COMET Assay for Detection of DNA Damage During Axolotl Tail Regeneration.

The purpose of this chapter is to evaluate DNA damage during axolotl tail regeneration using an alkaline comet assay. Our method details the isolation of cells from regenerating and non-regenerating tissues and the isolation of peripheral blood for single-cell gel electrophoresis. Also, we detail each of the steps for the development of the comet assay technique which includes mounting the isolated cells on an agarose matrix, alkaline electrophoresis, and DNA damage detection.

Antileishmanial activity of a new chloroquine analog in an animal model of Leishmania panamensis infection.

Leishmania panamensis is a relevant causative agent of tegumentary leishmaniasis in several Latin American countries. Available antileishmanial drugs have several limitations including relatively high toxicity, difficult administration, high production costs and the emergence of resistance in circulating strains. Therefore, the identification of new molecules as potential therapeutics for leishmaniasis is of great relevance. Here, we developed a murine model of L. panamensis infection and evaluated the effect of a new compound in vivo. After treatment of animals with the compound, we observed a significant reduction of inflammation and parasite load at the inoculation site, in a dose-dependent manner. We observed a reduction in IL-10 production by popliteal lymph nodes cells of infected mice. These results pave the way for future evaluation of this compound as a potential antileishmanial drug or as a suitable scaffold for lead optimization strategies.

Avaliação dos conhecimentos para o tratamento não farmacológico da dor / Evaluación de conocimientos para el manejo no farmacológico del dolor / Evaluation of knowledge for the non-pharmacological management of pain

Las intervenciones para el alivio del dolor se clasifican en farmacológicas y no farmacológicas; las primeras incluyen la administración de fármacos analgésicos y las segundas utilizan terapias complementarias. El objetivo de este estudio fue diseñar y validar un instrumento de medición denominado "Conocimiento sobre intervenciones no farmacológicas para el alivio del dolor" para evaluar el nivel de conocimientos sobre intervenciones no farmacológicas para el manejo del dolor en un grupo de enfermeras profesionales y auxiliares de enfermería de una institución de salud de Bucaramanga, Colombia. Métodos: El instrumento se desarrolló en dos fases; la primera fase consistió en la búsqueda bibliográfica para el diseño, la validación facial y de contenido por expertos. En la segunda fase se evalúo la confiabilidad prueba- reprueba de la versión final del cuestionario. Resultados: La versión final del cuestionario contempla seis dimensiones propuestas por el Instituto Nacional de Medicina Alternativa de Estados Unidos con un total de 30 ítems. La confiabilidad de este instrumento fue baja (Índice Kappa-Cohen <0.60) en el 80% de los ítems. Conclusión: Este es el primer instrumento diseñado para medir los conocimientos sobre intervenciones no farmacológicas para el alivio del dolor en enfermeras y auxiliares de enfermería en nuestro país, sin embargo, se requiere continuar con procesos que permitan mejorar su confiabilidad y evaluar su validez.

As intervenções para o alivio da dor classificam-se em farmacológicas e não farmacológicas; as primeiras, consideram a administração de analgésicos, as segundas, empregam-se terapias complementarias. Objetivo: desenvolver e validar um questionário para à avaliação do nível de conhecimentos sob intervenções não farmacológicas para o tratamento da dor num grupo de enfermeiras padrão e auxiliares de enfermagem numa instituição de saúde de Bucaramanga, Colômbia. Materiais e métodos: O instrumento desenvolveu-se em duas fases: a primeira constou da busca de literatura, o desenho, a validação facial e de conteúdo por expertos. Na segunda fase, o questionário aplicou-se duas vezes a 30 enfermeiras e auxiliares de enfermagem com um intervalo de 30 dias para avaliar a confiabilidade. Calculou-se a pontuação total do instrumento e o índice Kappa-Cohen. Resultados: A versão final do questionário tem 30 questões avaliando seis dimensões propostas pelo Instituto Nacional de Medicina Alternativa dos Estados Unidos. Mais do 80% dos participantes teve o 70% das perguntas acertadas, entretanto, a confiabilidade do instrumento foi baixa (Kappa-Cohen <0,60) no 80% dos itens. Conclusão: Este é o primeiro instrumento desenvolvido para aferir conhecimentos sobre terapia não farmacológica para o alivio da dor em enfermeiras e auxiliares de enfermagem na Colômbia, entretanto requer continuar aprimorando a sua confiabilidade e a avaliação da sua validez.

Interventions to alleviate pain are classified in two categories, pharmacological and non/pharmacological; the first ones include the administration of pain relievers and the second ones use complementary therapies. Objective. Design and validate an instrument of measurement to evaluate the level of knowledge about non-pharmacological interventions to manage pain in a group of professional nurses and auxiliary nurses from a health institution in Bucaramanga, Colombia. Materials and Methods: The instrument was developed in two stages: the first stage consisted of the bibliographic research, the design and the facial and content validation by experts. In the second phase, the instrument was applied twice to 30 nurses and auxiliary nurses with an interval of 30 days to evaluate reliability. The total score of the instrument and the Cohen's kappa coefficient were calculated. Results: The final version of the questionnaire contemplates six dimensions proposed by the National Center for Complementary and Integrative Health (NCCIH), with a total of 30 items. More than 80% of the participants had 70% of the answers correct, however, the reliability of this instrument was low (Cohen's kappa < 0.60) in 80% of the items. Conclusion: This is the first instrument designed to measure knowledge about non-pharmacological interventions to alleviate pain for nurses and auxiliary nurses in our country, however, it is required to continue with processes that allow the instrument to improve its reliability and evaluate its validity.

Environmental Conditions May Shape the Patterns of Genomic Variations in Leishmania panamensis.

Due to the absence of transcriptional regulation of gene expression in Leishmania parasites, it is now well accepted that several forms of genomic variations modulate the levels of critical proteins through changes in gene dosage. We previously observed many of these variations in our reference laboratory strain of L. panamensis (PSC-1 strain), including chromosomes with an increased somy and the presence of a putative linear minichromosome derived from chromosome 34. Here, we compared the previously described genomic variations with those occurring after exposure of this strain to increasing concentrations of trivalent antimony (SbIII), as well as those present in two geographically unrelated clinical isolates of L. panamensis. We observed changes in the somy of several chromosomes, amplifications of several chromosomal regions, and copy number variations in gene arrays after exposure to SbIII. Occurrence of amplifications potentially beneficial for the Sb-resistant phenotype appears to be associated with the loss of other forms of amplification, such as the linear minichromosome. In contrast, we found no evidence of changes in somy or amplification of relatively large chromosomal regions in the clinical isolates. In these isolates, the predominant amplifications appear to be those that generate genes arrays; however, in many cases, the amplified arrays have a notably higher number of copies than those from the untreated and Sb-treated laboratory samples.

Characterization of an HPV33 natural variant with enhanced transcriptional activity suggests a role for C/EBPß in the regulation of the viral early promoter.

The Long Control Region (LCR) of the human papillomavirus (HPV) genome encompasses the early promoter (EP) that drives expression of the viral oncogenes in infected cells and HPV-associated cancers. Here, we report on a natural variant of HPV33 that displays higher EP activity than the prototype in transfected C33A and HeLa cervical carcinoma cells, and in the osteosarcoma U2OS cell line which supports replication of HPV episomes. This increased promoter activity was ascribed to a single nucleotide variation in the LCR, T7791C, in a putative binding site for the transcription factor C/EBPß. T7791C abrogated binding of recombinant C/EBPß to this site in vitro and stimulated the EP in vivo, suggesting that it abrogates a negatively-acting regulatory element. A second C/EBPß binding site was identified in vitro that activated the EP in vivo and whose function and location in the epithelial-specific enhancer is shown to be conserved in the highly prevalent HPV18. These results suggest that C/EBPß is both an activator and a repressor of the HPV33 EP, acting via two distinct binding sites. Prediction of C/EBPß sites in the LCR of 186 HPV types suggests that C/EBPß regulation of the EP is common among high-risk viruses from the α genus.

Naturally Occurring Variations Modulate the Activity of the HPV33 Early Promoter and its Affinity for the E2 Transcription Factor.

The human papillomavirus (HPV) Long Control Region (LCR) encompasses the early promoter (EP) that drives transcription of the E6 and E7 oncogenes in keratinocytes and HPV-associated cancers. In this study, the transcriptional activities of the HPV33 EP from the prototype LCR and from eight variants representative of the worldwide diversity of the virus were examined in primary human keratinocytes (PHK) and in the HeLa cervical carcinoma cell line by luciferase reporter-gene assays. Remarkably, the two variations with the greatest effect on the EP in PHK were C7732G and a 79-bp deletion that were associated with high-grade cervical lesions and persistent infections, respectively, in epidemiological studies. In contrast, the three variations most active in HeLa cells were C7537A, A7874C and A7879G. A7874C, which lies within an E2-binding sequence, is also shown to increase the activity and binding of E2 at this site. Collectively, these results indicate that naturally-occurring variations affect the HPV33 EP differentially in PHK than in cancer cells and, furthermore, that they can also alter its regulation by E2. These findings provide a molecular basis for rationalizing the results of previous epidemiological studies and for understanding the contribution of LCR polymorphisms to the oncogenicity and persistence of HPV33 infections.

The impact of changes in distress tolerance on PTSD symptom severity post-treatment among veterans in residential trauma treatment.

Given that rates of PTSD, particularly among military populations, are increasing, it is critical to gain a better understanding of factors associated with treatment response. Low distress tolerance (DT), conceptualized as the perceived or actual inability to tolerate negative emotional states, may impacts veterans' responses to PTSD treatment. Low DT has been associated with more severe PTSD symptoms in clinical and non-clinical samples; however, its impact on PTSD symptomatology across treatment has yet to be assessed. We examined the impact of changes in DT, from intake to discharge, on post-treatment PTSD symptom severity within two samples of veterans recruited from Veterans Affairs residential PTSD treatment facilities in the northwestern and southern United States (Total N=86; 87% male; 46% White, 39% Black, 9% Latino, 6% Other). Veterans completed the Distress Tolerance Scale and PTSD Checklist (PCL) at intake and discharge from residential PTSD treatment. Regression analyses revealed that, within each veteran sample, those with the greatest improvements in DT had the lowest PCL total and subscale scores at discharge after controlling for respective intake PCL scores. This suggests increases in DT across treatment help explain the degree of benefits experienced by veterans following PTSD treatment.

Generation of Signs Within Semantic and Phonological Categories: Data from Deaf Adults and Children Who Use American Sign Language.

Two key areas of language development include semantic and phonological knowledge. Semantic knowledge relates to word and concept knowledge. Phonological knowledge relates to how language parameters combine to create meaning. We investigated signing deaf adults' and children's semantic and phonological sign generation via one-minute tasks, including animals, foods, and specific handshapes. We investigated the effects of chronological age, age of sign language acquisition/years at school site, gender, presence of a disability, and geographical location (i.e., USA and Puerto Rico) on participants' performance and relations among tasks. In general, the phonological task appeared more difficult than the semantic tasks, students generated more animals than foods, age, and semantic performance correlated for the larger sample of U.S. students, and geographical variation included use of fingerspelling and specific signs. Compared to their peers, deaf students with disabilities generated fewer semantic items. These results provide an initial snapshot of students' semantic and phonological sign generation.

Firearm Ownership Among Military Veterans With PTSD: A Profile of Demographic and Psychosocial Correlates.

Post-traumatic stress disorder (PTSD), a condition that disproportionately affects military veterans, is associated with heightened rates of aggression and suicide. Although experience with firearms is common among this population, virtually nothing is known regarding who is more likely to own a firearm and whether firearm ownership is differentially associated with psychological and behavioral risk factors among veterans with PTSD. Of 465 veterans (79% male) entering PTSD treatment, 28% owned a firearm (median number of firearms among owners = 3, range = 1-40). Firearm owners reported higher income were less likely to be unemployed, and were more likely to be male, Caucasian, married, and living in permanent housing. Ownership was associated with higher combat exposure and driving aggression, yet lower rates of childhood and military sexual trauma, suicidal ideation, and incarceration. Ownership was not associated with previous suicide attempt, arrest history, number of traumas experienced, PTSD symptoms, or depression. Together, among a sample of treatment-seeking military veterans with PTSD, those who owned a firearm appeared to demonstrate greater stability across a number of domains of functioning. Importantly though, routine firearm safety discussions (e.g., accessibility restrictions; violence risk assessments) and bolstering of anger management skills remain critical when working with this high-risk population.

Longitudinal Receptive American Sign Language Skills Across a Diverse Deaf Student Body.

This article presents results of a longitudinal study of receptive American Sign Language (ASL) skills for a large portion of the student body at a residential school for the deaf across four consecutive years. Scores were analyzed by age, gender, parental hearing status, years attending the residential school, and presence of a disability (i.e., deaf with a disability). Years 1 through 4 included the ASL Receptive Skills Test (ASL-RST); Years 2 through 4 also included the Receptive Test of ASL (RT-ASL). Student performance for both measures positively correlated with age; deaf students with deaf parents scored higher than their same-age peers with hearing parents in some instances but not others; and those with a documented disability tended to score lower than their peers without disabilities. These results provide longitudinal findings across a diverse segment of the deaf/hard of hearing residential school population.

Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking.

Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseous impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9-GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body following insult or injury. Alternatively, this method might find utility in delivering therapeutic genes for neuroinflammatory conditions.

Preservice Teacher and Interpreter American Sign Language Abilities: Self-Evaluations and Evaluations of Deaf Students' Narrative Renditions.

In deaf education , the sign language skills of teacher and interpreter candidates are infrequently assessed; when they are, formal measures are commonly used upon preparation program completion, as opposed to informal measures related to instructional tasks. Using an informal picture storybook task, the authors investigated the receptive and expressive narrative sign language skills of 10 teacher and interpreter candidates in a university preparation program. The candidates evaluated signed renditions of two signing children, as well as their own expressive renditions, using the Signed Reading Fluency Rubric (Easterbrooks & Huston, 2008) at the completion of their fifth sign language course. Candidates' evaluations were compared overall and across 12 sign language indicators to ratings of two university program professors. Some variation existed across ratings for individual indicators, but generally the candidates were aware of and could accurately rate their own abilities and those of two signing children.

Seated movement indexes emotion and its regulation in posttraumatic stress disorder.

This paper reports the results of a study that administered an emotion regulation task to Operation Enduring Freedom/Operation Iraqi Freedom veterans diagnosed with posttraumatic stress disorder (27) and to healthy controls (23). Seated movement and postural responses were transduced with a sensitive accelerometer attached to the underside of a low-mass cantilevered chair. Consistent with prior studies in which subjects stood on force plates, aversive photographs induced attenuation of nondirectional movement in patients and controls. Regarding seated postural responses, controls leaned towards neutral photographs and away from aversive ones, while participants with PTSD did the opposite. Regulation had no impact on seated movement but was associated with a seated postural withdrawal from the computer screen.

A psychophysiological investigation of emotion regulation in chronic severe posttraumatic stress disorder.

There have been few direct examinations of the volitional control of emotional responses to provocative stimuli in PTSD. To address this gap, an emotion regulation task was administered to 27 Operation Enduring Freedom/Operation Iraqi Freedom combat veterans and 23 healthy controls. Neutral and aversive photographs were presented to participants who did or did not employ emotion regulation strategies. Objective indices included corrugator electromyogram, the late positive potential, and the electrocardiogram. On uninstructed trials, participants with PTSD exhibited blunted cardiac reactivity rather than the exaggerated cardioacceleratory responses seen in trauma cue reactivity studies. On interleaved regulation trials, no measure evidenced group differences in voluntary emotion regulation. Persons with PTSD may not differ from normals in their capacity to voluntarily regulate normative emotional responses to provocative stimuli in the laboratory, though they may nevertheless respond differentially on uninstructed trials and endorse symptoms of dyscontrol pathognomonic of the disorder outside of the laboratory.

Deaf students' receptive and expressive american sign language skills: comparisons and relations.

This article presents receptive and expressive American Sign Language skills of 85 students, 6 through 22 years of age at a residential school for the deaf using the American Sign Language Receptive Skills Test and the Ozcaliskan Motion Stimuli. Results are presented by ages and indicate that students' receptive skills increased with age and were still developing across this age range. Students' expressive skills, specifically classifier production, increased with age but did not approach adult-like performance. On both measures, deaf children with deaf parents scored higher than their peers with hearing parents and many components of the measures significantly correlated. These results suggest that these two measures provide a well-rounded snapshot of individual students' American Sign Language skills.

Technology intervention research with deaf and hard of hearing learners: levels of evidence.

The authors evaluated the research base relative to technology use with deaf and hard of hearing students, examining 29 peer-reviewed studies published January 2000-August 2013 that used technology-based intervention (multimedia instructional applications/software) and investigating its effects on academic variables (academic skills used in instructional settings). They then evaluated the studies according to quality indicators for evidence-based research, both individually and as a body of evidence supporting intervention for deaf and hard of hearing students (Gersten et al., 2005; Horner et al., 2005; Institute of Education Sciences, 2013; Kratochwill et al., 2010, 2013). One of 24 group design studies met all Essential Quality Indicators; 3 of 5 single-case design studies did (Horner et al., 2005). No reviewed technology intervention met criteria for an established evidence base. Interventions are presented across technology types and academic areas to facilitate discussion of implications for researchers and practitioners.

Increasing children's ASL classifier production: a multicomponent intervention.

The Authors examined classifier production during narrative retells by 10 deaf and hard of hearing students in grades 2-4 at a day school for the deaf following a 6-week intervention of repeated viewings of stories in American Sign Language (ASL) paired with scripted teacher mediation. Classifier production, documented through a multiple-baseline-across-participants design, was followed by visual analysis and calculation of the percentage of non-overlapping data (Scruggs, Mastropieri, & Casto, 1987). Following the intervention, most students increased specific elements within their classifier production during narrative retells of stories they had watched and showed improved performance from pre- to postintervention measures. Picture support and fading of teacher mediation during retells variably affected classifier production. The results suggest that students may benefit from tailored intervention, including repeated viewings of ASL models paired with teacher mediation and possibly picture support, to increase classifier production during narrative retells.

Diverse activation of microglia by chemokine (C-C motif) ligand 2 overexpression in brain.

BACKGROUND: The chemokine (C-C motif) ligand 2 (CCL2) is a monocyte chemoattractant protein that mediates macrophage recruitment and migration during peripheral and central nervous system (CNS) inflammation. METHODS: To determine the impact of CCL2 in inflammation in vivo and to elucidate the CCL2-induced polarization of activated brain microglia, we delivered CCL2 into the brains of wild-type mice via recombinant adeno-associated virus serotype 9 (rAAV-9) driven by the chicken ß-actin promoter. We measured microglial activation using histological and chemical measurement and recruitment of monocytes using histology and flow cytometry. RESULTS: The overexpression of CCL2 in the CNS induced significant activation of brain resident microglia. CD45 and major histocompatibility complex class II immunoreactivity significantly increased at the sites of CCL2 administration. Histological characterization of the microglial phenotype revealed the elevation of "classically activated" microglial markers, such as calgranulin B and IL-1ß, as well as markers associated with "alternative activation" of microglia, including YM1 and arginase 1. The protein expression profile in the hippocampus demonstrated markedly increased levels of IL-6, GM-CSF and eotaxin (CCL-11) in response to CCL2, but no changes in the levels of other cytokines, including TNF-α and IFN-γ. Moreover, real-time PCR analysis confirmed increases in mRNA levels of gene transcripts associated with neuroinflammation following CCL2 overexpression. Finally, we investigated the chemotactic properties of CCL2 in vivo by performing adoptive transfer of bone marrow-derived cells (BMDCs) isolated from donor mice that ubiquitously expressed green fluorescent protein. Flow cytometry and histological analyses indicated that BMDCs extravasated into brain parenchyma and colabeled with microglial markers. CONCLUSION: Taken together, our results suggest that CCL2 strongly activates resident microglia in the brain. Both pro- and anti-inflammatory activation of microglia were prominent, with no bias toward the M1 or M2 phenotype in the activated cells. As expected, CCL2 overexpression actively recruited circulating monocytes into the CNS. Thus, CCL2 expression in mouse brain induces microglial activation and represents an efficient method for recruitment of peripheral macrophages.

Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1.

The bovine papillomavirus type 1 (BPV1) E2 protein binds as a dimer to the viral genome to promote its transcription, replication and maintenance in keratinocytes. Although BPV1 E2 dimerizes primarily through its DNA-binding domain, it was shown previously that its transactivation domain (TAD) can also dimerize in vitro through formation of a disulfide bond between cysteine 57 (C57) of adjacent monomers and of an ion pair between arginine 172 (R172) and aspartic acid 175 (D175). The function of this TAD dimerization interface in vivo remains unknown. Here, we report the effects of substituting C57, R172 and D175 by alanine on the transactivation activity of BPV E2 as well as on its ability to support viral DNA replication using a novel luciferase-based assay. Results for this mutational analysis suggest that the TAD dimerization interface is not essential for either process but may contribute to the DNA replication activity of BPV1 E2.

Anxiety sensitivity and sleep quality: independent and interactive predictors of posttraumatic stress disorder symptoms.

A cardinal feature of posttraumatic stress disorder (PTSD) is decreased sleep quality. Anxiety sensitivity (AS) is one factor that has shown early theoretical and empirical promise in better understanding the relation between sleep quality and PTSD outcomes. The current study is the first to test the independent and interactive effects of sleep quality and AS on PTSD symptoms. Consistent with hypotheses, AS and sleep quality were found to be independent and interactive predictors of PTSD symptom severity in our sample of male military veterans seeking treatment for PTSD. Slope analyses revealed that AS was differentially related to PTSD symptom severity as a function of quality of sleep. The veterans with good sleep quality and relatively lower levels of AS had the lowest level of PTSD symptoms, whereas the veterans with poor sleep quality and low AS evidenced severity of PTSD symptoms similar to those with high AS.