Spontaneous Unfolding and Refolding of FNIII Domains Assayed by Thiol Exchange

Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of

type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble

supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that

are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.

The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and

provide a comprehensive analysis of stability and unfolding/folding kinetics of each

domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.

A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate

constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we

use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.

Age model and sedimentation rate of core M174/KL11 (Table 1)

Stable isotope and faunal records from the central Red Sea show high-amplitude oscillations for the past 380,000 years. Positive delta18O anomalies indicate periods of significant salt buildup during periods of lowered sea level when water mass exchange with the Arabian Sea was reduced due to a reduced geometry of the Bab el Mandeb Strait. Salinities as high as 53 per mil and 55 per mil are inferred from pteropod and benthic foraminifera delta18O, respectively, for the last glacial maximum. During this period all planktonic foraminifera vanished from this part of the Red Sea. Environmental conditions improved rapidly after 13 ka as salinities decreased due to rising sea level. The foraminiferal fauna started to reappear and was fully reestablished between 9 ka and 8 ka. Spectral analysis of the planktonic delta18O record documents highest variance in the orbital eccentricity, obliquity, and precession bands, indicating a dominant influence of climatically - driven sea level change on environmental conditions in the Red Sea. Variance in the precession band is enhanced compared to the global mean marine climate record (SPECMAP), suggesting an additional influence of the Indian monsoon system on Red Sea climates.

Effect of Earnings Volatility on Cost of Debt: The case of Swedish Limited Companies

The paper empirically tests the relationship between earnings volatility and cost of debt with a sample of more than 77,000 Swedish limited companies over the period 2006 to 2013 observing more than 677,000 firm years. As called upon by many researchers recently that there is very limited evidence of the association between earnings volatility and cost of debt this paper contributes greatly to the existing literature of earnings quality and debt contracts, especially on the consequence of earnings quality in the debt market. Earnings volatility is a proxy used for earnings quality while cost of debt is a component of debt contract. After controlling for firms’ profitability, liquidity, solvency, cashflow volatility, accruals volatility, sales volatility, business risk, financial risk and size this paper studies the effect of earnings volatility measured by standard deviation of Earnings Before Interest, Taxes, Depreciation and Amortization (EBITDA) on Cost of Debt. Overall finding suggests that lenders in Sweden does take earnings volatility into consideration while determining cost of debt for borrowers. But a deeper analysis of various industries suggest earnings volatility is not consistently used by lenders across all the industries. Lenders in Sweden are rather more sensitive to borrowers’ financial risk across all the industries. It may also be stated that larger borrowers tend to secure loans at a lower interest rate, the results are consistent with majority of the industries. Swedish debt market appears to be well prepared for financial crises as the debt crisis seems to have no or little adverse effect borrowers’ cost of capital. This study is the only empirical evidence to study the association between earnings volatility and cost of debt. Prior indirect research suggests earnings volatility has a negative effect on cost debt (i.e. an increase in earnings volatility will increase firm’s cost of debt). Our direct evidence from the Swedish debt market is consistent for some industries including media, real estate activities, transportation & warehousing, and other consumer services.

Rate of Belowground Carbon Allocation Differs with Successional Habit of Two Afromontane Trees

Background: Anthropogenic disturbance of old-growth tropical forests increases the abundance of early successional tree species at the cost of late successional ones. Quantifying differences in terms of carbon allocation and the proportion of recently fixed carbon in soil CO2 efflux is crucial for addressing the carbon footprint of creeping degradation. Methodology: We compared the carbon allocation pattern of the late successional gymnosperm Podocarpus falcatus (Thunb.) Mirb. and the early successional (gap filling) angiosperm Croton macrostachyus Hochst. es Del. in an Ethiopian Afromontane forest by whole tree (CO2)-C-13 pulse labeling. Over a one-year period we monitored the temporal resolution of the label in the foliage, the phloem sap, the arbuscular mycorrhiza, and in soil-derived CO2. Further, we quantified the overall losses of assimilated C-13 with soil CO2 efflux. Principal Findings: C-13 in leaves of C. macrostachyus declined more rapidly with a larger size of a fast pool (64% vs. 50% of the assimilated carbon), having a shorter mean residence time (14 h vs. 55 h) as in leaves of P. falcatus. Phloem sap velocity was about 4 times higher for C. macrostachyus. Likewise, the label appeared earlier in the arbuscular mycorrhiza of C. macrostachyus and in the soil CO2 efflux as in case of P. falcatus (24 h vs. 72 h). Within one year soil CO2 efflux amounted to a loss of 32% of assimilated carbon for the gap filling tree and to 15% for the late successional one. Conclusions: Our results showed clear differences in carbon allocation patterns between tree species, although we caution that this experiment was unreplicated. A shift in tree species composition of tropical montane forests (e. g., by degradation) accelerates carbon allocation belowground and increases respiratory carbon losses by the autotrophic community. If ongoing disturbance keeps early successional species in dominance, the larger allocation to fast cycling compartments may deplete soil organic carbon in the long run.

Child-to-child interaction and corrective feedback during a tandem chat exchange project

L’objectif principal de cette recherche était d’examiner l’interaction entre élèves d’ALS et de FLS de sixième année du primaire du Québec et de l’Ontario communicant par clavardage et utilisant l’approche en Tandem. Plus spécifiquement, elle visait à vérifier si ces apprenants offraient de la rétroaction corrective et, le cas échéant, si cette dernière suscitait de la réparation. L’opinion des participants par rapport à leur expérience de communication en Tandem a été examinée. Les sessions de clavardage ont eu lieu sur une période de quatre mois produisant 16 séances de clavardage (8 en anglais et 8 en français). Les résultats montrent que des apprenants de langue seconde ont offert de la rétroaction corrective à 370 occasions. L’examen des données révèle que les sujets ont utilisé les trois types de rétroaction: la rétroaction explicite, la reformulation et la négociation de la forme. Contrairement à ce qui a pu être observé dans d’autres études antérieures impliquant de jeunes apprenants, une des découvertes majeures de cette recherche fut l’utilisation, par les participants, de la rétroaction explicite plutôt que la reformulation. Par contraste avec la recherche de Morris (2005) qui impliquait également de jeunes apprenants de niveau primaire utilisant le clavardage, le taux de réparation a été plutôt bas. La grande majorité des élèves d’ALS ont aimé utiliser l’ordinateur comme moyen d’apprentissage de leur L2 et de leur L1 contrairement aux élèves de FLS. Les implications pédagogiques des résultats de cette recherche ainsi que des suggestions pour de futures recherches sont également discutées.

Do simple ventilation and gas exchange measurements predict early successful weaning from respiratory support in unselected general intensive care patients?

Background. The value of respiratory variables as weaning predictors in the intensive care unit (ICU) is controversial. We evaluated the ability of tidal volume (Vtexp), respiratory rate ( f ), minute volume (MVexp), rapid shallow breathing index ( f/Vt), inspired–expired oxygen concentration difference [(I–E)O2], and end-tidal carbon dioxide concentration (PE′CO2) at the end of a weaning trial to predict early weaning outcomes. Methods. Seventy-three patients who required .24 h of mechanical ventilation were studied. A controlled pressure support weaning trial was undertaken until 5 cm H2O continuous positive airway pressure or predefined criteria were reached. The ability of data from the last 5 min of the trial to predict whether a predefined endpoint indicating discontinuation of ventilator support within the next 24 h was evaluated. Results. Pre-test probability for achieving the outcome was 44% in the cohort (n¼32). Non-achievers were older, had higher APACHE II and organ failure scores before the trial, and higher baseline arterial H+ concentrations. The Vt, MV, f, and f/Vt had no predictive power using a range of cut-off values or from receiver operating characteristic (ROC) analysis. The [I–E]O2 and PE′CO2 had weak discriminatory power [areaunder the ROC curve: [I–E]O2 0.64 (P¼0.03); PE′CO2 0.63 (P¼0.05)]. Using best cut-off values for [I–E]O2 of 5.6% and PE′CO2 of 5.1 kPa, positive and negative likelihood ratios were 2 and 0.5, respectively, which only changed the pre- to post-test probability by about 20%. Conclusions. In unselected ICU patients, respiratory variables predict early weaning from mechanical ventilation poorly.

Volatility Spillover and Multivariate Volatility Impulse Response Analysis of GFC News Events

This paper applies two measures to assess spillovers across markets: the Diebold Yilmaz (2012) Spillover Index and the Hafner and Herwartz (2006) analysis of multivariate GARCH models using volatility impulse response analysis. We use two sets of data, daily realized volatility estimates taken from the Oxford Man RV library, running from the beginning of 2000 to October 2016, for the S&P500 and the FTSE, plus ten years of daily returns series for the New York Stock Exchange Index and the FTSE 100 index, from 3 January 2005 to 31 January 2015. Both data sets capture both the Global Financial Crisis (GFC) and the subsequent European Sovereign Debt Crisis (ESDC). The spillover index captures the transmission of volatility to and from markets, plus net spillovers. The key difference between the measures is that the spillover index captures an average of spillovers over a period, whilst volatility impulse responses (VIRF) have to be calibrated to conditional volatility estimated at a particular point in time. The VIRF provide information about the impact of independent shocks on volatility. In the latter analysis, we explore the impact of three different shocks, the onset of the GFC, which we date as 9 August 2007 (GFC1). It took a year for the financial crisis to come to a head, but it did so on 15 September 2008, (GFC2). The third shock is 9 May 2010. Our modelling includes leverage and asymmetric effects undertaken in the context of a multivariate GARCH model, which are then analysed using both BEKK and diagonal BEKK (DBEKK) models. A key result is that the impact of negative shocks is larger, in terms of the effects on variances and covariances, but shorter in duration, in this case a difference between three and six months.

Raman microscopy of formamide-intercalated kaolinites treated by controlled-rate thermal analysis technology

Raman spectroscopy of formamide-intercalated kaolinites treated using controlled-rate thermal analysis technology (CRTA), allowing the separation of adsorbed formamide from intercalated formamide in formamide-intercalated kaolinites, is reported. The Raman spectra of the CRTA-treated formamide-intercalated kaolinites are significantly different from those of the intercalated kaolinites, which display a combination of both intercalated and adsorbed formamide. An intense band is observed at 3629 cm-1, attributed to the inner surface hydroxyls hydrogen bonded to the formamide. Broad bands are observed at 3600 and 3639 cm-1, assigned to the inner surface hydroxyls, which are hydrogen bonded to the adsorbed water molecules. The hydroxyl-stretching band of the inner hydroxyl is observed at 3621 cm-1 in the Raman spectra of the CRTA-treated formamide-intercalated kaolinites. The results of thermal analysis show that the amount of intercalated formamide between the kaolinite layers is independent of the presence of water. Significant differences are observed in the CO stretching region between the adsorbed and intercalated formamide.

Physiological and symptomatic responses to cycling and walking in intermittent claudication

To shed light on the potential efficacy of cycling as a testing modality in the treatment of intermittent claudication (IC), this study compared physiological and symptomatic responses to graded walking and cycling tests in claudicants. Sixteen subjects with peripheral arterial disease (resting ankle: brachial index (ABI) < 0.9) and IC completed a maximal graded treadmill walking (T) and cycle (C) test after three familiarization tests on each mode. During each test, symptoms, oxygen uptake (VO2), minute ventilation (VE), respiratory exchange ratio (RER) and heart rate (HR) were measured, and for 10 min after each test the brachial and ankle systolic pressures were recorded. All but one subject experienced calf pain as the primary limiting symptom during T; whereas the symptoms were more varied during C and included thigh pain, calf pain and dyspnoea. Although maximal exercise time was significantly longer on C than T (690 +/- 67 vs. 495 +/- 57 s), peak VO2, peak VE and peak heart rate during C and T were not different; whereas peak RER was higher during C. These responses during C and T were also positively correlated (P < 0.05) with each other, with the exception of RER. The postexercise systolic pressures were also not different between C and T. However, the peak decline in ankle pressures from resting values after C and T were not correlated with each other. These data demonstrate that cycling and walking induce a similar level of metabolic and cardiovascular strain, but that the primary limiting symptoms and haemodynamic response in an individual's extremity, measured after exercise, can differ substantially between these two modes.

Hydrazine-hydrate intercalated halloysite under controlled-rate thermal analysis conditions

The thermal behaviour of halloysite fully expanded with hydrazine-hydrate has been investigated in nitrogen atmosphere under dynamic heating and at a constant, pre-set decomposition rate of 0.15 mg min-1. Under controlled-rate thermal analysis (CRTA) conditions it was possible to resolve the closely overlapping decomposition stages and to distinguish between adsorbed and bonded reagent. Three types of bonded reagent could be identified. The loosely bonded reagent amounting to 0.20 mol hydrazine-hydrate per mol inner surface hydroxyl is connected to the internal and external surfaces of the expanded mineral and is present as a space filler between the sheets of the delaminated mineral. The strongly bonded (intercalated) hydrazine-hydrate is connected to the kaolinite inner surface OH groups by the formation of hydrogen bonds. Based on the thermoanalytical results two different types of bonded reagent could be distinguished in the complex. Type 1 reagent (approx. 0.06 mol hydrazine-hydrate/mol inner surface OH) is liberated between 77 and 103°C. Type 2 reagent is lost between 103 and 227°C, corresponding to a quantity of 0.36 mol hydrazine/mol inner surface OH. When heating the complex to 77°C under CRTA conditions a new reflection appears in the XRD pattern with a d-value of 9.6 Å, in addition to the 10.2 Ĺ reflection. This new reflection disappears in contact with moist air and the complex re-expands to the original d-value of 10.2 Å in a few h. The appearance of the 9.6 Å reflection is interpreted as the expansion of kaolinite with hydrazine alone, while the 10.2 Å one is due to expansion with hydrazine-hydrate. FTIR (DRIFT) spectroscopic results showed that the treated mineral after intercalation/deintercalation and heat treatment to 300°C is slightly more ordered than the original (untreated) clay.