Asplenium antiquum and Haplopteris zosterifolia, by examining their co-occurrence and size-class association in the field. To elucidate factors controlling their interactions, we conducted reciprocal-removal and greenhouse-drought experiments, and nutrient and isotope analyses. Forty-five percent of H. zosterifolia co-occurred with A. antiquum, whereas only 17% of A. antiquum co-occurred with H. zosterifolia. Removing the fronds plus substrate of A. antiquum reduced the relative frond length and specific leaf area of H. zosterifolia, but removing fronds only had little effect. Removing H. zosterifolia had no significant effects on the growth of A. antiquum. H. zosterifolia co-occurring and not co-occurring with A. antiquum had similar foliar nutrient concentrations and δ15N values, suggesting that A. antiquum does not affect the nutrient status of H. zosterifolia. Reduced growth of H. zosterifolia with the removal of A. antiquum substrate, together with higher foliar δ13C for H. zosterifolia growing alone than those co-occurring with A. antiquum, suggest that A. antiquum enhances water availability to H. zosterifolia. This enhancement probably resulted from water storage in the substrate of A. antiquum, which could hold water up to 6.2 times its dry weight, and from reduced evapotranspiration due to shading of A. antiquum fronds. Greater water loss occurred in the frond-clipped group than the unclipped group between days 3–13 of the drought treatment. Our results imply that drought mitigation by substrate-forming epiphytes is important for maintaining epiphyte diversity in tropic and subtropic regions with episodic water limitations, especially in the context of anthropogenic climate change.Positive species interactions (facilitation) play an important role in shaping the structures and species diversity of ecological communities, particularly under stressful environmental conditions. Epiphytes in rainforests often grow in multiple-species clumps, suggesting interspecies facilitation. However, little is known about the patterns and mechanisms of epiphyte co-occurrence. We assessed the interactions of two widespread epiphyte species,  Positive species interactions (i.e. facilitation) play an important role in community dynamics Epiphytes contribute up to 30% of species richness in some tropical rainforests and are thus an important component of the extraordinarily high biodiversity in tropical rainforests Asplenium antiquum Makino  E. H. Grane  is a subtropical rainforest in northeastern Taiwan . The area has 65 vascular epiphytes belonging to 20 families and 42 genera The Fushan Experimental Forest , A. antiquum frond removal (13 pairs), A. antiquum frond plus substrate (frond+substrate) removal (14 pairs), and control (15 pairs in which neither species is removed). H. zosterifolia may benefit from co-occurring with A. antiquum through enhanced availability of nutrient and water provided by the substrate of A. antiquum. In addition, H. zosterifolia growing underneath A. antiquum may benefit from reduced evapotranspiration due to the shading provided by the fronds of A. antiquum. Therefore, we had two different A. antiquum removal treatments, a frond removal treatment and a frond+substrate removal treatment. Because the roots of H. zosterifolia often grow into the substrate of A. antiquum, it was not possible to remove the substrate completely without damaging the H. zosterifolia growing underneath. We removed as much of the substrate as possible while trying to avoid physically damaging the roots of H. zosterifolia. The treatments were assigned randomly except for eight pairs growing right next to the trails that were open to the public, which were assigned to the control group to minimize interference from tourists. One-way analysis of variance (ANOVA) showed no systematic difference between these eight pairs and the other seven pairs in the control group in all of the pre-treatment measurements taken in this study . Thus we combined the measurements from the two control sub-groups.To examine the interaction between the two epiphytic ferns, we conducted a removal experiment on December 20, 2009 on 55 pairs of co-occurring A. antiquum and H. zosterifolia. The frond length was determined on three randomly selected un-grazed and mature green fronds from each individual. A frond was considered mature when all sporangia were brown. The SLA of the frond samples collected in January and August was determined by scanning the fronds and calculating the areas with Image J . During each of the five post-treatment measurements, we collected one complete frond of A. antiquum and 4–5 fronds of H. zosterifolia for nutrient analysis. The frond samples were stored at 4°C without preservatives, cleaned with de-ionized water to remove dust, fungi and other materials, oven-dried at 80°C for 72 hours, and ground. The concentrations of K, Ca, Mg, and P were determined after ashing and dissolving ash in HCl using ICP-OES . Total C and N were determined by dry-combustion using an elemental analyzer .To assess the growth response to reciprocal removal, prior to and 5 times after the removal treatment , we measured the length and number of fronds of A. antiquum fronds but above the H. zosterifolia fronds. We determined direct site factor (DSF) and indirect site factor (ISF) for each photograph using HemiView 3.43 To estimate light availability, we took hemispherical photographs for each pair of the two epiphyte species, in October 2009 prior to, and in December 2009, following the removal treatment at the same positions. The two sets of photographs were taken two months apart because hemispherical photographs cannot be taken when it is raining and it rains very often  to mimic the environmental condition under the canopy of Fushan Experimental Forest. All plants, with or without fronds, were not watered for 20 days to simulate a drought. Air temperature inside the greenhouse was modulated by an automatic ventilation system; mean daytime and nighttime temperatures were 25.1°C and 23.0°C, respectively. After the drought treatment, the fronds in the unclipped group were clipped. The substrates of both groups were re-saturated with water. After measuring the weight of each substrate under field capacity, all substrates and fronds were dried separately to constant weight.To test the effect of A. antiquum probably plays an important role on its interaction with H. zosterifolia we measure water-holding capacity of the substrate. The maximum water-holding capacity of each substrate was determined by subtracting the dry weight of the substrate from that of the substrate under field capacity. The fresh weights of the fronds in the unclipped group were determined by subtracting the substrate weight under field capacity from the total weight at day 0 (maximum water holding status of the whole plant).Because water stored in the substrate of v/Fm) was measured daily around 10 am on one randomly selected frond of each frond-unclipped plant using pulse-amplitude modulated photosynthesis yield analyzer . Leaves were dark-adapted for 40 minutes before measurements. The Fv/Fm was calculated as Fv/Fm = (Fm−F0)/Fm, where F0 is ground fluorescence level of dark adapted leaf and Fm is the maximum fluorescence yield of leaf when a saturating light pulse of 10000 µmol m−1 s−1 is applied for 0.8 second.The potential quantum yield  the fronds of nine pairs of co-occurring H. zosterifolia and A. antiquum, (2) the fronds of nine individuals of H. zosterifolia without co-occurring A. antiquum, and (3) the substrate of each of the nine A. antiquum. Two fronds and approximately 100 g of substrate were collected from each sampled individual. Four of the nine samples of each of the three groups were collected on March 2011 and the other five collected on June 2012. These samples were dried at 80°C and ground to powder for elemental and isotopic analyses in the laboratory. About 3 mg of each dried sample was weighed into a tin capsule. Total carbon and nitrogen percentages were measured with a Costech 4010 Elemental Analyzer. Stable isotopes of carbon and nitrogen were determined with a Delta V Advantage, Thermo Fisher isotope ratio mass spectrometer. Isotopic ratios were reported in δ notation (δ = ([Rsample/Rstandard]–1) • 1000‰, where R = 13C/12C or 15N/14N). Analytical precision was better than 0.3‰ for both δ13C and δ15N on laboratory standards and on duplicate samples. Isotope ratios were expressed relative to the international standards: Vienna Peedee Belemnite (VPDB) for δ13C and atmospheric N2 (AIR) for δ15N.To examine the effects of A. antiquum and H. zosterifolia differed among the three groups . We used one-way ANOVA to compare the frond length and number of the two epiphytes before the removal treatment among the treatments. We compared light availability prior to the removal among the four treatments and between above and below A. antiquum using two-way ANOVA with treatments and positions as the independent variables. A similar analysis was conducted for light availability after the removal treatment. Because there was a significant treatment x position effect, we compared light availability above and below A. antiquum for each treatment using paired t-test. We compared changes in light availability below A. antiquum following the removal among the four treatments using one-way ANOVA.We used a Chi-square test to examine if the frequency and size-class distributions of A. antiquum between the control group and H. zosterifolia removal group, and 2) H. zosterifolia among the control group, the A. antiquum frond removal group and A. antiquum frond+substrate removal group for the five post-treatment measurements. In the analysis, the frond number and length were expressed as the ratio between each of the five post-removal measurements to the pre-removal measurement. This adjusts for pre-existing differences prior to the treatment although the pre-existing differences were insignificant as will be shown in the result section. We also used one-way ANOVA with repeated measurements to compare nutrient concentration of 1) A. antiquum between the control and H. zosterifolia removal groups, and 2) H. zosterifolia among the three treatments. When among-group differences were significant, we used Fisher’s least significant difference (LSD) tests for post-hoc comparisons. We compared SLA of 1) A. antiquum between the control and H. zosterifolia removal groups using a t-test, and 2) H. zosterifolia among the three treatments in January using one-way ANOVA. We used t-tests to examine the difference in SLA of H. zosterifolia and A. antiquum between January and August 2010 for each of the treatment groups. We did not use paired-t test because the SLA in the two different months were measured on different fronds.We used one-way ANOVA with repeated measurements to compare frond number and length of 1) A. antiquum to fully saturated condition between fronds clipped and founds unclipped groups was examined using t-test. We assessed the temporal trend of Fv/Fm using simple linear regression. The differences of both δ13C and δ15N among four groups  were tested using one-way ANOVA followed by Fisher’s LSD tests for post-hoc comparisons when the among-group differences were significant.The daily difference of the relative weight in A. antiquum plants, 19 did not co-occur with any other epiphyte species, and the other 81 co-occurred with a total of 18 epiphyte species. H. zosterifolia was the second  most common epiphyte co-occurring with A. antiquum, following Pothos chinensis (Raf.) Merr. (Araceae) (28 plants) and tying with Procris laevigata Bl. (Urticaceae) . Similarly, only 12 of the 100 H. zosterifolia plants did not co-occur with other epiphytes, and the other 88 H. zosterifolia co-occurred with a total of 18 epiphyte species. A. antiquum was the most common epiphyte co-occurring with H. zosterifolia. A total of 45 of the 100 H. zosterifolia plants existed underneath A. antiquum. The frequency distribution of the two species in the three groups: growing alone, co-occurred with each other, and co-occurred with other species, differed significantly . The number of A. antiquum co-occurring with H. zosterifolia was significantly lower than expected from evenly distributed among the three groups, whereas the number of H. zosterifolia co-occurring with A. antiquum was significantly greater than expected.Of the 100 A. antiquum . The H. zosterifolia in the group co-occurring with A. antiquum had 77% plants in the size classes 3 (120–160 cm) and 4 (>160 cm), whereas those in the other two groups had more than 80% plants in the size classes 1 (40–80 cm) and 2 (80–120 cm) (H. zosterifolia was in the size class 4 (the largest), and only one was in size class 3.The size-class distribution did not differ among the three groups for –120 cm) . None ofA. antiquum (0.05–0.07) were approximately 50% lower than the values above it (0.12–0.15) and did not differ among the treatments  . The post-removal light availability (both ISF and DSF) did not differ between below and above A. antiquum in the A. antiquum frond removal and frond+substrate removal groups . However, after the treatment light availability was significantly higher above A. antiquum than below (p<0.001), in both the control and H. zosterifolia removal groups  . Followil groups .A. antiquum did not differ between the control and the H. zosterifolia removal group  = 2.35, p = 0.14 for frond number and F = 0.144, p = 0.71 for frond length). Similarly, prior to the removal, the frond number and length of H. zosterifolia did not differ among the control, A. antiquum frond removal group, and the frond+substrate removal group  = 1.11, p = 0.34 for frond number and F = 2.59, p = 0.09 for frond length). Following the treatment, the relative frond number and length of A. antiquum (as proportions of the pre-treatment measurements) did not differ between the control group and the H. zosterifolia removal group  = 0.008, p = 0.93 for frond number and F = 0.034, p = 0.86 for frond length)  . Throughantiquum . Thus thH. zosterifolia were consistent and parallel among the three treatments: control, A. antiquum frond removal, A. antiquum frond+substrate removal. There was an initial decrease between October 2009 and January 2010 and a further decrease between April and August 2010  = 2.11, p = 0.13) although the frond+substrate removal group had consistently lower relative frond number. However, the relative frond length of H. zosterifolia following the treatments differed significantly among the three treatments  = 5.98, p = 0.005). In June and August 2010, frond length was significantly greater in the control group than the frond removal group, which in turn was significantly longer than the frond+substrate removal group . Thus A. antiquum had a positive effect on the growth of H. zosterifolia, and the substrate of A. antiquum was an important facilitative factor.The changes in the relative frond number of ust 2010 . The relA. antiquum fronds did not differ significantly between the control group and the H. zosterifolia removal group    = 13, p = 0.002) (Concentrations of N and Ca and C/N ratios of s >0.05) . Concents <0.05) . Carbon = 0.002) .H. zosterifolia, concentrations of all analyzed nutrients except Ca did not differ among the three groups   (For the fronds of s >0.05) . Ca concs <0.05) .A. antiquum  = 0.13, p = 0.72) and H. zosterifolia  = 0.32, p = 0.73). Between January and August 2010, the SLA of A. antiquum increased from 83 to 96 cm2 g−1 in the control group and from 87 to 92 cm2 g−1 in the H. zosterifolia removal group. However, these increases were insignificant both for the control group  = 2.94, p = 0.098) and the H. zosterifolia removal group  = 0.51, p = 0.48). The SLA of H. zosterifolia decreased between January and August 2010 in all three groups but the differences were only significant in the frond+substrate removal group  = 17.8, p<0.001). In this group, the SLA decreased more than 50% from 230 cm2 g−1 in January 2010 to 94 cm2 g−1 in August 2010. Thus A. antiquum had a positive effect on the SLA of H. zosterifolia, but the effect was significant only when both the substrate and fronds of A. antiquum were present.The SLA did not differ significantly among treatments in January 2010 for both p values <0.05)  and dropped thereafter to 0.69 by day 20   for the 10 plants. Water loss was significantly greater in the frond-clipped group than in the frond-unclipped group from day 3 to 13 of the drought experiment (s <0.05) . Thereaf<0.0001) .13C clearly distinguished A. antiauum from H. zosterifolia , which were significantly higher than those of H. zosterifolia growing alone (-31.6‰) and co-occurring (-32.4‰) with A. antiquum (p<0.001). For δ15N, no significant differences among groups were found (p = 0.21). The two types of H. zosterifolia, growing alone and co-occurring with A. antiquum, had similar mean values (approximately -0.54‰), which were lower than those of A. antiquum fronds (-0.14‰) and substrates (0.12‰) (δerifolia . The fro (0.12‰) .H. zosterifolia co-occurred with A. antiquum, and A. antiquum was the epiphyte with which H. zosterifolia co-occurred most commonly. The individuals of H. zosterifolia co-occurring with A. antiquum were significantly larger than those growing alone  co-occurring with A. antiquum in our studied forest may also benefit from drought mitigation by A.antiquum. Thus substrate-forming epiphytes may function as keystone species, and the microhabitats where they grow are biodiversity hotspots within the forest canopy. A study of forest canopy biomass indicates that five individuals of A. nidus hosted a total of 205,428 invertebrates from 27 orders, and the biomass of invertebrates on individual A. nidus plants was positively correlated with their number of fronds A. nidusIctinaetus malayensis, is known to nest on Asplenium. Thus, through habitat cascades Crown humus accumulated by epiphytes has been proposed to be an important source of moisture for canopy organisms during dry period A. antiquum through drought mitigation on H. zosterifolia. Such positive interaction between epiphyte species may play an important role in maintaining species diversity in tropical and subtropical ecosystems and in determining their responses to climate change.Interspecies facilitation through drought mitigation may be vital for maintaining epiphyte diversity in the context of anthropogenic climate change. The frequency and severity of drought stress are anticipated to increase in tropical moist forests


II signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.The widespread P II proteins represent one of the most widespread and most ancient signal transduction protein families in nature. They are found in bacteria, archea and in chloroplasts of green algae and plants. PII proteins act as signal processing units by integrating the cellular energy and nitrogen supply through binding of the central metabolites 2-oxoglutarate (2-OG), ATP and ADP in an interdependent manner nSAAA), and stiff helical linkers (A[EAAAK]nA). The latter have shown to be able to efficiently separate protein domains [II proteins fused to the FPs should form a large complex with a molecular mass of approx. 619 kDa upon interaction , whereas the use of the long helical linker resulted in the lowest energy transfer (greenish graphs). Regarding the PII-Venus fusions, the use of the Strep‑tag as linker yielded the best results whereas again the helical linker fusions did show the lowest energy transfer. Possibly the stiffness of the helical linkers resulted in unfavorable conformations or a larger distance between the FPs. Accordingly, the combination of NAGK‑HL25‑C + PII-HL15‑V‑ST  interaction assays and determination of catalytic properties of NAGK in presence or absence of PII.To compare the interaction between PII-ST‑V or PII-wt as analyte. Interestingly, no binding signal was measurable with any PII variant, even at flow rates as low as 5 µl/min (data not shown), despite FRET experiments clearly showing interaction. These results indicate that either the interaction of PII with Cerulean-fused NAGK is too slow to be detected by SPR or binding of NAGK‑FL25‑C with its His-tag to the chip surface shields the PII interaction site. By contrast, if the His-tagged NAGK-wt was bound to the sensor chip, interaction was measurable with PII-wt but also with the Venus-tagged PII, although to a lower extent . In the process of these experiments we noticed that it is beneficial for the NAGK activity to preincubate the enzyme in the reaction buffer. Only after a preincubation of at least 20 min at 25 °C NAGK reaches its maximum activity during the assay. Similar effects could be observed in FRET and SPR experiments. Possibly, NAGK needs time to reestablish its active hexameric conformation after storage in 50 % glycerol buffer at -20 °C. Due to this preincubation step, Kcat values were approximately 3 times higher and Km values 2 to 3 times lower compared to those published earlier [Next we studied the interaction of the FP-tagged Penhanced . We deteII-ST‑V binding to NAGK is very similar to the one achieved by PII-wt. This demonstrates that the FPs do not affect the efficiency of complex formation. Contrary to our initial expectations, but confirmed by the FRET data  of free NAGK-wt is approx. 20 µM [II the IC50  increases 25 times to 502 (± 20) µM for the wt proteins, which is very similar to the values published before [50 of 11 (± 1) µM and PII-ST‑V is able to relieve arginine inhibition to an IC50 of 561(± 32) µM, similar to the effect observed for the wild-type proteins. Together these results show that the fluorescence-tags, despite their size, have only a minor impact on PII - NAGK interaction. NAGK‑FL25‑C is as active as the wild-type enzyme, the activity is enhanced by PII binding and PII also relives NAGK from arginine inhibition. As a second parameter for complex formation, the inhibition of NAGK activity by arginine and the relief of arginine inhibition by Panalyzed . Arginind before . Free NAII-ST‑V seems to interact less strongly with NAGK than non-tagged PII in SPR spectroscopy, no difference was recorded between tagged and non-fluorescence tagged proteins when measuring kinetic constants and relief from arginine inhibition, a sensitive indicator of complex formation. A further striking difference is the behavior of the PII-S49G variant: whereas the FRET measurements indicated the formation of a complex, no complex formation was seen by SPR, neither with the Venus-tagged form nor with the untagged variant. To resolve this issue, interaction of the PII-S49G variant with NAGK was also measured by the enzyme assay. The PII-S49G variant was indeed able to partially relief NAGK from arginine inhibition, raising the IC50 8.5 times to 170 (± 21) µM. Thus PII-S49G is by far not as efficient in preventing arginine inhibition as PII-wt is, an intriguing difference to the almost wild-type like activation of NAGK in the absence of arginine. The differential effect of the PII-S49G mutation, on catalytic activation on one hand, and relief from arginine inhibition on the other, indicates that these two effects are transmitted by different intermolecular interactions. The comparison also shows that the different assays used report different aspects of complex formation. SPR is apparently very sensitive to steric hindrance caused by surface fixation of one interaction partner. Moreover, SPR fails to detect the interaction of the PII-S49G variant, indicating that the hydrogen bond of Ser49 is needed to stably fix PII on surface-bound NAGK, although this variant is perfectly able to interact in solution with NAGK. On the other hand, efficient relief from arginine inhibition seems to require the formation of this hydrogen bond, whereas it seems dispensable for the basic activation of NAGK.Comparing the SPR data with the results of enzyme analysis and FRET measurements reveals a striking difference: although PII-wt and PII-ST‑V proteins. The high binding affinity of PII-wt towards ATP has been shown before by equilibrium dialysis, ultrafiltration techniques [II-ST‑V towards ATP is doubled at the first binding site, compared to PII-wt, and is about 40 % higher at the second and third site.To investigate whether the fusion of the FPs affects the effector molecule binding properties, isothermal titrations calorimetric (ITC) experiments were performed with Pchniques  and ITC chniques  and coulII. On the contrary, the tags lead to a higher affinity for ATP. As shown in the structure of liganded PII [II flips towards the ATP biding site and shields it from the surface. The fused Venus protein may further constrict the ATP exit channel and therefore keep the bound ATP with higher affinity.Overall these analyses show that the fluorescence-tags have no inhibitory influence on effector molecule binding of Pnded PII , the C‑tII-ST‑V pair was investigated in more detail. In the following experiments, Cerulean was excited with 433 nm light and the Cerulean and Venus fluorescence peak emission was recorded at 475 and 525 nm. The relative FRET ratio was calculated, which equals the emission at 525 nm (mainly Venus) divided by the emission at 475 nm (mainly Cerulean). First of all, we tested the impact of the ATP concentration on the protein interaction. NAGK‑FL25‑C and PII-ST‑V were incubated 30 min in the presence of different ATP concentrations and then FRET was measured. As shown in II - NAGK interaction that has an influence on the energy transfer efficiency. In previously conducted SPR experiments, PII and NAGK showed the same interaction with or without ATP if enough Mg2+ was present [II and NAGK still interact but are present in a less favorable conformation regarding FRET. In the following experiments we consequently used ATP concentrations of 0.1 mM.After having characterized the principal properties of the FP tagged proteins and their mode of interaction, the FRET capabilities of the NAGK‑FL25‑C + P present . The FREII and the PII mutants. The FRET ratio was recorded every 8 s over 13 min as shown in II-ST‑V, the ratio is approx. 0.4, displaying pure Cerulean fluorescence. After recording the zero FRET response for 30 s, PII-ST‑V was added to the reaction. It immediately binds to NAGK‑FL25‑C, resulting in a fast increase in energy transfer. Following this initial raise the FRET ratio gradually saturates, reaching a maximum after 5 to 10 min.Next we took a closer look at the association kinetics of FP-tagged NAGK, PII variants PII-S49G‑ST‑V, PII-E85A‑ST‑V and PII-S49G‑E85A‑ST‑V. The steady state FRET ratios are a lot lower in these experiments . In a parallel setup, 2-OG was already supplied to the reaction mix when PII and NAGK were added and subsequently incubated for 30 min (dashed line). Both setups lead to the same endpoint FRET ratios, indicating that a preassembled PII - NAGK complex responds towards 2-OG in the same manner as PII before having bound to NAGK. The dynamic range for 2-OG detection is between 0.01 and 1.0 mM for the used conditions. The IC50, the 2-OG concentration that led to a 50 % drop in FRET, is 0.1 mM . The vector pASK-IBA3  with tet-promotor and a Strep‑tag coding sequence was used for PII expression. The vector pET-15b  with T7-promotor and a His-tag coding sequence was used for the expression of NAGK. DNA sequences for the GFP variants Venus and Cerulean were amplified from pSW41, a generous gift from Sabrina Wend, University of Freiburg, Freiburg, Germany. The linker sequences were generated by overlapping oligo PCRs. E. coli strain RB9060 [II plasmids and the proteins were purified using Strep‑tag affinity chromatography as described earlier [E. coli BL21 cells were transformed with the NAGK plasmids and the proteins were purified using His-tag affinity chromatography as previously described [Cloning was conducted using standard PCR techniques. Oligonucleotides were synthesized by Eurofins MWG GmbH  or Sigma Aldrich Chemie GmbH . Restriction enzymes were purchased from Fisher Scientific . PCR products and digested DNA were purified using QIAquick gel extraction kits . Plasmid DNA from n RB9060  was tranescribed .2, 0.5 mM DTT and 0.1 mM ATP. The fusion proteins were used in concentrations of 100 nM. Prior measurements NAGK was carefully transferred into the reaction buffer and incubated for 10 min on ice and after that 20 to 40 min at 37 °C. To measure association kinetics PII-Venus fusions were added, the mixture was excited by 433 nm and Cerulean and Venus emission peaks were measured at 475 and 525 nm wavelengths every 8 s. Between the measurements the shutter was closed to prevent bleaching. Fluorescence spectra were measured using an emission scan ranging from 445 to 650 nm with excitation at 433 nm wavelength. The bandpass for emission and excitation was set to 1.5 nm in all experiments.Fluorescence measurements were performed using a FluoroMax‑2 . If not otherwise described the reaction buffer contained 50 mM imidazole (pH 7.5), 50 mM KCl, 20 mM MgClII, then both proteins were preincubated together. 50 µl of the preincubation mix were added to the reaction mix resulting in final concentrations of 50 mM imidazole, pH 7.5, 50 mM KCl, 20 mM MgCl2, 0.4 mM NADH, 1 mM phosphoenolpyruvate, 10 mM ATP, 0.5 mM DTT, 11 U lactate dehydrogenase, 15 U pyruvate kinase and 25 nM of NAGK and PII. The reaction was started by adding varying concentrations of the substrate NAG, resulting in a final reaction volume of 1 ml, and the NADH oxidation was immediately assessed by measuring the absorbance at 340 nm with a SPECORD 200 photometer  for 60 s. The slope of the change of absorbance over time was calculated. One unit of NAGK catalyses the conversion of 1 µmol NAG min−1, calculated with a molar absorption coefficient of NADH of ɛ340 = 6178 L mol−1 cm−1. GaphPad Prism 6  was used to calculate the enzymatic parameters Km, Kcat, and IC50.The NAGK activity assay was performed in similar ways as described before regarding buffers and the measurement setup ,24. But 2+ loaded NTA surface of flow cell (FC) 1 until a signal of ~1500 resonance units was reached. FC 2 acted as a control for unspecific binding and the signal was subtracted from FC 1. Variants of PII were injected to the sensor chip and, after binding, removed again by injecting a 1 mM ADP solution.SPR experiments were performed using a using a BIAcore X biosensor system (Biacore) as previously described . His-tag2HPO4, 1.8 mM KH2PO4, 25 mM NaCl, 10 mM KCl and 1 mM MgCl2 (pH 7.5). 1 mM of the titrant ATP was dissolved in the same buffer. After an initial 2 µl injection 4 µL of the titrant were injected 70 times into the 1.4285 ml cell with stirring at 200 rpm at a temperature of 25 °C. The PII concentration in the cell was 33 µM (trimer concentration). The binding isotherms were calculated from received data and fitted to a three-site binding model with the MicroCal ORIGIN software  as indicated.ITC experiments were performed using a VP‑ITC microcalorimeter  in buffer containing 10 mM NaFigure S1II variants.Determination of kinetic constants for NAGK-wt and NAGK‑FL25-C with and without various P (A) NAGK enzyme activity assays of the indicated PII - NAGK combinations were performed using increasing amounts of the substrate NAG. (B) NAGK enzyme activity assays of the indicated PII - NAGK combinations were performed with increasing concentrations of Arginine and 50 mM NAG. The mean of three replicas with error bars displaying the standard deviation is shown. The results are summarized in (TIF)Click here for additional data file.Figure S2II-wt and PII-ST‑V.Two exemplary Isotherms for the determination of ATP binding properties of P The upper panel shows the raw data, the lower panel shows the calculated, blank subtracted, binding isotherms. The results are summarized in (TIF)Click here for additional data file.Figure S3II-ST‑V.Effect of various ATP concentrations on FRET efficiency of NAGK‑FL25‑C with P The FRET ratio (525 nm / 475 nm emission) was measured after incubating NAGK-FL25-C and PII-ST-V together for 20 min at 37 °C in the presence of different ATP concentration. The mean of three replicas with error bars displaying the standard deviation is shown.(TIF)Click here for additional data file.Figure S450 values for 2-OG and ADP on NAGK‑FL25‑C + PII-ST‑V.Determination of IC The relative FRET ratio (525 nm / 475 nm emission) was measured after incubating NAGK-FL25-C and PII-ST-V together for 30 min in the presence of different 2-OG concentration (A) or ADP/ATP ratios (B). All signals were normalized to values from control experiments without 2 OG or ADP. The mean of three replicas with error bars displaying the standard deviation is shown. Fitting and IC50 calculation was conducted using GaphPad Prism 6.(TIF)Click here for additional data file.Figure S5II - NAGK complex sensitivity towards 2-OG.Influence of arginine on P PII-ST-V and NAGK-FL25-C were incubated together for 40 min at 37 °C with different concentrations of 2-OG and with or without 1 mM arginine, before the FRET ratio was determined. All reactions were performed as triplicates; standard deviation is indicated by error bars.(TIF)Click here for additional data file.


Mild streptococcal infections infection are extremely common, however invasive infections can lead to high mortality rates. Described in 1982, post streptococcal reactive arthritis (PSRA) has been reported with increasing frequency.We aimed to look at the number of children presenting to a Tertiary Children’s Hospital, with serological significant streptococcal illnesses, diagnoses and antibiotic treatment of those patients.Over a One year period of ASOT ( anti streptolysin O) results from a tertiary Children’s Hospital were reviewed. The ASOT results documented, paired data of ASOT & Anti DNase B (anti Deoxyribonuclease B antibodies) reviewed for correlation. Diagnoses were obtained using clinical notes.Throat swabs results were reviewed where performed. The antibiotic treatment advised documented.ASOT January 2009- December 2010 = 645N° patients=359N° ASOT’s >400 = 100 (28 %)Notes reviewed = 95/100 patientsWhere both ASOT and Anti DNAse B were performed simultaneously (No =194), results correlated in 88%Throat Swab performed in the group with ASOT >400 = 43/10065 % patients with ASOT >400 were prescribed antibioticsNearly 1/5 of patients with positive streptococcal serology had a rheumatological diagnosis, suggesting it is a significant trigger in rheumatological conditions.This study highlights vigilance and alertness that patients with Streptococcal Infections may evolve or contribute to the development of a rheumatogical condition.There is a need for a consensus opinion on treatment and eradication of streptococcal infection.


Western blot analysis revealed that omentin-1 induced activation of Akt (phosphatidylinositol-3 kinase downstream effector) and such effect was impeded by transfection of hOB with Akt-siRNA. Furthermore, LY294002 (a selective PI3K inhibitor) and HIMO (a selective Akt inhibitor) abolished the omentin-1-induced hOB proliferation. These findings indicate that omentin-1 induces hOB proliferation via the PI3K/Akt signaling pathway and suggest that osteoblast is a direct target of omentin-1.It has been presumed that adipokines deriving from adipose tissue may play important roles in bone metabolism. Omentin-1, a novel adipokine, which is selectively expressed in visceral adipose tissue, has been reported to stimulate proliferation and inhibit differentiation of mouse osteoblast. However, little information refers to the effect of omentin-1 on human osteoblast (hOB) proliferation. The current study examined the potential effects of omentin-1 on proliferation in hOB and the signal pathway involved. Omentin-1 promoted hOB proliferation in a dose-dependent manner as determined by [ Briefly, cells were plated at a density of 2 × 104 cells/well in 24-well plates and treated with 25–200 ng/mL omentin-1 for 48 h, in the presence of [3H]thymidine. 24 hours later, the plates were washed with PBS, and 10% TCA solution was added to the wells. Incorporated [3H]thymidine was released by washing with 0.2 N of NaOH, and radioactivity was measured using a β-scintillation counter. Results are expressed as counts per minute.HOB proliferation was assessed using thymidine incorporation by cells to determine the proliferation of hOB, we confirmed that omentin-1 stimulated hOB proliferation in a dose-dependent manner. Compared to the control group, cells treated with omentin-1 at concentrations of 25, 50, 100, and 200 ng/mL increased the [3H]thymidine incorporation of hOB by 23.66%, 53.29%, 93.93%, and 140.36%, respectively, with statistical significance  (Using thymidine incorporation assay was adopted. Our data have demonstrated that omentin-1 stimulated the proliferation of hOB in a dose-dependent manner. The proliferative effect of omentin-1 on hOB is therefore consistent with the report by Xie et al. [In the present experiment, 25–200 ng/mL omentin-1 has been chosen to treat the hOB based on the previous research . To detee et al. . To gain further insight into the underlying mechanism about how omentin-1 stimulated hOB proliferation, we evaluated signaling pathway that was potentially involved. Multiple signaling pathways, such as MAPK, Wnt, AMP, and PI3K-Akt, were found to participate in the modulation of osteoblast proliferation –38. AmonIn summary, our present study identified omentin-1 as an important regulator in human bone remodeling by promoting the hOB proliferation through the PI3K/Akt signaling pathway. These findings revealed the relationship between adipokines and bone metabolism and provided us a better understanding of the involved signaling mechanisms. Further investigation of omentin-1 and osteoblast may offer us a new target for osteoporotic prevention and treatment.


We directly demonstrate the importance of saturating the Si dangling bonds at the graphene/SiC(0001) interface to achieve high carrier mobility. Upon successful Si dangling bonds elimination, carrier mobility increases from 3 000 cm2V−1s−1 to >11 000 cm2V−1s−1 at 0.3 K. Additionally, graphene electron concentration tends to decrease from a few 1012 cm−2 to less than 1012 cm−2. For a typical large (30 × 280 μm2) Hall bar, we report the observation of the integer quantum Hall states at 0.3 K with well developed transversal resistance plateaus at Landau level filling factors of ν = 2, 6, 10, 14… 42 and Shubnikov de Haas oscillation of the longitudinal resistivity observed from about 1 T. In such a device, the Hall state quantization at ν = 2, at 19 T and 0.3 K, can be very robust: the dissipation in electronic transport can stay very low, with the longitudinal resistivity lower than 5 mΩ, for measurement currents as high as 250 μA. This is very promising in the view of an application in metrology.We investigate the magneto-transport properties of epitaxial graphene single-layer on 4H-SiC(0001), grown by atmospheric pressure graphitization in Ar, followed by H Graphene is a monoatomic layer of carbon atoms that possesses unique electronic properties. It is a zero band gap semiconductor, therefore ambipolar; it has extremely high carrier mobility at room temperature and charge carriers behave as massless Dirac fermionsAb initio calculations have shown that the buffer layer is a warped carbon layer with periodic inclusions of pentagon-hexagon-heptagon  complexes, covalently bond to the Si atoms of the substrateThe quality and number of layers in epitaxial graphene on SiC depend on the growth conditions , SiC polytype, face and orientation. Atmospheric pressure graphitization of a SiC(0001) substrate can be used to produce large terraces fully covered by a single layer graphene482 annealing as a route to obtain high mobility epitaxial graphene. In this case hydrogen can saturate the silicon dangling bonds without a real decoupling of the interface12 cm−2, we find, more importantly, a clear increase of the mobility by about a factor of 2 to 5. The treatment is robust against nanofabrication and thermal cycling, from room temperature down to cryogenic temperature. For hydrogen treated graphene, we find motilities as high as 11 300 cm2V−1s−1 at 0.3 K and the quantum Hall effect confirms the high quality of the graphene obtained, making the developed growth process a promising route for demanding applications like metrologyHere we propose post-growth gentle H2 at 820°C for 10 mn, in the following we will refer to this sample as to BH2. The goal here is to saturate the Si dangling bonds from the substrate without decoupling the interface layer from the substrate  showed three components at 283.7, 284.6, and 285.4 eV in binding energy42 sample, the C 1s spectrum shows the same three components . However, the SiC bulk component in the C1s core level spectra shifts 0.2 eV towards lower binding energy. This variation in the band bending in the sample BH2, confirms the partial saturation of Si dangling bondThe graphene used in this study is obtained by annealing 4H-SiC(0001) at 1550°C in 800 mbar argon for 10 minubstrate . We cann2). Several intense peaks are observed between 1200 and 1800 cm−1; they correspond to second-order Raman bands originated from the SiC substrate15−1, ii) the G band (symmetric Eg2 phonon mode) at 1582 cm−1 and iii) the 2D band at 2696 cm−1. In the two spectra (B and BH2) the D band which corresponds to the disorder is weak in comparison with G and 2D band (double resonant electron-phonon process). The low intensity of this peak shows that there is only a small amount of defects/disorder in the graphene structure. This is an indication for the high quality of the produced epitaxial graphene before and after hydrogenation.Assuming the graphene-SiC sample can be modeled as a semi-infinite SiC substrate with a uniform graphene overlayer, the thickness of the graphitic film can be calculated from the ratio between the intensity of the G and SiC components of the XPS spectrum. This ratio fits well with an exponential decay of roughly single-layer of graphene covering for the two samples. Additionally, we used Raman spectroscopy to study the effect of hydrogen adsorption. Electron transport in graphene is sensitive to the amount and type of disorder, and when combined with structural and electronic property analysis techniques, it allows in-depth probing of the graphene properties. For this purpose, [see Methods] large 30-μm wide multi-terminal Hall bars were fabricated from hydrogen exposed graphene sample (BH2) and from intrinsic graphene samples (A and B); the latter to be used as reference to evaluate the effect of the hydrogen exposure. Hall bars were designed with the drain-source channel oriented along the step edges of graphene and rotated by 45 and 90 degrees in order to test any anisotropy of charge transport in epitaxial graphene related to the step-structured character of the SiC surface. The graphene terraces are not seen under normal reflection optical microscopy, and while is possible to use AFM this is time consuming, especially to obtain information about large samples as in our case. We found that the graphene terraces can be clearly detected in an optical microscope by using polarized light2 and B is significant. It cannot be explained by the irreproducibility of the growth process, since the samples are from the same growth process and this rather suggests the impact of gentle hydrogenation on charge transport in graphene. Finally, it appears that hydrogenation does not deteriorate the homogeneity of the transport property since the width of the resistivity value distribution is not worse in the case BH2 than for B. This finite width is predominantly ascribed to contaminations coming from the sample fabrication and/or to inhomogeneity in the graphene growthxx) and transverse (ρxy) magnetoresistivity measurements were carried out. Such Hall measurements allow the extractions of the carrier concentration and the carrier mobility separately, whereas it is not possible to disentangle both quantities in a Drude resistivity measurement solely. Moreover, at higher magnetic field, the relativistic quantum Hall effect, the hallmark of massless Dirac fermions, gives a finer insight of the electronic transport properties quality. We present on 2 (top to down panel). For the device from sample A, the relative angle between the SiC step edges and the Hall bar channel is 10°, for the devices from B and BH2, it is 45°, the same orientation making the comparison easier. The zero magnetic field peaks in the longitudinal resistivity is due to the weak localization correction to the conductivity, and is well known in disorder metals, in 2DEGs, and graphene19xx starts to oscillate when the magnetic field is increased. These oscillations, known as Shubnikov de Haas oscillations, appear with the Landau quantization of the density of states. At higher magnetic fields, up to 11 T, the QH effect develops: plateaus are observable in the transverse resistivity ρxy evolution with the magnetic field in coincidence with the ρxx minimaxy versus B, in the regime of the classical Hall effect; it is equal to n = B/(ρxy.e). The mobility μ is then extracted from the zero field Drude resistivity σ0 = 1/ρ0 = neμ. To determine the Drude resistivity, we subtracted from the zero field value the weak localization peak. The electron-electron contribution to the conductivity is low and can be neglected because of the large value of the diffusion constant (ref e-e interaction)2.We now discuss the results of electronic transport property measurements of a certain number of devices fabricated with the three produced graphene samples . We first characterized the room temperature Drude resistivity via 4 terminals probe station measurements. The resistivity is 2.3 and 3.6 kΩ in average for devices from B, and BH2 respectively as shown in 12 cm−2. The typical devices made from A and B present carrier concentrations which are different by a factor of two. That difference could be explained by the slight irreproducibility of the growth process, since these samples are from 2 subsequent growths, and/or by differences in the fabrication process, the preparation or the conditioning of the samples assuming that the carrier concentration would be over all determined by extrinsic contamination. More interestingly, the carrier density is twice lower in the device made from hydrogenated graphene BH2 when compared to the device made from B. The facts that the two devices originate from the same growth process and were prepared in the same conditions make the interpretation of the carrier concentration comparison between B and BH2 devices more clear and less obscured by the effect of external contamination (ad-atoms). Given that carrier density in the samples is relatively high as compared to the charge neutrality point, in a range where the mobility is not expected to depend too much on density, the comparison between the values of the mobility of the three samples is meaningful and straightforward. For intrinsic graphene the mobility values of the two samples A and B are very close, around 2300 cm2V−1s−1, despite the difference in the orientation of the devices with respect to the step edges  estimated for these samples. About the impact of the gentle hydrogenation, it is interesting to remark that the sequence of the transverse resistance ρxy plateaus observed in the QHE regime at higher magnetic fields is unchanged for samples B and BH2  (with i > 0 an integer)23The carrier density for the three n-type doped device ranges from 1 to 2 × 10ep edges . The mob and BH2 . In both2. The goal of a first series of experiments with these devices was to quantify the degree of anisotropy of the magnetotransport properties with respect to the angle formed between the Hall bars source-drain channel direction and the substrate step edge overall direction. For that purpose, three devices were compared: one device was aligned parallel to the step edges, the second was rotated by an angle of 45° and the third by 90°. The devices are 30 μm wide and the typical width of the SiC terraces after growth is 4–5 μm as shown in 2 devices show a mobility, ranging from 4 300 to 6 300 cm2V−1s−1 at 1.7 K, that is about two to three times larger than what was found on devices from samples A and B. Our results show that, as expected, the carrier density is not significantly affected by the alignment, since the Hall bar to Hall bar variation is less than 20%, on the same order of the variation between different parts of the same Hall bar. On the other hand, the carrier mobility shows a clear tendency: decreasing at higher Hall bar misalignments, which is consistent with what is already reported in literature for single layer graphene coupled to the SiC substrate via a buffer layer25In the following of the manuscript we concentrate on devices produced with hydrogenated graphene sample BH2 large Hall devices fabricated from BH2 and oriented parallel to the step edges were characterized at low temperature (0.3 K) several times. They show typical electron doping around 1 × 1012 cm−2 (with a minimum of 7 × 1011 cm−2) and electron mobility up to 11 300 cm2V−1s−1. These transport properties (n and μ) are reproducible upon thermal cycling up to room temperature and can be spatially homogeneous within 20% in a given of these large device. To demonstrate the potential of hydrogenated graphene, more detailed transport properties measurements have been performed in one of these devices that presents high and homogeneous carrier mobility (8000–9600 cm2V−1s−1) for carrier density of 1 × 1012 cm−2  up to very high measurement currents of 250 μA  are not observed along the whole hydrogenated graphene sample. Whereas on one given edge of the device, ρxx value was measured below 5 mΩ, on the opposite edge it was measured equal to 150 Ω at current as low as 10 nA. The very high breakdown current measured in a certain configuration confirms the local high quality of the graphene grown and post-hydrogenated. Homogeneity and improvement of the transport properties will probably be achieved by fine tuning the growth parameters and optimisation of the device process, e.g. by using e-beam lithography which is known to leave less residues on the graphene surface. More generally, the post-growth annealing of graphene under hydrogen, as proposed in this article, is interesting for the metrological application of the graphene QHE in the sense that it would reduce the carrier concentration and increase the carrier mobility, two inescapable prerequisite for implementing the QHE at low magnetic field which is the base of the graphene promise for that application.Three 30 × 280 μm012 cm−2 . At low 012 cm−2 . The Half 250 μA , what is2 wafer, mild annealing experiments have been carried out under vacuum (10−5 mbar) (see methods)12 cm−2 to 7 × 1012 cm−2 with a final value in the range of the typically measured values for non-intentionally treated graphene, coupled to the Si-face SiC via a buffer layer, like sample B. Heating at higher temperature on longer time scale would certainly lead to higher electron doping up to the order of 1013 cm−2. A decrease of the mobility is also importantly observed from 4520 cm2V−1s−1 to 3000 cm2V−1s−1 at low temperature, approaching the mobility measured for intrinsic graphene samples A and B. With a closer analysis of the data, it appears that the spatial dispersion of the mobility values at low temperature first increases and then decreases. It seems to reach an intrinsic limit. The temperature dependence of the mobility between 300 K and 0.3 K is rather high before annealing, displaying an increase by a factor of about 3 when decreasing the temperature and then supporting the rather good quality of graphene with a limited quantity of defects. Along the annealing process, it tends to decrease, reaching a minimum after the first annealing step at 400 K. It is compatible with a degradation of the graphene quality. For the electron density, we observe a continuous increase with a final value which does no more depend on temperature, as expected for an intrinsic high doping by charge transfer from the graphene/SiC substrate interface. The annealing step at 400 K during 0.5 h has a crucial impact. It can be interpreted as yielding a partial hydrogen desorption at the interface between the graphene layer and the SiC substrate. In this hypothesis, the complete hydrogen removal would be achieved with longer annealing at higher temperatures. The degradation of the charge transport properties after the annealing gives an additional proof of the benefits of the post-growth treatment by hydrogen. After each annealing step, the longitudinal and transversal magneto-resistivities have also been measured up to 19 T at 0.3 K. The Hall resistance plateau sequence characteristic of single graphene is preserved. After the annealing at 450 K, we note the disappearance of the longitudinal magnetoresistivity asymmetry with the magnetic field direction that is observed in one particular measurement configuration with this modest mobility sample before the annealing. Such asymmetry is also observed in other devices of this study  by hydrogen is particularly attractive for electronic and metrology device applications, thanks to its high mobility and its demonstrated quality for low-dissipative transport in the QHE regime.In summary, we have presented research that directly shows the importance of introducing a hydrogen intercalation step following epitaxial graphene on 4H-SiC(0001). A gentle hydrogenation that does not lead to decoupling of the graphene film, as evidenced by XPS and transport data, allowed us to increase the charge carrier mobility. Hydrogenation results in the saturation of the dangling bonds of the graphene/SiC(0001) interface layer and provides a means for significant improvements in carrier mobility from approximately 3000 cm2), with a typical step height of about 4–6 nanometers. The SiC sample is heated to 1000°C in a semi UHV and then further heated to 1525°C in an Ar atmosphere. This graphitization process results in the growth of an electrically active graphene layer on top of the interface layer. The graphene sample was characterized using Raman spectroscopy, x-ray photoemission spectroscopy and atomic force microscopy. The micro-Raman spectroscopy was performed at room temperature with a Renishaw spectrometer using 532 nm excitation wavelength lasers light focused on the sample by a DMLM Leica microscope with a 50× objective and a power of 5 mW with spot size of about 1 μm. The AFM measurements were realised under ambient conditions, and the images were recorded in noncontact mode. The XPS measurements were performed on the Tempo beamline of the Soleil synchrotron radiation . For transport measurements, we fabricate Hall devices by optical lithography, reactive ion etching and metal deposition. The graphene Hall bars were produced using standard optical lithography. We used the image reversal AZ5214 photoresist, palladium/gold (20/100 nm) was employed as a contact material in order to ensure good contact transparency. Our Hall bars are 30 microns wide, the distance between the voltages probes are either 70 or 110 μm, the number of voltage probes for each bar is either 4 or 8. The largest devices are 30 × 280 μm2.The single layer graphene studied in this paper were produced via a two-step process from substrate of 4H-SiC(0001) (Si-face). Prior to graphitization, the substrate was hydrogen etched (100% H2) at 1550°C to produce well-ordered atomic terraces of SiC. The terraces cover the whole wafer (about 1 cm-5 mbar), in the helium 3 refrigerator, heating is achieved by Joule effect, dissipating electrical power in a resistive discrete component attached to the sample holder. The temperature is monitored with a calibrated Pt100 thermometer also mounted on the ceramic sample holder.The electronic magnetotransport measurements were carried out at low temperature, either at 1.7 K in Oxford VTI helium 4 refrigerator with magnetic field perpendicular to the sample up to 11 Tesla, or at 0.3 K in an Oxford Heliox helium 3 refrigerator equipped with a Cryogenic Ltd superconducting magnet capable to generate magnetic fields up to 20 Tesla. The final annealing experiment was performed under vacuum (10A.O. grow the graphene sample and conducted the measurements by XPS, E.P. and V.C. characterized it by AFM and Raman spectroscopy. E.P. and V.C. produced the Hall bars and conducted the measurements at 1.7K, F.L., F.S., W.P. carried experiments at 0.3K. E.P., F.L., F.S., W.P. and A.O. analyzed the data, and D.M. supported the experiment. E.P. and F.S. wrote the paper with all authors contributing to the final version. A.O. planned the experiments and supervised the project.


Cells adapt to various chronic toxic exposures in a multitude of ways to minimize further damage and maximize their growth potential. Expression of L1 elements in the human genome can be greatly deleterious to cells, generating numerous double strand breaks (DSBs). Cells have been reported to respond to chronic DSBs by altering the repair of these breaks, including increasing the rate of homology independent DSB repair. Retrotransposition is strongly affected by proteins involved in DSB repair. Therefore, L1 expression has the potential to be a source of chronic DSBs and thus bring about the changes in cellular environment that could ultimately restrict its own retrotransposition.We demonstrate that constitutive L1 expression leads to quicker DSB repair and decreases in the retrotransposition potential of L1 and other retrotransposons dependent on L1 expression for their mobility. This cellular adaptation results in reduced sensitivity to L1 induced toxicity. These effects can be induced by constitutive expression of the functional L1 ORF2 alone, but not by the constitutive expression of an L1 open reading frame 2 with mutations to its endonuclease and reverse transcriptase domains. This adaptation correlates with the relative activity of the L1 introduced into the cells.The increased number of DSBs resulting from constitutive expression of L1 results in a more rapid rate of repair. The cellular response to this L1 expression also results in attenuation of retrotransposition and reduced sensitivity of the cells to negative consequences of L1 ORF2 expression. The influence does not appear to be through RNA interference. We believe that the increased rate of DSB repair is the most likely cause of the attenuation of retrotransposition. These alterations act as a fail safe mechanism that allows cells to escape the toxicity associated with the unchecked L1 expression. This gives cells that overexpress L1, such as tumor cells, the ability to survive the high levels of expression. However, the increased rate of break repair may come at the cost of accuracy of repair of the lesion, resulting in increased genomic instability. Mammalian cells often evolve adaptive responses to deal with chronic exposure to various toxic agents, including ethanol and opiates -4. CellsLong interspersed element-1 (LINE-1 or L1) is the most numerous and only currently active family of human autonomous, non-long terminal repeat (LTR) retrotransposons. They constitute 17% of the genome, with approximately 500,000 copies. Most of these L1 elements do not contain functional copies of the two open reading frames (ORF1 and ORF2) required for efficient retrotransposition -8. Only The L1 ORF2p has been demonstrated to contain both endonuclease and reverse transcriptase activities, which are crucial for retrotransposition -12. ThesL1 expression is associated with DSB formation ,17, and High levels of endogenous full length L1 mRNA expression have been detected in multiple tissues and cell lines . The endUsing cells stably expressing both functional and non-functional L1 ORF2p, we show that cells adapt to the constitutive expression of the functional L1 ORF2p in a dose dependent manner, by repressing the retrotransposition of both L1 and Alu. Furthermore, this cellular adaptation, which limits retrotransposition, also diminishes the toxicity typically associated with the expression of L1 ORF2p. We demonstrate that cells exposed to constitutive L1 ORF2p expression have altered DNA DSB repair kinetics, which provides a potential explanation for the reduction of L1 and Alu mobilization, and the diminished toxicity associated with L1 expression.Endogenous L1 mRNA is subject to splicing and premature polyadenylation ,22,23, wP ≤ 0.05) elevated levels of 53 BP1 foci compared with controls  or luciferase  lower than in corresponding control cells    fewer G418 resistant colonies in HeLa ORF2 ER-- cells than in HeLa ORF2 cells  induced DSBs on HeLa ORF2 and HeLa ORF2 ER-- cells. Significantly different sensitivity to IR between these cell lines was not found in either a proliferation or a colony formation assay Figure .Together, these data suggest that the reduced retrotransposition rates observed in the long term L1 expressing cells are not due to an impaired growth rate, and additionally cannot easily be explained by differences in the viability or general health of the cells. The cells seem to have adapted to the stable expression of L1 ORF2 by specifically diminishing their sensitivity to L1 ORF2 as a source of DSBs.Because several of the known activities of L1 ORF2p were repressed by chronic exposure of ORF2p, it was possible that the ORF2 mRNA was being specifically targeted by RNA interference (RNAi) for degradation, a mechanism previously suggested to repress L1 activity -30. To t2O2 or L1 ORF2 expression. 53BP1 foci returned to background levels significantly(P ≤ 0.05) more rapidly in HeLa ORF2 cells than in HeLa ORF2 ER-- cells after both treatments, indicating that exposure to constitutive L1 ORF2 expression induces a cellular adaptation that more rapidly repairs DSBs induced by exposure to either H2O2 or transient L1 ORF2p expression . Because the RNA from the mutant ORF2 should only differ by two bases from the wild type RNA, it is very unlikely that it would influence RNAi differentially.P ≤ 0.05) faster repair of DSBs and greater protection from toxicity associated with transient ORF2p expression  increase in DNA repair associated with a decrease in retrotransposition. The change in DSB repair timing was measured by the formation and clearance of 53BP1 foci. 53BP1 foci format DSBs and are associated with NHEJ mediated repair of these lesions [We found a significant . We then carried out chloroform extraction and isopropanol precipitation. We selected poly(A) RNA species using a commercial kit  as instructed by the manufacturer. We resuspended the poly(A) selections and precipitated RNA in 30 μl of RNase-free water and fractionated it in a single lane of agarose formaldehyde gel.We combined the contents of four 75 cmWe transferred RNA to a nylon membrane  by capillary transfer overnight at room temperature in a standard 5× sodium chloride/sodium citrate (SSC) solution. We crosslinked the RNA to the membrane with ultraviolet light and prehybridized it in 30% formamide, 1x Denhardt's solution, 1% SDS, 1 mol/l NaCl, 100 μg/ml salmon sperm DNA and 100 μg/ml yeast tRNA at 60°C for at least 6 hours. Hybridization with a strand specific probe was carried out overnight in the same solution at 60°C. We carried out several 10-minute washes at high stringency  at 60°C.We generated  the strand specific probe used for the northern blot assay. Primer sequences for generating the template are available on request.5 cells per flask. Transient transfections were performed the next day , in accordance with the manufacturer's protocol. Cells were grown under selection media containing 400 μg/ml G418  for 14 days. Colonies were fixed, stained and visually scored.Transient L1  or Alu  retrotraCells were plated onto a 96 well imaging plate 16 hours before visualization, and treated with 3.7% formaldehyde followed by 90% cold methanol. After washing, cells were blocked with 1% bovine serum albumin, before being incubated with 2 μg/ml of a 53bp1 antibody  for 1 hour. Cells were incubated with 5 μg/ml Alexa 488 conjugated secondary antibodies  and 5 μg/ml Hoechst stain (Molecular Probes) before being visualized.g, washed twice with phosphate-buffered saline (PBS), and finally resuspended in 400 uL of PBS. Cells were lysed with an equal volume of 2× Laemmli buffer and boiled for 5 minutes. Extracts were fractionated on a 3 to 8% Tris-acetate gel (Invitrogen) and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour in 5% milk in PBS-Tween and incubated with α-ORF2 antibody  overnight at 4°C. Detection was carried out using horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology) and a substrate kit . Membranes were exposed to BioMax Light Film (Kodak) and developed with a processor . Equal loading was confirmed using β-actin antibodies  using the same protocol.HeLa cells stably integrated with the L1 constructs were grown to confluence in T75 flasks. Cells were treated with trypsin and pelleted at 300 5 cells per flask. After 1, 2 or 3 days of growth, the cells were collected by trypsinization and quantified using a hemocytometer.Growth rate was measured as previously described  with min5 cells per flask. Transient transfections of a neomycin resistance plasmid were performed the next day  in accordance with the manufacturer's protocol. Cells were grown under selection media containing 400 μg/ml Geneticin (Fisher Scientific) for 14 days. Colonies were fixed, stained and visually scored.Colony formation assays were performed as previously described  with somAfter exposure to ionizing radiation, a colony formation assay was conducted as described both above and in previous works .After ionizing radiation exposure, the cell growth assay described above was conducted, with the minor alteration that cell growth was measured after 7 days instead of 1 to 3 days.BamHI and HinDIII.All expression vectors used to generate stable cell lines and to drive Alu retrotransposition  were cloned into the pBud CE 4.1 vector by ligation after digestion with http://www.evolvingcode.net) in a manner that preserved the amino acid sequence of the protein.The ORF1 sequence of the L1 optimized for expression has previously been described . The L1 t-test to compare values to the control for each experiment.In all cases, p-values were determined using the student The authors declare that they have no competing interests.NAW carried out primary studies, including the principle data presented in Figures Figure 1A. Cells stably expressing L1 ORF2 have an increased number of endogenous DSBs. (A) Nuclei of untreated HeLa ORF2 and HeLa ORF2 ER-- cells were stained with Hoechst, and 53BP1 foci revealed by immunofluorescence. (B) 53BP1 foci of untreated HeLa ORF2 and HeLa ORF2 ER-- cells were quantified six times. Data are means and SD (error bars) of these six independent measurements. The two cell lines were significantly different (P ≤ 0.05). (C) Western blots with an antibody to the N-terminal portion of ORF2 were carried out on transiently transfected HeLa cells (M), HeLa cells transformed with the L1 endo./RT double-mutant , HeLa cells transformed with ORF2 expression vector  or an optimized L1 vector  and untransfected HeLa (lane 4). The ORF2 band is marked, as well as a blot of actin on the same membrane.Click here for fileFigure 2. Repression of Alu retrotransposition is not influenced by the source of L1 ORF2. The retrotransposition of a tagged Alu element driven by a vector expressing full length L1 or L1 ORF2 was measured in HeLa ORF2 and HeLa ORF2 ER-- cells. Asterisks signify a statistically significant (Data are means and SD (error bars) of three independent measurements. ≤ 5 × 10-6) difference from 53BP1 foci levels seen in HeLa ORF2 ER-- cells.Click here for fileFigure 3. Constitutive expression of L1 ORF2 does not affect sensitivity to 1 Gy of ionizing radiation. The sensitivity of HeLa ORF2 and HeLa ORF2 ER-- cells to 1 Gy of ionizing radiation was measured by transfecting the cells with a neomycin resistance vector after exposure to 1 Gy of ionizing radiation. Data are means and SD (error bars) of five independent measurements.Click here for fileFigure 4A, B. (A) Percentage similarity between synthetic L1 ORF2s. Similarity between the synthetic L1 ORF2s is listed in table form. This percentage was calculated as total unchanged nucleotides divided by total nucleotides. (B) Sequence alignments of synthetic L1 ORF2s. The row labels refer to the synthetic L1 ORF2 sequence being displayed. (A) signifies L1 ORF2A, (B) signifies L1 ORF2B. C signifies L1 ORF2C. The L1 ORF2 sequences are aligned with L1 ORF2A. Nucleotides matching the L1 ORF2A sequence are shaded.Click here for file


OsPOP5 gene from rice by nested-PCR. Sequence analysis showed that the cDNA encodes a protein of 596 amino acid residues with Mw ≈ 67.29 kD. In order to analyze the protein function under different abiotic stresses, OsPOP5 was expressed in Escherichia coli. OsPOP5 protein enhanced the tolerance of E. coli to high salinity, high temperature and simulated drought. The results indicate that OsPOP5 is a stress-related gene in rice and it may play an important role in plant tolerance to abiotic stress.The prolyl oligopeptidase family, which is a group of serine peptidases, can hydrolyze peptides smaller than 30 residues. The prolyl oligopeptidase family in plants includes four members, which are prolyl oligopeptidase , dipeptidyl peptidase IV , oligopeptidase B , and acylaminoacyl peptidase . POP is found in human and rat, and plays important roles in multiple biological processes, such as protein secretion, maturation and degradation of peptide hormones, and neuropathies, signal transduction and memory and learning. However, the function of POP is unclear in plants. In order to study POP function in plants, we cloned the cDNA of the  In order to overcome these adverse stress factors, plants respond and adapt to these adverse circumstance at physiological, biochemical, and molecular levels. Various functional and regulatory proteins play an important role in controlling abiotic stress tolerance . Some stProlyl oligopeptidase  was first discovered in 1979. The prolyl oligopeptidase family of serine proteases  includes four members in plants, these members are prolyl oligopeptidase , dipeptidyl peptidase IV , oligopeptidase B , and acylaminoacyl peptidase , and they have been identified and their enzymatic properties have been characterized . So far,The homology of amino acid sequence between the four basic peptidases is quite low, but recent crystal structure determination and modeling analysis suggests that they have a similar three-dimensional (3D) structure, and the crystal structure showed that POP contains a catalytic (peptidase) domain with an α/β hydrolase fold and a 7-bladed β-propeller domain . The β-pPOP was found in human as an oxytocin-degrading enzyme  were placed in eppendorf tubes and boiled for 8 min. The samples were fractionated on 12% SDS-PAGE, and finally gels were stained with Coomassie brilliant blue.Expression of OsPOP5 in 3.7.600 value of 1.0. To measure salt tolerance of transformed E. coli cells, the samples were diluted by 50-fold, 100-fold, 200-fold and then 10 μL of the diluted samples was spotted on IPTG LB agar plates containing 50 μg/mL ampicillin and 1.0 mM IPTG and supplemented with additional 400, 500, and 600 mM concentration gradient of NaCl, 500 mM, 800 mM and 1 M mannitol for drought treatment, respectively. For the assay of the thermophylactic experiments, 1 mL samples were transferred to 50 ºC. Aliquots (100 μL) were taken at different periods 0.5, 1, 1.5, 2, 2.5, and 3 h successively, and then 10 μL of samples diluted by 50-fold, 100-fold and 200-fold was spread onto IPTG LB agar plates. For the assay of cryophylactic experiments, the samples were kept frozen for 24 h at −80 ºC and allowed to thaw at ambient temperature (32 ºC) for 1 h, which constituted one freeze—thaw cycle. Aliquots (100 μL) were taken after 2, 4, 6, 8, 10, and 12 cycles successively, and then 10 μL of dilutions (1:10) was spread onto IPTG LB agar plates.Stress tolerance assays of BL/OsPOP5 and BL/pET32a were analyzed. IPTG induction and cell cultures were prepared as described above. The concentration of all induced cultures in liquid LB was adjusted to OD4.OsPOP5 was cloned and characterized in rice for the first time. Analysis of the gene structure shows that OsPOP5 encodes a 596 amino acid protein with a PI of 6.00. There are ABRE, G-boxes, GCC-boxes, MBSs, TGA-motif, TGAGC-motif and motif IIb cis-elements in the OsPOP5 promoter. Semi-quantitative RT-PCR analysis results indicate OsPOP5 is a stress related gene. Heterologous expression of OsPOP5 protein in E. coli enhanced E. coli abiotic stress tolerance, and thus implies that OsPOP5 may plays an important role in plant tolerance to abiotic stress.In this study, the gene encoding a prolyl oligopeptidase family gene,


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.