Nullomer Derived Anticancer Peptides (NulloPs): Differential Lethal Effects on Normal and Cancer Cells \u3cem\u3ein vitro\u3c/em\u3e

We demonstrate the first use of the nullomer (absent sequences) approach to drug discovery and development. Nullomers are the shortest absent sequences determined in a species, or group of species. By identifying the shortest absent peptide sequences from the NCBI databases, we screened several potential anti-cancer peptides. In order to improve cell penetration and solubility we added short poly arginine tails (5Rs), and initially solubilized the peptides in1M trehalose. The results for one of the absent sequences 9R (RRRRRNWMWC), and its scrambled version 9S1R (RRRRRWCMNW) are reported here. We refer to these peptides derived from nullomers as PolyArgNulloPs. A control PolyArgNulloP, 124R (RRRRRWFMHW), was also included. The lethal effects of 9R and 9S1R are mediated by mitochondrial impairment as demonstrated by increased ROS production, ATP depletion, cell growth inhibition, and ultimately cell death. These effects increase over time for cancer cells with a concomitant drop in IC-50 for breast and prostate cancer cells. This is in sharp contrast to the effects in normal cells, which show a decreased sensitivity to the NulloPs over time

Safeguarding Forensic DNA Reference Samples with Nullomer Barcodes

Unintended transfer of biological material containing DNA is a concern to all laboratories conducting PCR analysis. While forensic laboratories have protocols in place to reduce the possibility of contaminating casework samples, there is no way to detect when a reference sample is mislabeled as evidence, or contaminates a forensic sample. Thus there is public concern regarding the safeguarding of DNA submitted to crime labs. We demonstrate a method of introducing an internal amplification control to reference samples, in the form of a nullomer barcode which is based upon sequences absent or rare from publically accessible DNA databases. The detection of this barcode would indicate that the source of analyzed DNA was from a reference sample provided by an individual, and not from an evidence sample. We demonstrate that the nullomers can be added directly to collection devices (FTA paper) to allow tagging during the process of sample collection. We show that such nullomer oligonucleotides can be added to existing forensic typing and quantification kits, without affecting genotyping or quantification results. Finally, we show that even when diluted a million-fold and spilled on a knife, the nullomer tags can be clearly detected. These tags support the National Research Council of the National Academy recommendation that “Quality control procedures should be designed to identify mistakes, fraud, and bias” in forensic science (National Academy of Sciences, 2009)

Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive Immune Response

Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages

Emergence of Anthrax Edema Toxin as a Master Manipulator of Macrophage and B Cell Functions

Anthrax edema toxin (ET), a powerful adenylyl cyclase, is an important virulence factor of Bacillus anthracis. Until recently, only a modest amount of research was performed to understand the role this toxin plays in the organism’s immune evasion strategy. A new wave of studies have begun to elucidate the effects this toxin has on a variety of host cells. While efforts have been made to illuminate the effect ET has on cells of the adaptive immune system, such as T cells, the greatest focus has been on cells of the innate immune system, particularly the macrophage. Here we discuss the immunoevasive activities that ET exerts on macrophages, as well as new research on the effects of this toxin on B cells

IL-10 administration reduces PGE-2 levels and promotes CR3-mediated clearance of Escherichia coli K1 by phagocytes in meningitis

Ineffectiveness of antibiotics in treating neonatal Escherichia coli K1 meningitis and the emergence of antibiotic-resistant strains evidently warrants new prevention strategies. We observed that administration of interleukin (IL)-10 during high-grade bacteremia clears antibiotic-sensitive and -resistant E. coli from blood of infected mice. Micro-CT studies of brains from infected animals displayed gross morphological changes similar to those observed in infected human neonates. In mice, IL-10, but not antibiotic or anti-TNF antibody treatment prevented brain damage caused by E. coli. IL-10 administration elevated CR3 expression in neutrophils and macrophages of infected mice, whereas infected and untreated mice displayed increased expression of FcγRI and TLR2. Neutrophils or macrophages pretreated with IL-10 ex vivo exhibited a significantly greater microbicidal activity against E. coli compared with cells isolated from wild-type or IL-10−/− mice. The protective effect of IL-10 was abrogated when CR3 was knocked-down in vivo by siRNA. The increased expression of CR3 in phagocytes was caused by inhibition of prostaglandin E-2 (PGE-2) levels, which were significantly increased in neutrophils and macrophages upon E. coli infection. These findings describe a novel modality of IL-10–mediated E. coli clearance by diverting the entry of bacteria via CR3 and preventing PGE-2 formation in neonatal meningitis

Sublethal Doses of Anthrax Lethal Toxin on the Suppression of Macrophage Phagocytosis

BACKGROUND: Lethal toxin (LT), the major virulence factor produced by Bacillus anthracis, has been shown to suppress the immune system, which is beneficial to the establishment of B. anthracis infections. It has been suggested that the suppression of MEK/MAPK signaling pathways of leukocytes contributes to LT-mediated immunosuppressive effects. However, the involvement of MAPK independent pathways has not been clearly elucidated; nor has the crucial role played by LT in the early stages of infection. Determining whether LT exerts any pathological effects before being enriched to an MEK inhibitory level is an important next step in the furtherance of this field. METHODOLOGY/PRINCIPAL FINDINGS: Using a cell culture model, we determined that low doses of LT inhibited phagocytosis of macrophages, without influencing MAPK pathways. Consistent low doses of LT significantly suppressed bacterial clearance and enhanced the mortality of mice with bacteremia, without suppressing the MEK1 of splenic and peripheral blood mononuclear cells. CONCLUSION/SIGNIFICANCE: These results suggest that LT suppresses the phagocytes in a dose range lower than that required to suppress MEK1 in the early stages of infection

AB Toxins: A Paradigm Switch from Deadly to Desirable

To ensure their survival, a number of bacterial and plant species have evolved a common strategy to capture energy from other biological systems. Being imperfect pathogens, organisms synthesizing multi-subunit AB toxins are responsible for the mortality of millions of people and animals annually. Vaccination against these organisms and their toxins has proved rather ineffective in providing long-term protection from disease. In response to the debilitating effects of AB toxins on epithelial cells of the digestive mucosa, mechanisms underlying toxin immunomodulation of immune responses have become the focus of increasing experimentation. The results of these studies reveal that AB toxins may have a beneficial application as adjuvants for the enhancement of immune protection against infection and autoimmunity. Here, we examine similarities and differences in the structure and function of bacterial and plant AB toxins that underlie their toxicity and their exceptional properties as immunomodulators for stimulating immune responses against infectious disease and for immune suppression of organ-specific autoimmunity

Bacterial Toxins as Pathogen Weapons Against Phagocytes

Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favor microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signaling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed.This work was supported by FEDER funds through Programa Operational Factores de Competitividade - COMPETE and by national funds through FCT - Fundacao para a Ciencia e a Tecnologia (project PTDC/BIA-MIC/3463/2012 FCOMP-01-0124-FEDER-028364; to AV). Research in the groups of Molecular Microbiology and Fish Immunology and Vaccinology is supported by national funds through FCT Fundacao para Ciencia e a Tecnologia/MEC - Ministerio da Educacao e Ciencia and co-funded by FEDER within the partnership agreement: PT2020 related with the research unit number 4293. The Group of Molecular Microbiology also receives support from a Research Grant 2014 by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (to SS) and the PT2020 research project Infect-ERA/0001/2013 PROANTILIS. AdV received the FCT fellowship SFRH/BPD/95777/2013 by national funds through FCT - Fundacao para a Ciencia e a Tecnologia/MEC - Ministerio da Educacao e Ciencia and co-funded by QREN and POPH (Programa Operational Potential Humano). SS is supported by FCT-Investigator program

Study of domino effect in an industrial area

Les effets dominos ou cascade d’événements dans les industries et particulièrement dans les industries chimiques et de transformation, sont reconnus comme des scénarios d’accidents possibles depuis environ trois décennies. Ils représentent une préoccupation croissante, car ils ont le potentiel de provoquer des conséquences dévastatrices. L’effet domino, comme phénomène, est un sujet controversé lorsque son évaluation est nécessaire. L’examen de la bibliographie a démontré l’absence d’une définition commune et d’une procédure simple d’utilisation et précise pour son appréciation. C’est pourquoi l’un des objectifs de cette recherche est de formaliser les connaissances relatives aux effets dominos afin de comprendre les mécanismes de leurs occurrences. Pour ce faire nous avons étudié les paramètres à examiner pour déterminer la possibilité de cascade et être en mesure d’identifier les scénarios dominos. L’enjeu étant de permettre l’amélioration de la prévention du risque d’effet domino. L’autre objectif est donc de produire une méthode pour l’identification et l’analyse des effets dominos. Nous avons développé une méthodologie globale pour l’étude des effets dominos en chaîne initiés par des pertes de confinement. Elle permet l’identification et la hiérarchisation des chemins de propagation des accidents. Cette méthode facilite la prise de décision pour la prévention des effets dominos, tout en proposant un outil efficace et simple d’utilisation. Les résultats de l’étude sont fournis sous forme d’une hiérarchisation quantitative des équipements impliqués dans les scénarios dominos, en tenant compte des effets des conditions météorologiques et des mesures de maîtrise des risques existantes ou proposées.Cette hiérarchisation donne une idée claire des dangers que représentent les équipements par rapport aux accidents en cascade, en précisant si la dangerosité de l’équipement provient de sa capacité à initier ou à propager un effet de cascade.La méthode est basée sur une description topographique de la zone étudiée, incluant les caractéristiques de chaque équipement, et prend en compte les mesures de maîtrise des risques mises en œuvre par l’industriel. Elle repose sur deux phases principales : La première, est l’identification des chemins de propagation des accidents. Pour ce faire, la méthode d’analyse par arbre d’événements est utilisée. Les cibles potentielles sont déterminées en combinant les valeurs seuils d’escalade et les modèles de vulnérabilité (pour l’estimation de la probabilité d’endommagement). Cette première phase est implémentée sous MATLAB® et Visual Basic for Applications (VBA) afin de faciliter l’entrée des données, et l’analyse des résultats dans Microsoft Excel®. La deuxième phase est l’identification des équipements les plus dangereux vis-à-vis des effets dominos. Elle consiste à hiérarchiser les équipements impliqués dans les chemins de propagation, en fonction de leur vraisemblance à causer ou à propager un effet domino. L’algorithme qui effectue cette phase est codé sous VBA. La méthode a été conçue de façon à ce qu’elle puisse être utilisée sans qu’il soit nécessaire de s’appuyer sur les résultats des études de dangers. Néanmoins, si ces résultats sont disponibles, il est alors possible d’alléger certaines étapes de la méthode. Elle s’est révélée facile à utiliser, cela a été constaté lors de son application dans le cadre de projets et stages d’étudiants.Domino effects or cascading events in the chemical and process industries are recognized as credible accident scenarios since three decades. They are raising a growing concern, as they have the potential to cause catastrophic consequences. Domino effect, as phenomenon, is still a controversial topic when coming to its assessment. There is still a poor agreement on the definition of domino effect and its assessment procedures. A number of different definitions and approaches are proposed in technical standards and in the scientific literature. Therefore, one of this research objectives is to formalize domino effects knowledges in order to comprehend their occurrence mechanisms. Thus, the parameters that should be looked at so as to understand the escalation possibility and in order to identify domino scenarios, were analyzed. The aim is to improve domino effect hazards prevention, through the development of a methodology for the identification and the analysis of domino effects.We developed a method for the analysis of domino accident chain caused by loss of containments. It allow the identification and prioritization of accident propagation paths. The method is user-friendly and help decision making regarding the prevention of cascading events. The final outcomes of the model are given in form of quantitative rankings of equipment involved in domino scenarios, taking into account the effect of meteorological conditions and safety barriers. The rankings give a clear idea of equipment hazard for initiating or continuing cascading events.The methodology is based on a topography of the industrial area of concern, including the characteristics of each unit and accounting for protection and mitigation barriers. It is based on two main stages. The first is the identification of accident propagation paths. For this, the event tree method is used. The possible targets are identified combining the escalation thresholds and vulnerability models (to estimate damage probability). This first stage was implemented using the MATLAB® software and Visual Basic for Applications (VBA) to enable an easy input procedure and output analysis in Microsoft Excel®.The second stage is the identification of the most dangerous equipment. It consists in prioritizing equipment involved in the propagation paths according to their likelihood to cause/propagate domino effect. The algorithm that performs this phase was coded in VBA.The method was designed so as it can be used without the need to rely on the results of safety reports. However, if such results are available, it is possible to lighten some steps of the method. It revealed easy to apply, this was confirmed through projects and student internships.Gli effetti domino, in cui un primo incidente causa in cascata altri scenari incidentali, sono tragli scenari incidentali più severi che avvengono nell’industria chimica. Nonostante l’attenzioneche anche la normativa dedica a tali scenari, la valutazione dell’effetto domino è un soggettocontroverso. L’analisi della letteratura tecnica e scientifica ha mostrato l’assenza di unadefinizione comune di « effetto domino » e di una semplice procedura per l’identificazione ditali scenari. È per tale motivo che uno degli obiettivi di questo lavoro di ricerca è diformalizzare le conoscenze relative agli effetti domino al fine di meglio comprendere imeccanismi che possono provocarli. A tal proposito sono stati studiati i parametri necessariper determinare la possibilità dell’insorgere di cascate di eventi e per essere in grado diidentificare i possibili scenari incidentali dovuti ad effetto domino. L’obiettivo finale del lavoroè stato di sviluppare un metodo per l’identificazione e l’analisi quantitativa della propagazionedi incidenti primari nell’ambito di scenari dovuti ad effetto domino.E’ stata sviluppata una metodologia generale per l’analisi degli effetti domino causati daperdite di confinamento. Tale metodologia permette l’identificazione e la classificazione deipercorsi di propagazione degli incidenti. Tale metodo facilita inoltre la prevenzione deglieffetti domino, proponendo uno strumento efficace e semplice da utilizzare.I risultati di questo studio sono forniti in forma di una classificazione delle apparecchiaturecoinvolte in scenari dovuti ad effetto domino, tenendo conto degli effetti delle condizionimeteorologiche e delle misure esistenti per la gestione del rischio. Tale classificazione fornisceanche un chiara idea dei pericoli rappresentati dalle singole apparecchiature nel caso diincidenti in cascata, in quanto precisando se la pericolosità delle attrezzature proviene dallaloro capacità di innescare o propagare un reazione a catena.Il metodo è basato su una descrizione topografica del sito studiato, che comprende anche lecaratteristiche di ogni attrezzatura, che tiene conto delle misure di gestione dei rischi e dellebarriere di sicurezza presenti, basato su due fasi principali. La prima è l’identificazione deipercorsi di propagazione degli incidenti. A tale scopo è stato utilizzato un metodo basatoVIsull’albero degli eventi. I potenziali bersagli vengono determinati combinando i valori di sogliaper la propagazione degli eventi ed i modelli di vulnerabilità delle apparecchiature. Questaprima fase è implementata in MATLAB® e Visual Basic for Applications (VBA) in modo dafacilitare la gestione dei dati e l’analisi dei risultati in Microsoft Excel®.La seconda fase è l’identificazione delle apparecchiature più pericolose per gli effetti domino.Tale fase consiste nel classificare le apparecchiature coinvolte nei percorsi di propagazione infunzione della loro capacità di causare o propagare un effetto domino. L’algoritmo dedicato inquesta fase è eseguito su VBA.I risultati ottenuti anche nell’applicazione ad un caso di studio hanno evidenziato le potenzialitàdel metodo, che rappresenta un significativo progresso nell’analisi quantitativa dell’effetto domino