Hemoglobin/O2 systems: mechanistic discrimination based on Ackers' model

The kinetics of the reaction of hemoglobin with molecular oxygen, in which rapid mixing is followed by a fast temperature jump, is numerically simulated. We use the system of Ackers (1998) which distinguishes four forms of bi-ligated hemoglobin. The data suggest the involvement of isomerization steps for bi- and triliganded hemoglobin. Our first model assumes a linear addition of oxygen with one path to and from each bi-ligated species. Our second model allows cross-overs between paths, as described by Ackers (1998). Our third model exploits the observation (Perrella et al., 1990) that two of the four bi-ligated forms are at low concentration. We explore whether these models can be distinguished experimentally. We find a narrow oxygen concentration range where Models 1 and 2 can be distinguished by rapid flow experiments. The distinction between Models 2 and 3 is larger in stopped flow experiments within a limited oxygen concentration range but not easily detectable in chemical relaxation following rapid flow. The detection of two special states of free hemoglobin may be possible at low oxygen concentration. However, the step reaction free enthalpy (or Gibbs free energy) values make it more likely that two special states are present in fully ligated hemoglobin.     

Identification of anti-repressor elements that confer high and stable protein production in mammalian cells

The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number-dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.     

Trends in Expanded-Spectrum Cephalosporin-Resistant Escherichia coli and Klebsiella pneumoniae among Dutch Clinical Isolates,from 2008 to 2012

We investigated time trends in extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from different patient settings in The Netherlands from 2008–2012. E. coli and K. pneumoniae isolates from blood and urine samples of patients > = 18 years were selected from the Dutch Infectious Disease Surveillance System-Antimicrobial Resistance (ISIS-AR) database. We used multivariable Poisson regression to study the rate per year of blood stream infections by susceptible and resistant isolates, and generalized estimating equation (GEE) log-binomial regression for trends in the proportion of extended-spectrum cephalosporin-resistant isolates. Susceptibility data of 197,513 E. coli and 38,244 K. pneumoniae isolates were included. The proportion of extended-spectrum cephalosporin-resistant E. coli and K. pneumoniae isolates from urine and blood samples increased in all patient settings, except for K. pneumoniae isolates from patients admitted to intensive care units. For K. pneumoniae, there was a different time trend between various patient groups (p<0.01), with a significantly higher increase in extended-spectrum cephalosporin-resistant isolates from patients attending a general practitioner than in isolates from hospitalized patients. For E. coli, the increasing time trends did not differ among different patient groups. This nationwide study shows a general increase in extended-spectrum cephalosporin-resistant E. coli and K. pneumoniae isolates. However, differences in trends between E. coli en K. pneumoniae underline the importance of E. coli as a community-pathogen and its subsequent influence on hospital resistance level, while for K. pneumoniae the level of resistance within the hospital seems less influenced by the resistance trends in the community.     

The efficacy of computer reminders on external quality assessment for point-of-care testing in Danish general practice: rationale and methodology for two randomized trials

Background

Effective provider-parent communication can improve childhood vaccination uptake and strengthen immunisation services in low- and middle-income countries (LMICs). Building capacity to improve communication strategies has been neglected. Rigorous research exists but is not readily found or applicable to LMICs, making it difficult for policy makers to use it to inform vaccination policies and practice. The aim of this project is to build research knowledge and capacity to use evidence-based strategies for improving communication about childhood vaccinations with parents and communities in LMICs.

Methods and design

This project is a mixed methods study with six sub-studies. In sub-study one, we will develop a systematic map of provider-parent communication interventions for childhood vaccinations by screening and extracting data from relevant literature. This map will inform sub-study two, in which we will develop a taxonomy of interventions to improve provider-parent communication around childhood vaccination. In sub-study three, the taxonomy will be populated with trial citations to create an evidence map, which will also identify how evidence is linked to communication barriers regarding vaccination. In the project's fourth sub-study, we will present the interventions map, taxonomy, and evidence map to international stakeholders to identify high-priority topics for systematic reviews of interventions to improve parent-provider communication for childhood vaccination. We will produce systematic reviews of the effects of high-priority interventions in the fifth sub-study. In the sixth and final sub-study of the project, evidence from the systematic reviews will be translated into accessible formats and messages for dissemination to LMICs.

Discussion

This project combines evidence mapping, conceptual and taxonomy development, priority setting, systematic reviews, and knowledge transfer. It will build and share concepts, terms, evidence, and resources to aid the development of communication strategies for effective vaccination programmes in LMICs.     

In vivo suppression of coding associated with bacteriophage-induced S-adenosylmethionine hydrolase

The methylation of ribosomal and transfer ribonucleic acid (RNA) synthesized after the induction of a hydrolase for S-adenosylmethionine by phage T3 infection is reducible to 50% of the methylation of RNA in uninfected cells. Hypomethylated ribosomal RNA is found in 70S particles that dissociate in 100 mum Mg(++) to yield only 30S and 50S subunits. By this criterion, the omitted methyl groups apparently are not required for ribosomal maturation or stability. The rate of production of alkaline phosphatase in a phosphatase amber mutant was examined after phage infection in the presence and in the absence of streptomycin to determine the effect on the translation process consequent to S-adenosyl-l-methionine (SAM) hydrolase induction. Significant increases in the rates of phosphatase production were found when ultraviolet-inactivated T3 or streptomycin was added. The effects were cumulative when the cells were treated with both bacteriophage and the drug. Ultraviolet-inactivated T7, a phage closely related to T3 but which does not produce the SAM hydrolase, did not enhance the rate of alkaline phosphatase production. We suggest that the production of SAM hydrolase affects the stability of the translation process by the observed hypomethylation or by mechanisms concerning polyamine metabolism.     

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.     

The association between low-grade inflammation, iron status and nucleic acid oxidation in the elderly

This study applied a case-control approach to investigate the association between low-grade inflammation, defined by high values within the normal range of C-reactive protein (CRP) and interleukin-6 (IL-6), and urinary markers of nucleic acid oxidation. No differences in excretion of urinary markers of nucleic acid oxidation between cases and controls were found and multivariable linear regression analysis showed no association between urinary markers of nucleic acid oxidation and inflammatory markers. Post-hoc multivariable linear regression analysis showed significant associations between nucleic acid oxidation and various iron status markers and especially a close relationship between nucleic acid oxidation and ferritin. This study shows no association between low-grade inflammation and urinary markers of nucleic acid oxidation in a population of elderly Italian people. The results suggest that low-grade inflammation only has a negligible impact on whole body nucleic acid oxidation, whereas iron status seems to be of great importance.     

Malaria in the Post-Partum Period; a Prospective Cohort Study

Background

Several studies have shown a prolonged or increased susceptibility to malaria in the post-partum period. A matched cohort study was conducted to evaluate prospectively the susceptibility to malaria of post-partum women in an area where P.falciparum and P.vivax are prevalent.

Methods

In an area of low seasonal malaria transmission on the Thai-Myanmar border pregnant women attending antenatal clinics were matched to a non-pregnant, non-post-partum control and followed up prospectively until 12 weeks after delivery.

Results

Post-partum women (n = 744) experienced significantly less P.falciparum episodes than controls (hazard ratio (HR) 0.39 (95%CI 0.21–0.72) p = 0.003) but significantly more P.vivax (HR 1.34 (1.05–1.72) p = 0.018). The reduced risk of falciparum malaria was accounted for by reduced exposure, whereas a history of P.vivax infection during pregnancy was a strong risk factor for P.vivax in post-partum women (HR 13.98 (9.13–21.41), p<0.001). After controlling for effect modification by history of P.vivax, post-partum women were not more susceptible to P.vivax than controls (HR: 0.33 (0.21–0.51), p<0.001). Genotyping of pre-and post-partum infections (n⊕ = ⊕10) showed that each post-partum P.falciparum was a newly acquired infection.

Conclusions

In this area of low seasonal malaria transmission post-partum women were less likely to develop falciparum malaria but more likely to develop vivax malaria than controls. This was explained by reduced risk of exposure and increased risk of relapse, respectively. There was no evidence for altered susceptibility to malaria in the post-partum period. The treatment of vivax malaria during and immediately after pregnancy needs to be improved.