Accumulation of advanced glycation end (AGEs) products in intensive care patients: an observational, prospective study

<p>Abstract</p> <p>Background</p> <p>Oxidative stress plays an important role in the course and eventual outcome in a majority of patients admitted to the intensive care unit (ICU). Markers to estimate oxidative stress are not readily available in a clinical setting. AGEs accumulation has been merely described in chronic conditions, but can also occur acutely due to oxidative stress. Since AGEs have emerged to be stable end products, these can be a marker of oxidative stress. Skin autofluorescence (AF) is a validated marker of tissue content of AGEs. We hypothesized that AGEs accumulate acutely in ICU patients.</p> <p>Methods</p> <p>We performed an observational prospective study in a medical surgical ICU in a university affiliated teaching hospital. All consecutively admitted ICU patients in a 2 month period were included. Skin AF was measured using an AGE reader in 35 consecutive ICU patients > 18 yrs. As a comparison, historical data of a control group (n = 231) were used. These were also used to calculate age-adjusted AF-levels (AF_adj). Values are expressed as median and interquartile range [P₂₅-P₇₅]. Differences between groups were tested by non parametric tests. P < 0.05 was considered statistically significant.</p> <p>Results</p> <p>AF_adjvalues were higher in ICU patients (0.33 [0.00 - 0.68]) than in controls (-0.07 [-0.29 - 0.24]; P < 0.001). No differences in skin AF_adjwere observed between acute or planned admissions, or presence of sepsis, nor was skin AF_adjrelated to severity of disease as estimated by APACHE-II score, length of ICU, hospital stay or mortality.</p> <p>Conclusion</p> <p>Acute AGE accumulation in ICU patients was shown in this study, although group size was small. This can possibly reflect oxidative stress in ICU patients. Further studies should reveal whether AGE-accumulation will be a useful parameter in ICU patients and whether skin AF has a predictive value for outcome, which was not shown in this small study.</p

AmrZ is a major determinant of c-di-GMP levels in Pseudomonas fluorescens F113

The transcriptional regulator AmrZ is a global regulatory protein conserved within the pseudomonads. AmrZ can act both as a positive and a negative regulator of gene expression, controlling many genes implicated in environmental adaption. Regulated traits include motility, iron homeostasis, exopolysaccharides production and the ability to form biofilms. In Pseudomonas fluorescens F113, an amrZ mutant presents a pleiotropic phenotype, showing increased swimming motility, decreased biofilm formation and very limited ability for competitive colonization of rhizosphere, its natural habitat. It also shows different colony morphology and binding of the dye Congo Red. The amrZ mutant presents severely reduced levels of the messenger molecule cyclic-di-GMP (c-di-GMP), which is consistent with the motility and biofilm formation phenotypes. Most of the genes encoding proteins with diguanylate cyclase (DGCs) or phosphodiesterase (PDEs) domains, implicated in c-di-GMP turnover in this bacterium, appear to be regulated by AmrZ. Phenotypic analysis of eight mutants in genes shown to be directly regulated by AmrZ and encoding c-di-GMP related enzymes, showed that seven of them were altered in motility and/or biofilm formation. The results presented here show that in P. fluorescens, AmrZ determines c-di-GMP levels through the regulation of a complex network of genes encoding DGCs and PDEs

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de-novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized

Dramatic pain relief and resolution of bone inflammation following pamidronate in 9 pediatric patients with persistent chronic recurrent multifocal osteomyelitis (CRMO)

<p>Abstract</p> <p>Background</p> <p>Chronic recurrent multifocal osteomyelitis (CRMO) is an inflammatory, non-infectious osteopathy that affects predominantly patients ≤ 18 years of age. There is no uniformly effective treatment. Our objective is to describe clinical, magnetic resonance imaging (MRI), and bone resorption response to intravenous pamidronate in pediatric CRMO.</p> <p>Methods</p> <p>We report our prospectively documented experience with all CRMO patients treated with pamidronate between 2003 and 2008 at a tertiary pediatric centre. Pamidronate was administered as intravenous cycles. The dose of pamidronate varied among subjects but was given as monthly to every 3 monthly cycles depending on the distance the patient lived from the infusion center. Maximum cumulative dose was ≤ 11.5 mg/kg/year. Pamidronate treatment was continued until resolution of MRI documented bone inflammation. Visual analog scale for pain (VAS) and bone resorption marker urine N-telopeptide/urine creatinine (uNTX/uCr) were measured at baseline, preceding each subsequent pamidronate treatment, at final follow-up, and/or at time of MRI confirmed CRMO flare. MRI of the affected site(s) was obtained at baseline, preceding every 2^ndtreatment, and with suspected CRMO recurrence.</p> <p>Results</p> <p>Nine patients (5 F: 4 M) were treated, with a median (range) age at treatment of 12.9 (4.5–16.3) years, and median (range) duration of symptoms of 18 (6–36) months. VAS decreased from 10/10 to 0–3/10 by the end of first 3–day treatment for all patients. The mean (range) time to complete MRI resolution of bone inflammation was 6.0 (2–12) months. The mean (confidence interval (CI)) baseline uNTX/uCr was 738.83 (CI 464.25, 1013.42)nmol/mmol/creatinine and the mean (CI) decrease from baseline to pamidronate discontinuation was 522.17 (CI 299.77, 744.56)nmol/mmol/creatinine. Median (range) of follow-up was 31.4 (24–54) months. Four patients had MRI confirmed CRMO recurrence, which responded to one pamidronate re-treatment. The mean (range) uNTX/uCr change as a monthly rate from the time of pamidronate discontinuation to flare was 9.41 (1.38–19.85)nmol/mmol/creatinine compared to -29.88 (-96.83–2.01)nmol/mmol/creatinine for patients who did not flare by the time of final follow-up.</p> <p>Conclusion</p> <p>Pamidronate resulted in resolution of pain and MRI documented inflammation in all patients. No patient flared while his/her uNTX/uCr remained suppressed. We propose that pamidronate is an effective second-line therapy in persistent CRMO.</p

Pseudomonas aeruginosa PAO1 Preferentially Grows as Aggregates in Liquid Batch Cultures and Disperses upon Starvation

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or ‘suspended biofilms’, by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10–400 µm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa

Blueprint for a minimal photoautotrophic cell: conserved and variable genes in Synechococcus elongatus PCC 7942

Background: Simpler biological systems should be easier to understand and to engineer towards pre-defined goals. One way to achieve biological simplicity is through genome minimization. Here we looked for genomic islands in the fresh water cyanobacteria Synechococcus elongatus PCC 7942 (genome size 2.7 Mb) that could be used as targets for deletion. We also looked for conserved genes that might be essential for cell survival.Results: By using a combination of methods we identified 170 xenologs, 136 ORFans and 1401 core genes in the genome of S. elongatus PCC 7942. These represent 6.5%, 5.2% and 53.6% of the annotated genes respectively. We considered that genes in genomic islands could be found if they showed a combination of: a) unusual G+C content; b) unusual phylogenetic similarity; and/or c) a small number of the highly iterated palindrome 1 (HIP1) motif plus an unusual codon usage. The origin of the largest genomic island by horizontal gene transfer (HGT) could be corroborated by lack of coverage among metagenomic sequences from a fresh water microbialite. Evidence is also presented that xenologous genes tend to cluster in operons. Interestingly, most genes coding for proteins with a diguanylate cyclase domain are predicted to be xenologs, suggesting a role for horizontal gene transfer in the evolution of Synechococcus sensory systems.Conclusions: Our estimates of genomic islands in PCC 7942 are larger than those predicted by other published methods like SIGI-HMM. Our results set a guide to non-essential genes in S. elongatus PCC 7942 indicating a path towards the engineering of a model photoautotrophic bacterial cell.Financial support was provided by grants BFU2009-12895-C02-01/BMC (Ministerio de Ciencia e Innovación, Spain), the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement number 212894 and Prometeo/2009/092 (Conselleria d’Educació, Generalitat Valenciana, Spain) to A. Moya. Work in the FdlC laboratory was supported by grants BFU2008-00995/BMC (Spanish Ministry of Education), RD06/0008/1012 (RETICS research network, Instituto de Salud Carlos III, Spanish Ministry of Health) and LSHM-CT- 2005_019023 (European VI Framework Program). Dr. González-Domenech was supported by grant from the University of Granada. LD, thanks to financial support from Facultad de Ciencias, Universidad Nacional Autónoma de México

Indicators of "Healthy Aging" in older women (65-69 years of age). A data-mining approach based on prediction of long-term survival

<p>Abstract</p> <p>Background</p> <p>Prediction of long-term survival in healthy adults requires recognition of features that serve as early indicators of successful aging. The aims of this study were to identify predictors of long-term survival in older women and to develop a multivariable model based upon longitudinal data from the Study of Osteoporotic Fractures (SOF).</p> <p>Methods</p> <p>We considered only the youngest subjects (<it>n </it>= 4,097) enrolled in the SOF cohort (65 to 69 years of age) and excluded older SOF subjects more likely to exhibit a "frail" phenotype. A total of 377 phenotypic measures were screened to determine which were of most value for prediction of long-term (19-year) survival. Prognostic capacity of individual predictors, and combinations of predictors, was evaluated using a cross-validation criterion with prediction accuracy assessed according to time-specific AUC statistics.</p> <p>Results</p> <p>Visual contrast sensitivity score was among the top 5 individual predictors relative to all 377 variables evaluated (mean AUC = 0.570). A 13-variable model with strong predictive performance was generated using a forward search strategy (mean AUC = 0.673). Variables within this model included a measure of physical function, smoking and diabetes status, self-reported health, contrast sensitivity, and functional status indices reflecting cumulative number of daily living impairments (HR ≥ 0.879 or RH ≤ 1.131; P < 0.001). We evaluated this model and show that it predicts long-term survival among subjects assigned differing causes of death (e.g., cancer, cardiovascular disease; P < 0.01). For an average follow-up time of 20 years, output from the model was associated with multiple outcomes among survivors, such as tests of cognitive function, geriatric depression, number of daily living impairments and grip strength (P < 0.03).</p> <p>Conclusions</p> <p>The multivariate model we developed characterizes a "healthy aging" phenotype based upon an integration of measures that together reflect multiple dimensions of an aging adult (65-69 years of age). Age-sensitive components of this model may be of value as biomarkers in human studies that evaluate anti-aging interventions. Our methodology could be applied to data from other longitudinal cohorts to generalize these findings, identify additional predictors of long-term survival, and to further develop the "healthy aging" concept.</p

Addison's disease symptoms - a cross sectional study in urban South Africa

BACKGROUND: Addison's disease is a potentially life-threatening disorder, and prompt diagnosis, and introduction of steroid replacement has resulted in near normal life-expectancy. There are limited data describing the clinical presentation of Addison's disease in South Africa. It is hypothesised that patients may present in advanced state of ill-health, compared to Western countries. Patients A national database of patients was compiled from primary care, referral centres and private practices. 148 patients were enrolled (97 white, 34 mixed ancestry, 5 Asian and 12 black). METHODS: Demographic and clinical data were elicited using questionnaires. Biochemical data were obtained from folder reviews and laboratory archived results. RESULTS: The majority of the cohort was women (62%). The median and inter-quartile age range (IQR) of patients at enrolment was 46.0 (32.0-61.0) years, with a wide range from 2.8-88.0 years. The median and IQR age at initial diagnosis was 34.0 (20.0-45.0) years (range 0.02-77.0) years, indicating that at the time of enrolment, the patients, on average, were diagnosed with Addison's disease 12 years previously. Hyperpigmentation was observed in 76%, nausea and vomiting occurred in more than 40%, and weight loss was noted in 25%. Loss of consciousness as a presenting feature was recorded in 20 %. with a 95% confidence interval [CI] of (14-28%) and shock occurred in 5% CI (1.5-8.5%). Case-finding was recorded at 3.1 per million. CONCLUSIONS: The usual constellation of hyperpigmentation, nausea, vomiting and weight loss suggests Addison's disease, but a significant proportion present with an advanced state of ill-health and Addisonian crises. A lower prevalence rate, compared to Western countries is suggested

Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

BACKGROUND: The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4. METHODOLOGY/PRINCIPAL FINDINGS: We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E(a)) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T(i)) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E(a) of 278 kJ/mol (66.5 kcal/mol), and a T(i) of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules. CONCLUSIONS/SIGNIFICANCE: Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors