This highlights the possible role of phosphoproteins in the temporal aspects of striatal signal transduction.In reward learning, the integration of NMDA-dependent calcium and dopamine by striatal projection neurons leads to potentiation of corticostriatal synapses through CaMKII/PP1 signaling. In order to elicit the CaMKII/PP1-dependent response, the calcium and dopamine inputs should arrive in temporal proximity and must follow a specific  order. However, little is known about the cellular mechanism which enforces these temporal constraints on the signal integration. In this computational study, we propose that these temporal requirements emerge as a result of the coordinated signaling via two striatal phosphoproteins, DARPP-32 and ARPP-21. Specifically, DARPP-32-mediated signaling could implement an input-interval dependent gating function, via transient PP1 inhibition, thus enforcing the requirement for temporal proximity. Furthermore, ARPP-21 signaling could impose the additional input-order requirement of calcium and dopamine, due to its Ca A response towards an environmental stimulus could be reinforced if it elicits a reward. On the subcellular level, the environmental stimulus and the reward signal lead to a transient increase in striatal calcium- and dopamine-signaling, respectively. The integration of calcium and dopamine signals, which is important for reward-learning, could elicit a downstream response only if they are close in time and arrive in correct order . This study proposes that the requirement for the input signals to be temporally close and in correct order could emerge due to the coordinated signaling via two striatal phosphoproteins, DARPP-32 and ARPP-21. The DARPP-32 signaling implements an input-interval dependent gating function and ARPP-21 implements an input-order dependent threshold-like function. Thus, a molecular mechanism has been presented here which could explain the emergence of important temporal aspects of subcellular signal integration in reward-learning. Reinforcement learning plays an important role in building the learned-behavioral repertoire of an organism. It operates by updating the salience of an environmental cue or a cue-response association which has elicited a reward in the past. Basal ganglia are critical for reward learning and the input nucleus, striatum, is the locus of integration for the environmental and the reinforcement signals . The env2+-Calmodulin-dependent kinase II (CaMKII) and Protein-Phosphatase 1 (PP1) signaling plays an important role in this process [The postsynaptic integration of NMDAR-mediated calcium and dopamine-dependent D1R signaling leads to the potentiation of corticostriatal synapses on D1R-expressing MSNs, thus resulting in reward-learning –9. Ca2+- process ,11. HoweDopamine and cAMP-regulated Phosphoprotein 32kDa) could play an important role in the dopamine-dependent gating of the calcium signaling, which aligns with previous experimental observations [AMP-Regulated Phosphoprotein 21kDa), has the potential to introduce the input-order dependency into the integration process. ARPP-21, upon dopamine-dependent phosphorylation, has the ability to bind with Ca2+/calmodulin, thus affecting the calcium signaling [In this computational study, we investigated the integration of calcium and dopamine signals by D1R-expressing MSNs to explain the emergence of the aforementioned temporal constraints, using quantitative kinetic modeling. Our results suggest that DARPP-32  with a maximum timestep of 0.01s. This model assumes a well-mixed approximation in a single compartment. Thus, no spatial or molecular heterogeneity has been taken into account.If a reaction is an irreversible reaction then the backward rate constant, kThe topology of the signaling network is built using biochemical reactions which have experimentally known interpretations for the signaling pathways. While it is possible that there are additional reactions and pathways in the actual system, we selected a subset which is consistent with the known striatal literature and pertinent to the currently-studied behavior. In cases where biochemical details were incomplete, we assumed simple mass action reactions that could account for the known molecular dependencies. A detailed description of the signaling network is provided below. After fixing the topology, the free variables left in the system are the reaction kinetic parameters and starting amount for different proteins. The parameters have been constrained using various types of neuronal and, in many cases, MSN-specific data. A list of target phenotypes used to constrain the model is also described below. The model SBML file could be downloaded from BioModels database (Model Id: MODEL1603270000) [It is known that the integration of transient calcium and dopamine signals leads to synaptic potentiation of the corticostriatal synapses and this process is mediated by CaMKII activity ,9,17. Ca2+/calmodulin. The transition of calmodulin to Ca2+/calmodulin is modeled as a sequential two-step reaction. Each step results in the binding of calcium to one of the calcium-binding domains in calmodulin. The parameters for each of the steps are factored assuming a double binding-site Adair-Klotz’s model fitted to the known calcium calmodulin binding data [The sequence of calcium-triggered signaling events modeled in this study is shown in ing data . At eaching data .2+/calmodulin target is CaMKII. Under the basal condition, the inactive CaMKII is known to be enriched in the F-actin rich cytoplasmic compartment [In our model, an important Capartment . On the partment . In our partment . These o2+/calmodulin [2+/calmodulin [The activation of CaMKII leads to its autophosphorylation thereby further increasing its affinity to Calmodulin . The autlmodulin . We modelmodulin . Additiolmodulin , which ilmodulin ,26,27. TThe active CaMKII leads to the phosphorylation of its substrates and many of these substrates are dephosphorylated by PP1 ,11. To colf G-proteins [olf further predominantly activates Adenylyl Cyclase type V (AC5) which leads to an increased level of cAMP [The sequence of dopamine-triggered signaling events modeled in this study is shown in proteins ,30. The  of cAMP ,31. The  of cAMP –37. Thusolf-dependent AC5 activation. The parameters for the cAMP degradation by phosphodiesterases (PDEs) have also been refined in the current version of the model to match the known PDE kinetics observed in live cells [Under the basal condition the cAMP levels are expected to be low in the D1R expressing MSNs due to the low basal receptor activity. The basal cAMP level estimated for different types of neurons with low AC activity is around 30–90 nM ,39. In sve cells . It is ave cells . The eleve cells . Similarve cells . Furtherve cells . These MPKA activation in MSNs leads to the phosphorylation of DARPP-32 at its Thr-34 residue and this turns DARPP-32 into a potent inhibitor of protein phosphatase PP1 . DARPP-3As described in the “Results” sections, our results indicate that the DARPP-32-mediated dopamine signaling alone is insufficient to explain the emergence of certain temporal constraints of the calcium-dopamine integration  dopamine and (2) calcium. The dopamine input represents increased extracellular dopamine concentration due to the burst activity of dopaminergic neurons in response to an unexpected reward . Such a As described above, the integration of striatal calcium and dopamine inputs leads to the phosphorylation of downstream CaMKII/PP1 substrates in MSNs. This, however, requires that the inputs should fulfill certain temporal requirements . SpecifiTo investigate the integration of dopamine and calcium inputs by the subcellular signaling processes in the D1R expressing MSNs, we developed a kinetic model of the standard striatal signaling network, We first looked at how dopamine could affect the calcium triggered CaMKII signaling. To this end, we measured the difference between the CaMKII/PP1 substrate phosphorylation produced by a transient calcium input , where tcalcium and tdopamine are the start time of calcium and dopamine transients, respectively. A small absolute value of Δt indicates that the calcium and dopamine inputs are close in time. A positive value of Δt means that the calcium input is followed by the dopamine input and vice-versa. This relation between the substrate response and Δt could shed light on whether the input-interval and input-order constraints exist in this signaling network. If the substrate response is higher for smaller absolute values of Δt compared to large Δt values then this suggests the existence of an input-interval constraint. In other words, calcium and dopamine should be close in time for effective downstream activation. Similarly, if substrate response is higher for positive values of Δt compared to negative values then this indicates the existence of input-order constraint, i.e. the calcium input should be followed by the dopamine input for effective downstream response.Using the aforementioned signaling network, we then investigated how the substrate phosphorylation depends on the temporal relation between calcium and dopamine inputs. The interval between the inputs is referred as Δt, such that Δt =  and Ser-55 mutant of ARPP-21 (A21S55A) shows that loss-of-function mutation in ARPP-21 abolishes the input-order interval by significantly affecting the response in the negative Δt region, As shown above, the addition of ARPP-21 into the signaling network could introduce the input-order constraint by significantly reducing the downstream response for the negative Δt regime, We looked at how active CaMKII and PP1 levels are affected for different Δt values to understand the relation between substrate phosphorylation and Δt for this updated signaling network, 2+/calmodulin [2+/calmodulin sequestration by ARPP-21 thus reducing the CaMKII activation. To ascertain this possibility we first considered the case of Δt = -2s as an example, 2+/calmodulin. A significant part of the Ca2+/calmodulin binds to the pARPP-21 produced by the preceding dopamine, 2+/calmodulin available for CaMKII activation, 2+/calmodulin leads to a lower CaMKII activation. Thus, the sequestration of Ca2+/calmodulin by pARPP-21, thereby reducing its availability, indeed explains the low CaMKII activation in the negative Δt regime.We then investigated how the ARPP-21 could reduce the CaMKII activation in the negative Δt regime, i.e. only when dopamine input precedes the calcium input. It is known that dopamine leads to the phosphorylation of Ser-55 residue on ARPP-21 and this phosphorylation turns ARPP-21 into an excellent binding partner of Calmodulin . This co2+/calmodulin by pARPP-21 is also reduced, 2+/calmodulin available for CaMKII activation, 2+/calmodulin by ARPP-21 is negligible, 2+/calmodulin available for CaMKII activation is not reduced, As the interval between dopamine and calcium decreases in the negative Δt region, e.g. Δt = -1s, the sequestration of CaAs highlighted in the previous section, the dopamine-dependent phosphorylation of ARPP-21 results in the dampening of a subsequent calcium-triggered CaMKII activation. In a two-trial scenario, this could mean that the ARPP-21 which is phosphorylated in the first trial may affect the CaMKII activation in the second trial. To test this cross-talk between successive trials we considered the calcium-dopamine integration in a two-trial scenario. The two trials are separated in time by an inter-trial interval (ITI). Each of the trials is represented by a pair of calcium and dopamine inputs with Δt = 1s.2+/calmodulin-dependent downstream response in an ITI dependent fashion.We first tested a scenario with an ITI = 10s. In this scenario, the substrate response produced by the first trial was similar to the response observed in a single trial case (as in the previous sections), ITI = t is the area under the curve for substrate phosphorylation in the second trial which is separated by the first trial with an ITI = t seconds. ITI = 100 is the activation area for second trial with an ITI = 100s. The trials with ITI = 100s are considered as well separated trials because with this ITI the second trial response is not affected by the first trial. Thus, if REF = 1, then the response in the second trial is fully eliminated due to the inhibitory effect of the first trial and if REF = 0, then there is no effect of the first trial on the response of the second trial. On the other hand, a negative value for REF indicates a potentiating effect of the first trial on the second trial.We define a metric called refractoriness (REF) to quantify the ITI dependent effect of the first trial on the response of the second trial. REF is defined as follows:The simulations suggest that the REF decreases as a function of ITI, i.e. REF is higher for lower ITI values and it decreases as ITI increases It could be possible that in a more complex scenario  the downstream response may integrate over the trials thereby overcoming the refractoriness imposed by pARPP-21. To test this possibility, we implemented a 10 trial scenario with ITI = 10s. Similar to the two trial scenario, each trial consists of 10 calcium pulses at 10 Hz followed by a dopamine input with Δt = 1s. The simulation for this 10 trial scenario suggests that there appears to be a small build of substrate response over the trials, Amplitude of the striatal dopamine represents the reward prediction error and it could vary depending on the difference between the expected and the acquired reward . In a siThe quantitative model built in this study is quite detailed and contains several parameters. Many of the parameters which might be important are not directly measured for the system of interest. They are rather optimized to fit experimental observations/phenotypes which emerge from the interactions of multiple parameters. However, it is important that the biological phenomena and predictions in the study should be robust with respect to changes in parameters around the selected point in the parameter space. Here, the parameters of interest are the kinetic rate constants of individual reactions and the starting amount of various conserved species. As mentioned above, the model output appears to be quite robust with respect to different parameter perturbation, The top sensitive-parameters list for the positive Δt region includes several DARPP-32 related parameters, In the case of negative Δt, the top sensitive parameters include the total amount of ARPP-21 and the kinetic parameter for ARPP-21 phosphorylation by PKA, 2+/calmodulin. It is also interesting to note that the total amount of calmodulin appears to be a sensitive parameter for both positive and negative Δt region. This could be because calmodulin has several interactions with the DARPP-32, ARPP-21 and CaMKII mediated signaling. The combined effects of calmodulin on the DARPP-32 and CaMKII significantly affect the gating potential of DARPP-32 signaling. Moreover, the core ARPP-21 mediated signaling relies on the calmodulin sequestration/competition thus adding further to the sensitivity of the model/predictions towards calmodulin.Additionally, the total amounts of CaMKII and PP2B also appear in the list of sensitive parameters in this case. This becomes clear by considering that both CaMKII and PP2B are also the downstream targets of calmodulin similar to ARPP-21 and changes in their total amounts could affect the level of binding between phospho-ARPP-21 and CaThe subcellular integration of the NMDA-dependent calcium and dopamine by D1R-expressing MSNs is believed to be an important biological process for reward learning. In this kinetic modeling study, we propose a novel mechanism which could explain the emergence of the observed input-interval and input-order constraints on the integration of calcium and dopamine. These temporal constraints could emerge as a result of the coordinated signaling through two striatally enriched phosphoproteins, namely DARPP-32 and ARPP-21, In our simulations, dopamine acts as a gate for the calcium-triggered CaMKII/PP1 signaling and this aligns with known observations ,9,17. ThThe transient nature of both CaMKII activation and PP1 inhibition limits the possible time-interval (Δt) between the respective calcium and dopamine inputs, which could be synergistically integrated. Moreover, the PP1 inhibition kinetics should be fast enough to match the temporal characteristics of CaMKII activation. In other words, the PP1 inhibition should occur before the transient CaMKII activation fades away if these two were to interact. This requires particularly fast kinetics for the cAMP/PKA signaling axis which is already known to be the case in striatal neurons . Recent 2+/calmodulin, thus reducing the available Ca2+/calmodulin for CaMKII activation. Similar to the DARPP-32 mediated phenomenon, this ARPP-21 mediated effect also demands fast phosphorylation kinetics.Even though the transient DARPP-32-dependent effect could implement the observed input-interval constraint, according to our results, DARPP-32 signaling alone may not distinguish between the order of calcium and dopamine incidence. Moreover, the molecular identity of the substance which could make the striatal CaMKII/PP1 signaling sensitive to the temporal order of calcium and dopamine is not clear. However, as presented above, ARPP-21 has the potential to implement an input-order dependent filtering mechanism by acting as a competitor to CaMKII. This could enable the striatal signaling machinery to distinguish between the incidence order of calcium and dopamine. When ARPP-21 is phosphorylated by a preceding dopamine it turns into a potent sequestering agent for subsequent calcium-triggered Ca2+/calmodulin. This competition stipulates that the amount of calmodulin be lower than the collective amount of its possible binding partners, i.e. Ca2+/calmodulin is a limiting resource. If Ca2+/calmodulin is not a limited resource then pARPP-21 and CaMKII do not have to compete with each other, thereby reducing the inhibitory effect of ARPP-21 on CaMKII activation. This in turn will eliminate the input-order sensitivity of the calcium-dopamine integration. Even though the absolute amount of calmodulin could be high in many cell types, various studies suggest that free calmodulin is indeed a limiting resource [2+/calmodulin seems to exist also in MSNs [As aforementioned, the ARPP-21 mediated mechanism rests on the idea that ARPP-21, upon phosphorylation, competes with CaMKII for the available Caresource –69. The resource . Moreove in MSNs  thus sup2+/calmodulin is also responsible for the activation of PP2B which dephosphorylates DARPP-32 Thr34. However, unlike CaMKII, pARPP-21 does not have any significant effect on the level of PP2B activation, for transient input signals, in our signaling model because the affinity of Ca2+/calmodulin towards PP2B is assumed to be very high . Thus, the current study does not predict any significant ARPP-21 mediated effect on DARPP-32 dephosphorylation within the assumed parameter space for transient input signals. However, this does not rule out such a regulation. Rather this could be due to the insufficiency of data to tune this aspect of the current signaling model.In addition to CaMKII, Car range; ) compareEven though the role of striatal ARPP-21 is not clear, it seems to be involved in addiction and motivational behavior ,72. The Apart from imposing the input-order constraint on the calcium-dopamine integration, our simulations also suggest that ARPP-21 could impose an inter-trial refractoriness as a function of the ITI in a multi-trial conditioning scenario. This could imply that an MSN, or its dendritic segment, which is potentiated in the recent past would resist a further potentiation within a refractory period. This could mean that multiple trials with shorter ITIs may not be completely independent to each other. As the ITI increases the trials start to become decoupled. Such, an inverse relation between conditioned response and ITI is well known for classical conditioning ,74. Alth2+/calmodulin stimulated Adenylyl cyclase, which exhibits calcium-dependent priming on the G-protein activation [2+/calmodulin stimulated Adenylyl cyclase, Adenylyl cyclase type I (AC1), has also been suggested to be involved in the emergence of striatal input-interval and input-order constraints [s (Golf) G-protein and Ca2+/calmodulin and is known to act as a coincidence detector for these two signals [olf and does not display any calcium-dependent synergy [This study highlights the possible role of phosphoproteins in controlling the temporal aspects of striatal signal transduction. Furthermore, we also attempt to illustrate the idea that different aspects of a single phenomenon  could be distributed among parallel signaling modules (DARPP-32 and ARPP-21) and do not necessarily have to be implemented by a single signaling node. One such single-signaling node which is believed to be responsible for similar temporal constraints in other systems, like Aplysia serotonin response, is Cativation ,76. A sistraints . AC1 is  signals . Even th signals ,79. Duri signals ,79,80. A synergy . It coulOn the other hand, the DARPP-32/ARPP-12 dependent mechanism proposed here is quite generalizable to various systems consisting of different AC isoforms. Even though we focused on the potential role of DARPP-32 and ARPP-21, they might just be part of a much wider signaling mechanism to produce the temporal constraints on striatal calcium-dopamine integration. For example, the activation of other signaling processes, like PKA, have also been observed to display the input-interval and input-order constraints . Thus, fIt is interesting to note that there are brain regions other than the striatum, e.g. the amygdaloid complex and the bed nucleus of the stria terminalis (BNST), where the co-expression of DARPP-32 and ARPP-21 is known to exist ,85–87. BS1 Text(PDF)Click here for additional data file.


The expression of microRNAs (miRNAs) is primarily regulated during their transcription. However, the transcriptional regulation of miRNA genes has not been studied extensively owing to the lack of sufficient information about the promoters and transcription start sites of most miRNAs.DROSHA knockout cells. DROSHA knockout resulted in increased accumulation of pri-miRNAs and facilitated the precise mapping of their 5′ end nucleotides using the rapid amplification of cDNA ends (RACE) technique. By analyzing the promoter region encompassing the transcription start sites of miRNAs, we found that the unrelated miRNAs in their sequences have many common elements in their promoters for binding the same transcription factors. Moreover, by analyzing intronic miRNAs, we also obtained comprehensive evidence that miRNA-harboring introns are spliced more slowly than other introns.In this study, we identified the transcription start sites of human primary miRNAs (pri-miRNAs) using The precisely mapped transcription start sites of pri-miRNAs, and the list of transcription factors for pri-miRNAs regulation, will be valuable resources for future studies to understand the regulatory network of miRNAs.The online version of this article (doi:10.1186/s12864-016-3252-7) contains supplementary material, which is available to authorized users. Most of the biological pathways in cells are influenced by microRNAs (miRNAs) . TherefoThe transcription of miRNA genes produces primary miRNAs (pri-miRNAs), which are used as a substrate of the DROSHA-DGCR8 protein complex. Through the action of DROSHA, the pri-miRNAs are cleaved into hairpin-like precursor miRNAs (pre-miRNAs). The pre-miRNAs serve as the substrate for DICER, which is the nuclease that produces mature miRNAs. Interestingly, the genomic location of about one-half of pri-miRNAs overlaps with other transcription units , 5; moreCompared to that of intronic miRNAs, the transcriptional regulation of intergenic miRNAs is not easy to understand. The primary reason for this difficulty is that the gene structure of intergenic miRNAs is not well established. In general, the genomic elements near the intergenic miRNAs, other than the location of pre-miRNA sequences, are largely unknown. Therefore, it is hard to determine the gene structure of intergenic miRNAs and the analysis of their promoter elements is not feasible.To understand the elements in the miRNA promoters, transcription start sites of miRNAs need to be identified. Although several studies have analyzed the transcription start sites of miRNAs and the promoter elements of intergenic miRNAs, these analyses were limited due to the low resolution of the experiments –17. MostTo identify the transcription start sites of miRNAs, RNA-seq data, with higher signal near the 5′ end of pri-miRNAs, is essential. Due to the continuous cleavage of pri-miRNAs by DROSHA and the rapid degradation of the remaining fragments after processing, the number of RNA fragments containing the information of the 5′ end of pri-miRNAs remains low in the cells. This makes the identification of the gene structure of pri-miRNAs difficult Fig. .Fig. 1RNDROSHA knockout cells to analyze the gene structure of pri-miRNAs. By analyzing the data from RNA-seq from DROSHA knockout cells and confirming it through rapid amplification of cDNA ends (RACE), we mapped the 5′ end of pri-miRNAs precisely. In addition, we analyzed the promoter regions of miRNA genes and identified the transcription factors that may regulate pri-miRNA expression. Our data will be very useful for future analysis of the transcriptional regulation of miRNAs.In this study, we used human DROSHA knockout human colorectal cancer cell lines that we established recently [DROSHA knockout cells showed increased accumulation  to more than 200 kilobases (kbs). For example, the transcription of miR-200c, which is a member of the pri-miR-200c ~ 141 cluster (tilde indicates transcript spanning the indicated gene cluster), is initiated 472 base pairs (bps) upstream of the hairpin sequence. The genomic distances between the transcription start sites and pre-miRNA hairpins for 16 out of 29 pri-miRNAs are less than 10 kbs Fig. . In someWe analyzed the distribution of RNA polymerase II (Pol II) and TATA-binding protein (TBP) signals near the transcription start sites of pri-miRNAs using the public data from the Encyclopedia of DNA Elements (ENCODE) project . As a coAlthough the regulatory relationship between miRNAs and their target mRNAs has been studied extensively, the transcriptional regulation of miRNAs themselves by transcription factors is not well understood. To analyze the transcriptional regulation of miRNAs, we utilized the data generated by the ENCODE project, which contains the chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for 161 transcription factors . To idenWe found the binding sites of diverse transcription factors to be clustered in the promoter region of pri-miRNAs that we selected Figs.  and 3a. Upon comparing the binding fractions of transcription factors between the pri-miRNA and RefSeq mRNA promoters, we found that several transcription factors were significantly enriched in pri-mRNA promoters Fig. . Among tIt is plausible that a similar set of transcription factors would bind to the promoters of miRNAs that need to be regulated together. To identify such pairs of miRNAs, which might be regulated by common transcription factors, we calculated the correlation value between transcription factor binding to promoters of both the pri-miRNAs of each pair; these correlation values differed considerably among pri-miRNA pairs Fig. . The corDROSHA ablation  to hinder the processing of pri-miRNAs, thereby increasing the amount of pri-miRNAs in the cells [DROSHA knockout cells can enrich the transcribed regions of pri-miRNAs more strongly because no DROSHA activity exists in the cells.Several studies have investigated the structure of miRNA genes and analyzed miRNA promoters. One of the recent studies used sequencing data from NA genes . In thishe cells . The trehe cells . Based ohe cells , the useDROSHA knockout cells that we used in this study offer several advantages for the identification of pri-miRNA gene structure because the processing of canonical pri-miRNAs is completely abolished in this cell line. By using the DROSHA knockout human cells, we could obtain more in-depth information about the transcribed regions of human pri-miRNAs with more reliability. Importantly, we confirmed the RNA-seq data through 5′ RACE, which enabled us to identify the transcription start sites of pri-miRNAs at the nucleotide level. This study is the first one to confirm the transcription start sites of many intergenic miRNAs using biochemical assays.The The analysis of the promoters of intergenic miRNAs shows that there are several transcription factors that prefer to bind to the promoters of miRNAs over those of RefSeq mRNAs Fig. . AlthougDROSHA knockout cells reported in this study will be a valuable resource for future studies. For example, this data can be used to identify transcriptions start sites of other pri-miRNAs not included in this study. In addition, this data will be helpful to understand the transcript structure of pri-miRNAs, such as the analysis of splicing patterns or transcription termination sites.The RNA-seq data of DROSHA knockout cells and confirming it through RACE experiments. We also identified transcription factors for pri-miRNAs regulation. These data will be valuable resources for future studies to understand the regulatory network of miRNAs.From this study, we identified the transcription start sites of pri-miRNAs precisely by analyzing the RNA-seq data from DROSHA knockout cell line was established, as described previously [The colorectal cancer cell line, HCT116 (Korean Cell Line Bank), was maintained in McCoy’s 5A medium supplemented with 10 % fetal bovine serum (WelGENE). The eviously .DROSHA knockout HCT116 cells. To enrich pri-miRNAs, which contain poly(A) tails, oligo(dT) Dynabeads (Life Technologies) was used. The sequencing library was made using TruSeq Stranded Total RNA Library Prep Kit (Illumina), and the quality of the library was checked using Agilent 2100 Bioanalyzer (Agilent). Finally, the library was sequenced on HiSeq 2500 (Illumina) for 50 sequencing cycles. FASTQ sequences obtained from the sequencer were trimmed to remove nucleotides with lower quality values. And then, the sequences were aligned into the human genome using Bowtie2 algorithm with default parameters [TRIzol reagent (Life Technologies) was used to extract total RNA from wild-type or rameters . Among tfastx_trimmer -Q 33 -f 2 -l 37 -i seq.fastq | bowtie2 -x ./hg19 -q - | grep "^@\|XM:i:0" - | samtools view -q 10 -Sb - > aligned.bamDROSHA knockout cells were compared. For the selection of pri-miRNA candidates of RACE analysis, we examined the signals between predicted transcription start sites of pri-miRNAs and 5′ end of pre-miRNAs. We selected the pri-miRNAs whose peaks in bedGraph are higher than 10. In addition, the pri-miRNAs whose peaks in the DROSHA knockout library are more than 50 % higher than those in the wild-type library were selected.After converting the results into bedGraph files, the graphs representing RNA signals of wild-type and The generation of cDNA for 5′ RACE was carried out according to the manufacturer’s protocol . From the analysis of transcribed region based on the RNA-seq data, we designed primers to amplify the 5′ end region of pri-miRNAs. The sequences of primers are included in the Additional file We assumed the promoter of miRNAs to be the region from 2000 nts upstream to 500 nts downstream from the transcription start site of pri-miRNAs. To analyze the binding of transcription factors to promoters of intergenic miRNAs, we downloaded the genomic coordinates of the ChIP-Seq data of transcription factors generated by the ENCODE project . By comp0.5Correlation value = (Number of transcription factors common for both miR-A and miR-B) / ((Number of transcription factors for miR-A) X (Number of transcription factors for miR-B))http://genome.ucsc.edu/). For each intron or exon, the number of sequencing reads from wild-type and DROSHA knockout cells were counted. If the read number for an intron or an exon was too low (<10), that region was excluded from further analysis. After this filtering, the fold changes in read numbers (DROSHA knockout/wild-type) for each region was calculated.The genomic coordinates of introns and exons of each host gene having intronic miRNAs were obtained from The UCSC Genome Browser (


LEAFY-like gene has been demonstrated to play a key role in the flowering process regulation of angiosperms. Expression analysis of EjLFY-1, a LEAFY (LFY) homolog of loquat (Eriobotrya japonica Lindl.), indicated its participation in the regulation of flowering in loquat. To verify its function and potential value in the genetic engineering to shorten the juvenile phase, ectopic expression of EjLFY-1 in strawberry (Fragaria × ananassa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the loquat EjLFY-1 gene driven by the CaMV 35S promoter. Totally 59 plantlets were verified to be the transformants. The presence, expression and integration of EjLFY-1 in the transformants were assessed by PCR, quantitative real-time PCR and Southern blot, respectively. Constitutive expression of EjLFY-1 in strawberry accelerated the flowering process in strawberry with the shorten necessary period for flowering induction, development of flower and fruit set. While vegetative growth habits of the transformants in the first cropping season were consistent with the WT ones. Meanwhile, both the flowers and fruits of the transformants were also as same as those of the WT ones. Furthermore, the early-flowering habit was maintained in their asexual progeny, the runner plants. While with continuous asexual propagation, the clones showed a more strengthen early-flowering phenotype, such as the reduced vegetative growth and the abnormal floral organs in individual plantlets. These results demonstrated the function of this gene and at the same time provided us new insights into the utilization potential of such genes in the genetic engineering of perennial fruits.As a master regulator involved in flower development,  Flowering is one of the most important events in the life cycle of plants. Flowering process of plant is affected by both endogenous factors, such as hereditary characters or plant hormones levels, and environmental conditions, such as temperature or day length. Flowering at right time accommodating to the seasonal and endogenous signals is vital for the reproductive success of all plants. The flowering time determination in plants is controlled by complicated gene networks.Arabidopsis provides us a better understanding about it. Numerous genes involved in the flowering regulation were demonstrated. In Arabidopsis, for example, it has been reported that at least 180 genes are implicated in flowering-time control (FLOWERING LOCUS T (FT), which then activate other floral homeotic genes. For plant, although the organ where developmental decision leading to flowering occurs is shoot apex, leaf is the organ which sensing environmental signals. In Arabidopsis, the mobile signal FT has been demonstrated to be the long-distance signal moved from leaf to shoot apex and then induce the flowering .In the past decades, a great deal of progress has been accomplished in understanding the molecular basis of the regulatory mechanisms underlying flowering time in plants, especially studies in the molecular regulatory networks of  control , regardl control . Such silowering . Expressactor FD . At SAM LEAFY and its homologs in other plants encode a kind of unique transcription factors that assign the floral fate of meristems, being thought as a meristem identity gene which determines floral identity  is an evergreen fruit tree native to China and cultivated mainly in tropical and subtropical regions. Fruits of loquat can be consumed fresh or processed for jam, juice, wine, syrup, or as candied fruits .Most gene function analysis in fruits were carried out with ectopic expression in model plants, such as  species . In addiWithout the gene’s recombination and segregation, propagation with stolon buds in strawberry makes it feasible to sufficiently investigate the transgenic effects of the exogenous genes in their asexual progeny, which is especially important for fruit trees. In production, fruit species must be propagated in asexual ways, such as grafting or layering, to maintain their characters stable. The stability of the transgenic effect in the asexual propagated progeny is crucial for the application of genetic engineering in fruit trees.EjLFY-1 by investigating the ectopic expression effects of this gene on the flowering process in strawberry, and at the same time to investigate the stability of the effects of such transgene in the successive asexual propagated generations of the transformants.The main objective of the this study was to evaluate the function of Table 1), were designed based on the reported EjLFY-1 sequence (GenBank accession no. AB162033). Restriction enzyme sites were added additionally at the 5′-end and 3′-end of the primers for the subsequent vector construction. Flower bud and leaf of loquat cv. ‘Zaozhong No.6’ were used for the extraction of RNA and DNA, respectively. Full-length cDNA and DNA sequence of EjLFY-1 were obtained by PCR. PCR products were cloned into pGEM-T easy vector  and then sequenced.Gene-specific forward and reverse primers, EjLFYfwd and EjLFYrev  were collected with 10-day intervals. 10 μg RNA from each sample was electrophoresed in 1.2% agarose gel and transferred to Hybond N+ nylon membranes . A gene-specific fragment (498 bp) amplified by a pair of primers  at the conserved region of its 3′ end was used as the probe for hybridization. The specific methods of probe labeling and hybridization are as described in the instruction offered by Roche DIG High Prime DNA labeling and Detection Starter Kit I .To determine the involvement of EjLFY-1 was subcloned into Xba I-Sac I sites of the binary vector pBI121 plasmid so that EjLFY-1 was under the control of CaMV 35S promoter . Recombinant plasmid pBI121-35S::EjLFY-1 was then introduced into the Agrobacterium tumefaciens strain EHA105.Full-length cDNA sequence of Fragaria × ananassa) cv. Tudla was used for Agrotransfection. A. tumefaciens-mediated transformation was performed with the leaf-disk procedure  containing kanamycin (30 mg L-1). Regenerated buds were excised and transferred to 1/2MS medium containing kanamycin (50 mg L-1) for rooting. Resistant plants from separate leaf explants were defined as independent transgenic events.Strawberry  which specially belongs to Agrobacterium was also checked and excluded by PCR. Primers (AGRIF and AGRIR) and amplification methods are listed in Table 1.Genomic DNA were extracted from young leaves of kanamycin-resistant plants and the WT plants following a modified CTAB method , and theEjLFY-1, Southern blotting was performed using a randomly selected plantlet which had been confirmed by PCR. About 20 μg of genomic DNA was digested with EcoR I and then electrophoresed on a 0.8% (W/V) agarose gel. The digested DNA in the gel was then transferred to an Immobilon-Ny+ membrane . A fragment of NPT II gene obtained by PCR  was labeled as the probe using the DIG DNA Labeling and Detection Kit II . Hybridization was carried out according to the manufacturer’s instructions.For further verification of the integration of Agrobacterium strain containing recombined plasmid was used as a control for detection of the existence of ChvA gene.Non-transgenic strawberry (WT) was used as the control, except that the EjLFY-1 in the first generation transgenic plantlet was detected by RT-PCR. Plantlets grown in greenhouse were used to extract the total RNA using an improved CTAB method as described by 18 primer using Superscript II reverse transcriptase , according to the protocols provided by the manufacturers. Primers EjLFYfwd and EjLFYrev were used for the RT-PCR confirmation and 2 μl of the cDNA product was used as the template.Expression of EjLFY-1 in the third-generation propagated clones originated from stolon buds of the transformants. Leaf was used as the material for the detection of EjLFY-1, shoot apex meristem was used to examine the expression level of FaAP1, a homolog of APETALA1 (AP1) in strawberry. Expression level of FaTFL1, homolog of the main floral repressor TERMINAL FLOWER 1 (TFL1) in strawberry, was also detected. Gene-specific primers used in qRT-PCR were also listed in Table 1. RNA extraction and cDNA synthesis were carried out following the method mentioned above. The qRT-PCR was performed using SYBR Green qPCR Kit  following the manufacturer’s instructions. qPCR was repeated with three biological replicates, and each sample was assayed in triplicate by PCR. The 26S rRNA gene of strawberry was used as a internal control.Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of Independent transgenic lines and WT plants were rooted, transferred into pots and grown in a solar greenhouse in Shenyang Agricultural University  at December of 2009. The agamic propagated plants from stolon buds of the transgenic plantlets were rooted and planted into new nutritional pots, and then separated from the donor plants.At the end of each cropping seasons, old leaves and roots of the plantlets were partially removed and the plantlets were then transferred into a new nutritional pots separately.The plantlets were successively flowering after growing in the greenhouse for 3–4 months and their horticultural traits were recorded individually. Flower timing of each sample was measured as the time period from transferring into pots to formation of the first flower. Plant height, flower numbers were also recorded.The amino acid sequences were aligned and a phylogenetic tree was generated using the Clustal W method . Boot stEjLFY-1 sequence which reported by EjLFY-1 and other LFY-like genes showed that they share the conserved domains with each other in the N-terminal and C-terminal, especially in the C-terminal conserve motif .With a pair of gene-specific primers, a segment of 1,227 bp was isolated and sequenced. Sequence alignment showed that it is similar with the A phylogenetic tree was constructed by using the deduced protein of EjLFY-1 and the other reported LFY-like proteins with Neighbor-Joining Method  was 2,602 bp in length, indicating the existing of intron(s). Alignment of gEjLFY-1 with its cDNA sequence showed the existing of two introns. The first intron is 464 bp and the second one is 911 bp in length. The amount of introns in gEjLFY-1 is also same as those reported LFY-like genes of other Rosaseae plants such as strawberry, dog rose and apple , except in the earliest detected vegetative bud . Transcription level of EjLFY-1 was increased gradually during the swelling period of the buds when the inflorescence meristems were initiated . Then the transcription level reached the topmost point when the inflorescence meristems started to generate flower meristems . Once the flowers came into being, expression of EjLFY-1 decreased . No transcripts were detected in little flowers or blooming flowers .Northern blotting data suggested that the transcription of EjLFY-1 CDS under the control of CaMV 35S promoter was introduced into strawberry cv. Tudla. Totally 59 resistant plantlets belonging to three independent lines  were got and rooted on MS medium containing kanamycin. The resistant plantlets and the WT ones were then transferred into pots and grown in greenhouse . While for the detection of ChvA gene, target fragment couldn’t be detected in any tested transformants , indicating the detected fragments of EjLFY-1 or NPT II were caused by transformation event, excluded the possibility of Agrobacterium continuation.To verify the genomic integration of the target gene, presence of NPT II gene. Southern hybridization with the NPT II probe showed that two bands were visible in L2-2 while none was observed in the WT control . This result provides a further evidence of the EjLFY-1 insertion in strawberry genotypes.L2-2, a random selected PCR-positive transgenic plantlet, was further tested by using Southern blotting detection for the integration of EjLFY-1 in independent transgenic line was detected by RT-PCR . Results showed that transcripts of EjLFY-1 could be detected in the tested transgenic lines , but not in WT ones.Expression level of EjLFY-1 on the regulation of flowering process in strawberry, transgenic plantlets belonging to three independent lines  and the WT lines grown in small pots filled with nutrient soil were transferred into experimental greenhouse at December of 2009. It was found that plantlets belonging to L1 and L2 lines developed their inflorescences early and continuously at March of 2010, while the WT plants were still in vegetative growth at the meantime . The early flowering habit of the transformants was very remarkable. Even at the April of 2010, when more than half plantlets of the L1 and L2 came into bloom, the WT ones didn’t develop inflorescences yet. Over-expression of EjLFY-1 promoted flowering process of the plants belongs to L1 and L2, their average flowering time were 23 and 41 days shorter than that of the WT, respectively . While for plants of line L3, the change of flowering formation period was negligible in compare with WT.To evaluate the effect of over-expression of EjLFY-1 affected not only the flowering time of strawberry but also the flower quantities. Compared with WT plants, the transformants produced more flowers including the line L3 ones in which flowering time showed no significantly changes . Even though, most flowers or fruits of the transformants showed no obviously changes compared with the WT ones, except that individual transgenic plantlets showing doubled sepals .Other horticultural traits of the transformants were also tested at 2010. Over-expression of EjLFY-1 promoted the flowering process of the transgenic lines, it didn’t reduce the vegetative growth habit of those transformants at the first growing season. For example, three transgenic lines even showed a little higher average height phenotype than the WT ones at each investigated point except the beginning .Although over-expression of Almost all the fruit species in commercial production must be propagated by asexual ways so as to keep their stable traits. Normally the nursery stock of strawberry is runner plant which comes from stolon bud of the donor plant.Figure 8A). And unlike the almost unchanged vegetative growth habits of the transformants in the first cropping season, runner clones showed an obviously reduction in vegetative growth. For example, plant showed a dwarf phenotype and their leaf were also extremely smaller than those of the WT ones .Early-flowering habit was also maintained in the runner plants originated from L1 and L2. And with the continuous asexual propagation, the clones showed a more strong early-flowering phenotype: the third-generation runner clones came into flowering at a very young stage . In addition, variation of flower organs was also found in individual plantlets of the progeny, such as the multiplicated sepals  in one runner plantlet originated from L1.Flowering of the runner clones were not only early but also continuously. Some runner plantlets of the transformants even could develop into flower in the hot summer, when the WT ones were still in their vegetative growth phase and only produced stolons . As the downstream target gene of LFY homologs, expression of FaAP1, the AP1 homolog in strawberry, was also detected. Results showed that although the EjLFY-1 expression were higher in all the transgenic runner plants than that of the WT ones (in which were zero), expression of FaAP1 in those plants differed: its expression in OXRP-1 (Over-expression runner plant 1) was higher than not only the WT ones but also the OXRP-2 and OXRP-3 . The varied expression levels were also detected in FaTFL1, the main repressor in flowering process . Generally, the FaTFL1 expression in WT was lower than those in all the three tested transgenic runner plants.qRT-PCR detection showed that expression of Although the crop improvement of loquat is being carried out both by breeding programs and selection accessions from germplasm resources, the varieties grown mostly come from selections . One reaLEAFY-like gene exists not only in angiosperms but also in plant species which don’t produce flowers , suggesting its possible flowering regulator role in the flowering process of loquat. flowers . As a ki flowers . One pos flowers . Two cony itself , and they itself . Such chEjLFY-1 started to increase its expression level in the buds following the transition from vegetative bud to inflorescence bud, decreased its expression level before the forming of flowers and eventually vanished after flowering . Comparing with expression of EjAP1, the AP1 homolog of loquat, EjLFY-1 started and ended its expression earlier . Unlike the reduced vegetative development phenotype in the other reported transgenic plant species over-expressing LFY-like genes, strawberry over-expressing EjLFY-1 didn’t show obvious changes in the vegetative development habit at the first cropping season. Meanwhile the flowers and fruits of the transformants were almost as same as the wild ones . Such results suggest the potential value of EjLFY-1 in genetic engineering for the fast breeding of loquat. For plantlets belonging to line 3, although the integration and expression were also verified by PCR, they didn’t show the similar early-flowering phenotype as the other two transformants lines. One possible reason for that is the insertion site effect of the transgene. However, it requires further examination.To verify the function of The long juvenile phase of fruit trees limits their genetic improvement and prevents full domestication of them . One of EjLFY-1 could be maintained. Interestingly, with the continuous asexual propagation, the transgenic clones showed even a more consolidated early-flowering trait compared with their ancestor transformants of the first cropping season, such as the reduced vegetative growth habit and even abnormal floral organs . Reduction of the vegetative growth traits in those clones maybe caused by the continuous shorten of the vegetative growth, such as the earlier break of dormancy caused by accelerated flowering process. Such finding provides us new insights into the utilization potential of genetic engineering in the breeding of fruits.In this study, the observation results about the transgenic asexual clones lasting for three cropping seasons showed that the early-flowering habit induced by over-expression of EjLFY-1 may result in an excessive expression of the downstream floral organ related genes in strawberry, such as the AP1 homolog which may also control the outer two whorls of floral organs in strawberry just like what AP1 does in Arabidopsis  suggested that the ectopic expression of EjLFY-1 still exists in those runner clones, following a varied FaAP1 expression changes compared with the WT ones. TFL1 is a floral repressor which also binds to FD and suppresses the expression of floral promoters LEAFY and AP1  1 and 2 , except the plant OXRP-3 in which the FaAP1 expression was not at a high level although the EjLFY-1 was also detected.The existing of abnormal floral organs in their clones suggests that continuously over-expression of bidopsis . Actuall and AP1 . With thLFY has been demonstrated to function in distantly related species (AFL1 or AFL2, the LFY homologs in apple, can induce the early-flowering in Arabidopsis (AtLFY in apple didn’t show the same results instead of a columnar phenotype with shorten internodes (LFY-like genes and their interacting factors among different species. In this study, we confirmed that the early-flowering habit existed in strawberry over-expressing EjLFY-1 and such habit could be maintained in their clones. For the closely evolutional relationship between strawberry and loquat, both belongs to Rosaceae family, it is promising that over-expression of EjLFY-1 in loquat could also accelerate its flowering process and break its juvenile stage.Although  species , this isbidopsis . In otheternodes . Similarternodes . PerhapsZZ and SL conceived and designed research. YL, QZ, NM, HS, and CL conducted experiments. GH and JW contributed plant materials. YL and QZ analyzed data. YL wrote the manuscript. All authors read and approved the manuscript.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.


This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in In vytometry  and fluoytometry , as wellytometry , 9. Fluoerformed . It has erformed  that the minutes . In this minutes , 15 is u In vitro visualizations of VWF dynamics have employed microfluidic devices and focused largely on wall-bound VWF i levels leading to the presence of Ca2+-bound Indo-1 inside platelets and/or to the presence of activated platelets showing increased [Ca2+]i but still not expressing P-Selectin and, therefore, not detectable by the flow cytometer. The elevated baseline of the S7 Indo-1 sample in the flow cytometry results (9.3%) is likely caused by the additional manipulation required for the double-labeling.A repeatability test, aimed at confirming that consistent readings from the flow cytometer correspond to consistent results from the imaging system, is conducted using six P-Selectin labeled samples (S1-P to S6-P). In addition, to demonstrate the viability of using Indo-1 loaded platelets for detecting activation, one sample is double-labeled with the anti-P-Selectin antibody and with Indo-1. The fluorescence from the FITC-conjugated anti-P-Selectin antibody is employed in the flow cytometry measurements, while, in the imaging system, activation is detected by recording the light emitted at 405 nm, the peak wavelength for Cad Indo-1 . The resTo demonstrate the ability of the proposed techniques to detect varying levels of activated platelets consistent with flow cytometry readings, the samples are stimulated with different thrombin concentrations, as detailed in Methods. Results presented in The imaging technique can also readily detect platelet aggregates, a sample of which is shown in μg/ml, and for samples S3 and S4 it is 5.0 μg/ml. The difference in concentration is a result of using two batches of rVWF-eGFP prepared at different times. After shearing, an expected loss of protein is observed. The concentration of samples S1 and S2 decreases to 2.0 μg/ml, while that of S3 and S4 decreases to 4.0 μg/ml. For all cases, application of shear markedly increases the number of detected multimers. The increase in sample S1, the only one which would allow mechanoenzymatic cleavage, is not significantly different from that of sample S2, where mechanoenzymatic cleavage is inhibited by the presence of EDTA. This trend suggests that this increase is not caused by cleavage. Indeed, no cleavage has been detected as indicated by the absence of proteolytic fragments in multimer analyses performed on the samples containing ADAMTS13 . Furthermore, the increase in the number of signals occurs also in samples S3 and S4 that do not contain ADAMTS13. A likely explanation for these observations involves conformational changes associated with breakup of noncovalent bonds linking VWF aggregates but not covalent bonds within the multimers. The absence of cleavage even in the presence of ADAMTS13 suggests that the shear exposure time is much shorter than that required for exposing the A2 domain and for ADAMTS13 to cleave the protein. In other studies [Four samples of rVWF-eGFP have been imaged before and after application of short bursts of shear stress to test the ability of the present techniques to detect changes in the number of rVWF-eGFP multimers as an indication of aggregation/disaggregation and/or cleavage. These tests have been performed in the presence/absence (±) of EDTA and/or ADAMTS13.  studies , exposurμm2), as well as medium (85 < area < 675 m2) and large aggregates (area > 675 μm2). The size range representing isolated VWF-coated platelets is determined from the initial images recorded in the PRP sample with ristocetin, where no aggregates are present. To confirm the absence of aggregates due to platelet agglutination, the size of the platelets' signals from the PRP sample with ristocetin is compared with the ones from the platelets labeled with P-Selectin. Both samples show the same size distribution; therefore confirming the absence of platelet agglutination. As expected, the number of isolated VWF-coated platelets after excitation is still several orders of magnitude higher than those of the aggregates. Yet, the presence of medium- and large-scale aggregates is evident. In another sample prepared and stimulated the same way, the formation of what appeared to be a fibrin mesh is observed. This claim is made since the images become hazy, and the fibrin mesh is clearly visible by visual inspection. The VWF-platelet aggregates still form in this sample, but they are slowed down by the fibrin mesh compared to those in the mesh-free solution, even when they are of similar size. A PRP sample containing VWF : IgG : FITC is employed to detect VWF-platelet aggregates. To visualize isolated VWF-coated platelets, the sample is imaged first after adding ristocetin. Subsequently, thrombin is added, and the sample is imaged after one vortexing step lasting 10 s at 1500 RPM . In both cases, 2000 images per dataset are recorded. The vortexing is aimed at providing enough mechanical excitation to promote, together with thrombin, platelet activation and aggregation. As shown in 2+-bound Indo-1. A series of tests show that the average number of activated platelets per image positively correlates with flow cytometry measurements. The results are repeatable, proving the reliability of the new techniques. Recombinant VWF-eGFP multimers in different structural conformations are readily detected and tracked as they move in suspension. Detecting variations in the number of free-moving multimers in solution is an effective means of quantifying rVWF-eGFP conformational changes. Although the present measurements are restricted to breakup of aggregates due to the short shear exposure times, the same procedure could likely detect changes to the number of rVWF-eGFP multimers due to cleavage. The short exposures are meant to mimic scales relevant to passage through valves, left ventricular assist devices, and so forth. The use of VWF : IgG : FITC for detecting platelet-decorated VWF strands as they move in the solution allows for the quantification of changes to their shape and size, including the number of platelet aggregates. Consequently, this technique can visualize the effects of pro- and anticoagulant processes induced by shear stresses and/or chemical agonists/antagonists on the number and motion of aggregates. The latter is demonstrated here by showing the impact of fibrin mesh formation on the movement of VWF-platelet aggregates. These results are consistent with prior studies performed in microfluidic chambers and stenosis replica [This paper introduces space and time resolved fluorescence imaging techniques for detecting in situ platelet activation, VWF structural conformational changes, and VWF-platelet aggregates moving in deep suspensions. These procedures expand currently available methodologies for observing coagulation-related processes and blood damage in static samples  and shal replica , 43, 44,Detection of the phenomena discussed in this paper in deep suspensions introduces several challenges, primarily the low intensity of the fluorescent signals, which results in intensities that are just few gray levels above the noise floor. The presence of partially out-of-focus objects and the spatially and temporally varying background add further complexity. Consequently, data analysis requires ad hoc image enhancement and tracking algorithms aimed at increasing the low SNR. Edge-detection-based procedures are also needed for identifying the most in-focus signal within a track and for calculating geometric determinants, such as size, aspect ratio, and shape.The present techniques can be applied in sample volumes with a half-depth falling within the working distance of the long-range microscope objectives employed.Due to the high blood flow velocities encountered, for example, in stenosis, prosthetic heart valves, and left ventricular assist devices, the ability to record events using very short exposure times (≤1 ms) is fundamental. Application of high-sensitivity EM-CCD imaging enables us to achieve this goal, although most of the data presented in this paper involve longer exposure times. As a demonstration, Finally, despite the fact that the presentation of the technique's capabilities has been focused on one cell and protein type, the introduced methods can be readily applied to other cell, protein, and flow types by appropriately choosing the postprocessing parameters in a way that best suits the particular application.Platelet activation, VWF conformational changes, and VWF-platelet strands moving in deep suspensions are visualized and quantified using high-sensitivity fluorescent imaging. Several image enhancement and tracking procedures are applied to overcome low SNR conditions. The number of detected activated platelets is positively validated against standard flow cytometry measurements. The VWF conformational changes are readily quantified based on the number of multimers. Future tests will involve (i) systematic measurements of the effect of exposure time and shear level on the platelet and VWF response, (ii) comparison of the size and number of VWF-platelet aggregates to measurement obtained by standard aggregation tests , and (iivon-Willebrand Factor (VWF) multimer analysis of the four rVWF-eGFP imaged samples. NPP: normal pooled plasma.


S)-norcoclaurine and its derivatives. Specifically, we will detail the current status of synthesis of BIAs in microbial hosts as well as using isolated and recombinant enzymes.Benzylisoquinoline alkaloids (BIAs) are among the most important plant secondary metabolites, in that they include a number of biologically active substances widely employed as pharmaceuticals. Isolation of BIAs from their natural sources is an expensive and time-consuming procedure as they accumulate in very low levels in plant. Moreover, total synthesis is challenging due to the presence of stereogenic centers. In view of these considerations, green and scalable methods for BIA synthesis using fully enzymatic approaches are getting more and more attention. The aim of this paper is to review fully enzymatic strategies for producing the benzylisoquinoline central precursor, ( Benzylisoquinoline alkaloids (BIAs) are a nitrogen-containing group of plant secondary metabolites with a centenary history of investigation . The intad hoc database of benzylisoquinolines compounds, called BIAdb, helpful for all those who are working in the field of drug discovery, to explore the pharmacological potential of BIAs   . The novstituent . Many biR)- and (S)-diastereoisomers of norcoclaurine 4′-O-β-d-glucoside were synthesized  as the asymmetric catalyst yielded the (S)-N-Cbz-6,7-di-O-benzylnorcoclaurine, which, after the attachment of a glucosyl moiety to the p-hydroxybenzyl unit and subsequent deprotection gave the (S)-norcoclaurine-4′-O-β-d-glucoside as trifluoroacetate (TFA) salt in 93% yield and 95% diastereomeric excess.Briefly, the Bischler-Napieralski cyclization/reduction sequence of a proper amide  by emploAlthough many elegant syntheses of BIAs have been proposed, the development of more efficient synthetic routes is still a great challenge to the chemists due to the intricate molecular architecture of these molecules. Usually, chemical synthesis of alkaloids is complex due to the presence of multiple stereogenic centres and requires multistep procedures, many of which involve the use of protective groups resulting in moderate to poor yields.As a matter of fact, one of the major limitations in the chemical synthesis of BIAs is the scale up of the process that, even if feasible, it still remains not economically convenient.Moreover, direct extraction of alkaloids from plants is often impractical, as they are often produced in very small amounts by their natural source. For all these reasons, in this last decade new synthetic processes have been developed joining the expertise of chemists with the new tools emerging in the field of biotechnology . These aThe growing interest in plant alkaloids and their use for therapeutic purposes has led to the development of different extraction methods. In fact, due to their complex chemical structures, when feasible BIAs are directly extracted from plants. However, very often these methods consist in expensive and time-consuming procedure and the isolation yields are generally too low. Moreover, the secondary metabolite patterns of plants grown at different times and places with different physical and chemical stimuli can vary greatly and this represents a major obstacle in using plants as a drug producing source. Hence, the production of plant derived drugs requires genetically uniformed monocultures grown in completely standardized conditions.S-adenosylmethionine (SAM)-dependent N- and O-methyltransferases, cytochromes P450, NADPH dependent dehydrogenases/reductases, FAD-dependent oxidoreductases, O-acetyltransferases, and O-demethylase ..79].Aldehyde species were generated in situ by transaminase (TAm) starting from dopamine that in its turn reacts with the newly synthesized aldehyde in the presence of NCS to give norlaudanosoline with good conversion and excellent enantioselectivity.Lathyrus cicera diamine oxidase (LCAO) that shows a broad substrate specificity towards several ethyl amines [T. flavum norcoclaurine synthase (TfNCS)  . In this2O2 produced by LCAO adding catalase. Before the second step, LCAO was inactivated with phenylhydrazine and then NCS and dopamine were added to the reaction pot. The newly synthesized BIAs were obtained in very good yields (>72%) and excellent enantioselectivity (ee > 99%).The first step was optimized removing HThe first example of a three-step enzymatic cascade for the stereoselective synthesis of 1,3,4-trisubstituted-THIQ was reported in 2017  Scheme .The first reaction step was catalyzed by carboligase (CL) that converted 3-hydroxybenzaldehyde and pyruvate in acyloin. In the second step, a transaminase (TAm) was used to form 2-amino-1-(3-hydroxyphenyl) propan-1-ol that in its turn reacted with phenylacethaldehyde in a NCS catalyzed Pictet-Spengler cyclization. The 1,3,4-trisubstituted-THIQ thus obtained was isolated excellent yield (92%).TfNCS variants with improved ketone tolerance that were able to accept non-aldehyde carbonyl substrates were identified [In the same year, entified . These eThe (chemo)-enzymatic methods here reported for the synthesis of natural and non-natural BIAs demonstrate the great potential of in vitro biocatalysis for the formation of complex chiral compounds. The main advantage of these domino processes is that they do not require intermediate isolation or functional group protection strategies. Moreover, all these reactions are performed in aqueous media and exhibit high atom economy.However, the critical step is represented by the lab scale production that needs to be improved at least to gram scale. This improvement can be in principle achieved by finely tuning the enzyme activity and/or by increasing its stability and recycling. In fact, many enzymes lack of long-term stability under the experimental conditions and it is also difficult to recover and recycle them in order to make the overall process economically and environmentally advantageous. Enzyme immobilization can be a useful tool for enhancing stability, reducing costs and modulating catalytic properties.


The optimal duration of firstline chemotherapy in metastatic esophagogastric cancer is unknown. In most clinical trials therapy was given until tumour progression or limiting toxicity. Maintenance concepts aiming to prolong the duration of response and maintain quality of life have been established in other tumour types but not in esophagogastric cancer. S-1 is an oral fluoropyrimidine with proven efficacy in metastatic esophagogastric cancer.The Maintenance Teysuno® (S-1) in esophagogastric cancer (MATEO) trial is a multinational, randomized phase II study that explores the role of S-1 maintenance therapy in Her-2 negative, advanced esophagogastric adenocarcinoma. After a 12-week firstline platinum-fluoropyrimidine-based chemotherapy patients without tumour progression are randomized in a 2:1 allocation to receive S-1 alone or continue with the same regimen as during the primary period. The primary endpoint is overall survival. Secondary endpoints include safety and toxicity, progression-free survival and quality of life.Correlative biomarker analyses focus on the identification of a subgroup of patients with a prolonged benefit from S-1 based maintenance therapy.MATEO will be the first trial to define the role of a S-1 based maintenance therapy in patients having received a platinum-based firstline chemotherapy.NCT02128243 (date of registration: 29–04-2014). Gastric cancer is the third most common cause of cancer related death worldwide. Every year around 725,000 people die due to gastric cancer . A largeIn the beginning of the twentieth century most gastric cancers were located in the distal parts of the stomach. While during the last decades the overall incidence of gastric cancer has declined, especially in Western countries, a strong increase in tumours located at the esophagogastric junction  occurred , 3.Due to a lack of early symptoms and no effective screening programs most gastric cancers and AEGs are diagnosed in irresectable advanced or metastatic stages in Western countries where even in localized stages long term survival is below 50% , 5. RelaIn patients with metastatic disease, palliative chemotherapy remains the mainstay of treatment. This therapy aims to prolong survival and to improve or maintain quality of life by ameliorating tumour-related symptoms.Superiority in terms of overall survival compared with best supportive care was shown in a meta-analysis published by Wagner and colleagues . CombinaTrastuzumab for Gastric Cancer (ToGa) trial [Until now only Her-2 overexpressing tumours, a subgroup of ~16% of esophagogastric cancers, are eligible for biologically targeted therapy in the firstline treatment of advanced disease. The combination of trastuzumab with cisplatin and a fluoropyrimidine has shown superiority in the phase III a) trial . Followia) trial , 9.For patients with Her-2 negative gastric cancers undergoing firstline treatment for metastatic disease no approved targeted agents are available, thus chemotherapy remains the treatment of choice.Platinum compounds combined with fluoropyrimidines are the mainstay of polychemotherapy for Her-2 negative esophagogastric cancer having shown superiority over older regimens containing methotrexate and etoposide , 11.For several years the ECF regimen, consisting of epirubicin, cisplatin and 5-FU (5-fluorouracil), has been considered to be a reference regimen in the metastatic setting. The REAL2 phase III trial has shown non-inferiority of oxaliplatin in comparison with cisplatin and capecitabine in comparison with 5-FU. In addition, overall survival was modestly longer in the EOX group  compared with the ECF group. Therefore, EOX is nowadays considered a standard regimen for metastatic esophagogastric cancer .Adding a taxane to cisplatin and 5-FU (DCF regimen) significantly improved the response rate, time-to-tumour-progression (TTP) and overall survival (OS) in the TAX 325 trial . AlthougThe optimal duration of firstline chemotherapy in advanced esophagogastric cancer is unknown. In most phase III trials chemotherapy was given until tumour progression or the occurrence of inacceptable toxicity .As an exception, in the REAL2 trial, chemotherapy was given for a maximum of 8 cycles, each with duration of 21 days . In the Dose reductions of chemotherapeutical components due to toxicity are not uncommon in patients receiving two- or three-drug combinations. For example, dose reductions were necessary in 35–42% of all patients in the REAL2 trial .So far no randomized trials have been performed to determine the optimal duration of firstline chemotherapy in metastatic esophagogastric cancer. In clinical routine, firstline chemotherapy is mostly given until tumour progression or inacceptable toxicity occurs.Prolonged periods of chemotherapy in responding patients often lead to increased cumulative toxicity. Therefore, concepts of therapy de-escalation and the idea of a maintenance therapy have been addressed in several tumour entities.The goal of a maintenance therapy is to consolidate the tumour regression that has been achieved during the first months of combination chemotherapy (primary period) and at the same time to avoid excessive toxicity which often occurs with prolonged polychemotherapy. Maintenance concepts have been established in lung cancer and in colorectal cancer , 21.In this trial S-1 was chosen for assessing the role of maintenance therapy in advanced esophagogastric cancer. S-1 is an oral fluoropyrimidine consisting of tegafur , gimeracil , and oteracil .S-1 administered in combination with cisplatin has been recently approved in Europe for the treatment of advanced gastric cancer based on the results of the S1301/FLAGS study . In thisThe primary objective of the MATEO trial is to assess the relative efficacy of S-1 maintenance therapy vs. continuation of polychemotherapy after a primary phase in patients with metastatic esophagogastric cancer in terms of overall survival.Secondary objectives include the comparison of S-1 maintenance therapy vs. continuation of polychemotherapy after primary therapy with respect to safety/toxicity, progression-free survival and quality of life. Translational analyses focus on identifying a molecularly defined subgroup of patients with a sustained benefit from S-1 based maintenance therapy.The MATEO trial is an open-label, multi-centre, controlled randomized, parallel-group phase II non-inferiority trial in patients with metastatic esophagogastric cancer. A randomized control group was incorporated into the design since reliance on historic data alone for assessing the relative efficacy of de-escalation with S-1 and continuation of polychemotherapy is not appropriate. 297 patients in six European countries are planned to be randomized.Patients with metastatic, Her-2 negative esophagogastric adenocarcinoma will be registered before or after the initiation of a firstline chemotherapy regimen. Patients who have previously been treated with curative surgery with or without perioperative (neoadjuvant and/or adjuvant treatment) are eligible if primary treatment for metastatic disease starts at least 6 months after the end of perioperative treatment. Details of the in- and exclusion criteria can be found in Table All patients start with a firstline treatment period of 12 weeks during which a polychemotherapy regimen can be chosen by the local investigator, the following regimens being eligible: cisplatin/capecitabine, cisplatin/5-FU, cisplatin/S-1, FLO/modified Folfox-6, EOX/EOF or FLOT .Treatment in the maintenance period will be continued until tumour progression or limiting toxicity.Quality of life (QoL) is assessed using the EORTC QLQ-C30 and STO22 questionnaires . BaselinThe nutritional state will be evaluated using the Nutritional Risk Screening (NRS) 2002 . EvaluatThe translational part of this trial aims to identify subgroups of patients with a sustained, beneficial response during S-1 maintenance therapy. Blood samples are taken at different time points during the study, polymorphisms of genes related to the fluoropyrimidine metabolism as well as microRNA profiles and cytokine profiles will be measured to identify predictive markers for S-1 maintenance therapy. Formalin-fixed paraffin-embedded tumour tissue will be used for molecular analyses, to correlate clinical efficacy with recently published molecular defined subtypes.The statistical goal of this randomized phase II trial is to formally assess whether de-escalation with S-1 is not substantially inferior to continuation of polychemotherapy in terms of overall survival. If S-1 can be demonstrated to be non-inferior based on the pre-specified non-inferiority margin and the selected significance level, de-escalation with S-1 can be considered to be promising for further evaluation and subsequent phase III studies, taking into consideration additional potential benefits like decreased toxicity, cost or ease of application. Statistically, the goal is to test whether the ratio of the hazard of death with S-1 divided by the hazard of death with continued polychemotherapy is less than the pre-specified non-inferiority margin.Selection of a non-inferiority margin is based upon a combination of clinical judgement and statistical reasoning. Literature data suggests that continuation of polychemotherapy will lead to a median overall survival of roughly 8 months  , 18. WitFurthermore, a power of 80% under the alternative hypothesis that both treatment arms are in fact associated with the same underlying hazard rates, i.e. HR = 1  was pre-specified.Based on these assumptions and assuming exponential distribution of survival times, 250 observed deaths are required for confirmatory statistical analysis. Since the power of the study is determined by the number of deaths rather than the number of patients, there are a range of sample sizes that could meet the objectives of this trial. Assuming that 10 patients can be randomized per month, with 297 patients (198 treated with S-1 and 99 treated with continued polychemotherapy), the total study duration  will be about 39 months.To account for the fact that merely roughly 75% of patients treated with primary chemotherapy will qualify for randomization including patients who were enrolled in the trial after completion of primary therapy, a total of 400 patients will be expected to be enrolled to the trial.0.Overall survival (OS) will be defined as the time length between randomization and the date of death from any cause or the date of last follow-up in case of no documentation of death. Kaplan-Meier methods stratified by treatment arm will be applied for estimating the probability of survival over time. For confirmatory analysis of non-inferiority of S-1 a Cox proportional hazards regression model for overall survival with treatment as the only factor and response to primary therapy and polychemotherapy as stratification variables will be fitted. Based on the resulting adjusted hazard ratio for the treatment effect, significant non-inferiority will be claimed if the upper limit of the associated one-sided 90% confidence limit does not exceed the non-inferiority margin θProgression-free survival (PFS) will be defined as the time length between the date of randomization and the date of first disease progression or death (whichever occurs first). Patients alive with no documented progression will be censored at the last documented visit. For statistical analysis, the distribution of progression-free survival time will be described using Kaplan-Meier methods. To adjust for stratification factors used at time of randomization, Cox proportional hazards models will be applied.When de-escalation with S-1 can be shown to be associated with non-inferior overall survival, assessment whether de-escalation improves QoL compared to continuation of chemotherapy in maintenance phase therapy is of special interest. Therefore, the following statistical approach –exploratory in nature- is foreseen:Quality of life will be evaluated using the validated EORTC QLQ-C30 questionnaire and the gastric module STO22.Cumulative distribution of absolute changes  will be presented. Furthermore, average scores taken over the first 9 weeks of maintenance treatment and taken over the whole maintenance phase will be calculated for global health status/QoL and all other functional and symptom scales of QoL-questionnaires.Non-parametric (exact) Wilcoxon-Mann-Whitney tests will be applied for treatment comparisons. To focus on clinically relevant individual changes in QoL measures only, responder analyses showing the rate of patients experiencing a specific change that is meaningful to patients are also pre-specified in the study protocol. To reduce statistical multiplicity issues, a subset of QoL-parameters of special interest is predefined in the study protocol.This trial is accomplished in conformity with the principals of the Declaration of Helsinki and the Guidelines for Good Clinical Practice in their current revision. The trial will be carried out in keeping with national and international legal and regulatory requirements.At the time of submitting this manuscript 104 patients have been initially registered, 56 patients have been randomized.The optimal duration of systemic polychemotherapy for metastatic esophagogastric cancer is unknown. In many clinical trials chemotherapy was given until tumour progression or limiting toxicity, whereas in some other trials treatment was stopped after a pre-defined period of time.No formal comparison of these two different strategies has been performed.Given the increasing toxicity rate with a prolonged application of systemic polychemotherapy, the patients’ quality of life could be negatively affected.During the past years maintenance and de-escalation strategies have been examined in different tumour types like colorectal or lung cancer. Maintenance strategies led to acceptable therapy-related toxicity on the one hand and prolonged the time-to-tumour-progression and overall survival on the other hand , 21. So Our study is designed to show non-inferiority of S-1 maintenance therapy in comparison to continuation of polychemotherapy. This comparison was chosen instead of an observational arm without treatment due to the fact that in clinical routine treatment of metastatic esophagogastric cancer is often performed until tumour progression or until limiting toxicity occurs.In accordance to other clinical trials exploring a platinum-based firstline therapy in metastatic gastric cancer, patients with previous curative surgery with or without perioperative systemic treatment are eligible, if the primary therapy for metastatic disease starts at least six months after the end of perioperative therapy, whereas the subgroup of patients with an early relapse (<6 months after the end of perioperative systemic treatment) is excluded, anticipating a platinum-refractory disease in these patients.Analysis of changes in the quality of life will be performed during the primary period and maintenance period to examine changes with the Qol in a de-escalated therapeutic setting.Translational analyses focus on the identification of a subgroup of patients with a sustained benefit of S-1 based maintenance therapy.This trial is conducted by the Young Medical Oncologists (YMO) Group in cooperation with the Gastric Cancer Working Group of the Arbeitsgemeinschaft Internistische Onkologie (AIO) within the German Cancer Society (Sponsor: AIO-Studien-gGmbH).


Allergic asthma is characterized by the presence of large numbers of eosinophils in the lungs and airways. Due to their capacity to rapidly release inflammatory mediators such as cytokines, chemokines, growth factors, and cytotoxic granule proteins upon stimulation, eosinophils play a critical role in pro-inflammatory processes in allergen-exposed lungs. Identifying key players and understanding the molecular mechanisms directing eosinophil trafficking and recruitment to inflamed airways is a key to developing therapeutic strategies to limit their influx. Recent studies have brought to light the important role of glycans and glycan binding proteins in regulating recruitment of eosinophils. In addition to the role of previously identified eosinophil- and endothelial-expressed adhesion molecules in mediating eosinophil trafficking and recruitment to the inflamed airways, studies have also indicated a role for galectins  in this process. Galectins are mammalian lectins expressed by various cell types including eosinophils. Intracellularly, they can regulate biological processes such as cell motility. Extracellularly, galectins interact with β-galactosides in cell surface-expressed glycans to regulate cellular responses like production of inflammatory mediators, cell adhesion, migration, and apoptosis. Eosinophils express galectins intracellularly or on the cell surface where they interact with cell surface glycoconjugate receptors. Depending on the type  and location , galectins differentially regulate eosinophil recruitment, activation, and apoptosis and thus exert a pro- or anti-inflammatory outcome. Here, we have reviewed information pertaining to galectins  that are expressed by eosinophils themselves and/or other cells that play a role in eosinophil recruitment and function in the context of allergic asthma and their potential use as disease biomarkers or therapeutic targets for immunomodulation.Eosinophils are differentiated granulocytes that are recruited from the bone marrow to sites of inflammation  Eosinophils are the predominant granulocytic leukocytes present in allergic airways, and eosinophilia is the hallmark of airway inflammation in asthma –3. Althox epitopes, such as CD34, to play a role in recruitment of eosinophils to allergic lungs in mice , CBP35, or Mac-2] was first identified in rat basophilic leukemia cells . This leth Bcl-2  as well th Bcl-2 . Studiesth Bcl-2 –58 incluth Bcl-2  and atopth Bcl-2 .via its CRD and showed co-localized expression with α4 on the cell surface of eosinophils from allergic subjects. In addition, eosinophil-expressed Gal-3 interacted with endothelial Gal-3. Self-association to homodimerize or form multivalent complexes is a characteristic feature of Gal-3 (via its CRD (based on the blockade of these interactions by lactose) after exiting the cell where it is then able to mediate eosinophil rolling and adhesion on VCAM-1 and Gal-3 as depicted in the schematic shown in Figure Acute allergen exposure was shown to result in increased recruitment of Gal-3-expressing inflammatory cells (macrophages and eosinophils) to the airways and elevated levels of soluble Gal-3 in the bronchoalveolar lavage fluid of wild-type mice . On the of Gal-3 . Since gin vitro  generates intermediate N-glycans that are further extended by the addition of N-acetyllactosamine units resulting in high affinity ligands for Gal-3  and that changes in glycosylation can affect these functions  and enhs review . Gal-1 bs review . Althougs review , it is uization) . Remodelization) . The inhization) . This isization)  and propThe use of Gal-1 as a potential therapeutic (immunosuppressive) agent in Th1- and Th17-mediated inflammatory responses has been investigated in various disease models –93. Whilin vitro and for murine eosinophils when administered intraperitoneally  galectin strongly expressed in human, but not mouse, eosinophils . This lein vivo .+ Tregs and is necessary for limiting cell proliferation and Treg-cell-mediated suppression of cocultured CD4+ T cells .Eosinophils play a critical role in mediating inflammatory processes in asthmatic lungs by virtue of their ability to release pro-inflammatory cytokines, chemokines, and growth factors that promote development of the hallmark features of asthma. The recruitment of primed mature eosinophils from the blood stream to sites of inflammation in the lung is a multistep paradigm involving initial rolling in the lumen of the blood vessels followed by activation-dependent firm adhesion to the vessel wall and chemoattractant-induced transmigration across the vascular endothelium to extravascular sites, a process driven by cell adhesion molecules (integrins and selectins), chemokines (eotaxin and other chemoattractants), and metalloproteases. In recent years, studies have indicated a regulatory role for galectins in eosinophil trafficking, migration, and activation and thus impact the pathogenesis of allergic asthma. As detailed in this review, Gal-3 plays a pro-inflammatory role in allergic asthma by promoting eosinophil trafficking and migration, while Gal-1 exerts an anti-inflammatory effect due to its ability to limit eosinophil migration and induce apoptosis, wherein decreased expression or absence of this galectin results in increased eosinophilia and exacerbated asthmatic immune responses. The role of Gal-9 in asthma is more intricate; on the one hand, this galectin functions as a chemoattractant for eosinophils and activates these cells but also appears to exert a regulatory function by inducing apoptosis of only activated eosinophils. In disease models, absence of Gal-9 results in increased eosinophilia and Th2 cells but low Foxp3Studies at a cellular level and in animal models clearly indicate a role for Gal-1, -3, and 9 in regulating (positively or negatively) eosinophil recruitment and the pathogenesis of allergic asthma; however, further studies to elucidate expression patterns of these galectins in relation to different phenotypes and endotypes of allergic asthma in humans are necessary to define whether they can serve as disease biomarkers or therapeutic targets for pharmacological modulation. Currently, there are no therapeutic agents that are commercially available for targeting these galectins endogenously for treatment of asthma. However, considerable progress has been made with respect to Gal-3. Gal-3 has been suggested as a reliable biomarker to predict the modulation of airway remodeling in severe asthma patients before they begin omalizumab therapy  and seleAll the authors listed contributed to the review in close collaboration.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.