Role of Acetyl-Phosphate in Activation of the Rrp2-RpoN-RpoS Pathway in Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ54–σS sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans–C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay

When simulated environments make the difference: the effectiveness of different types of training of car service procedures

An empirical analysis was performed to compare the effectiveness of different approaches to training a set of procedural skills to a sample of novice trainees. Sixty-five participants were randomly assigned to one of the following three training groups: (1) learning-by-doing in a 3D desktop virtual environment, (2) learning-by-observing a video (show-and-tell) explanation of the procedures, and (3) trial-and-error. In each group, participants were trained on two car service procedures. Participants were recalled to perform a procedure either 2 or 4 weeks after the training. The results showed that: (1) participants trained through the virtual approach of learning-by-doing performed both procedures significantly better (i.e. p < .05 in terms of errors and time) than people of non-virtual groups, (2) the virtual training group, after a period of non-use, were more effective than non-virtual training (i.e. p < .05) in their ability to recover their skills, (3) after a (simulated) long period from the training—i.e. up to 12 weeks—people who experienced 3D environments consistently performed better than people who received other kinds of training. The results also suggested that independently from the training group, trainees’ visuospatial abilities were a predictor of performance, at least for the complex service procedure, adj R2 = .460, and that post-training performances of people trained through virtual learning-by-doing are not affected by learning styles. Finally, a strong relationship (p < .001, R2 = .441) was identified between usability and trust in the use of the virtual training tool—i.e. the more the system was perceived as usable, the more it was perceived as trustable to acquire the competences

Spontaneous extracranial carotid atherosclerosis evolution in asymptomatic individuals: a three-year prospective study Evolução espontânea da aterosclerose carotídea extra craniana em indivíduos assintomáticos: estudo prospectivo de três anos

OBJECTIVE: To evaluate the spontaneous evolution of extracranial carotid atherosclerosis in asymptomatic patients who did not present the main risk factors associated to the disease. METHOD:A prospective study including patients of both genders, age ranging from 40 to 70 years, not presenting any signs and symptoms of cerebrovascular disease and without the main atherosclerosis risk factors were included. Patients who were using or had used medication during the follow-up period that could potentially influence in the spontaneous course of atherosclerosis were excluded. The evaluation of the plaque and degree of stenosis were acquired using mode B, 7.5 MHz Doppler ultrasonography (USG). The follow-up was carried out for 36 months, with clinical, neurological, and USG exams repeated in a period of 6 to 8 months. Ninety-six individuals (48 women) completed the study with the presence of plaque, and 52 (26 women) with a degree of stenosis. RESULTS: As to the degree of stenosis, 25% of the patients had worsening, 69% remained stable and 6% improved. When only the presence or absence of plaque was considered, 20% showed worsening (plaque developed during follow-up), 7% improved (disappearance of plaque), and 73% remained stable. No differences were found between the male and female patients. CONCLUSION: These results confirm the dynamic characteristics of plaque. In asymptomatic individuals without specific treatment, spontaneous improvement may occur, however, rarely. These findings may contribute as an assessment criterion when a decision is to be made in high-risk patients.<br>OBJETIVO: avaliar a evolução espontânea da aterosclerose carotídea. MÉTODO: estudo prospectivo com pessoas de ambos os sexos, idade de 40 a 70 anos, sem sinais e sintomas de doença cerebrovascular e sem os principais fatores de risco para aterosclerose. Foram excluídos os doentes que estavam em uso ou os que, durante o período de acompanhamento, usaram medicações que potencialmente pudessem influir no curso espontâneo da aterosclerose. As avaliações da placa e do grau de estenose foram obtidas por ultrassonografia com Doppler (USG). Investigou-se separadamente a presença de placa e o grau de estenose. O seguimento foi feito por 36 meses, com exame clínico, neurológico e novo USG repetidos com intervalo de 6 a 8 meses. Completaram o estudo 96 indivíduos (48 mulheres) quanto à presença da placa e 52 (26 mulheres) quanto ao grau de estenose. RESULTADOS: Quanto ao grau de estenose, 25% dos indivíduos pioraram, 69% permaneceram estáveis e 6% melhoraram. Quando se considerou apenas a presença ou ausência da placa, 20% pioraram (desenvolveram placa durante o seguimento), 7% melhoraram (desaparecimento da placa) e 73% permaneceram estáveis. Não houve diferença em relação ao sexo. CONCLUSÃO: Estes resultados confirmam as características dinâmicas da placa. Em indivíduos assintomáticos e sem tratamento específico, a melhora espontânea ocorre, porém é pequena. Estes dados podem contribuir para auxiliar na tomada de uma decisão, em doentes de alto risco