Chronic myeloid leukemia (CML) is a myeloid clonal proliferation disease defining by the presence of the Philadelphia chromosome that shows the movement of BCR-ABL1. In this study, the critical role of the Musashi2-Numb axis in determining cell fate and relationship of the axis to important signaling pathways such as Hedgehog and Notch that are essential for self-renewal pathways in CML stem cells will be reviewed meticulously..In this review, a PubMed search using the keywords of Leukemia, signaling pathways, Musashi2-Numb was performed, and then we summarized different research worksAlthough tyrosine kinase inhibitors such as Imatinib significantly kill and remove the cell with BCR-ABL1 translocation, they are unable to target BCR-ABL1 leukemia stem cells. The main problem is stem cells resistance to Imatinib therapy. Therefore, the identification and control of downstream molecules/ signaling route of the BCR-ABL1 that are involved in the survival and self-renewal of leukemia stem cells can be an effective treatment strategy to eliminate leukemia stem cells, which supposed to be cured by Musashi2-Numb signaling pathway.The control of molecules /pathways downstream of the BCR-ABL1 and targeting Musashi2-Numb can be an effective therapeutic strategy for treatment of chronic leukemia stem cells. While Musashi2 is a poor prognostic marker in leukemia, in treatment and strategy, it has significant diagnostic value. Cancer refers to a group of diseases, which irregular cell growth is their outstanding feature; it can predominantly invade and spread cells from the primary site to other parts of the body , 2. WhenTyrosine kinase that is an important mediator of the signaling cascade plays a key role in diverse biological processes like growth, differentiation, metabolism and apoptosis. For decades, it had been postulated that Imatinib mesylate, an inhibitor of the specific BCR-ABL1 tyrosine kinase, is the most effective and selective therapeutic strategy, which had been developed as the first molecularly targeted therapy -19. ImatAccumulating evidence has demonstrated that all of the important signaling pathways related to survival of LSCs can be activated by oncogene BCR-ABL1; therefore, this oncogene creates self-renewal property in LSCs , 23. HowThe following data was collected by electronic databases, including PubMed, Scopus, ScienceDirect and Cochrane library, Google Scholar, and Web of Science. All types of relevant studies, original articles, books and abstracts were included and their results were reviewed and reported until 2018.The resistance of CML stem cells to BCR-ABL1 kinase inhibitor called ImatinibThe development of Imatinib mesylate, as a tyrosine kinase inhibitor, that is used as a first-line treatment in newly diagnosed CML patients had brought significant successes in the treatment of patients with CML -42. UsinMusashi2-Numb signaling axisMusashi family involves a group of RNA-binding proteins that are expressed in stem cells and invasive tumors . In mammet al. and Kharas et al. in 2010 were the first studies that have shown the relationship between the Msi2 inducer of the Msi2-Numb pathway and hematopoietic malignancy, and their investigations indicated the Msi2 portion and its regulative interactions with leukemia in a stable and definitive manner  is related directly to decreased survival  and the results showed that Msi2 expression at progressive CML stages (blast crisis) is placed on the higher levels to primary levels of CML (chronic phase) and this overexpression of Msi2 has an inverse relationship with the Numb gene in the both blast crisis and chronic phase. Ito and coworkers  in LAMA-84 cell line (which is a cell line derived from blastic phase of CML patients) after transfection of siRNA against Msi2  and Gli that are respectively the components of Notch and Hh pathways. It is worth noting that Hh and Notch pathways are two important pathways that play significant roles in the self-renewal of resistant cancer stem cells. Therefore, by inhibiting these molecular ways, it is expected that cancer cell growth will be decreased and LSCs can be directed to apoptosis , 74, 95 rly high . Kharas nst Msi2 . In theiHedgehog signaling pathway Hh signaling pathway was first identified in Drosophila for patterning the early embryo. Several studies show that Hh signaling pathways may regulate the cell fate and maintain the stem cell/progenitor cells , 98. ThiNotch signaling pathway et al. pointed that in the CML mouse models, Msi2/Numb expression level and Notch signaling pathway may alter and there is a relationship between them. They also mentioned that the continued suppression of Numb gene is required to maintain the blast crisis in CML. While, the increasing of Numb expression or the inhibition of Msi2 expression might lead to inhibition of the Notch signaling pathway and direct the cell toward differentiation; therefore, the CML blastic phase is suppressed. The new findings of the Msi2/Numb/Notch signaling pathway may be a key to understanding a new insight to hematopoietic malignancies, especially in the advanced stages of CML blastic crisis  . Following the blocking of Msi2 gene, Zhang et al. showed early apoptosis induction by flowcytometry staining with Annexin-PI, and the late apoptosis through staining with Wright-Giemsa (for detection of apoptotic cell morphology) , the LAMA-84 and AR230 (CML) cell lines  . They rephology) . To confll lines . The importance of targeting the Musashi2-Numb signaling pathway to eradicate leukemia stem cellsCML is a malignant disease having chronic and blast crisis phases. More recently, appropriate therapies such as kinase inhibitors, Imatinib, were used in clinical base, but this process is not always a successful treatment for patients who are resistant to the medication. As a result, treatment with this drug has some limitations, so new molecular treatment methods are considered . IncreasIn recent years, considerable progress has been made for treatment of patients with CML. However, providing an approach that is specifically able to reduce LSCs held promise for the effective treatment of CML. The inappropriate activity of Msi2-Numb pathway in LSCs has been reported. Imatinib in the treatment of patients with CML has a significant role, but in spite of this, it has been observed that there are still BCR-ABL1 positive cells that Imatinib is unable to target them; these cancer stem cells (CSCs) are believed to be the cause of resistance to Imatinib therapy. Msi2 is a factor that is expressed at the high levels in CSCs; Msi2 inhibition leads to an increase in the Numb gene expression pattern (as the gene that determines cell fate). This increment of Numb through the key genes of Hh and Notch signaling pathways can decrease the number of LSCs. Therefore, it is obvious that inhibition of Msi2 may be a promising therapeutic approach in reducing human LSCs in CML patients resistant to the drug.


Danio rerio). In addition, we highlight the potential of Wif1 research to understand and possibly influence mechanisms underlying eye diseases and regeneration.The coordinated interplay between extrinsic activating and repressing cell signaling molecules is pivotal for embryonic development and subsequent tissue homeostasis. This is well exemplified by studies on the evolutionarily conserved Wnt signaling pathways. Tight temporal and spatial regulation of Wnt signaling activity is required throughout lifetime, from maternal stages before gastrulation until and throughout adulthood. Outside cells, the action of numerous Wnt ligands is counteracted and fine-tuned by only a handful of well characterized secreted inhibitors, such as for instance Dickkopf, secreted Frizzled Related Proteins and Cerberus. Here, we give an overview of our current understanding of another secreted Wnt signaling antagonist, the Wnt inhibitory factor Wif1. Wif1 can directly interact with various Wnt ligands and inhibits their binding to membrane bound receptors. Epigenetic promoter methylation of Wif1, leading to silencing of its transcription and concomitant up-regulation of Wnt signaling, is a common feature during cancer progression. Furthermore, an increasing number of reports describe Wif1 involvement in regulating processes during embryonic development, which so far has not received as much attention. We will summarize our knowledge on Wif1 function and its mode of action with a particular focus on the zebrafish ( Even in the presence of pathway components within a cell, the temporal control of signaling activity needs to be tightly controlled during development and tissue homeostasis. This can occur outside the cells such that ligands are prevented to bind receptors by secreted inhibitory molecules. These repressor proteins can act by directly binding to the ligand or by interacting with their receptor. Studies of the Wnt signaling cascades have been particularly informative as to the mechanisms of extrinsic control of signal activation by either of the nineteen Wnt ligands in mammals and twenty-six in teleosts . The secin vitro -like repeats, and a 45 aa long hydrophilic tail  and its interaction with Wnt3a revealed that the EGF domains of Wif1 are required for Wnt ligand binding , lateral line and corneal epithelium (zebrafish) and discrete domains of the brain  are located  that appears to provide neuroprotection in cellular and animal models of neurodegenerative and neurological disorders (Dynamic changes in d retina . The capd retina . Intrigual cells  and alsoagonists . These dpability . The difpability . Curcumiisorders . The capisorders .Wif1 expression. Combining the knowledge of the genetic and epigenetic feedback regulation of Wif1 with the regeneration capacities of the zebrafish nervous system has great potential to complement and aid research progress on neural regeneration in mammals.In summary, the structure and mechanistic underlying Wif1 function is rather well described. Epigenetic silencing of the Wif1 promoter is a common feature, frequently resulting in cancer progression when uncontrolled. Wif1 appears to fine-tune cellular processes, perhaps also temporally controlling them, by dynamically regulating the delicate balance of Wnt signaling via feedback loop activation. Indeed, an increasing number of studies link Wif1/Wnt signaling to different comparably subtle, yet important processes in development and disease, particularly in the eye. Furthermore, Wif1 downregulation and concomitant upregulation of Wnt signaling has been connected to regeneration processes in the zebrafish CNS, while spinal cord lesions in mammals with limited regeneration potential has the opposing effect on LP and MC had the original idea and outlined the review. All authors contributed to writing the article.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.


CTNNB1 gene or the APC gene. The established role of the Wnt/β-catenin pathway in DTF forms an attractive therapeutic target, however, drugs targeting this pathway are still in an experimental stage and not yet available in the clinic. Only few studies address other signaling pathways which can drive uncontrolled growth in DTF such as: JAK/STAT, Notch, PI3 kinase/AKT, mTOR, Hedgehog, and the estrogen growth regulatory pathways. Evidence for involvement of these pathways in DTF tumorigenesis is limited and predominantly based on the expression levels of key pathway genes, or on observed clinical responses after targeted treatment. No clear driver role for these pathways in DTF has been identified, and a rationale for clinical studies is often lacking. In this review, we highlight common signaling pathways active in DTF and provide an up-to-date overview of their therapeutic potential.Desmoid-type fibromatosis (DTF) is a rare, soft tissue tumor of mesenchymal origin which is characterized by local infiltrative growth behavior. Besides “wait and see,” surgery and radiotherapy, several systemic treatments are available for symptomatic patients. Recently, targeted therapies are being explored in DTF. Unfortunately, effective treatment is still hampered by the limited knowledge of the molecular mechanisms that prompt DTF tumorigenesis. Many studies focus on Wnt/β-catenin signaling, since the vast majority of DTF tumors harbor a mutation in the  It has s et al.  and Lipss et al. ]. The cus et al. .CTNNB1 mutations  gene, making the protein more resistant to proteolytic degradation  of DTF tumors harbor a mutation in exon 3 of the radation –41. Lesst of FAP . In botht of FAP . The groe enough , 44.Interestingly, the β-catenin mutations observed in DTF are almost exclusively confined to residues T41 and S45, while alterations at other N-terminal phosphorylation residues, that is D32-S37, are rarely observed. Recently, Rebouissou et al. showed in liver cancers that the T41 and S45 mutants activate the pathway only weakly compared to others . In accoCTNNB1 mutated DTF compared to CTNNB1 WT, and hypothesized a correlation between MMP7 and prognosis, as previously was demonstrated in pancreatic cancer  to regulate target genes. Expression of TCF7 (TCF1) and LEF1 could not be identified, while solely a minority of DTF samples expressed TCF7L2 (TCF4) . Others c cancer , 55. Thec cancer , 53. In ecreased  52)..CTNNB1 mcyclooxygenase (COX), a member of the COX enzyme family (COX1 and COX2) and the vascular endothelial growth factor (VEGF), a protein that regulates angiogenesis. A role of COX in DTF has been indicated by the expression of COX2 and by the expression of phosphorylated, and thus activated, associated growth factors receptors, such as the platelet derived growth factor receptor α and ß (PDGF-α and PDGF-ß)  which works by inhibiting both prostaglandins and the COX enzyme  , 73. Act PDGF-ß) , 73. Inh PDGF-ß) , 57. HalX enzyme  58)..cyclooxyApc+/Apc1638N mice , and reported a decrease in ß-catenin levels and cellular proliferation rate, as well as a reduction in tumor number and volume in 38N mice  60). Th. ThApc+/38N mice  (60).Overexpression of VEGF has been correlated with ß-catenin nuclear staining in DTF . AdditioThe blockage of Wnt/β-catenin signaling with the truncated Frizzled 9 receptor fused to the IgG1 Fc region , was recently tested in a Phase 1 study for solid tumors . In thisWhile the above-mentioned treatments, targeting Wnt/β-catenin targets constitute attractive therapeutic possibilities, no prospective clinical trials using these treatment strategies in sporadic DTF have been designed. Experimental inhibitors of Wnt/β-catenin signaling have been developed, however, systemic abolition of Wnt secretion is not preferable since this will result in defects in gut homeostasis, affects the immune system and affects both ß-catenin-dependent and independent Wnt signaling . AberraGLI1, PTCH1, and Hedgehog interacting protein (HHIP) in human DTF samples compared to adjacent normal tissues. Additionally it was demonstrated that expression of Gli1, Gli2, and Ptch1 in mouse (ApcN+/1638) tumors was upregulated compared to normal tissue. In vivo, pharmacological inhibition of Hh with triparanol, which works by interference with the post-translational modification of Hh signaling molecules and with the sterol-sensing domain of the receptor PTCH1, led to a reduction in tumor volume in ApcN+/1638 mice. Genetic approaches to reduce Hh signaling in DTF, using Apc+/1638N; Gli2+/− mouse models, gave rise to the development of fewer and smaller tumors , 98GLI1,2+-stabilized heterodimer containing three domains: an extracellular (NECD), a transmembrane (NTMD) and an intracellular domain (NICD) . Particin vitro study performed by Shang et al. which demonstrated a significant higher expression of nuclear HES1 in DTF tissues compared to scar tissue by IHC and reported expression of NOTCH1, JAGGED1, and HES1 in DTF cells by Western Blot analysis. Additionally, it was demonstrated that PF-03084014, decreased NICD and HES1 expression in a dose dependent manner in DTF cells, and that Notch signaling inhibition contributed to impaired DTF cell proliferation by inducing a cell cycle G1 arrest and decreasing migration and invasion . Tw. Twin viinvasion  77, 78), 78in viThe Janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway regulates cell proliferation, survival, differentiation, migration and apoptosis, and has a role in pathogen resistance. The JAK/STAT pathway is the main signaling mechanism for many cytokines and growth factors. A variety of ligands e.g., interferon-α/interferon-ß (IFN), secreted by leukocytes (IFN-α), fibroblasts (IFN-ß), and various other cells involved in immune responses, and interleukins (IL) together with their cognate receptors, stimulate the pathway. Ligand binding causes receptor dimerization and subsequent activation of the JAK tyrosine kinases associated with the cytoplasmic domains of the receptor. JAKs in close proximity are trans-phosphorylated and phosphorylate the receptors. The phosphorylated receptor sites can then serve as docking sites for cytoplasmic transcriptions factors (STATs). These STATs become phosphorylated by JAKs, dimerize and are translocated into the nucleus where they activate or repress the transcription of target genes [reviewed by O'Shea et al. ].JAK mutations or mutations in other upstream oncogenes, and activating mutations in STAT . The paDysregulation of the PI3 kinase/AKT signaling pathway is frequently encountered in cancers and facilitates tumorigenesis. Over-activation of AKT may be caused by the presence of gain-of-function mutations in PI3K subunits, or loss-of- function mutations in Phosphatase and Tensin homolog deleted from chromosome 10 (PTEN) or PTEN expression loss. PTEN, a tumor suppressor is a lipid phosphatase, negatively regulating AKT by dephosphorylation of PIP3. Alternatively, overexpression or activating mutations in tyrosine kinase receptors and their ligands, as well as the interaction of Ras with PI3K, can excite AKT activity .The role of TGF-ß in DTF has been established by the expression of TGF-ß target genes  and by the upregulation of TGF-β signaling pathway components  in comparison to normal fascia . MignemiSMAD4), changes in β-catenin levels, and increased production of glycosaminoglycan and collagen. Additionally, treatment with EGF increased DTF cell motility  on DTF cell lines and stimulation with these cytokines caused up- and down regulation of various target genes .–68.SMAD4Cross-talk between the insulin-like growth factor (IGF) and the estrogen receptor (ER) mediated signaling has been demonstrated in breast-cancer cells. Activation of MAPK, which is located downstream of IGF-1, enhances ER induced transcription via ER phosphorylation. Therefore, it is presumed that growth factor signaling pathways and the estrogen pathways complement and overlap each other [reviewed by Dhingra ]. ToremiEstrogens affect various physiological processes that concern the development of the reproductive system and several reproductive functions. The role of estrogens in cancer has been described in breast cancer and uterine tumors [reviewed by Dhingra ]. Two su125I-TGF-ß1 , 70125I-I-TGF-ß1 ]. RaloxiI-TGF-ß1 . DespiteI-TGF-ß1  89)..125I-TGFThis review aims to provide a summary of the current knowledge of important, cancer-related signaling pathways in the setting of DTF. The role of Wnt/β-catenin signaling in DTF has been firmly established in numerous studies, showing the presence of β-catenin signaling enhancing mutations in the vast majority of tumors. Therapeutic options targeting the Wnt/β-catenin signaling pathway remain scarce and are not yet widely tested in the clinical setting for DTF. Several clinical trials, targeting other signaling pathways, like Notch and Hedgehog, are currently ongoing, but few study the contribution of these pathways to DTF tumorigenesis.CTNNB1) in relevant tissues are difficult to generate and expensive to maintain. The existing Apc+/ApcN1638 mouse model has already been proven as a useful model for FAP-associated DTF and is often used as a tumor model for non-FAP related DTF (Xenopus tropicalis model harboring a mutated APC, may yield another DTF tumor model that can be exploited as a platform to define novel therapeutic targets and preclinical validation studies (A major challenge remains to study DTF in the preclinical setting. This is partly due to the rarity of the tumor type, but also to the limited availability of DTF cell lines and other cell and animal models . Culturiated DTF  61, 146CTNNB1 in studies . Well-deAdditionally, signaling pathways are often seen as separate entities, however, in reality cross-talk occurs between different pathways. The precise interactions between different signaling- and biochemical pathways is complex and still poorly understood. Aberrant signaling of one pathway can often be corrected via compensatory mechanisms in another pathway . In DTF Furthermore, clinical studies to evaluate the efficacy of systemic and targeted treatments without any randomization procedures remain challenging in current clinical practice as they are often difficult to interpret. Due to the unpredictable growth behavior of DTF with reports of spontaneous regression without treatment and stable disease, it is difficult to distinguish the true treatment effects from natural growth behavior. Moreover, the design of randomized controlled trials might be restricted by the rarity of the disease and the small number of DTF patients with an indication for systemic treatment. Despite these challenges, future studies should include signaling pathways other than Wnt/β-catenin signaling to uncover additional driver genes and pathways in DTF and to clarify the potential working mechanisms of target drugs in the setting of DTF.This article does not contain any studies with human participants or animals performed by any of the authors. For this type of study formal consent is not required.MT, DG, CV, SS, and EW conceptualized and designed the study. MT wrote the first draft of the manuscript. MT, RS, DG, CV, SS, and EW wrote sections of the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version.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.


Wideband excitation and control of Fano resonance and electromagnetically induced transparency (EIT), both of which rely on coherent interaction between two excitation paths, is challenging. It requires precise control and tuning of interacting resonances or coupling between different resonant structures over a wide frequency range. Gain (Stokes) and absorption (anti-Stokes) resonances associated with the stimulated Brillouin scattering (SBS) process can be excited and controlled over a wide frequency range by tuning the pump frequency, its power and profile. We exploit coherent interaction between the Brillouin Stokes and anti-Stokes resonance, in radio frequency domain, to demonstrate Fano and EIT-like resonance over a wide frequency range and control their shape and strength optically and electrically. For the Fano resonance, the asymmetry and polarity are electrically controlled over an unprecedented frequency range (100 MHz–43 GHz) by varying the bias to the intensity modulator whereas, the strength is varied by tuning the Brillouin pump power and/or the bias. The depth and 3 dB linewidth of the transparency window in the EIT-like resonance are controlled using pump and probe parameters. The flexibility of the SBS process that allows wideband electrical and optical control of Fano and EIT-like resonance opens up the potential for applications that range from low-power switching, sensing to tunable RF delay. The origin of such asymmetry is devoted to the quantum interference of a discrete (resonant state) with continuum (non-resonant). Since the criterion for the occurrence of Fano resonance involves coherent interaction between two excitation paths, the phenomenon is considered quite generic. Till date, Fano resonances have been observed in many complex dynamical systems such as atomic-plasmonic coupling system2; mechanical excitations in nano-optomechanical structures4; biased semiconductor super lattices5; Fano effect in quantum dots7; Weyl fermions8; waveguide-cavity systems11; perovskites12; optically driven atomic force microscopy13; and forward stimulated Brillouin scattering (FSBS)17. Fano resonance finds tremendous applications in microwave spectroscopy of high absorption matter19; bio-sensors20; ultrafast switching21; and Fano laser23. Electromagnetic induced transparency (EIT), on the other hand, arises from the destructive quantum interference between two transition amplitudes and manifests itself as a transparency in an absorption profile24. Like Fano resonance, EIT-like resonance has been demonstrated in optomechanical systems26, photonic crystals27, Tera-hertz metamaterials28, plasmonic metamaterials29, microresonators using FSBS31, and coupled resonators and microcavities33. However, controlled wideband excitation of both the phenomenon, which is critical for enabling a number of above-mentioned applications, is challenging due to the requirement of precise control of structural or atomic resonance over a large frequency range. It is, therefore, important to harness these phenomena in systems where the interacting pathways can be easily excited and controlled over a wide frequency range.Asymmetric shape resonances were observed and studied by Fano for the first time in atomic systems owing to auto-ionization processes36, sensing37, Brillouin cooling38, Brillouin lasers40 to wideband microwave photonic signal processing45. These applications are enabled using backward or forward SBS process in optical fibers, photonic chips, silicon nanostructures and waveguides, hybrid circuits and optical resonators. In most of these applications, either a Stokes wave (ωs = ωp − ΩB) or an anti-Stokes wave (ωas = ωp + ΩB) interacts with a Brillouin pump (ωp) to generate an acoustic wave (ΩB). The observed radio frequency (RF) response in these cases, therefore, has a Lorentzian profile associated with a gain or loss resonance. However, simultaneous excitation of the Stokes (gain) and anti-Stokes (loss) resonance and its effect has remained largely unexplored because anti-Stokes wave undergoes strong absorption and therefore does not generate significant output signal. While the amplitude of output anti-Stokes signal is negligible compared to the Stokes wave, which sees gain, its phase response is of the same order as that of Stokes and result in coherent interaction between the Stokes and anti-Stokes wave whenever their amplitudes are nearly equal.Stimulated Brillouin scattering (SBS), which occurs as a result of coherent interaction between two optical waves and an acoustic wave, offers tremendous flexibility in terms of wavelength/frequency of operation, bandwidth and spectral profile of the gain and loss resonances associated with it. Depending on the nature of the acoustic mode involved, SBS can be excited using two counter-propagating optical waves, in which case it is termed as backward SBS (BSBS) or using two co-propagating optical waves where it is known as (forward) SBS (FSBS). SBS is also known to be the strongest nonlinearity, which is nearly 100 times stronger than Raman scattering in optical fibers. The flexible nature of the Brillouin process and its high efficiency has made SBS the natural choice for a large number of applications that range from slow-lightHere we demonstrate controlled wideband excitation of Fano and EIT-like resonances exploiting the coherent interaction between the Stokes and the anti-Stokes resonance of the BSBS process in the microwave domain. We control the profile, strength, and polarity of the Fano resonance using optical and electrical means over an unprecedented frequency range that extends from 100 MHz to 43 GHz. Our novel approach enables control of the 3 dB linewidth of EIT-like resonance while maintaining the transparency depth. Here we demonstrate that the linewidth can be tuned from 14 MHz to 20 MHz with a fixed transparency depth of 45 dB. Similarly the depth can be tuned with a fixed 3 dB linewidth. For a fixed linewidth of ∼14 MHz, we show that the depth changes from 25 dB to 45 dB for a 4 dB increase in gain. Dynamic control of the Fano resonance and induced transparency over a wide frequency range, using optical and electrical means, opens way for a number of applications in the area of low-power switching, sensing, and microwave photonic signal processing.E(t), radio frequency response is given as: 46. In the BSBS process, Stokes (ωc − ΩB) and anti-Stokes (ωc + ΩB) probe signals are generated by modulating a carrier (ωc) with an RF signal at the Brillouin frequency (ΩB). By filtering out the anti-Stokes field along the probe arm, Stokes and carrier signals , the resulting RF response at the Brillouin shift arises from the beating of the carrier and the Stokes signal Ec(t) and Es(t) are the carrier and Stokes field respectively. The RF spectrum at νB exhibits a Fano-like asymmetry  beat signal between the carrier and Stokes wave and (ii) beat signal between the carrier and anti-Stokes wave and the net beat signal is given by Eas(t) is the anti-Stokes signal. Since the carrier is common to the individual beat signals, the RF spectral response results from the coherent interaction between the Brillouin Stokes and anti-Stokes signals.For an optical signal with electric field als Fig.  are let ape Fig. , which iape Fig. . When thtry Fig. . The mea47, which causes a shift in the phase response associated with the Stokes and anti-Stokes resonance  fiber, which is used as the Brillouin medium. To observe Fano resonance, a probe with equal amplitude Stokes and anti-Stokes sidebands is used. Carrier with a larger amplitude is used to generate a large beat signal.To generate the Stokes and anti-Stokes sidebands, we use an off-the-shelf single drive Z-cut intensity modulator (Thorlabs), where the electrode is closer to one of the arms of the Mach-Zehnder modulator (MZM). In this case, the upper and lower modulation sidebands see a constant bias dependent phasence Fig.  while kence Fig. . By tuni. π Fig. . On the νmin, at which the minima occurs increases. The increase in extinction and νmin with pump power occurs because it affects both the phase and amplitude of the Stokes and anti-Stokes resonances, which optimizes the condition for destructive interference. Figure α parameter fixed.To demonstrate optical control of the Fano profile, we vary the Brillouin pump power at a fixed bias voltage. For a fixed probe signal, the Fano resonance becomes stronger as the pump power is increased Fig.  and the α in Eq. 48. The electrical control of Fano resonance has the potential for low power, ultra-fast switching as it changes the RF amplitude at a given frequency by 30 dB for a small change in applied bias voltage ∼0.2 V (solid and dots)  at a fixed pump power, equivalent to a Brillouin gain of 2.3 dB. Figure ts) Fig. . From Fi48:ωc, and ϕ01,02 denote laser (carrier) frequency and bias induced phase shifts, respectively, in two arms of the MZM. The modulation frequency and amplitude for each arm are represented by ωm, m1 and m2. For small signal modulation, the output of the MZM can be written as47:m = m2 − m1.In order to model the coherent interaction between the Stokes and anti-Stokes resonances, we assume that the optical field emerging from a Z-cut intensity modulator is given according toα that depends on m1 and m2 as follows:We define a parameter ωm is scanned around ΩB. The resulting output probe field is then given as41:When the modulated signal is propagated through a Brillouin active medium, the amplitude and phase of the upper and lower sidebands are modified by Brillouin loss and gain, respectively, when ωm, which results from the beating of the carrier with Stokes and anti-Stokes and is calculated according to:Detection of the probe field using a broadband detector gives the signal at frequency ωm can then be obtained using:R are detector responsivity and load resistance. From Eqs ,,ωm can tmentclass2pt{minimmentclass2pt{minimωRF) other than the Brillouin frequency (ΩB), we modulate the laser (ωc) in the pump arm with frequency (ωRF + ΩB) or (ωRF − ΩB) to create pumps for the Brillouin gain and loss resonance as ωRF is varied up to 43 GHz. Using pump frequency tuning, we achieve controlled excitation of Fano resonance over a wide radio frequency range of 100 MHz to 43 GHz  as shown in Fig. ωRF is tuned around ΩB. This results in a transparency window within the rejection band when observed on the VNA  spectrum on Brillouin pump parameters, makes it a suitable system to study wideband excitation and control of Fano and induced transparency.Unlike EIT in atomic systems, where destructive interference between two transition amplitudes introduces transmission in a narrow spectral region, the destructive interference between the Stokes and anti-Stokes amplitudes creates symmetrically placed rejection bands with large depth that amplifies the transparency amplitude introduced due to the Brillouin gain resonance. The depth of these rejection bands and 3 dB linewidth of the transparency window are highly sensitive to ratio of the anti-Stokes to Stokes signal amplitude and pump power. By controlling this ratio and Brillouin gain, large change (>20 dB) in the transparency depth is achievable with a small change in the gain without affecting the linewidth. Tuning of anti-Stokes to Stokes amplitude ratio and gain parameter allows control of the linewidth at fixed transparency depth. In this demonstration, induced transparency with unprecedented 3 dB linewidth ∼10 MHz is observed at 17 GHz, which can be further reduced by controlling the anti-Stokes to Stokes amplitude ratio and Brillouin pump power.In order to understand the Fano and EIT-like resonance using coherent interaction of Brillouin pathways, we developed a model that accurately predicts the frequency response for different pump powers, bias values and anti-Stokes to Stokes power ratio. The model gives an understanding of the amplitude and phase conditions required to control the shape, polarity, extinction, and linewidth of Fano and EIT-like resonances.49, SBS50 and other components used in our work have, recently, been demonstrated on optical chip which opens up the development of photonic integrated circuit devices16 for controlled wideband excitation of Fano and EIT-like resonance and their application to low-power switching, microwave photonic signal processing, and sensing.Demonstration of Fano resonance and induced transparency using SBS and their control over a wide frequency range with electrical and optical means offers potential for applications in low-power switching and microwave photonic signal processing. Intensity modulatorsB), frequency of the VNA is scanned around ΩB.To demonstrate Fano resonance, we set up the pump-probe experimentFig.  that is ωRF) other than ΩB, the laser output is amplified using an EDFA (PriTel) before splitting it into the pump and the probe arm. Carrier suppressed double sideband modulation (CS-DSB) is used in the pump arm to tune the pump frequency. The carrier in the pump arm is suppressed by adjusting the bias to an intensity modulator and using a fiber Bragg grating (FBG) before the HP-EDFA (Amonics). The modulation frequency in the pump arm is kept at ωRF + ΩB or ωRF − ΩB using an RF signal generator (Keysight) whereas probe is generated by modulating the laser (carrier) at frequency ωRF with a VNA. Pump and probe are then counter-propagated in a 50 m long PM fiber through FPCs, PBSs and PM circulators. The probe power is controlled using a low-noise EDFA (Pritel) for different experiments. Optical and electrical response of probe is then recorded using OSA, photodetector and VNA.In order to demonstrate Fano resonance at frequencies  is used as the Brillouin medium. The setup utilised for observing induced transparency is similar to that of Fano. Unlike Fano, where equal amplitude Stokes and anti-Stokes sidebands are used, observation of induced transparency requires control of the anti-Stokes to Stokes power ratio. Fiber Bragg gratings (FBGs) are used to control Stokes power in the probe arm. To obtain the loss profile in Fig. B utilises the similar modified approach as used in wideband Fano. The laser output is fed into a high power erbium-doped fiber amplifier (HP-EDFA: PriTel) and split using a 50:50 splitter. One arm of the splitter is used to generate the pump using CS-DSB modulation. The other arm generates the probe and FBGs are utilised in this path to suppress the sideband that would see gain. The ratio of Stokes and anti-Stokes is tuned by tuning the FBGs to obtain the required transparency depth and the probe signal is recorded on OSA and VNA.The setup for EIT at frequencies other than ΩData sets and simulation codes generated during the current study are available from the corresponding author on a reasonable request.


AKT1, BAD, FOXP3, RORγt and IFNλ3 as the host factors were investigated.Adult T-cell leukemia/lymphoma (ATLL) is a lymphoproliferative disorder of HTLV-1-host interactions in infected TCD4+ cells. In this study, the HTLV-1 proviral load (PVL) and HBZ as viral elements and The study was conducted in ATLLs, HTLV-1-associated myelopathy/tropical spastic paraparesis patients (HAM/TSPs) and HTLV-1-asympthomatic carriers (ACs). The DNA and mRNA from peripheral blood mononuclear cells were extracted for gene expression assessments via qRT-PCR, TaqMan assay, and then confirmed by western blotting.P = 0.002) and HAM/TSP (P = 0.041). The HBZ expression in ATLL (101.76 ± 61.3) was radically higher than in ACs (0.12 ± 0.05) and HAM/TSP (0.01 ± 0.1) (P = 0.001). Furthermore, the AKT1 expression in ATLLs (13.52 ± 4.78) was higher than ACs (1.17 ± 0.27) (P = 0.05) and HAM/TSPs (0.72 ± 0.49) (P = 0.008). However, BAD expression in ATLL was slightly higher than ACs and HAM/TSPs and not significant. The FOXP3 in ATLLs (41.02 ± 24.2) was more than ACs (1.44 ± 1) (P = 0.007) and HAM/TSP (0.45 ± 0.15) (P = 0.01). However, RORγt in ATLLs (27.43 ± 14.8) was higher than ACs (1.05 ± 0.32) (P = 0.02) but not HAM/TSPs. Finally, the IFNλ3 expression between ATLLs (31.92 ± 26.02) and ACs (1.46 ± 0.63) (P = 0.01) and ACs and HAM/TSPs (680.62 ± 674.6) (P = 0.02) were statistically different, but not between ATLLs and HAM/TSPs.As it was expected, the HTLV-1-PVL were higher in ATLLs than ACs (HBZ and host AKT1 and Rad 51 are novel candidates for molecular targeting therapy of ATLL. However, high level of RORγt may inhibit Th1 response and complicated in ATLL progressions.The present and our previous study demonstrated that HTLV-1-PVL and  Retroviridea family, which infects about 10–20 million people in the world. Although, after a long period of time, a small percentage (3–5%) of infected individuals will progress to HTLV-associated myelopathy-tropical spastic paraparesis (HAM/TSP) or Adult T-cell leukaemia/lymphoma (ATLL), the large number of infected carriers remains asymptomatic [The human lymphotropic virus type 1 (HTLV-1) is a member of the The HTLV-1 endemic areas include, Southwestern Japan, the Caribbean basin, Central Africa, the Melanesian Islands, South America and the Middle East . In IranTax and HTLV-1 bZIP factor (HBZ). These regulatory proteins have a close interaction through mutual transcriptional regulation interference in the infected T cells. HBZ as regulatory protein through inhibition of Tax can help to evade host immune responses. Therefore, the over-expression of HBZ is implicated on the development of malignancy in the late stages of cell transformation in HTLV-1-infected cells [HBZ mRNA expression and HAM/TSP disease severity [The HTLV-1 activity and infectivity in asymptomatic carriers and HTLV-1 associated diseases is quantified by the number of infected peripheral blood mononuclear cells (PBMCs), which expressed as the proviral load (PVL) . The inced cells . Moreoveseverity , 8.The Tax and HBZ may exert their oncogenic and pathogenicity effects by changing the outcome of signalling pathways, such as NFκB and phosphoinositide 3-kinase (PI3K), and apoptotic event , 9. AKT Likewise, BAD and BAX are members of the BCL-2 family proteins and are the main molecules in regulating cell survival and apoptosis . HTLV-1 Interferons  are the most important antiviral agents in innate and adaptive immune responses. Type III IFNs, includes 3 subtypes, IFN-λ1, λ2 and λ3, known as interleukin-29 (IL-29), IL-28A, and IL-28B, respectively . IFN-λ iHBZ overexpresses to suppress the expression of Tax as a virus activator and dominated antigen to escape the immune response. Therefore, the persistence and pathogenesis of HTLV-1 depends on the host immune response such as Th1, cytotoxic T lymphocytes (CTLs), natural killer cells (NK-cells) and Th17 [FOXP3) transcription factor, play a critical role in the maintenance of the immune system homeostasis. An immunosuppressive micro-environment enables HTLV-1-infected cells to escape the host immune responses. Besides, the most HTLV-1-infected cells have been suggested to be the Treg lymphocytes. Many reports have been conducted on the role of Tregs in HTLV-1-associated disease progression [To lower the host immune pressure, and Th17 . Regulatgression . Moreovegression . In the HBZ as viral elements, AKT1 as a regulator of the cell cycle progression and cell survival, BAD as proapoptetic agent and finally RORγt, FOXP3 genes as the main transcription factor in Th17 and Treg cells, and IFN-λ3 as an antiviral agent in the innate immune responses.In the present study, the host-virus interactions in the manifestation of ATLL and HAM/TSP were investigated by assessment of HTLV-1-PVL and Tax and LTR DNA fragments of provirus.In this cross-sectional study subjects consisted of 18 ATLL, 10 HAM/TSP newly diagnosed patients, and 18 HTLV-1 ACs from the hematology/oncology and neurology wards of Ghaem referral university Hospital, Mashhad, Iran, between May-2014 and November-2015. Diseases, ATLL and HAM/TSP, in patients were confirmed by two neurologist and oncologist. All participants had a seropositive test for HTLV-1  which infection was confirmed by PCR for The PBMCs were isolated from whole-blood by Ficoll density gradient . Total RNA and protein extraction from PBMCs was isolated from TriPure  treated samples according to the manufacturer’s instructions. The extracted RNA was then reverse-transcribed to cDNA by the AccuPower ®RT PreMix cDNA synthesis kit . The extracted protein was kept at − 70 °C for subsequent western blotting analysis.HBZ, AKT1, BAD, FOXP3, RORγt, IFNλ3 and the cellular reference gene beta-2 microglobulin (β2M) using the TaqMan method. For assessment of gene expression five standards were prepared using five folds serial dilution of a concentrated sample for the gene of interest and reference gene. Then, standard curves were generated for the target and reference genes, and the data were analyzed using the relative method by Rotor Gene 6000 software . The relative quantity of the interested gene was normalized to the relative quantity of β2M as the reference gene and expressed as the expression index. Briefly, the relative AKT1, BAD, FOXP3, RORγt, IFNλ3 and HBZ expression levels for each sample were calculated by an equation of: normalized Index = copy number of gene of interest /copy number of reference gene (β2M).Primers and probes were designed by Beacon Designer software . Table 4 and expressed as the number of HTLV-1 proviruses per 104 PBMCs.HTLV-1 PVL was assessed on extracted DNA from PBMCs and an absolute real-time PCR was performed using a commercial quantification kit  by a Rotorgen real-time PCR machine. The HTLV-1 copy number was reported as an actual amount of cellular DNA by using quantification of the albumin gene as the reference gene. HTLV-1 and albumin DNA concentrations were calculated from two 5-point standard curves. The normalized value of the PVL was calculated as the ratio of  × 10AKT1 and RORγt genes. Shortly, the proteins were separated on 12% polyacrylamide gel electrophoresis (SDS-PAGE) performance , and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membrane . After electrotransfring, PVDF membranes were blocked by BSA 2% (overnight at 4 °C). For identification of AKT1 or RORγt protein, membranes were incubated with mouse IgG monoclonal antibody to human beta Actin protein  as reference protein at a dilution of 1:500 and mouse IgG monoclonal antibody to human AKT1  or RORγt protein  for 1 h at room temperature. Thereafter, PVDF membranes were incubated for 1 h at room temperature with goat anti-mouse IgG-HRP antibody  as secondary antibody at a dilution of 1:10,000. Finally, the specific AKT1 and RORγt proteins were detected by enhanced chemiluminescence detection system .Protein expression was confirmed by western blotting for P-value was considered statistically significant if P ≤ 0.05.pt?>Statistical analysis was performed using SPSS software ver.16.0 . The results have been shown as mean ± standard error of mean (SEM). Nonparametric statistical tests including Kruskal Wallis (one-way ANOVA) analysis for comparing more than two-independent samples or Mann-Whitney U test for two-independent samples were used for statistical analysis of data. Correlation analysis was performed using Spearman’s test to detect the association between the variables. The The study investigated 18 seropositive ATLL patients , 10 seropositive HAM/TSP patients  and 18 seropositive HTLV-1 ACs . There was no significant difference between age and gender in the HTLV-1 ACs, ATLLs and HAM/TSP patients. Place of birth for 85% of the ATLL subjects were Razavi Khorasan province, including 60% in Mashhad, 15% in Neyshabur, 10% in Sabzevar and 15% remaining from other provinces.Among the patients, 61% had lymphadenopathy, 21% had immunodeficiency disorders, and 18% had skin lesion. Two patients had lymphadenopathy and skin lesion, simultaneously, and one patient had immunodeficiency and skin lesion. Note that none of the patients showed three clinical symptoms simultaneously.4 PBMCs in ATLL patients, 562.4 ± 119.2 copies/104 PBMCs in ACs and 649 ± 190.4 copies /104 PBMCs in HAM/TSP patients. The PVL in ATLL patients was significantly higher than that in ACs (P = 0.002). The difference of PVL between ATLL and HAM/TSP patients was statistically significant (P = 0.041). No significant difference was found between ACs and HAM/TSP patients . The expression of this viral factor was considerably higher in the ATLL group than in the HAM/TSP group . No significant difference was found between ACs and the HAM/TSP group  and HAM/TSP patients (P = 0.008). A significant difference was found in the AKT1 gene expression between the HAM/TSP patients and ACs (P = 0.027) , ACs (1.07 ± 0.32) and HAM/TSP patients (1.57 ± 0.36). However, a significant difference in the BAD expression was found between ACs and HAM/TSP patients (P = 0.027)  and 91 times more than HAM/TSP patients . No significant difference was found between ACs and the HAM/TSP group . No significant difference was found in the RORγt expression between ATLL and HAM/TSP patients, or between ACs and HAM/TSP patients . Moreover, a significant difference was observed between ACs and HAM/TSP patients . However, no significant difference was found between ATLL and HAM/TSP patients in the IFNλ3 expression  was used as reference gene. According to Fig. High-level mRNA expression of HBZ mRNA level in ATLL, HAM/TSP and ACs groups . There was a significant association between PVL and IFNλ3 mRNA level in the three groups .The correlation between variables was evaluated in the three groups and in each group separately. Significant correlation observed between PVL and HBZ and AKT1 , HBZ and FOXP3 , also between HBZ and IFNλ3 mRNA levels  in the three groups.Furthermore, a significant association was observed between AKT1 and IFNλ3 mRNA expression levels . Moreover, strong significant correlation was observed between HBZ and IFNλ3 mRNA expression levels  in ACs.In ATLL patients there was a significant association between AKT1 and IFNλ3 mRNA expression levels .In HAM/TSP patients there was significant association between It is well known that only 3–5% of infected subjects develop ATLL or HAM/TSP , therefoAlthough, the previous studies have suggested a possible important role for PVL in the development of ATLL and HAM/TSP, the association between PVL and disease development remains unclear. According to Iwanaga et al. study, the role of high PVL level remains uncertain because the majority of ACs with a high PVL level remained asymptomatic . In the Tax expression to escape the host immune pressure for virus elimination by Tax specific CTLs. Therefore, it produces as a second oncogenic signal for the maintenance of the leukemic cells. HBZ is expressed in nearly all malignant cells of the ATLL patients, and its expression is correlated positively with PVLs and negatively with tax/rex and gag/pol expression [HBZ expression gene since it is possible with an increase of PVL, HBZ increment happens in infected cells.In addition to PVL, Tax is produced in the early phases of infection to establish viral replication and induce T cell transformation, proliferation and DNA damage. HBZ is subsequently expressed to inhibit pression . In the HBZ expression surprisingly showed an 800-fold higher increase in ATLL patients compared with ACs and HAM/TSP patients. Thus, these results are consistent with those of previous studies on the role of HBZ in the progression toward leukemogenesis, and support the critical role of HBZ in HTLV-1 oncogenesis. In the most cases, HBZ is the only HTLV-1 molecule that is expressed and may be involved in the maintenance of most ATLL cells which has an important role in leukemogenesis. Taken together, these studies have suggested that, although HBZ has growth-promoting activity in ATLL, it does not participate in transformation [Our findings showed that ormation , 26.HBZ expression between HAM/TSP patients and ACs. This finding is consistent with previous studies that were shown more effective role of Tax than HBZ increment in progression toward HAM/TSP [Our findings show no significance difference in  HAM/TSP , 27.AKT1 expression in the ATLLs is 12–18 times more than the ACs and HAM/TSP patients. These findings showed that the PI3K/AKT1 pathway may have stronger effects on ATLL than on HAM/TSP. In the present study, a significant association was found between HBZ and AKT1 genes expression, thus HBZ increment can impact on AKT1 expression and its subsequent effects on maintenance of HTLV-1 infected cells and ATLL progression. Also, lower HBZ gene expression in HAM/TSP patients than ACs may affect low expression of AKT1 in HAM/TSP patients compare with ACs.Apart from these viral factors in the immortalisation of host-infected cells, survival and apoptotic signalling pathways are pivotal in promoting malignancy, such as PI3K pathway and BCL-2 family. The present study focuses on the AKT1 molecule from the PI3K signalling pathway and the pro-apoptotic factor from the intrinsic programmed cell death pathway as an antagonist for BAD. For instance, AKT inactivates BAD through phosphorylation and therefore inactivates its ability to induce apoptosis for promoting cell survival. Conversely, the dephosphorylation of BAD leads to the targeting of BAD to the mitochondrial membrane in which BAD interacts with anti-apoptotic proteins, such as BCL-2 and BCL-xL, to induce apoptosis. Nakahata et al. showed that the PI3K/AKT signalling pathway has a critical role in the induction of ATLL and other malignancies . HoweverBCL expression is high and that the level of pro-apoptotic proteins Bax, BAD and Bak are not considerably changed in HTLV-1 infected T cells [BAD were not significant between ATLL patients and ACs. Conversely, the AKT1 expression had a significant difference between these groups in Jeong et al. study. Therefore, AKT1 may inhibit the BAD pro-apoptotic molecule and contribute to cell survival [BAD expression between HAM/TSPs and ACs) and previous studies, BAD which involves in cell death, preventing the BAD apoptotic pathway may exacerbate as well as induce the HTLV-1 associated diseases.Signalling pathways such as NF-κB and PI3K/AKT1 can somehow regulate proteins from the BCL-2 family. Nicot et al. found that the anti-apoptotic  T cells . In the survival . Previousurvival , 32. The+ and CD25+) T-cell phenotype, they are not regarded as having helper functions. Instead, ATLL cells demonstrate a strong immunosuppressive activity in vitro [FOXP3 and/or GITR mRNA is expressed in almost all of the primary ATLL samples [FOXP3 expression level in ATLL cells was significantly higher than that in ACs and HAM/TSP patients. Consistent with this finding Satou et al. showed in HBZ-transgenic mice CD4+ CD25+ Treg cells increased and also they showed HBZ expression directly induced FOXP3 gene transcription in T cells, however these cells functionally impaired while their proliferation was enhanced, thereby impairing the suppressive function of Treg cells [Although, ATLL cells display an activated helper/inducer  and IFNλ3 gene expression was found. Besides, significant association between AKT1 expression and IFN λ3 was observed expression in both ATLL and HAM/TSP patients, thus this finding indicates high expression of AKT1 in HTLV-1 infected cell may induce high IFNλ3 mRNA expression in ATLL and HAM/TSP patients. In the present study, the elevated IFNλ3 level was observed in HTLV-1 but in infected individuals could not prevent to disease development.The IFNλ3 was considered after observing the presence of specific polymorphisms near the IFNλ3 gene is associated with an increased response to treatment or spontaneous clearance of those that developed chronic hepatitis C , 42. Morll lines . Also, wThis study has some limitations; the incidence of ATLL even in our endemic region is rare for large sampling. Furthermore, to obtain high and stable RNA extraction for gene expression using quantitative real-time PCR, the PBMCs were treated by TriPure. Therefore, the protein extraction from TriPure for western blot assay did not come to the proper results for all of studied protein, except AKT1 and RORγt.Our last bioinformatics study using networking also showed that PI3K/AKT/mTOR, in cell cycle and anti-apoptotic pathways and BCRA-1 as DNA damage repair signalling pathways are main players in ATLL development and progression . The pre


The MIPMs were also employed as the stationary phase to fabricate molecularly imprinting solid-phase extraction column, and the spike recovery was demonstrated to be not significantly decreased even after nine cycles. Furthermore, the reliability of the method was also evidenced through the comparison of the MIPMs prepared from different batches.The noxious clenbuterol misapplied as the feed additive has posed an enormous threat to humans who actively rely on the food chains with high potential of contamination by clenbuterol, such as pork and beef. It is, therefore, highly desirable to develop novel materials and strategies for dealing with the clenbuterol. Herein, functional polymer microspheres prepared by Pickering emulsion polymerization were explored for the selective enrichment of the clenbuterol, and their structure and oxygen functionalities could be tailor-made by a molecular imprinting process. The clenbuterol imprinting was adequately demonstrated to not only increase the particle size (~52 nm vs. ~42 nm) and create cavities for the accommodation of the clenbuterol molecules, but also reduce the oxygen functionalities of the resulting molecularly imprinted polymer microspheres (MIPMs) by approximately 4 at.%, which is believed to correlate with the high specificity of the MIPMs. Various characterization methods were employed to evidence these findings, including scanning electron microscopy, BET measurements, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and elemental mapping examination. More importantly, the MIPMs showed a markedly superior enrichment capability towards clenbuterol to the counterpart, that is, non-molecularly imprinted polymer microspheres (NIPMs). Compared to the NIPMs without specificity for clenbuterol, the MIPMs exhibited an impressive selectivity to clenbuterol, with the relative selectivity coefficient ( Even nchitis) . TherefoBefore the monitoring of clenbuterol, enrichment processing is usually needed to obtain more accurate results and lower limits of detection by the subsequent instrumental investigation of the pre-concentrated clenbuterol , liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS) ,5,6). ThMolecular imprinting technologies are widely employed to render diverse materials selective to a target species ,24. Mole3)3) at one end and a hydrophilic moiety (–NH2) at the other might also help to improve the stability of the emulsion and consequently the quality of the emulsion polymerization products. The subsequent solvent extraction of clenbuterol leads to the generation of artificial recognition sites being complementary to clenbuterol [Although clenbuterol-imprinted polymers have been researched over these years, there still lacks an in-depth comparison between MIPs and non-molecularly imprinted polymers (NIPs) on the specific structural and property variation imparted via the clenbuterol imprinting treatment . The polymerization in the presence of clenbuterol will alter the solubility of the polymer chains , eventuanbuterol , indicatIn this study, we systematically explored the impact of the clenbuterol molecular imprinting on the polymer microspheres with respect to the microstructure, surface functionalities, and adsorption performance towards clenbuterol. Through a comparison between the resulting MIP microspheres (MIPMs) and NIP microspheres (NIPMs), the molecular imprinting effect on the structure and functionalities of MIPMs was clarified. Pickering emulsion polymerization was adopted to synthesize MIPMs rather than the conventional strategies such as bulk polymerization , precipiFumed silica with a mean diameter of 12 nm  was supplied by Evonik Degussa . The initiator, 2,2′-azoisobutyronitrile , and the monomer, methacrylic acid  were purchased from Tianjin Fucheng Chemical Reagent Factory . Clenbuterol, terbutaline, salbutamol, and methyl red were analytical reagents and supplied by Sigma Chemicals Co. Ltd. . The cross-linking agent, ethylene glycol dimethyl acrylate , was purchased from Aladdin Reagent . All of the other chemicals were of the analytical grade and obtained from Tianjin Fucheng Chemical Reagent Factory . All of the chemicals were used without further purification unless otherwise stated.The procedures for the synthesis of MIPMs are illustrated in The characterizations of the structural functionalities of the MIPM and NIPM samples were carried out by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) using an IRAffinity-1S spectrometer  and XSAM800-XPS equipment , respectively. Scanning electron microscopy (SEM) images and elemental mapping images of the MIPMs and NIPMs were obtained using a HITACHI S-4800 Scanning Electron Microscope equipped with an energy dispersive X-ray (EDX) spectrometer . The Brunauer–Emmett–Teller (BET) measurements of the MIPMs and NIPMs were carried out with a Micromeritics ASAP 2020 nitrogen adsorption apparatus . Before the BET test, the MIPMs and NIPMs samples were pre-degassed at 150 °C. The corresponding pore size distribution was determined by analyzing the desorption branch based on the Barrett–Joyner–Halenda (BJH) method. The mass spectrometric analysis was conducted using a 6540 UHD mass spectrometer .C0 and Ct represent the initial clenbuterol concentration and the concentration at time t, respectively.To a glass vial, the MIPMs or the NIPMs (50 mg) were added, followed by the addition of a water solution of clenbuterol . After the homogenization of the mixture imparted by shaking, the glass vial was equipped with a thermostatic water bath oscillator at an oscillation rate of 120 rpm for the adsorption processing. At different durations, the mixed solution was drawn for the measurements of the clenbuterol concentration in the solution, and the adsorbed amount was calculated on the basis of Equation (1) by LC-MS. The amount of the supernatant injected in the HPLC/MS system was 5 μL, and the test conditions were adopted as follows: electrospray ionization as the ion source, dry gas flow rate of 40 L/min, atomization pressure of 10 psi, and methanol as the mobile phase at the flow rate of 0.5 mL/min.qe represents the equilibrium adsorption amount (mg/g), qt (mg/g) is adsorption amount at time t, and k1 and k2 designate the pseudo-first-order rate constant (1/min) and pseudo-second-order rate constant (g/mg min), respectively.The pseudo-first-order and pseudo-second-order kinetic models were adopted to analyze the adsorption kinetics according to Equations (2) and (3), respectively.eq, mg/g) was calculated based on Equation (4).C0 and Ce are the initial and equilibrium concentrations (mg/L) of clenbuterol, respectively, m represents the mass of the MIPM and NIPM adsorbents, and V is the volume (L) of the clenbuterol solution.To each of five glass vials, the MIPMs or the NIPMs (50 mg) were added. Then, the clenbuterol solutions at a volume of 10 mL, with different concentrations , were respectively charged into the five vials pre-loaded with polymer microspheres. After shaking for homogenization, these mixtures were placed into a thermostatic water bath oscillator, and the adsorption interactions proceeded at the oscillation rate of 120 rpm for 6.5 h which was sufficient for reaching equilibrium as proven by the test on the adsorption kinetics. Three different temperatures were considered, namely 30, 45 and 60 °C corresponding to the temperatures in Kelvin of 303, 318 and 333 K, respectively. After the equilibrium was reached, the resulting mixtures were centrifuged at 4000 rpm for 5 min before the measurement of the supernatants by LC-MS with the same test conditions as those described in the qe and Ce are the equilibrium adsorption amount (mg/g) and clenbuterol concentration (mg/L), respectively, kL and kF are the Langmuir constant (L/g) and Freundlich constant (L/g), respectively, maxq is the maximum adsorption capacity (mg/g), and 1/n is an empirical parameter related to adsorption intensity.The commonly-used adsorption isotherm models were considered to analyze the present adsorption test results, in this case, Langmuir and Freundlich isothermal models that were described by Equations (5) and (6), respectively.kL is the Langmuir constant (L/g), C0 presents the initial maximum clenbuterol concentration (mg/L), and RL can be used to indicate the favorability of adsorption process, which are described below:RL > 1, unfavorable;RL < 1, favorable;RL = 1, Linear;RL = 0, irreversible.To assess whether the adsorption is favorable or unfavorable, Equation (7) can be referenced.Ce is the equilibrium concentration of clenbuterol in solution (mg/L), qe (mg/g) is the adsorbed amount at equilibrium. T represents the temperature in Kelvin (K), and R is the universal gas constant (8.314 J/(mol K)).To study the inherent energy changes in the adsorption course, three basic thermodynamic parameters were measured, i.e., enthalpy change qe is the equilibrium adsorption amount (mg/g), Ce is the equilibrium concentration of the adsorbate in the solution (mg/L), kd represents the distribution coefficient (L/g), k is the selectivity coefficient, x represents competing species against clenbuterol, including terbutaline, salbutamol, and methyl red, and k′ is the relative selectivity coefficient.A glass vial was loaded with the MIPMs or NIPMs (20 mg), and then a 10 mL mixed solution containing clenbuterol, terbutaline, salbutamol, and methyl red (each with a concentration of 1 mg/L) was poured into the glass vial pre-loaded with MIPMs or NIPMs. After homogenization imparted by shaking, the mixture was placed into a thermostatic water bath oscillator, and the adsorption interactions proceeded at the oscillation rate of 120 rpm for 6.5 h which was sufficiently long for making the adsorption system reach equilibrium. Then, the resulting mixture was centrifuged at 4000 rpm for 5 min, and the supernatant was drawn for the LC/MS measurement with the same test conditions as those described in the Various media were employed to prepare 10 mg/L clenbuterol solutions, including water, a water solution of ammonium acetate (4.6 mmol/L), a water solution of 0.3%TX-100 (4.6 mmol/L), acetonitrile, and an acetonitrile solution of ammonium acetate (4.6 mmol/L). These prepared solutions were separately added into the vial pre-loaded with 50 mg MIPMs or NIPMs. After the mixtures had been homogenized by shaking, they were placed in a thermostatic water bath oscillator with an oscillation rate of 120 rpm/min for the isothermal adsorption for 5 h. The processed clenbuterol solutions were centrifuged for 5 min, and the supernatants were withdrawn for the LC-MS measurement. Based on the equilibrium adsorption quantity calculated, the imprinting factor (IF) was estimated according to the following Equation (14).A new batch of the clenbuterol-imprinted polymer microspheres was prepared to prove that the present method for the fabrication of MIPMs was reliable, and the newly prepared sample was designated as MIPMs-2. The adsorption isotherm was then measured for MIPMs-2 according to the isothermal adsorption test on MIPMs. Through comparison of the parameters obtained by fitting the adsorption isotherms for the adsorption systems with MIPMs and MIPMs-2, the reliabilities of the present fabrication method and isothermal adsorption test could be assessed.v/v) at a volume of 10 mL and then with 2 mL methanol. A water solution of clenbuterol  subsequently passed through the washed MISPE column at a flow rate of about 5 mL/min under negative pressure. Finally, the MISPE column adsorbed with clenbuterol was successively eluted with a mixture of methanol/acetic acid  at volumes of 1 mL, 2 mL, and 2 mL. After the residual solution had been drained out of the MISPE column, the eluted solution was examined using LC-MS based on the test conditions as mentioned earlier. The MISPE test was repeated nine times, and the spike recovery was calculated for each cycle. Consequently, the recycling performance of the MISPE column with the MIPMs was evaluated based on the comparison of the spike recoveries calculated.The prepared MIPMs were used as the stationary phase of the molecularly imprinted solid-phase extraction (MISPE) column. Briefly, the deionized water and methanol were firstly used to thoroughly wash the MISPE column walls which were then dried in air. Sieve plates were employed to seal the open ends of the MISPE column, and thus the falling of the MIPMs was prevented. The filled MISPE column was further washed with a mixture of methanol and acetic acid ) at one end and a hydrophilic moiety (–NH2) at the other exist in the molecular structure of clenbuterol . Such a discrepancy between the adsorption kinetics as for the adsorption systems with the MIPM and NIPMs was assumed to stem from the reduced content of oxygen-containing functional groups in the MIPMs relative to the NIPMs, which weakened the chemical interactions between oxygen groups  values in comparison with those of the NIPMs, together with the relative selectivity coefficient (k′) values largely exceeding 1. This result is an indication that, in comparison with the NIPMs, the MIPMs exhibited an enhanced selectivity to clenbuterol among potential competing analogs with high structural similarities  on the MIPMs, relative to those on the NIPMs, which is beneficial for the selective uptake of the clenbuterol in the subsequent adsorption tests since too many oxygen groups will normally debase the specificity. The MIPMs show much better adsorption performance towards clenbuterol in comparison with the NIPMs, and more importantly, they also exhibit an excellent selectivity to clenbuterol among some potential analogs with high structural similarities. The recyclability of the MIPMs in the clenbuterol enrichment is also manifested through running the MISPE column equipped with the MIPMs nine times. Furthermore, the reliability of the fabrication method is also verified by the comparison of two batches of the MIPMs. Therefore, these MIPMs possess great potential for environmental monitoring and purification applications towards clenbuterol and other threatening species posing a severe threat to human life safety.


Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation. Engagement of T cell receptors (TCRs) induces the formation of microclusters that mediate the downstream signalling events. Here the authors show, using high resolution TIRF-SIM and live cell imaging, that ZAP70 and LAT are recruited to TCR with distinct kinetics, with the delayed ZAP70-TCR association modulated by TCR-induced calcium flux. LAT contains several tyrosines, which, after phosphorylation, can bind Src homology (SH2)-containing molecules, notably GADS, GRB2, and PLCγ1. This LAT complex subsequently recruits other adapters and enzymes, including SLP76, VAV1, NCK, and ADAP. Thus, LAT serves as an important scaffold for the recruitment of multiple downstream effectors involved in TCR signal transduction.T cell activation is mediated by engagement of the TCR, which consists of the α and β chains, the CD3δ, ε, γ, and TCRζ subunits. Recognition and binding of peptide-loaded major histocompatibility complex protein (pMHC) by the α and β TCR chains initiates the signal transduction cascade by recruiting Src-family protein tyrosine kinases (PTKs), predominantly Lck, or Fyn, to phosphorylate the immunoreceptor-based tyrosine activation motifs (ITAMs) on the intracellular CD3 and TCRζ subunits of the TCR. Phosphorylation of the ITAMS leads to the binding and activation of a Syk-family PTK, zeta-chain-associated protein kinase 70 (ZAP-70), which in turn phosphorylates key adapter proteins, including the transmembrane protein, linker of activation of T cells, or LAT4. This sensitivity is thought to be, in part, the result of signal amplification from the transiently engaged TCRs through a multi-protein structure at the membrane called the TCR microcluster5. Within seconds of T cell engagement to an activating surface, submicron-sized clusters marked by the TCR and other signaling molecules form at the contact site and act as a platform for the recruitment and activation of downstream effector molecules6. Studies using anti-TCR-coated coverslips or pMHC-containing lipid bilayer to activate T cells have shown concentrated tyrosine phosphorylation activity, as well as dynamic localization of TCRζ, ZAP70, and LAT to these microclusters, indicating that the TCR microcluster functions as a basic signaling unit during T cell activation7. Moreover, the dynamic interaction between TCR microclusters, actin cytoskeleton, and adhesion molecules leads to the formation of an immunological synapse between the T cell and APC to facilitate lysis of target cells, directed cytokine secretion, and other effector functions9.T cells display remarkable sensitivity to antigen despite the relatively weak affinity of TCRs for pMHCs and low numbers of stimulatory ligand on the antigen presenting cell (APC) surface10. In similar fashion, oligomerization of the LAT signaling complex was shown to be induced by multivalent interactions between GADS, ADAP, SLP76, and LAT, suggesting that a combination of adapter interactions drives microcluster formation11. Expanding on the crosslinking model, an in vitro reconstitution study has proposed that microclusters form due to a phase transition mediated by crosslinked LAT, GRB2, and SOS1 molecules12. In addition, Lillemeier and colleagues have proposed a “protein island” mechanism, whereby TCR and LAT naturally exist as nanoscale clusters (‘islands’) and become linked to neighboring “islands” after TCR ligation to form microclusters13.TCR microcluster formation is thought to involve non-covalent crosslinking between adapter and receptor proteins downstream of TCR ligation. One such mechanism involves cooperative interactions between LAT, SOS1, c-Cbl, and GRB2 molecules, in which multiple binding sites on LAT and SOS1 or c-Cbl for the SH2 and SH3 domains of GRB2 enable oligomerization of LAT-associated signaling molecules14. These observed differences come as no surprise as SMLM images are prone to drift correction and registration errors16, and can yield varying results depending on the reconstruction parameters18. In addition, SMLM studies utilize fixed cell samples and cannot observe the evolution of microcluster structures during T cell activation. To avoid these issues, we performed live, super-resolution, multicolor imaging of activated T cells using total internal reflection fluorescence structured illumination microscopy (TIRF-SIM).Single molecule localization microscopy (SMLM) studies of activated T cells have produced different observations regarding the arrangement of TCR signaling molecules in microclusters. Studies proposing the “protein island” model have observed strong segregation between 50 and 200 nm-sized TCR and LAT “islands” while another study observed a relatively heterogenous mixture of nanoclusters and transient interactions of TCR and LAT19. Moreover, kinetic studies of T cell activation have focused on the sequence of events downstream of microcluster formation such as vesicle recruitment, MTOC repositioning, and immune synapse formation23. The earliest study describing TCR-activated signaling microclusters reported that microcluster formation and calcium flux were detected immediately after contact between the T cell membrane and the anti-TCR-coated coverslip6. Extending these observations, Huse et al used photo-activatable pMHC to show that GRB2 clustering and calcium flux were observed at 4 s and 6–7 s after TCR stimulation24.Whereas the spatial localization of microcluster components has been studied in great detail, imaging recruitment kinetics has been challenging due to the rapid recruitment and internalization of signaling molecules at microclustersHere we report that TCR microclusters contain two spatially distinct domains, and that these domains assemble in discrete, sequential kinetic steps. These kinetic steps are regulated by intracellular calcium flux dependent on T cell stimulation. The results suggest the presence of a previously unrecognized feedback mechanism that regulates T cell activation.25. However, in addition to the limitations of SMLM mentioned above, we and others have reported artifactual clustering of photoactivatable fluorescent proteins used in SMLM27 and poor resolution in antibody-based SMLM methods due to inadequate labeling of targeted structures29. Given these issues, we sought to use TIRF-SIM, which yields ~100 nm resolution, and is compatible with well characterized, monomeric fluorescent proteins to perform live imaging of microcluster formation in T cells30.As some TCR microclusters are smaller than the diffraction limit (~250 nm) of conventional light microscopes, previous studies have utilized SMLM techniques to gain further insight into microcluster structure and composition. To date, SMLM studies have revealed patterns of molecular assembly within microclusters, the nanoscale clustering of molecular components, and the 3D distribution of concentrated receptors in resting T cellsJurkat T cells expressing TCRζ-Halo conjugated to Janelia Fluor 646 ligand (JF646) and ZAP70-Emerald were activated on anti-CD3ε-coated glass and imaged using TIRF-SIM. TCRζ-Halo and ZAP70-Emerald signals co-localized at each microcluster Fig.  as expecoC), and imaged cells using TIRF microscopy. As expected, activated Jurkat cells co-expressing CD3ε-YFP and TCRζ-Halo-JF646 showed simultaneous recruitment of both integral TCR components to microclusters , and the kinetic lag between TCRζ-GRB2 equaled the combined kinetic lags between TCRζ-ZAP70, and ZAP70-GRB2  showing that the several lags in recruitment were not affected by the choice of tagged fluorescent protein or the combination of microcluster molecules used to measure it. The kinetic lags between TCRζ-ZAP70, and ZAP70-GRB2 were affected by temperature, though the ZAP70-GRB2 kinetic lag appeared to stabilize at ~15 s above 30 °C  and at 37 °C (~10 s), presumably the temperature at which most previous studies were done. Taken together, the above results provide evidence for a non-stochastic, stepwise recruitment of the kinase ZAP70 to the TCR, followed by recruitment of signaling proteins Grb2 and then c-Cbl to the ZAP70-bound TCR.Discrete, sequential kinetic lags were also observed in a Jurkat T cell triple-expressing TCRζ-Halo-JF646, ZAP70-Apple, and GRB2-Emerald Fig. . Switchi °C Fig. . The tem+ cells were purified and expanded from human peripheral blood and transfected with either TCRζ-Emerald and ZAP70-Scarlet or ZAP70- Emerald and Grb2-Scarlet. Transfected cells were then dropped on stimulatory coverslips coated with anti-CD3 and anti-CD28 antibodies and imaged at 37 °C. As observed in Jurkat cells, primary human T cell blasts exhibited delays in the TIRF time-lapse images between both the recruitment of ZAP70-Scarlet to TCRζ-Emerald and Grb2-Scarlet to ZAP70-Emerald  as between ZAP70-GRB2, suggesting that LAT, SLP76, and GRB2 are recruited simultaneously to ZAP70-marked microclusters . Consistent with the simultaneous recruitment of LAT, SLP76, and GRB2 adapter molecules, co-expression of PLCγ1-CFP, GADS-YFP, and GRB2-Apple or SLP76-CFP, NCK-YFP, and GRB2-Apple showed coincident appearance of PLCγ1, GADS, GRB2, SLP76, and NCK at individual microclusters , indicating that LAT-associated adapters and enzymes become recruited to the signaling domain simultaneously. Overall, these results show that whereas signaling domain formation is delayed relative to the receptor domain, signaling domain components are recruited simultaneously during its formation, consistent with the highly cooperative nature of protein-protein interactions10.Upon TCR triggering, LAT phosphotyrosine motifs serve as binding sites for SH2 domain-containing proteins, including PLCγ1, Grb2, and Gads. Gads recruits the adapter SLP-76 to LAT and SLP-76 in turn recruits NCK, ADAP, and VAV1ers Fig. . Moreove32. Consistent with these results, we observed a short kinetic lag between recruitment of Grb2 and c-Cbl that is visually apparent between the red (Grb2) and green (c-Cbl) curves during the recruitment phase . Given that multivalent crosslinking of LAT is thought to drive overall microcluster formation12, these results are somewhat surprising, and indicates that receptor domain formation can be independent of LAT crosslinking. In addition, molecules that can be directly recruited to proteins in the receptor domain  can bypass the LAT crosslinking mechanism.Though TCRζ, ADAP, SLP76, GADS, VAV1, PLCγ1, NCK, GRB2, and c-Cbl localized to microclusters in E6.1 Jurkat cells, only TCRζ, GRB2, NCK, and c-Cbl signals were apparent at ZAP70-marked microclusters in J.LAT KO cells, with a partial localization for NCK Fig. . The res33. In particular, the WASP family member, WAVE/Scar is critical for ARP2/3-driven F-actin polymerization downstream of TCR activation, leading to rapid, radial lamellipodial growth in T cells41. Of the three isoforms, we chose WAVE1 for its selective localization to the leading edge of lamellipodia42 to report ARP2/3-driven cell expansion in activated T cells. Whereas mEmerald-WAVE1 localized strongly to the leading edge of stimulated E6.1 cells, WAVE1 was not detected at the cell periphery in J.LAT KO cells  and longer at physiological concentrations (1.0–2.0 mM CaCl2), with the transition to longer kinetic lags taking place between 0.5 and 1.0 mM CaCl2  or preformed clusters45 are within the resolution limit of TIRM-SIM (80–120 nm), preformed TCR and LAT clusters were not observed in resting cells or during the early stages of T cell activation. In contrast, other super-resolution studies have reported significantly smaller sizes for TCR and LAT complexes, as oligomers of 2–5 TCR and LAT molecules14, or strictly as monomeric TCRs46. As such nano-scale sizes are beyond the resolution limit of TIRF-SIM, imaging techniques with higher resolution will need to be utilized to further investigate the nano-scale arrangement of molecular components within the receptor and signaling domains, such as correlative super-resolution imaging using both TIRF-SIM and SMLM.Super-resolution imaging of activated Jurkat T cells revealed that microclusters contain spatially distinct receptor and signaling domains at the sub-diffraction scale. This spatial separation of microcluster compartments is supported by previous biochemical studies showing that LAT molecules are corralled within distinct membrane domains22. However, the domains described here are at smaller length scales and occur in seconds rather than minutes after activation. The spatial separation and the kinetics of molecular recruitment of the receptor and signaling domains we observed suggest distinct molecular mechanisms driving the oligomerization of TCR and LAT complexes. Our results showed that TCR clustering and ZAP70 recruitment to TCR were independent of LAT. This is possibly due to crosslinking from the coverslip-bound anti-TCR antibody, or to self-oligomerization of ligand-bound TCRs47. In addition, as cooperativity of defined protein–protein interactions drives the oligomerization of LAT complexes, the simultaneous recruitment of signaling domain molecules is consistent with stochastic crosslinking of multi-protein complexes11. However, two key questions are raised regarding the segregated but neighboring microcluster domains. First, if the signaling domain is composed of LAT-associated complexes and is formed by their mutual crosslinking, why does signaling domain formation take place directly adjacent to the receptor domain? One possibility is that the ZAP70 kinase is activated while tethered to the TCR within the receptor domain, becomes detached and phosphorylates substrates in nearby domains as described previously48. However, the released pool of active ZAP70 will soon encounter phosphatases surrounding the microcluster and become inactivated. Thus, substrates such as LAT and SLP76 are required to be in proximity of the receptor domain to be robustly phosphorylated by activated ZAP70. In support of this idea, we note that the previously reported length scales of ZAP70 movement at the membrane (~100 nm) correlates to distances between the receptor and signaling domains48.Spatial segregation between receptor and signaling domains is reminiscent of secretion and signaling domains reported in a CTL synapse33, molecular binding between the receptor and signaling domain components would need to counteract the pulling forces from the inward actin flow. Indeed, over time a portion of peripheral signaling domain clusters succumbed to the pulling force, and became detached from microclusters as evident from time-lapse TIRF-SIM images  before the ligand disassociates from TCR, the large kinetic delays between the ensemble recruitment of ZAP70 to TCRζ and the LAT complex to ZAP70 may need to be considered when calculating the kinetic parameters. In particular, the long, calcium-dependent delay in the recruitment of ZAP70 to TCR clusters  is surprising given the high affinity of ZAP70 for phosphorylated ITAMs54. In addition to the stochastic inhibition from phosphatase activity, and molecular competition for ITAM binding, this non-stochastic, calcium flux-induced kinetic delay may indicate a novel negative feedback mechanism to prevent ZAP70 recruitment to activated TCR.Contrary to the stochastic enlargement of microclusters predicted by the “crosslinking” and “protein island” models, we observed a non-stochastic process of microcluster assembly through stepwise recruitment of TCR, ZAP70, LAT-associated signaling complex, and c-Cbl. As the stepwise recruitment pattern follows the known biochemical sequence of TCR signaling, and as disruption of signaling domain recruitment impeded the progress of downstream pathways in J.LAT KO cells, our kinetic results have multiple implications for models of TCR signal transduction such as the kinetic proofreading model52. As the functional signaling domain is required for downstream signaling events such as calcium flux and actin polymerization-driven cell spreading, and as the kinetic lags are modulated by calcium-dependent mechanisms, this active signaling window is a potential target for downstream regulation of TCR signaling.Second, the kinetic delays between recruitment of microcluster components has implications beyond the scope of the kinetic proofreading model. For instance, the delayed recruitment of c-Cbl relative to the signaling domain, which represents a time window of active signaling before signaling domain disassociation, is not included in such T cell activation models53, TCR signaling would be dampened by intracellular calcium flux. In support of this, a previous study showed increased phosphorylation of TCR signaling components and enlargement of TCR microclusters after calcium chelation20. On the other hand, artificially increasing calcium in Jurkat cells promoted actin polymerization and reduced TCR mobility on the T cell surface55, suggesting that calcium flux may alter TCR microcluster kinetics through manifold pathways. Overall, our results point to an important distinction between TCR activation thresholds before and after calcium flux, and to a new role for calcium in downregulating TCR signaling. Additionally, whereas previously reported negative feedback mechanisms have focused on the role of phosphatases such as SHP-1 in reversing key phosphorylation events during TCR activation57, the calcium-dependent kinetic delays in microcluster assembly represents a potentially new mode of TCR regulation.Third, our results show that calcium signaling has an inhibitory effect on TCR signaling. As the induction of calcium flux was shown to significantly increase kinetic lags, and as the kinetic proofreading model predicts a correlation between activation threshold for TCR ligand interactions and kinetic delays between the signaling stepsWe note here that the spatial and kinetic results were obtained using a specific in vitro system at room temp to study T cell activation. To further substantiate these findings, microcluster kinetics should be probed under other activation settings. Furthermore, future experiments should test the effect of agonist strength on kinetic recruitment parameters. Having stated this, we reiterate the strength of our in vitro system, which allowed consistent imaging of TCR activation with high spatial and temporal resolution. Whereas activating T cells at room temp revealed the large kinetic lags in microcluster recruitment, significant kinetic lags were observed at physiological temp as shown in Fig. 20. ZAP70-Scarlet and GRB2-Scarlet were generated by cloning ZAP-70 or Grb2 cDNAs into HindIII-BamHI or NheI-AgeI digested pLifeAct_mScarlet_N1 plasmid respectively . TCRζ-Halo, LAT-Halo, SLP76-Halo, GRB2-Halo, and ZAP70-Halo were generated by cloning PvuI-SbfI  or NheI-ApaI  digested Halo sequence from pHTC-Halotag CMV-Neo vector (Promega) into plasmids containing the respective cDNA sequences. mEmerald-WASP1-N-14 was a gift from Michael Davidson (Addgene plasmid # 54315). pGP-CMV-GCaMP6m (GCaMP6) was a gift from Douglas Kim (Addgene plasmid # 40754). Generation of Nck-GFP, Nck-YFP, ADAP-GFP, Vav1-GFP, Vav1-YFP, c-Cbl-YFP, c-Cbl-CFP, PLCγ1-CFP, CD3ε-YFP, GADS-YFP, and SLP76-CFP were described previously33.Constructs used in this paper were generated as follows: LAT-Emerald, GRB2-Emerald, ZAP70-Emerald, SLP76-Emerald, GADS-Emerald, and TCRζ-Emerald were generated by cloning AgeI-NotI  or AgeI-BsrGI  or AgeI-NheI  digested Emerald sequence from Emerald-zyxin6  into plasmids containing the respective cDNA sequences. Generation of ZAP-Apple and Grb2-Apple were described previously226  and mouse monoclonal anti-TCRζ (pY142) antibody . BAPTA-AM, Indo-1-AM and Fluo-4-AM were from Invitrogen. EGTA and ionomycin were purchased form Sigma. Halo-tag ligand conjugated to Janelia Fluor (JF)-646 and -549 were gifts of Luke Lavis at Janelia Research Institute.Human anti-CD3 (HIT3a clone) monoclonal antibodies were purchased from Pharmingen and were used to coat coverslips for imaging assays and OKT3 (produced in the laboratory) was used to trigger T cell activation in calcium flux assayed by flow. The following antibodies were used for immunostaining: mouse anti-pLAT32. J.LAT KO and J.CaM2.5 cells were gifted from Art Weiss and have been described previously36. E6.1 Jurkat cells were cultured in RPMI , 10% fetal bovine serum , and 1% penicillin–streptomycin (Life Technologies). For transient transfections, 1e6 Jurkat cells were transfected with 2 μg DNA using the Nucleofector Kit V , Program X-001 24 h prior to imaging. Prior to imaging Jurkat cells were spun down and resuspended in imaging buffer . For calcium chelation and calcium titration experiments imaging buffer without CaCl2 was used.Jurkat E6.1 cells have been described previously+ cells were isolated using the MACS Pan T Cell Isolation Kit . Lymphoblasts were generated by activation with human T cell TransAct  in RPMI supplemented with 10% FBS, 1% penicillin–streptomycin and 50 U/ml of human IL-2. T lymphoblasts were cultured at 37 °C in 5% CO2 for 6 days in exponential growth phase. Cells were then cultured for an additional day in the presence of 10 U/ml of IL-2. Cells were transfected with a LONZA electroporator using LONZA solution for primary human T cells  and protocol T-23. Primary human T cells were imaged 5 h after transfection.Primary human peripheral blood T cells were obtained without donor identifiers from the National Institutes of Health Blood Bank. Mononuclear cells were isolated by Ficoll density gradient centrifugation. Untouched CD358. Jurkat cells were added to HIT3a antibody-coated 8-well coverslip chambers  and imaged at RT (21 °C) or other specified temperature in imaging buffer. Jurkat cells expressing Halo-tagged constructs were labeled with 100 nM Janelia Fluor Halo-647 ligand for 30 min at 37 °C. Cells were imaged after three washes in complete medium and then resuspended in imaging buffer. All chambers used for live cell imaging contained imaging buffer. TIRF images from live cells were collected with a Nikon Ti-E inverted microscope, using a 100× SR Apochromat TIRF objective lens , and an Andor iXon Ultra 897 EM charge-coupled device camera . Time-lapse images were collected at 3 s/frame except for experiments with short kinetic lags such as Figs. 30. In brief, images were acquired using an inverted microscope  fitted with a 100 × 1.49 NA objective (Olympus) and a spatial light modulator to provide structured illumination. TIRF-SIM image in Fig. Preparation of antibody-coated coverslip has been described previouslyd at RT 2 °C or otJurkat T cells were allowed to adhere to the HIT3a antibody-coated coverslip for 2 min at 37 °C in imaging buffer and then fixed for 30 min in 4% (wt/vol) PFA solution (Electron Microscopy Sciences). Samples were permeabilized in 0.1% Triton-X-100 for 3 min and then incubated in a blocking solution consisting of 10% FBS (Sigma-Aldrich), 0.01% sodium azide (Sigma-Aldrich), 1× PBS for 1 h at room temperature (RT). After three washes in 1× PBS, the cells were stained with Alexa 647-conjugated antibody in blocking solution (20 μg/ml) for 1 h at RT. The cells were washed 3× in PBS and imaged using TIRF-SIM or TIRF microscope as detailed above.R2 was monitored for proper fitting of normalized intensity data. The Kinetic lag was obtained by calculating the difference in the half-max of non-linear fitted curves. Kymographs in Fig. Image J was used to measure the distribution of molecules at microclusters in multi-color TIRF-SIM images as shown in Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6