Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results

Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.     

Mangrove-sponge associations: a possible role for tannins

A positive correlation between sponge coverage and tannin concentrations in prop roots of Rhizophora mangle L. has previously been reported. However, the ecological role of tannins within the mangrove sponge association remains speculative. This study investigated whether tannins play a role in sponge recruitment and assessed tannin and polyphenol production in R. mangle roots in response to sponge colonization. We demonstrated in a field experiment using artificial substrates with different tannin concentrations that tannins are positively involved in larval recruitment of the sponge Tedania ignis and that roots significantly enhanced tannin and polyphenolic content in response to natural and experimental sponge fouling. Differential recruitment in response to tannins may have been the result of a behavioral response in sponge larvae. It is also possible that tannins affected the structure of the fouling microbial biofilm on the artificial substrate, or tannins affected the post-settlement dynamics of sponge recruits. Elevations in concentrations of tannins and polyphenolic compounds upon coverage with sponges, combined with differential recruitment of T. ignis in response to differences in tannin concentrations, may indicate a positive feedback in recruitment. This may in part explain the typical heterogeneity in sponge coverage and community composition among roots.     

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation,Barrier Function,and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.     

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: Implications for a proteasome-to-autophagy switch

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS).     

Dynamic shear behavior of mandibular condylar cartilage is dependent on testing direction

Little information is available on the direction-dependency of shear behavior in mandibular condylar cartilage. Therefore, we tested the hypothesis that such a dependency of the dynamic shear properties is present in mandibular condylar cartilage. From each of 17 condyles, two cartilage-bone plugs were dissected and tested in a simple shear sandwich configuration under a compressive strain of 10%. Sinusoidal shear strain (frequency range: 0.01-10 Hz) was applied in the medio-lateral or antero-posterior direction with an amplitude of 1.0%, 2.0%, and 3.0%. The magnitudes of the dynamic shear moduli, as calculated from the resulting shear stress, were found to increase with applied frequency and the shear strain amplitude. The values |G*|, G' and G' for a medio-laterally applied shear were about 20-33% of those in the antero-posterior shear, although the loss tangent (elasticity/viscosity ratio) was almost the same. In conclusion, the present results clearly show the direction-dependent characteristic of the mandibular condylar cartilage in dynamic shear.     

Organisational Factors Induce Prolonged Emergency Department Length of Stay in Elderly Patients – A Retrospective Cohort Study

Study objective

To assess the association of patient and organisational factors with emergency department length of stay (ED-LOS) in elderly ED patients (226565 years old) and in younger patients (<65 years old).

Methods

A retrospective cohort study of internal medicine patients visiting the emergency department between September 1^st 2010 and August 31^st 2011 was performed. All emergency department visits by internal medicine patients 226565 years old and a random sample of internal medicine patients <65 years old were included. Organisational factors were defined as non-medical factors. ED-LOS is defined as the time between ED arrival and ED discharge or admission. Prolonged ED-LOS is defined as ≥75^th percentile of ED-LOS in the study population, which was 208 minutes.

Results

Data on 1782 emergency department visits by elderly patients and 597 emergency department visits by younger patients were analysed. Prolonged ED-LOS in elderly patients was associated with three organisational factors: >1 consultation during the emergency department visit (odds ratio (OR) 3.2, 95% confidence interval (CI) 2.3–4.3), a higher number of diagnostic tests (OR 1.2, 95% CI 1.16–1.33) and evaluation by a medical student or non-trainee resident compared with a medical specialist (OR 4.2, 95% CI 2.0–8.8 and OR 2.3, 95% CI 1.4–3.9). In younger patients, prolonged ED-LOS was associated with >1 consultation (OR 2.6, 95% CI 1.4–4.6). Factors associated with shorter ED-LOS were arrival during nights or weekends as well as a high urgency level in elderly patients and self-referral in younger patients.

Conclusion

Organisational factors, such as a higher number of consultations and tests in the emergency department and a lower seniority of the physician, were the main aspects associated with prolonged ED-LOS in elderly patients. Optimisation of the organisation and coordination of emergency care is important to accommodate the needs of the continuously growing number of elderly patients in a better way.