Role of the Dinaric Karst (western Balkans) in shaping the phylogeographic structure of the threatened crayfish Austropotamobius torrentium

1. This study examines phylogeography and phylogeny of the threatened stone crayfish, Austropotamobius torrentium, in order to elucidate the role of the Dinaric Karst geology in shaping the evolutionary history and genetic diversity of aquatic fauna in the western Balkans. Mitochondrial 16S rRNA and COI genes were partially sequenced from 188 and 159 crayfish, respectively, sampled from 70 localities. Phylogenetic relationships were reconstructed using four methods of phylogenetic inference. Divergence times between phylogroups were estimated in a Bayesian framework, and their demographic history was examined using neutrality tests and mismatch distribution analysis. 2. Seven geographically localised phylogroups separated by pronounced genetic gaps were found. Five of them have a distribution range within the northern‐central Dinaric (NCD) region, while the remaining two include populations from the southern Balkans (SB) and central and south‐eastern Europe (CSE). The oldest divergence event separated two NCD lineages from the rest of populations in the Late Miocene or Early Pliocene. Divergences amongst the five NCD phylogroups and SB + CSE occurred in the Pliocene. The most recent split separated SB and CSE phylogroups during the Late Pliocene. For both genes, uncorrected pairwise divergences between most of the phylogroups (4.1–8.7% for COI and 1.6–4.8% for 16S rRNA) were of the same range as, or higher than, some of the interspecific distances previously reported for the genus Austropotamobius. 3. Geographically isolated and deeply divergent cryptic monophyletic phylogroups within A. torrentium in the NCD region arose in the course of intensification of Neotectonic movements during the Pliocene and the beginning of the Pleistocene and the development of karstification that has heavily fragmented the palaeohydrography of the area. The results confirm a gradual north–south expansion of stone crayfish during the pre‐Pleistocene that preceded the rapid northward post‐glacial re/colonisation of central Europe (CSE phylogroup) through the Danube drainage. 4. Austropotamobius torrentium comprises morphologically cryptic but molecularly distinct taxa. Considering the relatively small geographical areas they inhabit, the NCD phylogroups of stone crayfish should be given the highest conservation priority.     

Accuracy of pulse oximetry to estimate HbO2 fraction of total Hb during exercise

The accuracy of two pulse oximeters (Ohmeda 3700 and Biox IIa) was evaluated during cycle ergometer incremental exercise in 10 healthy subjects. The exercise protocol began at 30 W with the power output being increased 15 W.min-1 until volitional fatigue. Ear and finger probe pulse oximetry measurements of available hemoglobin (%Spo2) were compared with arterial oxyhemoglobin fraction of total hemoglobin (%HbO2) measured directly from arterial blood samples using a CO-oximeter. To provide a wide range of %HbO2 values, four subjects exercised under hypoxic conditions [inspired partial pressure of O2 (PIo2) = 107 Torr], while the remaining six subjects exercised under normoxic conditions (PIo2 = 150 Torr). Because carboxyhemoglobin (HbCO) or methemoglobin (MetHb) is not measured by pulse oximeters, %HbO2 was corrected for HbCO and MetHb and expressed as percent arterial O2 saturation of available Hb (%Sao2). Small and insignificant differences (P greater than 0.05) existed between SpO2 (all 3 instruments) and %SaO2 at the lowest work rate and the highest power output achieved. Regression analyses of %SpO2 vs. %SaO2 produced correlation coefficients of r = 0.82 [standard error of the estimate [(SEE) = 1.79], r = 0.89 (SEE = 1.48), and r = 0.93 (SEE = 1.14) for the Biox IIa, Ohmeda 3700 (ear), and the Ohmeda 3700 (finger) pulse oximeters, respectively. We conclude that pulse oximetry, within the above limits of accuracy, is useful in estimating %SaO2 during exercise in healthy subjects.     

Health Insurance Status as a Barrier to Ideal Cardiovascular Health for U.S. Adults: Data from the National Health and Nutrition Examination Survey (NHANES)

Background

Little is known about the association between cardiovascular (CV) health and health insurance status. We hypothesized that U.S. adults without health insurance coverage would have a lower likelihood of ideal cardiovascular health.

Methods and Results

Using National Health and Nutrition Examination Survey (NHANES) data from 2007–2010, we examined the relationship between health insurance status and ideal CV health in U.S. adults aged ≥19 years and <65 (N = 3304). Ideal CV health was defined by the American Heart Association (AHA) as the absence of clinically manifested CV disease and the simultaneous presence of 6–7 “ideal” CV health factors and behaviors. Logistic regression modeling was used to determine the relationship between health insurance status and the odds of ideal CV health. Of the U.S. adult population, 5.4% attained ideal CV health, and 23.5% were without health insurance coverage. Those without health insurance coverage were more likely to be young (p<0.0001), male (p<0.0001), non-white (p<0.0001), with less than a high school degree (p<0.0001), have a poverty-to-income ratio less than 1 (p<0.0001) and unemployed (p<0.0001) compared to those with coverage. Lack of health insurance coverage was associated with a lower likelihood of ideal CV health; however, this relationship was attenuated by socioeconomic status.

Conclusions

U.S. adults without health insurance coverage are less likely to have ideal CV health. Population-based strategies and interventions directed at the community-level may be one way to improve overall CV health and reach this at-risk group.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Mechanical strength of the cement-bone interface is greater in shear than in tension.

The objective of this study was to determine the relative mechanical properties of the cement-bone interface due to tensile or shear loading. Mechanical tests were performed on cement-bone specimens in tensile (n = 51) or shear (n = 55) test jigs under the displacement control at 1 mm/min until complete failure. Before testing, the quantity of bone interdigitated with the cement was determined and served as a covariate in the study. The apparent strength of the cement-bone interface was significantly higher (p < 0.0001) for the interface when loaded in shear (2.25 MPa) when compared to tensile loading (1.35 MPa). Significantly higher energies to failure (p < 0.0001) and displacement before failure (p < 0.01) were also determined for the shear specimens. The post-yield softening response was not different for the two test directions. The data obtained herein suggests that cement-bone interfaces with equal amounts of tensile and shear stress would be more likely to fail under tensile loading.     

Introduction of unnatural amino acids into proteins using expressed protein ligation

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.