Evidence concerning rate-limiting steps in protein folding from the effects of trifluoroethanol

The refolding kinetics of 13 proteins have been studied in the presence of 2,2,2-trifluoroethanol (TFE). Low concentrations of TFE increased the folding rates of all the proteins, whereas higher concentrations have the opposite effect. The extent of deceleration of folding correlates closely with similar effects of guanidine hydrochloride and can be related to the burial of accessible surface area during folding. For those proteins folding in a two-state manner, the extent of acceleration of folding correlates closely with the number of local backbone hydrogen bonds in the native structure. For those proteins that fold in a multistate manner, however, the extent of acceleration is much smaller than that predicted from the data for two-state proteins. These results support the concept that for two-state proteins the search for native-like contacts is a key aspect of the folding reaction, whereas the rate-determining steps for folding of multistate proteins are associated with the reorganization of stable structure within a collapsed state or with the search for native-like interactions within less structured regions.     

Evidence for a mechanism of amyloid formation involving molecular reorganisation within native-like precursor aggregates

The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive beta-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3-5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and beta-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.     

A classification model of grazing areas in southern Italy using Remote Sensing data

Detection of vegetation typologies is of particular interest in epidemiological studies on animal diseases. This paper is focused on the automatic detection of grazing areas in southern Italy browsed by equines, oxen and sheep, mostly sedentary, with many in fenced areas for overnight stays. Results revealed Satellite Remote Sensing was an indispensable tool in area surveys for vegetation cover characterization. Our classification model shows an accuracy level of 90.21% and a precision of 92.69%. Results suggest similar application protocols can be used in areas with different vegetation cover to characterizing potential infection risk areas for geohelminths and other diseases.     

Advances in satellite remote sensing of pheno-climatic features for epidemiological applications

Geographical Information Systems (GIS) and Remote Sensing (RS) technologies are being used increasingly to study the spatial and temporal patterns of diseases. They can be used to complement conventional ecological monitoring and modelling techniques, and provide a means to portray complex relationships in the ecology of diseases with strong environmental determinants. In particular, satellite technology has been extraordinarily improved during recent years, providing new parameters useful to understand the epidemiology of parasites, such as vegetation indices, land surface temperatures, soil moisture and rainfall indices. In the present review, Normalized Difference Vegetation Index (NDVI) is primarily considered, since it is the index characterizing vegetation that is most used in epidemiological studies. Multi-temporal study of RS data allows collection of bio-climatic information about risk area distribution, along with predictive studies and anticipatory models of diseases, at different geographic scales ranging from global to local. The main physical and technological basis of a mathematical model, effective at different scales, for identification of landscape pheno-climatic features is described in the current paper.     

Bioactivity studies of extracts from Tridax procumbens.

An updated review on the biological activity of Tridax procumbens is presented. A detailed biological screening comprised of gram-positive and gram-negative bacteria, yeasts and fungi using crude extracts of this plant was undertaken. The n-hexane extract of the flowers showed activity against Escherichia coli. The same extract of the whole aerial parts was active against Mycobacterium smegmatis, Escherichia coli, Salmonella group C and Salmonella paratyphi. The ethyl-acetate extract of the flowers was active against Bacillus cereus and Klebsiella sp. The aerial parts extract also showed activity only against Mycobacterium smegmatis and Staphylococcus aureus, while the aqueous extract showed no antimicrobial activity. None of the tested extracts was active against the yeasts, Candida albicans, Candida tropicalis and Rhodotorula rubra; or the fungi: Aspergillus flavus, Aspergillus niger, Mucor sp. and Trichophyton rubrum.     

Spermatozopsis acidophila Kalina (Chlorophyta,Volvocales), a little known alga from highly acidic environments

Abstract

A strain of the alga Spermatozopsis acidophila from highly acidic environments in the solfatara at Pisciarelli (Naples, Italy) has been studied from the ecological, physiological, morphological and ultrastructural points of view.

Its pH optimum (1.0) and its upper limit of pH (1.8) are the lowest recorded for any algal species.

Our ultrastructural investigation has confirmed that it belongs to the family Poly-blepharidaceae (Volvocales). A conspicuously developed chloroplast was noted, along with a remarkable quantity of microtubules and E. R. cisternae.     

KRAS mutations testing in colorectal carcinoma patients in Italy: from guidelines to external quality assessment

Background

Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients.

Methodology/Principal Findings

In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass.

Conclusions

The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers.     

Effect of sub-optimal neuromotor control on the hip joint load during level walking

Skeletal forces are fundamental information in predicting the risk of bone fracture. The neuromotor control system can drive muscle forces with various task- and health-dependent strategies but current modelling techniques provide a single optimal solution of the muscle load sharing problem. The aim of the present work was to study the variability of the hip load magnitude due to sub-optimal neuromotor control strategies using a subject-specific musculoskeletal model. The model was generated from computed tomography (CT) and dissection data from a single cadaver. Gait kinematics, ground forces and electromyographic (EMG) signals were recorded on a body-matched volunteer. Model results were validated by comparing the traditional optimisation solution with the published hip load measurements and the recorded EMG signals. The solution space of the instantaneous equilibrium problem during the first hip load peak resulted in 10(5) dynamically equivalent configurations of the neuromotor control. The hip load magnitude was computed and expressed in multiples of the body weight (BW). Sensitivity of the hip load boundaries to the uncertainty on the muscle tetanic stress (TMS) was also addressed. The optimal neuromotor control induced a hip load magnitude of 3.3 BW. Sub-optimal neuromotor controls induced a hip load magnitude up to 8.93 BW. Reducing TMS from the maximum to the minimum the lower boundary of the hip load magnitude varied moderately whereas the upper boundary varied considerably from 4.26 to 8.93 BW. Further studies are necessary to assess how far the neuromotor control can degrade from the optimal activation pattern and to understand which sub-optimal controls are clinically plausible. However we can consider the possibility that sub-optimal activations of the muscular system play a role in spontaneous fractures not associated with falls.     

Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast

A general feature of the nucleus is the organization of repetitive deoxyribonucleic acid sequences in clusters concentrating silencing factors. In budding yeast, we investigated how telomeres cluster in perinuclear foci associated with the silencing complex Sir2-Sir3-Sir4 and found that Sir3 is limiting for telomere clustering. Sir3 overexpression triggers the grouping of telomeric foci into larger foci that relocalize to the nuclear interior and correlate with more stable silencing in subtelomeric regions. Furthermore, we show that Sir3's ability to mediate telomere clustering can be separated from its role in silencing. Indeed, nonacetylable Sir3, which is unable to spread into subtelomeric regions, can mediate telomere clustering independently of Sir2-Sir4 as long as it is targeted to telomeres by the Rap1 protein. Thus, arrays of Sir3 binding sites at telomeres appeared as the sole requirement to promote trans-interactions between telomeres. We propose that similar mechanisms involving proteins able to oligomerize account for long-range interactions that impact genomic functions in many organisms.     

Drosophila vitelline membrane cross-linking requires the fs(1)Nasrat, fs(1)polehole and chorion genes activities

Abstract. During the final step of Drosophila vitelline membrane formation, the structural proteins composing this layer become cross-linked by covalent bonds. In the present report, we analyzed the vitelline membrane cross-linking in mutants having defects either in this layer or in the chorionic layers. In the fs(1)Nasrat and fs(1)polehole mutant alleles conferring defects in vitelline membrane formation, disruption of vitelline membrane cross-linking was observed, indicating the involvement of these two genes in the process. On the contrary, in the fs(1)Nasrat and fs(1)polehole alleles showing defects only at the termini of the embryo the vitelline membrane is properly formed, confirming a multifunctional activity of their gene products. Altered vitelline membrane cross-linking was also detected in a mutant of the chorion protein gene Cp36and in the chorion amplification mutant fs(1)K1214, suggesting a role of the structural components of chorion layers in the process of vitelline membrane hardening.