Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both  Tbx4 and Tbx5, in three processes essential to the development of the respiratory system: the specification of the lung and trachea primordia, the growth and branching of the airways to form the lung, and formation of cartilage rings of the trachea and bronchi. In the absence of Tbx5, only one lung is specified, and no trachea. Both Tbx4 and Tbx5 regulate the process of lung branching by controlling the expression of the secreted growth factor Fgf10 and activation of Fgf10 signaling. In the trachea, both Tbx4 and Tbx5 are important for condensation of cells to form cartilage rings, although this is regulated by a distinct pathway that does not involve Fgf10.Defective development of the mammalian respiratory system can lead to tracheal, bronchial, or pulmonary malformations causing severe consequences at birth or during postnatal life. Studies using mouse genetics have begun to reveal complex regulatory mechanisms that guide the development of the respiratory system, but understanding how disruption of these mechanisms leads to malformations is far from complete. In this study, we analyze the role of two T-box transcription factors,  The development of the respiratory system represents an evolutionary hallmark that allowed vertebrates to survive on land utilizing air as a source of oxygen. Because the respiratory system is dispensable for embryonic survival in mammals, defects in development of the respiratory system manifest at or after birth. Indeed, abnormal development of the respiratory system in humans is associated with multiple disorders such as tracheal/bronchial atresia, tracheoesophageal fistula, bronchogenic cysts, pulmonary/lobar atresia and pulmonary hypoplasia Nkx2.1 has been identified as the earliest marker of lung endoderm specification. At E9.25 (25–28 somites), the primary lung buds appear as ventro-lateral outpouchings of the foregut connected ventrally by the tracheal primordium. The lung buds grow in a ventro-posterior direction and continue to elongate until E11.5. The point of connection of the lung buds is thought to be the origin of the tracheal tube, which separates from the esophagus in a caudal to cranial direction by E11.5 In the mouse embryo, the endodermal foregut tube is patterned by signals from the lateral plate mesoderm leading to specification of the lung and trachea at embryonic day (E) 9.0 (19–24 somites). Wnt2, Fgf10, Bmp4, Shh and retinoic acid synthesis genes, have been shown to play important roles in lung specification and branch formation. Complete absence of both Wnt2 and Wnt2b in mesenchyme surrounding the anterior foregut or absence of β-catenin in the foregut epithelium leads to a loss of specification of lung primordia as seen by the absence of Nkx2.1 expression Fgf10, which is normally expressed in mesenchyme surrounding the epithelial branching tips, form a short trachea but have no lungs Bmp4 signaling by overexpression of Xnoggin leads to a decrease in lung size and irregularly shaped lung lobes Shh null mutant mice have only a rudimentary lung sac due to branching severe branching defect Shh in lung epithelial cells leads to the formation of hypoplastic lungs with reduced branching of the peripheral tubules Genes involved in different signaling pathways, including Sox9 has been implicated as an important regulator of mesenchymal condensation and chondrocyte differentiation Sox9, FGF2, Igf1, Tgfβ2 and Bmp2 enhance chondrocyte formation Shh, Sox2, retinoic acid synthesis genes and Fgf signaling pathway genes have been shown to affect cartilage ring formation Fgf10 mutants form a partial tracheal tube in spite of the failure of lung formation Fgf10 leads to defects in tracheal ring formation and that overexpression of Fgf10 between E11.5 and E13.5 disrupts tracheal rings by altering the periodic expression of Shh in the trachea After E11.5, mesenchyme surrounding the dorsal aspect of the trachea differentiates into the trachealis smooth muscle. Mesenchyme surrounding the ventral aspect of the trachea and lateral aspect of the main stem bronchi segments and differentiates into C shaped rings composed of chondrocytes. Ventral tracheal cartilage is formed by migration of cells that undergo mesenchymal condensation Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing chick lung buds and trachea between stages 15–21 Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later Tbx1 homozygous null mutants die at birth due to severe heart defects; the lungs are never fully inflated Tbx4 homozygous mutants, lung buds form but the embryos die at E10.5 due to failure of allantois development and the subsequent lack of chorio-allantoic fusion leading to placental insufficiency Tbx5 mutants die around E10 due to defects in heart development Tbx4 and Tbx5, but not Tbx2 and Tbx3, in lung organ cultures results in inhibition of branching and loss of Fgf10 expression Tbx4 function leads to a reduction in Fgf10 expression in lung mesenchyme and inhibits lung bud formation. Ectopic expression of Tbx4 leads to ectopic expression of Fgf10 and Nkx2.1 and lung bud formation in the esophagus. Additionally, ectopic expression of Tbx4 at the boundary between the trachea and the esophagus can lead to lack of separation of these two structures, resulting in a tracheoesophageal fistula Tbx5 mutations cause Holt Oram syndrome characterized by heart and forelimb abnormalities. A single de-novo mutation in TBX5 has been linked to right lung agenesis The T-box transcription factor genes are important during embryonic development. All members of this gene family contain a conserved DNA-binding T-box domain, which binds to a conserved sequence, the T-box binding element, to activate or repress transcription of specific target genes Tbx4 and Tbx5 in lung and trachea development in the mouse, we made use of conditional alleles to bypass early embryonic lethality. We studied three distinct processes, namely 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation. We show that during early stages of development, Tbx5 is important for specification of the lung buds and the trachea. After specification, Tbx4 and Tbx5 interact during lung growth and branching and the regulation of branching is dependent on Fgf10 signaling. Additionally, Tbx4 and Tbx5 interact in the formation of mesenchymal condensations, which ultimately form the tracheal/bronchial cartilage rings independent of Fgf10 signaling.To study the roles of Tbx5 expression is first detected using in situ hybridization (ISH) at E9.0 (24 somites) in the mesenchyme of the lung and trachea primordia, concurrent with Nkx2.1 expression in the ventral foregut epithelium (Tbx4 expression is detected in the lung buds when they first appear a few hours later at E9.25 (28 somites) in a pattern similar to Tbx5 , the foregut tube is devoid of Nkx2.1 expression and the lung buds are not present Nkx2.1 expression  and removal of Tbx4 in addition to Tbx5 did not exacerbate the Tbx5 phenotype , late enough to bypass lethality but well before lung branching begins. Embryos with CreER and different combinations of the flTbx4 and flTbx5 alleles were examined at E12.5 and E13.5. Conditional Tbx4 null lungs were similar to controls at E12.5 but had fewer branching tips at E13.5 . We hypothesize that the variability in phenotype is due to variable extent of recombination of the flTbx5 allele  became cyanotic at birth and died shortly thereafter due to respiratory distress. Unlike the variable lung size in the lung-specific Tbx5 null mutants, the lungs of these mutants were consistently smaller than controls at E13.5  nor Tbx4 single heterozygous lungs show a branching defect, double heterozygous lungs are smaller at E13.5 and E18.5 with a reduced number of branching tips at E13.5. Because these genes are closely related Tbx4 and Tbx5 could physically interact with each other as heterodimers to activate or repress transcription of downstream targets, as has been suggested for other T-box genes Tbx4;Tbx5 double heterozygous lungs are smaller than conditional Tbx4 nulls. Unequal and distinct functions could be explained by domains outside of the T-box. For instance, the C terminal domain of both Tbx4 and Tbx5 has transcription activating capability which has been correlated to the shared limb outgrowth promoting activity of these genes In lung branching morphogenesis,  another . AlthougTbx4 nulls;Tbx5 heterozygous and lung-specific Tbx4 heterozygous;Tbx5 null lungs, reduction of Tbx4 or Tbx5 leads to a severe decrease in branching, a defect in formation of the lobes and failure of lobe separation, while absence of Tbx4 and Tbx5 leads to branching arrest. These results are in line with the published observations that antisense RNAs used against both Tbx4 and Tbx5 inhibit lung branching in culture and that this affect is explained by loss of Fgf10 expression Fgf10 promoter in vitro suggesting that Fgf10 is a direct downstream target Fgf10 and Wnt2 in the lung mesenchyme. The Fgf10 targets Bmp4 and Spry2 show very low levels of expression in the epithelium of Tbx4 and Tbx5-deficient mutants and Etv5, another Fgf10 target, shows greatly reduced expression in the conditional double null lungs, leading to the hypothesis that the Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. Lungs that are triple heterozygous for Tbx4, Tbx5 and Fgf10 are reduced in size compared to Tbx4;Tbx5 double heterozygous lungs, which supports this hypothesis. Lung-specific Tbx4 heterozygote;Tbx5 null lungs are severely retarded but lung size is not affected by further loss of Fgf10, demonstrating epistasis and supporting the hypothesis that Fgf10 lies downstream of Tbx4 and Tbx5. Lack of rescue of branch formation and lack of activation of the Fgf10 signaling pathway by exogenously supplied Fgf10 in culture suggests that there are other factors downstream of Tbx4 and Tbx5 that affect Fgf10 signaling. Although, lungs deficient in Tbx4 and Tbx5 retain the ability to respond to Fgf10 coated beads . The extracellular matrix (ECM) molecules, heparan sulfate (HS) proteoglycans aid in Fgf10-Fgfr2 interactions during lung development Tbx4 and Tbx5-deficient mutants. Inhibition of heparanase decreases submandibular gland morphogenesis in culture due to deficiency of Fgf signaling Tbx4 regulates ECM molecules in the developing allantois, specifically the chondroitin sulfate proteoglycan versican (R. Arora and V. E. Papaioannou unpublished observations), suggesting that ECM might be one of the targets for T-box genes in regulating development of other organs as well.One possibility for such a factor is a mesenchymal signaling molecule that communicates with the epithelium and activates downstream target(s) required for the activation of the Fgf10 pathway. We examined expression of Tbx4 and Tbx5 causes defects in both formation of the tracheal cartilage rings and development of the trachealis smooth muscle indicating interactions between Tbx4 and Tbx5. Tbx4 and Tbx5 heterozygous tracheas have 10–11 cartilage rings but Tbx4;Tbx5 double heterozygous tracheas have 6–8 cartilage rings, some of which are incomplete. Additional reduction of Tbx4 and Tbx5 in the lung-specific Tbx4 heterozygous;Tbx5 null lungs leads to a complete disruption of cartilage ring formation supporting a genetic interaction between Tbx4 and Tbx5 in trachea formation.Appropriate dorsal smooth muscle development and ventral tracheal/bronchial cartilage development is important for the normal functioning of the trachea. Smooth muscle provides tracheal flexibility and the cartilaginous rings prevent tracheal collapse. Defects in trachea formation may result in tracheomalacia or tracheal stenosis. Reduction of Sox9, a master regulator of the process of chondrogenesis, is expressed normally at E12.5 throughout the ventral mesenchyme in lung-specific Tbx4 heterozygous;Tbx5 null tracheas but at E13.5 these tracheas show a lack of characteristic mesenchymal condensations and reduced Sox9 expression. Either Tbx4 and Tbx5 control of Sox9 expression becomes more sensitive to dosage at E13.5 or Tbx4 and Tbx5 control another factor, possibly an ECM molecule, which is important for formation of mesenchymal condensations and, in the absence of these condensations, there is a down regulation of Sox9 expression  at a concentration of 20 mg/ml in sunflower oil (Sigma) was administered to pregnant females by intraperitoneal injection between 15.30 and 19.30 hours on E8.5 or between 23.00 and 24.00 hours on E9.0.Whole-mount ISH, immunohistochemistry (IHC), immunofluorescence (IF) and ISH on cryosections was performed as described previously For histology embryos were removed from the uterine horns, dissected out of the decidua and fixed in Bouin's fixative (Sigma). After dehydration in ethanol, embryos were embedded in paraffin wax, sectioned at 8 µm thickness and stained with hematoxylin and eosin (H & E).Alcian blue staining was performed according to standard protocols 2 on Transwell-Col filters (Fisher Scientific) containing 1.5 ml BGJb media (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml vitamin C (Sigma) and 2 µM 4-OH tamoxifen (Sigma). For lung bud culture, lung buds were dissected at E11.5 in phosphate buffered saline (PBS) with 0.1% bovine serum albumin (Sigma) and cultured in media containing DMEM (Invitrogen) with 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen) and 1 µM 4-OH tamoxifen on 3.0 µm filters (Millipore) or 0.4 µm Transwell filters (Fisher Scientific). Similar results were obtained using both types of filter; results reported are for experiments with Millipore filters. Where specified, Fgf10 (R&D) was added after 1 day of culture at a concentration of 500 ng/ml. In some experiments heparin beads coated with Fgf10 (100 µg/ml) or BSA (100 µg/ml) were placed near the branching tips of the explants after 1 day of culture. Transwell filters were used for the bead experiments.Foregut culture was carried out as described previously E-cadherin RNA probe using ISH. In case of whole mount lungs, lobes were separated and photographed to count the number of branching tips. The cultured lungs were photographed and the branching tips were counted. For some experiments, after E-cadherin ISH, lungs were post fixed in 4% PFA, washed with PBT, dehydrated in 100% methanol, cleared in BABB and then photographed.Lungs were stained with either E-cadherin antibody using IHC or Figure S1flTbx4 and flTbx5. Females carrying embryos with flTbx4, flTbx5 and CreER alleles were injected with either 8 mg tamoxifen at E9.0 and dissected at E12.5  or 7 mg tamoxifen at E8.5 and dissected at E13.5 . fl/flTbx4 was completely excised to mutant −/−Tbx4 at both doses and times  whereas the single conditional allele of fl/+Tbx5 was incompletely excised by injection at E9.0 (E) but completely excised by injection at E8.5 (F). In addition when lungs with the genotype fl/flTbx4; fl/flTbx5; CreER were treated with 1 µm tamoxifen in culture, the Tbx4 locus was nearly completely excised at the end of 1 day of culture (C) but the Tbx5 locus was only partially excised (G). At the end of a 4 day culture both Tbx4 (D) and Tbx5 (H) loci achieved virtually complete excision. fl, floxed conditional PCR band; WT, wild type PCR band; Mut, excised PCR band.Analysis of excision efficiency of (TIF)Click here for additional data file.Figure S2Tbx4 and Tbx5 does not affect alveolar differentiation.  T1α IHC on cryosections of control and lung-specific Tbx4 heterozygous;Tbx5 null lungs at E18.5 shows comparable staining in the lung epithelium. Nuclear fast red was used as a counter stain. A′ and B′ are higher magnification views of boxed regions in A and B.  Prosurfactant protein C (Pro-SPC) IF on cryosections of control and lung-specific Tbx4 heterozygous;Tbx5 null lungs at E18.5 shows comparable staining in the lung epithelium. DAPI was used to stain the nuclei. Scale bars represent 100 µm.Loss of (TIF)Click here for additional data file.Figure S3Tbx4 and Tbx5 has multiple affects on branching morphogenesis.  H & E sections of lung-specific Tbx4 heterozygous;Tbx5 null lungs (B) show lack of separation of the cranial (cr), medial (m) and caudal (cd) lobes in the right lung as compared to control lungs (A). Black arrowheads point to the space created between the lobes in the control lungs (A) and to the corresponding regions of the mutant lungs (B).  Control lungs stained for E-cadherin at E13 show greater outgrowth (yellow parentheses) of lateral branches (C) than the lung-specific Tbx4 heterozygous;Tbx5 null lungs (D). Scale bars represent 100 µm.Loss of (TIF)Click here for additional data file.


Our experimental findings demonstrate that the ability to control the degree of functionalisation of graphene is instrumental to engineer different electronic properties in graphene materials.We demonstrate the possibility to tune the electronic transport properties of graphene mono-layers and multi-layers by functionalisation with fluorine. For mono-layer samples, with increasing the fluorine content, we observe a transition from electronic transport through Mott variable range hopping (VRH) in two dimensions to Efros-Shklovskii VRH. Multi-layer fluorinated graphene with high concentration of fluorine show two-dimensional Mott VRH transport, whereas CF We have fabricated transistor structures with fluorinated graphene mono-layers and multi-layers and studied their electrical transport properties in the temperature range from 4.2 to 300 K. We show that the electronic transport properties of fluorinated graphene can be tuned by adjusting the fluorine content, so that different transport regimes can be accessed, like Mott variable range hopping (VRH) in two dimensions [Here, we explore the electronic transport properties of functionalised graphene with a fluorine content ranging from 7%  substrates. The fluorinated graphite was synthesised via two routes: graphite fluorides (using F2 gas) and fluorine graphite intercalation compounds (FGIC) (using XeF2 as fluorinating agent), see Section 6. The samples produced using F2 gas that we investigate here are multi-layers and have the concentration of fluorine of 28 and 100%, whereas the samples synthesised using XeF2 gas are all mono-layers and have the fluorine content of 7, 24 and 28%.Fluorinated graphene mono-layers and multi-layers were mechanically exfoliated from fluorinated graphite and deposited onto conventional Si/SiO-1 which is typical for pristine mono-layer graphene [0.28. In total, four mono-layer, five CF and five CF0:28 multi-layer flakes were processed into four-terminal transistor devices, where the electrical contacts were defined by e-beam lithography, deposition of Cr/Au (5/50 nm) and lift-off procedure, see Figure Flakes of fluorinated graphene are located using an optical microscope , see Figure -1. The G band is associated with the double degenerate E2g phonon mode at the Brillouin zone center, while the 2D mode originates from a second-order process, involving two intervalley phonons near the K point, without the presence of any kind of disorder or defect [-1. These Raman peaks originate from double-resonance processes at the K point in the presence of defects, involving, respectively, intervalley (D) and intravalley (D') phonons [Figure r defect . In the  phonons -54.sp2 bonded carbon atom rings, we believe that the D peak is mainly activated by the F atoms which act as vacancies in these sp2 rings.In exfoliated pristine graphene, the D peak can only be observed at the edges of the flakes where there is a large concentration of structural defects and its intensity is typically much lower than the intensity of the G peak ,56. In oD/IG for the D and G bands. It has recently been shown that in graphene ID/IG has a non-monotonic dependence on the average distance between defects LD, increasing with increasing LD up to LD ~ 4 nm and decreasing for LD > 4 nm [A better understanding of the level of disorder in our samples is reached when analysing the intensity ratio ID > 4 nm ,54,57. S0.24 exhibit very distinct G and D' peaks. Based on the aforementioned phenomenological model [0.07 and CF0.28 samples are in the regime where the intensity ratio ID/IG increases with increasing LD (i.e. decreasing the concentration of F) whereas the CF0.24 samples are in the opposite regime. This scenario is confirmed when comparing the intensity ratio ID/IG for fluorinated mono-layers extracted from graphite with different fluorine content: ID/IG = 1.74 for CF0:28 and ID/IG = 2.16 for CF0.07. From the LD dependence on ID/IG we estimate LD ~ 1.5 nm for CF0.28 and LD ~2 nm for CF0.07 [0.24 samples, we estimate LD ~ 5.3 nm for device 1 and LD ~ 6.1 nm for device 2. These values of LD are in agreement with the observed frequencies of the D, G, D' and 2D peaks as well as with the FWHM values of the 2D peaks [The Raman spectra of fluorinated mono-layer samples produced from graphite with fluorine content of 7 and 28%  as a function of gate voltage (Vg) for the fluorinated mono-layer samples for different temperatures. The resistivity exhibits a non-monotonous dependence on Vg with a maximum at Vg = +10 V, stemming for a doping level of n = 0.74 1012 cm-2 commonly seen also in pristine graphene devices and attributed to doping by atmospheric water. In all cases, the resistivity of fluorinated graphene shows a pronounced temperature dependence. Indeed, the maximum of resistivity changes over two orders of magnitude as T decreases from 300 to 4.2 K. Away from the maximum of resistivity region, the temperature dependence remains weak, with the mobility of carriers of 150 cm2/Vs. Furthermore, at low temperature the resistance shows strong mesoscopic fluctuations, as expected for samples of small size [ρ(Vg) curves using a moving average filter.Having characterised the level of disorder from Raman spectroscopy, we now proceed to address the role of disorder on the electrical transport properties of fluorinated graphene materials. Figure all size . In the Δε: ρ(T) = ρ0exp(Δε/2kBT), see Figure lnρ (1/T) versus 1/T decreases with decreasing T, which is a signature of hopping conduction via localised states [ρ on temperature is ρ(T) = ρ0exp(T0/T)1/3, where kBT0 = 13.6/a2g(Fε), g is the density of localised states at the Fermi level F εand a is the localisation length [ρ(T) for the samples produced from CF0.07 and CF0.24 graphite  is described well by the 2D-VRH model.To examine the presence of the energy gap, we analyse the temperature dependence of the maximum of resistivity by an exponential law describing thermal activation of carriers across an energy gap d states . The facd states ,48. In tn length ,48. ExpeT0 as a function of carrier concentration for these three samples. The value of T0 approaches zero at a carrier concentration of ±1.2 1012 cm-2. This value gives the concentration of the localised electron states in the energy range from ε = 0 to the mobility edge, see Figure Vg ± 20V and indicates the transition from hopping to metallic conduction.Figure Δε, one needs to know the exact energy dependence of the density of states in the gap. For estimations, we will use the linear relation for the density of extended states above the mobility edge g(ε) = 2ε/πħ2v2 (v = 106 m/s is the Fermi velocity), and a constant value for the density of localised states below the mobility edge, Figure Δε = 60 meV and twice this value for the full mobility gap. In this approximation, the density of the localised states in the gap is ~1036 J/m2. Using the obtained value of the hopping parameter at the maximum of resistivity, we can then estimate the localisation length at ε = 0 as a = 40 nm for CF0.24 (device 1), a = 81 nm for CF0.24 (device 2) and a = 265 nm for CF0.07.In order to relate the obtained concentration of the localised states to the energy gap 0.28 graphite. For this sample, characterised by the largest disorder LD ~1.5 nm, the experimental data cannot be described by thermally activated law nor Mott VRH. In this case, the ln(ρ) follows a T-1/2 dependence characteristic of the Efros-Shklovskii VRH in the presence of Coulomb interaction between the localised states (ρ(T) = ρ0exp(T0/T)1/2) [T0 is related to the localisation lengths through T0 = 2.8e2 / 4 rεπε0kBa and for our sample we estimate T0 = 52 K. Assuming that r εis the dielectric constant of SiO2 we obtain the localisation length a = 282 nm.Figure 0/T)1/2) . T0 is r0.28 prepared by exposure to fluorine gas. The fully fluorinated multi-layer show systematically a very large resistance (more than 100 GΩ) independently of the specific thickness and no gate-voltage control of the resistivity. To achieve gate modulation in these samples, we reduced the fluorine content by annealing the samples at 300°C, in a 10 % atmosphere of H2/Ar for 2 h. This annealing process restores a partial gate-voltage control of the resistance  gives a gap of only 25 meV, which is significantly smaller than the expected energy gap for fully fluorinated graphene. Similarly to the fluorinated mono-layer graphene, the resistivity dependence on temperature is fitted well by VRH with the value of T0 = 20000 K. This confirms that the previously found activation energy of 25 meV is not the activation energy Δε that separates the localised states from extended states at the mobility edge, but is an activation energy δε of hopping between localised states within the mobility gap, see Figure Resistance measurements of CF0.28 prepared by exposure to fluorine gas. The I-V characteristics of these samples are strongly non-linear (T) dependence is rather well described by a thermally activated law at elevated temperatures (regime A), with a Δε = 0.25 eV, followed by a temperature regime where the resistivity decreases with lowering the temperature (regime B). A 1/T dependence of ln(ρ) could be the consequence of either intrinsic transport through thermally excited carriers above the bandgap or conduction through NNH via localised states within the gap. Since the I-V characteristics of our devices show a non-linearity on a Vsd range much larger than the estimated Δε = 0.25 eV  the graphene multi-layers exhibit a stronger temperature dependence and a larger transport gap than what is observed in mono-layers. This difference could originate from the different fluorination processes used for the multi-layers (direct fluorination with F2 gas at high temperature to produce graphite fluorides) and for the monolayers (FGIC synthesised at lower temperatures), see Section 6. Indeed, different fluorination processes may lead to different concentrations of localised states. Even though in both materials, F-GIC and graphite fluorides, the nature of the bonding between fluorine and carbon atoms is covalent, the C-F bond order is slightly lower in F-GIC [Finally, for the same degree of fluorination  and CF0.28 synthesised with this method at 530°C. However, due to the harsh fluorination conditions, many structural defects are formed, which makes it very difficult to exfoliate large enough mono-layer flakes that can be identified by optical microscopy and easily processed into devices. To prepare larger fluorinated graphene samples, we have used a second fluorination method where graphite is exposed to a fluorinating agent, i.e. XeF2. In this case, the functionalisation process is carried out at T ≤ 120°C, as XeF2 easily decomposes on the graphite surface into atomic fluorine [2 was prepared in a glove box in an Ar atmosphere. Owing to its reactivity and diffusion, the fluorination results in a homogenous dispersion of fluorine atoms that become covalently bonded to carbon atoms [2 gas that we investigate here have the concentration of fluorine of 7, 24 and 28%.To produce fluorinated graphite, we have used two distinct methods -62. In tdisation . The layfluorine . The mixon atoms ,62,63. Aon atoms . The con2) and C2F4. However, the decomposition of graphite was found to start as a secondary reaction close to 600°C, with fluorination being the main reaction. Since the reactions with F2 and XeF2 have been carried at lower temperatures than the graphite decomposition temperature, the underestimation of F:C ratio in our fluorinated graphite samples is likely to be less than 0.02.The fluorine concentration (i.e. F:C molar ratio) of fluorinated graphite was determined by gravimetry (weight uptake). The concentration obtained by weight uptake was confirmed by solid-state NMR measurements on samples fabricated under identical conditions and the accuracy of gravimetry was estimated to be 0.02 -67. The μm in diameter. An incident power of 5 mW was used. We ensured that this power does not damage the graphene by performing Raman measurements on a similarly sized pristine graphene flake which shows the common spectra of mechanically exfoliated graphene: the G band and 2D band  at 1580 and 2700 cm-1, see Figure We have characterised all the exfoliated flakes by Raman spectroscopy using an excitation light with a wavelength of 532 nm and a spot size of 1.5 The resistance of the transistor devices was measured both in dc, by means of Keithley 2400 Source-meter, and in ac at low frequency (34 Hz) with a lock-in amplifier in a voltage-biased configuration. For the ac-measurements, the excitation current was varied to ensure that the resulting voltage was smaller than the temperature to prevent heating of the electrons and the occurrence of nonequilibrium effects. The comparison of 2- and 4-probe measurements shows that the contact resistance in our devices is negligible as compared to the sample resistance. This experimental finding insures that even 2-probe transport measurements are probing the electrical properties of the bulk fluorinated graphene rather then simply the Cr/graphene interface.2D-VRH: two-dimensional Mott variable range hopping; AFM: atomic force microscopy; F-GIC: fluorine graphite intercalation compounds; FWHM: full width at half maximum; NMR: nuclear magnetic resonance; NNH: nearest neighbour hopping; VRH: variable range hopping.The authors declare that they have no competing interests.FW fabricated the fluorinated graphene devices and carried out the measurements. MD synthesised the fluorinated graphite. FW and MFC performed the data analysis. FW, SR and MFC wrote the manuscript. All authors discussed the results, read and approved the final manuscript.


A new international statement defines usual interstitial pneumonia (UIP) which is a histological and radiological form of idiopathic pulmonary fibrosis (IPF) more precisely than previously. In the diagnosis of IPF, either in high resolution computed tomography (HRCT) a UIP pattern must be present or alternatively specific combinations of HRCT and surgical lung biopsy findings can be accepted. In about two third of the cases IPF can be diagnosed by clinical and radiological criteria. Thus surgical lung biopsy is needed in about one third of cases to achieve the ultimate diagnosis, which requires multidisciplinary cooperation. In large clinical trials conducted during the last decade, lung biopsy was performed in about 30–60% of the cases. The most serious complication of lung biopsy is mortality within 30 days after the procedure, with a frequency of about 3–4% reported in most studies. Because of the histological variability, surgical lung biopsy should be taken from a minimum of two lobes. The number of fibroblast foci in surgical lung biopsy has been shown to correlate with survival in several studies. Until recently, surgical lung biopsy (SLB) has been regarded as the golden standard in the diagnosis of idiopathic pulmonary fibrosis (IPF) and other types of idiopathic interstitial pneumonias (IIP). During the past decades, the pathological and clinical terms for the IIPs have been at least partly different which has been responsible for misinterpretations between different specialists working with the patients with IIP.The new international statement on the diagnosis and management of IPF has recommended adopting a multidisciplinary approach for the ultimate diagnosis. On the In a new statement, IPF is defined as a progressive interstitial pneumonia of unknown cause, occurring in adults, limited to the lungs and associated with the histological and/or radiological pattern of usual interstitial pneumonia (UIP). In the diagnosis of IPF, other known causes must be excluded, in HRCT, a UIP pattern must be present, or specific combinations of HRCT and SLB findings can be accepted. Further, there are no longer the so-called major or minor criteria present in the previous guidelines, and BAL or transbronchial biopsy is no longer required as was the case in the statement released in 2000,4.The new diagnostic criteria for radiology and histopathology are more precise than previously and now include 3 HRCT categories and 4 histological categories, namely 1) UIP, 2) possible UIP and 3) inconsistent with UIP for HRTC, and 1) UIP, 2) probable UIP, 3) possible UIP and 4) not UIP for histopathology. The histological categories are presented in detail in Table The 2011 statement recommends adoption of the following diagnostic algorithm for patients with suspected IPF.The study of Raghu et al. indicated that IPF can be diagnosed by clinical and radiological criteria in about two third of all cases. SubsequThe most common differential diagnostic problem is nonspecific interstitial pneumonia (NSIP). Flaherty and co-authors claimed that patients with an HRCT pattern of UIP are likely to exhibit a histological pattern of UIP, but the patients with an HRCT pattern other than UIP may have either histological UIP or NSIP in the SLB specimen, and that HRCT has limited specificity in identifying histological proven NSIP. Silva aThere are some published data about the frequency of SLB in different countries, which have shown the actual incidence of SLB in routine clinical diagnostics during the 1990’s and 2000’s. Epidemiologic studies in Italy, Belgium, Greece, Spain, United States and Finland have revealed that the diagnosis has been confirmed by SLB in 28–38% of the cases-22. QuesSeveral studies have shown that SLB taken by either open lung thoracotomy (OLB) or video-assisted thoracoscopic surgical (VATS) operation is an efficient diagnostic procedure for ILD-36. MourThe most common complication of SLB is prolonged air-leak which occurs in about 6–12% of the cases; other common complications can be a need for mechanical ventilation, pneumonia, pneumothorax, hemothorax, pleural effusion, empyema and prolonged ventilation-41. In aTable The study of Park et al. showed that SLB performed at the time of acute exacerbation of IPF resulted in higher 30-day mortality (28.6%) compared to the patients without acute exacerbation (3.0%). Utz andBecause of the retrospective and descriptive nature of the studies, it is difficult to draw any firm conclusions on the risk factors or limits for SLB. On the other hand, it is probable that no controlled clinical trial will be performed on this particular topic in the future. If one wishes a more accurate estimation of the risks of SLB, then larger patient material from multicenter studies may be required. A new method for performing SLB has been recently presented, such as awake thoracoscopic biopsy, which was shown to be feasible requiring only regional anesthesia and it has resulted in low morbidity, excellent diagnostic yields, short hospital stays, and low costs.It has been shown that there exists an inter-observer variability between pathologists when analyzing SLB samples. The study of Nicholson et al. revealed that there was only fair agreement among pathologists who had been provided with very little clinical information of the patients. The preSome studies have indicated that the histological features may be variable in some cases of IPF since in some lobes or segments of lung NSIP-like features can exist in addition of UIP features. The patients with the coexistence of both NSIP and UIP patterns seemed to behave similarly to those with only a UIP pattern,52. SampNSIP is nowadays a recognized entity exhibiting, however, overlapping features with other types of IIPs and hypersensitivity pneumonia. Unlike IPF, the prognosis of NSIP is good and it occurs mostly in middle-aged women who are never-smokers. At presIn addition of the diagnosis of ILD, SLB can be used also as source of a biomarker for the prognosis in IPF patients. The number of fibroblast foci (FF) i.e. active centers of fibrogenesis consisting of myofibroblasts, fibroblasts and extracellular matrix (ECM) proteins and lined by regenerative hyperplastic or metaplastic alveolar epithelium, has been shown to correlate with patient survival in several studies,55-59, bDespite this quite convincing evidence, the counting of FF in biopsies has not yet become a common routine clinical practice. This might be attributable to the fact that the number of FF has been evaluated with variable methods in the different studies. At present FF has proved to be the only reproducible histological factor that correlates with the prognosis in IPF. There are very few studies, which have evaluated the value of immunohistochemical markers from SLB specimens on survival, since during the past decade most studies focusing on biomarkers have been conducted by using serum or BAL samples. Nearly two decades ago it was reported that a high expression level of the ECM protein, tenascin-C as detected by immunohistochemistry in SLB samples, correlated with shortened survival in patients with UIP.Neither the previous nor the current statement has recommended TBB for the diagnosis of IPF; this technique has been used mainly to exclude other ILDs such as sarcoidosis, or malignancies and infections,4. The sIt is probable that approximately one third of the IPF patients would need SLB in order to obtain an accurate diagnosis. Several remarkable points should be considered when making a decision whether or not to perform the SLB. The mortality, which can occur shortly after the procedure, has probably been the major reason to refrain from performing SLB. The precise risk limits for complications of SLB procedure are not well known. Thus a thorough deliberation taking into consideration age, other diseases, medication, lung function and the stage of the pulmonary fibrosis are needed before proceeding to a procedure of SLB. As it has been noted in the new statement, in patients with severe physiologic impairment or substantial comorbidity, the risks of SLB may outweigh the benefits of establishing a secure diagnosis of IPF, and moreover, clinicians must spend adequate time with patients to discuss patients’ values and preferences.The accuracy of the histological diagnosis might be problematic, and thus it seems to be fundamental that SLB are analyzed by experienced pulmonary pathologists. It is probable that the histological diagnosis may become even more challenging than previously when only the patients with non-typical HRCT features are biopsied, and UIP may be at risk of remaining obscured by other concomitant diseases or confounding factors. Moreover, the challenge of pulmonary pathology will be greater because of the fact that more complex and less classical lung diseases will be investigated that will make classification of histological patterns more difficult and may limit the role of pathology as a routine predictor of prognosis. Evaluation of the pathological archives from previous decades might help in the training of pulmonary pathologists and for gathering experience from a large amount of samples including also the typical cases representing IPF/UIP. It is likely that more SLB may be needed for the diagnosis of NSIP and for the exclusion of IPF, especially after the preliminary results of the Panther-study which revealed a harmful effect of triple therapy treated IPF patients.Since many IPF patients are at an older age and suffer from many other diseases, or their lung disease has already progressed to an advanced stage, SLB cannot be considered as a safe procedure for all individuals. Thus, there is a definite need for developing new diagnostic tools for the diagnosis of IPF and other ILDs. According to the current statement, BAL or TBB are no longer required for the diagnosis of IPF. A new BAL guideline has been recently published as has aThe author declares that she has no competing interests.


In this theoretical research work, the fracture characteristics of graphene-modified polymer nanocomposites were studied. A three-dimensional representative volume element-based multiscale model was developed in a finite element environment. Graphene sheets were modeled in an atomistic state, whereas the polymer matrix was modeled as a continuum. Van der Waals interactions between the matrix and graphene sheets were simulated employing truss elements. Fracture characteristics of graphene/polymer nanocomposites were investigated in conjunction with the virtual crack closure technique. The results demonstrate that fracture characteristics in terms of the strain energy release rate were affected for a crack lying in a polymer reinforced with graphene. A shielding effect from the crack driving forces is considered to be the reason for enhanced fracture resistance in graphene-modified polymer nanocomposites. Nanocomposites composed of nanofiller reinforcement and a polymer matrix are currently subject to intense research due to possible improvements in physical, mechanical, and/or electrical properties compared to neat polymer. Graphene is emerging as a potential candidate for nanoscale reinforcement of polymer nanocomposites. Stankovich et al.  introducIn their experimental works, Rafiee et al. ,3 observThe number of theoretical/numerical works published on graphene-based nanocomposites has so far been limited. Existing works in this field are, for example, the molecular dynamics-based simulation techniques employed by Awasthi et al. , who stuTo the present authors' knowledge, no theoretical studies are available in the technical literature on the fracture behavior of graphene/polymer nanocomposites. In the present paper, an attempt has been made to develop a multiscale model to investigate the fracture characteristics of graphene-modified epoxy nanocomposites. The proposed multiscale modeling technique was developed in the ANSYS (version 13) finite element software environment  in conjunction with the virtual crack closure technique (VCCT). A multiscale approach employing the VCCT provides an efficient numerical analysis scheme in terms of the involved degrees of freedom. As a consequence, the analysis can be performed with widely available computational systems.In the present work, considering the polymer matrix and graphene nanofiller as a continuum and atomistic phase, respectively, a multiscale model in conjunction with finite element analysis was developed. The bond interaction between carbon atoms in graphene was simulated with the help of beam elements. In the current finite element simulation, the modified Morse potential was employed to model the bonded interaction between C-C bonds. The Morse potential has already been applied in a number of research works ,9, whereES) and the angle-bending component (EB) as given in Equations 1 to 3. The variables and parameters required for Equations 1 to 3 can be found in [According to the modified Morse potential, the potential energy of the isolated graphene sheet can also be expressed as the sum of the bond-stretching component , which was further employed in conjunction with Equation 4 to model the non-linear stress–strain behavior of C-C bonds  of the multiscale model as shown in Figure tructure , but in tructure , epoxy wThe most significant part of the proposed multiscale model is the interphase region between the atomistic graphene model and the continuum polymer representation  was employed as illustrated in Figure Mechanical properties for the truss elements were determined in a similar manner to that presented in the research work by Odegard et al. , namely r is the distance between two atoms, ϕ is the hard sphere radius (ϕ = 0.34 m), and ϕ is the potential well depth . The mechanical properties for the truss elements were obtained by equating the LJ ‘6-12’ potential with the structural strain energy of truss elements. The resulting expression is provided in Equation 6 and was used for assigning material stiffness values to each truss element with a cross-sectional area of 0.0907 nm2:where Figure GI) was considered as the characterizing parameter in this paper. The mathematical formulation based on Figure GI[In this work, the virtual crack closure technique was employed in conjunction with a multiscale model to study the fracture characteristics of graphene/polymer nanocomposites under opening-mode crack propagation. The strain energy release rate (SERR) mode I  for subsequent investigations.Initially, SERR values were estimated for a crack propagating in neat polymer with boundary conditions as shown in Figure GI cohesive crack growth was investigated, considering boundary conditions as illustrated in Figure GI was reduced with the increase in graphene volume fraction. In comparison to the neat epoxy model , an improvement of up to 6% in fracture toughness (when the crack passes in the vicinity of the graphene sheet) was observed for a graphene volume fraction of 2.125%, which increased to 18% for a volume fraction of 8.5%. Note that in the current modeling approach, an increase in graphene volume fraction is associated with a reduction of the graphene sheet aspect ratio, which is defined as the length to width ratio. The influence of graphene aspect ratio will further be discussed in the following sections.In the first part of this study, the effect of graphene volume fraction on G1R values in neat epoxy can be attributed to the absence of this shielding effect.Observed improvements in fracture toughness of graphene nanocomposites were attributed to the change in stress distribution in the (continuum) polymer epoxy phase due to the graphene inclusion in the vicinity of the crack. It is postulated that graphene with its space frame structure and high stiffness bears most of the applied load and shields the crack tip from opening loads or crack-driving forces, whereas higher SERR GIR values obtained according to Figure GI values employing the conditions defined in Figure To further investigate the influence of graphene on fracture characteristics and the aforementioned shielding effect of graphene in graphene/polymer nanocomposites, modeling was performed with all three models as described in Figure GI values were reduced by up to 24% (for crack propagation near the graphene sheet) compared with the GIR values. Modeling results therefore indicate that the crack tip shielding effect from crack driving forces is more pronounced when graphene is present next to the crack on both sides of the crack plane. It shall be mentioned at this point that an improvement in SERR also occurs when a ‘Single graphene’ model with a graphene volume fraction identical to that of the ‘Twin graphene’ case is considered, which is shown in the subsequent section.It can be inferred from the SERR values plotted in Figure Earlier experimental works ,3 showedGr) employed in the above models was kept constant at 4.1209 nm. The sheet width, however, was adjusted, that is, for the model with high aspect ratio and uniform dispersion  of the graphene sheet was employed compared to the model with low aspect ratio (Figure The multiscale models as defined in the schematics in Figure n Figure , half tho Figure . The anaFirst, modeling was performed for a graphene volume fraction of 8.5%. Corresponding SERR values are plotted with respect to crack length in Figure To corroborate the above findings, modeling was performed next for a graphene volume fraction of 4.25%, and results are shown in Figure A multiscale modeling technique in conjunction with a representative volume element approach was successfully employed in this investigation to study the fracture characteristics of graphene/polymer nanocomposites. Substantial improvements in the fracture characteristics of graphene/polymer nanocomposites were observed for higher volume fractions of graphene. It was concluded from this research that shielding of the crack tip from driving forces occurred in the vicinity of graphene sheets. In addition to graphene volume fraction, a uniform distribution of graphene in the polymer matrix as well as high aspect ratio of graphene sheets were found to enhance fracture toughness of graphene nanocomposites. Consequently, crack growth retardation can be achieved in graphene nanocomposites even without diverting or increasing the path of crack propagation.The authors declare that they have no competing interests.Both authors made equally valuable contributions to this paper. Both authors read and approved the final manuscript.


MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1), Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements. LINE1s, the only active autonomous mobile DNA in humans, occupy at least 17% of our genome. It is believed that about 100 L1s are potentially active in any individual diploid genome. The L1 has also been responsible for genomic insertion of processed pseudogenes and more than a million non-autonomous retrotransposons, mainly Alus and SVAs. Together, this mass of genomic baggage has had, and continues to have, profound effects on gene organization and expression. Consequently a number of molecular mechanisms have evolved to prevent the unchecked expansion of endogenous retroelements. We demonstrate that the putative RNA helicase MOV10, recently discovered to limit production and infectivity of retroviruses, also profoundly inhibits retrotransposition of L1s, Alus, and SVAs in cell culture. Microscopy and immunoprecipitation show a close association of MOV10 protein with the L1 ribonucleoprotein particle. This study reveals a novel factor that interacts with the L1 retrotransposon to modulate its activity, and it increases our understanding of the means by which the cell coexists with these genomic “parasites.” Arabidopsis, armitage, a protein involved in RISC assembly and piRNA control of double-strand RNA viruses and endogenous retroelements in Drosophila, and ERI-6/7, which acts with Argonaute protein ERGO-1 to generate specific subsets of siRNAs in Caenorhabditis elegansMOV10 was originally identified as a protein that prevents infection of mice by Moloney murine leukemia virus MOV10 interacts with Argonaute 1 (AGO1) and AGO2 in mRNA processing (P-) bodies and stress granules (SGs), dynamic cytoplasmic aggregates that participate in mRNA decay and sequester stalled mRNA translation complexes during times of cellular stress Several reports have demonstrated that overexpression of MOV10 severely impairs infectivity of HIV-1 and other lentiviruses. However, evidence is conflicted as to the effects of depleting endogenous MOV10, claiming that inhibiting MOV10 variously reduces HIV-1 infectivity by 50% Over two-thirds of the human genome may be repeat-derived, mostly from transposable elements LINE-1 (L1) non-LTR retrotransposons comprise at least 17% of the human genome, and are its only autonomously active mobile DNA. Up to 5% of newborn children are estimated to have a new retrotransposon insertion in trans for their insertion into the genome  CCCTCT hexameric repeats, 2) sequence with homology to two antisense Alu fragments, 3) variable number of tandem repeats (VNTR), and 4) sequence derived from the ENV gene and right LTR of an extinct HERV-K (SINE-R). There are roughly 2700 SVA copies in the human genome, most of which are full-length (2–3 kb), and some of which are active Here we reveal a close association of both exogenous and endogenous MOV10 with the L1 ribonucleoprotein particle (RNP). Using established cell culture reporter assays, we demonstrate that MOV10 protein, first shown to inhibit retroviruses, has an expanded repertoire that includes suppression of non-LTR retrotransposition. Some phylogenetic analyses suggest that non-LTR retrotransposons are the likely progenitors of retroviruses and LTR retrotransposons L1 expresses a 6-kb bicistronic RNA that encodes a 40 kD RNA-binding protein (ORF1p) of essential but uncertain function for retrotransposition, and a 150 kD ORF2 protein with endonuclease and reverse transcriptase (RT) activities. Epitope-tagging of either of these proteins permits the immunoprecipitation (IP) of L1 RNP particles from cells, along with associated cellular proteins in vitro PCR-based assay We tagged the C-terminus of ORF1 in L1-RP  analyses. Among approximately 100 non-ribosomal proteins unique to the pc-L1-1FH sample and identified by at least two predicted peptides (paper in preparation), we detected Moloney leukemia virus 10, homolog (mouse), or MOV10 .We cloned MOV10 cDNA with N-terminal V5-epitope tag and showed by Western blotting that its protein could co-IP with tagged pc-L1-IFH RNPs after transfection in 293T cells. This association was RNA-dependent and was lost upon treatment with RNase , lane 4.FLAG-tagged pc-L1-1FH was also able to pull-down endogenous MOV10 protein from 293T cells . We nextTherefore, both exogenously expressed and endogenous MOV10 proteins associate with the L1 RNP and ORF1p in multiple cell lines.ORF1p is a predominantly cytoplasmic protein, but can be detected in nuclei and enters nucleoli of a minor percentage of cells Meister et al. To assess MOV10 association with the L1 RNP, we cotransfected V5-tagged MOV10 together with ORF1-GFP L1-RP, a construct containing a CMV promoter, ORF1 C-terminally tagged with EGFP, and intact downstream L1 sequence We previously reported We next assayed for colocalization of endogenous ORF1p and MOV10 proteins. ORF1p-EGFP overlaps endogenous MOV10 protein in almost all visible cytoplasmic granules . ConversFinally, we examined 293T cells for association of the L1 RNP and FLAG-tagged mouse MOV10L, the MOV10 testis-specific paralog To summarize, both exogenous and endogenous ORF1p and MOV10 proteins are directed to the same cytoplasmic compartments.mneoI-reporter assay Cognizant of the ability of MOV10 to inhibit retroviral infection, and of the related MOV10L protein to suppress transcription of L1s and IAP retrotransposons in male germ cells of knock-out (KO) mice The duration of the cell culture retrotransposition assay is necessarily long: in 293T cells the first retrotransposition events appear after 24 hours, and slowly accumulate in number over the course of the experiment. Therefore, it is important to determine if coexpressed proteins cause toxicity that might bias interpretation of results. Both to determine transfection efficiency and to assay for potential MOV10-related toxicity, we transfected pCEP-EGFP, a vector that constitutively expresses EGFP, together with empty or V5-tagged MOV10, and after 4 days performed flow cytometry. Loss of fluorescence due to MOV10 cotransfection was minimal, and could not account for the severe reduction in levels of retrotransposition  silenced MOV10 protein expression by 90%, while a third (shE3) had no apparent effect .Tet was cotransfected with either empty vector or a retrotransposition “driver” plasmid: i.e. pCEP 5′UTR ORF2 No Neo  were stained and counted. Alu-neoTet with empty vector generated almost no colonies, but in the presence of a driver plasmid numerous neomycin-resistant foci were detected. Introducing MOV10 reduced foci number by almost 95 percent , for effect on L1 retrotransposition in cell culture. Mutations in all motifs except Ia and V  and its mutant proteins were expressed at similar levels in 293T cells, and all co-IPed efficiently with pc-L1-1FH , which is involved in maintaining some genes in a transcriptionally repressed state during development. El Messaoudi-Aubert In addition to its role in histone modification, CBX7 recruits DNA methyltransferases (DNMTs) to chromatin How CBX7 or CBX8 might inhibit retrotransposition is unclear. Perhaps recruitment of PcG proteins to nascent L1 transcripts induces transcriptional silencing. Alternatively, association of these proteins with the L1 RNP might alter chromatin structure at sites of genome insertion, and so interfere with resolution of the L1 integrant. Chromatin immunoprecipitation (CHIP) and qPCR analyses of multiple L1 loci may determine if MOV10, together with PRC1 proteins, are recruited to L1s to suppress expression. Little information exists concerning the effects of chromatin structure on mammalian retrotransposon expression, although two studies have proposed roles for HDAC1 and HDAC2 In summary, MOV10 inhibits L1 expression and the number of its cellular RNPs available for retrotransposition through an as yet undetermined mechanism. However, possible mechanisms include MOV10-mediated 1) sequestration and silencing of L1 RNPs in stress granules, 2) RISC inhibition of L1 transcription or translation, and 3) PcG protein recruitment for suppression of L1 expression.Here we demonstrate that the putative RNA helicase MOV10 is a potent inhibitor of LINE1-mediated retrotransposition when overexpressed in cells. Conversely, knockdown of endogenous MOV10 results in a significant increase in levels of L1 retrotransposition. These results parallel the recently described capacity of MOV10 to diminish viral production and infectivity of HIV and other pathogenic retroviruses The mechanism by which MOV10 inhibits retrotransposition remains unclear, but in light of MOV10 association with RISC, it is reasonable to consider that RNAi silencing is involved. Two studies have reported that L1-derived small (sm) RNAs participate in its inhibition de novo methylation of their regulatory sequences de novo methylation and silencing of retrotransposons piRNAs appear to inhibit retrotransposons by stimulating As noted above, MOV10 localizes with AGO1 and AGO2 in stress granules and P-bodies Of course, helicases are involved in many other cellular processes, including transcription, pre-mRNA processing, RNA export, translation, RNA storage, RNA decay, and ribosome biogenesis  and synonymous (dS) nucleotide substitutions per site and dN/dS (ω) ratios over seven primate species using the PAML 4.5 software package Phylogenetic analyses suggest that eukaryote non-LTR retrotransposons predate all LTR retrotransposons, which in turn gave rise to retroviruses through the acquisition of an envelope (env) gene Cells were lysed in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with protease and phosphatase inhibitor cocktails II and III, vanadyl ribonucleoside complexes, PMSF (Sigma), and RNasin (Promega). Nuclear extracts were prepared with NE-PER Nuclear Protein Extraction Kit (Pierce). RNase inhibitors were omitted from samples treated with 15 µg/ml DNase-free RNase (Roche). FLAG-tagged L1 RNP complexes were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma), and the Johns Hopkins University Mass Spectrometry and Proteomics Facility analyzed immunoprecipitates.Protein identification of complex samples by LC-MS/MS was performed using an LTQ ion trap MS (Thermo Fisher Scientific) interfaced with a 2D nanoLC system (Eksigent). Peptides were fractionated by reverse-phase HPLC and sequences were identified using Proteome Discoverer and Mascot software (Matrix Science).IP of V5-tagged proteins from 2102Ep cells  utilizedTo generate pc-RP-1FH, we introduced by Kunkel mutagenesis neoI retrotransposition assays, MOV10 cDNA was recloned in a modified Gateway system vector lacking the neo gene .ORF1 TAP-tagged L1 and ORF1-GFP-L1-RP constructs have been described RNAi Consortium shRNA library (Open Biosystems) clones were shGAPDH (TRCN0000221342), shE1 (TRCN0000049978), shE2 (TRCN0000049981), and shE3 (TRCN0000049980). These were packaged in lentiviral particles in 293T cells by standard methods.Tet and SVA.2 retrotransposition reporter constructs have been described 99 PUR RPS EGFP, 99 PUR JM111 EGFP, Alu neoHuman embryonic kidney HEK293T cells, HeLa-HA cells, and 2102Ep cells were grown in DMEM medium with 10% FBS (Hyclone), GlutaMax, and Pen-strep (Invitrogen). All transfections used FuGENE HD (Promega) reagent. 2102Ep and HeLa-HA cells were gifts from P. K. Andrews  and J.V. Moran (U. of Michigan), respectively.5 HeLa or 293T cells/well were seeded in 6-well dishes. The following day, 1.0 µg of 99 PUR RPS EGFP, a plasmid containing L1-RP and EGFP retrotransposition reporter cassette, was cotransfected together with 0.5 µg empty vector  or test plasmid. All transfections were in quadruplicate. Five days post-transfection, cells having a retrotransposition event and hence expressing EGFP were assayed by flow cytometry. Gating exclusions were based on background fluorescence of plasmid 99 PUR JM111 EGFP, an L1 construct containing two point mutations in ORF1 that completely abolish retrotransposition The EGFP L1 cell culture retrotransposition assay was conducted as previously described Tetneo was cotransfected with pcDNA3 or retrotransposition driver plasmids pCEP 5′UTR ORF2 No Neo 75 flasks, and four days later selection for retrotransposition events with 600 µg/ml of G418 was begun. After 15 days of selection, cells were fixed, stained with Giemsa, and colonies were counted.Alu retrotransposition assays were carried out essentially as described in Dewannieux et al. mneoI was cotransfected with both pCEP.ORF2 and pcDNA.ORF1-RP in HeLa-HA cells, expanded to T75 flasks, and selected with G418 as described above for the Alu assay. SVA.2 EGFP was cotransfected in 293T cells together with pCEP.ORF2 or pcDNA.ORF1-RP, and retrotransposition was examined by FACs after 5 days SVA retrotransposition assays were conducted as previously described. SVA.2 Immunofluorescence techniques have been described Western blot antibody incubation was in blocking solution (PBS/0.05% Tween 20/5% dry milk) overnight at 4°C. Membranes were washed 3× for 15 min with PBS/0.05% Tween 20, incubated with secondary antibody at room temperature in blocking solution for 2 hr and washed again. Detection used SuperSignal West Pico Chemiluminescent Substrate (Pierce).Reverse transcriptase analysis followed the LEAP protocol of Kulpa and Moran GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN3′RACE adapter NV: GCGAGCACAGAATTAATACGACT3′RACE outer: CACCGCATATTCTCACTCATAGGaORF2-end1: Two µl of IP sample were added to each cDNA extension reaction. PCR amplification of cDNA utilized Expand Long Template PCR System (Roche). Products were separated on 2% agarose gels.GATGAGTTCATATCCTTTGTAGGGGGGAGTGGCACCTTCCAGGGTC.For RT–PCR analyses, immunoprecipitates were directly treated with TURBO DNase (Invitrogen), and cDNA generated using iScript cDNA Synthesis Kit (Bio-Rad). Standard PCR reactions used the Expand Long Template PCR System (Roche) and the following primers: bORF2-end2, Bisulfite analysis was performed exactly as described http://www.ebi.ac.uk/goldman-srv/webPRANK/; http://www.jalview.org; Protein sequences of Figure S16 mutant constructs , Chimp , Gorilla , Orangutan , Gibbon , Rhesus macaque ; Marmoset , and Bushbaby . Unaligned insertions and deletions in the gibbon sequence are likely assembly errors. Conserved helicase motifs are blocked in pink, and residues altered in MOV10-V5-Hisnstructs  are unde(TIF)Click here for additional data file.Figure S2Construct pc-L1-1FH-EGFP is retrotransposition-competent, but at a reduced level compared with a similar construct lacking the FLAG-HA tag on ORF1 (pc-L1-RP-EGFP). The EGFP reporter cassette (TIF)Click here for additional data file.Figure S3Methylation analyses of the 5′ UTR promoter of endogenous L1 elements show no effect of MOV10. (A) The individual methylation status of 36 L1 sequences in the presence (right) or absence (left) of V5-MOV10 protein. Open circles, closed circles and lines represent unmethylated, methylated, and mutated CpG positions, respectively. (B) The percentage of methylation of the 20 CpG residues in the absence (white) or presence (black) of V5-MOV10. Applying Fisher's Exact Test, no significant effect of ectopic MOV10 expression on L1 5′ UTR methylation status was found. CpG residues are numbered according to the retrotransposition-competent element L1.3 (accession number L19088.1).(TIF)Click here for additional data file.Figure S4Analysis of primate MOV10 genes for positive selection using maximum likelihood estimation and PAML4 software. (A) Pair-wise comparisons of dN and dS among the eight primate MOV10 sequences shown in (TIF)Click here for additional data file.Table S1RNA helicase proteins identified after LC-MS/MS and Mascot protein/peptide analyses of immunoprecipitated L1 RNPs.(TIF)Click here for additional data file.


Attentional processes in children play a critical role in daily school demands and accomplishments. Studies on the association of attentional processes with school achievement and age in healthy school children are scarce. The aim of the present study was to identify correlations between dimensions of attentional performance, scholastic achievement and age.An extensive testing battery was used to assess a wide range of attentional dimensions. A principal component analysis revealed three factors that are related to attentional performance . Age was negatively associated with distractibility, lapses of attention and cognitive speed, indicating that distractibility and lapses of attention decreased with age in healthy children and resulted in lower cognitive speed.Attentional processes in healthy children should be measured in relation to distractibility, lapses of attention and cognitive speed. Attentional processes are the foundation for a variety of cognitive functions, such as perception, learning, the integration of information to establish working memory processes, and the active recall and integration of information Attentional processes are apparent in the context of the intensity and selectivity of stimuli, which can be thought of as quantitative and qualitative processing Other concepts assume a supervisory attentional system that controls attentional processes that are responsible for focusing on an object To focus on a specific task, one must neglect distractions that could trigger an external or internal attentional shift. For this reason, the active inhibition of distracters is required, and ignoring distractions while focusing on a specific task are separate subcomponents of attentional processes Scholastic achievement is positively correlated with attention-related capabilities and the development of attentional processes. However, in most investigations, attentional performance has been assessed using teacher and parent ratings of children's ability to focus and shift attention. Attentional deficits in children have also been assessed with teacher and parent observations xy = −0.532; p<0.01; see xy = −0.537; p<0.01) and cubic trend . As expected, we did not detect significant associations between age and scholastic achievement in our sample of healthy children. DS was negatively correlated with age , DS  and LA .As hypothesized, there were significant correlations between age and the three aspects of attentional performance . CS and age were negatively correlated and followed a linear  must be taken into account although the spectrum of school grades in the present sample ranged from 1 to 5 . Finally, although the KITAP provides a reliable way of measuring attentional performance, other methods of assessment may be taken into consideration in future studies.In summary, the present study provides preliminary evidence that aspects of attentional performance improve with age, possibly at the cost of reduced cognitive speed.Data were obtained from 61 healthy school children, between 6.1 and 10.69 years old , who attended a German primary school. Exclusion criteria were attentional and motor deficits as well as neurological and psychiatric conditions, which were assessed during parent interviews. Prior to participation in the study parents were asked if their children had a traumatic brain injury or intellectual disabilities, and such children were not included into the study. Intellectual capacity was not assessed in detail as the sample consisted of healthy children without any reported behavioral or school problems. In addition the parents were asked if their children had behavioral problems or other acute diseases. Children with severe untreated vision impairments (children with glasses or lenses) or hearing impairments did not participate in the study. The study was approved by the local school authorities  and carried out in accordance with the Helsinki Declaration. Parents, the head of the primary school and children gave their written informed consent to participate in the study. Characteristics of the study sample are provided in Sub-dimensions of attentional performance were assessed using the KITAP alertness, distractibility and sustained attention with the following factor loads: MD of RT : 0.79; MD of RT (distractibility): 0.738; and MD of RT (sustained attention): 0.708. Factor loads for DS were also identified: SD of RT (Go/No-go): 0.738; SD of RT : 0.717; SD of RT (flexibility): 0.271; SD of RT (distractibility): 0.61; and errors (flexibility): 0.683. Factor loads for LA were 0.535 for omissions during divided attention, 0.57 for omissions in the Go/No-go task, and 0.511 for omissions during the visual scanning task.A confirmatory factor analysis was conducted to identify the relevant attentional domains assessed in the KITAP. The analysis showed that CS, DS and LA were the main dimensions of attention that explained most of the intraindividual variance. CS was accumulated by the median (MD) of the reaction times (RT) of the KITAP subtests School grades in the subjects “German” and “Mathematics” were used to assess scholastic achievement . These two subjects were chosen as they are taught throughout the entire school period in Germany, which would also allow future follow-up assessments. In addition, in the first school year students only get grades in the subjects “German” and “Mathematics”. Moreover, these particular subjects cover different aspects of scholastic performance, i.e. as regards verbal capabilities and mathematical reasoning.p = 0.086). The items were loaded on a single factor, which was derived by a confirmatory factor analysis, that explained 73.35% of the variance.To determine the reliability and internal consistency, Cronbach's alpha (0.915) and Guttman split-half reliability were computed (r (tt) = 0.87). A Kolmogorov-Smirnov goodness-of-fit test indicated that the data were normally distributed . The level of statistic significance was set at


Objective(s): To investigate the expression of human papilloma virus (HPV) L1 capsid protein, and human telomerase RNA component (hTERC) in cervical cancer and the role of detection of both genes in screening of cervical cancer. Materials and Methods: A total of 309 patients were recruited and cervical exfoliated cells were collected. Immunocytochemistry was employed to detect HPV L1 capsid protein, and fluorescent in situ hybridization (FISH) was performed to detect the hTERC. Results: The expression of HPV L1 capsid protein reduced with the increase of the histological grade of cervical cells and was negatively related to the grade of cervical lesions. However, the expression of hTERC increased with the increase of the histological grade and positively associated with the grade of cervical lesions. The proportion of patients with L1(-)/hTERC(+) was higher in patients with histological grade of CIN2 or higher than that in those with histological grade of CIN1. The L1(+)/hTERC(-) and L1(-)/hTERC(-) were negatively related to the grade of cervical lesions. L1(-)/hTERC(+) was positively associated with the grade of cervical lesions. The L1/hTERC ratio increased. The negative predictive value of both HPV L1 and hTERC was higher than that of HPV L1 or hTERC, but there was no marked difference in the screening efficacy of cervical cancer among HPV L1, hTERC and HPV L1+hTERC. Conclusion: HPV L1 capsid protein and hTERC gene may serve as markers for the early diagnosis and prediction of cervical lesions. The increase in L1/hTERC ratio reflects the progression of cervical lesions to a certain extent.  Cervical cancer is the second leading common cause of malignancies in women -2. WorldSample collection A total of 309 patients were recruited from the Shenzhen Hospital of Beijing University from January 2010 to May 2011, and cervical exfoliated cells were collected. The average age was 37.3±8.9 years (range: 18~67 years). Cytological examination was performed according to the Bethesda System (TBS) 2001. On the basis of cytological findings, for intraepithelial lesion or malignancy (NILM) was found negative in 72 patients, atypical squamous cells of undetermined significance (ASC-US) in 71, atypical squamous cells-cannot exclude high-grade squamous intraepithelial (ASC-H) in 19, Low grade squamous intraepithelial lesion (LSIL) in 80, high grade cervical squamous intraepithelial lesion (HSIL) in 49 and squamous-cell carcinoma (SCC) in 18. On the basis of pathological findings, cervical intraepithelial neoplasia (CIN) grade 1 was found in 168 patients, CIN grade 2/3 in 84, SCC in 24, normal cervical cells/cells of chronic cervicitis (absence of CIN or NILM) in 33. All patients had no conization of cervix, ureterectomy and a history of radiotherapy and/or chemotherapy. Patients with abnormal cells (≥ASC-US) or positive for HPV DNA also received vaginoscopy and biopsy. Diagnostic curettage was performed if necessary.Immunocytochemistry for HPV L1 Capsid ProteinCytoReact cell/tissue HPV L1 detection kit  was used to detect HPV L1 capsid protein according to manufacturer’s instructions. In brief, the smeared cells were fixed in 96% ethanol for 20 min and then dried. Subsequently, these cells underwent dehydration in 96%, 75% and 50% ethanol series and then in distilled water (2 min for each). Following washing with acid, pre-hybridization was done and inactivation of endogenous peroxidase was performed to remove the non-specific staining. Exogenous HPV L1 antibody was used to treat these cells followed by DNA hybridization and treatment with exogenous antibody again. Following hybridization, treatment with RNAase and peroxidase was performed. Staining was done with alkaline phosphatase and immunohistochemistry by AEC staining. Counterstaining and mounting were performed and sections were preserved at room temperature in dark. These sections were observed under a microscope. Sections with one nucleus stained red were regarded as positive for HPV L1. In addition, the negative and positive samples provided by the manufacture served as negative and positive controls, respectively and observed under a microscope. Sections with one nucleus stained red were regarded as positive for HPV L1.Detection of hTERC gene with fluorescent in situ hybridization (FISH) Probes (hTERC/CSP3 DNA) were provided by the Beijing Jinpujia Medical Technology Company. These probes can hybridize into 3q26.3, and red fluorescence (Tetramethylrhodamine) was presented. CSP3 served as a control probe, can hybridize into the centromere of chromosome 3 and presented green fluorescence. In brief, the cervical exfoliated cells were digested in collagenase B, treated with hypotonic solution and then fixed. Then cells were digested in pepsin. Following drying, cells were denaturated in formamide. The sections were hybridized with pre-denaturated probes overnight. After washing, the sections were observed under a fluorescence microscope. Normal cells had single nucleus and presented one red and one green signal. Cells with abnormal hTERC amplification had single nucleus with more than two red signals and no less than two green signals. A total of hundred cells with complete signals were counted and was defined positive when six presented cells were increased in hTERC copies . Positive represented increase in hTERC amplification.Statistical analysis Z) and compared with chi square test and Z test. A value of P<0.05 was considered statistically significant. Statistical analysis was performed with SPSS version 13.0 for Windows. The HPV L1 capsid protein expression and hTERC level were compared with chi square test and Spearman’s rank test. The sensitivity, specificity, positive predictive value and negative predictive value and diagnostic accuracy of HPV L1 capsid protein expression in cervical cells in prediction of CIN2/3 cervical lesions were determined. Receiver operating characteristic curve (ROC) was delineated and the area under curve (AUC) was calculated . In patients with CIN1, the HPV L1 capsid protein expression was markedly higher than that in patients with CIN2/3  or SCC . The HPV L1 capsid protein expression was negatively related to the grade of cervical lesions , , . Detection of hTERC gene 2=115.66,P<0.001). In patients with CIN1, the hTERC level was markedly lower than that in patients with CIN2/3 or SCC . The hTERC level was positively related to the grade of cervical lesions   . Detection of HPV L1 capsid protein and/or hTERC in diagnosis of CIN grade 2 or higherP<0.001). When diagnosis was done with findings in detection of HPV L1 capsid protein and hTERC, the negative predictive value was relatively high (P<0.05). When diagnosis was done with findings in detection of HPV L1 capsid protein, hTERC and HPV L1 capsid protein+ hTERC, the AUC was 0.704, 0.782 and 0.714, respectively, and they could be used to diagnose the CIN grade 2 or higher (P<0.001). However, z test confirmed that there was no difference in the screening of cervical cancer (P>0.05)  .Correlation of cervical lesions with HPV L1 capsid protein expression and hTERC2=14.628, P<0.001) or SCC . The L1(-)/hTERC(+) was positively related to the grade of cervical lesions (P<0.001), but L1(+)/hTERC(-) and L1(-)/hTERC(-) were negatively associated with the grade of cervical lesions (P<0.001). In addition, the constituent ratio of different forms was related to the grade of cervical lesions. The constituent ratio of L1(-)/hTERC (-)→L1(+)/hTERC (-)→L1(+)/hTERC(+)→L1 (-)/hTERC(+) had an increasing tendency with the increase in the grade of cervical lesions (The HPV L1 capsid protein expression and hTERC had four combining forms: L1(+)/hTERC(+), L1(+)/hTERC(-), L1(-)/hTERC(+) and L1 (-)/hTERC (-). In patients with CIN1, the proportion of patients with L1(-)/hTERC(+) was lower than that in patients with CIN2/3 .et al . In CIN1 patients, the proportion of L1(-)/hTERC(+) patients was lower than that in patients with CIN2/3 (42.9%) or SCC (100%), and this was the unique form in SCC patients. This indicates the loss of HPV L1 expression, and hTERC amplification plays an important role in the progression of cervical lesions. L1(-)/hTERC(+) also demonstrates that the HPV DNA has been integrated into host genome, and loss of HPV L1 expression and hTERC amplification may predict a high risk for cervical cancer. L1(+)/hTERC(+) was only found in CIN patients but not in SCC patients and those with normal cervical cells or chronic cervicitis. L1(+)/hTERC(+) means the presence of hTERC amplification, replication of HBP DNA, synthesis of HPV L1 and assembly of virosome. This form predicts high infectivity but can not be used to differentiate low grade from high grade. Thus, under the condition of L1(+)/hTERC(+), the grade of cervical lesions are uncertain. L1(+)/hTERC(-) was found in patients with normal cervical cells or chronic cervicitis or CIN1 or CIN2/3 but not in those with SCC. This implies that the HPV replication can induce cellular immunity but not hTERC amplification. L1(+)/hTERC(-) was negatively related to the grade of cervical lesions , and thus patients with L1(+)/hTERC(-) are regarded as having low risk for cervical cancer. L1(-)/hTERC(-) was not found in SCC patients. L1(-)/hTERC(-) means latent HPV infection, no or low HPV replication and no hTERC amplification. L1 (-)/hTERC(-) was negatively associated with the grade of cervical lesions  and patients with L1(-)/hTERC(-) can be regarded as having low risk. The L1/hTERC expression was in the following order with the increase in grade of cervical lesions: L1(-)/hTERC(-), L1(+)/hTERC(-), L1(+)/hTERC(+) and L1(-)/hTERC(+), which also reflects the progression of cervical lesions. Thus, we speculate that patients with L1(-)/hTERC(+) and CIN1 or lower should be closely monitored and cytological examination and biopsy may be regularly performed. For patients with L1(-)/hTERC(-) or L1(+)/hTERC (-) and CIN grade 1 or lower, the interval of follow up can be prolonged. For patients with L1(-)/hTERC(-) or L1(+)/hTERC(-) and CIN grade 2/3, physicians should be cautious about over-diagnosis and over-treatment. The findings in the present study required to be further confirmed in further studies. In addition, our results also revealed that L1 (-)/hTERC(+) was positively related closely to the grade of cervical lesions (The expression of HPV L1 capsid protein reduced with the increase of the histological grade of cervical cells and was negatively related to the grade of cervical lesions. However, the expression of hTERC increased with the increase of the histological grade and positively associated with the grade of cervical lesions. HPV L1 capsid protein and hTERC gene may serve as markers for the early diagnosis and prediction of cervical lesions. The increase in L1/hTERC ratio reflects the progression of cervical lesions to a certain extent.


The Southern Chiapas focus of onchocerciasis in Southern Mexico represents one of the major onchocerciasis foci in Latin America. All 559 endemic communities of this focus have undergone semi-annual mass treatment with ivermectin since 1998. In 50 communities of this focus, ivermectin frequency shifted from twice to four times a year in 2003; an additional 113 communities were added to the quarterly treatment regimen in 2009 to achieve a rapid suppression of transmission.Simulium ochraceum s.l. collected from this focus during the dry season of 2011 were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in an upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.06/2,000 flies examined. Serological assays testing for Onchocerca volvulus exposure conducted on 4,230 children 5 years of age and under  revealed that 2/4,230 individuals were exposed to O. volvulus .In-depth epidemiologic and entomologic assessments were performed in six sentinel communities (which had undergone 2 rounds of ivermectin treatment per year) and three extra-sentinel communities (which had undergone 4 rounds of ivermectin treatment per year). None of the 67,924 The in-depth epidemiological and entomological findings from the Southern Chiapas focus meet the criteria for interruption of transmission developed by the international community. Onchocerca volvulus in the black fly vector of this parasite and reduction of exposure to and new infections with O. volvulus are the criteria currently used to certify focal interruption of parasite transmission. In the present study, we report entomological and epidemiological assessments in the Southern Chiapas focus of Mexico that together indicates that transmission of O. volvulus has been interrupted in this focus. None of the Simulium ochraceum s.l. vector black flies collected from sentinel and extra-sentinel communities in this focus during the 2011 transmission season was found to contain parasite DNA when tested by PCR-ELISA, suggesting vector parasite contact was nearly nonexistent. In addition, there was a minimal exposure to the parasite in children 5 years of age and under, as measured by circulating antibody to a parasite-specific antigen. The Southern Chiapas focus was the major focus in Mexico and one of the largest in Latin America, with well-documented history of active transmission prior to the commencement of Mectizan mass distribution. This study demonstrates the interruption of transmission in geographically large focus in Latin America with a historically high intensity of transmission.The absence of infective larvae of  Onchocerca volvulus, which in Latin America is transmitted by new world black flies (Simulium spp.) in six countries , where 525,543 individuals are at risk 2) and well-documented history of intense transmission. The Southern Chiapas focus contains 559 affected communities, 39 of which were hyperendemic for onchocerciasis before control efforts began, while 209 of the communities were mesoendemic, and 311 were hypoendemic. The population at risk in the Southern Chiapas focus  comprised about 21% of the total at-risk population in the Americas.Human onchocerciasis is caused by the filarial parasitic nematode Since the 1990s, onchocerciasis control in Mexico has relied on the mass distribution of Mectizan (ivermectin) to the at-risk communities. Annual mass ivermectin distribution treating to all eligible residents from the at-risk communities began in 1994. In 1998 the strategy was modified to provide mass treatments every 6 months. In 50 communities from the Southern Chiapas focus, the ivermectin distribution frequency again shifted in 2003, from twice to four times a year. An additional 113 communities were added to the quarterly treatment regimen in 2009.O. volvulus and eventually eliminate transmission of the parasite in all 13 foci in Latin America. The World Health Organization (WHO) O. volvulus in the vector population . Practically, because polymerase chain reaction (PCR) using O. volvulus-specific DNA probes are generally applied to examine pools of flies, parity cannot be easily determined, so the threshold used is less than one infective fly per 2,000 flies tested (assuming a 50% parity rate) O. volvulus transmission has been interrupted in the Southern Chiapas focus.The goal of the Onchocerciasis Elimination Program for the Americas In 1995, local health authorities selected six sentinel communities of the Southern Chiapas focus . These iThese communities were the ones evaluated in this study. These communities have to varying extents been included in previous in-depth epidemiological evaluations (EEP) which have been carried out in the Southern Chiapas focus . The EEPApart from the sentinel and extra-sentinel communities, a large-scale serological study was performed during 2010 on 4,230 children resident in 110 extra-sentinel communities with high historical endemicity. This sampled group represented 41% of all children five and under who were residents in these historically highly endemic communities.In the Southern Chiapas focus, ivermectin distribution was commenced in 1990, but was initially only offered to registered onchocerciasis “clinical cases”. In 1994, every eligible resident in the hyperendemic and mesoendemic communities was treated, but only 25% of the eligible residents from hypo-endemic communities were treated. From 1995 to 1997, the 40% of the eligible population in hypo-endemic communities was treated. Beginning in 1998, the entire eligible populations in all communities, regardless of their level of endemicity, were provided with semi-annual ivermectin treatments. In 50 communities of this focus, treatments were increased from twice to four times per year in 2003; the quarterly treatment regimen was extended to 113 communities in 2009. A total of 22 consecutive treatment rounds reaching >85% of the eligible population have been provided in the Southern Chiapas focus over the past 11 years. Ivermectin coverage of the eligible population has remained at a level greater than 85% every year from 1998 through 2011 .S. ochraceum s.l) were collected in the sentinel and extra-sentinel communities using standardized procedures O. volvulus transmission season, lasting from December to March. Collections were carried out during the first 50 minutes of each hour, beginning at 11:00 AM and ending at 4:50 PM Black flies  have been published elsewhere Flies were combined into pools containing a maximum of 50 individuals per pool and the heads and bodies separated as previously described Seasonal transmission potentials [STP] for each sentinel and extra-sentinel village were calculated as the product of the seasonal biting rate, the proportion of flies carrying L3 larvae in the transmission season (from December through March), and the average number of L3 larvae in each infective fly. As previously discussed, after multiple rounds of Mectizan treatment, the number of infective larvae present in each infective fly was assumed to be one S. ochraceum s.l. females were not collected throughout the year, it was not possible to precisely calculate the annual transmission potential (ATP). However, given the paucity of vector black flies present outside the normal transmission season, the transmission potential outside of the peak transmission period is probably zero or near zero. The STP (transmission occurring during the peak transmission season of December through March) thus likely represented a fairly accurate estimate of ATP.The seasonal biting rate was calculated as the product of the geometric mean O. volvulus, was determined from two populations of children in the Southern Chiapas focus: residents in the sentinel and extra-sentinel villages, and school children selected from the overall population sample in the focus. In 110 communities , blood spots were collected from 4,313 children 5 years of age and under. Sera from 4,230 children were screened for Ov16 antibodies.The prevalence of IgG4 antibodies to Ov16 O. volvulus positive individuals). The cutoff was chosen as 40 arbitrary units by identifying the value that optimized both sensitivity and specificity. Any positive results were repeated before being reported as positive.Blood spots were collected by finger prick from each individual enrolled in the study, dried in the field, transported to the laboratory at 4°C, and kept refrigerated in sealed bags containing silica gel at −20°C until use, which occurred within a month of collection. Two 6-mm punches of blood saturated filter paper were placed in a phosphate-buffered saline-Tween (PBS-T) 0.05% and bovine serum albumin (BSA) 5% buffer and eluted overnight at 4°C. The elution was then run in duplicate in a standard ELISA O. volvulus microfilariae in the cornea (MFC) and/or the anterior chamber of the eye (MFAC). Before the exam the patients kept a “head down position” (forehead in the lap) for 5 minutes to allow MFC and/or MFAC to settle in a visible position. In 2007 and 2008, a population of 1,418 and 326 residents, representing about 72% and 74% of the total population in the six sentinel and the three extra-sentinel communities were examined.Ocular examinations were carried out by an ophthalmologist experienced in onchocerciasis ocular evaluations for OEPA. The examinations were done using a Topcon Optical SL-3D slit lamp . Exams focused on finding Nodulectomy campaigns have been undertaken in Mexico since 1932. Individuals that undergo nodulectomy received beneficial effects as their mf loads and skin pathology were reduced. Hence, nodulectomy was considered a routine treatment provided by the Mexican elimination program. The number of individuals with nodules of the Southern Chiapas focus was registered from 1995 through 2010. In parallel, the number of new clinical cases  were also registered from 1989 through 2010. The skin biopsies were taken from each patient using a 1.5- to 2.0-mm corneoscleral biopsy punch. Skin biopsies were incubated overnight in buffered saline, and emerging microfilariae were visualized and counted using an inverted microscope.The procedures were reviewed and approved by the Presidents of the Ethical and Bio-security Committees of the National Institute of Public Health  and the Health Secretariat of México . These are the equivalents of the Institutional Review Boards for Human Subject Research in the USA. Community meetings were held in all selected villages within the focus to explain the research procedures, and the right of each individual to decide whether or not to participate was explained. The individuals were also informed that they would be provided with the results of the tests upon request. Before each examination, each adult who had voluntarily come to the examination point and agreed to participate were provided with a capsule summary of the project and process and oral consent was obtained. Parents or guardians provided oral consent on behalf of all child participants. The Ethical Committee of the Health Secretariat of México approved the use of oral consent, given that the studies were conducted as part of the national onchocerciasis surveillance program and were therefore part of a routine public health monitoring program conducted by the Mexican government.Community leaders were also consulted and approved the use of the selected locations in the village and river banks as vector catching points.PoolScreen v2.0 was used to calculate a prevalence of infection and associated 95% CIs in the vector populations. The prevalence of infective flies was then combined with estimates of the biting rate  to calculate an estimated STP. The proportion of individuals with mf in the cornea and/or anterior chamber of the eye was calculated as the number of positive individuals divided by the total number examined, and expressed as a percentage. The associated 95% exact CIs of the proportion of individuals harboring Ov16 antibodies from the large-scale serology study were determined using the method of Thompson S. ochraceum s.l.  were subjected to analysis by PCR. The number of flies tested in each community was sufficient to comply with the WHO guideline of having at least 10,000 flies tested from each community. The results are summarized in In 2008, a total of 103,610 O. volvulus DNA. Similarly, flies were collected in two communities (Brasil and Mexiquito) where evidence of O. volvulus DNA in the vector was seen in entomological studies carried out in 2009–2010. None of the pools collected in 2011 were positive  to assess the status of onchocerciasis transmission, because these metrics take into account the biting rate and the prevalence of infective flies. Estimates of the ATP necessary to maintain the parasite population (the transmission breakpoint) range from 5 to 54 L3/person/year using mathematical modeling O. volvulus antibodies in children in the Southern Chiapas focus was conducted, assessing over 40% of the population of children of age 5 and under who were residents of the afflicted communities. Only two children harbored Ov16 antibodies from 4,230 blood spot samples examined leading to a point estimate of exposure of 0.05% and a one-sided 95% confidence interval on the estimate of the prevalence of exposure of 0.08%, which was below the cutoff established by OEPA of 0.1%.In 2010, a detailed assessment of the prevalence of Additional data suggest that that clinical onchocerciasis has also been eliminated in the Southern Chiapas focus. A total of 1,719 individuals were identified with nodules in this focus in 1995; this declined to 82 individuals in 2010 . This deChecklist S1STROBE Checklist.(DOCX)Click here for additional data file.