Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy

Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from stimulated emission depletion microscopy to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.     

A promising 31P NMR-multivariate analysis approach for the identification of milk phosphorylated metabolites and for rapid authentication of milk samples

A fast and reliable method for the identification of milk from different mammalians was developed by using ³¹P NMR metabolite profile of milk serum coupled to multivariate analysis (PCA and classification models UNEQ, SIMCA and K-NN). Ten milk samples from six different mammalians, relevant to human nutrition (human, cow, donkey, mare, goat, sheep), were analyzed and eight monophosphorylated components were identified and quantified: phosphocreatine (PCr), glycerophosphorylcholine (GPC), glycerophosphorylethanolamine (GPE), N-acetylglucosamine-1-phosphate (NAcGlu-1P), lactose-1-phosphate (Lac-1P), galactose-1-phosphate (Gal-1P), phosphorylcholine (PC), glucose-6-phosphate (Glu-6P). PCA showed interesting clustering based on the animal genus. K-NN can be successfully used to discriminate between donkey and cow samples while UNEQ class-modeling resulted more suitable for compliance verification. Results confirm the natural variability of milk samples among different species. These data highlight the great potentials of NMR/multivariate analysis combined method in the rapid analysis of phosphorylated milk serum metabolites for milk origin assessment and milk adulteration detection.     

Predictive markers of safety and immunogenicity of adjuvanted vaccines

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.     

Kinematic and electromyographic analysis of the Nordic Hamstring Exercise

The Nordic Hamstring Exercise (NHE) has been introduced as a training tool to improve the efficiency of eccentric hamstring muscle contraction. The aim of this study was to perform a biomechanical analysis of the NHE. Eighteen participants (20.4 ± 1.9 years) performed two sets of five repetitions each of the NHE and maximal eccentric voluntary contraction (MEVC) of the knee flexors on an isokinetic dynamometer whilst knee angular displacement and electrical activity (EMG) of biceps femoris were measured. EMG was on average higher during the NHE (134.3% of the MEVC). During the forward fall of the NHE, the angle at which a sharp increase in downward velocity occurred varied between 47.9 and 80.5 deg, while the peak knee angular velocity (pVelocity) varied between 47.7 and 132.8 deg s^?1. A significant negative correlation was found between pVelocity and peak EMG (r = ?0.62, p < 0.01) and EMG at 45 deg (r = ?0.75, p < 0.01) expressed as a percentage of peak MEVC EMG. Some of the variables analyzed exhibited good to excellent levels of intra- and inter-session reliability. This type of analysis could be used to indirectly monitor the level of eccentric strength of the hamstring muscles while performing the NHE and potentially any training- or injury-related changes.     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.