Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS- to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio.

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

New insights on transcriptional responses of genes involved in carbon central metabolism, respiration and fermentation to low ATP levels in Escherichia coli.

Adenosine-5-triphosphate (ATP) plays a fundamental role in many cellular processes such as transport, central carbon metabolism, biosynthetic reactions, macromolecular synthesis, signal transduction and cellular division. In addition, the intracellular [ATP]/[ADP] ratio in Escherichia coli plays an important role in controlling the specific rates of growth (µ), glucose consumption (qGlc ) and oxygen uptake (qO2), as well as the transcriptome pattern in the cell, as was recently reported. In the current study, the energetic level (expressed as [ATP]/[ADP] ratio) was substantially reduced in E. coli strains by either over-expressing the F1 -ATPase activity (JMAGD(+)) or inactivating ATP synthase (JMat(-)). The physiological characterization of the wild-type JM101 strain and its derivative JMAGD(+) and JMatp(-) strains was conducted in bioreactors containing minimal medium with glucose. The inactivation of the atp operon and F1 -ATPase overexpression significantly diminished the energetic level and cAMP concentration in derivative strains. Relative transcription levels of 105 genes involved in glucose transport, glycolysis, tricarboxylic acid (TCA) cycle, fermentation, respiration, transcriptional regulators, transcription and genes involved in stress were determined by using qPCR. Interestingly, in the JMAGD(+) and JMatp(-) strains, having a reduced energetic level, many transcripts of glycolysis, TCA cycle and respiratory genes were down-regulated when compared to wild type JM101. The transcriptional responses, detected in the strains with reduced energetic level show down-regulation of genes involved in central carbon metabolism and respiration, these results are apposite to the observed trends of increased metabolic fluxes in glucose consumption, glycolysis, acetate synthesis, TCA cycle and respiration. Regulation mediated by CRP-cAMP complex may explain some observed transcriptional responses of TCA cycle genes, since cAMP concentration and crp transcript level were significant reduced in the JMatp(-) mutant. Therefore, the substantial reduction of [ATP]/[ADP] ratio had a relevant effect on the CRP-cAMP regulatory system (among other global regulators), which may trigger an extensive transcriptional response.

Cost-effective smoke-free multiunit housing media campaigns: connecting with local communities.

Presented are cost-effective paid media strategies to educate Californians to advocate for stronger smoke-free multiunit housing (SF-MUH) policies between 2006 and 2008. Included is a summary of general market and specific ethnic market costs that correspond to SF-MUH attitudes and home smoking bans. Statewide questionnaires indicated that half of the intended general market saw an antitobacco TV ad and half of the intended ethnic markets heard radio ads. Analyses indicated that it cost $0.67 and $0.78 per person to see Caution Tape and Apartment TV ads, respectively. Slightly higher per capita costs corresponded with positive attitudes toward SF-MUH: $0.87 for Caution Tape and $1.00 for Apartment. Lessons learned from this campaign included effectiveness of specific ads in ethnic markets, impact on SF-MUH work plan policy objectives, and the need for collaborations among state and local partners throughout the message development process.

Oral health status in pediatric patients with cerebral palsy fed by oral versus enteral route.

AIMS: Cerebral palsy (CP) is a chronic, nonprogressive disorder affecting movement, posture, and tone, caused by injuries in the central nervous system during the early stages of life. Patients with CP have swallowing disorders, which make oral feeding difficult and necessitate the use of external feeding devices. The objective of this research was to study the oral health status of pediatric patients affected with CP fed by either oral or enteral route. METHODS: A cross-sectional observational clinical study of the oral health of two groups of patients with CP, fed either orally or enterally (via percutaneous endoscopic gastrostomy, PEG). RESULTS: Patients fed by enteral route via PEG presented lower caries scores (DMFT: PEG: 1.09, non-PEG: 2.81) and higher percentages of supragingival dental calculus than the oral feeding group (PEG: 86%, non-PEG: 57.6%). CONCLUSION: Oral health status differed in pediatric patients with CP fed enterally via PEG and those fed orally. Specific preventive measures in both groups will be required to minimize the risk of complications.

Effects of Hand Configuration on the Grasping, Holding, and Placement of an Instrumented Object in Patients With Hemiparesis.

Objective: Limitations with manual dexterity are an important problem for patients suffering from hemiparesis post stroke. Sensorimotor deficits, compensatory strategies and the use of alternative grasping configurations may influence the efficiency of prehensile motor behavior. The aim of the present study is to examine how different grasp configurations affect patient ability to regulate both grip forces and object orientation when lifting, holding and placing an object. Methods: Twelve stroke patients with mild to moderate hemiparesis were recruited. Each was required to lift, hold and replace an instrumented object. Four different grasp configurations were tested on both the hemiparetic and less affected arms. Load cells from each of the 6 faces of the instrumented object and an integrated inertial measurement unit were used to extract data regarding the timing of unloading/loading phases, regulation of grip forces, and object orientation throughout the task. Results: Grip forces were greatest when using a palmar-digital grasp and lowest when using a top grasp. The time delay between peak acceleration and maximum grip force was also greatest for palmar-digital grasp and lowest for the top grasp. Use of the hemiparetic arm was associated with increased duration of the unloading phase and greater difficulty with maintaining the vertical orientation of the object at the transitions to object lifting and object placement. The occurrence of touch and push errors at the onset of grasp varied according to both grasp configuration and use of the hemiparetic arm. Conclusion: Stroke patients exhibit impairments in the scale and temporal precision of grip force adjustments and reduced ability to maintain object orientation with various grasp configurations using the hemiparetic arm. Nonetheless, the timing and magnitude of grip force adjustments may be facilitated using a top grasp configuration. Conversely, whole hand prehension strategies compound difficulties with grip force scaling and inhibit the synchrony of grasp onset and object release.

Detección y prevalencia de patógenos periodontales de una población con periodontitis crónica en Uruguay mediante metodología convencional y metagenómica / Detection and prevalence of periodontal pathogens in a Uruguayan population with chronic periodontitis using conventional methodology and metagenomics

Las enfermedades periodontales son un significativo problema mundial a nivel de salud humana. Décadas de investigaciones, evidencian que en la mayoría de los casos la periodontitis crónica es la más común, caracterizada por ser de evolución lenta, con formación de bolsas periodontales, posterior reabsorción del hueso alveolar, pérdida y destrucción de piezas dentarias y tejido óseo. Si bien se reconoce el origen multifactorial en el desarrollo de la periodontitis, es relevante la participación de la microbiota subgingival en la etiología de la enfermedad periodontal. Algunas de las especies bacterianas patógenas que han sido asociadas con el desarrollo de la enfermedad periodontal son Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, entre otras. En este estudio, nos propusimos investigar cuáles de éstas cinco especies estaban presentes en las bolsas periodontales de 51 pacientes uruguayos con periodontitis crónica. Para alcanzar éste objetivo se utilizó una técnica convencional microbiológica y metagenómica (multiplex-PCR). Los resultados de la técnica convencional microbiológica evidenciaron la presencia de A. actinomycetemcomitans (33%) y de bacterias negras pigmentadas anaerobias (100%) en las muestras. De los resultados obtenidos en la multiplex-PCR, se demostró que las especies de mayor prevalencia fueron F. nucleatum (100%), T. forsythia (92%) y P. gingivalis (88%). Por el contrario, las especies de menor prevalencia fueron P. intermedia (39%) y A. actinomycetemcomitans (33%)...

Periodontal diseases are a major problem in human health. Decades of research have shown that the most common disease is chronic periodontitis, characterized by a slow evolution with the formation of periodontal pockets, subsequent alveolar bone resorption, loss and destruction of teeth and bone tissue. While we know the multifactorial origin of the development of periodontitis, the participation of subgingival microbiota is relevant in the etiology of periodontal disease. Some pathogenic bacteria species that have been associated with the development of periodontal disease are Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, among others. In this work we studied which of these five species were present in the periodontal pockets of 51 Uruguayan patients with chronic periodontitis. To achieve the results a conventional microbiological technique and metagenomics (multiplex-PCR) were used. The results of the microbiological conventional technique showed the presence of A. actinomycetemcomitans (33%) and black pigmented anaerobic bacteria (100%) in the samples. From the results obtained in the multiplex-PCR we saw that the most prevalent species were F. nucleatum (100%), T. forsythia (92%) and P. gingivalis (88%). In contrast, lower prevalence species were P. intermedia (39%) and A. actinomycetecomitans...(33%)

Roles of Saccharomyces cerevisiae RAD17 and CHK1 checkpoint genes in the repair of double-strand breaks in cycling cells.

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.