Exploring the Boundaries for In Vitro-In Vivo Extrapolation: Use of Isolated Rat Hepatocytes in Co-culture and Impact of Albumin Binding Properties in the Prediction of Clearance of Various Drug Types.

Prediction of hepatic clearance of drugs (via uptake or metabolism) from in vitro systems continues to be problematic, particularly when plasma protein binding is high. The following work explores simultaneous assessment of both clearance processes, focusing on a commercial hepatocyte-fibroblast co-culture system (HµREL) over a 24-hour period using six probe drugs (ranging in metabolic and transporter clearance and low-to-high plasma protein binding). A rat hepatocyte co-culture assay was established using drug depletion (measuring both medium and total concentrations) and cell uptake kinetic analysis, both in the presence and absence of plasma protein (1% bovine serum albumin). Secretion of endogenous albumin was monitored as a marker of viability, and this reached 0.004% in incubations (at a rate similar to in vivo synthesis). Binding to stromal cells was substantial and required appropriate correction factors. Drug concentration-time courses were analyzed both by conventional methods and a mechanistic cell model prior to in vivo extrapolation. Clearance assayed by drug depletion in conventional suspended rat hepatocytes provided a benchmark to evaluate co-culture value. Addition of albumin appeared to improve predictions for some compounds (where fraction unbound in the medium is less than 0.1); however, for high-binding drugs, albumin significantly limited quantification and thus predictions. Overall, these results highlight ongoing challenges concerning in vitro hepatocyte system complexity and limitations of practical expediency. Considering this, more reliable measurement of hepatically cleared compounds seems possible through judicious use of available hepatocyte systems, including co-culture systems, as described herein; this would include those compounds with low metabolic turnover but high active uptake clearance. SIGNIFICANCE STATEMENT: Co-culture systems offer a more advanced tool than standard hepatocytes, with the ability to be cultured for longer periods of time, yet their potential as an in vitro tool has not been extensively assessed. We evaluate the strengths and limitations of the HµREL system using six drugs representing various metabolic and transporter-mediated clearance pathways with various degrees of albumin binding. Studies in the presence/absence of albumin allow in vitro-in vivo extrapolation and a framework to maximize their utility.

Neonatal Group B Streptococcal Infection in Australia: A Case-control Study.

BACKGROUND: To determine maternal and neonatal risk factors for, and incidence of, neonatal early-onset group B streptococcus (EOGBS) and late-onset (LOGBS) infection in South Australia (SA) and the Northern Territory (NT). METHODS: A case-control study with 2:1 matched controls to cases. The study included tertiary hospitals in South Australia and the Northern Territory, Australia. Retrospective data were collected from a 16-year epoch (2000-2015). RESULTS: Of a total of 188 clinically suspected or confirmed cases, 139 were confirmed, of which 56.1% (n = 78) were EOGBS and 43.9% (n = 61) were LOGBS. The incidence of clinically suspected and confirmed cases of EOGBS was 0.26/1000 live births in SA and 0.73/1000 live births in the NT, and the incidence of confirmed cases was 0.19/1000 for SA and 0.36/1000 for the NT. The incidence of clinically suspected or confirmed LOGBS was 0.18/1000 live births in SA and 0.16/1000 for the NT. The majority of infants with GBS presented with sepsis, pneumonia, or meningitis. Developmental delay was the most commonly recorded long-term complication at 1 year old. Risk factors for EOGBS included maternal GBS carriage, previous fetal death, identifying as Aboriginal and/or Torres Strait Islander, and maternal fever in labor/chorioamnionitis. CONCLUSIONS: GBS remains a leading cause of neonatal morbidity and mortality. Adding previous fetal death to GBS screening guidelines would improve GBS prevention. The introduction of maternal GBS vaccination programs should be guided by country-specific disease epidemiology.

Healthcare practitioners' experiences of breaking bad news: A critical interpretative meta synthesis.

OBJECTIVE: To provide an overview of healthcare practitioners' experiences of breaking bad news. METHOD: Interpretative meta synthesis of 14 qualitative papers identified through a systematic search. Data were extracted and constructs elicited via reciprocal translational analysis. RESULTS: Findings comprised four inter-connected themes: (1) Discomfort, particularly difficult emotional and physical responses to breaking bad news; (2) Relational distress, connected to attachment and identification with patients; (3) Inadequate clinician, indicating the fear and rumination about the possibility of getting it wrong and the subsequent self-blame; and (4) Only human, highlighting a culture of invulnerability where practitioner self-care is deprioritised. CONCLUSIONS: Breaking bad news provokes significant distress for practitioners, particularly where they experience strong attachment to or identification with a patient. Breaking bad news was understood as an isolating experience and staff's non-disclosure of difficulties appeared to sustain a culture of invulnerability in which admission of distress was not encouraged. PRACTICE IMPLICATIONS: Given the adverse impacts and potential for practitioner burnout, organisations should acknowledge ramifications and offer time for staff to process the delivery of bad news, promote a culture of collective care and provide space for reflective practice.

Epidemiology of early and late-onset neonatal sepsis in an Australian regional special care nursery with a high proportion of Aboriginal and Torres Strait Islander births.

AIM: To describe the incidence and aetiology of early and late-onset neonatal sepsis and compare rates in Aboriginal and Torres Strait Islander infants against non-Indigenous infants in the Top End of the Northern Territory. METHODS: This was a retrospective case series of infants with positive blood or cerebrospinal fluid cultures at Royal Darwin Hospital between 2012 and 2016. Cultures from infants during initial hospital admission up to 120 days of age were included for analysis. Demographic, clinical, laboratory and treatment data were collected from medical records. Published definitions of sepsis and criteria for organism pathogenicity and were used to determine cases of sepsis. RESULTS: There were 52 episodes of sepsis in 45 infants. There were eight cases of early onset sepsis, with an incidence of 0.51 per 1000 live births. The incidence was similar for Aboriginal and non-Indigenous infants. The case fatality rate was 25%. Late-onset sepsis occurred in 44 cases, comprising 1.3% of all infants admitted to the special care nursery. Coagulase-negative Staphylococcus was the most frequently cultured organism. Case fatality rate was 11%. Aboriginal and Torres Strait Islander infants had a five-time higher risk of late-onset sepsis compared with non-Indigenous infants; however, their increased risk was not independent of other sepsis risk factors of low rates and prematurity. CONCLUSIONS: The incidence of culture-confirmed early and late-onset sepsis was low, but case fatality was high. Bacteraemia is an important contributor to neonatal and infant mortality in our setting.

Single-View Echocardiography by Nonexpert Practitioners to Detect Rheumatic Heart Disease: A Prospective Study of Diagnostic Accuracy.

BACKGROUND: Echocardiographic screening can detect asymptomatic cases of rheumatic heart disease (RHD), facilitating access to treatment. Barriers to implementation of echocardiographic screening include the requirement for expensive equipment and expert practitioners. We aimed to evaluate the diagnostic accuracy of an abbreviated echocardiographic screening protocol (single parasternal-long-axis view with a sweep of the heart) performed by briefly trained, nonexpert practitioners using handheld ultrasound devices. METHODS: Participants aged 5 to 20 years in Timor-Leste and the Northern Territory of Australia had 2 echocardiograms: one performed by an expert echocardiographer using a GE Vivid I or Vivid Q portable ultrasound device (reference test), and one performed by a nonexpert practitioner using a GE Vscan handheld ultrasound device (index test). The accuracy of the index test, compared with the reference test, for identifying cases with definite or borderline RHD was determined. RESULTS: There were 3111 enrolled participants; 2573 had both an index test and reference test. Median age was 12 years (interquartile range, 10-15); 58.2% were female. Proportion with definite or borderline RHD was 5.52% (95% CI, 4.70-6.47); proportion with definite RHD was 3.23% (95% CI, 2.61-3.98). Compared with the reference test, sensitivity of the index test for definite or borderline RHD was 70.4% (95% CI, 62.2-77.8), specificity was 78.1% (95% CI, 76.4-79.8). CONCLUSIONS: Nonexpert practitioners can be trained to perform single parasternal-long-axis view with a sweep of the heart echocardiography. However, the specificity and sensitivity are inadequate for echocardiographic screening. Improved training for nonexpert practitioners should be investigated.

Integrator-Dependent and Allosteric/Intrinsic Mechanisms Ensure Efficient Termination of snRNA Transcription.

Many RNA polymerases terminate transcription using allosteric/intrinsic mechanisms, whereby protein alterations or nucleotide sequences promote their release from DNA. RNA polymerase II (Pol II) is somewhat different based on its behavior at protein-coding genes where termination additionally requires endoribonucleolytic cleavage and subsequent 5'→3' exoribonuclease activity. The Pol-II-transcribed small nuclear RNAs (snRNAs) also undergo endoribonucleolytic cleavage by the Integrator complex, which promotes their transcriptional termination. Here, we confirm the involvement of Integrator but show that Integrator-independent processes can terminate snRNA transcription both in its absence and naturally. This is often associated with exosome degradation of snRNA precursors that long-read sequencing analysis reveals as frequently terminating at T-runs located downstream of some snRNAs. This finding suggests a unifying vulnerability of RNA polymerases to such sequences given their well-known roles in terminating Pol III and bacterial RNA polymerase.

The first 2 months of COVID-19 contact tracing in the Northern Territory of Australia, March-April 2020.

The Northern Territory (NT) Centre for Disease Control (CDC) undertook contact tracing of all notified cases of coronavirus disease 2019 (COVID-19) within the Territory. There were 28 cases of COVID-19 notified in the NT between 1 March and 30 April 2020. In total 527 people were identified as close contacts over the same period; 493 were successfully contacted; 445 were located in the NT and were subsequently quarantined and monitored for disease symptoms daily for 14 days after contact with a confirmed COVID-19 case. Of these 445 close contacts, 4 tested positive for COVID-19 after developing symptoms; 2/46 contacts who were cruise ship passengers (4.3%, 95% CI 0.5-14.8%) and 2/51 household contacts (3.9%, 95% CI 0.5-13.5%). None of the 326 aircraft passengers or 4 healthcare workers who were being monitored in the NT as close contacts became cases.

Utilising Magnetically Isolated Lysosomes for Direct Quantification of Intralysosomal Drug Concentrations by LC-MS/MS Analysis: An Investigatory Study With Imipramine.

Lysosomes are acidic intracellular organelles that can extensively sequester basic lipophilic drugs due to pH and membrane partitioning, and therefore may significantly influence subcellular drug concentrations. Current in vitro methods for lysosomal drug sequestration evaluation typically lack the ability to accurately and sensitively quantify drug concentrations directly within the lysosome. In the current study, magnetic lysosomal isolation was used in the lysosome rich rat NR8383 cell line and combined with LC-MS/MS analysis to quantify intralysosomal concentrations and lysosomal partitioning (KpLysosome) values of imipramine. The purity of the isolated lysosomes was validated by enzymatic and electron microscopy analysis. Lysosomal imipramine accumulation was explored using 2 methods: addition of imipramine to cells followed by lysosomal isolation (Method 1), and direct addition of imipramine to isolated lysosomes (Method 2). This work highlighted that both experimental buffers and ATP influence intralysosomal drug concentrations, and non-specific drug binding and re-distribution limits the use of Method 1. Method 2 may benefit future lysosomal drug accumulation studies, as imipramine demonstrated high KpLysosome values (3500), comparable to in silico predictions. This study reports a novel method for the direct quantification of intralysosomal drug concentrations that has the ability to be adapted to other cell types.

A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes.

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.

C9orf72 expansion within astrocytes reduces metabolic flexibility in amyotrophic lateral sclerosis.

It is important to understand how the disease process affects the metabolic pathways in amyotrophic lateral sclerosis and whether these pathways can be manipulated to ameliorate disease progression. To analyse the basis of the metabolic defect in amyotrophic lateral sclerosis we used a phenotypic metabolic profiling approach. Using fibroblasts and reprogrammed induced astrocytes from C9orf72 and sporadic amyotrophic lateral sclerosis cases we measured the production rate of reduced nicotinamide adenine dinucleotides (NADH) from 91 potential energy substrates simultaneously. Our screening approach identified that C9orf72 and sporadic amyotrophic lateral sclerosis induced astrocytes have distinct metabolic profiles compared to controls and displayed a loss of metabolic flexibility that was not observed in fibroblast models. This loss of metabolic flexibility, involving defects in adenosine, fructose and glycogen metabolism, as well as disruptions in the membrane transport of mitochondrial specific energy substrates, contributed to increased starvation induced toxicity in C9orf72 induced astrocytes. A reduction in glycogen metabolism was attributed to loss of glycogen phosphorylase and phosphoglucomutase at the protein level in both C9orf72 induced astrocytes and induced neurons. In addition, we found alterations in the levels of fructose metabolism enzymes and a reduction in the methylglyoxal removal enzyme GLO1 in both C9orf72 and sporadic models of disease. Our data show that metabolic flexibility is important in the CNS in times of bioenergetic stress.

Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control.

Cell-based studies of human ribonucleases traditionally rely on methods that deplete proteins slowly. We engineered cells in which the 3'→5' exoribonucleases of the exosome complex, DIS3 and EXOSC10, can be rapidly eliminated to assess their immediate roles in nuclear RNA biology. The loss of DIS3 has the greatest impact, causing the substantial accumulation of thousands of transcripts within 60 min. These transcripts include enhancer RNAs, promoter upstream transcripts (PROMPTs), and products of premature cleavage and polyadenylation (PCPA). These transcripts are unaffected by the rapid loss of EXOSC10, suggesting that they are rarely targeted to it. More direct detection of EXOSC10-bound transcripts revealed its substrates to prominently include short 3' extended ribosomal and small nucleolar RNAs. Finally, the 5'→3' exoribonuclease, XRN2, has little activity on exosome substrates, but its elimination uncovers different mechanisms for the early termination of transcription from protein-coding gene promoters.

Astrocyte adenosine deaminase loss increases motor neuron toxicity in amyotrophic lateral sclerosis.

As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.

Lon recognition of the replication initiator DnaA requires a bipartite degron.

DnaA initiates chromosome replication in bacteria. In Caulobacter crescentus, the Lon protease degrades DnaA to coordinate replication with nutrient availability and to halt the cell cycle during acute stress. Here, we characterize the mechanism of DnaA recognition by Lon. We find that the folded state of DnaA appears crucial for its degradation, in contrast to the well-known role of Lon in degrading misfolded proteins. We fail to identify a single degradation motif (degron) sufficient for DnaA degradation, rather we show that both the ATPase domain and a species-specific N-terminal motif are important for productive Lon degradation of full-length DnaA. Mutations in either of these determinants disrupt DnaA degradation in vitro and in vivo. However, analysis of truncation products reveals that appending other extensions to the ATPase domain is sufficient to trigger degradation, suggesting plasticity in Lon recognition. Our final working model is that Lon engages DnaA through at least two elements, one of which anchors DnaA to Lon and the other acting as an initiation site for degradation.

In Vitro and in Silico Tools To Assess Extent of Cellular Uptake and Lysosomal Sequestration of Respiratory Drugs in Human Alveolar Macrophages.

Accumulation of respiratory drugs in human alveolar macrophages (AMs) has not been extensively studied in vitro and in silico despite its potential impact on therapeutic efficacy and/or occurrence of phospholipidosis. The current study aims to characterize the accumulation and subcellular distribution of drugs with respiratory indication in human AMs and to develop an in silico mechanistic AM model to predict lysosomal accumulation of investigated drugs. The data set included 9 drugs previously investigated in rat AM cell line NR8383. Cell-to-unbound medium concentration ratio (Kp,cell) of all drugs (5 µM) was determined to assess the magnitude of intracellular accumulation. The extent of lysosomal sequestration in freshly isolated human AMs from multiple donors (n = 5) was investigated for clarithromycin and imipramine (positive control) using an indirect in vitro method (±20 mM ammonium chloride, NH4Cl). The AM cell parameters and drug physicochemical data were collated to develop an in silico mechanistic AM model. Three in silico models differing in their description of drug membrane partitioning were evaluated; model (1) relied on octanol-water partitioning of drugs, model (2) used in vitro data to account for this process, and model (3) predicted membrane partitioning by incorporating AM phospholipid fractions. In vitro Kp,cell ranged >200-fold for respiratory drugs, with the highest accumulation seen for clarithromycin. A good agreement in Kp,cell was observed between human AMs and NR8383 (2.45-fold bias), highlighting NR8383 as a potentially useful in vitro surrogate tool to characterize drug accumulation in AMs. The mean Kp,cell of clarithromycin (81, CV = 51%) and imipramine (963, CV = 54%) were reduced in the presence of NH4Cl by up to 67% and 81%, respectively, suggesting substantial contribution of lysosomal sequestration and intracellular binding in the accumulation of these drugs in human AMs. The in vitro data showed variability in drug accumulation between individual human AM donors due to possible differences in lysosomal abundance, volume, and phospholipid content, which may have important clinical implications. Consideration of drug-acidic phospholipid interactions significantly improved the performance of the in silico models; use of in vitro Kp,cell obtained in the presence of NH4Cl as a surrogate for membrane partitioning (model (2)) captured the variability in clarithromycin and imipramine Kp,cell observed in vitro and showed the best ability to predict correctly positive and negative lysosomotropic properties. The developed mechanistic AM model represents a useful in silico tool to predict lysosomal and cellular drug concentrations based on drug physicochemical data and system specific properties, with potential application to other cell types.

ClpAP is an auxiliary protease for DnaA degradation in Caulobacter crescentus.

The Clp family of proteases is responsible for controlling both stress responses and normal growth. In Caulobacter crescentus, the ClpXP protease is essential and drives cell cycle progression through adaptor-mediated degradation. By contrast, the physiological role for the ClpAP protease is less well understood with only minor growth defects previously reported for ΔclpA cells. Here, we show that ClpAP plays an important role in controlling chromosome content and cell fitness during extended growth. Cells lacking ClpA accumulate aberrant numbers of chromosomes upon prolonged growth suggesting a defect in replication control. Levels of the replication initiator DnaA are elevated in ΔclpA cells and degradation of DnaA is more rapid in cells lacking the ClpA inhibitor ClpS. Consistent with this observation, ClpAP degrades DnaA in vitro while ClpS inhibits this degradation. In cells lacking Lon, the protease previously shown to degrade DnaA in Caulobacter, ClpA overexpression rescues defects in fitness and restores degradation of DnaA. Finally, we show that cells lacking ClpA are particularly sensitive to inappropriate increases in DnaA activity. Our work demonstrates an unexpected effect of ClpAP in directly regulating replication through degradation of DnaA and expands the functional role of ClpAP in Caulobacter.

Validating trial-based functional analyses in mainstream primary school classrooms.

There is growing evidence to support the use of trial-based functional analyses, particularly in classroom settings. However, there currently are no evaluations of this procedure with typically developing children. Furthermore, it is possible that refinements may be needed to adapt trial-based analyses to mainstream classrooms. This study was designed to expand the trial-based functional analysis literature by implementing the procedure in 2 mainstream primary school classrooms and validating the analysis through comparison of multiple treatment options, including some that were not indicated by the functional analysis. We also extended the procedure by including a peer-attention condition and obtaining data from teachers regarding the feasibility of the procedures. For all participants, functional analysis results helped to identify effective treatments. Furthermore, relative effects among treatments were accurately predicted by the functional analysis outcomes. Teachers reported that they understood the logic of functional analysis and found both analysis and treatment procedures to be easy and effective.

Mec1 is one of multiple kinases that prime the Mcm2-7 helicase for phosphorylation by Cdc7.

Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms that modulate origin firing and replication fork assembly during cell cycle progression.

Pause-2-Play: a pilot schoolbased obesity prevention program / Pause-2-Play: um programa piloto escolar de prevenção de obesidade

OBJECTIVES: Pause-2-Play is an obesity prevention program targeting screen-related sedentary behaviours and increasing physical activity among elementary school students. The program consisted of a Behavioural Modification Curriculum and a Health Promoting Afterschool Program. This pilot study reports program feasibility, practicability, and impact. METHODS: the 12-week pilot program was implemented with 32 grade five and six students. Program feasibility and practicability were assessed using a qualitative approach. Intervention effects were assessed by comparing pre-post changes in BMI, body composition, fitness scores, screen time, and cognitive variables related to screening viewing behaviours. RESULTS: Pause-2-Play was perceived as a useful, fun program with numerous benefits including: children trying new snacks, feeling fitter and better about one's own body shape, and becoming more aware of a healthy lifestyle. The intervention resulted in a statistically significant reduction in percent body fat and an increase in fat-free mass index in overweight children; a decrease in waist circumference and an increase in fat-free mass index were observed in normal weight children. The intervention also statistically improved fitness scores in both normal weight and overweight children. CONCLUSIONS: Pause-2-Play was feasible, practical, and favourably changed body composition and fitness level.

OBJETIVOS: Pause-2-Play é um programa de prevenção da obesidade direcionado aos comportamentos sedentários relacionados ao uso de monitores de computador e televisores, visando promover a atividade física entre estudantes de escolas de educação básica. O programa trata-se de um currículo de modificação comportamental e um programa extracurricular de promoção de saúde. Este estudo-piloto relata sobre a viabilidade, praticidade e impacto do programa. MÉTODOS: o programa de doze semanas foi implementado com 32 alunos escolares do quinto e sexto grau. A viabilidade e a praticidade do programa foram avaliadas a partir de uma abordagem qualitativa. Os efeitos da intervenção foram avaliados por meio de comparação de mudanças de IMC pré e pós, composição corporal, escores de capacidade física, tempo passado diante um monitor ou televisor, e variáveis cognitivos relacionados aos comportamentos que envolvem o uso de monitor ou televisor. RESULTADOS: percebeu-se que Pause-2-Play foi um programa útil e lúdico cujos vários benefícios incluíam: o fato das crianças ter experimentado novos alimentos, uma sensação de melhor forma física e uma auto-imagem da forma do corpo mais positiva, e uma conscientização aumentada em relação a estilos de vida saudáveis. A intervenção resultou numa redução estatisticamente significativa na porcentagem de gordura corporal e um aumento no índice de massa livre de gordura em crianças portadoras de sobrepeso; um decréscimo na circunferência da cintura e um aumento no índice de massa livre de gordura foram observados em crianças eutróficas. Além disso, a intervenção levou a uma melhoria nos escores de capacidade física tanto entre as crianças eutróficas como entre as portadoras de sobrepeso. CONCLUSÕES: Pause-2-Play mostrou se viável e praticável e mudou a composição corporal e os níveis de capacidade física de forma favorável.

Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation.

The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2-7 replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase preferentially targets chromatin-associated Mcm2-7 complexes that are assembled on the DNA during prereplicative complex (pre-RC) formation. Here we address the mechanisms that control the specificity of DDK action. We show that incorporation of Mcm2-7 into the pre-RC increased the level and changes the specificity of DDK phosphorylation of this complex. In the context of the pre-RC, DDK preferentially targets a conformationally distinct subpopulation of Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent manner. Importantly, we find that DDK association with and phosphorylation of origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. Our findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the origin-bound Mcm2-7 complexes that will drive replication fork movement during S phase and suggest new mechanisms to regulate origin activity.

Chronic post-ischemia pain (CPIP): a novel animal model of complex regional pain syndrome-type I (CRPS-I; reflex sympathetic dystrophy) produced by prolonged hindpaw ischemia and reperfusion in the rat.

A neuropathic-like pain syndrome was produced in rats following prolonged hindpaw ischemia and reperfusion, creating an animal model of complex regional pain syndrome-Type I (CRPS-I; reflex sympathetic dystrophy) that we call chronic post-ischemia pain (CPIP). The method involves placing a tourniquet (a tight fitting O-ring) on one hindlimb of an anesthetized rat just proximal to the ankle joint for 3 h, and removing it to allow reperfusion prior to termination of the anesthesia. Rats exhibit hyperemia and edema/plasma extravasation of the ischemic hindpaw for a period of 2-4 h after reperfusion. Hyperalgesia to noxious mechanical stimulation (pin prick) and cold (acetone exposure), as well as mechanical allodynia to innocuous mechanical stimulation (von Frey hairs), are evident in the affected hindpaw as early as 8 h after reperfusion, and extend for at least 4 weeks in approximately 70% of the rats. The rats also exhibit spontaneous pain behaviors (hindpaw shaking, licking and favoring), and spread of hyperalgesia/allodynia to the uninjured contralateral hindpaw. Light-microscopic examination of the tibial nerve taken from the region just proximal to the tourniquet reveals no signs of nerve damage. Consistent with the hypothesis that the generation of free radicals may be partly responsible for CRPS-I and CPIP, two free radical scavengers, N-acetyl-L-cysteine (NAC) and 4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl (Tempol), were able to reduce signs of mechanical allodynia in this model.