Computer-assisted mutagenesis of ecotin to engineer its secondary binding site for urokinase inhibition

Inhibitors of urokinase-type plasminogen activator (uPA) were selected in vitro from two ecotin phage-display libraries to study the effect on binding of amino acid substitutions at critical positions 108, 110, 112, and 113 within the 100s loop (RNKL, respectively, in wild type ecotin). The first, a focused library, was the result of a computation-assisted approach using the three-dimensional structure of the ecotin-trypsin complex to guide the modeling of amino acid substitutions predicted to increase affinity for uPA. The second, a complete library, allowed for all substitutions at the above identified positions. The consensus sequences selected from the focused, and complete libraries were RRWS and R(R/N)QL, respectively. Inhibition constant determinations showed ecotin variants containing these sequences to be similarly potent (K(i) = 1-2 nm). These substitutions were combined with previously identified substitutions in another critical region of ecotin. One of these combinations (D70R/M84R/RRQL) is the tightest (K(i) = 50 pm) ecotin variant inhibitor of uPA. The blending of combinatorial methods and computer algorithms designed to predict stronger binders has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. This technology can be applied to select for enhanced binding interactions at protein-protein interfaces and accelerate the process of protease inhibitor development.     

A common polygenic basis for quinine and PROP avoidance in mice

Inbred strains of mice (Mus musculus) differ greatly in ability to taste various bitter compounds. For some compounds, the differences result from allelic variation at a single locus. However, segregation patterns incompatible with monogenic inheritance have been found for quinine avoidance. The Soa bitter sensitivity locus exerts some influence on this phenotype, but an unknown number of other loci also contribute. Relative avoidance patterns for quinine sulfate in panels of naive inbred strains resembled avoidance patterns for 6-n-propyl-2- thiouracil (PROP), suggesting a common genetic basis. In particular, C57BL/6J mice strongly avoided both 0.1 mM quinine sulfate and 1 mM PROP in two-bottle preference tests, whereas C3H/HeJ mice were indifferent to both. Therefore, 12 BXH/Ty recombinant inbred strains, derived from these strains, were tested with both solutions to begin identification of the unknown bitter loci. Naive mice were tested for four consecutive days with each compound (order counterbalanced). Some BXH/Ty strain means resembled those of the parent strains, but others were intermediate. This indicated recombination among loci affecting avoidance, and therefore polygenic inheritance. The strain means were highly correlated across compounds (r = 0.98), suggesting that the same polygenes controlled both phenotypes. The BXH/Ty means for both compounds were then compared with the strain genotypes at 212 chromosome position markers distributed throughout the genome. Eight markers on five chromosomes (3, 6, 7, 8 and 9) yielded significant correlations. Six of the markers were correlated with both phenotypes, again suggesting common polygenic inheritance. The marker with the highest correlation was Prp, tightly linked to Soa on chromosome 6. The correlated marker regions likely contain quantitative trait loci affecting bitter avoidance. The phenotypic similarity of PROP to quinine, rather than to phenylthiourea, apparently stemming from a common polygenic basis, indicates a difference between mice and humans in gustatory organization related to bitters.      

Can we consider the stocks of alfonsinos Beryx splendens and Beryx decadactylus from the Azores a discrete fishery management unit?

This paper summarises the detailed information on catch, fork length, weight, sex and maturity of alfonsinos Beryx splendens and Beryx decadactylus collected from targeted surveys in Azores waters, complemented by fishery data, over the past c. 20 years. To date, it was not possible to define if the Azorean component of the population can be considered as a discrete local management unit. The reason for this is the lack of available information for these resources covering the entire spatial distribution of the species in the North Atlantic Ocean. Additionally, there are some conflicts between the different genetic results available, poor understanding about the species movements and data from the Azores show some discrepancies in aspects of reproduction. Consequently, there is no analytical assessment for this resource and the stock of Beryx spp. is currently managed based on the precautionary approach. Outputs of recent analyses are presented in this study and possible assumptions and strategies for the assessment are discussed.     

Assessment of neuropathic pain in leprosy patients with relapse or treatment failure by infrared thermography: A cross-sectional study

BackgroundNeuropathic pain (NP) is one of the main complications of leprosy, and its management is challenging. Infrared thermography (IRT) has been shown to be effective in the evaluation of peripheral autonomic function resulting from microcirculation flow changes in painful syndromes. This study used IRT to map the skin temperature on the hands and feet of leprosy patients with NP.Methodology/Principal findingsThis cross-sectional study included 20 controls and 55 leprosy patients, distributed into 29 with NP (PWP) and 26 without NP (PNP). Thermal images of the hands and feet were captured with infrared camera and clinical evaluations were performed. Electroneuromyography (ENMG) was used as a complementary neurological exam. Instruments used for the NP diagnosis were visual analog pain scale (VAS), Douleur Neuropathic en 4 questions (DN4), and simplified neurological assessment protocol. The prevalence of NP was 52.7%. Pain intensity showed that 93.1% of patients with NP had moderate/severe pain. The most frequent DN4 items in individuals with NP were numbness (86.2%), tingling (86.2%) and electric shocks (82.7%). Reactional episodes type 1 were statistically significant in the PWP group. Approximately 81.3% of patients showed a predominance of multiple mononeuropathy in ENMG, 79.6% had sensory loss, and 81.4% showed some degree of disability. The average temperature in the patients’ hands and feet was slightly lower than in the controls, but without a significant difference. Compared to controls, all patients showed significant temperature asymmetry in almost all points assessed on the hands, except for two palmar points and one dorsal point. In the feet, there was significant asymmetry in all points, indicating a greater involvement of the lower limbs.ConclusionIRT confirmed the asymmetric pattern of leprosy neuropathy, indicating a change in the function of the autonomic nervous system, and proving to be a useful method in the approach of pain.     

Serology of paracoccidioidomycosis

The purposes of the present work were: i) to study the positivity indices and compare titers obtained with the indirect immunofluorescence (II), tube precipitation (TP), complement fixation (CF) and double immunodiffusion on agar gel (ID) tests in the sera of 196 patients with paracoccidioidomycosis before treatment, and ii) to compare the initial titers of II with those obtained 1 year or more after treatment. II was the most sensitive serologic reaction (85.2%), and the positivity indices for CF, ID and TP were 67.7%, 66.0% and 50.0%, respectively. The sera tended to show parallel mean titers in II, CF and TP tests. One year after treatment there was a fall in titers of II in 66.2% of patients. The data, taken as a whole, demonstrate the usefulness of the indirect immunofluorescent test and the importance of using 2 or more serologic tests for the diagnosis and monitoring of patients with paracoccidioidomycosis.     

HbA1c Test as a Tool in the Diagnosis of Gestational Diabetes Mellitus

Aims

Gestational diabetes mellitus (GDM) is a prevalent and potentially serious condition which may put both mothers and neonates at risk. The current recommendation for diagnosis is the oral glucose tolerance test (OGTT). This study aimed to determine the usefulness of HbA1c test as a diagnostic tool for GDM as compared to the traditional criteria based on the OGTT.

Methods

This was a diagnostic test accuracy study. We performed OGTT and HbA1c test in women attending prenatal visits at a tertiary hospital. GDM was defined according to WHO1999 or ADA/WHO 2013 criteria. ROC curve was used to evaluate the diagnostic performance of HbA1c. Sensitivity, specificity and likelihood ratios for different HbA1c cut-off points were calculated.

Results

Of the 262 women in the third trimester of gestation enrolled in the study, 86 (33%) were diagnosed with GDM. Only five of these women presented HbA1c ≥48 mmol/mol (6.5%). This cut-off point presented 100% specificity but very low sensitivity (7%). Based on ROC curve, and considering OGTT as the reference criterion, HbA1c ≥40 mmol/mol (5.8%) showed adequate specificity in diagnosing GDM (94.9%) but low sensitivity (26.4%). Unlike, HbA1c values of 31 mmol/mol (5.0%) presented adequate sensitivity (89.7%) but low specificity (32.6%) to detect GDM. For women with HbA1c ≥40 mmol/mol (5.8%), the positive and negative likelihood ratios were 5.14 (95%CI 2.49–10.63) and 0.78 (0.68–0.88), respectively. The post-test probability of GDM was about 40%, representing a 4.0-fold increase in the mean pre-test probability. This cut-off point could eliminate the need for the unpleasant and laborious OGTT tests in almost one third of cases, as 38% of patients with GDM may be diagnosable by HbA1c test alone.

Conclusions

Our results show that combined HbA1c and OGTT measurements may be useful in diagnosing GDM.     

Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study

Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91^phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process.     

A Review of Methods for Non-Invasive Heart Rate Measurement on Wrist

When evaluating general health condition on a patient, heart rate is an essential indicator as it is directly representative of the cardiac system state. Continuous measurement methods of heart rate are required for ambulatory monitoring involved in preliminary diagnostic indicators of cardiac diseases or stroke. The growing number of recent developments in wearable devices is reflective of the increasing demand in wrist-worn activity trackers for fitness and health applications. Indeed, the wrist represents a convenient location in terms of form factor and acceptability for patients. While most commercially-available devices are based on optical methods for heart rate measurement, others methods were also developed, based on various physiological phenomena. This review is focused on heart rate measurement methods located on forearm and more specifically on the wrist. For each method, the physiological mechanism involved is described, and the associated transducers for bio-signal acquisition as well as practical developments and prototypes are presented. Methods are discussed on their advantages, limitations and their suitability for an ambulatory use. More specifically, the superposition of motion artefacts over the signal of interest is one of the largest drawbacks for these methods, when used out of laboratory conditions. As such, artefact reduction techniques proposed in the literature are also presented and discussed.