Transcriptional Bursting and Co-bursting Regulation by Steroid Hormone Release Pattern and Transcription Factor Mobility.

Genes are transcribed in a discontinuous pattern referred to as RNA bursting, but the mechanisms regulating this process are unclear. Although many physiological signals, including glucocorticoid hormones, are pulsatile, the effects of transient stimulation on bursting are unknown. Here we characterize RNA synthesis from single-copy glucocorticoid receptor (GR)-regulated transcription sites (TSs) under pulsed (ultradian) and constant hormone stimulation. In contrast to constant stimulation, pulsed stimulation induces restricted bursting centered around the hormonal pulse. Moreover, we demonstrate that transcription factor (TF) nuclear mobility determines burst duration, whereas its bound fraction determines burst frequency. Using 3D tracking of TSs, we directly correlate TF binding and RNA synthesis at a specific promoter. Finally, we uncover a striking co-bursting pattern between TSs located at proximal and distal positions in the nucleus. Together, our data reveal a dynamic interplay between TF mobility and RNA bursting that is responsive to stimuli strength, type, modality, and duration.

Echocardiographic image collection and evaluation in infants with CHD: lessons learned from the imaging core lab for the Residual Lesion Score study.

Many factors affect patient outcome after congenital heart surgery, including the complexity of the heart disease, pre-operative status, patient specific factors (prematurity, nutritional status and/or presence of comorbid conditions or genetic syndromes), and post-operative residual lesions. The Residual Lesion Score is a novel tool for assessing whether specific residual cardiac lesions after surgery have a measurable impact on outcome. The goal is to understand which residual lesions can be tolerated and which should be addressed prior to leaving the operating room. The Residual Lesion Score study is a large multicentre prospective study designed to evaluate the association of Residual Lesion Score to outcomes in infants undergoing surgery for CHD. This Pediatric Heart Network and National Heart, Lung, and Blood Institute-funded study prospectively enrolled 1,149 infants undergoing 5 different congenital cardiac surgical repairs at 17 surgical centres. Given the contribution of echocardiographic measurements in assigning the Residual Lesion Score, the Residual Lesion Score study made use of a centralised core lab in addition to site review of all data. The data collection plan was designed with the added goal of collecting image quality information in a way that would permit us to improve our understanding of the reproducibility, variability, and feasibility of the echocardiographic measurements being made. There were significant challenges along the way, including the coordination, de-identification, storage, and interpretation of very large quantities of imaging data. This necessitated the development of new infrastructure and technology, as well as use of novel statistical methods. The study was successfully completed, but the size and complexity of the population being studied and the data being extracted required more technologic and human resources than expected which impacted the length and cost of conducting the study. This paper outlines the process of designing and executing this complex protocol, some of the barriers to implementation and lessons to be considered in the design of future studies.

Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting.

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

Degraded RNA from Human Anterior Cruciate Ligaments Yields Valid Gene Expression Profiles.

Correlating gene expression patterns with biomechanical properties of connective tissues provides insights into the molecular processes underlying the tissue growth and repair. Cadaveric specimens such as human knees are widely considered suitable for biomechanical studies, but their usefulness for gene expression experiments is potentially limited by the unavoidable, nuclease-mediated degradation of RNA. Here, we tested whether valid gene expression profiles can be obtained using degraded RNA from human anterior cruciate ligaments (ACLs). Human ACL RNA (N = 6) degraded in vitro by limited ribonuclease digestion resemble highly degraded RNA isolated from cadaveric tissue. PCR threshold cycle (Ct) values for 90 transcripts (84 extracellular matrix, 6 housekeeping) in degraded RNAs variably ranged higher than values obtained from their corresponding non-degraded RNAs, reflecting both the expected loss of target templates in the degraded preparations as well as differences in the extent of degradation. Relative Ct values obtained for mRNAs in degraded preparations strongly correlated with the corresponding levels in non-degraded RNA, both for each ACL as well as for the pooled results from all six ACLs. Nuclease-mediated degradation produced similar, strongly correlated losses of housekeeping and non-housekeeping gene mRNAs. RNA degraded in situ yielded comparable results, confirming that in vitro digestion effectively modeled degradation by endogenous ribonucleases in frozen and thawed ACL. We conclude that, contrary to conventional wisdom, PCR-based expression analyses can yield valid mRNA profiles even from RNA preparations that are more than 90% degraded, such as those obtained from connective tissues subjected to biomechanical studies. Furthermore, legitimate quantitative comparisons between variably degraded tissues can be made by normalizing data to appropriate housekeeping transcripts.

Evaluation of Left Ventricular Outflow Gradients During Staged Exercise Stress Echocardiography Helps Differentiate Pediatric Patients With Hypertrophic Cardiomyopathy From Athletes and Normal Subjects.

BACKGROUND: Elevated left ventricular outflow tract (LVOT) gradients during exercise can occur in patients with hypertrophic cardiomyopathy (HCM) as well as in athletes and normal controls. The authors' staged exercise protocol calls for imaging at rest and during each stage of exercise to evaluate the mechanism of LVOT obstruction at each stage. They investigated whether this staged approach helps differentiate HCM from athletes and normal controls. METHODS: They reviewed pediatric exercise stress echocardiograms completed between January 2009 and October 2017 at their center and identified those with gene-positive HCM, athlete's heart, and normal controls. Children with inducible obstruction (those with no LVOT gradient at rest who developed a LVOT peak gradient > 25 mm Hg during exercise) were included. LVOT peak gradient, velocity time integral, acceleration time, and deceleration time were measured at rest, submaximal stages, and peak exercise. RESULTS: Compared with athletes, HCM patients had significantly higher LVOT peak gradients at rest (P = .019), stage 1 of exercise (P = .002), and peak exercise (P = .051), as well as a significantly higher change in LVOT peak gradient from rest to stage 1 (P = .016) and from rest to peak (P = .038). The acceleration time/deceleration time ratio of the LVOT Doppler was significantly lower in HCM patients compared with normal controls at peak exercise. CONCLUSIONS: The HCM patients who develop elevated LVOT gradients at peak exercise typically manifest early obstruction in the submaximal stages of exercise, which helps to differentiate them from athletes and normal controls.

Ribonucleoprotein purification and characterization using RNA Mango.

The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.

Evaluation of the Tibial Tubercle to Posterior Cruciate Ligament Distance in a Pediatric Patient Population.

BACKGROUND: Evaluation of distal extensor mechanism alignment continues to evolve in children with patella instability. Prior studies support the use of the tibial tubercle to trochlear groove (TT-TG) distance but limitations exist for this measurement including: changes in the TT-TG distance with knee flexion, difficulty with finding the deepest part of a dysplastic trochlea, and limitations regarding identification of the site of the anatomic abnormality. The tibial tubercle-posterior cruciate ligament (TT-PCL) distance has been introduced as an alternative measure to address the shortcomings in the TT-TG distance by quantifying the position of the TT independent of the trochlea and with respect to the tibia only. The objectives of this study were to (1) confirm that TT-PCL measurements in the pediatric population are reliable and reproducible; (2) determine whether normal TT-PCL distance changes with age; and (3) compare TT-PCL distances in patients with and without patellar instability to assess its utility in the workup of pediatric patellar instability. METHODS: All knee magnetic resonance imaging performed for patients from birth to 15.9 years of age at our institution between December 2004 and February 2012 were retrospectively collected (total 566). Eighty-two patients had patellar instability and 484 patients did not have patellar instability. Two magnetic resonance imaging reviewers measured TT-PCL distance on T2-weighted axial images in a blinded manner. Intraobserver and interobserver agreement was measured. Correlation between TT-PCL distance and age as well as group differences between mean TT-PCL distances was evaluated. RESULTS: Intraobserver and interobserver agreement was excellent (0.93) and very good (0.80), respectively. The mean TT-PCL distance was 20.1 mm with a range of 5.8 to 32.1 mm. The mean age was 12.6 years with a range of 0.8 to 15.9 years. The average TT-PCL distance was 21 mm for the instability group and 19.9 mm for the control group. TT-PCL distance increased significantly as subject age increased; however, there was no significant measurement difference shown between the patellar instability group and the control group. CONCLUSIONS: TT-PCL distance increased with age in the pediatric population but did not correlate with recurrent patella instability in this pediatric cohort. LEVEL OF EVIDENCE: Level III-diagnostic.

Lung function measures following simulated wildland firefighter exposures.

Across the world, biomass smoke is a major source of air pollution and is linked with a variety of adverse health effects. This is particularly true in the western U.S. where wood smoke from wildland forest fires are a significant source of PM2.5. Wildland firefighters are impacted as they experience elevated PM2.5 concentrations over extended periods of time, often occurring during physical exertion. Various epidemiological studies have investigated wood smoke impacts on human health, including occupational field exposures experienced by wildland firefighters. As there are numerous challenges in carrying out these field studies, having the ability to research the potential health impacts to this occupational cohort in a controlled setting would provide important information that could be translated to the field setting. To this end, we have carried out a simulated wildland firefighter exposure study in a wood smoke inhalation facility. Utilizing a randomized crossover trial design, we exposed 10 participants once to clean filtered-air, 250 µg/m3, and 500 µg/m3 wood stove-generated wood smoke PM2.5. Participants exercised on a treadmill at an absolute intensity designed to simulate wildland firefighting for 1.5 hr. In addition to measured PM2.5 smoke concentrations, mean levels of CO2, CO, and % relative humidity were continuously monitored and recorded and were representative of occupational "real-world" exposures. Pulmonary function was measured at three time points: before, immediately after, and 1-hr post-exposure. Although there were some reductions in FVC, FEV1, and FVC:FEV1 measures, results of the spirometry testing did not show significant changes in lung function. The development of this wood smoke inhalational facility provides a platform to further address unique research questions related to wood smoke exposures and associated adverse health effects.

The stoichiometry of scaffold complexes in living neurons - DLC2 functions as a dimerization engine for GKAP.

Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.

Diets Containing Shiitake Mushroom Reduce Serum Lipids and Serum Lipophilic Antioxidant Capacity in Rats.

BACKGROUND: We previously reported that dietary intake of shiitake mushroom (SM; Lentinus edodes) decreased serum concentrations of polar lipids in male rats. OBJECTIVE: This study evaluated the dietary effects of SM on serum cholesterol-related and serum antioxidant indexes in rats of both sexes. METHODS: Sprague-Dawley rats [38 dams and their offspring (20 males and 20 females/diet)] were fed diets containing 0 (control), 1%, 4%, or 10% (wt:wt) SM powder from gestation day 4 through to postnatal day (PND) 126. Biochemical indexes were monitored during the midgrowth phase (PNDs 50-66). RESULTS: The food consumption by offspring fed the control diet and diets supplemented with SM was not different when measured on PND 65. However, the 4% and 10% SM diets resulted in male rats with 7% lower body weights than those of the other 2 groups on PND 66. SM consumption dose-dependently decreased the concentrations of lipidemia-related factors in sera, irrespective of sex. At PND 50, serum concentrations of total cholesterol, HDL cholesterol, and non-HDL cholesterol in SM-fed male and female rats were generally lower (3-27%) than those in the corresponding control groups. Consumption of the 10% SM diet resulted in significantly decreased (55%) serum triglyceride concentrations relative to the control groups for both sexes. The 10% SM diet elicited a 62% reduction of serum leptin concentrations in females but not in males, and this same diet increased serum insulin (137%) and decreased serum glucose (15%) in males compared with controls. Serum lipophilic antioxidant capacity in males and females fed SM diets was generally lower (31-86%) than that in the control groups. CONCLUSION: SM decreased the concentrations of lipidemia-related factors in rat sera irrespective of sex. The SM-elicited reduction of lipophilic antioxidant capacity irrespective of sex may reflect a lower pro-oxidative state and, hence, improved metabolic profile.

Tissue Doppler imaging during exercise stress echocardiography demonstrates a mechanism for impaired exercise performance in children with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is associated with decreased exercise tolerance in children, presumably due to diastolic dysfunction. Modern imaging techniques to assess myocardial function during active exercise have not been applied to this population. We hypothesized that impaired contractile reserve, as assessed by tissue Doppler imaging (TDI) and strain, contributes to reduced exercise capacity in affected individuals. METHODS: Children (<18 years) with HCM and healthy age- and sex-matched controls were prospectively enrolled. Resting echocardiograms and staged upright cycle ergometry with simultaneous echocardiograms were performed. During exercise, left ventricular outflow tract (LVOT) gradients and color Doppler maps of apical four-chamber and parasternal short-axis views were obtained. Post processing of images was performed to obtain TDI velocities, and measurements of strain were attempted. Exercise parameters and staged TDI values were compared. RESULTS: The study population consisted of 58 subjects (22 with HCM and 36 controls). Patients with HCM had significantly higher peak LVOT gradients compared to controls at baseline and at each exercise stage. TDI revealed that diastolic function, as assessed by E' velocities at septal and lateral mitral annuli, normalized with exercise in HCM patients. Further, systolic function (S' velocity) of HCM patients at rest was normal but failed to augment normally at peak exercise. CONCLUSIONS: Children with HCM have decreased TDI velocities at rest. With exercise, they may increase their E' velocities but fail to augment S' velocities, demonstrating decreased contractile reserve. In the patient with suspected HCM but equivocal findings, exercise TDI assessment may complement the diagnostic evaluation.

A new measurement protocol to differentiate sources of halitosis.

OBJECTIVE: Three sources of halitosis exist, potentially in any combination: mouth, nasal cavity or alveolar breath. There has been no universally accepted protocol which differentiates and quantifies each odour source separately. In this study a new gas measurement protocol is described and tested to determine whether each odour source can be separately detected without contamination. MATERIALS AND METHODS: Ninety healthy volunteers were divided into three groups. Hydrogen sulphide (H2S), volatile organic compounds (VOCs) and hydrogen (H2) were artificially generated in the mouth, nose and pulmonary alveoli, respectively. VOC, ammonia (NH3), sulphur dioxide (SO2), H2S and H2 gas readings from mouth, nose and alveolar air were measured and compared. Measurements were taken before and during gas generation. RESULTS: Contamination of nasal air (2.8%) and alveolar air (5.0%) by oral H2S; alveolar air (2.06%) and oral air (4%) by nasal organic gas; nasal air (18.43%) and oral air (9.42%) by alveolar H2 was calculated. CONCLUSION: The results demonstrated that artificially generated oral H2S nasal VOC and alveolar H2 can be individually quantified. This gas measurement protocol can be used diagnostically or to gauge response to therapy in any medical or dental setting.

Phospholipid/aromatic thiol hybrid bilayers.

Gold-supported hybrid bilayers comprising phospholipids and alkanethiols have been found to be highly useful in biomembrane mimicking as well as biosensing ever since their introduction by Plant in 1993 (Plant, A. L. Langmuir 1993, 9, 2764-2767). Generalizing the mechanism (i.e., hydrophobic/hydrophobic interaction) that primarily drives bilayer formation, we report here that such a bilayer structure can also be successfully obtained when aromatic thiols are employed in place of alkanethiols. Four aromatic thiols were studied here (thiophenol, 2-naphthalene thiol, biphenyl-4-thiol, and diphenylenevinylene methanethiol), all affording reliable bilayer formation when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes were incubated with self-assembled monolayers of these thiols. Characterization of the resultant structures, using cyclic voltammetry, impedance analysis, and atomic force microscopy, confirms the bilayer formation. Significant differences in electrochemical blocking and mechanical characteristics of these new bilayers were identified in comparison to their alkanethiol counterparts. Taking advantage of these new features, we present a new scheme for the straightforward biorecognition of a lipolytic enzyme (phospholipase A2) using these phospholipid/aromatic thiol bilayers.

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states.

Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell-to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.

Female mice lacking p47phox have altered adipose tissue gene expression and are protected against high fat-induced obesity.

The current study was designed to determine if the NADPH-oxidase NOX2 plays a role in development of obesity after high fat feeding. Wild-type (WT) mice and mice lacking the essential cytosolic NOX2 system component p47(phox) (P47KO mice) were fed AIN-93G diets or high-fat diets (HFD) containing 45% fat and 0.5% cholesterol for 13 wk from weaning. Fat mass was increased to a similar degree by HFD in males of both genotypes (P < 0.05). However, female P47KO-HFD mice had no increase in adiposity or adipocyte size relative to female WT-HFD mice. Resistance to HFD-driven obesity in P47KO females was associated with increased expression of hepatic TFAM and UCP-2 mRNA, markers of mitochondrial number and uncoupling, and increased expression of hepatic mitochondrial respiratory complexes and whole body energy expenditure in response to HFD. Microarray analysis revealed significantly lower expression of mRNA encoding genes linked to energy metabolism, adipocyte differentiation (PPARγ), and fatty acid uptake (CD36, lipoprotein lipase), in fat pads from female P47KO-HFD mice compared with WT-HFD females. Moreover, differentiation of preadipocytes ex vivo was suppressed more by 17ß-estradiol in cells from P47KO compared with cells from WT females in conjunction with overexpression of mRNA for Pref-1 (P < 0.05). HFD mice of both sexes were resistant to the development of hyperglycemia and hepatic steatosis (P < 0.05) and had reduced serum triglycerides, leptin, and adiponectin relative to WT-HFD mice (P < 0.05). These data suggest that NOX2 is an important regulator of metabolic homeostasis and diet-induced obesity.

Comparison of how ambient PMc and PM2.5 influence the inflammatory potential.

Airborne particulate matter (PM) is one of the six criteria air pollutants currently regulated by the U.S. Environmental Protection Agency (EPA), with existing ambient standards for PM2.5 and PM10. Currently there are no health-based regulations for the size fraction between 2.5 and 10 µm, commonly known as the coarse fraction (PMc). The present study investigates current gaps in knowledge for PMc including exposure toxicity and PM ratios (PMc:PM2.5) in PM10. Throughout the world, all the three PM size fractions have been shown to be associated with adverse impacts. Recent studies have shown that PMc can be more detrimental to susceptible populations when directly compared to PM2.5, and that the PMc fraction in PM10 can account for the majority of the inflammatory response from PM10 exposure. In our studies we utilized a bone marrow-derived mouse macrophage in vitro system to compare the inflammatory potential of PMc, PM2.5 and mixtures of the two. The result was a linear increase in interleukin(IL) -1ß with increasing levels of exposure to winter and summer PMc, as compared to PM2.5, which exhibited logarithmic growth. Also, exposure to PM10 as a function of PM2.5 and PMc mass ratios showed that IL-1ß and TNF-α levels increased synergistically with a greater burden of PMc. Endotoxin content in the PM did not correlate with these results, suggesting that other activators in PMc are likely responsible for activating the NF-κB pathway and the inflammasome.

Calculating the risk benefit equation for aggressive treatment of non-convulsive status epilepticus.

OBJECTIVE: To address the question: does non-convulsive status epilepticus warrant the same aggressive treatment as convulsive status epilepticus? METHODS: We used a decision model to evaluate the risks and benefits of treating non-convulsive status epilepticus with intravenous anesthetics and ICU-level aggressive care. We investigated how the decision to use aggressive versus non-aggressive management for non-convulsive status epilepticus impacts expected patient outcome for four etiologies: absence epilepsy, discontinued antiepileptic drugs, intraparenchymal hemorrhage, and hypoxic ischemic encephalopathy. Each etiology was defined by distinct values for five key parameters: baseline mortality rate of the inciting etiology; efficacy of non-aggressive treatment in gaining control of seizures; the relative contribution of seizures to overall mortality; the degree of excess disability expected in the case of delayed seizure control; and the mortality risk of aggressive treatment. RESULTS: Non-aggressive treatment was favored for etiologies with low morbidity and mortality such as absence epilepsy and discontinued antiepileptic drugs. The risk of aggressive treatment was only warranted in etiologies where there was significant risk of seizure-induced neurologic damage. In the case of post-anoxic status epilepticus, expected outcomes were poor regardless of the treatment chosen. The favored strategy in each case was determined by strong interactions of all five model parameters. CONCLUSIONS: Determination of the optimal management approach to non-convulsive status epilepticus is complex and is ultimately determined by the inciting etiology.

AM1-receptor-dependent protection by intermedin of human vascular and cardiac non-vascular cells from ischaemia-reperfusion injury.

Intermedin (IMD) protects rodent heart and vasculature from oxidative stress and ischaemia. Less is known about distribution of IMD and its receptors and the potential for similar protection in man. Expression of IMD and receptor components were studied in human aortic endothelium cells (HAECs), smooth muscle cells (HASMCs), cardiac microvascular endothelium cells (HMVECs) and fibroblasts (v-HCFs). Receptor subtype involvement in protection by IMD against injury by hydrogen peroxide (H2O2, 1 mmol l⁻¹) and simulated ischaemia and reperfusion were investigated using receptor component-specific siRNAs. IMD and CRLR, RAMP1, RAMP2 and RAMP3 were expressed in all cell types.When cells were treated with 1 nmol l⁻¹ IMD during exposure to 1 mmol l⁻¹ H2O2 for 4 h, viability was greater vs. H2O2 alone (P<0.05 for all cell types). Viabilities under 6 h simulated ischaemia differed (P<0.05) in the absence and presence of 1 nmol l⁻¹ IMD: HAECs 63% and 85%; HMVECs 51% and 68%; v-HCFs 42% and 96%. IMD 1 nmol l⁻¹ present throughout ischaemia (3 h) and reperfusion (1 h) attenuated injury (P<0.05): viabilities were 95%, 74% and 82% for HAECs, HMVECs and v-HCFs, respectively, relative to those in the absence of IMD (62%, 35%, 32%, respectively). When IMD 1 nmol l⁻¹ was present during reperfusion only, protection was still evident (P<0.05, 79%, 55%, 48%, respectively). Cytoskeletal disruption and protein carbonyl formation followed similar patterns. Pre-treatment (4 days) of HAECs with CRLR or RAMP2, but not RAMP1 or RAMP3, siRNAs abolished protection by IMD (1 nmol l⁻¹) against ischaemia-reperfusion injury. IMD protects human vascular and cardiac non-vascular cells from oxidative stress and ischaemia-reperfusion,predominantly via AM1 receptors.

Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex.

The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, fluorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer-dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer-dimer contacts. We conclude that inducer-dependent dimer-dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members.

Absolute quantification of gene expression in individual bacterial cells using two-photon fluctuation microscopy.

Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 µM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks.