The interface between bone and connective tissues such as the Anterior Cruciate Ligament (ACL) constitutes a complex transition traversing multiple tissue regions, including non-calcified and calcified fibrocartilage, which integrates and enables load transfer between otherwise structurally and functionally distinct tissue types. The objective of this study was to investigate region-dependent changes in collagen, proteoglycan and mineral distribution, as well as collagen orientation, across the ligament-to-bone insertion site using Fourier transform infrared spectroscopic imaging (FTIR-I). Insertion site-related differences in matrix content were also evaluated by comparing tibial and femoral entheses. Both region- and site-related changes were observed. Collagen content was higher in the ligament and bone regions, while decreasing across the fibrocartilage interface. Moreover, interfacial collagen fibrils were aligned parallel to the ligament-bone interface near the ligament region, assuming a more random orientation through the bulk of the interface. Proteoglycan content was uniform on average across the insertion, while its distribution was relatively less variable at the tibial compared to the femoral insertion. Mineral was only detected in the calcified interface region, and its content increased exponentially across the mineralized fibrocartilage region toward bone. In addition to new insights into matrix composition and organization across the complex multi-tissue junction, findings from this study provide critical benchmarks for the regeneration of soft tissue-to-bone interfaces and integrative soft tissue repair. The interface between soft tissue and bone is essential for physiologic musculoskeletal motion, and serves to integrate and minimize stress concentrations between distinct tissue types. A functional interface with bone is especially important for the Anterior Cruciate Ligament (ACL), the primary tibiofemoral intra-articular ligament and joint stabilizer . The bioThis complex ligament-to-bone transition, however, is not maintained or re-established following ACL reconstruction. Absence of this functional interface may compromise graft stability and long-term clinical outcome –16. DeveSpectroscopic imaging methods such as FTIR-I –24 and RThe objective of this study is to characterize the complex matrix organization at the ACL insertions using FTIR-I, mapping region-dependent changes in collagen, proteoglycan, and mineral content as well as collagen orientation across the multi-tissue transition. As this is the first reported study to utilize FTIR-I for the characterization of the ACL-to-bone interface, image acquisition as a function of sample preparation  will be compared. The second objective of this study is to identify any insertion site-dependent changes in matrix content and distribution by comparing spectroscopic mapping of the femoral and tibial insertions. Both region- and insertion site-dependent differences are anticipated across the interface regions. Findings from this study will lead to new insights into matrix composition and organization at this critical soft tissue-to-bone junction, while providing the much needed benchmarks for interface regeneration and integrative soft tissue repair.The insertion site samples were isolated from neonatal bovine tibiofemoral joints obtained from a local abattoir . After removal of surrounding muscle, subcutaneous fascia, and collateral ligaments, the patella and patellar tendon were removed, followed by the underlying adipose tissue as well as both medial and lateral menisci. The cruciate ligaments were transected, and both the femoral and tibial ACL insertions were identified and excised. Transverse or sagittal incisions approximately 7 mm apart were made through the insertion specimens to isolate samples containing regions of ligament, fibrocartilage, and bone.Both decalcified and non-decalcified samples were evaluated in this study. To prepare the decalcified samples, transverse and sagittal samples of the insertions (n=3 per insertion site from each of three specimens) were immediately fixed with 80% ethanol and 1% cetylpyridinium chloride  for 24 hours following the methods of Bi et al. , while CNon-decalcified samples were used for mineral analysis. To this end, transverse insertion samples (n=3 per insertion site from each of three specimens) were fixed after isolation with 90% ethanol for 24 hours ,32. Foll-1 with a spectral resolution of 8 cm-1 and a spatial resolution of ~6.25 μm [Fourier transform infrared imaging (FTIR-I) analysis was performed using an FTIR spectrometer  coupled to an FTIR microscope imaging system . Spectra were acquired between 2000-800 cm~6.25 μm ,34. Usin-1), and glycosaminoglycan (GAG) content was estimated by integrating the area under a carbohydrate band associated with C-O-C and C–OH vibrations (1140-985 cm-1), according to previous studies that have correlated collagen and GAG content to these respective band areas  and MATLAB 7.10 R2010b . Prior to analysis, spectra were corrected by baseline subtraction using the ISYS software. Collagen content was estimated by integrating the peak area under the Amide I band  and amide II bands (1590-1480 cm-1), and numerical indices for collagen orientation were obtained by calculating the amide I: amide II band area ratio. Collagen orientation was categorized according to the parameters previously reported for cartilage analysis and a 0° polarizer setting, whereby amide I: amide II ratio values ≥2.7 and ≤1.7 indicate fibrils parallel and perpendicular to the bone-fibrocartilage interface, respectively, while a ratio ranging from 1.7-2.7 indicates mixed or random fibril orientation [Additionally, collagen alignment was determined by scanning the demineralized samples with a gold-wire polarizer grid (PerkinElmer) inserted into the path of the IR light, with the polarizer aligned at 0° with respect to the interface between ligament and bone. As Amide I and amide II bond vibrations are approximately orthogonal , previouentation .-1) and dividing by the amide I band area (-1). Sample spectra were likewise baseline corrected and normalized, and a pure PMMA spectrum was subtracted from the sample spectra to eliminate the PMMA background. This method of normalization and subtraction was implemented in order to compensate for different degrees of PMMA penetration into the multiple tissue types evaluated across the insertions.The relative mineral-to-matrix ratio was calculated by integrating the area under the ν1, ν3 phosphate band contour  was performed to determine region- and insertion-dependent differences in matrix or mineral content. Tukey HSD post-hoc tests were performed for all pair-wise comparisons with significance declared for p<0.05. Statistical analysis was performed using the Minitab 16 statistical software package .Results are presented in the form of mean ± standard deviation, with -1) and amide II (1590-1480 cm-1) peaks were evident in all three tissue regions. The carbohydrate band related to proteoglycans (1140-985 cm-1) was present in the fibrocartilage spectra, and an intense phosphate ν1, ν3 band (1200-900 cm-1) was evident in the bone spectra. No difference in collagen distribution was observed in the PMMA-embedded, non-decalcified sections when compared to the paraffin-embedded, decalcified samples, but the phosphate band was absent from the decalcified samples (data not shown). In addition, similar trends in collagen  to the fibrocartilage-bone interface. This orientation changed continuously, with a more mixed or random collagen fibril orientation (Amide I:II ratio) observed toward the center and MFC region of the fibrocartilage interface for both femoral  and tibiThe proteoglycan content and distribution at the femoral and tibial insertions were highly variable. Specifically, proteoglycan content varied laterally throughout the fibrocartilage regions spectroscopically in both transverse  and sagi2 = 0.98) with an exponential constant of (5.8 ± 0.3) x 10-2. In addition, the mineral-to-matrix ratio was significantly higher in the mineralized fibrocartilage and bone regions compared to the ligament and non-mineralized fibrocartilage regions , both region- and insertion-dependent variations in collagen, proteoglycan, and mineral content, as well as collagen orientation, were detected and quantified across the multi-tissue regions of the interface. Characterizing these compositional changes, as summarized in Systematic analysis of femoral and tibial ACL insertions revealed region-dependent changes in collagen distribution, which is in agreement with published histological characterizations ,4,6–8,10Region-dependent differences in proteoglycan content were also detected at the ACL-to-bone insertions. In particular, changes in proteoglycan content were evident laterally throughout the insertion fibrocartilage, although on average, little difference was seen across the fibrocartilage region from ligament to bone. The absence of large differences in proteoglycan content among the tissue regions or variations within the fibrocartilage itself may be related to the neonate state of the tissue. In addition, the type of proteoglycan and glycosaminoglycan chain length may vary between different regions and tissues, possibly also accounting for changes in total GAG content found laterally in the insertions. For example, Vogel et al. have shown that the primary proteoglycan in the tensile-loaded region of the human tibialis posterior tendon is decorin, which is a small proteoglycan with only one GAG chain of varying length, whereas compressed fibrocartilaginous regions of the tendon contain small proteoglycans including decorin and biglycan, as well as large proteoglycans . This diAn exponential gradient or relatively step-wise change in mineral content was observed between the non-mineralized and mineralized fibrocartilage regions. This finding was in contrast to the linear increase in mineral content reported for mature rat supraspinatus-bone interface, as determined using Raman spectroscopy ,26. GiveThe controlled spatial distribution of collagen, proteoglycans, and mineral content across the ACL-bone interface  can resuInsertion site-dependent differences were also observed here. While lateral variations in proteoglycan distribution were observed in both femoral and tibial insertions, the differences were more pronounced in the femoral insertion. Conversely, collagen distribution was more consistent in the femoral fibrocartilage compared to the tibial insertion. These disparities may reflect differences in the mechanical loading profile between insertions as well as throughout each insertion site. In addition, although not statistically significant, average relative mineral content was found to be higher in the tibial mineralized fibrocartilage and bone regions when compared to the femoral insertion, which may contribute to the higher apparent Young’s modulus of the tibial insertion reported by Moffat et al. .This study examined the ligament-to-bone interface in an immature model due to the need for an understanding of this stage of enthesis formation as interface regeneration will likely require the initial formation of a similar immature enthesis prior to maturation. In this study, collagen fiber orientation in neonatal insertion fibrocartilage was found to vary minimally within the insertion, and did not yet exhibit the orientation reported for the mature enthesis . CollageSimilar to collagen distribution and orientation, it is likely that proteoglycan content will change as the interface remodels with age. Wang et al. observed markedly low staining intensity for proteoglycans in the ACL-to-bone insertions of mature animals compared to neonatal, especially as the interface progressed from a hyaline-like cartilage to fibrocartilage . Region--1) [While this is the first study to apply FTIR-I for interface characterization, it is noted that collagen content across the fibrocartilage insertion was estimated based solely on the amide I band area, under the assumption that variations in section thicknesses are minimal. This assumption is likely valid given the large number of data points collected and consistency among the specimens. It is emphasized that the analysis performed in this study is intended to provide an estimate of the relative amounts of matrix and mineral content rather than absolute amounts. While the proteoglycan and collagen spectra overlap within the fingerprint region (1800-800 cm-1) , it has -1) . In addi-1) ,28, boviAs the soft tissue-bone interface is crucial for musculoskeletal functionality, the findings of this study are particularly relevant for current efforts to achieve biological graft fixation by engineering a functional interface between ACL grafts and bone –46. FromThis study focused on quantitative mapping of changes in matrix and mineral distribution across the ACL-to-bone insertion sites using the FTIR-I. Both region- and insertion site-dependent differences in collagen, proteoglycan, and mineral distributions were observed. These controlled spatial changes in matrix content and distribution likely contribute to the reported increases in mechanical properties across the insertion site, and facilitate the transition of complex loads from ligament to bone.


Despite successful scale up of anti-retroviral therapy (ART) in Malawi, the number of children initiating ART early in their life is low and mortality of HIV infected infants not accessing ART is high. Availability of PCR remains limited and despite presumptive diagnosis (PD) is recommended in the national guidelines, it is rarely used as a reason to start HIV-exposed infants on ART. Reasons may include factors at health worker and clinic level. Knowledge of these barriers is important to increase access to ART for this high risk group.A structured questionnaire with 11 closed questions was administered to one ART clinic staff member for each site (nurse or clinician) by team-members of the regular national ART supervision for Quarter 1/2009 in the central and southern region. Questions addressed specific reasons for low uptake of PD and tested providers' knowledge of the PD definition. Finally, the interviewer explained how to make a PD correctly using a job-aid.Over 2 weeks, respondents of 49 of 173 ART-sites completed the questionnaire. They were mainly clinical officers (22) and nurses (17) from a central hospital, 5 district hospitals, 3 rural hospitals, 13 health centers, and 27 other facilities such as mission hospitals, smaller clinics run by NGOs, armed forces or privately. Forty-one clinics provided HIV testing and counseling for pregnant women on-site and 35 clinics offered antiretrovirals for PMTCT. Most providers felt that children <18 month of age are under-represented in their ART-cohort, mentioned that generally few HIV exposed children are eligible for ART in their catchment area and that limited access to PCR are reasons for low uptake of PD (34/49 respondents each). Twenty-nine saw the presence of maternal HIV-antibodies as a barrier that makes the diagnosis difficult, and 20 noted that staff at the under 5 clinic or postnatal ward rarely identifies and refers HIV exposed infants to HIV-services. Twelve providers said they generally don't register children at that age. Of the 49 providers, 15 defined presumptive diagnosis correctly and 15 partly correct.Providers seem to be aware of the need to start more HIV-infected infants on ART and of structural barriers to access this service. Improved knowledge and sensitization of providers on how to diagnose severely ill HIV-exposed infants presumptively may increase access to ART for children in this age-group.


Here, we report the complete genome sequences of two tembusu virus strains, ZC-1 and LQ-1, isolated from ducks and geese, respectively, in 2012. Phylogenetic analysis showed that the nucleotide and amino acid sequences of the two strains are closely related to those of the TMUV isolates around Shandong province. The full-length genome sequences of two waterfowl-origin TMUVs provided herein will help to understand the molecular epidemiology of tembusu virus in China, which deserves further investigation. The flavivirus tembusu virus (TMUV) was originally isolated from a duck in China in 2010 (Ex Taq polymerase. The PCR products were purified and sequenced (Invitrogen). The sequences were assembled and manually edited to produce the full-length genomic sequence of the TMUV isolates.In October 2012, two TMUV strains were isolated from a duck farm with an outbreak of egg-drop syndrome in Peking ducks and another goose farm with an outbreak of neurological symptoms in wulong geese in Shandong province, China. Samples from the theca folliculi from the affected ducks and the brain from the affected geese were used for virus isolation, and the two isolated virus strains were named ZC-1 and LQ-1. Seven pairs of overlapping PCR primers were designed in our lab using Primer Premier 5.0 software, according to two published complete genome sequences of TMUV, SD001 and the sparrow-isolated strain SDMS (GenBank accession no. KC333867) . AccordiThe full-length genome sequences of the two waterfowl-origin tembusu virus strains are 10,990 nucleotides (nt) in length, contain a long open reading frame (ORF) , encode a polyprotein of 3,425 amino acids (aa), and contain three structural proteins  and eight nonstructural proteins . Comparative sequence analysis revealed that ZC-1 and LQ-1 are 98.1% to 99.0% identical to other TMUV strains. The percent identity of the nucleotide sequences of the genomic sequences of these two isolates is 99.7%. The two strains were simultaneously isolated from two farms that are in two adjacent cities. Specifically, the nucleic acid and amino acid sequences of the two strains are more closely related to the strains BYD-1 , isolateAlthough waterfowls infected with TMUV exhibited heavy egg-drop syndrome or encephalitis as their main symptom, the layer hens and sparrows infected with TMUV did not exhibit any symptoms or pathological changes. Serum samples collected from people who worked in the TMUV-infected farms were tembusu virus positive . WhetherWe report herein the complete genome sequences of two tembusu virus strains, ZC-1 and LQ-1. The data contribute to the exploration of the molecular epidemiology and evolutionary of the tembusu virus. Moreover, the mutation sites of different strains might reveal the mechanisms of molecular epidemiology and molecular pathogenesis.KF557894 and KF557893, respectively.The complete genome sequences of the duck and goose tembusu virus isolates have been submitted to GenBank under the accession no.


Ordered arrays of light-emitting conjugated polymer nanofibers are realized by near-field electrospinning. The authors report on the realization of ordered arrays of light-emitting conjugated polymer nanofibers by near-field electrospinning. The fibers, made from poly, have diameters of a few hundreds of nanometers and their emission peaked at 560 nm. The observed blue-shift compared to the emission from reference films is attributed to different polymer packing in the nanostructures. Optical confinement in the fibers is also analyzed through self-waveguided emission. These results open interesting perspectives for the realization of complex and ordered architectures by light-emitting nanofibers, such as photonic circuits, and for the precise positioning and integration of conjugated polymer fibers into light-emitting devices. The scientific and technological relevance of polymer nanofibers is stimulating a lot of research efforts to develop novel methods for the fabrication of 1D organic nanostructures with controlled shape, composition and assembly. Electrospinning (ES) is the most versatile and widely used technique for the fabrication of polymer nanofibers.10 The elongation of a polymer solution by an electrostatic field is the key feature of ES, easily allowing the fabrication of continuous polymeric and hybrid fibers with diameters down to a few tens of nanometers. Typically, these nanostructures are collected in non-woven mats or uniaxial arrays,11 and several regimes of jet instability can limit the precise positioning of individual fibers. This is particularly relevant when single active nanofibers have to be integrated into devices such as organic light-emitting diodes,12 field-effect transistors,13 and optical sensors.3 Recently, scanning tip ES14 and near-field electrospinning (NF-ES)16 have been demonstrated to be suitable for the synthesis and positioning of single nanofibers with high precision. In particular NF-ES, utilizing the stable region of the electrospun jet close to the metallic needle, has been exploited to fabricate polyethylene oxide (PEO),16 poly(vinylidene fluoride),17 polycaprolactone18 and composite (PEO/carbon nanotubes)19 fibers, with diameters down to 16 nm and assembled even in complex three-dimensional patterns.21 SnO2 and TiO2 nanofibers have also been fabricated by depositing and annealing well-ordered polymeric fibers embedding suitable precursors.23 However, the application of NF-ES to light-emitting conjugated polymers is still basically unexplored, notwithstanding the clear added value of these materials for a large variety of optoelectronic applications, many of which have already reached commercialization.Polymer nanofibers are flexible 1-dimensional (1D) nanostructured materials that are increasingly utilized in many fields such as tissue engineering,27 However the integration of individual light-emitting nanofibers into photonic and electronic devices is still difficult, due to the limited ability of standard ES systems to control the spatial deposition of individual polymer nanofibers between electrodes or other functional interfaces. NF-ES can largely outperform conventional ES for depositing conjugated polymer fibers with high spatial control. Moreover, NF-ES can enable also an improved control of fiber morphology,20 which is essential for the exploitation of single active fibers for photonic or sensing applications. We realize well-aligned light-emitting fibers by a prototype conjugated polymer, poly (MEH-PPV), producing ordered arrays. The fibers exhibit blue-shifted photoluminescence (PL) compared to reference films, and self-waveguiding of the emission. These results open the way for the fabrication of complex arrays and photonic circuits composed of nanofibers, exploiting the unique features of conjugated polymers for emitting, guiding and sensing UV-visible light.In this paper, we report on light-emitting conjugated polymer nanofibers realized by NF-ES. Conjugated polymers cannot be easily electrospun due to their limited solubility, chain rigidity and low molar mass. Despite such difficulties, nanofibers made of these polymers have been produced by standard ES systems.Mw = 300 000) in either 3.5 mL of an acetonitrile–toluene mixture (65/35 v/v) or 3 mL of acetic acid/toluene (17/83 v/v), and 10 mg of MEH-PPV  in 2 mL of toluene. Among the different tested solvent combinations, those reported in the following allow the formation of a stable jet and uniform fibers to be obtained. Typically, fibers spun from acetic acid/toluene solutions feature more uniform emission, whereas the presence of acetonitrile favors the formation of a higher number of fluorescent bead-like structures . The MEH-PPV solution is stirred for 4 hours until complete dissolution of the polymer. After stirring vigorously the PEO solution for 8 hours, 500 μL of MEH-PPV solution is added. The resulting relative concentration of MEH-PPV/PEO is ∼1 : 100 (w/w), and this solution is stirred and ultrasonically agitated, thus becoming homogeneous. SiO2/Si (oxide thickness = 800 nm), cleaned with acetone in an ultrasonic bath and dried with pure air, is used as a substrate. The substrate is placed on a grounded metallic plate (collector), 500 μm below the needle tip to utilize the stable liquid jet region for a controllable deposition of the fibers.The light-emitting fibers are electrospun by mixing MEH-PPV with PEO, used as a polymer carrier. We dissolve separately 250 mg of PEO  and z stages (C-862 Mercury DC-motor Controller), allowing to control the collector movement by using dedicated software. The solution is loaded into the syringe, whose metallic needle is connected to the positive electrode of the power-supply and fed through the syringe needle at a constant rate (50 μL h–1). The applied electrostatic voltage is about 1.3 kV. During the deposition, the x–y stage velocity is 50 cm s–1. With these values of voltage and stage velocity, and at a tip–substrate distance of 500 μm, straight, continuous and uniform fibers are deposited. Lower stage velocities determine the formation of spiraling and buckling nanofibers, whereas non-continuous nanofibers would be deposited by using higher velocities. As a general rule, smooth and continuous fibers are formed when the stage translational velocity is as much as possible comparable to the deposition rate.16 The experiments are carried out at room temperature and in air atmosphere. The morphology of the electrospun nanofibers is examined by Scanning Electron Microscopy (SEM) using a FEG-SEM (LEO 1530) at 5–10 kV beam energy. Atomic force microscopy is performed by using a Multimode system equipped with a Nanoscope IIIa electronic controller (Veeco Instruments). Si cantilevers with a resonance frequency of 250 kHz are used to image the nanofiber surface topography in tapping mode.The aligned fibers are deposited using a home-built NF-ES apparatus, schematized in λ = 408 nm). The emission is collected by a 20×  or a 60×  objective and the intensity is measured by either a photomultiplier or a spectral detection unit equipped with a multi-anode photomultiplier (Nikon).Fluorescence micrographs of the nanofibers are acquired using a laser confocal microscope composed of a scan head  and an inverted microscope . Samples are excited by a CW diode laser , coupled to the microscope by a dichroic mirror and focused on the sample through the microscope objective (spot diameter about 3 μm). The fiber emission is collected by the same microscope objective and analyzed using a monochromator , equipped with a Charged Coupled Device (CCD) detector . This μ-PL system is also used for characterizing fiber waveguiding. Part of the light emitted by the fluorescent conjugated polymer, excited by the tightly focused laser beam, is coupled into the fiber and waveguided. The fiber optical losses can be evaluated by measuring the intensity of the PL signal diffused by the fiber surface, as a function of the distance, d, from the exciting laser spot.26 To this aim, the spatially resolved emission intensity map of a single light-emitting fiber is measured using a CCD camera (Leica DFC 490).The single-nanofiber emission is analyzed by using a micro-photoluminescence (μ-PL) set-up, based on an inverted microscope  equipped with a 60× oil immersion objective . The PL is excited by a diode laser , normally incident on samples. The beam polarization can be adjusted with respect to the nanofiber longitudinal axis by a h = 500 μm in our experiments, The key factor of NF-ES is the exploitation of the stable region of the extruded jet close to the metallic needle (2). The realized parallel fibers feature a very high degree of mutual alignment. The distribution of the angles formed by the fiber axes and the stage translational axis is characterized by a standard deviation <1°, much lower than the typical values reported in uniaxially aligned fibers realized by standard electrospinning systems.28 In –1) showing a more intense emission. We point out that these features can be observed only by confocal microscopy, which is sensitive to the active component of the fiber (MEH-PPV), whereas no internal structure is appreciable in fibers by SEM , or shared by neighboring polymer chains (interchain).37 Compared to intrachain excitons, the emission from interchain species is typically red-shifted and characterized by a lower quantum yield. The formation of interchain excitons can be favored by aggregation effects, generally occurring in solid state samples or in highly concentrated solutions.37 The observed blue-shift of the single fiber emission suggests a decrease of the emission from aggregates and interchain states in the nanofibers compared to the films, as observed also in MEH-PPV nanostructures produced by the standard electrospinning process.38However, the spectral shift measured here in this work between the emission from single fibers and from the reference film  is larger than in previous studies.39 leading to a distribution of the excited state energies. Once the exciton is created by absorption of light, it can undergo electronic energy transfer towards the lower energy sites,41 resulting in red-shifted emission.This effect can also be due to a reduced energy migration towards low energy sites. In fact, conjugated polymers are characterized by a broad absorption and emission spectrum, which is in part due to the inhomogeneous distribution of excited state energies, strongly correlated with the distribution of the conjugation lengths. The packing of the conjugated polymer backbones in different shapes breaks the conjugation length in sub-segments (conjugation lengths),43 The observed large blue-shift could in part be due to a reduced energy migration in the nanofiber that can be related to the molecular packing of the conjugated polymers in the fibers induced by near-field electrospinning. Several studies have evidenced the peculiar packing of the polymer macromolecules in electrospun nanofibers, induced by the applied electric field and by the stretching related to the high strain rate characteristic of the process.44This electronic energy migration depends also on sample morphology.I(θ), excited by a laser beam, whose linear polarization direction forms an angle, θ, with respect to the fiber axis. By this method the polarization dependence of the absorption process through the emission intensity can be probed.45θ, evidencing anisotropic polarized absorption and a predominant alignment of MEH-PPV optical transition dipoles along the nanofiber axis, since emission (absorption) is maximized for incident excitation polarization parallel to the fiber axis. For comparison, spincast films, where MEH-PPV molecules are expected to be randomly distributed, do not show any dependence of the emission intensity on the incident polarization direction [The packing and possible alignment of MEH-PPV macromolecules within the electrospun nanofibers are investigated through polarized excitation and PL spectroscopies. The polarized excitation measurements are accomplished by collecting the nanofiber fluorescence intensity, irection .M = (IMax – IMin)/(IMax + IMin) , are in the range 0.1–0.3, close to values calculated for an ensemble of MEH-PPV molecules with uncollapsed, defect-coil conformation, whereas in ensembles of single molecules spincast from solution higher values are typically measured that can be related to the presence of both collapsed and uncollapsed conformations.39 These results are substantiated by the analysis of the fluorescence polarization [inset of 16 In fact, the short needle–collector distance can disfavor the complete evaporation of the solvents from the spun jet. As a consequence, the presence of solvent residues in the deposited fibers can allow the polymer molecules to relax further to reach a more isotropic configuration following deposition and during solidification,32 thus decreasing the fluorescence degree of polarization. For light-emitting macromolecules, this eventual relaxational dynamics should also be related to the solvent quality, with good or poor solvents for the used conjugated polymer favoring typically more elongated or aggregated configurations, respectively.37Interestingly, the measured fluorescence intensity modulation depths, inset of , evidenc3 cm–1, comparable to similar active systems.45 We attribute optical losses mainly to scattering from the surface fiber microstructure evidenced in –1 level, suitable for on-chip waveguiding applications, by using specifically designed host–guest donor–acceptor systems.46 These experiments are currently in progress in our laboratories.Finally, we investigate the waveguiding properties of the light-emitting nanofibers, in view of their possible exploitation as active waveguides. In summary, we demonstrate the possibility of realizing ordered arrays of light-emitting conjugated polymer fibers by NF-ES. We realize arrays of parallel and crossed fibers, composed of nanostructures with a diameter of about 500 nm, emitting at 560 nm. The fibers show self-waveguiding of the emission with losses mainly limited by self-absorption and scattering from the fiber surface and internal nanostructure. The achieved high degree of control of fiber positioning would allow arrays of uniaxially aligned fibers to be realized, inheriting the anisotropic optical properties of single fibers. In perspective, the technique can be used to fabricate arrays of optically active nanofibers for sensing and photonic circuit applications and to deposit single active nanofibers in light-emitting devices.


MafB has important roles in cell differentiation and regulation of target gene expression; however, the mechanisms of this regulation and the identities of other transcription factors with which MafB interacts remain uncertain. Bioinformatics methods provide a valuable approach for elucidating the nature of these interactions with transcriptional regulatory elements from a large number of DNA sequences. In particular, identification of patterns of co-occurrence of regulatory Mafb deficient macrophages. GATA-1 motifs and MafB motifs could regulate expression of these genes in both a negative and positive manner, respectively. The validity of this conclusion was tested with data from a luciferase assay that used a C1qa promoter construct carrying both the GATA-1 motifs and MAREs. GATA-1 was found to inhibit the activity of the C1qa promoter with the GATA-1 motifs and MafB motifs.Here, the directional relationships among several functional motifs were evaluated using the Log-linear Graphical Model (LGM) after extraction and search for evolutionarily conserved motifs. This analysis highlighted GATA-1 motifs and 5’AT-rich half Maf recognition elements (MAREs) in promoter regions of 18 genes that were down-regulated in C1qa. In addition, these findings show that analysis of combinations of evolutionarily conserved motifs can be successfully used to identify patterns of gene regulation.These observations suggest that both the GATA-1 motifs and MafB motifs are important for lineage specific expression of  In recent years, genomic analyses have identified many short DNA sequences that function as transcriptional regulatory elements and also show evolutionary conservation. These signature sequences are usually referred to as ”motifs”. There is considerable interest in these motifs because variations in gene expression play crucial roles in many biological functions and are also of importance in disease etiology. Much of the information on the roles of these motifs has been obtained using microarray or qRT-PCR analyses to investigate the dynamics of gene expression. These technologies also provide insights into variation in cell fate decision, such as (re)differentiation or (dys)function. The genomic elements that control variations in gene expression, and hence cell fate, are those associated with transcription factors. As a consequence, considerable efforts are being made to detect and characterize these motifs.Three procedures are widely employed to identify motifs: sequence alignment, motif extraction and motif search. Sequence alignments make it possible to identify biologically meaningful regions 2]. In o. In o2].Many approaches have been employed to investigate motif interaction, such as regression methods . HoweverMafb KO mice  from Step1-3.p-value were also calculated with the defined formula. The inclusion of 10 functional motif candidates increased drastically the number of degrees of freedom, making it difficult to assess a final RM by tests based on model deviance. A final model was therefore selected when the p-value became lower than 0.001 was written owing to the long length of the motif name. The deviance and 01Figure . The finh Figure . Ten comh Figure . The posh Figure . AP2, ZIGenes with a combination of functional motif candidates in their promoters are indicated by a star in Table . SeveralMafb deficient macrophages, respectively”.”GATA-1 motifs and MafB motifs negatively and positively regulate expression of 18 genes that are down-regulated in The analysis provided insight into the cooccurrence of the functional motifs required for the regulation of MafB target gene expression. A hypothesis on motif combinations was constructed and the modeling of this hypothesis is shown in Figure . The hypC1qa and Cd5l  promoters considerably impaired promoter activity in RAW264.7 macrophage cells. Cotransfection of the luciferase reporters driven by the gene promoters along with MafB expression vectors dramatically decreased luciferase gene expression.The results from biochemical experiments were used to confirm the binding of MafB to the MafB motif and its regulation of transcription. Two of the mouse genes present in the set of 18 down-regulated genes were subjected to a luciferase assay. Mutations of MARE in C1qa (data not shown), and -54bp upstream of the start site in Cd5l (data not shown). These results indicate that the MARE immediately upstream of the transcription start site is required to regulate C1qa and Cd5l  promoter activity. Additionally, this confirms that MafB binds to the MafB motif with MARE to regulate transcription, as the MAREs were located in the ID 10-10 and ID 10 motifs. Another luciferase assay was performed with the same experimental methods using C1qa promoter to determine whether the GATA-1 and MafB motifs were positioned within 400bp upstream and 100bp downstream of the transcription start site   Table . To inver Figure .The 4 nucleotides in GATA-1 motif are often the same as the 6 nucleotides of MAREs. This suggests that MafB binding may be prevented by GATA-1, or that both of motifs with these proteins may bind each other for transcriptional or translational repression. GATA-1 and MafB may influence the level of mRNA expression in MafB target genes and thereby cause the generation of abnormal protein levels. As a consequence, defective MafB regulation may underlie abnormal functions in macrophages. GATA-1 has been shown to suppress monocyte differentiation in bone marrow, and to inhibit macrophage differentiation and apoptosis ,27. Thesα, GATA-1, or GATA-2 strongly enhances the macrophage differentiation potential of Pax5 KO pro-B cells under lymphoid culture conditions, whereas GATA-1 expression induces erythroblast development [http://macpak.csml.org/click/index.php?q=d:18472258). The transdifferentiation of pro-B cells to macrophages or erythrocytes may be determined by their MafB and GATA motifs. Thus, through investigation of co-occurring motifs it may be possible to identify the switches for cell differentiation.The GATA-1 is generally required for the differentiation and proliferation of erythrocytes, and the MafB is known to be essential for maintaining macrophage function. The co-occurrence of GATA-1 and MafB motifs initially seems to be contradictory with respect to macrophages. However, it has been reported that in macrophages MafB is a repressor of Ets-1, an inhibitor of erythrocyte differentiation in the chicken . Anotherelopment ,35(MACPAMafb deficient macrophages, respectively. This result was verified experimentally. By combining the bioinformatics analysis and the experimental approach, it was suggested that both the GATA-1 motifs and the MafB motifs are important for expression of C1qa. Through the use of the bioinformatics methods, the regulatory motifs related to MafB have been identified and their interactions were suggested. The findings here also suggest that combinations of evolutionarily conserved motifs are capable of predicting gene regulation. It is to be expected that future studies will elaborate on and develop these findings. For example, further research on the control of gene expression may discover rules for the co-occurrence of particular motifs in the genomes. Eventually, these new techniques for identifying regulatory elements should help to accelerate development of applications in medical sciences and lead to elucidation of the causes of different diseases.Identification of combinations of transcriptional regulatory elements from large amounts of DNA sequences can benefit greatly from proposed bioinformatics methods as the multiple alignments and the LGM. In particular, the pattern of co-occurrence of motifs is proposed to provide a strong means for directing potential interaction of transcription factors not known from biochemical studies. The hypothesis postulates that GATA-1 motifs and MafB motifs negatively and positively regulate expression of 18 genes that are down-regulated in C1qa: complement component 1, q subcomponent, a chain; DBTSS: database of transcriptional start sites; DNR: doubly-nested randomized iterative; DNA: deoxyribonucleic acid; FM: full model; GM-CSFR: granulocyte/macrophage-colony stimulating factor receptor; GATA: gata-binding protein; HDAC: histone deacetylase; KO: knockout; LGM: log-linear graphical model; Maf: musculoaponeurotic fibrosarcoma; MARE: maf recognition element; MEME: motif-based sequence analysis tools; MZF1: myeloid zinc finger gene 1; N: nucleotides; NCBI: national center for biotechnology information; ORI: over-representation index; RM: reduced model; ZID: zinc fingerprotein with interaction domain; PRRN: multiple sequence alignment program; SWG: Smith-Waterman-Gotoh algorithm.AML1: acute myeloid leukemia 1 gene; AP4: transcription factor ap4; cDNA: complementary dna; The authors declare that they have no competing interests.MM developed the bioinformatics methods, implemented the algorithms, carried out the data analysis, and drafted and wrote the manuscript. MH had made substantial contributions to acquisition of microarray data, and was involved in critical study of important intellectual content. MH designed, MN and MM carried out the experiments. ST conducted the supervision of the study and manuscript preparation, and gave final approval of the version to be published. All authors read and approved the final manuscript.


While new drugs aimed at BRAF-mutated cancers are entering clinical practice, cells and tumors with activating Ras mutations are relatively resistant to those and quite a few other anti-cancer agents. This inspires the effort to reverse this resistance or to uncover new vulnerabilities in such resistant cancers. IPA3 has been originally identified as a small molecule inhibitor of p21-activated protein kinase 1 (PAK1), a candidate therapeutic target in human malignancies. We have tested a battery of melanoma and colon carcinoma cell lines that carry mutations in BRAF, NRAS and KRAS genes and have observed that those with NRAS and KRAS mutations are more sensitive to killing by IPA3. Genetic manipulations suggest that the differential response depends not just on these oncogenes, but also on additional events that were co-selected during tumor evolution. Furthermore, sublethal doses of IPA3 or ectopic expression of dominant-negative PAK1 sensitized Ras-mutated cells to GDC-0897 and AZD6244, which otherwise have reduced efficiency against cells with activated Ras. Dominant-negative PAK1 also reduced the growth of NRAS-mutated cells in confluent cultures, but, unlike IPA3, caused no significant toxicity. Although it remains to be proven that all the effects of IPA3 are exclusively due to inhibition of PAK1, our findings point to the existence of selective vulnerabilities, which are associated with Ras mutations and could be useful for better understanding and treatment of a large subset of tumors. Discovery of unique vulnerabilities in cancer cells has emerged as a major starting point for anti-cancer drug development. Serendipitous discovery of cancer-specific toxins is now supplemented, if not yet supplanted, by targeted development of small molecule modulators of specific biochemical functions. Synergistic advances in cancer genetics and in general knowledge of cellular metabolism and signal transduction produce the ever-growing list of desirable therapeutic targets. The corresponding small molecule inhibitors, either empirically discovered or rationally designed, are currently transiting from pre-clinical pipelines into the armamentarium of clinical oncology.A prominent example of this phenomenon is the emergence of candidate drugs that target various components of MAP kinase cascade. BRAF gene product is targeted by several of such compounds , 2 and iP21-activated kinases or PAKs are a family of evolutionary conserved enzymes that were originally identified as downstream effectors of Rho GTPases. Group I PAKs, which include PAK1, PAK2 and PAK3, have the same general architecture, but distinct and, occasionally, opposite biological roles. PAK1 in particular has been associated with a variety of pathological conditions, including cancer (reviewed in ). PAK1 iGenetic data suggests that mutations in Ras and BRAF oncogenes are somewhat equivalent during the early stages of tumor development. This is inferred from, generally, mutually exclusive occurrence of these changes , 28. NRAWe examined five melanoma lines, three of which were BRAF- and two NRAS-mutated, and four colon carcinoma lines, of which three were KRAS- and one BRAF-mutated. The cells were treated with various doses of IPA3 and the response was recorded by microphotography and by methylene blue staining and extraction method Figure . We obseThe connection between oncogenic Ras and such downstream effectors as ERKs is believed to be principally mediated by CRAF kinase, which, in turn, supports the activity of ERK activators, MEKs. However, other Raf kinases, such as BRAF, could still be present in the same cell. Although some functions of CRAF and BRAF are similar, the contribution of BRAF to growth and survival of such cells is expected to be overshadowed by that of Ras/CRAF axis. This is best attested by insensitivity of such cells to BRAF inhibitors. We have examined whether IPA3 treatment affects the response of Ras-mutated lines to BRAF inhibitor GDC-0879. As expeIn a reciprocal experiment, ectopic expression of activated NRAS protected cells from BRAF inhibitor GDC-0879 Figure , but faiWe used a dominant-negative form of PAK1 (PAK1-K299R) as an alternative way of affecting the function of PAK1 in Ras-mutated cells. In comparison with an empty vector, PAK1-K299R sensitized cells to GDC-0879 Figure , AZD6244Differential sensitivity to IPA3 in Ras-mutated cell lines and the ability of this compound, even in sublethal concentrations, to sensitize these cells to other impacts point to an important vulnerability, which might be clinically explored in a single-agent or combined therapy. The biochemical basis of the difference in drug response between BRAF- and Ras-mutated cells remains unclear. Our observations suggest that this phenomenon is not a direct consequence of the activation of these oncogenes: when we attempted to transfer the oncogenes between the cell lines, the status of IPA3 resistance did not follow the transgene. It is tempting to speculate, that a PAK1-like function is critical to these cancers, but in Ras-mutated cells the signal to PAK1 originates directly from Ras, while BRAF needs a cooperating event to achieve the same goal. Assuming that the cooperating event is IPA3 insensitive, this hypothesis agrees with our observations, although alternative explanations are possible as well. The scenario, in which genetic events that provide alternative solutions to the same hurdle in the early cancer development steer the evolution of the disease into significantly different paths, is well-known from other systems (e.g. )An important issue in this and other works that utilize IPA3 is how specific this compound is. Its original characterization included a diligent study of more than two hundred other kinases, of which only a handful were affected . HoweverIn our experiments, dominant-negative PAK1 resembled IPA3 in that it was able to sensitize Ras-mutated cells to a BRAF inhibitor. Although GDC-0897 is very specific against BRAF-mutated cells , we cannThe same transgene also sensitized to IPA3 itself, which would be expected if the two impacts target the same biochemical process. Sensitization to AZD6244 (aka Selumetinib) is in particular important, as it points to an avenue of increasing the efficacy of the compound, which is currently entering clinical practice. This may be especially interesting in the view of the earlier reports that Ras-mutated cells are, generally, more resistant to MEK inhibitors than the BRAF-mutated ones , 32, andIt is important to note that expression of PAK1-K299R did not recapitulate the pronounced toxicity of IPA3. The most trivial explanation for our results is that the expression of the transgene varied between individual infected cells and, on average, was insufficient to achieve complete inhibition of the endogenous PAK1. This may explain why confluent cultures of those cells appeared more heterogeneous according to the measurements of cell density Figure  and visuAn unbiased genetic screen  for the The cancer cell lines used in this study included: BRAF-mutated melanomas , NRAS-mutated melanomas , BRAF-mutated colon carcinoma (COLO-205), KRAS-mutated colon carcinomas . All cells were cultured in humidified chambers at 37°C and 5% CO2 in high-glucose DMEM supplemented with L-glutamine (4 mM), fetal bovine serum (10%), penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were free of mycoplasma contamination, as tested using MycoAlert Mycoplasma Detection Kit (Lonza).Recombinant retroviral stocks were produced by co-transfecting 293T cells with an appropriate vector and a packaging construct . Transfections were carried out using Lipofectamine Plus (Life Technologies Corporation) according to the manufacturer's recommendations. Viral stocks were applied as previously described .Dominant-negative PAK1 expression vector was described earlier . ConstruRelative cell numbers were compared using methylene blue staining/extraction method. Briefly, cells were rinsed with phosphate-buffered saline, fixed in methanol and stained with a 2% solution of methylene blue in a 1:1 mix of water and methanol. Unincorporated dye was washed away with deionized water; the remaining dye was extracted in 1.0 M HCl and quantified by spectrometry at 600 nM.


Ecosystem-based forest management strives to develop silvicultural practices that best emulate natural disturbances such as wildfire to conserve biodiversity representative of natural forest ecosystems. Yet, current logging practices alter forest structure and reduce the proportion of old-growth forest and, consequently, can exert long-term effects on the dynamics of forest biota. The stand- and landscape-scale factors driving bird community dynamics in post-disturbance environment remain poorly understood. In this study, we examined bird community dynamics along successional gradients in boreal ecosystems originating from fire and logging in landscapes dominated by old-growth forest. We tested if bird species richness and community compositions in clear-cutting stands became comparable to those in natural stands after 70 years, and identified the relative contributions of stand- and landscape-scale forest attributes in bird community dynamics. Based on records of bird occurrences at 185 field sites in natural and clearcutting stands, we demonstrate that (1) both forest structures and bird communities underwent evident changes along successional gradients in post-clearcutting environment; (2) bird species richness and community composition in 60- to 70-years-old clearcutting stands still differed from those in 50- to 79-years-old natural stands, in spite of the fact that most forest attributes of clearcutting stands became comparable to those of natural stands after 40 years; and (3) landscape disturbances contributed more than stand characteristics in explaining the lack of convergence of mature forest species, residents, and short-distance migrants in post-clearcutting environment. Our study points out that more regards should be paid to improve the landscape configuration of the managed forests, and implies that old-growth forest retention within logged areas, combined with selection cutting and prolonged logging rotations, can better emulate fire and alleviate forest harvesting effects on bird community assemblages typical of natural boreal ecosystem. Sustainable forest management strategies strive to maintain the biodiversity of forest ecosystems Clearly, such differences between logging- and fire-originated forests will have significant effects on the assemblage and dynamics of the forest fauna. Indeed, birds are known to be sensitive to the variation in habitat characteristics and successional stages An additional and more fundamental question for long-term conservation of biodiversity is whether an altered community in managed forests would approach the status of a natural community after a period of time. Disturbances, whether they are natural or anthropogenic, typically lead to the abrupt loss of biomass or biodiversity, but they also can generate novel environments that trigger ecological succession The current study characterises bird community dynamics in boreal forests originating from fire and logging in the Côte-Nord region of Québec, Canada. This region has a relatively long fire cycle, which ranges from 270 years in the west to >500 years in the east Picea mariana (Mill.) BSP) and balsam fir  Mill.). Several other tree species are frequently observed, including white spruce (Picea glauca (Moench) Voss), white or paper birch (Betula papyrifera Marsh.), trembling aspen (Populus tremuloides Michx.), and jack pine (Pinus banksiana Lamb.) The study was conducted in boreal forest of the Côte-Nord region of Québec, Canada . The region is characterized by a humid climate, where mean annual temperature ranges from −2.5 to 0.0°C and annual precipitation ranges from 1000 to 1400 mm, with 35% falling as snow Our sampling sites were distributed in natural forest stands versus stands originated from clearcutting (hereafter clearcutting stands). The natural stands consist of forest stands that originated from fire and which had not been logged. The ages of the natural stands ranged from 50 to 225 years (mean  = 135 years), of which 65% were >120-years-old We surveyed breeding birds using the fixed-radius point count method 2 circular plot (radius: 11.3 m) centred on each sampling site and recorded the diameter at breast height (DBH) of every living tree (species identified) and snag having DBH ≥9 cm. Then we calculated the basal areas of conifers and snags based on these measures. We also treated living trees with DBH <9 cm as saplings and counted the number of them within each plot. Then we calculated the sapling densities. We also measured canopy covers using spherical densiometer. These variables were chosen to represent stand-level forest characteristics because previous studies conducted in the same area have shown that these variables can explain the majority of forest structure We characterized sampling sites at the stand and landscape scales. At the stand scale, we established a 400 mAt the landscape scale, we used forest inventory maps, managed with ArcGIS 10 , to characterize the landscape mosaic at different spatial scales. In the forest inventory maps stands were digitalized as polygons which constituted baseline information such as stand origin  and stand age for the planning of logging operations by forestry companies. We then quantified the proportion of logged areas within buffer areas centred on the sampling sites at four spatial scales, each being matched to a radius of 200 m, 500 m, 1 km, and 2 km, respectively.We compiled bird species presence-absence in each sampling site. First, we examined whether stand structure  differed among any age classes of natural and clearcutting stands, using one-way Multivariate Analysis of Variance (MANOVA). Age class included three categories of natural stand and four categories of clearcutting stand (see above). If a significant difference in stand structure was detected, we conducted one-way Analysis of Variance (ANOVA) for different stand characteristics to examine which ones differed among age classes. Holm–Bonferroni method was applied in the ANOVAs to reduce the probability of making type I error so that the family-wise type I error probability was less than 0.05. Each ANOVA was followed by Tukey multiple comparisons to examine (1) if there was a difference between individual age classes of clearcutting stands, and (2) if there was a difference between 60- to 70-years-old clearcutting (C-4) stands and 50- to 79-years-old natural (N-I) stands. A difference between any age classes of clearcutting stands would indicate evident changes in stand structure along successional gradients in post-clearcutting environment, and a difference between C-4 and N-I stands would indicate poor comparability in stand structure between clearcutting and natural stands of similar ages.We further conducted constrained ordination to understand the relative positions of sampling sites in a multi-dimensional space characterized by environmental variables, including stand characteristics, landscape disturbances , and geographic positions (i.e. latitude and longitude). Community variations across sampling sites were projected to a multi-dimensional space “constrained” by these environmental variables. Each of the ordination axes was a linear combination of the environmental variables and all the ordination axes were orthogonal to each other.Second, we compared mean species richness  between different age classes of natural and clearcutting stands. To do so, we first applied a hierarchical modelling approach to estimate species richness for each site while accounting for imperfect detection of individual species F-statistic and corresponding P-value based on permutations of species compositional data. A significant PERMANOVA Pseudo-F-value may indicate a difference in dissimilarity among the treatments, different within-treatment dispersion, or both conditions. We used a principal component ordination plot based on species composition dissimilarity matrix (Sørensen) to visualize compositional differences between treatments and the composition dispersion of treatments from their respective community centroids F-statistics based on any distance measure and following permutation of the observations. We permuted the data 1000 times for each test. When significant results were obtained from PERMDISP, we carried out pairwise tests using Tukey multiple comparisons (Tukey's Honestly Significant Difference test).We also examined the compositional differences (beta diversity) among treatments using a non-parametric test, viz., Permutational Multivariate Analysis of Variance (PERMANOVA) K possible models, in which K is the total number of explanatory variables. The increase in model fit generated by an explanatory variable is estimated by averaging the pairwise improvements of the 2K−1 models that include this variable over the 2K−1 models without this variable. This process is repeated for K times to partition the variances so that the total independent contribution of each explanatory variable can be estimated. By these means, hierarchical partitioning distinguish those variables that have high independent, not partial, correlation with a response variable from variables that have little independent effect on the response variable. The latter may still have a high pair-wise correlation with the response variable but this is due to joint action with other explanatory variables. We used the log-likelihood of the models as the measures of goodness-of-fit. These two approaches were likely to alleviate multicollinearity problems that were otherwise ignored by using any single-model technique.Third, we conducted multiple regressions and model averaging to examine the relative importance of the environmental variables in explaining total- or group-richness. Because the estimated richness, unlike the observed richness, was not a count number, it was log transformed and Gaussian models were conducted. In each regression analysis, total- or group-richness was treated as the dependent variable, and the environmental variables (see above) were the independent variables. In the model averaging protocol the relative importance of each explanatory variable was calculated as a sum of the Akaike weights over all of the models in which the parameter of interest appears All analyses were performed in R2.15.2 (R Development Core Team 2012).No specific permissions were required for our study area and locations because it was not a protected area or private land under restriction from entering. Our field studies did not involve endangered or protected species. No approval was necessary for the data collection procedure because no captures were done; the study was only observational.F24,712 = 14.96, P<0.001). One-way ANOVA, followed by Tukey multiple comparisons, indicated significant increase in basal area of conifer , whereas canopy cover was higher in C-4 stands than in N-I stands (P = 0.007). In addition, Tukey multiple comparisons showed that C-4 and N-I stands were indistinguishable in their sampling density (P = 0.543) and basal area of snag  were known to be associated with mature forest, 14 (20.0%) with young forest, 12 (17.1%) with shrub land, and 16 (22.9%) were generalists. These species included 13 (18.6%) residents, 23 (32.9%) short-distance migrants, and 34 (48.6%) Neotropical migrants .F6,178 = 14.30, P<0.001), and Tukey multiple comparison indicated that total richness did not change after clearcutting (P>0.544), and remained lower than that in natural stands after 60 to 70 years . The richness of species associated with shrub land was high immediately after clearcutting, and became as low as that in natural stands after 20 years . There were differences in species richness between age classes for young forest species , and the richness of young forest species was lower in 40- to 59-year-old or 60- to 70-year-old clearcutting stands than in 80- to 120-year-old natural stands (Tukey multiple comparison: P<0.001), and lower in 60- to 70-year-old clearcutting stands than in natural stands that were older than 120 years , and the richness of Neotropical migrants was higher in 20- to 39-year-old clearcutting stands than in natural stands that were older than 120 years  and Neotropical migrants (P = 0.999). There was no difference in the richness of generalists between any age classes , indicating that some age classes had higher variability in bird composition than others. Tukey multiple comparisons showed that C-4 clearcutting stands had higher dispersion than 5- to 19-year-old (C-1) clearcutting stands , whereas the centroid of bird composition did not show a significant shift in post-clearcutting environment . Likewise, basal area of conifer was significantly negatively correlated with the richness of generalists and Neotropical migrants, and stand characteristics had greater independent contribution than landscape disturbance in explaining the variance of these two groups of species . Canopy cover, basal area of snag, and the proportion of disturbed area at 200 m scale were significantly negatively correlated with the richness of young forest species, and stand characteristics had greater independent contribution than landscape disturbance in explaining the variance of the richness of young forest species, although the difference seemed to be less evident .Note that latitude and longitude were included in these analyses to control for the effects of geographic positions. Thus the relative independent contributions of stand characteristics and landscape disturbance reported above were not a result of the geographic positions of the sampling sites. More details concerning the log-likelihood and Akaike information criterion (AIC) values of the models could be seen in Our study demonstrates that both forest structures and bird communities underwent evident changes along successional gradients in post-clearcutting environment. However, bird species richness and community composition in 60- to 70-years-old clearcutting stands still differed from those in 50- to 79-years-old natural stands, in spite of the fact that most forest attributes of clearcutting stands became comparable to those of natural stands after 40 years. Although both stand characteristics and landscape disturbances contribute in explaining the variance of species richness, landscape disturbances contribute much more than stand characteristics in explaining the variance of the richness of mature forest species, residents, and short-distance migrants, which are the species groups whose richness were maintained at lower levels in clearcutting stands than in natural stands after 60 to 70 years.Our study shows that the early post-clearcutting stands supported bird assemblages with low variability in species composition, which may reflect the fact that clearcutting creates a relatively homogeneous habitat for the few bird species that are typically associated with disturbed, early-successional environments Although bird community composition underwent evident changes along successional gradients in post-clearcutting environment, such changes mainly reflected the increase in compositional variability rather than a shift of compositional centroid. In addition, the centroid of bird community composition in clearcutting stands remained different than that in natural stands after 60 to 70 years. Such finding is consistent with the group-specific patterns in species richness that, although the richness of young forest species, shrub land species, generalists, and Neotropical migrants in late post-clearcutting stands were comparable to those in natural stands, the richness of mature forest species, residents, and short-distance migrants in clearcutting stands remained lower than those in natural stands after 60 to 70 years.It is increasingly recognized in the ecological literature that different groups of species may respond differently, and even in opposite manners, to the same underlying causal factor Some of the previous studies are apt to classify species into groups according to their migratory status It is evident that logged areas support bird communities that are easily distinguishable from those of natural forest Our study shows that neither species richness nor community composition of bird in the clearcutting stands approached that of natural stands, even though 70 years had elapsed following harvest. Further, our study shows that such differences mainly reflected the lack of convergence of some, but not all, characteristic groups, namely mature forest species, residents, and short-distance migrants. Other studies likewise found that abundances of bird species associated with early-successional habitats were maintained at high levels, whereas abundances of mature forest birds remained low in managed forest landscapes As a matter of fact, stand characteristics could only explain a small proportion of variance of species richness in compare with landscape disturbances. This is particularly true for mature forest species, residents, and short-distance migrants, which are the groups that lack of convergence in post-clearcutting environment. Thus our study shows that the effects of clearcutting are at landscape scale. In particular, the proportion of disturbed area at 200 m to 1 km scales negatively affected the richness of residents, indicating that the effects of logging may well extend to 1 km. Further, the result holds when the effect of stand age is controlled by limiting the analysis to 60- to 70-year-old clearcutting stands and 50- to 79-year-old natural stands. Although late post-clearcutting stands largely regain their pre-logging characteristics in term of forest structure, they are embedded in a disturbed matrix and probably suffer from matrix-effects from surrounding disturbed areas Our study shows that, in landscapes dominated by old-growth forest, clearcutting would have a profound influence on bird community composition and species richness, and ultimately, on ecosystem functioning Because forest stands experienced clearcutting are highly distinguishable from natural stands in terms of forest structure and biodiversity patterns, silvicultural practices have attempted replacing clearcutting with harvesting system such as selection cutting that may better protect stand structure Another stream of prescription to improve the current forest practices is the one that suggests prolonging logging rotation. Socioeconomic values create strong incentives for reducing as much possible the time required before a given forest stand can be reharvested. A rotation cycle as short as 40 years has been commonly suggested Studies suggest that old-growth forest stands should be retained within the logged area Our study provides information that can aid the development of forest management strategies that should be effective in alleviating effects of forest harvesting on bird assemblages in boreal landscapes dominated by old-growth forests. The basic principle is to develop forest practices that can well simulate natural disturbance regimes at both stand and landscape scales. So far many prescriptions have been put forward and each of them can help to achieve the above goal in one way or another. However, it is highly needed to integrate these prescriptions so that their positive effects could be combined. In particular, more regards should be paid to improve the landscape configuration of the managed areas.Table S1The proportion (in %) of sampling sites in which each bird species was recorded in natural and clearcutting stands, and the species' habitat association and migratory status.(DOCX)Click here for additional data file.Table S2The relative importance and independent contribution of environmental variables in explaining the variance of total- and group-richness calculated from (a) all data and (b) 60- to 70-year-old clearcutting stands and 50- to 79-year-old natural stands only.(DOCX)Click here for additional data file.Table S3The basic information of models used in model averaging and hierarchical partitioning procedures for (a) all data and (b) 60- to 70-year-old clearcutting stands and 50- to 79-year-old natural stands only.(DOCX)Click here for additional data file.


Neurofibromatosis type-1 is a common genetic disorder which often affects the skeleton. Skeletal manifestations of neurofibromatosis type-1 include scoliosis, congenital pseudarthrosis of the tibia and intraosseous cystic lesions. Dislocation of the hip associated with neurofibromatosis type-1 is a rare occurrence and is underreported in the literature.We report a case of hip dislocation resulting from an intra-articular neurofibroma in an 18-year-old Caucasian woman following minor trauma. This was originally suggested by the abnormalities on early radiographs of her pelvis and later confirmed with computed tomography and magnetic resonance imaging. Treatment was successful with skeletal traction for six weeks with no further hip dislocations at a 12-year follow-up.This case illustrates the radiological features of this rare complication of neurofibromatosis type-1 using the modalities of plain radiograph, magnetic resonance imaging and computed tomography reconstruction. The radiological images give a clear insight into the mechanism by which neurofibromatosis type-1 leads to hip dislocation. It also demonstrates one treatment option with excellent results on long-term follow-up. Neurofibromatosis type 1 (NF-1) is one of the most common autosomal dominant disorders affecting humans. It is estimated to affect 1 in 3,000 newborns and 1,000,000 people worldwide [The clinical features of NF-1 include: cafβ-au-lait spots, Lisch nodules, axillary freckling, optic gliomas and peripheral neurofibromas. NF-1 is a disease deeply relevant to orthopaedic surgery. Patients with NF-1 may present with characteristic orthopaedic manifestations such as scoliosis, congenital pseudoarthrosis of the tibia and limb hypertrophy. IntraosDislocation of the hip associated with NF-1 is a rare occurrence. A comprehensive review of the literature revealed 12 cases of hip dislocation attributed to NF-1. We report a case of recurrent hip dislocation resulting from an intra-articular neurofibroma in an 18-year-old woman.An 18-year-old Caucasian woman with a history of NF-1 presented to the emergency department with pain in her left hip following minor trauma. She had tripped over her dog and landed on her left side. NF-1 had been diagnosed clinically in childhood. She had a histologically proven neurofibroma excised from her right forearm four years previously. She had a strong family history of NF-1, her mother and three second degree relatives exhibited clinical features.On examination, we saw that her left leg was shortened, internally rotated, and adducted. There was decreased range of movement. She had diffuse swelling of her left lower limb with a distinct soft tissue mass above her right lateral malleolus. She had six cafβ-au-lait patches on her trunk and bilateral axillary freckling. A radiograph of the pelvis revealed a superior dislocation of her left hip with an abnormal appearing femoral neck Figure .Her hip was relocated under general anaesthetic and this was maintained with skin traction. A computed tomography (CT) of her pelvis displayed a smooth erosion of the lateral margin of her left ileum and femoral neck, markedly increased femoral neck offset, a concave abnormality superior to the left acetabulum and thinning of the left inferior pubic ramus. These changes, which appeared to be long standing, were accepted to be a result of a local neurofibroma causing bone erosion. Eight days post-operatively she experienced a sudden onset of left hip pain on attempting to move in bed. A radiograph of her hip revealed repeat dislocation. Relocation of her hip was performed under general anaesthetic and balanced skeletal traction was maintained by inserting a pin to her left proximal tibia. Skeletal traction was maintained for six weeks. She was mobilizing without aids at eight weeks and follow-up at three months revealed a normal hip examination.She presented to the orthopaedic clinic six years later complaining of disfiguring hypertrophy of her left lower limb which had worsened markedly over the preceding two years. She was referred to plastic surgeons with a view toward performing a cosmetic debulking procedure. A pre-operative magnetic resonance imaging (MRI) scan of her lower limb demonstrated soft tissue swelling of her entire lower limb consistent with plexiform neurofibromatosis Figure . An MRI Neurofibromatosis type-1 is a common genetic disorder that can have both focal and generalized skeletal manifestations. Generalized skeletal manifestations such as osteoporosis and short stature are common. Focal abnormalities such as tibial dysplasia, short angle scoliosis and sphenoid wing dysplasia, although less common, are well documented in the literature. However, there is a relative paucity of reported cases of pathological hip dislocation in patients with NF-1, with only 12 documented cases found in the published literature. Six dislocations occurred following trivial trauma -8and sixThe suggested mechanism for dislocation for the majority of cases has been related to the intra-articular growth of neurofibromas ,8,10,13.Treatment described has ranged from conservative approaches to definitive surgical intervention, including Girdlestone resection  and totaFor our patient her hip dislocation was accepted to be a result of a neurofibroma impinging on the hip joint. This was originally suggested by the abnormalities on early radiographs of her pelvis and later confirmed with CT and MRI. The CT reconstructions of the hip area clearly demonstrate the mechanism by which hip dislocation has occurred Figure . The smoThis case illustrates the radiological features of this rare complication of NF-1 using the modalities of plain radiograph, MRI and CT reconstruction. The radiological images give a clear insight into the mechanism by which NF-1 leads to hip dislocation. It also demonstrates one treatment option with excellent results on long-term follow-up.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.JG and JB collected data and drafted the manuscript. JH conceived the report. All authors critically appraised the manuscript and approved the final text.


Mangalica breeds are indigenous to Hungary and their breeding history dates back to about 200–250 years ago. They are fat-type pigs and have a rare curly hair phenotype. The aim of our study was to establish the relationships between these unique breeds and other European breeds.Based on a core sequence of 382 bp present in 2713 mitochondrial D-loop sequences from pigs belonging to 38 local breeds from nine countries, five cosmopolitan breeds and wild boars from 14 countries, we identified 164 haplotypes. More than half of the 2713 sequences belonged to either four haplotypes characteristic of continental European breeds or two haplotypes characteristic of British/cosmopolitan breeds; each haplotype is present in more than 100 individuals. Most Mangalica individuals belonged either to one of these common continental European haplotypes or to two Mangalica-specific haplotypes that were absent in all other breeds. In addition, we identified the ancestral mitochondrial D-loop signature present in these 2713 sequences and found that ~ 80% carried the European ancient signatures, ANC-Aside and ANC-Cside or their closely related signatures, while most of the remaining sequences carried a modern Asian signature, ANC-Easia. Mangalica individuals carried the ANC-Aside signature, but not the ANC-Cside or ANC-Easia signatures.In all the Mangalica individuals, a unique ancient European signature was found in the mitochondrial DNA D-loop region, but they belonged almost exclusively to either certain very abundant European or two Mangalica-specific D-loop haplotypes. This indicates that the present-day Mangalica population in Hungary evolved either by introgression of other European breeds and wild boars or via total isolation after the divergence of European ancient porcine bloodlines. There are hundreds of modern pig breeds and many of these are local breeds whereas only a few cosmopolitan breeds are used in the meat industry . The thrAlthough a number of studies have been conducted to examine the diversity, relationship and introgression between European pigs -16, MangFor this purpose, mtDNA D-loop sequences of 195 Mangalica individuals from a previous study  and 2518Six major haplotypes, each one observed in more than 100 individuals, were identified i.e. HAP07, HAP08, HAP09, HAP13, HAP56 and HAP57. Haplotypes HAP07 and HAP08 correspond to the core European haplotypes C and A, respectively, as described previously ,18. In tAmong the 195 Mangalica individuals from our previous study , 56 beloST and Nei’s genetic distance indices for breeds for which mtDNA D-loop sequences were available for ten or more individuals. The results revealed that the within-breed diversity of the Mangalica breeds was similar to that of local Spanish and Italian breeds and that the Mangalica breeds were more homogeneous than the cosmopolitan and British local breeds and certain South-European wild boars .We computed the Fs Figure . The Swaes Table . The Redes Table . With thAn analysis on 208 European archaeological pig specimens identified five mtDNA D-loop signatures: ANC-Aside, ANC-Cside, ANC-Italy, ANC-Y1-6A and ANC-Y2-5A . Of thesOur analysis revealed that individuals that carried the major European haplotypes HAP07 or HAP09, HAP08 or HAP13 and HAP56 or HAP57 also carried ANC-Cside, ANC-Aside or ANC-Easia signatures, respectively [see Additional file While Mangalica breeds can belong to several haplotypes, almost 50% of the breeds analyzed displayed only two haplotypes, HAP15 and HAP16, which were not present in any of the other breeds and wild boars studied. Furthermore, all 203 Mangalica individuals in this study carried the ANC-Aside signature.Both the ANC-Aside and ANC-Cside signatures and the haplotypes HAP08 and HAP07 that carry them differ by one nucleotide substitution. Due to the difference between the lengths of the signature and the haplotype sequences [see Additional file There are several possible explanations for the unique signature/haplotype characteristics of the Mangalica breeds. One hypothesis is that ancestors of the current Mangalica population in Hungary that carried the ANC-Aside signature and of the European wild boar populations that carried the ANC-Cside signature became geographically isolated during prehistoric times. Another possibility is that the ANC-Aside signature evolved from the ANC-Cside signature in the Mangalica ancestor species after isolation. It cannot be excluded that either the population bottleneck that occurred in the Mangalica population during the 1960’s  or breedIn summary, we conclude that the present-day Mangalica population in Hungary may have originated by two different mechanisms: either introgression of common European bloodlines into the Mangalica breeds or by total isolation of some Mangalica ancestor species, as suggested by the presence of both common European and Mangalica-specific mtDNA D-loop haplotypes in the population.The authors declare that they have no competing interests.FM conceived the study, performed the haplotype and network analyses, and drafted the manuscript; JM performed the diversity analyses; GT participated in data analyses and helped to draft the manuscript; VS performed data collection and management; EB helped to analyse data and to draft the manuscript. All authors read and approved the final manuscript.Breeds used in this study with their location and number of individuals. The file provides information about the breeds, their location and GenBank accession number of the individuals’ mitochondrial D-loop sequence with references [ferences ,20,23-33Click here for fileMaterials and methods. The file provides information about the methods with references [ferences ,34-41 thClick here for filePolymorphic positions in the mtDNA D-loop haplotypes identified in this study. The table shows the polymorphic positions in the determined haplotypes relative to the reference sequence AJ002189 [AJ002189 . VerticaClick here for fileDistribution of haplotypes and ancient signatures among European modern pig breeds and wild boars. The file provides information about the abundance of the D-loop haplotypes in breeds and pig types and of the ancient signatures in breeds, and about the correlation between the major European haplotypes and their ancient signature.Click here for file


Idiotype (Id)-based immunotherapy has been exploited as cancer treatment option. Conceived as therapy for malignancies bearing idiotypic antigens, it has been also extended to solid tumors because of the capacity of anti-idiotypic antibodies to mimic Id-unrelated antigens. In both these two settings, efforts are being made to overcome the poor immune responsiveness often experienced when using self immunoglobulins as immunogens. Despite bearing a unique gene combination, and thus particular epitopes, it is normally difficult to stimulate the immune response against antibody variable regions. Different strategies are currently used to strengthen Id immunogenicity, such as concomitant use of immune-stimulating molecules, design of Id-containing immunogenic recombinant proteins, specific targeting of relevant immune cells, and genetic immunization. This review focuses on the role of anti-Id vaccination in cancer management and on the current developments used to foster anti-idiotypic B and T cell responses. L and VH) that form a unique surface for antigen binding. V regions are generated during B cell ontogeny by the so-called VDJ rearrangement of the germ-line Ig genes. This genetic rearrangement allows for the tremendous initial diversity of human Igs in naïve B cells, a critical feature of the immune system, which is further increased and reshaped by somatic hyper-mutation of V regions in antigen-stimulated mature B cells.Immunoglobulins (Ig) are glycoproteins formed by two identical heavy and two identical light polypeptide chains. The N-terminal ends of each pair of heavy-light chains consist of two variable (V) immunoglobulin (Ig) domains . Despite being self-proteins Ids can be immunogenic. For this reason Ids have been exploited as therapeutic immunogens in cancer treatment in two well-defined and clearly distinct contexts: (i) directly as a tumor-specific target on membrane Ig-positive malignant B cells as a consequence of their clonotypic origin, and (ii) as surrogate of tumor-associated antigen (TAA) to induce specific immune responses .The association of the two V domains generates the idiotype (Id), a distinctive structure and a unique collection of antigenic determinants called idiotopes. Idiotopes derive mainly from the CDR regions of the Ig V domains, frequently found to be of a conformational nature and derived from somatic mutations . These strategies are essentially based on the generation of an anti-Id antibody and/or T cell-based immune response by means of protein or DNA-based vaccines. The success of these approaches to eradicate established tumors or prevent their development has been largely demonstrated in animal models.In the first case the Id itself represents the immunogen and the target on the surface of the malignant B cell. Because of their clonotypic origin the Id expressed by malignant B cells represents the only true example of tumor-specific antigen . TherapeFigures C).The second relevant context focuses on the use of a defined Id to induce, through a mechanism of molecular mimicry, a specific immune response against a TAA. In this case the system entails the selection of an anti-idiotypic antibody (Ab2) generated against the Id of an Ab1 specific for the TAA. The Id of the Ab2 is, in turn, used to induce an anti-idiotypic anti-Ab2 response (Ab3) that will not only recognize the immunizing Id of Ab2, but very frequently also the antigen for which the Ab1 is specific . The Id Figure 2B). While in some examples structural homology between antigen and the Ab2 Id has been demonstrated to a certain extent , then the paratope of the Ab2 would structurally resemble the antigen , and (ii) the Id as a mimic of a tumor antigen, with the anti-Id response ultimately intended to target the latter . There is, however, an important distinction between the two cases. While the Id expressed on a tumor cell is a self-antigen, and therefore immunological tolerance is expected, this is not the case when it is used as a mimic of another antigen. In addition, Ab2 are frequently obtained in mice, for which the whole Ig is a foreign antigen for the patient. The response against the Id can thus be favored, and the constant region can act as a xenogeneic carrier. Nevertheless, in cases when the mouse antibody is not highly immunogenic immune-stimulating compounds are required to enhance the response  the Id itself as a target antigen on tumor cells, as in the case of B cell lymphomas , was significantly more efficient in inducing anti-anti-idiotypic antibodies when conjugated to KLH and administered in association to rin BCG; . Similarrin BCG; . Finallyrin BCG; .Apart from KLH, GM-CSF has been extensively tested in numerous protein immunization studies , which aAlum, QS-21, and BCG are adjuvants that have been used in the clinics in anti-idiotypic vaccine formulations with different antigen-mimicking murine Ab2 mAbs . An inteL and VH region sequences directly from biopsy materials. The tumor-associated idiotypic VL and VH chains can then be cloned and assembled into different formats  for expression in mammalian, bacteria, insect, or plant cells  with family-based V gene primers allows identification and isolation of Vnt cells . As examnt cells . scFv mohallenge .E. coli , induced a clear anti-idiotypic response by gene gun DNA immunization. The adjuvant properties of bacterial DNA seemed to be responsible for this outcome, as when the same recombinant construct was administered through recombinant adeno-associated virus (rAAV) infection no response at all was detected  to address the vaccine efficacy of Id-encoding adenoviruses (Administration of naked DNA has the disadvantage that a large amount of the immunogen is degraded before entering the cells . The useoviruses . It was oviruses .BCL1) fused to the human IgG1 CH3 domain (IdBCL1/CH3), induced significant anti-Id antibody titers one month from rAAV injection  have been extensively explored as transgene delivery vectors for their capacity to infect mainly non-dividing cells, such as muscle cells, and to induce a long-term expression of the transgene. The potentiality of these vectors for immunotherapeutical purposes has been intensely explored . Overallnjection . Howeverin vivo targeting of antigen-presenting cells (APCs) were tested. The first approach produced encouraging results in different clinical trials  pulsed with the Id protein or l trials . A combil trials . A more l trials .Figure 3). In a pioneering study, two chemokines, IP-10 and MCP-3, were N-terminally fused to the Ids from two different lymphoma models in the scFv format, and delivered as plasmid DNA or purified proteins  or antibodies specific for APC-restricted molecules  responses only if the delivered DNA construct is expressed by APCs, which depends on the method of DNA delivery. However, by using a chimeric mouse/human IgG DNA vaccine delivered by intramuscular injection, it was shown that protection in the 38C13 murine B cell lymphoma model was not impaired by CD4+/CD8+ T cell depletion -transgenic mouse model specific for an MHC class II-restricted peptide of the λ2 Ig light chain derived from the MOPC315 plasmacytoma , were immortalized with Epstein–Barr virus to isolate anti-idiotypic antibodies . A pool  antigen . In anot antigen . The ind antigen . Interes antigen .Table 1). Treatment of several other malignancies can also involve Id-based vaccination, taking advantage of antigen mimicry by Ids. Solid evidences of clinical benefit are however still awaited, and more work is needed to unveil the mechanisms and factors that impact on the generation of therapeutic anti-Id immune responses.Although the Id of Igs is nominally a self-antigen and consequently poorly immunogenic, strategies intended to induce anti-Id immune responses have successfully demonstrated the feasibility of breaking tolerance against Ids, both in animal models and in the human context. The employment of adjuvants, carriers, viral vectors, or the direct recruitment of immune-related cells has allowed translating Id targeting by the immune system into therapeutic approaches for Ig-expressing tumors (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MLH1, MSH2 and MSH6.Hereditary nonpolyposis colorectal cancer  is a genetic disorder, where family members are at high risk of developing cancer of the colon, endometrium, small intestine and urinary tract. The cause for LS is due to constitutional mutations in several mismatch repair genes (MMR) mainly in denaturing high-performance liquid chromatography) or sequencing, whereas deletion of one or more exons  were detected mainly by MLPA (multiplex ligation-dependent probe amplification) [The Polish population has been well characterized by a large study comprising ~1000 LS-suspected families. Most nucleotide substitutions (about 90%) were discovered mainly by DHPLC (ication) .Over 60% families of the former study, which were affected by recurrent MMR mutations, were taken as a basis to design the following iPLEX/TaqMan test, that allows simultaneous testing of almost all recurrent mutations in only analysis  .Sequenom), for which a reasonable cost-effectiveness is achieved only for large series of probes. Thus the approach is convenient only for high-throughput laboratories.The drawback of this kind of analysis are the high costs of the machines (MLH1 and MSH2. An analogous test for recurrent mutations of the gene APC could, in a similar way, accelerate the molecular diagnostic of predisposition to FAP.However the present approach is cost-effective even for testing individual patients. The present test, based on a Taqman PCR analysis, allows a fast identification of the 20 most frequent mutations of the genes