From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease’s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as “spots” on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.     

Efficiency of population structures for mapping of Mendelian and imprinted quantitative trait loci in outbred pigs using variance component methods

In a simulation study different designs for a pure line pig population were compared for efficiency of mapping QTL using the variance component method. Phenotypes affected by a Mendelian QTL, a paternally expressed QTL, a maternally expressed QTL or by a QTL without an effect were simulated. In all alternative designs 960 progeny were phenotyped. Given the limited number of animals there is an optimum between the number of families and the family size. Estimation of Mendelian and parentally expressed QTL is more efficient in a design with large family sizes. Too small a number of sires should be avoided to minimize chances of sires to be non-segregating. When a large number of families is used, the number of haplotypes increases which reduces the accuracy of estimating the QTL effect and thereby reduces the power to show a significant QTL and to correctly position the QTL. Dense maps allow for smaller family size due to exploitation of LD-information. Given the different possible modes of inheritance of the QTL using 8 to16 boars, two litters per dam was optimal with respect to determining significance and correct location of the QTL for a data set consisting of 960 progeny. The variance component method combining linkage disequilibrium and linkage analysis seems to be an appropriate choice to analyze data sets which vary in marker density and which contain complex family structures.     

The key role of lamb presence in affecting flight response in Sardinian mouflon (Ovis orientalis musimon)

Observations of the flight response in mammals are commonly used to test the behavioural response to predation risk. Such a response is likely to be stronger in more sensitive individuals such as females, and females with offspring in particular, as well as during such periods as the birth season. From August 2005 to July 2006 we recorded the flight distances of different mouflon group types in a natural population in Sardinia. The comparison of the flight distances of the groups (n groups=375, n mouflons=1303) provided evidence for the key role of lamb presence. Female groups with lambs fled at greater distances than male groups and female groups without lambs. This difference was linked to the females' priority to secure the survival of their offspring. Indeed, the uniformity of the flight distances of males and non-reproductive females was in contrast to the general expectations regarding ungulate species. In fact, smaller females were expected to be more sensitive to predation risk than larger males. The absence of major differences is probably due to the low sexual dimorphism of mouflon. Flight distances were shorter during the rut than during the lambing season, arguably because during the mating less time and energies were available for anti-predator behaviour. However, during the rut females with lambs still fled at greater distances than females without lambs, arguably because mothers took it as a priority to protect their parental investment rather then to find a potential mate.     

Main roads and land cover shaped the genetic structure of a Mediterranean island wild boar population

Patterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar (Sus scrofa meridionalis) population inhabiting the island of Sardinia (Italy), and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow (and thus individual movements) within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.     

拉萨高原居民肺容量的测定

对年龄、身高和体重相同的拉萨男性世居藏族39人和男性移居汉族43人的肺容量进行了测定。结果显示:藏族组的肺活量(VC)、肺总容量、胸围均大于汉族组,残气量有大于汉族组之趋势(P=0.06)。胸围的大小与VC呈正相关。5岁前和18岁后移居高原者之肺容量无差别。结果提示,拉萨世居藏族具有较大的肺容量,这对提高肺弥散功能和维持运动时的血氧饱和度有重要意义。     

Habitat selection and group formation pattern of fallow deer Dama dama in a submediterranean environment

We analyzed the patterns of association between sex and age classes of fallow deer Dama dama in addition to the modification of aggregation patterns as a function or the degree of cover in different habitats in a coastal submediterranean environment in Italy. Deer preferred large open grasslands, small clearings, woods and marshes. Habitat selection varied strongly according to sex, and there were also ecological differences among males of different age classes. Adult females were mostly in family groups, with subadults and fawns. These groups were very stable in time and space. On the contrary bachelor herds were unstable social associations, adapting their size to the local degree of cover. In open grasslands males and females sometimes formed mixed groups with group size similarly being adapted to degree of cover. The possibility that random events fully explained fallow deer aggregation patterns was excluded, while the existence of non-linear mechanisms of aggregation was shown. The persistence of antipredator strategies in this managed population may account for these findings.     

A change in twist of actin provides the force for the extension of the acrosomal process in limulus sperm: the false-discharge reaction

One of the most spectacular motions is the generation of the acrosomal process in the limulus sperm. On contact with the egg, the sperm generates a 60-mum-long process that literally drills its way through the jelly surrounding the egg. This irresversible reaction takes only a few seconds. We suggested earlier that this motion is driven by a change in twist of the actin filaments comprising the acrosomal process. In this paper we analyze the so-called false discharge, a reversible reaction, in which the acrosomal filament bundle extends laterally from the base of the sperm and not anteriorly from the apex. Unlike the true discharge, which is straight, the false discharge is helical. Before extension, the filament bundle is coiled about the base of the sperm. In the coil, the bundle is not smoothly bent but consists of arms (straight segments) and elbows (corners) so that the coil looks like a 14-sided polygon. The extension of the false discharge works as follows: starting at the base of the bundle, the filaments change their twist which concomitantly changes the orientations of the elbows relative to each other; that is, in the coil, the elbows all like in a common plane, but after the change in twist, the plane of each elbow is rotated to be perpendicular to that of its neighbors. This change transforms the bundle from a compact coil into an extended left- handed helix. Because the basal end of the bundle is unconstrained, the extension is lateral. The true discharge works the same way but starts at the apical end of the bundle. The apical end, however, is constrained by its passage through the nuclear canal, which directs the extention anteriorly. Unlike the false discharge, during the true discharge the elbows are melted out, making the reaction irreversible. This study shows that rapid movement can be regenerated by actin without myosin and gives us insight into the molecular mechanism.     

Estimation of pack density in grey wolf (<Emphasis Type="Italic">Canis lupus</Emphasis>) by applying spatially explicit capture-recapture models to camera trap data supported by genetic monitoring

Background

Density estimation is a key issue in wildlife management but is particularly challenging and labour-intensive for elusive species. Recently developed approaches based on remotely collected data and capture-recapture models, though representing a valid alternative to more traditional methods, have found little application to species with limited morphological variation. We implemented a camera trap capture-recapture study to survey wolf packs in a 560-km² area of Central Italy. Individual recognition of focal animals (alpha) in the packs was possible by relying on morphological and behavioural traits and was validated by non-invasive genotyping and inter-observer agreement tests. Two types (Bayesian and likelihood-based) of spatially explicit capture-recapture (SCR) models were fitted on wolf pack capture histories, thus obtaining an estimation of pack density in the area.

Results

In two sessions of camera trapping surveys (2014 and 2015), we detected a maximum of 12 wolf packs. A Bayesian model implementing a half-normal detection function without a trap-specific response provided the most robust result, corresponding to a density of 1.21?±?0.27 packs/100 km² in 2015. Average pack size varied from 3.40 (summer 2014, excluding pups and lone-transient wolves) to 4.17 (late winter-spring 2015, excluding lone-transient wolves).

Conclusions

We applied for the first time a camera-based SCR approach in wolves, providing the first robust estimate of wolf pack density for an area of Italy. We showed that this method is applicable to wolves under the following conditions: i) the existence of sufficient phenotypic/behavioural variation and the recognition of focal individuals (i.e. alpha, verified by non-invasive genotyping); ii) the investigated area is sufficiently large to include a minimum number of packs (ideally 10); iii) a pilot study is carried out to pursue an adequate sampling design and to train operators on individual wolf recognition. We believe that replicating this approach in other areas can allow for an assessment of density variation across the wolf range and would provide a reliable reference parameter for ecological studies.

     

Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

The <Emphasis Type="Italic">Mycobacterium marinum mel2</Emphasis> locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species

Background  

Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood.