An Age-Wise Comparison of Human Airway Smooth Muscle Proliferative Capacity

We compared the proliferation of neonatal and adult airway smooth muscle cells (ASMC) with no/moderate lung disease, in glucose- (energy production by glycolysis) or glucose-free medium (ATP production from mitochondrial oxidative phosphorylations only), in response to 10% fetal calf serum (FCS) and PDGF-AA. In the presence of glucose, cell counts were significantly greater in neonatal vs. adult ASMC. Similarly, neonatal ASMC DNA synthesis in 10% FCS and PDGF-AA, and [Ca²⁺]i responses in the presence of histamine were significantly enhanced vs. adults. In glucose-free medium, cell proliferation was preserved in neonatal cells, unlike in adult cells, with concomitant increased porin (an indicator of mitochondrial activity) protein expression. Compared to adults, stimulated neonatal human ASMC are in a rapid and robust proliferative phase and have the capacity to respond disproportionately under abnormal environmental conditions, through increased mitochondrial biogenesis and altered calcium homeostasis.     

An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.     

The ABC’s of Suicide Risk Assessment: Applying a Tripartite Approach to Individual Evaluations

There is considerable need for accurate suicide risk assessment for clinical, screening, and research purposes. This study applied the tripartite affect-behavior-cognition theory, the suicidal barometer model, classical test theory, and item response theory (IRT), to develop a brief self-report measure of suicide risk that is theoretically-grounded, reliable and valid. An initial survey (n = 359) employed an iterative process to an item pool, resulting in the six-item Suicidal Affect-Behavior-Cognition Scale (SABCS). Three additional studies tested the SABCS and a highly endorsed comparison measure. Studies included two online surveys (Ns = 1007, and 713), and one prospective clinical survey (n = 72; Time 2, n = 54). Factor analyses demonstrated SABCS construct validity through unidimensionality. Internal reliability was high (α = .86-.93, split-half = .90-.94)). The scale was predictive of future suicidal behaviors and suicidality (r = .68, .73, respectively), showed convergent validity, and the SABCS-4 demonstrated clinically relevant sensitivity to change. IRT analyses revealed the SABCS captured more information than the comparison measure, and better defined participants at low, moderate, and high risk. The SABCS is the first suicide risk measure to demonstrate no differential item functioning by sex, age, or ethnicity. In all comparisons, the SABCS showed incremental improvements over a highly endorsed scale through stronger predictive ability, reliability, and other properties. The SABCS is in the public domain, with this publication, and is suitable for clinical evaluations, public screening, and research.     

Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR

We used a strategy based on long PCR (polymerase chain reaction) for detection and characterization of mitochondrial DNA (mtDNA) rearrangements in two patients with clinical signs suggesting Pearson syndrome and Kearns-Sayre syndrome (KSS), respectively, and one patient with myopathic symptoms of unidentified origin. Mitochondrial DNA rearrangements were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantitative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a common 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues tested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that was present in the form of deletions as well as duplications. In the patient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearranged mtDNA populations in skeletal muscle, a previously described 7-kb deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be the cause of the myopathic symptoms as they were present at low levels (10–40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA rearrangements in three patients. Received: 10 March 1997 / Accepted: 20 May 1997