Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.     

Mechanical stimulation of the plantar foot surface attenuates soleus muscle atrophy induced by hindlimb unloading in rats.

Unloading-induced muscle atrophy occurs in the aging population, bed-ridden patients, and astronauts. This study was designed to determine whether dynamic foot stimulation (DFS) applied to the plantar surface of the rat foot can serve as a countermeasure to soleus muscle atrophy normally observed in hindlimb unloaded (HU) rats. Forty-four mature (6 mo old), male Wistar rats were randomly assigned to ambulatory control, HU alone, HU with active DFS (i.e., plantar contact with active inflation), HU with passive DFS (i.e., plantar contact without active inflation), and HU while wearing a DFS boot with no plantar contact groups. Application of active DFS during HU significantly counteracted the atrophic response by preventing approximately 85% of the reduction in type I myofiber cross-sectional area (CSA) in the soleus while preventing approximately 57% of the reduction in type I myofiber CSA and 43% of the reduction in type IIA myofiber CSA of the medial gastrocnemius muscle. Wearing of a DFS boot without active inflation prevented myofiber atrophy in the soleus of HU animals in a fashion similar to that observed in HU animals that wore an actively inflated DFS boot. However, when a DFS boot without plantar surface contact was worn during HU, no significant protection from HU-induced myofiber atrophy was observed. These results illustrate that the application of mechanical foot stimulation to the plantar surface of the rat foot is an effective countermeasure to muscle atrophy induced by HU.     

The role of solar and UV radiation in environmental stress assessment

The purpose of this study was to evaluate the role of ultraviolet (UV) radiation in environmental assessment. Analysis of two meteorological databases, from Israel and New Zealand, used in this study revealed that the UV radiation component in the environmental stress assessment was not significant. However, because of the health hazards, an independent UV index should be implemented and used for preventing acute and chronic injuries. A prediction of UV radiation from solar radiation measurement would be a great benefit for such an index.     

EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasion

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Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax)

The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), β-actin (two regions of β-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), β2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization.     

The Obesity Epidemic and Future Emergency Responders

Emergency responders should be fit to safely perform strenuous duties. In particular, young recruits are expected to be at or near peak career fitness. We studied the prevalence and health associations of excess weight among 370 consecutive emergency responder candidates for fire and ambulance services in Massachusetts. The mean age and BMI of the recruits were 26.3 (3.8) years and 28.5 (4.9) kg/m², respectively. Seventy‐seven percent had BMI ≥25 kg/m², and 33% were obese (BMI ≥30 kg/m²). After multivariate adjustment, both higher BMI categories and unit increases in BMI were significantly associated with higher blood pressures, worse metabolic profiles, and lower exercise tolerance. Excess weight is highly prevalent and associated with elevated cardiovascular risk among future emergency responders. These findings in a population expected to perform demanding duties supporting public safety merit prompt public health intervention.     

Raman spectroscopy of blood and urine liquid biopsies for ovarian cancer diagnosis: identification of chemotherapy effects

Blood plasma and serum Raman spectroscopy for ovarian cancer diagnosis has been applied in pilot studies, with promising results. Herein, a comparative analysis of these biofluids, with a novel assessment of urine, was conducted by Raman spectroscopy application in a large patient cohort. Spectra were obtained through samples measurements from 116 ovarian cancer patients and 307 controls. Principal component analysis identified significant spectral differences between cancers without previous treatment (n = 71) and following neo-adjuvant chemotherapy (NACT), (n = 45). Application of five classification algorithms achieved up to 73% sensitivity for plasma, high specificities and accuracies for both blood biofluids, and lower performance for urine. A drop in sensitivities for the NACT group in plasma and serum, with an opposite trend in urine, suggest that Raman spectroscopy could identify chemotherapy-related changes. This study confirms that biofluids' Raman spectroscopy can contribute in ovarian cancer's diagnostic work-up and demonstrates its potential in monitoring treatment response.