Transcriptional and phenotypic characterization of novel Spx-regulated genes in Streptococcus mutans

CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOIn oral biofilms, two of the major environmental challenges encountered by the dental pathogen Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the S. mutans transcriptional regulators SpxA1 and SpxA2 (formerly SpxA and SpxB, respectively) are involved in stress survival by activating the expression of classic oxidative stress genes such as dpr, nox, sodA and tpx. We reasoned that some of the uncharacterized genes under SpxA1/A2 control are potentially involved in oxidative stress management. Therefore, the goal of this study was to use Spx-regulated genes as a tool to identify novel oxidative stress genes in S. mutans. Quantitative real-time PCR was used to evaluate the responses of ten Spx-regulated genes during H2O2 stress in the parent and Delta spx strains. Transcription activation of the H2O2-induced genes (8 out of 10) was strongly dependent on SpxA1 and, to a lesser extent, SpxA2. In vitro transcription assays revealed that one or both Spx proteins directly regulate three of these genes. The gene encoding the FeoB ferrous permease was slightly repressed by H2O2 but constitutively induced in strains lacking SpxA1. Nine genes were selected for downstream mutational analysis but inactivation of smu127, encoding a subunit of the acetoin dehydrogenase was apparently lethal. In vitro and in vivo characterization of the viable mutants indicated that, in addition to the transcriptional activation of reducing and antioxidant pathways, Spx performs an important role in iron homeostasis by regulating the intracellular availability of free iron. In particular, inactivation of the genes encoding the Fe-S biogenesis SUF system and the previously characterized iron-binding protein Dpr resulted in impaired growth under different oxidative stress conditions, increased sensitivity to iron and lower infectivity in rats. These results serve as an entryway into the characterization of novel genes and pathways that allow S. mutans to cope with oxidative stress.In oral biofilms, two of the major environmental challenges encountered by the dental pathogen Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the S. mutans transcriptional regulators SpxA1 and SpxA2 (formerly SpxA and SpxB, respectively) are involved in stress survival by activating the expression of classic oxidative stress genes such as dpr, nox, sodA and tpx. We reasoned that some of the uncharacterized genes under SpxA1/A2 control are potentially involved in oxidative stress management. Therefore, the goal of this study was to use Spx-regulated genes as a tool to identify novel oxidative stress genes in S. mutans. Quantitative real-time PCR was used to evaluate the responses of ten Spx-regulated genes during H2O2 stress in the parent and Delta spx strains. Transcription activation of the H2O2-induced genes (8 out of 10) was strongly dependent on SpxA1 and, to a lesser extent, SpxA2. In vitro transcription assays revealed that one or both Spx proteins directly regulate three of these genes. The gene encoding the FeoB ferrous permease was slightly repressed by H2O2 but constitutively induced in strains lacking SpxA1. Nine genes were selected for downstream mutational analysis but inactivation of smu127, encoding a subunit of the acetoin dehydrogenase was apparently lethal. In vitro and in vivo characterization of the viable mutants indicated that, in addition to the transcriptional activation of reducing and antioxidant pathways, Spx performs an important role in iron homeostasis by regulating the intracellular availability of free iron. In particular, inactivation of the genes encoding the Fe-S biogenesis SUF system and the previously characterized iron-binding protein Dpr resulted in impaired growth under different oxidative stress conditions, increased sensitivity to iron and lower infectivity in rats. These results serve as an entryway into the characterization of novel genes and pathways that allow S. mutans to cope with oxidative stress104CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES [6849-12-1]FAPESP [2012/032278-3, 2014/03816-4]6849-12-12012/032278-3; 2014/03816-

Contextual influences on health worker motivation in district hospitals in Kenya

<p>Abstract</p> <p>Background</p> <p>Organizational factors are considered to be an important influence on health workers' uptake of interventions that improve their practices. These are additionally influenced by factors operating at individual and broader health system levels. We sought to explore contextual influences on worker motivation, a factor that may modify the effect of an intervention aimed at changing clinical practices in Kenyan hospitals.</p> <p>Methods</p> <p>Franco LM, et al's (Health sector reform and public sector health worker motivation: a conceptual framework. Soc Sci Med. 2002, 54: 1255–66) model of motivational influences was used to frame the study Qualitative methods including individual in-depth interviews, small-group interviews and focus group discussions were used to gather data from 185 health workers during one-week visits to each of eight district hospitals. Data were collected prior to a planned intervention aiming to implement new practice guidelines and improve quality of care. Additionally, on-site observations of routine health worker behaviour in the study sites were used to inform analyses.</p> <p>Results</p> <p>Study settings are likely to have important influences on worker motivation. Effective management at hospital level may create an enabling working environment modifying the impact of resource shortfalls. Supportive leadership may foster good working relationships between cadres, improve motivation through provision of local incentives and appropriately handle workers' expectations in terms of promotions, performance appraisal processes, and good communication. Such organisational attributes may counteract de-motivating factors at a national level, such as poor schemes of service, and enhance personally motivating factors such as the desire to maintain professional standards.</p> <p>Conclusion</p> <p>Motivation is likely to influence powerfully any attempts to change or improve health worker and hospital practices. Some factors influencing motivation may themselves be influenced by the processes chosen to implement change.</p

Stakeholders' Participation in Planning and Priority Setting in the Context of a Decentralised Health Care system: the case of prevention of mother to child Transmission of HIV Programme in Tanzania.

In Tanzania, decentralisation processes and reforms in the health sector aimed at improving planning and accountability in the sector. As a result, districts were given authority to undertake local planning and set priorities as well as allocate resources fairly to promote the health of a population with varied needs. Nevertheless, priority setting in the health care service has remained a challenge. The study assessed the priority setting processes in the planning of the prevention of mother to child transmission of HIV (PMTCT) programme at the district level in Tanzania. This qualitative study was conducted in Mbarali district, south-western Tanzania. The study applied in-depth interviews and focus group discussions in the data collection. Informants included members of the Council Health Management Team, regional PMTCT managers and health facility providers. Two plans were reported where PMTCT activities could be accommodated; the Comprehensive Council Health Plan and the Regional PMTCT Plan that was donor funded. As donors had their own globally defined priorities, it proved difficult for district and regional managers to accommodate locally defined PMTCT priorities in these plans. As a result few of these were funded. Guidelines and main priority areas of the Ministry of Health and Social Welfare (MoHSW) also impacted on the ability of the districts and regions to act, undermining the effectiveness of the decentralisation policy in the health sector. The challenges in the priority setting processes revealed within the PMTCT initiative indicate substantial weaknesses in implementing the Tanzania decentralisation policy. There is an urgent need to revive the strategies and aims of the decentralisation policy at all levels of the health care system with a view to improving health service delivery

The Nature of Working Memory for Braille

Blind individuals have been shown on multiple occasions to compensate for their loss of sight by developing exceptional abilities in their remaining senses. While most research has been focused on perceptual abilities per se in the auditory and tactile modalities, recent work has also investigated higher-order processes involving memory and language functions. Here we examined tactile working memory for Braille in two groups of visually challenged individuals (completely blind subjects, CBS; blind with residual vision, BRV). In a first experimental procedure both groups were given a Braille tactile memory span task with and without articulatory suppression, while the BRV and a sighted group performed a visual version of the task. It was shown that the Braille tactile working memory (BrWM) of CBS individuals under articulatory suppression is as efficient as that of sighted individuals' visual working memory in the same condition. Moreover, the results suggest that BrWM may be more robust in the CBS than in the BRV subjects, thus pointing to the potential role of visual experience in shaping tactile working memory. A second experiment designed to assess the nature (spatial vs. verbal) of this working memory was then carried out with two new CBS and BRV groups having to perform the Braille task concurrently with a mental arithmetic task or a mental displacement of blocks task. We show that the disruption of memory was greatest when concurrently carrying out the mental displacement of blocks, indicating that the Braille tactile subsystem of working memory is likely spatial in nature in CBS. The results also point to the multimodal nature of working memory and show how experience can shape the development of its subcomponents

Repurposing NGO data for better research outcomes: A scoping review of the use and secondary analysis of NGO data in health policy and systems research

Background Non-government organisations (NGOs) collect and generate vast amounts of potentially rich data, most of which are not used for research purposes. Secondary analysis of NGO data (their use and analysis in a study for which they were not originally collected) presents an important but largely unrealised opportunity to provide new research insights in critical areas including the evaluation of health policy and programmes. Methods A scoping review of the published literature was performed to identify the extent to which secondary analysis of NGO data has been used in health policy and systems research (HPSR). A tiered analytic approach provided a comprehensive overview and descriptive analyses of the studies which: 1) used data produced or collected by or about NGOs; 2) performed secondary analysis of the NGO data (beyond use of an NGO report as a supporting reference); 3) used NGO-collected clinical data. Results Of the 156 studies which performed secondary analysis of NGO-produced or collected data, 64% (n=100) used NGO-produced reports (e.g. to critique NGO activities and as a contextual reference) and 8% (n=13) analysed NGO-collected clinical data.. Of the studies, 55% investigated service delivery research topics, with 48% undertaken in developing countries and 17% in both developing and developed. NGO-collected clinical data enabled HPSR within marginalised groups (e.g. migrants, people in conflict-affected areas), with some limitations such as inconsistencies and missing data. Conclusion We found evidence that NGO-collected and produced data are most commonly perceived as a source of supporting evidence for HPSR and not as primary source data. However, these data can facilitate research in under-researched marginalised groups and in contexts that are hard to reach by academics, such as conflict-affected areas. NGO–academic collaboration could help address issues of NGO data quality to facilitate their more widespread use in research. Their use could enable relevant and timely research in the areas of health policy, programme evaluation and advocacy to improve health and reduce health inequalities, especially in marginalised groups and developing countries

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Exploring the influence of trust relationships on motivation in the health sector: a systematic review.

BACKGROUND: Dedicated and motivated health workers (HWs) play a major role in delivering efficient and effective health services that improve patients' experience of health care. Growing interest in HW motivation has led to a global focus on pay for performance strategies, but less attention has been paid to nurturing intrinsic motivation. Workplace trust relationships involve fair treatment and respectful interactions between individuals. Such relationships enable cooperation among HWs and their colleagues, supervisors, managers and patients and may act as a source of intrinsic motivation. This paper presents findings from a qualitative systematic review of empirical studies providing evidence on HW motivation, to consider what these studies suggest about the possible influence of workplace trust relationships over motivation. METHODS: Five electronic databases were searched for articles reporting research findings about HW motivation for various cadres published in the 10-year period 2003 to 2013 and with available full free text in the English language. Data extraction involved consideration of the links between trust relationships and motivation, by identifying how studies directly or indirectly mention and discuss relevant factors. RESULTS: Twenty-three articles from low- and middle-income countries and eight from high-income countries that met predetermined quality and inclusion criteria were appraised and subjected to thematic synthesis. Workplace trust relationships with colleagues, supervisors and managers, employing organisation and patients directly and indirectly influence HW motivation. Motivational factors identified as linked to trust include respect; recognition, appreciation and rewards; supervision; teamwork; management support; autonomy; communication, feedback and openness; and staff shortages and resource inadequacy. CONCLUSION: To the authors' knowledge, this is the first systematic review on trust and motivation in the health sector. Evidence indicates that workplace trust relationships encourage social interactions and cooperation among HWs, have impact on the intrinsic motivation of HWs and have consequences for retention, performance and quality of care. Human resource management and organisational practices are critical in sustaining workplace trust and HW motivation. Research and assessment of the levels of motivation and factors that encourage workplace trust relationships should include how trust and motivation interact and operate for retention, performance and quality of care