Ensiling is a common method of preserving energy crops for anaerobic digestion, and many scientific studies report that ensiling increases the methane yield. In this study, the ensiling process and the methane yields before and after ensiling were studied for four crop materials.The changes in wet weight and total solids (TS) during ensiling were small and the loss of energy negligible. The methane yields related to wet weight and to volatile solids (VS) were not significantly different before and after ensiling when the VS were corrected for loss of volatile compounds during TS and VS determination. However, when the TS were measured according to standard methods and not corrected for losses of volatile compounds, the TS loss during ensiling was overestimated for maize and sugar beet. The same methodological error leads to overestimation of methane yields; when TS and VS were not corrected the methane yield appeared to be 51% higher for ensiled than fresh sugar beet.Ensiling did not increase the methane yield of the studied crops. Published methane yields, as well as other information on silage related to uncorrected amounts of TS and VS, should be regarded with caution. Biogas production using energy crops as the main feedstock is attracting increasing attention. Germany is leading the field, with almost 3, 900 biogas plants in operation in 2009, the majority using ensiled crops . EnsilinAt the beginning of the 20th century it was reported that oven drying gives inaccurate values of the DM when the sample contains volatile compounds. It should therefore not be applied to silage as it contains varying amounts of volatile fatty acids (VFAs), lactic acid, ammonia and alcohols formed during the ensiling process ,7. McDonAlthough the limitations of using oven drying without correction for volatile compounds have been known for many years in agricultural sciences, the method is still routinely used in research related to methane production through anaerobic digestion. The methane yield from anaerobic digestion is normally expressed per unit of VS. The amount of VS is based on the amount of TS, which is determined according to standard methods by oven drying, without correction for volatile compounds . After oVS losses of 18% to 35% due to ensiling have been reported . At the et al. [Although no biogas-related research has, until very recently , made uset al.  suggest et al. , neitheret al.  describeet al. -26 and Met al.  in a Geret al. . Unfortuet al. ). Two reet al. ,29 conceet al. ,28. Howeet al. [Among others, McDonald et al.  have poiet al. .The purpose of the current study was to examine how ensiling influences the methane potential, the mass and the total solids of crops. Furthermore, we wished to draw attention to the errors that can arise from using uncorrected, oven-dry-based values of TS and VS, and to highlight a previously presented method, for correcting oven-dry-based TS and VS values for losses of volatile fermentation products during oven drying . The metThe wet weight was found to decrease during ensiling by about 1% for all materials except beets, for which the decrease was about 4% Table . For sugEthanol and acetic acid were present in all silages Table . Lactic After drying the silages no ethanol could be detected, and lactic, acetic and butyric acid were found at lower concentrations. On average, 100% (± 0%) of the ethanol (n = 8), 53% (± 13%) of the lactic acid (n = 6), 72% (± 0.01) of the butyric acid (n = 2) and 89% (± 17%) of the acetic acid (n = 8) evaporated during TS determination. The average values are not significantly different from those presented by Porter and Murray , includeCorrected TS contents are presented in rows I and J in Table The production of energy-containing gases such as hydrogen and methane during ensiling was negligible in all cases: less than 0.1 ml per g VS for all substrates except hemp, which gave less than 2 ml hydrogen per g VS. The energy contained in the hydrogen produced by hemp during ensiling corresponded to about 2 ppm of the energy in the methane produced in the biochemical methane potential (BMP) test. For hemp, beets and beet tops, only hydrogen and no methane was detected; for maize, methane but no hydrogen was detected. The low production of energy-containing gases, together with the low pH in all the silages, except hemp, indicates that the silages were well preserved.For maize, hemp and beet tops, 67% to 89% of the total gas produced (including carbon dioxide) during ensiling was produced during the first 4 days. The gas produced by beet silage was higher than that produced by the other crops, with high gas production during the first 4 days, and a second gas production peak around days 9 to 13, giving 72% of the total gas production between days 6 and 17. All crops produced less than 6% of the total gas between days 30 and 60. After 60 days, the buckets were moved from storage at room temperature to 4°C. Very little gas was produced after this, less than 1% by all crops except hemp, which produced around 5% of the total gas during this time.The maximum mass loss due to aerobic degradation resulting from entrapped oxygen at the start of the ensiling process was calculated and found to be negligible, at most 0.025% of the wet weight. The calculation was based on the assumption that the maximum volume of entrapped air was the volume of the bucket minus the volume of the substrate at the start of ensiling (assuming a density of the substrate of 1 kg/l), 21% of the air being oxygen.The methane potential was determined and is expressed per unit wet weight Figure  and per When relating the methane yield from ensiled material to uncorrected VS, the results are noticeably different. The apparent methane yield from beets was significantly higher (51%) from ensiled material than from fresh frozen material when expressing the yield per unit uncorrected VS Figure . A signiet al. [et al. [et al. [Herrmann et al.  found th [et al.  found me [et al.  did not The overestimated methane yield of beet silage and beet top silage in the current study, and the fact that the TS losses appeared higher than the wet weight losses for beets and maize when using uncorrected TS and VS contents, demonstrate that methane yields of silages based on uncorrected TS and VS are unreliable.Ensiling was not found to increase the methane yield from any of the crop materials investigated in this study. Instead, it was shown that observations such as increased VS-based methane yields or TS losses during ensiling may be artifacts caused by errors in the standard methods commonly used for TS and VS determination. Oven-dry-based TS and VS determination without correction for the loss of volatile compounds is an unsuitable method for all substrates containing noteworthy amounts of volatile compounds. This applies to ensiled energy crops as well as other materials, and is important when using the substrate for anaerobic digestion as well as for other purposes. Caution should therefore be exercised when considering published information about silages, and other materials containing volatile compounds, based on TS and VS. The application of a method developed for grass silage for correcting TS and VS , to otheHemp (Futura 75), maize (Arabica) and sugar beet , a non-commercially available cultivar with lower sugar content and higher biomass yield than normal sugar beet) were cultivated in southern Sweden . The crops were harvested on the following dates: hemp on 5 September 2007, maize on 29 September 2008, and sugar beet on 21 October 2008. Hemp and sugar beet were harvested manually. Maize was harvested with a maize forager set at a chopping length of 10 mm. The hemp and sugar beet tops (leaves and the neck of the root) were chopped in a garden shredder  into pieces about 2 cm long. The sugar beets were cut into 1 cm slices and then into squares measuring 2 to 3 cm. Part of each crop material was ensiled directly and part was frozen for later analysis. The TS and VS contents were determined in fresh crops before ensiling with and without ensiling inoculant, in fresh crops after freezing, and in ensiled crops before and after freezing. TS corrected for volatile compounds were determined in frozen ensiled crops. (Frozen samples were used since the authors were not aware of the corrected method prior to freezing the silage.)Ensiling was carried out in 4.8 l plastic buckets with tightly fitting lids, normally used for food storage . Hemp, maize, sugar beets (beets) and sugar beet leaves including the upper part of the roots (beet tops) were ensiled separately, using four replicate buckets for each kind of crop material. A gas collection system was made by connecting Tygon tubing  to a balloon made of Transfoil El-OPET/PE  with a hose connector  in each lid. Silicone was used to seal the connection between the hose connector and the lid and between the bucket and the lid. The chopped plant material was sprayed with a bacterial ensiling inoculant, Lactisil Stabil . In all, 20 ml was added per kg wet plant material, according to the manufacturer's instructions (1.25 g powder in 5 l tap water). The decrease in weight was recorded by weighing the material in the buckets before and after the ensiling period. The decrease in TS was determined based on the wet weight and TS of the fresh crops with ensiling solution and of ensiled crops.The buckets were stored at room temperature (23 to 25°C) for 60 days; after which they were stored at 4°C for a minimum of 100 days. The gas volume and the contents of methane and hydrogen were monitored during the entire ensiling period. The results from one bucket of beets and two buckets of hemp were excluded due to gas leakage.The replicate samples of each crop material were mixed after ensiling before sampling for TS and VS determination, and for BMP tests. The mixed samples were also frozen for later analyses. TS determination and BMP tests were started immediately after sampling to minimize losses due to volatilization during sample handling. Contents of VFAs, lactic acid and ethanol were determined in silage samples that had been frozen, since this part of the study was included later. Prior to analysis, frozen silages were thawed at 8°C in buckets with tightly fitting lids.BMP tests were performed as reported elsewhere , with thThe total gas volume and the content of methane  were mon2SO4 to a pH of 1 to 3 and filtrated through 0.45 μm polypropylene filters . The content of C1-C6 VFAs , lactic acid, succinic acid and ethanol were determined using high performance liquid chromatography (HPLC)  with an Aminex HPX-87H column  and a refractive index detector . Sulfuric acid (5 mM) was used as the mobile phase (0.6 ml/min), and the oven temperature was 40°C. The concentration of VFAs, lactic acid and ethanol and were calculated for the wet silage according to Equations 1 and 2:TS and VS were determined in duplicate or quadruplicate according to standard methods , using s1 = original wet weight related to TS added, g; m2 = water added, g; m3 = substrate TS added, g; c1 = concentration of analyzed compound, g/kg; and D = dilution factor = 5.Where mThe TS and VS were corrected in two ways: (1) according to the volatilization coefficients for grass silage dried at 100°C presented by Porter and Murray : lactic Gas composition with respect to methane was determined using gas chromatography and a thermal conductivity detector, as described elsewhere . HydrogeAll statistical analyses were performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test using the statistical software Prism . The term 'significant' is only used where a statistical analysis of significance has been performed. The significance level of 5% was used throughout all statistical analyses. Values are given ± 1 SD. The SDs of weight losses during ensiling, of TS and VS determinations, of the concentrations of volatile compounds added to the corrected values of TS and VS and of tests and controls in BMP were combined according to standard statistical rules  to proviWhere σ = standard deviation; a, b, c, d = independent measured quantities; and k = constant.The authors declare that they have no competing interests.LB became aware of the methodological problem investigated, and secured financial support for this study. All authors participated in the design of the study, harvesting of the crops and reviewing of the literature. EK set up the ensiling method and performed most of the ensiling experiments, the TS and VS determinations and all analyses of the volatile compounds. IAN participated in the ensiling trials and carried out the BMP tests. EK performed the statistical analysis and prepared the major part of the manuscript. LB and IAN contributed to writing the manuscript, and all authors read and approved the final manuscript.


Drug addiction is considered a disorder of neuroplasticity in brain reward and cognition systems resulting from aberrant activation of gene expression programs in response to prolonged drug consumption. Non-coding RNAs (ncRNAs) are key regulators of almost all aspects of cellular physiology. MicroRNAs (miRNAs) are small (∼21–23 nucleotides) ncRNAs transcripts that regulate gene expression at the post-transcriptional level. Recently, miRNAs were shown to play key roles in the drug-induced remodeling of brain reward systems that likely drives the emergence of addiction. Here, we review evidence suggesting that one particular miRNA, miR-212, plays a particularly prominent role in vulnerability to cocaine addiction. We review evidence showing that miR-212 expression is increased in the dorsal striatum of rats that show compulsive-like cocaine-taking behaviors. Increases in miR-212 expression appear to protect against cocaine addiction, as virus-mediated striatal miR-212 overexpression decreases cocaine consumption in rats. Conversely, disruption of striatal miR-212 signaling using an antisense oligonucleotide increases cocaine intake. We also review data that identify two mechanisms by which miR-212 may regulate cocaine intake. First, miR-212 has been shown to amplify striatal cAMP response element binding protein (CREB) signaling through a mechanism involving activation of Raf1 kinase. Second, miR-212 was also shown to regulate cocaine intake by repressing striatal expression of methyl CpG binding protein 2 (MeCP2), consequently decreasing protein levels of brain-derived neurotrophic factor (BDNF). The concerted actions of miR-212 on striatal CREB and MeCP2/BDNF activity greatly attenuate the motivational effects of cocaine. These findings highlight the unique role for miRNAs in simultaneously controlling multiple signaling cascades implicated in addiction. Non-coding RNAs (ncRNAs) can be defined as biologically functional RNAs that do not encode proteins. This class of transcripts is characterized by the presence of an increased density of stop codons and lack of extensive open reading frames (ORFs). For decades, work on ncRNAs focused almost exclusively on transport RNAs (tRNAs) and ribosomal RNAs (rRNAs), which are key regulators of mRNA translation into encoded proteins. However, recent advances in high throughput sequencing technologies have revealed tremendous diversity in ncRNAs. Moreover, ncRNAs are playing an increasingly more recognized role in key aspects of cellular function, including the regulation of gene expression, often through novel mechanisms of action. ncRNAs have also been implicated in key cellular process such as DNA imprinting, RNA splicing, editing, transcription, mRNA degradation, and translational repression. Interestingly, analysis of ncRNA complexity through evolution reveals that the proportion of non-coding sequences in eukaryotic genomes correlates closely with the complexity of the organism, even when proportion of the protein coding genes remains relatively static across organisms  and short-interfering RNAs (siRNAs), which are generally 18–25 nucleotides in length. Small RNAs (smRNAs) such as the small nucleolar RNAs (snoRNAs), smRNAs, piwi-interacting RNA (piRNAs) are between 20 and 300 nucleotides in length. The long ncRNAs (>200 nucleotides) can further be grouped into different categories on the basis of their origin -induced translational repression or sequestering them for storage or degradation . Both of these proteins are important components of the miRNA biogenesis pathway. Dicer is an endoribonuclease that catalyzes the processing of precursor miRNA into its mature form. Characterization of Dicer1 mutants in Arognaute2, a member of the argonaute family of proteins, is the catalytically active component of the RISC that not only facilitates miRNA processing but also their regulatory actions on target mRNA transcripts , and Cdk5r1 (cyclin-dependent kinase 5 activator 1). In the same study, the effects on cocaine reinforcement of selectively ablating Ago2, the catalytic component of RISC involved in transducing the inhibitory actions of miRNAs on their target transcripts, was assessed. Specifically, Schaefer et al. investigated the effects knocking down Ago2 in medium spiny neurons (MSNs) of the striatum that express the dopamine D2 receptor (D2R)  affects cocaine-induced place conditioning  whose expression is sensitive to cocaine  using an approach termed Serial Analysis of chromatin occupancy (SACO)  was significantly increased . Dominant negative or phosphorylation-deficient mutant forms of CREB attenuated these stimulatory effects of miR-212 on CREB signaling. More importantly, we also found that miR-212 amplified CREB signaling in the striatum in vivo. Specifically, we found that rats with extended access to cocaine showed increased expression of the CREB-responsive gene Nurr1, and that expression levels were greatly increased by miR-212 overexpression in striatum , also known as TORC1 and TORC2. Further investigation of the mechanism by which miR-212 enhances CREB signaling revealed that the kinase Raf1 was activated by miR-212, and was found to play a key role in sensitizing adenylyl cyclase activity. Finally, computational and biochemical analysis showed that Sprouty-related EVH1 domain-containing protein 1 (SPRED1), a known negative regulator of Raf1 signaling, is a miR-212 target mRNA transcript and SPRED1 repression by miR-212 contributes to its stimulatory effects on Raf1 and CREB activity  on cocaine intake in rats with restricted or extended cocaine access. It is known that CRTC overexpression increases CREB activity and that miR-212 increases CRTC1 expression  expression in cultured cells  is known to bind to methylated DNA and can act as a gene repressor by recruiting other chromatin remodeling proteins that combine to form the so-called repressor complex  (Park et al., Besides cancer, miR-212 has also been implicated in regulating organogenesis by playing a key role in modulating epithelial stromal interactions (Ucar et al., Taken together, these studies highlight the fact that miR-212 plays a critical role in fine-tuning transcriptional and neuroplastic responses to drugs of abuse. Specifically, we found that miR-212 can control cocaine intake through two complementary mechanisms: amplifying CREB signaling and reducing MeCP2/BDNF transmission in striatum.Since miR-212 and miR-132 share the same seed region it is widely believed that they target the same mRNAs. However, only a few putative miR-212/132 targets have been verified experimentally, which include MeCP2, Rb1and HB-EGF. There is increasing evidence available now showing that the 5′ and 3′ regions of the miRNAs can form the basis of differential target recognition despite having the identical seed region (Brennecke et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections.Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8 The two human retroviruses HTLV-1 and HTLV-2 are similar in their structure, replication cycle and the manner through which they spread between and within individuals. They differ in their preferred host T-cell type and in their possible clinical outcomes. HTLV-2 has not been linked with a specific disease, whereas HTLV-1 infection can cause leukemia and profound neuropathology. It is well established that HTLV-1-infected cells undergo clonal expansion in infected individuals, but little is known about clonality in HTLV-2 infection. In this work, we demonstrate that the extent of HTLV-2-infected cell expansion significantly exceeds that of HTLV-1-infected cells in healthy carriers, approximating instead to that observed in patients with HTLV-1-associated leukemia. Furthermore, we show that HTLV-2 characteristically resides in a small number of expanded clones that persist over time, and that the degree of oligoclonality significantly correlates with viral burden in HTLV-2-infected individuals. These results highlight the distinction between in vivo clonal proliferation and malignant transformation, and suggest that the infected cell type may be a more important determinant of clinical outcome in retroviral infections. In contrast, HTLV-2 causes little disease. By following a large cohort of HTLV-1/2-infected and seronegative individuals for almost two decades, the HTLV Outcomes study (HOST) detected myelopathy and other neurologic abnormalities among HTLV-2-infected subjects + T-cells, whereas HTLV-2 favours CD8+ T-cells + or CD8+ T-cells driven by HTLV-1 or HTLV-2, respectively, leads to differential expansion of the two T-cell subsets An important distinction between HTLV-1 and HTLV-2 lies in their host cell predilection. Although they use the same cellular receptors It is known that HTLV-2, like HTLV-1, can immortalize human lymphocytes in culture + and CD8+ T-cells, comparing the clonal distribution of HTLV-2-infected peripheral blood mononuclear cells (PBMCs) to that observed in HTLV-1, and examining the genomic environment of integrated HTLV-2 proviruses. For these purposes, we adapted our recently described high-throughput method for the identification, mapping and quantification of retroviral integration sites, which we have used previously to study host factors associated with clonal abundance, proviral expression and disease progression by tracking infected clones using the genomic coordinates of retroviral integration sites In this work, we investigated natural HTLV-2 infection by quantifying the viral burden in CD4+ and CD8+ T-cells to the HTLV-2 proviral load, PBMCs from 28 HTLV-2-seropositive carriers were sorted by flow cytometry into separate CD3+ CD4+CD8− and CD3+CD4−CD8+ populations. Integration sites were then mapped and quantified in DNA extracted from both sorted and unsorted cells using our previously described To determine the relative contributions of CD4+CD8− or CD4−CD8+ T-cell, and attributed the integration sites found in the unsorted sample to either CD4+ or CD8+ cells based on the sorted sample in which they were found more frequently. Across all samples, 50% of sites (representing >99% of proviruses) could be attributed in this manner.We assumed that HTLV-2 infects a single-positive CD4+ cells isolated by flow sorting exceeded the number of CD8+ cells in the majority of samples, HTLV-2 was identified chiefly in the CD8+ fraction, whether quantified as the number of sequencing reads or the number of proviruses ; a mean of only 0.3% (median = 0%) was positively attributed to CD4+ cells , HTLV-1-infected subjects were estimated to carry a median of 31,710 distinct clones in the blood (consistent with previous estimates for HTLV-1), whereas HTLV-2-infected subjects were estimated to carry a median of only 976 clones  and that much of the load  is composed of low abundance clones The distribution of proviral load across identified clones also differed significantly between HTLV-1 and HTLV-2. To compare the two viruses, we used the oligoclonality index The difference in clonal distribution between HTLV-1 and HTLV-2 was also apparent when measuring the absolute abundance of each clone as copies per 10,000 PBMCs . In partWe reported previously that the genomic environment flanking the integration site appears to play a role in determining the equilibrium abundance of a given clone in vivo Whereas there was no significant correlation between the oligoclonality index and the proviral load in HTLV-1 infection , we observed a highly significant positive correlation  between these parameters in HTLV-2 infection . In contTo test whether the expanded clones observed in HTLV-2 infection were long-lived, we analysed integration site content in samples collected at an earlier time point  from 10 of the HTLV-2-infected individuals in our cohort. The vast majority of the load (median = 96%) at the later time-point was represented by clones already present at the earlier time-point . These cBoth HTLV-1 and HTLV-2 infect the susceptible host by the same routes, and propagate within the host by the same two non-mutually-exclusive routes: the infectious route, which results in proviral integration at a new genomic site; and the mitotic route, where the provirus is replicated passively when the infected cell undergoes DNA replication and mitosis. It therefore benefits these viruses to drive proliferation of the infected cell. Indeed, the Tax proteins from both HTLV-1 and HTLV-2 have been shown in vitro to accelerate progression through the cell cycle, inhibit apoptosis and transform cells HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals + T-lymphocyte (CTL) activity The host genomic environment flanking HTLV-2 integration sites in vivo closely resembles that of HTLV-1 integration sites. Similarities are evident at the nucleotide and the chromosome levels, and when examining particular genomic features known to be more frequent in proximity to HTLV-1 integration sites than expected by chance The selection forces that act upon HTLV-1-infected clones have been the subject of many previous studies The immune response to HTLV-2 proteins is less well understood. Oliveira and colleagues showed that high frequencies of CTLs specific for HTLV-2 Tax can be found in the circulation of HTLV-2 carriers Although abundant clones  represent only a small fraction of all infected clones in HTLV-1 infection, they represented approximately 20% of all HTLV-2 clones in this study . There aRegardless of the forces that drive this vigorous clonal proliferation of HTLV-2-infected cells, the observed correlation between the oligoclonality index and proviral load in HTLV-2 infection . Samples, with data linkage, were donated by HTLV-1 or HTLV-2-infected subjects attending the National Centre for Human Retrovirology, St Mary's Hospital, Imperial College Healthcare NHS Trust, London after giving written informed consent. HOST Study approved by the University of California San Francisco Committee on Human Research.Cryopreserved PBMCs from 16 HTLV-1-infected and 28 HTLV-2-infected individuals were used in this study (Supplementary All DNA extractions were carried out using a DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's protocol.HTLV-2 proviral load was quantified as reported elsewhere +CD8− and CD4−CD8+ populations. Data were analysed with FACSDiva v6 software (BD Biosciences). For each sample, DNA was extracted from both sorted populations and from a separate aliquot of unsorted PBMCs.Cells were thawed and washed, then surface-stained with directly-conjugated monoclonal antibodies specific for CD3, CD4 and CD8. Flow cytometric sorting was conducted to high purity (>98%) using a custom-modified FACSAria II (BD Biosciences). Lymphocytes were pre-gated on CD3, then sorted as CD4CTCGCATCTCTCCTTCACG, 5LTRrev – CTGGTGGAAATCGTAACTGGA; HTLV-2 primers: H2LTRfw – GACTCACCTTGGGGATCCAT, H2LTRrev – TTAGCCAAATGGGCGTTTTA). Identification of integration sites was performed via LM-PCR followed by high-throughput sequencing as described previously AAGGGCTAGGGCTTCCTGAACCTC and H2B5 – CTATAGGCAGGCCCGCCCCAGGAG (or variants thereof according to defined LTR polymorphisms).Ligation-mediated polymerase chain reaction (LM-PCR) primer binding site sequences were determined from Sanger-sequenced PCR amplicons . The resulting sequences were aligned to the human genome reference  and HTLV-1/2 upstream sequence using the eland_pair implementation of CASAVA 1.8.2 (Illumina).+ or CD8+ cell population. Six clones were initially detected in both CD4+ and CD8+ cells; in these cases the clone was ascribed to the cell type with the greater number of proviruses of that clone.Integration sites were quantified by enumerating unique shear sites as described previously In silico sites were derived by random selection of 190,000 sites from the human genome (hg18). To eliminate any potential bias due to alignment limitation, the DNA sequences at those sites were generated using Galaxy http://malnirav.github.com/hiAnnotator), kindly provided by Nirav Malani and Frederic Bushman .Annotations to the human genome (hg18) were retrieved as described previously . Here, individual-based rarefaction curves depict the expected number of clones against the number of infected cells sampled. Four numerical criteria are used to score each model in terms of how consistently it reproduces the total observed rarefaction curve from nested subsamples thereof. Using the geometric mean, estimates from the best-performing models are aggregated to produce the final estimate. Samples with near-linear rarefaction curve  were excluded from the analysis. Such curves imply a  linear relationship between the number of infected cells and the number of clones, which is indicative of severe under-sampling. Prohibitively small sample sizes of less than 150 proviruses were also excluded from the analysis. DivE requires an estimate of the number of cells in the blood Nblood, for which we assume: (i) a circulating blood volume of 5L; (ii) a PBMC count of 3×109 L−1; and (iii) each infected T-cell carries a single copy of the provirus 9. Proviral load (PVL) is defined as the number of viral copies per 100 PBMCs. Therefore, Nblood is given by PVL×5×3×109.The http://www.R-project.org/). The Gini coefficient was calculated using the reldist R package (http://CRAN.R-project.org/package=reldist). Two-tailed non-parametric tests including the Mann-Whitney and Fisher's exact test were used for all comparisons.Statistical analysis was carried out using R version 2.15.2  The total number of sequencing reads for each HTLV-2 sample. (B) The total number of infected cells (distinct proviruses) identified in each HTLV-2 sample. (C) The total number of unique integration sites identified in each HTLV-2 sample.(TIF)Click here for additional data file.Figure S2Integration bias at the sequence level. Nucleotide sequences at the genomic sites of HTLV-2 integration are summarized by position and base. The arrow denotes the position of proviral integration. Base 1 is the first nucleotide following the integrated provirus. For each position, the relative proportion of sites containing each base is noted. Remarkably favoured (red) or disfavoured (blue) bases compared to random (in silico) sites are highlighted.(TIF)Click here for additional data file.Table S1Details of subjects in study.(DOCX)Click here for additional data file.


Objective(s): Human T-cell lymphotropic virus type-1 is an oncornavirus that causes adult T cell leukemia (ATL) HTLV-I-associated myelopathy⁄tropical spastic paraparesis (HAM/TSP). Golestan province is located in North West of Khorasan province known as an endemic area for HTLV-I in Iran. This study aimed to evaluate seroprevalence of HTLV-I in Golestan province.Materials and Methods: In this cross-sectional descriptive study in 2007, blood samples were collected from 2034 healthy people residing in different parts of Golestan province. Sera were assessed for HTLV-I/II–specific antibodies by ELISA method and reactive samples were confirmed by Western blot. Demographic and serologic data were entered in SPSS version 11.5 and statistical analysis was performed.Results: An overall HTLV-I/II prevalence of 0.7% was observed in 15 cases by ELISA. Six out of 15 were confirmed as HTLV-I by western blot. Regional variation in the prevalence of HTLV-I was observed; 0%, 0%, 0.1%, 1.9%, 0.3%, 0%, and 2.6% tested HTLV-I-positive from west to east of Golestan Province regions, respectively. Seropositivity increased with age. No association between HTLV-I infection and sex status was detected.Conclusion: Highest rate of HTLV-I seroprevalence was shown in east of this region located in neighborhood with Khorasan province, the only confirmed endemic area in Iran. It seems that eastern area of our province is endemic for HTLV-I. Further comprehensive detailed epidemiological and molecular studies are recommended. Human T-cell lymphotropic virus type-1 (HTLV-I) is a member of Retroviridae family which has been discovered as the first human retrovirus. This oncornavirus causes the T cell malignancy associated with two main diseases; adult T cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) . WorldwiIran has been introduced as an endemic area based on studies reported from Mashhad in Khorasan (A province of Iran recently divided into three provinces) located in the northeast of Iran , 12. The◦C. Serum samples were screened for the presence of anti-HTLV-I antibodies with the HTLV I/II enzyme linked immunosorbent assay (ELISA)  according to the manufacturer’s instructions. All reactive samples on serologic screening were tested further through Western blot (WB) analysis according to the manufacturer’s instructions . Descriptive data was summarized as the mean, standard deviation, and/or percentages were analyzed by SPSS 11.5 using Chi-square and t-tests. A P-Value of <0.05 was considered statistically significant.From all of the seven main cities with an estimated population of 1.5 million, 2034 individuals were selected through multistage cluster sampling in 2007. Demographic information, such as sex, age, and residency status, was collected. The study was approved by Deputy of Research of Golestan University of Medical Sciences regarding scientific and ethical issues. Informed consent was obtained from all participants. Five ml of blood samples were obtained from each individual. Serum was separated through centrifugation and was stored at −20Out of 2034 people studied in this population, based seroepidemiology, 848 cases were male (41.7%) and 1186 participants were females (58.3%) with the mean age of 38.66±16.54 years. All serum samples were analyzed for anti-HTLV antibodies.In the primary screening of the samples by ELISA, 15 (0.7%) were positive for HTLV1/2 antibodies. The Western blot results confirmation demonstrated that 6 (0.3%) out of 15 ELISA positive specimens were HTLV-1 positive, but HTLV positivity was not confirmed in 9 cases (0.4%). According to the Western blot results, the overall prevalence of the HTLV-I infection in the population under study was 0.3% (6/2034). We did not find an indeterminate result by Western blot. The HTLV-I infection rate for females was 0.3% (3/1186) and for males was 0.4% (3/848). No significant difference in the seroprevalence was observed between males and females .Regional variation in the prevalence of HTLV-I was observed to be 0%, 0% 0.1%, 1.9%, 0.3%, and 2.6% tested HTLV-I-positive from west to east of Golestan regions, respectively . SeropreIn the present study, we have reported the seroepidemiology of HTLV-I infection in a representative sample of individuals from different cities of the province of Golestan. The overall prevalence of HTLV-I infection in our study is 0.3% (95% CI: 0.06-0.53%) lower than the previous study with 4.4% in Thalassemia patients in Gorgan . This isfrom different investigations in the USA 0.6% , Brazil Comparison of the regional distribution of the carriers in the present study revealed a significant increase of the HTLV-I carriers in Kalaleh area. The observed changes could be considered mainly due to the migration of people from other areas. This interpretation is supported by the observation of ethno-epidemiology studies on HTLV-I carriers who are either born in the endemic areas or the descendants of migrants from those areas. It has been reported that some ethnically defined factors are likely to be associated with HTLV-I diseases among HTLV-I endemic populations .Previous studies have demonstrated that HTLV-I infection increases with age -29. AlsoIn our study, the age distribution of carriers showed presence of HTLV-I carrier in age groups more than 20 years old and the largest number of carriers was observed in this age group. Recent studies showed the number of carriers in the age groups between 0–9 and 50–59 significantly decreased . This deIn conclusion, serological screening of pregnant women, and the blood donors, along with the prevention of mother-to-child infection transmission by stopping breast feeding will greatly reduce the vertical transmission. In addition, there still remain other modalities of HTLV-I infection, such as sexual transmission and possible transuterine infection -37. Furt


HTLV-1 predominantly transforms CD4+T cells in vitro and induces leukemia and neurological disease in vivo, whereas HTLV-2 shows a preference for CD8+T cell transformation in vitro with limited in vivo pathology. To better understand cellular tropism of HTLV-1 and HTLV-2 early after in vivo infection we determined proviral load and gene expression kinetics of these viruses in purified CD4+ and CD8+T cells from newly infected rabbits.New Zealand White rabbits (four/group) were inoculated intravenously with HTLV-1 (Ach), HTLV-2 (pH6neo), and control irradiated producer cells. Blood was collected pre- (week 0) and post-inoculation  for detecting antibody responses using line blot assay, and proviral load and viral gene expression in purified CD4+ and CD8+ T cells using real-time PCR.HTLV-1 and HTLV-2 infected rabbits seroconverted and had detectable proviral loads in both CD4+ and CD8+T cells by 1 wk post infection. HTLV-1 showed slightly higher CD4+T cell proviral loads early, but overtime the virus was detected at higher levels in CD8+T cells. In general HTLV-2 proviral loads were lower than HTLV-1 and throughout the experimental time course the predominant infected cell was the CD8+T cell. HTLV-1 gene expression levels  peaked early in CD4+T cells, but overall expression levels over time were higher in CD8+T cells. HTLV-2 gene expression was detected in both CD4+ and CD8+T cells, but was consistently higher in CD8+T cells throughout the study. Viral determinants of tropism with emphasis on Env will also be discussed.In the infected rabbit, HTLV-1 shows an early preference for CD4+T cells, but over the12 wk study the majority of cells harboring and expressing the virus are CD8+T cells. In contrast, in this early stage of infection, HTLV-2 reveals a preference for CD8+T cells. We speculate that this differential tropism between HTLV-1 and HTLV-2 contributes to the distinct pathobiology of these two related viruses.


At 1, 4, and 24 h after single-dose treatment with clenbuterol or after acute running exercise, the soleus and TA muscles were isolated and subjected to analysis. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) increased after single-dose clenbuterol treatment and acute exercise in the soleus muscle but not in the TA muscle. Although there was no change in the phosphorylation of Akt after acute exercise in either muscle, phosphorylation of Akt in the soleus muscle increased after single-dose clenbuterol treatment, whereas that in the TA muscle remained unchanged. These results suggest that p38 MAPK and Akt pathways play a functional role in the adaptation to clenbuterol treatment and exercise, particularly in slow-twitch muscles.The aim of this study was to clarify the intracellular β Numerous studies have shown that the selective β2-agonist clenbuterol, a doping drug, induces skeletal muscle hypertrophy, transformation of muscle fibers, and alterations in muscle metabolic enzymes [2-AR activation [2-agonist clenbuterol and exercise on the activity of β2-AR signaling pathways in skeletal muscles are unclear. Studying β2-AR signaling in skeletal muscles is able to identify potential therapeutic applications for muscle wasting conditions such as sarcopenia, cancer cachexia, denervation, and neuromuscular diseases, aiming to attenuate the muscle wasting and associated muscle weakness, and to enhance muscle growth and repair after injury [β enzymes , 2. On t enzymes . These ativation . Howeverr injury , 2.2-AR can couple to both Gαs and Gαi signaling pathways. The Gαs-adenylyl cyclase (AC)-cyclic adenosine monophosphate (cAMP) pathway is the best characterized of the β2-AR signaling pathways. Phosphorylated protein kinase A (PKA) induced by cAMP regulates the activity of proteins, including cAMP response element binding protein (CREB) [2-AR coupling to Gαi appears to activate Gαs-independent pathways, such as mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K)-Akt signaling pathways [It is known that βn (CREB) , 2. CREBn (CREB) . In addipathways , 2, 6–8.pathways . In partpathways  and playpathways , 12. Thepathways .2-AR mRNA resulting from clenbuterol treatment for 10 days in rats [2-AR signaling to clenbuterol treatment and exercise may differ between muscle fiber types. Therefore, in the present study, we investigated the acute effects of clenbuterol treatment and exercise on the phosphorylation of CREB, p38 MAPK and Akt in mouse skeletal muscles to clarify the intracellular signaling specificity in the typical slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles.On the basis of metabolic and contractile properties and myosin heavy chain isoforms, skeletal muscle fibers can be broadly classified as type I, oxidative, slow-twitch fibers and type II, glycolytic, fast-twitch fibers. We have previously reported that the changed expression of β in rats , 15 and  in rats  is depenMale 10-week-old C57BL/6J mice were obtained from CLEA Japan . These mice were housed in a temperature (23–25 °C) and humidity (50–60 %)-controlled room with a 12-h light–dark cycle. Regular rodent laboratory chow and water were available ad libitum. The experimental procedures and animal care were approved by the Committee on Animal Care, Ethics and Use, Waseda University (2011-A003), and conducted according to the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, the Physiological Society of Japan.2-agonist experiment, clenbuterol hydrochloride  dissolved in 0.9 % NaCl solution was injected intraperitoneally (i.p.) into mice (1.0 mg/kg body weight). At 1, 4, and 24 h after treatment, the soleus and TA muscles were isolated. For the exercise experiment, because moderate exercise has been previously defined as brief  bouts of treadmill running at 50–75 % maximum O2 consumption or 15–22 m/min [For the β22 m/min , mice we2-agonist clenbuterol treatment, or both. The animals were randomly divided into 4 groups: sedentary control (S) group, voluntary exercise-trained (EX) group, clenbuterol-treated (CL) group, and EX + CL group. EX and EX + CL mice were singly housed in stainless steel cages  and given 24-h access to a running wheel for 6 weeks. Wheel revolutions were counted daily and multiplied by the wheel circumference (628 mm) to obtain daily running distances. S and CL mice were singly housed in similar cages without a running wheel for 6 weeks. Further, CL and EX + CL mice were injected i.p. with clenbuterol  dissolved in 0.9 % NaCl solution. S and EX mice were also injected i.p. with the same volume of 0.9 % NaCl solution. All mice were sacrificed at 24 h after the last voluntary exercise or injection. Skeletal muscles were rapidly removed, frozen in liquid nitrogen, and stored at −80 °C.Mice were subjected to 6-week voluntary exercise, β2-AR expression in cytosol and membrane fractions of skeletal muscles has been described previously [2PO4, 50 mM K2HPO4, and 10 mM EDTA; pH 6.53, 1:20, w/v) containing a protease inhibitor cocktail . The homogenate was centrifuged at 100,000g for 1 h at 4 °C. The resultant supernatant was collected as the cytosol fraction. The pellet was homogenized in an ice-cold solubilization buffer . After incubation for 2 h, the homogenate was centrifuged at 100,000g for 1 h at 4 °C. The resultant supernatant was collected as the membrane fraction. Each protein sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride  membrane. The membrane was blocked using phosphate-buffered saline plus Tween 20 containing 5 % nonfat dry milk (w/v), and then probed with anti-β2-AR antibody . The bound antibody was detected using enhanced chemiluminescence .The measurement of βeviously , 18. Bri2-AR, 5′-TCT GTC TGT CTG TCT GGA TGA TG-3′ (forward), 5′-CCC ATT GTC ACA GCA GAA AGG-3′ (reverse); peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), 5′-TGA CAT AGA GTG TGC TGC TCT G-3′ (forward), 5′-TGG TTC TGA GTG CTA AGA CCG CTG-3′ (reverse); 18S, 5′-GAG AAA CGG CTA CCA CAT CC-3′ (forward), 5′-CCC AAG ATC CAA CTA CGA GC-3′ (reverse). The PCR product was electrophoresed in 1 % agarose gel (w/v) containing ethidium bromide. Fluorescence intensity was determined using the Lumino-Image Analyzer LAS-3000 System .Total RNA extracted from skeletal muscles using TRIzol Reagent  was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit  with random primers. The product was subjected to PCR using Taq DNA polymerase  with the oligonucleotide primers as follows: βTotal protein from skeletal muscles was extracted using Tissue Protein Extraction Reagent (Thermo Fisher Scientific), as previously described . Equal aP < 0.05.Results are expressed as mean ± SEM. The significance of the difference between means was assessed by the Tukey–Kramer test after significant differences between groups were established by analysis of variance. Differences were considered significant for 2-AR protein in cytosol and membrane fractions, or in the expression of β2-AR mRNA in the soleus and TA muscles at 1–24 h after single-dose clenbuterol treatment and acute exercise (data not shown). Next, to identify β2-AR-induced signaling pathways that are potentially involved in skeletal muscle adaptation to single-dose clenbuterol treatment and acute exercise, we performed western blot analysis using phospho-specific antibodies against these molecules. We first investigated whether the phosphorylation of CREB in skeletal muscles is changed by single-dose clenbuterol treatment and acute exercise, but there was no change in the phosphorylation of CREB in the soleus and TA muscles at 1–24 h after single-dose clenbuterol treatment and acute exercise (data not shown).In this study, we did not observe any changes in the expression of the β2-AR [2-AR plays a key role in clenbuterol- and exercise-induced skeletal muscle adaptation. Therefore, we next investigated whether the phosphorylation of p38 MAPK in skeletal muscles is changed by single-dose clenbuterol treatment and acute exercise. The phosphorylation of p38 MAPK in the soleus muscle was significantly increased, to 580 and 460 %, at 1 and 4 h, respectively, after single-dose clenbuterol treatment  and 160 % (P = 0.19), at 1 and 4 h, respectively, after single-dose clenbuterol treatment . It has been reported that exercise stimulates PGC1α transcription in skeletal muscles through the activation of the p38 MAPK pathway  and β2-A2-AR . These fNext, we investigated whether the phosphorylation of Akt in skeletal muscles is changed by single-dose clenbuterol treatment and acute exercise. The phosphorylation of Akt was significantly increased, to 360 and 240 %, at 1 and 4 h, respectively, after clenbuterol treatment in the soleus muscle, whereas that in the TA muscle remained unchanged . These results suggest that the acute effect of clenbuterol treatment and exercise on the phosphorylation of p38 MAPK and Akt in slow-twitch muscles is transient.In the present study, we observed an increase in the phosphorylation of p38 MAPK after single-dose clenbuterol treatment and acute exercise in the soleus muscle Fig. , and likThe present study clearly showed that the phosphorylation of p38 MAPK in the soleus muscle was significantly increased after single-dose clenbuterol treatment and acute exercise, with an occasional increase in phosphorylated p38γ subunits Fig. a. It hasThe present study is the first report that increased phosphorylation of p38 MAPK after single-dose clenbuterol treatment is observed to a much greater extent in slow-twitch muscles than fast-twitch muscles Fig. a. Howeve2-AR-mediated G-protein switching to activate MAPK; phosphorylation of MAPK by the β2-AR proceeds through receptor coupling to Gαs and activation of PKA. Activated PKA phosphorylates the β2-AR leading to receptor coupling to, and activation of, Gαi, and then Gβγ released from the Gαi-coupled β2-AR activates MAPK in a Src- and Ras-dependent pathway. The in vitro cell culture model suggests the possibility that β2-agonist clenbuterol-induced receptor phosphorylation through the activation of PKA may progress in slow-twitch soleus muscle, compared to fast-twitch TA muscle, which is responsible for the increase in the phosphorylation of p38 MAPK after clenbuterol treatment to a much greater extent in the soleus muscle than the TA muscle.Daaka et al.  demonstrOur study also demonstrated that the phosphorylation of Akt was significantly increased after clenbuterol treatment in the soleus muscle, whereas that in the TA muscle remained unchanged Fig. a. Kline We did not find any changes in the phosphorylation of Akt after acute exercise in the soleus and TA muscles Fig. b. Some si-linked Gβγ subunits activate the PI3K-Akt signaling pathway [2-AR stimulation, and these lead predominantly to hypertrophy in fast-twitch muscle [2-AR stimulation induced hypertrophy preferentially in fast-twitch muscles [2-agonist clenbuterol administration increases blood glucose and insulin concentrations [Specifically, in vitro cell culture experiments have revealed that the Gα pathway , 33, 34.h muscle . Therefo muscles , 15. The muscles . The fintrations  could be2-AR signaling specificity in mouse skeletal muscles in response to single-dose clenbuterol treatment and acute exercise. The current findings suggest that p38 MAPK and Akt pathways play a functional role in the adaptation to clenbuterol treatment and exercise, particularly in slow-twitch muscles. In particular, the β2-agonists and intracellular β2-AR signaling have important clinical significance for enhancing muscle repair and restoring muscle function after muscle atrophy. Thus, this scientific evidence about intracellular β2-AR signaling in skeletal muscle could contribute to the identification of new therapeutic targets for attenuating muscle wasting and to the eradication of sports doping.In conclusion, the present study shows the intracellular β


Airway remodeling is a repair process that occurs after injury resulting in increased airway hyper-responsiveness in asthma. Thymic stromal lymphopoietin (TSLP), a vital cytokine, plays a critical role in orchestrating, perpetuating and amplifying the inflammatory response in asthma. TSLP is also a critical factor in airway remodeling in asthma.invitro and invivo.To examine the role of TSLP-induced cellular senescence in airway remodeling of asthma invitro. Senescence-Associated β-galactosidase  staining, and BrdU assays were used to detect cellular senescence. In addition, the Stat3-targeted inhibitor, WP1066, was evaluated in an asthma mouse model to determine if inhibiting cellular senescence influences airway remodeling in asthma.Cellular senescence and airway remodeling were examined in lung specimens from patients with asthma using immunohischemical analysis. Both small molecule and shRNA approaches that target the senescent signaling pathways were used to explore the role of cellular senescence in TSLP-induced airway remodeling invitro. In addition, a mouse asthma model indicates that inhibiting cellular senescence blocks airway remodeling and relieves airway resistance.Activation of cellular senescence as evidenced by checkpoint activation and cell cycle arrest was detected in airway epithelia samples from patients with asthma. Furthermore, TSLP-induced cellular senescence was required for airway remodeling TSLP stimulation can induce cellular senescence during airway remodeling in asthma. Inhibiting the signaling pathways of cellular senescence overcomes TSLP-induced airway remodeling. Asthma is characterized by chronic inflammation and structural alterations in the airways  and airw Airway epithelia produce some inflammatory factors that play critical roles in regulating metabolic and immunologic responses within airways. These inflammatory factors are often involved in epithelial damage in patients with asthma . TSLP, a Senescent cells are frequently found at sites of chronic disease. Many older patients have histological and symptomatic features of chronic obstructive pulmonary disease (COPD) and asthma ,17. Alvein vitro and in vivo. We found that 1) senescent signaling pathways are activated in airway epithelium in patients with asthma, 2) TSLP stimulation induced cellular senescence and silencing of cellular senescence pathways inhibits TSLP-induced airway remodeling in vitro and 3) Stat3-targeted therapies overcome TSLP-induced airway remodeling by inhibiting cellular senescence in vitro and in vivo. To determine whether inhibiting cytokine-induced cellular senescence can overcome airway remodeling in asthma, we explored the role of senescence in TSLP-induced airway remodeling in asthma The study was approved by the Ethics Review Committee for Human Studies at Qilu Hospital, Shandong University. Each subject provided written, informed consent. Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee of Shandong University.The protocol was approved by the Committee on the Ethics of Animal Experiments of Shandong University. Each mouse was anesthetized with intraperitoneal ketamine (35 mg/kg). Cervical dislocation was used to euthanize mice. All efforts were made to minimize pain and discomfort. The study was approved by the Ethics Review Committee for Human Studies at Qilu Hospital, Shandong University. Each subject provided written, informed consent. Endobronchial biopsies from 10 asthma patients were obtained from Shandong Provincial Chest Hospital . Healthy control specimens were obtained from Qilu Hospital of Shandong University , and were from pneumoresections. Subject characteristics are shown in Immunohistochemical staining was performed as described . The immThe human bronchial epithelial cell line, BEAS-2B, was derived from human bronchial epithelia and was purchased from ATCC . TSLP was purchased from R&D Systems . WP1066 was purchased from Santa Cruz Bio-technology, Inc. . 2 and 1 mg/ml bromo-4-chloro-3-indolyl-β-D-galactoside). BEAS-2B cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 10 minutes at room temperature, washed in PBS, and incubated for 24 h at 37 °C with SA-β-Gal  in staining solution (40 mM sodium citrate (pH 6), 150 mM, NaCl, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgClBrdU Cell Proliferation Assay Kit was purchased from Chemicon International  and used following manufacturer’s protocol. These primary antibodies were used: anti-p16 , anti-p21, anti-TSLP , anti-α-SMA , anti-collagen I (ab34710) and α – tubulin . All these antibodies were purchased from Santa Cruz Bio-technology, Inc.  or Abcam Inc. . BEAS-2B cells plated in 96-well plates were treated with TSLP. Cell viability was assayed using the Vibrant® MTT Cell Proliferation Assay Kit (Molecular Probes) following the manufacturer’s protocol. TSLP Quantikine Elisa kit was purchased from R&D systems, and we carried out the protocol according to the manual instruction.BEAS-2B cells plated on microscope cover slides were treated for 24 h with 1.5 ng/ml TSLP. Cells to be stained with anti-Ki67 were fixed with 2% paraformaldehyde in PBS for 30 min at RT and permeabilized by Triton X-100 (0.2% in PBS with 1% BSA) for 15 min at RT. Samples were blocked using TBS/0.05% Tween-20 with 3% skim milk for 1 h at RT. The mouse anti- Ki67 antibody was diluted 1:200 in TBS/0.05% Tween-20 and applied to cover slides with incubation overnight at 4°C. After washing with TBS/0.05% Tween-20, the cover slides were incubated with secondary Rabbit F(ab')2 Anti-Goat IgG H&L (Alexa Fluor® 488) (ab169344) diluted 1:00 in TBS/0.05% Tween-20 for 2 h at RT and washed. DAPI was used to stain the nuclei.Short hairpin RNA duplexes that target p21and p16 were purchased from Open Biosystem . The specificity of these shRNAs have been evaluated previously . BALB/c mice were purchased from the Center of Experimental Animals of Shandong University School of Medicine and animal experiments were performed in accordance with the Institutional Animal Care and Use Committee of Shandong University.Mice  were either control, untreated, OVA -induced asthma model or WP1066-treated (n=10). Mice were sensitized with OVA as described previously . WP1066-Methacholine was used at 0, 3, 6, 9, 12 g/L and a dose-response curve was constructed. Measurements of AHR were made 1 min after each dose and 2 min between doses. Results are expressed as the maximum resistance after each dose minus baseline  resistance.t–test. Significance was defined for p<0.05.Statistical analysis was performed using the SPSS 12.0 software package. Data were analyzed using Student’s ARF/p53/p21 pathway and the p16INK4/CDK/pRb pathway [Cellular senescence is a state of irreversible growth arrest and is induced by a wide variety of conditions, including telomere shortening (replicative senescence) and telomere-independent signals (stress-induced senescence) ,30. Prev pathway . Several pathway –35. In a pathway . We examTo further explore the role of cellular senescence in airway remodeling of asthma, we tested whether TSLP, a critical cytokine in airway remodeling in asthma, induces senescence in human airway epithelial cells. Previous studies demonstrated that the neutralization of TSLP could inhibit airway remodeling in a murine model of allergic asthma induced by dust mites . In our in vitro  reduces gene expression of TGF-β1, EGFR, and p21 to influence airway epithelia and prevent airway remodeling in a asthma mouse model [Senescence is an important fail-safe mechanism often arising in response to telomere erosion and stress. Chronic inflammation is a major histological characteristic of asthma and plays a critical role in airway remodeling. Chronic inflammation also represents tissue stress. Therefore, it is not surprising that cellular senescence is induced in airway epithelia of asthma. Senescence arises through checkpoint activation and cell cycle arrest and is a barrier to cell proliferation and tumorgenesis . In factse model .in vivo and in vitro. TSLP is considered a pivotal cytokine linking innate and adaptive immune disorders –50. Envi Currently, TSLP-targeted therapies are being explored in asthma. One recent report demonstrates that neutralization of TSLP can inhibit airway remodeling in a murine model of allergic asthma induced by house dust mites . AntibodTargeted therapy has been widely explored and used in asthma. The use of biologics in asthma, includes the IgE-targeted antibody omalizumab, the IL‑5-targeted antibody mepolizumab, the IL -13-targeted antibody tralokinumab, the IL- 9-targeted monoclonal antibody MEDI-528 and TNFα-blocking antibodies such as infliximab, golimumab and soluble TNFα receptor fusion proteins . Here wePrevious reports have demonstrated the central role of Stat3 signaling in TSLP-regulated inflammation and airway remodeling in asthma . Specifiin vitro and in vivo (Small molecules that target Stat3 are well established . Several in vivo , 5 and t in vivo  . AHR is


Cilia, evolutionarily ancient structures that project from the cell surface into the extracellular environment, are critical sensors of external cues. In animals, they detect light, odors, soluble chemicals and mechanical forces, and defects in cilia cause a range of diseases. Vertebrate cilia respond to intercellular signaling molecules such as Hedgehog, a regulator of embryonic development. Despite its important roles in embryogenesis and disease, little is known about the evolutionary origins of ciliary signaling. Understanding how cilia evolved mechanisms to receive and transduce signals will help identify core ciliary functions fundamental to animal biology. To shed light on the evolution of ciliary signaling, I will characterize the signaling function of cilia in a close relative of animals, choanoflagellates. Choanoflagellates provide a simple biological system in which to elucidate mechanisms of ciliary signaling and identify signaling molecules that may be conserved in mammals. I will address two questions: (1) What signaling proteins are present in choanoflagellate cilia? And (2) what are the functions of choanoflagellate homologs of Hedgehog pathway components? I will isolate cilia from choanoflagellates to identify putative ciliary signaling proteins using mass spectrometry and carry out follow-up studies on candidate proteins prioritized based upon their homology to animal proteins. To understand the ancestry of a known ciliary signaling pathway, I will characterize choanoflagellate homologs of the Hedgehog pathway members Patched (receptor) and Hedgehog (ligand) using a heterologous expression system. This research will provide novel insights into the evolution of ciliary signaling and mechanisms of disease-associated signal transduction.


Uric acid (UA) may protect muscle function from oxidative damage due to reactive oxygen species through its powerful antioxidant capacity. However, several studies have demonstrated that hyperuricemia is closely related to systemic inflammation and has oxidant properties effects, both of which may increase the risk of muscle strength loss. The purpose of this study was to examine the association of serum UA concentration with grip strength and leg extension power in adult men.This study is a cross-sectional survey in which 630 Japanese male employees aged 30 years and older participated. Five hundred and eighty-six subjects participated in the measurement of grip strength, and 355 subjects participated in the measurement of leg extension power. Blood samples were obtained for serum UA analysis.P = 0.01). In addition, serum UA levels (quartiles) showed an inverted J-shaped curve with grip strength . The results in the leg extension power group were similar to those observed in the grip strength group.After adjustment for potential confounders, grip strength differed significantly between participants with and those without hyperuricemia (geometric mean and 95% confidence interval [CI]: 40.3 [39.2–41.3] kg vs. 41.9 [41.3–42.5] kg; This population-based cross-sectional study shows for the first time that hyperuricemia is associated with poor muscle strength. Moreover, the results indicate an inverted J-shaped association between serum UA quartiles and muscle strength. Sarcopenia is defined as a gradual decline in skeletal muscle mass with aging and is associated with poor muscle strength. IncreasNumerous antioxidants circulating in blood or present in tissues may help reduce oxidative stress; however, of these, uric acid (UA) has long been thought to be one of the most important compounds involved in ROS removal. Maxwell et al. have impIn contrast to its role as an antioxidant, high concentrations of UA have also been considered a trigger for a number of diseases. Hyperuricemia, usually defined as a UA concentration of >7.0 mg/dL in men and >6.0 mg/dL in women, is a riOn the basis of studies conducted during the last 3 decades, it is difficult to define UA as clearly beneficial,10,15 orIn light of these observations, we hypothesize that low or high serum UA concentration may be associated with poor skeletal muscle strength. Therefore, the aim of the present study was to investigate the association between serum UA and muscle strength, i.e., grip strength and leg extension power, in adult men.This cross-sectional analysis was performed as part of the Oroshisho Study, a study of lifestyle-related effects on illnesses and health status in Japanese adults. In this study, all of the 1263 participants were adult employees and received an annual health examination at the Sendai Oroshisho Center in Sendai, Japan, 2009. This study was carried out from August 3 to 7. In addition to the annual health examination, physical activities, daily nutrient intakes, and muscle strength were also assessed. Further details on the study have been provided elsewhere,19.The analysis in current study was limited to participants who agreed to provide written informed consent for their data to be analyzed . Subjects were excluded from the present study for the following reasons: 1) being female (n = 282); 2) having no UA data available (n = 136); 3) having a history of cardiovascular disease and renal failure, or using antihypertensive, lipid-lowering, or antidiabetic agents (n = 146) because of the concern that such diagnoses and agents may lead to changes in UA levels and/or muscular function and thus confound and obscure a true association; or 4) hBlood samples were drawn from the antecubital vein in the morning, after overnight fasting, while subjects were in the sitting position; blood was collected into siliconized vacuum glass tubes; and then the frozen serum was sent to Bio Medical Laboratories  for subsequent measurement of blood components. Serum UA levels were measured enzymatically by using a Pureauto S UA kit ; the lower limit of detection was 0.2 mg/dL. UA levels were then divided into quartiles based on the distribution: <5.4, 5.4–6.0, 6.1–6.8, and >6.8 mg/dL. Hyperuricemia was defined as a serum UA level of ≥7.0 mg/dL for men.Grip strength in kilograms was assessed using a handheld digital Smedley dynamometer  in the standing position. Participants were asked to hold the dynamometer in a comfortable position and were not allowed to see the figure displayed on the dial during the trial. Grip strength was measured 4 times, twice for each hand alternately, with a brief interval between trials. Participants were encouraged to exert their maximal grip, and the average value of the highest power of both hands was recorded as the grip strength.Leg extension power was determined with an isotonic apparatus . After warming up, the subjects sat back on the seat, placed both feet on the sliding foot plate with the knee angle adjusted to 90°, and then, the waist of each participant was firmly fastened with belts. Participants were urged to extend their feet with maximum effort. Five trials were measured at 15-s intervals, and the maximal value was recorded as the leg extension power (W/kg) for the analysis. The reliability and validity of the leg extension power measurement was described in detail elsewhere.2 (kg/m2). Blood pressure was measured twice on the right arm with an automatic device  while subjects were in the sitting position, with a 5-min interval between measurements. The mean of 2 measurements was used for the current study. Information on age, sex, smoking status , drinking frequency , education levels (<college or ≥ college), and occupation (desk-centric or not) were obtained using a questionnaire survey.Body mass index (BMI) was calculated as weight/heightThe estimation of physical activity was determined using the International Physical Activity Questionnaire. Total d-1·1.73 m-2) = 194 × serum creatinine − 1.094 × age − 0.287.Serum high-sensitivity C-reactive protein (hs-CRP) was measured with N-latex CRP-2 . The lower limit of detection was 0.02 mg/L, and the hs-CRP value was considered to be 0.01 mg/L when lower than the detection limit. Other serum parameters were analyzed by enzymatic methods, using appropriate kits for measuring the concentrations of creatinine , triglycerides, high-density lipoprotein cholesterol (HDL-C) , and fasting blood glucose . The estimated glomerular filtration rate (eGFR) in men was calculated from the serum creatinine level by using a new Japanese equation, in whicIn this study, continuous variables were summarized as geometric mean  and categorical variables were summarized as percentages. Except for leg extension power, all continuous variables were log-transformed because of their abnormal distribution and back-transformed for data presentation.P < 0.05 was used for 2-sided tests. All tests were performed using IBM SPSS Statistics 19.0 software .Analysis of covariance (ANCOVA) was conducted to examine age-adjusted participant characteristics according to serum UA levels. By ANCOVA analysis, differences in muscle strength between subjects with and those without hyperuricemia were analyzed after adjusting for potential confounders. The linear or quadratic trends between serum UA quartiles and muscle strength were analyzed by univariate  and multivariate analysis (ANCOVA). In multivariate analysis, adjustment for age and BMI was performed in model 1. Model 2 was adjusted for lifestyle-related factors, including smoking status, drinking frequency, education levels, occupation, physical activity, daily energy intakes, and protein intakes in addition to the variables in model 1. Model 3 was adjusted for variables in model 2 and health status–related factors such as metabolic syndrome, depressive symptoms, and eGFR . Finally, model 4 was additionally adjusted for the circulating inflammatory marker hs-CRP. A significance level of The population sample consisted of 586 men for grip strength analysis (mean age [SD]: 46.3 [9.3] years) and 355 men for leg extension power analysis (46.3 [9.5] years). In both populations, the mean (SD) serum UA value was 6.1 (1.2) mg/dL.P for linear trend <0.01, for all). In addition, the prevalence of metabolic syndrome was higher across the quartiles of serum UA (P for linear trend <0.01), whereas the proportion of nonsmokers, former smokers, never drinkers, and sometimes drinkers, and the eGFR demonstrated a negative association with serum UA levels . In contrast, the mean age; the proportion of those who had education levels ≥ college, self-reported physical activity ≥23 MET hours/week, and depressive symptoms; the proportion of desk workers; HDL-C; and fasting glucose did not differ significantly among the serum UA quartiles. In addition, none of the nutrients examined demonstrated any statistically significant associations with serum UA levels.The age-adjusted associations between serum UA and participant characteristics in the grip strength group are presented in Table P for linear trend = 0.01), whereas no association between the proportion of never drinkers and UA quartiles were observed.The results of the main characteristics of participants in the leg extension power group were approximately equal to that of the grip strength group, with the exception of HDL-C and the proportion of never drinkers Table . In the P = 0.01) .In our study, grip strength differed significantly between participants with and those without hyperuricemia  and leg extension power . The inverted J-shaped association between UA quartiles and grip strength  and leg extension power  were unchanged even after adjustment for potential confounding factors, including age, BMI, smoking status, drinking frequency, education levels, occupation, physical activity, daily energy and protein intakes, metabolic syndrome, depressive symptoms, eGFR, and hs-CRP in the multivariate model (Table P for linear trend = 0.05) (data not shown).Furthermore, we also investigated the association of serum UA quartiles with muscle strength by using univariate and multivariate analyses. In univariate analysis Figure , serum UOur population-based cross-sectional study has demonstrated that hyperuricemia is associated with poor muscle strength after adjustment for potential confounders in Japanese adult men aged 30–83 years. Furthermore, we have found a significant inverted J-shaped curve between serum UA quartiles and muscle strength.To the best of our knowledge, this study shows for the first time that muscle strength was much lower in persons with hyperuricemia than in those without hyperuricemia. This study supports previous findings of low relative skeletal muscle mass in persons with hyperuricemia, which reported that participants with serum UA levels >8 mg/dL had 2 times the risk of sarcopenia  compared with those with <6 mg/dL after adjustment for other covariates in a population of 40 years old and older. In addiOn the other hand, a most recent prospective cohort study was the first to report a linear association of circulating UA levels with grip strength or/and leg extension power in elderly persons during a 3-year follow-up period. Given iIn the current study, higher UA levels were associated with poor grip strength and leg extension power by the following possible mechanisms. First, elevated UA concentrations are related positively to systemic inflammation, particularly in the highest category of serum UA. Indeed,In addition, there is one hypothesis that may explain the lower muscle strength observed in the first UA quartile. A low serum UA level may reflect a decreased antioxidant capability, as UA was considered an important antioxidant in the blood-9. MoreoAlthough this study has revealed important insights into the association between serum UA levels and muscle strength, it does have limitations. First, the association between serum UA and muscle strength is temporarily due to the cross-sectional nature of this study, and thus, a prospective study is necessary to confirm the causality of this association further. Second, we did not perform direct measurement of the UA antioxidant capacity as well as the total antioxidant capacity in the collected samples. Third, because we could not ascertain the site of inflammation in the present study, systemic or chronic inflammation may be present independent of serum UA. Last, the analysis was carried out only in men, and thus, our results cannot be directly generalized to women.Our cross-sectional study has, for the first time, revealed a negative impact of hyperuricemia on grip strength and leg extension power among Japanese men, after adjustment for additional covariates. Furthermore, the current study demonstrated that the association between serum UA levels (quartiles) and muscle strength does not show a linear trend but an inverted J-shaped curve. Future research should focus on clarifying the causality in association between circulating UA levels and muscle strength.ANCOVA: Analysis of covariance; BDHQ: Brief self-administered diet history questionnaire; BMI: Body mass index; CI: Confidence interval; DNA: Deoxyribo nucleic acid; eGFR: Estimated glomerular filtration rate; HDL-C: High-density lipoprotein cholesterol; hs-CRP: High-sensitivity C-reactive protein; IL-6: Interleukin-6; MET: Metabolic equivalent; ROS: Reactive oxygen stress; SD: Standard deviation; UA: Uric acid.The authors declare that they have no competing interests.CH, KN, and RN: study concept and design; CH, KN, YK, LG, HM, YC, MC, AO, HG, and HT: acquisition of subjects and data; CH, KN, LG, HM, YC and RN: analysis and interpretation of data; CH: drafting of the manuscript; YK, MC, AO, HG, and HT: administrative support; CH, KN, and RN: study supervision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/258/prepub


Vibrio coralliilyticus shows specific chemotactic search pattern preference for oxic and anoxic conditions, with the newly identified 3-step flick search pattern dominating the patterns used in oxic conditions. We analyzed motile V. coralliilyticus cells for behavioral changes with varying oxygen concentrations to mimic the natural coral environment exhibited during light and dark conditions. Results showed that 3-step flicks were 1.4× (P = 0.006) more likely to occur in oxic conditions than anoxic conditions with mean values of 18 flicks  identified in oxic regions compared to 13  at anoxic areas. In contrast, run and reverse search patterns were more frequent in anoxic regions with a mean value of 15 , compared to a mean value of 10  at oxic regions. Straight swimming search patterns remained similar across oxic and anoxic regions with a mean value of 13 . V. coralliilyticus remained motile in oxic and anoxic conditions, however, the 3-step flick search pattern occurred in oxic conditions. This result provides an approach to further investigate the 3-step flick.The coral pathogen,  V. alginolyticus to access nutrient patches quicker than bacteria using the run and tumble strategy such as Escherichia coliBacteria use chemotaxis and motility search patterns to position themselves in chemical gradients, locate nutrient sources and initiate pathogenesis Vibrio species, including the coral pathogen V. coralliilyticus, are reliant on chemotaxis to invade and colonize host species. Abundant in the coral mucus Vibrios alter the microbial metabolism in corals V. coralliilyticus causes the bleaching and tissue lysis of corals at temperatures greater than 25°C, and is further implicated in the disease of other marine organisms including bivalves, during winter months when temperatures are lower et al.V. coralliilyticus is critical for infection, including the adhesion to the corals and chemotaxis towards coral mucus.Pathogenic and symbiotic V. coralliilyticus must move through the surrounding seawater, the coral mucus surface layer and into the coral tissue cells for establishment and growth. This infection route, indicative of most coral pathogens, may illustrate unique search pattern strategies to assist in moving through numerous environments of differing viscosity and nutrient complexity. Local marine environments and conditions are constantly changing due to turbulence et al.V. coralliilyticus in response to fluctuating oxygen levels. This will provide insight into how V. coralliilyticus behaves in fluctuating oxygen levels from the surrounding seawater, surface mucus layer or boundary layer and the coral tissue.During coral infection, V. coralliilyticus type strain, LMG20984 was used for all experiments. Pure cultures were stored at −80°C in 15% glycerol stock solutions. Strains grew on Thiosulphate citrate bile salts sucrose agar media (Sigma Aldrich) overnight at 28°C. Liquid cultures were prepared in 30 mL of Luria-Bertani broth (Sigma Aldrich), incubated 16 hrs overnight at 28°C with 160 rpm shaking and harvested during exponential growth, diluted with LB to 1/10 to adjust cell density to OD 0.1±0.01, and visualized within 1 minute.The single, polar flagellated V. coralliilyticus cells were observed under dark-field, 200× magnification video-microscopy  with the field of view at mid-depth in the microscope-slide chamber. The video image was recorded continuously for 10 seconds at 2.4 frames s−1 using a time-lapse module (Carl Zeiss AxioVision 4). Search strategies of V. coralliilyticus were recorded and plotted across spatial gradients. Cell swimming paths of V. coralliilyticus were measured frame-by-frame and traced off the computer screen using overhead transparencies. Cell paths were defined as in Barbara and Mitchell A volume of 50 µL of cell suspension was added to an observation chamber constructed from two glass coverslips on a glass slide and covered with a third coverslip . The disA volume of 50 µL of 1/10 dilution of overnight culture was added to the created observation chamber. Triplicate transects were collected ranging from the oxic to the anoxic center of the observation chamber. Each transect consisted of 40 square eyepiece quadrats of 172 by 172 µm placed next to each other. The transect line spanned from the oxic to anoxic regions across 6,880 µm. At each quadrat a 10 second, 49-frame video was recorded. All distinguishable cells in each eyepiece quadrant video were tracked and analyzed. Cells were considered distinguishable if they remained in focus for at least 5 frames. All distinguishable cell trajectories were classified into one of three search patterns: run and reverse, straight swimming or 3-step flick . Each trV. coralliilyticus was being viewed in oxic or anoxic conditions all videos were recorded well before the lower value in this time window.Cell cultures and the headspaces were bubbled with either pure air or nitrogen for 10 minutes, and grown in conditions as mentioned above. Methylene blue and resazurin indicators (Sigma-Aldrich) were used to confirm whether a solution was saturated throughout the experiment V. coralliilyticus over time. Slides were prepared as stated above. New observation chambers were prepared for each oxygen condition of the timed experiment, which was run in triplicate , D is the diffusion coefficient (m2/s), R is the reaction rate for the species (mol/(m3s)) and in this case is used to signify flux, u is the vector velocity (m/s). Low Reynolds number, incompressible flows are characterized byNumerical simulations of oxygen diffusion in the presence of bacteria were produced using the MatLab-derived COMSOL Multiphysics with the shown geometries using transport of dilute species with convection and diffusion models . Previou−7 m.All cell trajectories were classified into search patterns and the results were analyzed using SPSS Statistics (version 19.0). Independent sample t-tests were utilized to calculate P values to establish whether flick or non-flick search patterns differ in total numbers or speed across the two observation chamber locations. T-tests were also used to determine any statistical differences in search pattern behavior during the time series.V. coralliilyticus search paths were identified and classified into three search strategies, straight swimming, run and reverse and the 3-step flick  = 2.0, n = 53), 21 µm s−1  and 21 µm s−1  respectively, were observed , 20 µm s−1  and 18 µm s−1  respectively, were observed , from a mean value of 8 flicks  in oxic environments to a mean value of 0.5 flicks  in anoxic environments. Run and reverse search patterns decreased 5-fold (P = 0.048), from a mean value of 5 run and reverse search patterns  in oxic conditions through to 1 run and reverse  in anoxic conditions. Straight swimming search pattern numbers increased from a mean value of 5.7 straight swimming search patterns  in oxic conditions to a mean value of 9 straight swimming search patterns  in anoxic conditions  to 21.9 µm s−1  respectively, although a large variation in search pattern velocity was identified . Velocities did not differ significantly between the three types of search patterns identified, namely the flick, run and reverse and straight swimming (Search pattern velocities were shown to decrease 2-fold from oxic to anoxic environments (P = 0.002), from 63.2 µm sentified . Search swimming .V. coralliilyticus were observed in cultures grown under both aerobic and anaerobic conditions. Immediate visualization of these cell cultures showed that in aerobically grown cultures, 38 flicks, 13 run and reverses and 9 straight swimming patterns with mean speeds of 45 µm s−1 , 45 µm s−1  and 32 µm s−1  respectively, were observed , 48 µm s−1  and 46 µm s−1  respectively , whilst non-flick patterns increased by 8  over the 25-minute experiment, though this result was not significant (P = 0.230) . StraighV. coralliilyticus is capable of utilizing chemotactic search patterns in oxic and anoxic conditions whilst maintaining search pattern velocities across oxic to anoxic conditions. This indicates that regardless of oxygen concentrations V. coralliilyticus are capable of remaining motile.Oxygen concentrations can differ between the surrounding water layer and the various microbiomes present in coral organisms, namely the surface mucus layer and coral tissue V. coralliilyticus uses the newly discovered cyclic flick search pattern, first found in V. alginolyticus, which is morphologically distinct from the run and reverse or run and tumble patterns adopted by E. coliV. coralliilyticus may be the result of saturated oxic conditions, similar to that seen in Salmonella typhimurium and Escherichia coliOur results indicate that V. coralliilyticus in oxic and anoxic conditions may not be a response to the oxygen concentrations directly, or lack thereof. Instead, as suggested by Armitage et al.V. coralliilyticus infection, where V. coralliilyticus cells remain motile in both oxic and anoxic conditions. With V. coralliilyticus’s motility fundamentally linked to infection and establishment in corals Changes in search pattern behavior of Figure S1Vibrio coralliilyticus cells. The cells tracked can be identified by the open circles, and the specific search pattern trajectories used have been overlaid. The red and yellow lines are flicks, the orange and white lines are straight swimming, and the blue line is a run and reverse. Examples of the 90° flicking events are marked by an X.A sample video recording of (MOV)Click here for additional data file.Table S1V. coralliilyticus search pattern preference from oxic to anoxic conditions in Experimental number of recordings (n) for (DOCX)Click here for additional data file.Table S2V. coralliilyticus search pattern changes over time in Experimental number of recordings (n) for (DOCX)Click here for additional data file.


Scaffolding proteins add a new layer of complexity to the dynamics of cell signaling. Above their basic function to bring several components of a signaling pathway together, recent experimental research has found that scaffolds influence signaling in a much more complex way: scaffolds can exert some catalytic function, influence signaling by allosteric mechanisms, are feedback-regulated, localize signaling activity to distinct regions of the cell or increase pathway fidelity. Here we review experimental and theoretical approaches that address the function of two MAPK scaffolds, Ste5, a scaffold of the yeast mating pathway and KSR1/2, a scaffold of the classical mammalian MAPK signaling pathway. For the yeast scaffold Ste5, detailed mechanistic models have been valuable for the understanding of its function. For scaffolds in mammalian signaling, however, models have been rather generic and sketchy. For example, these models predicted narrow optimal scaffold concentrations, but when revisiting these models by assuming typical concentrations, rather a range of scaffold levels optimally supports signaling. Thus, more realistic models are needed to understand the role of scaffolds in mammalian signal transduction, which opens a big opportunity for systems biology. A diverse range of receptors initiates this pathway, which consists of a phosphorylation-dependent relay of protein activation, resulting in altered transcription, ultimately regulating processes such as cell proliferation and differentiation. The details of this signaling pathway are very well studied and detailed maps of the function of specific protein kinases and protein phosphatases within the pathway have been produced  is given by the ratio of the concentration of the complex SX and the total scaffold concentration ST. Because we assume that binding occurs independently, the probability that all three ligands are bound at the same time is simply given by the product of the single binding probabilities as shown in Figure Mathematical analysis so far has highlighted that there is a clear optimum at which scaffolds enhance signaling, and even small deviations from this optimum will lead to attenuated signaling  helps to assemble ERK1/2 dimers that are essential for interaction of ERK with cytoplasmic substrates  are the Xenopus oocyte maturation in response to progesterone , evaluation of the response in different ways (integrated response or response at a certain time after stimulation) and evaluation of the response at the single cell or population level. Ultrasensitivity at the single-cell response profile is potentially lost when averaging over a population of cells. For example, the response of JNK to progesterone in Xenopus oocytes is ultrasensitive at the single cell level. However, as the critical concentration for a switch to occur varies greatly between single oocytes, the JNK response of a population of cells appears to be graded (Bagowski and Ferrell, While more and more mechanistic details about the action of scaffolds emerge, the role for scaffolds on the dynamics of MAPK signaling is currently not well-established for mammalian cells. Although many recent studies show how KSR1 controls spatial routing of signals and facilitates signal initiation, we still lack a comprehensive understanding of the role of the scaffold. Detailed mechanistic mathematical modeling and quantitative experimentation has been useful in the past by shedding light on how signaling works dynamically. For the yeast scaffold Ste5, mathematical models to explore the action of the scaffold have come to some maturity and provide detailed understanding of the role of this scaffold in this pathway. Similarly, we think that it would be timely to quantitatively study the role of KSR1 on signaling dynamics in mammalian cells, with detailed quantitative experiments that estimate the concentrations of scaffolds and signaling molecules. While the rather abstract models of scaffolds in mammalian MAPK signaling were helpful to conceptualize how scaffolds act, they also led to misleading results: The commonly accepted notion of the presence of an optimal scaffold concentration has led to the conclusion that scaffolded signaling might be more sensitive to perturbations in the concentration of pathway components Ferrell, . HoweverWorking toward realistic models for scaffolds is challenging due to combinatorial explosion: a comprehensive model of signaling via Ste5 which involves all 27 possible scaffold configurations both in cytoplasm and when located to the membrane exemplifies this complexity (Shao et al., Differential expression of scaffolds in different cell types may be one of the main reasons why many cell-type-specific interactions and feedbacks exist. Hence, once we know more about how scaffolds shape signal transduction, we will most likely also understand how and why MAPK signaling gains different roles in different tissues. Again, one can learn from yeast, where the scaffold protein Ste5 has been modified using a synthetic biology approach to recruit molecules that are either positive or negative regulators. These regulators are either induced by the pathway or expressed constitutively, either alone or in combination, which affects the shape of the pathway response in a predictable manner. This study highlights not only a chance for potential therapeutic results by targeted engineering of a pathway but also documents the various functions a scaffold might bear within the cell (Bashor et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.