and few cases corresponded to MRSA. Antibiotic prophylaxis, immunosuppressive therapies, history of infections, previous surgeries, and length of admission in the previous year were risk factors for MR-GNB colonisation.This study was supported by Te Spanish Ministry of Science and Innovation-Carlos III Health Institute, and European Regional Development Funds; Grant No. PI18CIII/00372 [Fondo de Investigaciones Sanitarias-Spanish Health Research Fund (ISCIII)]; Grant Award “Jose María Corretger” from the Spanish Society for Paediatric Infectious Diseases; Grant Research Award from the Spanish Association of Paediatric Primary Care; and a small grant award from the European Society for Paediatric Infectious Diseases.S

Establishing a core outcome set for peritoneal dialysis : report of the SONG-PD (standardized outcomes in nephrology-peritoneal dialysis) consensus workshop

Outcomes reported in randomized controlled trials in peritoneal dialysis (PD) are diverse, are measured inconsistently, and may not be important to patients, families, and clinicians. The Standardized Outcomes in Nephrology-Peritoneal Dialysis (SONG-PD) initiative aims to establish a core outcome set for trials in PD based on the shared priorities of all stakeholders. We convened an international SONG-PD stakeholder consensus workshop in May 2018 in Vancouver, Canada. Nineteen patients/caregivers and 51 health professionals attended. Participants discussed core outcome domains and implementation in trials in PD. Four themes relating to the formation of core outcome domains were identified: life participation as a main goal of PD, impact of fatigue, empowerment for preparation and planning, and separation of contributing factors from core factors. Considerations for implementation were identified: standardizing patient-reported outcomes, requiring a validated and feasible measure, simplicity of binary outcomes, responsiveness to interventions, and using positive terminology. All stakeholders supported inclusion of PD-related infection, cardiovascular disease, mortality, technique survival, and life participation as the core outcome domains for PD