Breath-by-breath measurement of the volume displaced by diaphragm motion.

To develop an accurate method to measure the volume displaced by diaphragm motion (DeltaVdi) breath by breath, we compared DeltaVdi measured by a previously evaluated biplanar radiographic method (Singh B, Eastwood PR, and Finucane KE. J Appl Physiol 91: 1913-1923, 2001) at several lung volumes during vital capacity inspirations in 10 healthy and nine hyperinflated subjects with 1) DeltaVdi measured from the same chest X-rays by two previously described uniplanar methods (Petroll WM, Knight H, and Rochester DF. J Appl Physiol 69: 2175-2182, 1990; Verschakelen JA, Deschepper K, and Demendts M. J Appl Physiol 72: 1536-1540, 1992) and a proposed method that considered actual cross-sectional shape of the rib cage and spinal volume (DeltaVdi(S)); and 2) DeltaVdi(S) measured by lateral fluoroscopy in the same 10 healthy subjects. Relative to biplanar DeltaVdi, DeltaVdi(S) values from lateral chest X-rays and fluoroscopy were not different, whereas DeltaVdi values of Petroll et al. and Verschakelen et al. were increased by (means +/- SD) 1.98 +/- 1.59 and 1.16 +/- 0.82 liters, respectively (both P < 0.001). During quiet breathing, DeltaVdi(S) by lateral fluoroscopy was 66 +/- 16% of tidal volume and similar to that between functional residual capacity and one-half inspiratory capacity by the biplanar radiographic method. We conclude that accurate breath-by-breath measurements of DeltaVdi can be made by using lateral fluoroscopy.     

Linkage of Paget Disease of Bone to a Novel Region on Human Chromosome 18q23

Paget disease of bone (PDB) is characterized by increased osteoclast activity and localized abnormal bone remodeling. PDB has a significant genetic component, with evidence of linkage to chromosomes 6p21.3 (PDB1) and 18q21-22 (PDB2) in some pedigrees. There is evidence of genetic heterogeneity, with other pedigrees showing negative linkage to these regions. TNFRSF11A, a gene that is essential for osteoclast formation and that encodes receptor activator of nuclear factor-kappa B (RANK), has been mapped to the PDB2 region. TNFRSF11A mutations that segregate in pedigrees with either familial expansile osteolysis or familial PDB have been identified; however, linkage studies and mutation screening have excluded the involvement of RANK in the majority of patients with PDB. We have excluded linkage, both to PDB1 and to PDB2, in a large multigenerational pedigree with multiple family members affected by PDB. We have conducted a genomewide scan of this pedigree, followed by fine mapping and multipoint analysis in regions of interest. The peak two-point LOD scores from the genomewide scan were 2.75, at D7S507, and 1.76, at D18S70. Multipoint and haplotype analysis of markers flanking D7S507 did not support linkage to this region. Haplotype analysis of markers flanking D18S70 demonstrated a haplotype segregating with PDB in a large subpedigree. This subpedigree had a significantly lower age at diagnosis than the rest of the pedigree (51.2+/-8.5 vs. 64.2+/-9.7 years; P=.0012). Linkage analysis of this subpedigree demonstrated a peak two-point LOD score of 4.23, at marker D18S1390 (straight theta=0), and a peak multipoint LOD score of 4.71, at marker D18S70. Our data are consistent with genetic heterogeneity within the pedigree and indicate that 18q23 harbors a novel susceptibility gene for PDB.     

Basal Body-Associated Nucleation Center for the Centrin-Based Cortical Cytoskeletal Network in Paramecium

The infraciliary lattice, a contractile cortical cytoskeletal network of Paramecium, is composed of a small number of polypeptides including centrins. Its overall pattern reflects a hierarchy of structural complexity, from assembly and bundling of microfilaments to formation of polygonal meshes arranged in a continuous network subtending the whole cell surface, with local differentiations in the shape and size of the meshes. To analyse how the geometry of this complex network is generated and maintained, we have taken two approaches. Firstly, using monoclonal antibodies raised against the purified network, we have shown that all the component polypeptides colocalize, in agreement with previous biochemical data indicating that the infraciliary lattice is formed of large complexes comprising all the component polypeptides. Secondly, by taking advantage of different experimental conditions leading to disassembly of the network, we have followed its reassembly. Cytological analysis of the process revealed 1) that the network regrows exclusively from specific infraciliary lattice organizing centers (ICLOC), precisely localized near each basal body and, 2) that the global organization is not precisely controlled by genetic information but by the basal body pattern. Finally, slight ultrastuctural differences between reassembled and control lattices suggest that the organization of the filament bundles is partly templated by that of the preexisting ones.     

The viral oncoprotein LMP1 exploits TRADD for signaling by masking its apoptotic activity

The tumor necrosis factor (TNF)-receptor 1–associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-κB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-κB kinase β (IKKβ). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1′s TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-κB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function.     

Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10³ CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.     

Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi

An Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation has been cloned and sequenced. This gene codes for a periplasmic protein with disulphide isomerase activity that has 69% identity and 94% similarity with the E. coli DsbA protein. An E. chrysanthemi dsbA-uidA fusion mutant has been constructed. dsbA expression seems to be constitutive. This mutant has multiple phenotypes resulting from the absence of disulphide bond formation in periplasmic and secreted proteins. Pectate lyases and the cellulase EGZ are rapidly degraded in the periplasm of the dsbA mutant. E. chrysanthemi synthesizes another periplasmic protein with disulphide isomerase activity, namely DsbC. The dsbC gene introduced on a multicopy plasmid in a dsbA mutant was only partially able to restore EGZ secretion, indicating that even if DsbA and DsbC possess disulphide oxydoreductase activity, they are not completely interchangeable. Moreover, pectate lyases expressed in an E. coli dsbA mutant were very instable but their stability was unaffected in a dsbC mutant. These results indicate that DsbA and DsbC could have different substrate specificities.     

Introducing quality measures in an academic research consortium: Lessons and recommendation from implementing an ad hoc quality management system for organ model research

Lessons from implementing quality control systems in an academic research consortium to improve Good Scientific Practice and reproducibility. Subject Categories: Microbiology, Virology & Host Pathogen Interaction, Science Policy & Publishing

Low reproducibility rates within biomedical research negatively impact productivity and translation. One promising approach to enhance the transfer of robust results from preclinical research into clinically relevant and transferable data is the systematic implementation of quality measures in daily laboratory routines.

Although many universities expect their scientists to adhere to GSPs, they often neither systematically support, nor monitor the quality of their research activities.

Today''s fast‐evolving research environment needs effective quality measures to ensure reproducibility and data integrity (Macleod et al, ²⁰¹⁴; Begley et al, ²⁰¹⁵; Begley & Ioannidis, ²⁰¹⁵; Baker, ²⁰¹⁶). Academic research institutions and laboratories may be as committed to good scientific practices (GSPs) as their counterparts in the biotech and pharmaceutical industry but operate largely without clearly defined standards (Bespalov et al, ²⁰²¹; Emmerich et al, ²⁰²¹). Although many universities expect their scientists to adhere to GSPs, they often neither systematically support, nor monitor the quality of their research activities. Peer review of publications is still regarded as the primary validation of quality control in academic research. However, reviewers only assess work after it has been performed—often over years—and interventions in the experimental process are thus no longer possible.The reasons for the lack of dedicated quality management (QM) implementations in academic laboratories include an anticipated overload of regulatory tasks that could negatively affect productivity, concerns about the loss of scientific freedom, and importantly, limited resources in academia and academic funding schemes.     

Differential expression and tumor necrosis factor‐mediated regulation of TNFRSF11b/osteoprotegerin production by human melanomas

Tumors escape host immune responses, in part, through the release of immunomodulatory factors and decoy receptors into their microenvironment. Several cancers express surface‐bound and soluble members of the tumor necrosis factor (TNF) receptor superfamily, including TNFRSF11b/osteoprotegerin (OPG). In its physiologic role, OPG regulates bone remodeling through competition for osteoclast‐activating cytokines and protects newly forming bone from T cell‐mediated apoptosis. In multiple tumor types, OPG production is associated with an aggressive phenotype and increased metastasis to bone, but no study has examined OPG production in human metastatic melanoma. We demonstrate that a significant proportion of human metastatic melanomas constitutively produces OPG through a mechanism governed by membrane‐bound TNF‐α signaling through TNF receptor 1 (TNFR1). These observations both define a specific mechanism that regulates melanoma production of OPG and establish a new molecular target for the therapeutic regulation of OPG.     

Characterization of Metabolic,Diffusion, and Perfusion Properties in GBM: Contrast-Enhancing versus Non-Enhancing Tumor

BACKGROUND: Although the contrast-enhancing (CE) lesion on T₁-weighted MR images is widely used as a surrogate for glioblastoma (GBM), there are also non-enhancing regions of infiltrative tumor within the T₂-weighted lesion, which elude radiologic detection. Because non-enhancing GBM (Enh?) challenges clinical patient management as latent disease, this study sought to characterize ex vivo metabolic profiles from Enh? and CE GBM (Enh+) samples, alongside histological and in vivo MR parameters, to assist in defining criteria for estimating total tumor burden. Methods: Fifty-six patients with newly diagnosed GBM received a multi-parametric pre-surgical MR examination. Targets for obtaining image-guided tissue samples were defined based on in vivo parameters that were suspicious for tumor. The actual location from where tissue samples were obtained was recorded, and half of each sample was analyzed for histopathology while the other half was scanned using HR-MAS spectroscopy. Results: The Enh+ and Enh? tumor samples demonstrated comparable mitotic activity, but also significant heterogeneity in microvascular morphology. Ex vivo spectroscopic parameters indicated similar levels of total choline and N-acetylaspartate between these contrast-based radiographic subtypes of GBM, and characteristic differences in the levels of myo-inositol, creatine/phosphocreatine, and phosphoethanolamine. Analysis of in vivo parameters at the sample locations were consistent with histological and ex vivo metabolic data. CONCLUSIONS: The similarity between ex vivo levels of choline and NAA, and between in vivo levels of choline, NAA and nADC in Enh+ and Enh? tumor, indicate that these parameters can be used in defining non-invasive metrics of total tumor burden for patients with GBM.     

Preclinical Pharmacokinetic Studies of the Tritium Labelled D-Enantiomeric Peptide D3 Developed for the Treatment of Alzheimer′s Disease

Targeting toxic amyloid beta (Aβ) oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aβ1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aβ oligomers and has proven therapeutic potential in transgenic Alzheimer´s disease animal models. However, there is little information on the pharmacokinetic behaviour of D-enantiomeric peptides in general. Therefore, we conducted experiments with the tritium labelled D-peptide D3 (³H-D3) in mice with different administration routes to study its distribution in liver, kidney, brain, plasma and gastrointestinal tract, as well as its bioavailability by i.p. and p.o. administration. In addition, we investigated the metabolic stability in liver microsomes, mouse plasma, brain, liver and kidney homogenates, and estimated the plasma protein binding. Based on its high stability and long biological half-life, our pharmacokinetic results support the therapeutic potential of D-peptides in general, with D3 being a new promising drug candidate for Alzheimer´s disease treatment.