Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae  and female adult worms  as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model , which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter's ‘Metadrug’ tool screening predictions.As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of  Brugia malayi, B. timori and Wuchereria bancrofti and according to a recent report about 1 billion people (18% of the world's population) are at risk of infection . Although, the World Health Organization launched a global filariasis elimination programme Among the six neglected tropical diseases, lymphatic filariasis (LF) is one of the major health problems in 73 tropical and subtropical countries in Africa, Asia, South and Central America and the Pacific Islands. According to the World Health Organization (WHO) global report, over 120 million people are currently infected with LF Eucalyptus tereticornis were chemically and biologically investigated in details, which afforded an anti-filarial agent, Ursolic acid  as a major constituent. The in-vitro activity of UA against the mf and adult worms, in-silico docking studies on glutathione-s-transferase (GST) parasitic enzyme and in vivo activity against B. malayi in Meriones unguiculatus model have been discussed here in detail.As a part of our drug discovery program, we recently reported a pentacyclic triterpenoid, glycyrrhetinic acid 1H and 13C NMR spectra were recorded on a Bruker 300 MHz spectrometer in deuterated pyridine. ESI-MS was carried out on a LCMS-2010 V  simultaneously in positive (detector voltage 1.6 KV) ionization under scan mode. The scan speed of the mass analyzer was 2000 m/z per sec within the range of 400–1000 m/z. A positive full scan mode for screening and library assisted identification was used whereas time schedule selected-ion mode (SIM) in +ve ionization mode of the characteristic abundant adduct ions. Purity of UA was assessed by HPLC and was ≥95% 2 & CH3) in UA were determined. The vacuum liquid chromatographic separations (VLC) were carried out on TLC grade Silica gel H (average particle size 10 µm) purchased from Merck, . All the required solvents and reagents were purchased from Spectrochem  and Thomas Baker Pvt. Ltd., India. Pre-coated Silica gel (60F) TLC plates 2.5 mm (Merck) were used to determine the profiles of VLC fractions and their purity. The developed TLC plates were first observed at 254 nm in UV and then sprayed with Bacopa reagent [vanillin-ethanol sulphuric acid (1 g: 95 ml: 5 ml)] and spots were visualized after heating the TLC plate at 110°C for 5 minutes.The E. tereticornis were collected from the medicinal farm of Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, Uttar Pradesh, India during the month of January, 2008. A voucher specimen # 12470 was deposited in the Herbarium section of the Botany and Pharmacognosy Department of the institute.The leaves of E. tereticornis (1.3 kg) were powdered and defatted with n-hexane. The defatted leaves were further extracted with MeOH (4×5 L)  3 fraction (35.0 g) was found active hence subjected for chromatographic separation over VLC-1 using silica gel H (260 g). The gradient elution of VLC was carried out with mixture of hexane, CHCl3 and MeOH in increasing order of polarity.The air dried leaves of  (4×5 L) .The comb3 (1∶1) to CHCl3 – MeOH (99∶1) was a complex mixture. Hence a part of it (5 g) was further chromatographed over VLC-2, using TLC grade silica gel H (50 g). Gradient elution of VLC-2 was carried out with mixture of hexane, CHCl3 and MeOH in increasing order of polarity. Fractions 175–182 (1.5 g) eluted with CHCl3 (100%) afforded a white amorphous compound (95% pure) which on further crystallization with CHCl3 yielded UA (99% pure.). The 1H and 13C NMR and ESI-MS spectra of the homogenous compound (UA) were recorded and the spectroscopic data are presented as below:Fractions 3–42 (7.2 g) eluted with hexane- CHCl+, C30H48O3, 1H NMR : δ 0.77, 0.78, 0.98, 1.09, 1.14  0.92 & 0.96 , 2.82 , 3.20 . 13C NMR : C1- 39.5 (t), C2- 28.3 (t), C3- 78.7(d), C4- 39.9 (q), C5- 56.3 (d), C6- 19.1 (t), C 7- 33.9 (t), C8- 40.4 (q), C9- 47.0 (d), C10- 37.7 (q), C11- 23.9 (t), C12- 126.0 (d), C13- 139.6 (q), C14- 42.9 (q), C15- 28.9 (t), C16- 25.2 (t), C17- 48.5 (q), C18- 54.0 (d), C19- 30.5 (d), C20- 39.7 (d), C21- 31.3 (t), C22- 37.6 (t), C23- 29.0 (s), C24-15.8 (s), C25- 16.4 (s), C26- 17.5 (s), C27- 24.1 (s), C28- 179.7 (q), C29- 17.7 (s), C30- 21.4 (s) (ESI-MS m/z 457 [M+H]21.4 (s) .M. coucha were kept in climate  and photoperiod (12hr light-dark cycles) controlled animal room. They were fed standard rodent chow supplemented with dried shrimps (M. coucha) and had free access to drinking water.Animals: The study was approved by the Institute's Animal Ethics Committee (IAEC)  of CSIR-Central Drug Research Institute, Lucknow, India, under the provisions of CPCSEA , Government of India. All the experiments in animals were conducted in compliance with the IAEC guidelines for use and handling of animals. Throughout the study, jird and B. malayi infection in animals: The human sub-periodic strain of B. malayi was cyclically maintained in M. couchaMeriones unguiculatus) Aedes aegypti mosquitoes. Infective larvae of B. malayi isolated from experimentally infected A. aegypti mosquitoes which were fed on microfilaraemic M. coucha (150–200 mf/10 µl blood), were washed thoroughly with insect saline (0.6%). Each animal was inoculated with 100 (M. coucha) or 200 L3 (jirds), through subcutaneous (s.c.) and intraperitonial (i.p.) routes, respectively.B. malayi infection were washed thoroughly in medium Hanks Balanced Salt Solution  containing mixture of antibiotics  and used for the present study.Isolation of parasites: Mf and adult worms  isolated freshly from peritoneal cavity (p.c.) of jirds harboring 5–6 month old In vitro assays: Based on viability of the parasites, two in vitro motility and 3--2,5 diphenyltetrazolium bromide (MTT) reduction assays The UA and IVM were dissolved in DMSO whereas DEC-C was prepared in sterile triple distilled water (STDW). The antifilarial agents were used at 2-fold serial dilutions ranged from 15.63–1000 µM (DEC), 1.56–100 µM (UA) and 0.31–20 µM (IVM). The final conc. of DMSO in the incubation medium was kept below 0.1%. DMSO (<0.1%) was used in place of test agents solution for control.in vitro on mf and adult worms of B. malayi (as target parasites) using motility (Mf and adult parasite) and MTT (adult parasite only) reduction assay 2 atmosphere. The effect on motility of the parasite stages was examined under microscope and scored. The experiment was repeated twice. In case of mf, only motility assay was used.Motility assay: Efficacy of the UA and reference drugs was assessed minus the test agent at 37°C for 1 h. and was expressed as percentage (%) inhibition of control.Motility assessment: Parasite motility was assessed under a microscope after 24/48 h exposure to test substance and scored as: 0 = dead; 1–4 =  loss of motility . Loss of motility is defined as the inability of the worms to regain pretreatment level of motility even after incubating in fresh medium MTT- formazan colorimetric assay for viability of worms: The same female worms used in motility were then gently blotted and transferred to 0.1 ml of 0.5% MTT in 0.01 M phosphate-buffered saline (pH 7.2) and incubated for 1 h at 37°C. The formazan formed was extracted in 1 ml of DMSO for 1 h at 37°C and its absorbance was measured at 510 nm in spectrophotometer . The mean absorbance value obtained from 4 treated worms was compared with the controls. The viability of the treated worms was assessed by calculating per cent inhibition in motility and MTT reduction over DMSO control worms in vitro hits: 100% inhibition in motility of female adults or mf and or ≥50% inhibition in MTT reduction ability of female parasites was considered acceptable antifilarial  activity and picked up as hits and subjected to further testing in vivoCriteria for assessment of 50: For IC50 (the concentration at which the parasite motility was inhibited by 50%) determination of the parasites were incubated with two fold serial dilutions from 1.56–100 (UA), 0.31–40 (IVM) and 15.63–1000 µM (DEC-C) using triplicate wells of cell culture plate. Experiments were run in duplicate and incubations were carried out in replicates for 24/48 hr as above. After incubation, inhibition in motility  and MTT reduction potential of the parasites were assessed as above. The experiment was repeated twice.Determination of IC50): The cytotoxicity assay of the test substances was carried out broadly following the method of Pagé et al. 6 cells/ml (100 µl per well) in DMEM supplemented with 10% heat inactivated FBS. A three-fold serial dilution of the test substances (starting from >20 x LC100 conc. of the test agent) in test medium was added. The plates with a final volume of 100 µl/well were incubated in 5% CO2 atmosphere at 37°C. After 72 h incubation 10 µl of 0.025% Resazurin in phosphate buffered saline  was dispensed as indicator for viability followed by an additional incubation for 4 h and the plate was then read in a fluorescence reader  at excitation wavelength of 530 nm and an emission wavelength of 590 nm. The assay was run in replicates in each of two independent experiments.Determination of Cytotoxic concentration 50 (CC50 and CC50 transferred to a graphic program (Excel) were calculated as described by Page et al. Data of ICSelectivity Index (SI) of the UA was computed by the formula as: −1 Å. Re-optimization was done by MOPAC  method until the RMS gradient attained a value smaller than 0.0001 kcal mol−1 Å. The 3D chemical structure of known drugs DEC-c (CID:15432) and IVM (CID: 6321424) were retrieved from PubChem compound database . The theoretically solved structure of B. malayi glutathione-S-transferase (BmGST) was selected as the potential target for molecular docking simulation studies. The BmGST crystallographic protein 3D structure was retrieved from Protein Data Bank (PDB ID: 1SJO). The Ligsite program was used to identify the potential active site of BmGST model for molecular docking studies and was then cross-checked with template active site as shown in Molecular modeling and geometry cleaning of the UA was performed through ChemBioDraw-Ultra-v12.0 . The 3D structure was subjected to minimized the energy by using molecular mechanics-2 (MM2) force field until the root mean square (RMS) gradient value became smaller than 0.100 kcal molM. coucha was administered with UA and DEC-c at 100 and 50 mg/kg body weight respectively through i.p. route for 5 consecutive days. The suspensions/solutions of UA/DEC-c were prepared daily before administration to the animals. Control animals received vehicle only.Administration of UA and the reference drugs: The finely powdered UA was suspended in 0.1% Tween-80 prepared in sterilized tap water. Solution of DEC was made in plain STW. B. malayi -M. coucha model: Animals harboring 5–7 months old B. malayi infection and showing progressive increase in microfilraemia were used in this study. UA and reference drug treated groups and an equal number of infected untreated animals kept as vehicle treated control, consisted of 5 animals each in two experiments were used.Mf count in 10 µl blood drawn from tail of the animals between 12:00 noon and 1:00 PM Assessment of microfilaricidal efficacy: Microfilaricidal efficacy of UA was evaluated on day 7/8 and 14 p.i.t. and expressed as percent reduction in mf count over pretreatment level Assessment of macrofilaricidal and worm sterilization efficacy: Adult worms were recovered from heart, lungs and testes of treated and control animals All the surviving females were teased individually in a drop of saline to examine condition of intrauterine mf stages of the parasite Statistical analyses were carried out using Statistica version 7/GraphPad Prism 3.0 version software. Results were expressed as mean ± S.D. of data from 5–6 animals in two experiments. The data were subjected to One-way ANOVA analysis and the significance of the difference between means were determined by Newman-Keuls Multiple Comparison Test. P<0.05 was considered significant and marked as *, P<0.01 as highly significant and marked as **, and P<0.001 was very highly significant and marked as ***. The trend analysis was done by fitting the simple regression model (Y = A+BX) using the method of least squares. The slopes of the line were compared by Analysis of Variance.http://www.genego.com) The ADME/Toxicity parameters compliance was evaluated by screening through Metadrug™, a commercial tool of MetaDiscovery   and mf (LC100: 200 µM)   . The CHCcid (UA) . FinallyB. malayi using motility and or MTT assays and the results are summarized in 100: 50 µM) than adult worms (LC100: 100 µM) and its IC50 values were 35.36 and 8.84 µM against the respective parasite stages. UA exerted >86% inhibition in MTT reduction ability of the adult worms. It reduced the viability of female parasite in a gradual dose dependent manner as assessed by MTT reduction assay . The CC50 (>300 µM) and SI (>10) values of UA demonstrated that it is safe for carrying out in vivo screening , respectively. Its IC50 against adult worms was 3.05 µM and that of mf was 1.49 µM. However IVM was less effective when parasites were incubated for 24 hr. IVM failed to inhibit MTT reduction ability of female worms even after 48 hr incubation (100: 1000 µM) and mf (LC100: 500 µM); it inhibited MTT reduction skill of the adult parasite to the tune of 62.55%. The IC50 of DEC against the respective parasite stages were found to be 353.55 µM and 297.30 µM.The time point studied for the standard drug IVM was 24 hr and 48 hr as IVM has slow action on the parasites. After 48 hr post incubation IVM was effective in inhibiting motility of female adult worm and mf at a minimum conc. of 5.0 µM and 2.5 µM  and DEC-C  caused concentration dependent decrease in viability of the parasites. However, in case of IVM the viability was time and conc. dependent .Concentration-dependent' LCin vitro findings revealed that CHCl3 extract of E. tereticornis was microfilaricidal and macrofilaricidal, active against human filarial worm B. malayi and the active principal was localized to UA.In summary, BmGST theoretical protein structure 3D model was retrieved from PDB crystallographic database and later used for molecular docking simulation studies of UA, to explore the possible mechanism of action within the filarial worm (−1) similar to that of reference drugs, DEC-c  and IVM . Docking results of UA also showed formation of H-bond (length 3.0 Å) with aromatic hydrophobic residue TYR-106, this may be the reason of high binding affinity, stability and activity of UA. The other binding site amino acid residues within a selection radius of 4 Å from the bound UA against BmGST protein structure model were nucleophilic  e.g., threonine (THR-102), aromatic (hydrophobic) e.g., phenylalanine (PHE-8), tyrosine , polar amide e.g., asparagine (ASN-203), glutamine (GLN-49), hydrophobic e.g., leucine   is playing a significant role in the long-term existence of filarial worms in mammalian host. The GST enzyme is a well known potential molecular target to inhibit filarial parasite's growth ial worm . The doc LEU-50) . These rin silico ADME/Tox parameters screening study was performed through Discovery Studio v3.5 molecular modeling & drug discovery software . The UA, DEC and IVM were evaluated with standard descriptors and all the chemical descriptors and parameters of ADME were calculated  , which sAlthough, ADME results showed that UA violates Lipinski's rule of five due to high logP value (logP>5), hence may cause problem in absorption through biological membranes or intestinal absorption, but it still falls within the acceptable limit of rule of five, when compared with the reference drugs, DEC and IVM .viz., mutagenicity, tumorogenicity, skin irritation and reproductive/developmental toxicity parameters were evaluated for high doses or long term use toxicity. The toxicity screening results showed that UA and the reference drug IVM showed no features of risk of tumorogenicity, mutagenicity, reproductive toxicity and skin irritation, therefore UA is safe for human use, whereas DEC yielded a high risk of mutagenicity and reproductive toxicity.The toxicity risk assessment at high doses and/or long term use was evaluated through the OSIRIS web server for the reference drugs, DEC, IVM and the studied compound UA . In thisB. malayi in M. coucha at 100 mg/kg s.c. for 5 consecutive days. UA produced 4–33% lower microfilaremia  than 0 day throughout the post treatment observation period  caused around 54% (P<0.001) adulticidal action over the untreated control. A moderate embryostatic effect of UA  was also noticed in female worms . DEC-C tin vitro and in vivo with no adverse affect on health and general behavior of the treated animals.Together, the results of UA showed promising antifilarial activity The compound UA was evaluated through MetaDrug tool  for compliance to the standard ADME/Tox parameters. Results showed the information of metabolites, QSAR based prediction of ADME/Tox properties, therapeutic activities, information of analogues, pathways, potential targets and signaling pathway map by leveraging an extensive database of chemical structures and pharmacological activities and visualized in the context of pathways, cell processes, toxicity and disease networks that are perturbed by the compound and its metabolites. These results for UA are briefly discussed below:Prediction of therapeutic activities for UA: Large numbers of therapeutic activities for the compound UA were identified through MetaDrug tool . The evaluated therapeutic activities for UA were; allergy, Alzheimer, angina, arthritis, asthma, bacterial, cancer, depression, diabetes, HIV, heart failure, hyperlipidaemia, obesity, migraine, osteoporosis and many more. The predicted activities for UA were classified as active or non-active based on calculated values. The predicted properties of UA were calculated on the basis of Tanimoto Percentage [TP] values (standard cut-off ≥0.5) .Prediction of analogues, pathways and potential targets for UA: The chemical structures and the name of some known similar compounds or analogues were predicted by MetaDrug tool related to UA on the basis of structural similarity (in the range of 98–100%). MetaDrug tool also detected the potential biological pathways and the targets with experimentally known prior mode of action for UA .i.e., 1.834e-8 (7.736) with six network objects. TLR2 and TLR4 induce MyD88/IRAK/TRAF6-dependent pathway in target cells, leading to activation of transcription factors NF-kB, AP-1, CREB1 and IRF5, which induce production of various proinflammatory mediators including cytokines, chemokines, nitric oxide (NO) and prostaglandins, leading to inflammatory response .There are only a few medicinal plant extracts and the isolated molecules, which have shown good anti-filarial activity. The literature showed that some secondary metabolites such as triterpenoids and coumarins showed significant activity against filarial parasites. Our recent finding on the antifilarial activity of pentacyclic triterpenoid, glycyrrhetinic acid (GA) has given us advantage of exploring anti-filarial activity in UA, having similar pentacyclic triterpenoid chemical structure in vitro anti-filarial activity of UA against mf and the adult worms, prompted us to carry out it's in silico studies to investigate its possible mechanism of action. It is well known that filarial nematode's detoxify GST enzymes, which play a significant role in the survival of the parasites inside the host's body. This enzyme has effective ability to neutralize the reactive oxygen species (ROS) attack on membrane that acts as cytotoxic products and protect the helminths inside the host in silico molecular docking binding affinity of UA against the GST enzyme was studied. The docking experiments were performed, which showed high binding affinity of UA with BmGST enzyme.The in vitro and in vivo in its antifilarial activity. Further UA being a natural compound, has the possibility of lead optimization by QSAR approach. Thus, in view of potential antifilarial activity, absence of toxicity and favorable pharmacokinetics UA may be considered as a suitable lead for designing and development of a safe and effective antifilarial agent.It was observed that for killing the life stages of parasites in vitro, 10 times less concentration of UA was required than the drug DEC. Similarly, in vivo, UA treatment afforded 4–33% drop in microfilaraemia over 0 day throughout the post treatment observation period. While in the untreated control it was (progressively) higher than the pretreatment level and never equaled the 0 day level. The analysis trend in the DEC-treated animals showed that the mf count after an initial dramatic drop, increased gradually over time, while in UA treated animals microfilaraemia remained almost static. This clearly shows that UA was better than DEC in controlling microfilaraemia. Further UA exhibited 54% adulticidal and 56% embryostatic effect with static microfilaraemia while DEC produced ∼26% macrofilaricidal, 15% embryostatic and >85% microfilaricidal effect (on day 7 p.i.t.). These results indicate that UA is clearly superior to DEC with respect to macrofilaricidal and embryostatic effect though not with respect to microfilaricidal effect. It may be mentioned here that macrofilaricidal and embryostatic effect of UA were probably responsible for the low and static microfilaraemia. Thus, UA is better than DEC both Figure S1100 and IC50 of Ursolic acid (UA) for microfilariae and adult parasites of LCBrugia malayi. After incubation with UA for 24 h the viability of parasite was assessed in motility assay using mf (A) and adult female worms (B) and in MTT reduction assay using adult female worms (C).(TIF)Click here for additional data file.Figure S2100 and IC50 of diethylcarbamazine citrate (DEC-C) for microfilariae and adult parasites of LCB. malayi. After incubation with DEC-C for 24 h the viability of parasite was assessed in motility assay using mf (A) and adult female worms (B) and in MTT reduction assay using adult female worms (C).(TIF)Click here for additional data file.Figure S3100 and IC50 of ivermectin for microfilariae and adult parasites of LCB. malayi. After incubation with ivermectin for 24 h (A–C) and 48 h (D–F) the viability of parasite was assessed in motility assay using mf  and adult female worms  and in MTT reduction assay using adult female worms .(TIF)Click here for additional data file.File S1Linear Trend analysis of mf count data.(DOC)Click here for additional data file.


This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gα Stem cells are the cells that have the ability to make identical copies of themselves for the lifetime of the organism , and can also divide to generate progenitor or precursor cells that then differentiate into all cell types of a mature tissue (differentiation) . In geneSeveral studies have now confirmed the central role played by GPCRs in embryonic development and stem cell maintenance ,11,12. Ds, Gαi/o, Gαq/11 and Gα12/13, based on structural and functional similarities [s and Gαi/o families function to activate or inhibit the activity of adenylate cyclase with a consequential increase or decrease in cyclic AMP (cAMP) production [q/11 family activates phospholipase-Cβ (PLCβ) ultimately leading to intracellular Ca2+ mobilization from the edoplasmic reticulum [12/13 family is involved in activation of the Rho family of small GTPases [GPCRs are versatile signaling molecules that modulate the activities of diverse intracellular signaling via G proteins . G protelarities . Each Gαlarities . The Gαsoduction . Memberseticulum . In addi GTPases . These d GTPases ,28. The  GTPases ,30,31.Following activation of GPCRs, rapid attenuation or desensitization of receptor responsiveness is necessary to prevent uncontrolled signaling. Desensitization is initiated by phosphorylation of the receptor by GPCR kinases ,33 folloGPCR signaling is highly diverse and the engagement of different G proteins and the strength or duration of signaling differs not only between GPCRs, but also depending on the ligand and cellular environmental context of any given GPCR. Some GPCRs, such as sphingosine-1-phosphate receptor 1 (S1P1), exclusively couple to one G protein, whereas other GPCRs, such as lysophosphatidic acid receptors (LPA), can couple to multiple G proteins triggering diverse downstream signaling cascades ,41. Abers signaling has been shown to promote proliferation and pluripotency in self-renewing and differentiating mouse ESCs [i proteins is demonstrated to affect the morphology and organization of human induced pluripotent stem cells (iPSC) [in vitro neural differentiation  has revealed striking differences in GPCR expression within the different cell populations [The role of GPCR signaling in stem cell function, although undoubtedly important, has not been fully elucidated. Several lines of evidence are suggestive of a critical role, for instance many of the signaling cascades activated by GPCR signaling directly regulate, or are synergistic with, pathways that regulate ESC pluripotency and differentiation . Generaluse ESCs . Signalis (iPSC) . Dramatis (iPSC) . Comprehulations ,51.i.e., frizzled and rhodospin) will be discussed in this review exemplifying GPCR-mediated regulation of normal and malignant stem cells.The GPCR superfamily is divided into five sub-families, including glutamate, frizzled, adhesion, rhodopsin and secretin. The specific roles of GPCRs from two of these families (i/o protein inhibitor (pertussis toxin) [Drosophila [The frizzled (Fzd) family of GPCRs is activated by the Wnt family of lipoglycoproteins . Wnt sigs toxin) ,56. Furtosophila . As suchosophila .2+ pathway, Fzd/planar cell polarity (PCP) pathway and Fzd/β-catenin pathway. Agonist stimulation of the Fzd/Ca2+ pathway leads to elevated intracellular Ca2+ levels in a G protein-dependent manner that activates calcium-dependent protein kinase c (PKC) and Ca2+/calmodulin-dependent protein kinase [Nineteen Wnt proteins serve as the primary endogenous agonists for 10 Fzd receptors encoded in the human genome . There in kinase ,63. The n kinase . Agonistn kinase . The Wntn kinase .Oct-4) [Fzd receptors play an important role in mammalian development and stem cell self-renewal. The expression of Fzd5, 7 and 10 has been found in the gastrulating embryos of mice and is implicated in neural induction . EvidencOct-4) . In contOct-4) ,72,73 suWnt/β-catenin signaling is evolutionarily conserved and plays critical roles in development and disease . InapproDysregulation of Wnt/β-catenin signaling occurs in multiple types of CSCs and increasing evidence has demonstrated a crucial role for Wnt/β-catenin signaling in the self-renewal and malignant behavior of CSCs ,79,80,81q in the maintenance of AML LSCs [q regulates LSC growth and survival in vitro and in vivo, and controls β-catenin activity. Using a commercially available Gαq inhibitor, GP-antagonist 2A, our data indicates that ex vivo pre-treatment of LSCs with the antagonist impairs their proliferative capacity in mouse bone marrow and prolongs mouse survival [q expression leads to suppression of mitochondrial complex 1 subunits  and consequent disruptions in mitochondrial function and energy metabolism in leukemic cells [q signaling activation. Thus, targeting β-catenin signaling and energy metabolism by blocking Gαq signaling could represent a novel therapeutic approach to reduce leukemogenesis in aggressive AML.In support of this therapeutic rationale, we have recently shown an essential role for G protein subunit GαAML LSCs . By usinsurvival . Therefoic cells , providiIn vivo lineage tracing experiments using a heritable-inducible lacZ reporter gene introduced into Lgr5-expressing cells has shown that Lgr5 is a marker of adult intestinal stem cells. Further examination of Lgr5 expression patterns in mice has identified discrete populations of Lgr5-expressing cells in organs including skin, stomach, mammary gland, tongue, kidney and endometrium, indicating that Lgr5 may function as a universal epithelial stem cell marker [The rhodopsin class is by far the largest GPCR family, comprising almost 85% of GPCRs. The leucine-rich repeat-containing (Lgr) proteins are a distinct subset of evolutionarily conserved rhodopsin GPCRs containing a large extracellular domain with multiple leucine-rich repeats ,87. In vl marker ,89,90,91Epithelial homeostasis in the adult intestine is regulated by several signaling pathways and key among these is the Wnt signaling pathway . Hyperacet al. has demonstrated the role of Lgr5-expressing cells in the development of colon cancer. By applying in situ hybridization (ISH) to a panel of human normal colon, adenoma and carcinoma samples, significant increase in levels of Lgr5 mRNA is observed in all serrated lesions that is accompanied by expansion of proliferative and invasive compartments, suggesting that Lgr5 may support invasion and metastasis [A recent study by Baker tastasis . Within tastasis . Within tastasis . DeducinInterestingly, Lgr4 and Lgr6, two close family members of Lgr5, have also been implicated in the regulation of stem cell properties. Lgr6 expression is found to mark hair follicle epidermal stem cells, whereas Lgr4 expression plays a major role in prostate stem cell function ,105. Lgrq-coupled receptor that promotes calcium mobilization and PKC activation, whereas EP2 and EP4 couple to Gαs and stimulate cAMP accumulation and PKA activation [in vivo LSC function  [in vitro, it is shown that the EP ligand, PGE2, increases the activation of Tcf/Lef transcription factors, induces the loss of β-catenin phosphorylation and increases its nuclear accumulation [s with the β-catenin destruction complex member and Rgs protein, Axin [2-induced transcriptional activation of Tcf/Lef [s to Axin results in the displacement of APC and loss of β-catenin phosphorylation leading to its nuclear accumulation [The importance of GPCR signaling in driving LSC activity is further exemplified by the prostaglandin E (EP) receptors, another rhodospin class of GPCRs. Prostaglandins are a product of the cyclooxygenases Cox1 and Cox2 and their functions are initiated following binding of prostaglandins to their cognate GPCR receptors (EP1-4). EP1 is a Gαtivation . We haveequency) . Althougmulation . This prin, Axin . Overexp Tcf/Lef . Since tmulation   pathway, as reported in many cell types ,125,126.EGFR transactivaton can proceed via several mechanisms  leading to activation of a metalloprotease, inducing pro-HB-EGF processing . SubsequDespite the widespread overexpression of EGFR in cancer, EGFR-targeted therapies have produced only modest clinical responses in patients . The divGPCRs represent the largest class of cell surface receptors and are currently targeted by approximately 30%–40% of marketed drugs . SeveralEmerging evidence that the crucial CSC population, which drives resistance to therapy and patient relapse, is also reliant on GPCR-mediated signaling presents opportunities for therapeutic exploitation. Further characterization of key GPCR-mediated signaling cascades  that contribute to CSC self-renewal will facilitate the development of CSC-targeted therapeutic strategies. Given the important role of aberrant Wnt/β-catenin in promoting CSC activity, substantial efforts have focused on the development of therapeutic approaches to target this pathway. However, progress has been thwarted by the extremely complex nature of Wnt/β-catenin signaling, notably the crosstalk with various non-Wnt factors ,155. The


Lymphocyte signaling cascades responsible for anti-tumor cytotoxicity and inflammatory cytokine production must be tightly regulated in order to control an immune response. Disruption of these cascades can cause immune suppression as seen in a tumor microenvironment, and loss of signaling integrity can lead to autoimmunity and other forms of host-tissue damage. Therefore, understanding the distinct signaling events that exclusively control specific effector functions of “killer” lymphocytes (T and NK cells) is critical for understanding disease progression and formulating successful immunotherapy. Elucidation of divergent signaling pathways involved in receptor-mediated activation has provided insights into the independent regulation of cytotoxicity and cytokine production in lymphocytes. Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated. While the Fyn–PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn–ADAP pathway preferentially regulates cytokine production in NK and T cells. In this review, we discuss how the structure of Fyn controls its function in lymphocytes and the role this plays in mediating two facets of lymphocyte effector function, cytotoxicity and production of inflammatory cytokines. This offers a model for using mechanistic and structural approaches to understand clinically relevant lymphocyte signaling. Effector functions of lymphocytes are mediated through signaling cascades that transmit extracellular signals to intracellular response elements. These signaling pathways allow lymphocytes to respond to transformed or virally infected cells via cell-mediated cytotoxicity and inflammatory cytokine production. The identity of receptor–ligand interactions and downstream intracellular proteins responsible for inducing effector functions has been well established in NK and T cells. However, the divergent roles of signaling molecules in mediating distinct effector functions remain largely uncharacterized. Fyn is a membrane proximal, non-receptor tyrosine kinase responsible for initiating signaling cascades downstream of the TCR and multiple NK cell-activating receptors . By recrFyn is one of nine members  of the Src-family of non-receptor tyrosine kinases (SFKs) . Since tThrough its ability to recruit and phosphorylate the p85α subunit of PI(3)K, Fyn is able to activate signaling cascades responsible for a variety of cellular functions, including cell development, cell-mediated cytotoxicity, and production of inflammatory cytokines . This inFyn therefore represents a node of signaling divergence for two primary outcomes of lymphocyte activation, cytotoxicity and inflammatory cytokine production. In this review, we discuss how the structure of Fyn allows it to act as a critical regulator of divergent signaling pathways and multiple effector functions in lymphocytes. We discuss conformational changes necessary for substrate recognition, specifically PI(3)K and ADAP, as well as control of enzymatic activity. We also discuss how protein structure allows Fyn to act as the exclusive mediator of the ADAP signaling cascade and resulting inflammatory cytokine production, thereby regulating a portion of the systemic immune response.Src-family of non-receptor tyrosine kinases are pleiotropic in nature and multiple family members, including Fyn, are ubiquitously expressed in multiple cell types. This allows them to control a variety of cellular functions and highlights the need for maintaining signaling integrity both in terms of enzymatic activity and substrate specificity . Across in vitro characterization of the protein structure combined with in vivo characterization of Fyn function in different cell types provides a basis for understanding structure-based Fyn regulation in lymphocytes. The quaternary structure of Fyn and the consecutive arrangement of the SH4, SH3, and SH2 domains control the function of the kinase domain (SH1) through substrate recognition and regulation of enzymatic activity  that can be reversibly palmitoylated. Studies using fibroblast cell lines show that palmitoylation of these residues induces localization to lipid rafts in the plasma membrane and contributes to spatial control of signaling (420) that is auto-phosphorylated upon activation of enzymatic activity. Collectively, the SH domains and quaternary structure of Fyn enable it to act in multiple facets of killer cell effector function by providing the substrate specificity and control of enzymatic activity required for regulation of divergent signaling.The structure of Fyn is conserved across all cell types, and domain SH through domain SH through ignaling , 17. Theignaling . The SH3ignaling  and two ignaling . The SH2ignaling –23. The In vitro characterization of SFK structures show that the function of Fyn is regulated by intramolecular interactions between the SH domains that prevent unnecessary activation under resting state. Structures of the full-length Fyn protein in both the inactive and active conformations remain to be elucidated; however, sequence homology and structural data of individual domains of Fyn and other SFKs provide a model for the intramolecular interactions that regulate Fyn activity in lymphocytes  in the C-terminal regulatory region and the SH2 domain -binding protein (PAG/CBP) –33. ThisG Figure A 34). A. Ain vit163 or Tyr181 of PAG, which initiates binding of the SH2 domain of Fyn (317of PAG necessary for recruitment of the regulatory protein c-Src kinase (Csk) . T. T531 as420 in the kinase domain of Fyn. This creates an electrostatic interaction that moves the C-Helix of the N-terminal lobe K-p85α subunit and, through phosphorylation, initiate PI(3)K activity Figure . This biThe quaternary structure of Fyn also allows it to activate a divergent signaling cascade primarily responsible for cytokine production in lymphocytes. Recent work has elucidated the role of the Fyn–ADAP axis in mediating inflammatory cytokine production in activated NK and T cells –12, 14. The role of the Fyn SH2 domain in initiating the ADAP-CBM-Tak1 signaling cascade leading to cytokine production highlights the importance of the unique structure of the Fyn. Lack of ADAP in NK cells impairs only cytokine production but, not anti-tumor cytotoxicity . HoweverAll SFKs contain a highly conserved SH2 domain with a preference for binding pYEEI phosphopeptide motifs . RoughlySH2 domains also contain a specificity-determining region adjacent to the phosphotyrosine pocket that is required to ensure signaling integrity Figure A. Variat134) has a poorly defined position, and intramolecular nuclear overhauser effect (NOE) analyses suggest that this residue does not contribute directly to phosphotyrosine binding  interacts with backbone amide of the Glu residue in the +1 position from the phosphotyrosine K and ADAP, it is able to control cytotoxicity and cytokine production in NK cells. Data from our lab and others suggest that the ability of Fyn exclusively to activate ADAP is a result of structural differences between SFKs that allow Fyn to bind the pYDGI motif of ADAP. To date, no mechanistic basis for Fyn–ADAP specificity has been confirmed, and proving any underlying structural cause will likely require solving the structure of the Fyn SH2 domain complexed with a pYDGI containing peptide. Solving this structure has the potential to elucidate the mechanism behind the signaling specificity responsible for distinct effector functions in lymphocytes, and serve as a model for identifying other divergent signaling cascades.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.


In the peripheral blood leukocytes (PBL) infected with human T-cell leukemia virus type-1 (HTLV-1), which causes HTLV-1 associated diseases including adult T-cell leukemia (ATL), HTLV-1 associated myelopathy (HAM) and HTLV-1 uveitis (HU), NF-κB-mediated anti-apoptotic signals or inflammatory signals are constitutively activated primarily by the HTLV-1 encoded oncoprotein Tax.Tax interacts with the I-κB kinase regulatory subunit, NEMO, to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, Hsp90, and its co-chaperone Cdc37. The antibiotic geldanamycin (GA) inhibits Hsp90’s ATP binding for its proper interaction with client proteins. Administration of a novel water soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) to Tax-expressing ATL transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines while this treatment has no apparent effects on normal PBLs. 17-DMAG also down-regulated Tax-mediated intracellular signals including activation of NF-κB, AP-1 or HTLV1-LTR in Tax-transfected HEK293 cells.Oral administration of 17-DMAG to ATL-model mice xenografted with lymphomatous transgenic Lck-Tax cells or HTLV-1 producing tumor cells dramatically attenuated the aggressive infiltration into multiple organs, viral replication and improved survival periods. These observations identified 17-DMAG as a promising candidate for prevention of ATL progression.


In several areas of high endemicity, HTLV-1 can be transmitted from mother to child, through prolonged breast-feeding. This way of transmission is particularly linked to the development of Adult T Cell Leukemia (ATL). In this context, we studied the mechanisms of HTLV-1 transmission through the digestive tract. We previously demonstrated in an in vitro model of compartimentalized co-culture device of human enterocytes, lymphocytes and dendritic cells, that HTLV-1 was unable to infect enterocytes, or disrupt tight cell junctions, but could rather be transported by transcytosis to infect sub-epithelial dendritic cells . Since HTLV-1 infection has been shown to be more efficient through cell-associated virions, we also focused on the crossing of HTLV-1 infected lymphocytes through the intestinal barrier. We thus studied the migration of HTLV-1 infected lymphocytes across the intestinal epithelium, both ex vivo  and in vitro (on a monolayer of human enterocytic cell line cultured on Transwell devices). We demonstrate that, in both systems, infected lymphocytes are able to cross the epithelial barrier, with increased capacities compared to uninfected lymphocytes. Our work suggests that HTLV-1 infected lymphocytes efficiently cross the intestinal barrier, which could constitute an additional way for mother to child HTLV-1 transmission during breast-feeding.


The aim of this study was the examination of the superficial anatomy of palmar creases and their relation to deeper neuro-vascular structures.Four creases: distal wrist flexion crease, thenar crease, proximal palmar crease and distal palmar crease were evaluated with reference to the following structures: palmar cutaneous branch of median nerve, palmar cutaneous branch of ulnar nerve, the nerve of Henle, transverse palmar branches from ulnar nerve, recurrent motor branch of median nerve, radial proper palmar digital nerve to the index and the ulnar proper palmar digital nerve to the thumb, Berrettini’s communicating branch, ulnar nerve and artery, superficial palmar arch. We performed dissections of 20 cadaveric upper limbs derived from a homogenous Caucasian group. In our study we measured the location of surgically important structures with reference to palmar skin creases.Among the other observations we noticed that the palmar cutaneous branches of the median and ulnar nerves were located at least 0.5 cm away from the thenar crease. The superficial palmar arch was found between the thenar and proximal palmar crease and never crossed the proximal or distal palmar creases.These anatomical dissections will provide reference material for further ultrasound studies on the arrangements of neuro-vascular structures in reference to superficial palmar creases. The palmar creases have been the subject of interest to scientists for centuries. Nevertheless, there are very few current references addressing the anatomy of palmar creases and its clinical significance. So far in the literature there have been descriptions of the palmar creases in reference to osseous structures or genetically inherited disorders , 7, 28. The aim of this study was to examine the superficial anatomy of palmar creases and their relation to deeper structures. Four palmar creases were evaluated: distal wrist flexion crease, thenar crease, proximal palmar crease and distal palmar crease. The anatomy of palmar skin creases was evaluated with reference to the following structures: palmar cutaneous branch of median nerve (PCBMN), palmar cutaneous branch of ulnar nerve (PCBUN), the nerve of Henle, transverse palmar branches from ulnar nerve, recurrent motor branch of median nerve, the ulnar proper palmar digital nerves to the thumb, the radial proper palmar digital nerve to the index, Berrettini’s communicating branch, ulnar nerve and artery and superficial palmar arch.We performed dissections of 20 cadaveric upper limbs (11 right and 9 left). The specimens were obtained from the Department of Anatomy at the Medical University of Warsaw, Poland. Twelve limbs were fixed with formaldehyde solution and eight were fresh frozen. All specimens were derived from a homogenous Caucasian group. The dissections of the palmar surface of hands were performed with the aid of surgical microscope with magnification 2–5.5×. The incisions were performed within the natural palmar creases from the proximal to the distal end of the crease . The mean distance from thenar crease to proximal and to distal palmar crease evaluated along the radial axis of the ring finger Fig.  was 0.97The thenar crease, in our dissections, was never crossed by any cutaneous or motor branches of nerves. These structures were always located a few millimetres away from the thenar creases (at least 5 mm). The only structure to be found within the course of the thenar crease is the carpal tunnel Table , 3.TableThe superficial palmar arch Fig.  is locatThe communicating branch between the median and ulnar nerve, known as the Berrettini’s communicating branch Fig. , was alsAt the ulnar end of the nerve.At the median end of the nerve.The length of the branch was an average of 0.94 cm (range 0.8–1.2 cm).The distance from the thenar crease to the communicating branch was measured at two points:The superficial palmar arch and the Berrettini’s communicating branch were always found between the thenar crease and the proximal palmar crease Table ; Fig 5. The triad of ulnar artery, nerve and nerve of Henle Fig.  was alsoThe distance of this triad to thenar crease was an average of 1.71 cm (range 1–2.3 cm). The nerve of Henle, the sympathetic nervi vasorum of the ulnar artery, gives additional sensory innervations to the palm. The nerve lies alongside the artery and then branches off into smaller nerves alongside the superficial palmar arch.Although there are usually three wrist flexion creases, only the distal crease is of sufficient consistency to be used as a reliable landmark. The length of distal wrist crease was an average of 5.95 cm (range 4–7.5 cm).The location of anatomical structures crossing the distal wrist crease and their arraignment was also evaluated Fig. .Fig. 9PaFlexor carpi radialis tendon was located an average of 1.4 cm (range 0.7–2.1) from radial side of the hand crossing the line of the distal flexion wrist crease. Flexor carpi ulnaris tendon was identified an average of 1.35 cm (range 1.3–1.4 cm) from ulnar side of the hand. Palmaris longus tendon was absent in two cases and when present located an average of 2.95 cm (range 2.6–3.3 cm) from ulnar side of the hand and an average of 2.9 cm (range 2.4–3.3 cm) from radial side.Ulnar artery and nerve with nerve of Henle was detected an average of 1.6 cm (range 1.2–1.8 cm) from ulnar side of the hand. The nerve of Henle was present in 100 % of specimens.PCBMN was located an average of 3.11 cm (range 2.6–3.7 cm) away from radial side of the hand.PCBUN was located an average of 3.82 cm (range 2–5.8 cm) away from radial side of the hand.The length of proximal palmar crease was an average of 3.54 cm (range 0.7–7.7 cm) and the length of distal palmar crease was of an average of 4.42 cm (range 1.3–7.0 cm).We also measured the distance from proximal to distal palmar crease along the radial axis of the ring finger and it was an average of 0.87 cm (range 0.6–1.0 cm) Fig. .We evaluated the distance from superficial palmar arch and Berrettini’s communicating branch to proximal and distal palmar creases.The distance from superficial palmar arch to proximal palmar crease was an average of 1.33 cm (range 0.6–1.7 cm) and to the distal palmar crease an average of 2.17 cm (range 1.9–2.7 cm).The distance from the Berrettini’s communicating branch to the proximal palmar crease was an average of 1.35 cm (range 0.6–2.1 cm) and to the distal palmar crease an average of 2.21 cm (range 1.6–2.7 cm).Palmar creases, which are defined as the skin histologically attached to an underlying structure, are formed during the foetal period. The main creases of the palm may be morphologically constant, but with regard to length and position, they vary from person to person. The palmar creases naturally grow and develop with hand. They are unique for every human being. Palmar creases form at about 12 foetal weeks, although spontaneous hand movement does not occur before 11.5 foetal weeks. Therefore, although foetal hand movement may influence the development of the creases, genetic factors are considered most important to their formation , 19. OurFour palmar creases were evaluated in present study: distal wrist flexion crease, thenar crease, proximal palmar crease and distal palmar crease. These creases were consistently present in our specimens. We performed dissections of twenty cadaveric limbs from which twelve were fixed with formaldehyde solution and eight were fresh frozen. We are aware of the existence of some limitations to the present study. This anatomic study was performed in nonliving tissue and thus in vivo conditions could not be examined. Moreover, the cadaveric post mortem processing might have altered the neuro-vascular relationships. We do feel, however, that our sample of specimens preserved in two different conditions reflects the broad spectrum of patients encountered in a hand surgery practice.In 1953, the most famous surgical landmark of hand known as the Kaplan’s , 17 cardAs the later studies show, the line may be presented and used in four different ways by hand surgeons . This maThe Kaplan’s cardinal line has been used as a surface marker in various anatomical studies. Anand and Trzeciak  anatomicIn our study the superficial palmar arch was located between thenar crease and proximal palmar crease, which is similar to results of Anand and Trzeciak.Other investigators, including Kaplan himself, have described numerous variations to the cardinal line , 17, 27.In our anatomical study we examined and measured not only the palmar creases but also important neuro-vascular structures such as nerve of Henle, transverse branches of ulnar nerve and Berrettini’s communicating branch. The nerve of Henle was present in 100 % of our specimens. According to the literature the nerve serves as the nervi vasorum of the ulnar artery, and gives innervations to the forearm and palm, in addition providing sympathetic innervations to the artery , 29. ThiAccidental damage to this nerve can occur especially in the case of iatrogenic injury of the superficial palmar arch. This is not a benign occurrence and can result in complex regional pain syndrome .Multiple cutaneous nerves to the palm are noted from the ulnar nerve. Many of these nerves exited perpendicularly from the longitudinal direction of the ulnar nerve, thus prompting a description of them as transverse palmar cutaneous branches of the ulnar nerve. These nerves pierced the palmar carpal ligament to innervate the skin and subcutaneous tissue of the hypothenar eminence and midpalm, usually distal to that area innervated by either the nerve of Henle or the palmar cutaneous branch of ulnar nerve , 29, 34.The communicating branch of ulnar nerve, known as the Berrettini’s communicating branch, connecting the third common palmar digital branch of the median nerve with the fourth common palmar and proper palmar digital branches of the ulnar nerve was present in 100 % of our specimens. The earliest illustrations of the communicating branch between the digital branches of the median and ulnar nerves were the Berrettini drawings in 1741 . StudiesIn two cases this nerve was present in a plexiform nature. This Berrettini’s communicating branch was perforated by the superficial palmar arch. The unusual relationship of Berrettini anastomosis with the superficial palmar arch is very rare, and knowledge about such a variation is important when performing carpal tunnel release, flexor tendon surgery, Dupuytren’s fasciectomy and when dealing with arterial repairs and vascular graft applications in the hand .Endoscopic and open carpal tunnel releases, even if performed meticulously, still carry the potential for complications. A survey of American Society for Surgery of the Hand (ASSH) members published in 1999 noted a myriad of injuries, including damage to the superficial palmar arch (SPA) in both types of releases . The preAs we mentioned above our cadaveric study has some limitation, but in the future we are planning to expand it on living subjects by performing the ultrasound examination of palmar hand surface. We will try to localise ultrasonographically the neurovascular structures with their correlation to palmar skin creases.We hope this future study will add to that anatomical one and will help us to use the palmar skin creases as the reliable topographical landmarks for planning the safe surgical incisions.The palmar creases may serve as landmarks for the deeper neuro-vascular structures.The superficial palmar arch was found between the thenar and proximal palmar crease and never crossed the proximal palmar crease.The thenar crease, in our dissections, was never crossed by any cutaneous or motor branches of nerves.These anatomical dissections will provide reference material for further ultrasound studies on the location of the neuro-vascular structures in reference to superficial palmar creases.


Caenorhabditis elegans (C. elegans) and performed the approach of gene set enrichment analysis (GSEA) on each dataset between adjacent stages. As a result, multiple genetic pathways and targets were identified as significantly down- or up-regulated. Among them, 5 truly aging-dependent signaling pathways including MAPK signaling pathway, mTOR signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway and ErbB signaling pathway as well as 12 significantly associated genes were identified with dynamic expression pattern during aging. On the other hand, the continued declines in the regulation of several metabolic pathways have been demonstrated to display age-related changes. Furthermore, the reconstructed regulatory networks based on three of aging related Chromatin immunoprecipitation experiments followed by sequencing (ChIP–seq) datasets and the expression matrices of 154 involved genes in above signaling pathways provide new insights into aging at the multiple pathways level. The combination of multiple genetic pathways and targets needs to be taken into consideration in future studies of aging, in which the dynamic regulation would be uncovered.Numerous genetic targets and some individual pathways associated with aging have been identified using the worm model. However, less is known about the genetic mechanisms of aging in genome wide, particularly at the level of multiple pathways as well as the regulatory networks during aging. Here, we employed the gene expression datasets of three time points during aging in  C. elegans had already been established as a useful model organism for studies of aging since it has a relatively short life span and can easily propagate populations of synchronized individuals. Aging in C. elegans has been proposed to be caused by hyperfunction, that is, overactivity during adulthood of processes particularly biosynthetic that contribute to development and reproduction. Such hyperfunction can lead to hypertrophy-associated pathologies, which cause the age increase in death [C. elegans [C. elegans and may provide some insights into potential mechanisms [C. elegans aging have been uncovered from the statistical analysis of differentially expressed genes (DEG) in the expression profiling, including the methods of Analysis of Variance (ANOVA), Significance Analysis of Microarray (SAM) and Student t test [The nematode worm in death . Several elegans . However elegans . Togethe elegans . Undoubtchanisms . A lot ot t test -8. Howevt t test , 10.As we all know, DNA-binding proteins, especially the transcription factors (TFs), play crucial roles in many major cellular processes. Nowadays, ChIP–seq can successfully detect the binding sites of TFs in genome wide , 12. FroC. elegans. There were significantly 42 up- and 42 down-regulated pathways from the stage of L4 to D6, as well as 26 up- and 56 down-regulated pathways from the stage of D6 to D15 . Interestingly, 6 different signaling pathways were significantly par-ticipating in this dynamic regulation, including MAPK signaling pathway, mTOR signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, ErbB signaling pathway and Hedgehog signaling pathway.Based on the GSEA approach, we have identified several significantly related genetic pathways during aging in 5 Figure . By the C. elegans.Moreover, there were totally 36 overlapping pathways significantly down-regulated both from L4 to D6 and from D6 to D15, and most of them were related to Amino Acid Metabolism (9/36) and Carbohydrate Metabolism (12/36); there were totally 19 overlapping pathways significantly up-regulated both from L4 to D6 and from D6 to D15, and most of them were related to Translation (4/19) and Transcription (3/19) as well as Replication and Repair (5/19). All of these data revealed the dynamic re-gulation in multiple pathways during aging in C. elegans RTK/Ras/MAPK signaling pathway were also identified as the aging-dependent targets [ced-2, lin-45, ZK856.8 and F59D6.7, were significantly up-regulated from L4 to D6 and down-regulated from D6 to D15, showing dynamic expression patterns. Their significances of gene expression changes between two adjacent stages were shown in Table C. elegans, the ced-2 gene encodes a Src homology (SH) 2 and 3-containing adaptor protein, homologous to human CrkII, which is required for phagocytosis during programmed cell death and for migration of the distal tip cells of the somatic gonad. It was previously reported that ced-2 mutants may exhibit persistant corpses indicating defects in phagocytosis and distal tip cell migration defects in the gonadal arms [lin-45 gene encodes an ortholog of the vertebrate protein RAF which is required for larval viability, fertility and the induction of vulval cell fates [lin-45/RAF1, mek-2/MEK1/2, andmpk-1/ERK1/2) as well as lip-1 encoding a MAPK phosphatase that inactivates MPK-1, a novel ERK-MAPK-mediated signaling pathway has been identified, which promotes longevity through two candidate transcription factors, SKN-1 and DAF-16 [ZK856.8 and F59D6.7 would be two important targets for further study of aging in MAPK signaling pathway.In the study of Youngman MJ et al. , a decli targets -18, suchdal arms . The lind DAF-16 . Additiolin-45 mentioned above, the gene of pdk-1 was identified with dynamic expression pattern, which was significantly up-regulated from L4 to D6 (p=4.58E-04) and down-regulated from D6 to D15 (p=1.19E-02) , which is a component of the DAF-2/insulin receptor-like signaling pathway and accordingly, functions to regulate such processes as dauer larvae formation as well as longevity [daf-2 insulin/IGF-1 receptor mutants is accompanied by up-regulation of the MDL-1 Mad basic helix-loop-helix leucine zipper transcriptionfactor, which is an inhibitor of cell proliferation and growth that slows progression of an age-related pathology in C. elegans [The mammalian target of rapamycin (mTOR), also known as mechanistic target of rapamycin has been identified as a key modulator of ageing and age-related disease. The fact that inhibition of mTOR signaling pathway may extend lifespan in model organisms and confer protection against a growing list of age-related pathologies has been clearly approved . In our p=1.19E-0  to generate the vulva [ErbB signaling pathway was the last one of our truly aging-dependent signaling pathways with dynamic expression pattern during aging Figure . The Erbay Table . lin-45 he vulva . Expresshe vulva . Althougced-2, lin-45, ZK856.8, F59D6.7, pdk-1, cyd-1, sma-4, andskr-7, 8, 9, 13, 14, all of which were significantly up-regulated from L4 to D6 and down-regulated from D6 to D15. The information of significances and involved pathways were shown in Table skr gene family  were included. skr-7 encodes a homolog of Skp1 that functions within a particular SCF ubiquitin-ligase (E3) complex by binding both a cullin (a homolog of S. cerevisiae Cdc53) and an F box protein (through direct interaction with an F box motif), which is required for posterior body morphogenesis, embryonic and larval development, and cell proliferation [skr-7, -8 and -9 were highly similar, but it is not clear whether these genes comprise a functionally redundant set. The function of SKR-13 is not necessarily confined to, or even partially involved with C. elegans ubiquitin-ligase complexes. In two-hybrid assays, SKR-13 does not bind to any known C. elegans cullins (CUL-1 through CUL-6) [skr-13 in the C. elegans genome are skr-12 and skr-14.Generally, there were 12 significant genes with dynamic regulation patterns during aging in above five truly significant signaling pathways Figure . They weferation , 33. Theage-1 and daf-2 mutants compared to wild-type animals [mekk-3 knock-down, a mammalian MEKK3-like kinase gene in C. elegans [Besides signal transduction, several measurements of metabolic activity have been demonstrated to display age-related changes. For example, about 50% of declines in carbon dioxide generation by populations of live worms grown on plates measured by gas respirometry have been found from day 6 to day 12 . Additio animals . In our  elegans . An age- elegans .C. elegans FOS-1 has been shown to act in uterine and vulval development, which regulates plc-1 expression in the spermatheca to control ovulation [ruvb-1, let-60 and siah-1 in Figure cdc-42, kin-19, mom-5,ife-2, mek-2, C07E3.9, gpa-12, sos-1, let-23and skr-21 in Figure There were totally 154 and 158 individual genes in above five truly significant signaling pathways, respectively from L4 to D6 and from D6 to D15. The expression profiles of these genes were shown in C. elegans and performed the approach of GSEA on each dataset between two adjacent stages. As a result, multiple genetic pathways and targets were identified as significantly down- or up-regulated. Among them, 5 truly aging-dependent signaling pathways including MAPK signaling pathway, mTOR signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway and ErbB signaling pathway as well as 12 significantly associated genes were identified with dynamic expression pattern during aging. On the other hand, the continued declines in the regulation of several metabolic pathways have been demonstrated to display age-related changes. Furthermore, the reconstructed regulatory networks based on three of aging related ChIP–seq datasets and the expression matrices of 154 involved genes in above signaling pathways provided new insights into aging at the multiple pathways level. In conclusion, the identification of these genetic pathways and target genes by our integrated analysis would support the hyperfunction theory, which is a plausible alternative to the molecular damage theory to explain aging in C. elegans [In this study, we employed the gene expression datasets of three time points during aging in www.ncbi.nlm.nih.gov/geo/), which was contributed by Youngman MJ et al. [C. elegans strain Bristol N2 during aging and then prepared for hybridization to Affymetrix C. elegans Genome Array (GPL200) from synchronized populations of C. elegans at three points: the late larval L4 stage , day 6 of adulthood (D6), and day 15 of adulthood (D15). There are three biological replicates for each time point: 3 for L4 , 3 for D6 , as well as 3 for D15 . We then divided the datasets into two subsets, including the study of period from L4 to D6  and the study of period from D6 to D15 . It aims to uncover the diverse mechanism of aging process.The gene expression dataset of GSE21784 related to aging was collected from the database of GEO  algorithm in Affy package .http://www.genome.jp/kegg-bin/get_htext?br08901.keg). The annotation of significant C. elegans genes in each pathway was based on the WormBase database, of version WS236 (www.wormbase.org). Next, clustering on groups and genes was performed based on the identified genes' expression in each significant pathway using the method of hierarchical clustering with Euclidean distance. The significance of associated target genes from L4 to D6 or from D6 to D15 was calculated by unpaired t test.The approaches of Gene Set Enrichment Analysis (GSEA) were performed using Category package in version 2.6.0 of Bioconductor to identify the significantly enriched pathways and genes in both above two subsets , 43. In In the database of modENCODE, there are almost one hundred available ChIP–seq data associated with individual transcription factors at diverse developmental stages. Recently, Van Nostrand et al. have performed integrative analysis of these data sets to infer gene regulatory interactions . As a rehttp://bioinfo.icts.hkbu.edu.hk/cmgrn/) is based on a methodology combining Bayesian hierarchical model with Gibbs Sampling implementation to identify correct sets of regulator-gene interactions which explore underlying network structures [The tool of Constructing Multi-level Gene Regulatory Networks (CMGRN) was used to integrate NHR-28 (L4), FOS-1 (L4) and UNC-62 (YD) ChIP-seq information of transcription factor (TF) binding with expression profiling of signaling pathway genes that significantly enriched during aging. CMGRN(ructures . We aime


By drawing on mouse and human studies as a main source, this review focuses on the recent progress on understanding TGFβ signaling in the uterus. The review also considers the involvement of dysregulated TGFβ signaling in pathological conditions that cause pregnancy loss and fertility problems in women.Transforming growth factor β (TGFβ) superfamily is evolutionarily conserved and plays fundamental roles in cell growth and differentiation. Mounting evidence supports its important role in female reproduction and development. TGFBs1-3 are founding members of this growth factor family, however, the  These studies have yielded new insights into this growth factor superfamily in uterine development, function, and diseases. This review will focus on TGFβ signaling in the uterus, primarily using results from studies with mice and humans.Transforming growth factor β (TGFβ) superfamily proteins are versatile and fundamental regulators in metazoans. The TGFβ signal transduction pathway has been extensively studied. The application of mouse genetic approaches has catalyzed the identification of the roles of core signaling components of TGFβ superfamily members in reproductive processes. Recent studies using tissue/cell-specific knockout approaches represent a milestone towards understanding the Core components of the TGFβ signaling pathway consist of ligands, receptors, and SMA and MAD (mother against decapentaplegic)-related proteins (SMAD). TGFβ ligands bind to their receptors and impinge on SMADs to activate gene transcription. TGFβ superfamily ligands include TGFβs, activins, inhibins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), anti-Müllerian hormone (AMH), and nodal growth differentiation factor (NODAL). Seven type I  and five type II receptors  have been identified . SMADs aTo initiate signal transduction, a ligand forms a heteromeric type II and type I receptor complex, where the constitutively active type II receptor phosphorylates type I receptor at the glycine and serine (GS) domain. Subsequent phosphorylation of R-SMADs by the type I receptor and formation and translocation of R-SMAD-SMAD4 complex to the nucleus are critical steps for gene regulation , 8–10. ATGFβ signals through both SMAD-dependent  and SMAD-independent  pathways in a contextually dependent manner , 14–16  [Smad4 signaling in ovarian granulosa cells leads to premature luteinization [Smad4 causes minimal fertility defects in mice [Recent studies have uncovered the roles of key receptors and intracellular SMADs of this pathway in female reproduction.  fertile , 35. SMA fertile , 29. Smane (FSH) , 37. We ne (FSH) . Disruptnization . However in mice . SMAD7 m in mice  and anta in mice , suggest in mice , 44, whi in mice , 46.7a null females demonstrate defects in reproductive tract formation, suggesting a critical role of Wnt/β catenin signaling in myometrial development [The uterus develops from the Müllerian duct, which forms at embryonic day E11.75 in mice . Uterineelopment –53.Oxtr), cyclooxygenase 2 (Cox2), connexin 43 (Cx43), zinc finger E-box binding homeobox 1 (Zeb1), and Zeb2[Myometrial contractility is critical for successful pregnancy and labor. The myometrial cells transform from a quiescent to a contractile phenotype trigged by the decline of progesterone levels during late pregnancy. What has long puzzled scientists is how this transformation occurs during pregnancy, and how myometrial development and function are coordinately regulated. Uterine contraction is controlled by hormonal, cellular, and molecular signals –65. Receand Zeb2. HoweverTgfbr1 conditional knockout (cKO) mouse model created using anti-Müllerian hormone receptor type 2 (Amhr2)-Cre, we have shown that TGFβ signaling is essential for smooth muscle development in the female reproductive tract [Tgfbr1 cKO mice are infertile and embryos are unable to be transported to the uterus due to the presence of the physical barrier of oviductal diverticula [Tgfbr1 cKO mouse model can be further exploited to understand the pathogenesis of myometrium-associated diseases, such as adenomyosis that is present in these mice [TGFβ signaling plays a pleiotropic role in fundamental cellular and developmental events , 3, 8. Uve tract , 44. Theerticula . MeanwhiPre-implantation embryonic development refers to a period from fertilization to blastocyst implantation, which requires coordinated expression of maternal and embryonic genes. The fertilized egg undergoes dynamic genetic programming and divisions to reach the blastocyst stage. The pluripotent inner cell mass of the blastocyst will develop into the embryonic proper, while the trophectoderm and the primitive endoderm form extra-embryonic tissues during development . PreimplBlastocyst implantation is a complex event that is controlled by both intrinsic embryonic programs and extrinsic cues including hormonal and uterine signals. Implantation in the mouse can be divided into three phases: apposition, attachment, and penetration. Following attachment, uterine stromal cells extensively proliferate and differentiate into decidual cells  . The rolin vitro analysis of the ligand function. In the uterus, the in vivo role of TGFβ signaling remains elusive, partially because of the redundancy of the ligands [Tgfb1 null mice survive to reproductive age, they were bred onto the severe combined immunodeficiency (SCID) background [Tgfb1 mutant mice appears to be morphologically normal [TGFBs1-3 are founding members of the TGFβ superfamily. The majority of currently available studies are confined to the identification of tissue/cell-specific expression of TGFBs and  ligands , 74 and  ligands . To allockground . Althougy normal , embryosin vitro model has been used to determine the effect of growth factors on preimplantation development, and the results showed that TGFB1 or epidermal growth factor (EGF) dramatically improves the inferior development of singly cultured embryos between eight-cell/morula and blastocyst stages. This study suggests that embryo and/or reproductive tract-derived growth factors are involved in the development of preimplantation embryos [In vitro treatment of preimplantation stage embryos with TGFB1 increases total numbers of cells in expanded and hatching blastocysts [in vitro blastocyst outgrowth is blocked by an antibody directed to parathyroid hormone-related protein [in vitro expression of oncofetal fibronectin, an anchoring trophoblast marker, indicating a potential role of TGFβ in trophoblast adhesion during implantation [Tgfb1 null embryos, leading to perinatal survival of these mice [An  embryos . In vitrstocysts . Further protein , which santation . TGFB1 aantation . An elegese mice . As furtese mice .Leiomyoma, generally known as uterine fibroid, is a benign tumor arising from the myometrium . Although leiomyoma is commonly benign, it could be the cause of fertility disorders and morbidity and mortality in women .Tgfb3 transcript and protein levels are elevated in human leiomyoma cells, compared with myometrial cells in two-dimensional (2D) and 3D cultures [Increasing lines of evidence point to the involvement of TGFβ signaling in the development of leiomyoma. It has been shown that the expression of TGFBs and receptors is elevated in leiomyomata versus unaffected myometrium . Among acultures –90. In acultures , 89. Howcultures .Although a link between overexpression of TGFBs and leiomyoma has been recognized, the precise mechanisms of TGFβ signaling in leiomyoma are largely unknown. It has been demonstrated that TGFB1-stimulated expression of fibromodulin may contribute to the fibrotic properties of leiomyoma . Moreoveth week of gestation, characterized by hypertension and proteinuria. The causes of preeclampsia are complex and beyond the scope of this review. It has been shown that plasma TGFB1 [Preeclampsia often occurs in pregnant women after the 20ma TGFB1 –104 and ma TGFB1  levels ama TGFB1 . Interesma TGFB1 , 108. Bama TGFB1 . It is tma TGFB1 . Moreovema TGFB1 –112. In ma TGFB1 –116. ThuTgfb2 mutant fetuses weigh less than littermate controls [Nodal knockout mice show diminished decidua basalis due to reduced proliferation and enhanced apoptosis as well as defects in placental development, resulting in IUGR and preterm fetal loss [Bmpr2 in the uterus causes defects in decidualization, trophoblast invasion, and vascularization, which are causes of IUGR in the pregnant females [Intrauterine growth restriction (IUGR), also called fetal growth restriction (FGR), refers to a complication of fetal growth during pregnancy. The estimated weight of the fetus with IUGR is often less than 90% of other fetuses at the same stage of pregnancy . Circumscontrols . Solublecontrols , althougcontrols . It has controls . Mouse mtal loss . Conditi females .Pten tumor suppressor have been shown to be associated with endometrial hyperplasia [Amhr2-Cre enhances epithelial cell proliferation. The aberration culminates in endometrial hyperplasia. Further studies have uncovered potential TGFBR1-mediated paracrine signaling in the regulation of uterine epithelial cell proliferation, and provided genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia [Endometrial hyperplasia is a pathological condition where endometrial cells undergo excessive proliferation . Categorerplasia , 132. Elerplasia , 134. Therplasia . Our recerplasia . Furtherin vivo represents a challenging task. It remains unclear how specific or integrated signals act on the chromatin to shape the epigenetic landscape in physiological and/or pathological conditions of the uterus. Therefore, the interaction between TGFβ signaling and other regulatory pathways  and potential epigenetic mechanisms underlying specific reproductive processes and/or diseases in the uterus need to be clarified. This knowledge will help to design new treatment options for uterine diseases and fertility disorders.A precisely controlled endogenous TGFβ signaling system is of critical importance for the development and function of female reproductive tract. Mouse genetics has proven to be a powerful tool to address many of the fundamental questions posed in the field of TGFβ and reproduction. Conditional knockout approaches have been utilized over the last two decades to decipher the reproductive function of TGFβ superfamily in female reproduction. These studies are at an exciting stage and are advancing at a rapid pace. The functional role of TGFβ signaling in the uterus is beginning to be unveiled. We anticipate that the genetic approach will continue to have large impacts and lead to new breakthroughs in this field. However, understanding how the hormonal, cellular, and molecular signals induce a specific biological response and functional outcome in the context of the uterine microenvironment


Cytotoxic T-lymphocyte (CTL) against HTLV-1 Tax has been demonstrated to play a vital role in controlling HTLV-1 infected cells in the infected carrier people. It was also shown that DNA immunization of mice with Tax expression vector suppressed the tumor growth of EL-4 lymphoma cells expressing Tax (EL4/Gax) through the activation of anti-Tax CTL [1]. Recently we established an HTLV-1-infected humanized mouse model (hu-NOG) to recapitulate ATL development and HTLV-1-specific immunity [2]. Here the efficacy of Tax-peptide vaccination was examined in the hu-NOG mouse model. Mixture of twelve overlapping peptides of 40-42 amino acids long encompassing whole Tax protein was inoculated subcutaneously three times weekly to hu-NOG mouse and then -irradiated HTLV-1 producing Jurkat cells were intraperitoneally injected to infect HTLV-1. While the control mice without immunization died of leukemia within two months of infection, Tax-immunization retarded the out growth of human lymphocytes after HTLV-1 infection and two out offive mice survived with alimited number of infected T-cells. IL-12 induction and the enhanced expression of Tax-specific CD8 T-cell were observed in the peripheral blood of immunized mice before infection. Furthermore, it was shown that the intranasal inoculation of Tax peptides has comparable effect on the onset of leukemia in the hu-NOG systems. These results suggest that the Tax peptide vaccination can elicit protective immunity against HTLV-1 infection and/or ATL developments.


Regulatory networks that govern embryonic development have been well defined. While a common hypothesis supports the notion that the embryonic regulatory cascades are reexpressed following injury and tissue regeneration, the mechanistic regulatory pathways that mediate the regenerative response in higher organisms remain undefined. Relative to mammals, lower vertebrates, including zebrafish and newts, have a tremendous regenerative capacity to repair and regenerate a number of organs including: appendages, retina, heart, jaw and nervous system. Elucidation of the pathways that govern regeneration in these lower organisms may provide cues that will enhance the capacity for the regeneration of mammalian organs. Signaling pathways, such as the hedgehog pathway, have been shown to play critical functions during development and during regeneration in lower organisms. These signaling pathways have been shown to modulate multiple processes including cellular origin, positional identity and cellular maturation. The present review will focus on the cellular and molecular regulation of the hedgehog (HH) signaling pathway and its interaction with other signaling factors during appendage development and regeneration. Th. Th96]. r region . It has limb bud . In conti.e., Gli downstream targets); and whether the regulation of HH signaling is common in various tissues or strains or species. In this review, we have highlighted the current understanding of the HH signaling pathway and its interactions with other factors and signals. Future studies will be needed to further fine-tune its function and precisely demonstrate the mechanistic role of HH signaling in the regenerative process. These studies could provide new insights regarding previously unknown function of HH signaling which may prove beneficial to promote regeneration.Signaling factors have an essential role in regenerative biology. It is important to decipher the common regulatory networks and their interactions to define the overall regenerative blueprint or regenerative map that will serve as a platform for regenerative therapies. Recent studies have indicated that activation of HH signaling is critical for liver regeneration in mammals, as inhibition of HH signaling resulted in inhibition of hepatocyte proliferation, progenitor response and matrix remodelling . These f


It has recently been demonstrated that HTLV-1 and HTLV-2 utilise antisense transcription to express the viral proteins, HBZ and APH-2, respectively. Studies have shown that HBZ is a key player in the pathogenesis of HTLV-1 as its temporal expression appears to be critical for the development of ATL and HAM/TSP. To date very little is known about the role of APH-2 in HTLV-2 infection. Thus, to investigate its role in the pathogenesis of HTLV-2, we recently performed yeast two hybrid screens of several cDNA libraries using APH-2 as bait. We identified a member of the Nuclear Factors Associated with double-stranded RNA (NFAR) proteins as a potential interacting protein of APH-2. The NFARs have been shown to be involved in the regulation of gene expression and most notably; host anti-viral defence. Our research affords the opportunity to investigate the interaction of APH-2 and NFARs and determine how this interaction impacts on HTLV-2 lifecycle events. Our findings indicate that APH-2 and NFARs interact in vivo and in vitro. Immunofluorescence demonstrates that APH-2 and NFARs localise to the nucleus. We have also performed knockdown studies to determine the effect of NFAR proteins on HTLV-2 LTR promoter activity. Our results reveal that NFARs exert no effect on basal LTR activation, but inhibit Tax2B-mediated viral transcription. Additionally we report that in the presence of APH-2, NFARs enhance Tax2B-mediated LTR activation. Overall, this study will provide novel insights into the functional role of APH-2 and NFAR proteins in the pathogenesis of HTLV-2.


Ultrasensitivity allows filtering weak activating signals and responding emphatically to small changes in stronger stimuli. In the presence of positive feedback loops, ultrasensitivity enables the existence of bistability, which convert graded stimuli into switch-like, sometimes irreversible, responses. In this perspective, we discuss mechanisms that can potentially generate a bistable response in the phosphorylation/dephosphorylation monocycle that regulates the activity of cofilin in dynamic actin networks. We pay particular attention to the phosphatase Slingshot-1 (SSH-1), which is involved in a reciprocal regulation and a positive feedback loop for cofilin activation. Based on these signaling properties and experimental evidences, we propose that bistability in the cofilin signaling module might be instrumental in T cell responses to antigenic stimulation. Initially, a switch-like response in the amount of active cofilin as a function of SSH-1 activation might assist in controlling the naïve T cell specificity and sensitivity. Second, high concentrations of active cofilin might endow antigen-experienced T cells with faster and more efficient responses. We discuss the cofilin function in the context of T cell receptor triggering and spatial regulation of plasma membrane signaling molecules. Initial signaling events triggered by the T cell receptor (TCR) after the specific engagement of antigenic peptide–MHC complexes (pMHC) occur in dynamic TCR microclusters organized at the periphery of the immunological synapse (IS) . TCR micin vitro experiments  in live cells have demonstrated that the affinity of the TCR/pMHC interaction is higher but yet short-lived than previously detected by eriments . The acteriments , 7. TCR eriments , 9. Antieriments . Thus, ieriments , 11. Theeriments . Thus, i2 binding at membranes  Figure A. Phosphembranes  and by oembranes . Cofilinembranes –19. In tembranes , which iembranes . In factembranes . Despiteembranes , there iSignaling modules based on opposing enzymes, such as the cofilin phosphorylation/dephosphorylation monocycle Figure A, can exThe cofilin signaling module has several of the above-mentioned properties that generate ultrasensitivity Figure A, includ2 in antigen-specific T cells (We propose that the steady-state response of active cofilin as a function of SSH-1 activation may be an irreversible bistable switch Figure B. A fast T cells . Interes T cells . CofilinIn naïve T cells, ultrasensitivity might be instrumental in maintaining the peripheral tolerance to low signals emanating from self-peptides while endowing cells with enough sensitivity to foreign antigens. Switching the module to the “on” state will rapidly increase actin dynamics to assist on the early assembly of TCR microclusters after the engagement of antigenic pMHC. This will enable an efficient organization of initial signaling complexes.Slingshot-1 may participate in the molecular memory that keeps the cofilin signaling module in the “on” state in Ag-e T cells even when the antigenic stimulus is depleted. Although recent findings challenge the notion that Ag-e T cells have lower activation thresholds , there iIn summary, while the cofilin signaling module at the “off” state may guarantee the auto-tolerance and sensitivity of naïve T cells, the “on” state may mediate faster and more efficient responses exerted by Ag-e T cells. Regulation of SSH-1 activity is expected to be essential for switching the cofilin signaling module to the on state. Thus, regulators and dynamics of SSH-1 during the activation of primary T cells should be investigated. A described regulator of SSH-1 during insulin signaling is PI3K . PI3K isRR-M performed experiments, analyzed data, and revised the manuscript. PC-S setup experimental protocols, analyzed data, and revised the manuscript. PR-N contributed to the research design, analyzed data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.