Interestingly, the pronounced staining for A1Rs in CA2 is not apparent until rats are 4 weeks old, suggesting that developmental changes other than receptor distribution underlie the sensitivity of CA2 synapses to A1R antagonists in young animals. To evaluate the role of A1R-mediated postsynaptic signals at these synapses, we tested whether A1R agonists regulate synaptic transmission at Schaffer collateral inputs to CA2 and CA1. We found that the selective A1R agonist CCPA caused a lasting depression of synaptic responses in both CA2 and CA1 neurons in slices obtained from juvenile rats (P14), but that the effect was observed only in CA2 in slices prepared from adult animals (~P70). Interestingly, blocking phosphodiesterase activity with rolipram inhibited the CCPA-induced depression in CA1, but not in CA2, indicative of robust phosphodiesterase activity in CA1 neurons. Likewise, synaptic responses in CA2 and CA1 differed in their sensitivity to the adenylyl cyclase activator, forskolin, in that it increased synaptic transmission in CA2, but had little effect in CA1. These findings suggest that the A1R-mediated synaptic depression tracks the postnatal development of immunolabeling for A1Rs and that the enhanced sensitivity to antagonists in CA2 at young ages is likely due to robust adenylyl cyclase activity and weak phosphodiesterase activity rather than to enrichment of A1Rs.Immunolabeling for adenosine A Because A1Rs couple to Gi/o type G proteins to decrease the activity of adenylyl cyclase and constrain the production of cAMP  containing the dorsal hippocampus were taken from sections located within −2.30 and −4.30 mm posterior to Bregma  equipped with differential interference contrast optics, a 40× water-immersion objective and a near-infrared camera . Submerged slices were superfused with oxygenated ACSF at a rate of 2.0 ml/min at room temperature (~25°C).Methods for obtaining whole-cell voltage-clamp and current-clamp recordings were similar to those described previously  using a horizontal puller , and experiments on CA2 neurons were performed only when area CA2 could be distinguished visually from area CA1. Pipettes were placed in contact with somata of visually-identified pyramidal neurons in CA2 and CA1 and gentle suction was applied under voltage clamp to form a tight seal (1–3 GΩ). Whole-cell configuration was achieved by increased suction, and experiments began after cells stabilized . Electrophysiological properties of CA2 neurons such as the amplitude of the sag in response to hyperpolarizing currents, capacitance, or resting membrane potentials, while significantly different from neurons in CA1 or CA3 as a group : 120 K-gluconate, 10 KCl, 3 MgClh = −70 mV) were obtained using an Axopatch 200B amplifier  and displayed on a computer monitor using the software package WinLTP (WinLTP Limited). Recordings were filtered at 5 kHz and digitized at 20 kHz . No correction was applied to compensate for liquid junction potentials. Only cells with a series resistance (Rs) < 25 MΩ and a <20% change in Rs from baseline during an experiment were included for analysis. Synaptic responses in CA2 and CA1 pyramidal neurons were evoked using cluster-style electrodes  placed ~150 μm from recorded neurons in the stratum radiatum at a location intended to stimulate the Schaffer collaterals  was calculated by measuring the amplitude of the steady-state current evoked during a −5 mV voltage step (50 ms duration) delivered 100 ms before test stimulation, and Rs was calculated by measuring the peak amplitude of the fast capacitive transient observed at the onset of the voltage step. The coefficient of variation  was calculated on the mean amplitude of responses obtained during 10 min epochs of stable recordings of EPSCs using Excel (Microsoft Corporation). The stability of responses was first confirmed by testing the slope of a regression line fit through data points included for 1/CV2 analyses, and results for 1/CV2 analyses were normalized to the baseline for plotting. The effects of bath- or intracellularly-applied drugs during pharmacological experiments were assessed on the amplitude of averaged EPSCs obtained during 5-min-long epochs recorded at different times during an experiment (latencies specified below). Also, experiments were interleaved so that no single experiment was performed twice in slices prepared from the same animal. All data were expressed as the mean ± SEM and were normalized to baseline recordings for plotting. Changes in response properties were assessed with Prism (GraphPad Software Inc.) using (where appropriate) paired or unpaired samples t-tests, one-way ANOVAs, or repeated-measures ANOVAs. Post hoc comparisons were made using the Bonferroni or Tukey methods with the alpha level set to P < 0.05.Evoked synaptic currents were analyzed using the software applications WinLTP and AxoGraph X (AxoGraph Scientific). Peak amplitudes of EPSCs were measured relative to the prestimulus baseline (8–2 ms period before stimulation pulse), and paired-pulse facilitation (PPF) was determined by expressing the amplitude of the second response as a proportion of the amplitude of the first response. During synaptic recordings, input resistance  from a constant holding potential . Input resistance was calculated by measuring both the peak and the steady-state voltage responses to −100 pA current steps (500 ms in duration), and inward rectification was quantified by expressing the peak input resistance as a proportion of the steady-state resistance (rectification ratio). All data were expressed as the mean ± SEM for plotting, and changes in response properties were assessed using paired samples via the patch recording pipette.Unless stated, all compounds used for pharmacological experiments were obtained from Sigma and prepared as concentrated stock solutions  by dilution in (where appropriate) either distilled water or dimethyl sulfoxide and stored at −20°C until required. Compounds were either bath-applied or loaded intracellularly t(5) = 0.11, P = 0.92, n = 6; CA1, baseline vs. last 5-min, t(6) = 0.37, P = 0.72, n = 7). To determine whether activation of adenosine receptors induces a lasting depression of synaptic responses in CA2 neurons, EPSCs were monitored before, during, and after a brief 5-min-long bath-application of adenosine. We found that although 10 μM adenosine significantly suppressed synaptic transmission during the first 10 min following the onset of drug application  = 10.62, P < 0.01, Bonferroni P < 0.001, n = 5; P = 0.88). Unlike the potentiating effects of A1R antagonists, which were more pronounced in CA2  = 89.33, P < 0.0001, Bonferroni P < 0.0001, n = 4, P > 0.999). We note, however, that the adenosine-mediated suppression of EPSCs in both CA2 and CA1 was highly variable and depended on the commercial source of the adenosine, with effects ranging from no suppression of EPSCs  to brief potentiation  = 7.84, P < 0.05, Bonferroni P < 0.0001, n = 7, P < 0.001). The suppression of synaptic responses in CA2 was apparent across a range of stimulation intensities and concentrations of CCPA. The amplitude of synaptic responses in area CA2 increased in proportion to the intensity of electrical stimulation delivered to the Schaffer collaterals, and this was significantly reduced by CCPA when assessed 15 min into the washout period for all but the lowest stimulation intensity tested  = 27.72, P < 0.001, Bonferroni P < 0.001 for 25–200 μA, n = 10, n = 5; 100 nM, n = 7; and 1 μM, n = 1), but a depression of EPSCs lasting longer than 15 min was observed only with the two highest concentrations  = 22.32, P < 0.001, Bonferroni P < 0.0001, n = 5, P < 0.001). And as noted above, the magnitude of the suppression of synaptic responses induced by 100 nM CCPA did not differ between area CA2 and CA1 at the time points assessed  = 0.02, P = 0.893; peak, Bonferroni P = 0.939; last 5-min, Bonferroni P = 0.762; data not shown; see also F = 5.26, P < 0.001, Bonferroni P < 0.01 for 25 μA and P < 0.0001 for 50–200 μA, n = 9, F = 2.51, P < 0.05, Bonferroni P < 0.05 at 150 and 200 μA, respectively; compare 1R activation on EPSCs in CA1 was also tested following bath-application of multiple concentrations of CCPA , but similar to area CA2, a depression of synaptic responses lasting longer than 15 min was observed only with the two highest concentrations of CCPA  = 6.37, P < 0.05, n = 6) when assessed at the end of the experiment, whilst responses in CA1 returned to baseline levels  = 2.14, P = 0.099, n = 5, t(3) = 2.13, P = 0.122, n = 4, 2; Caruana et al., 2 was computed for 10-min epochs of stable EPSCs recorded during the baseline period and at the very end of the experiment. Response-to-response variability was increased by CCPA as indicated by a reduction in 1/CV2 in both CA2  = 2.23, P = 0.066) or CA1 (t(4) = 1.95, P = 0.123). Data for 1/CV2 analyses obtained during the last 10-min of the experiment were also expressed as a ratio of the baseline and plotted against the CCPA-mediated change in the amplitude of EPSCs for each individual experiment  = 0.03, P > 0.99, n = 7, t(6) = 1.89, P = 0.108, n = 7, F = 0.36, P = 0.95, n = 6, t(5) = 0.32, P = 0.759, n = 6, t(6) = 3.70, P < 0.05, n = 7, t(5) = 7.82, P < 0.001, n = 6, 1Rs has a minimal effect on the overall excitability of principal neurons in the hippocampus and that A1Rs may regulate the activity of transmembrane currents responsible for inward rectification at hyperpolarized membrane potentials . Previously, A1R activation has been shown to activate G-protein-activated K+ channels (or GIRKS) to inhibit the activity of CA1 neurons  = 26.78, P < 0.0001, n = 5, P < 0.0001), responses did not differ significantly from slices treated with CCPA alone (Tukey P = 0.206). Interestingly, in area CA2, the co-application of AP5 significantly enhanced the depression of synaptic transmission induced by CCPA  = 22.66, P < 0.0001, n = 5, Tukey P < 0.05). It is unclear why blockade of NMDARs facilitates the CCPA-mediated suppression of EPSCs in area CA2, but activation of NMDARs is not a requirement for induction of A1R-mediated synaptic depression in either CA2 or CA1.To determine whether the depression of EPSCs induced by CCPA was mechanistically similar to other forms of synaptic depression in the hippocampus, we applied treatments that have been shown to affect synaptic function in other models of synaptic plasticity. Because activity-dependent forms of long-term depression (LTD) can rely on activation of NMDA receptors  = 11.83, P < 0.001, n = 5; P < 0.01), pausing test stimulation did not affect the magnitude of the CCPA-mediated depression-induced in CA2 once synaptic stimulation was resumed . Similar results were obtained in recordings made from neurons in area CA1  = 17.51, P < 0.001, n = 5; control vs. Stim Paused, Tukey P < 0.001; Stim Paused vs. Stim Continuous, Tukey P = 0.999; 1R-dependent depression of EPSCs by CCPA.We have shown previously that constant synaptic stimulation of the Schaffer collaterals is not required for the induction of A1R-mediated synaptic depotentiation  = 7.21, P < 0.01, Tukey P < 0.05, n = 4, P = 0.651). In contrast to previous work, inhibition of p38 MAP kinase also did not block A1R-mediated synaptic depression in hippocampal area CA1. EPSCs were depressed to 64.65 ± 12.09% of baseline by CCPA relative to controls  = 16.78, P < 0.001, Tukey P < 0.05, n = 3, P = 0.535). Thus, in slices from juvenile animals, the activity of p38 MAP kinase does not appear to play a significant role in A1R-dependent synaptic depression induced by 100 nM CCPA in either recording site.Previous work has shown that the activity of p38 MAP kinase contributes to A1R-mediated synaptic depression induced by CCPA. Similar to the negative results described above for various other inhibitors, co-application of 100 nM fostriecin with CCPA did not block the A1R-mediated synaptic depression in either CA2 or CA1. The amplitude of EPSCs was reduced significantly to 63.19 ± 14.43% of baseline and to 58.63 ± 7.30% of baseline in CA2 and CA1, respectively, relative to untreated controls  = 5.03, P < 0.05, n = 6, Tukey P < 0.05, F = 19.57, P < 0.0001, n = 6, Tukey P < 0.001, P = 0.995; CA2, Tukey P = 0.789). Although we do not provide positive evidence that fostriecin was effective, we note that we used 5 times the concentration found effective for glutamate receptor internalization , we bath-applied the phosphodiesterase inhibitor, rolipram, and assessed its effects on evoked synaptic responses in CA2 and CA1. Continuous application of 10 μM rolipram for 50-min induced a slight facilitation of EPSCs in CA2–132.70 ± 13.59% of baseline, but this increase did not differ significantly from control responses  = 1.06, P = 0.3104, Adenosine A CA2 see . Inciden1R-mediated depression of synaptic responses typically induced by 5-min application of CCPA was blocked in Schaffer collateral inputs to CA1  = 19.55, P < 0.0001, n = 6, Tukey P = 0.432). However, in CA2, rolipram treatment had no effect on the depression induced by CCPA  = 6.78, P < 0.01, n = 6, Rolipram + CCPA vs. control, Tukey P < 0.05). In line with this, the depression of EPSCs was significantly greater in CA2 than in CA1 (t(10) = 2.40, P < 0.05, 1R agonists.Interestingly, rolipram’s ability to induce a significant potentiation of EPSCs in area CA1 was coincident with its ability to block the synaptic depression induced by CCPA. For these experiments, slices were first pre-incubated in oxygenated rolipram  for at least 40 min before being transferred to the recording chamber. This pre-incubation period was then followed by the continuous perfusion of 10 μM rolipram for the entire duration of the experiment. Under these conditions, the As to CA1 2, but nos to CA1 1. In are1Rs. This depends critically, though, on whether the depression of EPSCs involves the downregulation of cAMP-dependent protein kinase activity as opposed to regulation of other cAMP-dependent proteins, such as the exchange protein activated by cAMP, or EPAC  = 13.18, P < 0.0001, n = 5, t(8) = 0.651, P = 0.533, n = 3, t(6) = 3.92, P < 0.01, t(8) = 0.289, P = 0.780, n = 4, t(9) = 2.21, P = 0.054, t(6) = 1.86, P = 0.113, Although some phosphatases, such as protein phosphatase 2A and protein phosphatase 4, are not required for the decline in synaptic strength induced by CCPA see 1,2, it iAdcy1, Adcy5, and Adcy6 , and this facilitation was significant relative to responses in age-matched and untreated controls (t(10) = 6.62, P < 0.0001, t(11) = 0.866, P = 0.405, n = 7, t(11) = 2.39, P < 0.05, 1R expression does not differ substantially across the hippocampus at early postnatal ages, the activity of critical A1R-dependent intracellular signals—including adenylyl cyclase, protein kinase, and phosphodiesterase activity—differ considerably between CA1 and CA2, thus resulting in an overall effect that essentially mimics the adult state.Finally, because at least three isoforms of adenylyl cyclase are highly expressed in area CA2 in the mouse  on PPF or differences between CA1 and CA2 in slices from juvenile animals, Muñoz and Solís (1R antagonist) treatment in slices from older animals. The high variance in synaptic responses seen with whole-cell recordings compared with the low variance in responses recorded in field potentials (Muñoz and Solís, 1R staining at P14 (Ochiishi et al., i/o coupling in the recorded neuron or localized knockout of Adora1 may eventually resolve the question of the presynaptic effects of adenosine.Regarding the effects of And Solís  recently1R in area CA2 first shown by Ochiishi et al. (1R agonist was sufficient to cause an age-dependent change in synaptic function in both CA2 and CA1 directly supports the idea of a developmental shift in the distribution of A1Rs across the hippocampus (see 1Rs by caffeine or another A1R antagonist could induce a robust potentiation of synaptic responses in area CA2 at concentrations that do little in CA1 and CA3 (Simons et al., 1R-mediated intracellular signals differ between areas CA2 and CA1, and this suggests that differences in A1R-mediated synaptic plasticity observed in slices prepared from juvenile animals may be due to mechanisms other than those linked directly to the expression pattern of A1Rs in the hippocampus. Specifically, both kinase activity, required for maintaining synaptic responses (see 1R-mediated synaptic depression in CA1 but not in CA2 (see 1R antagonists in area CA1. Importantly, though, the two are not likely to be mutually exclusive and both may contribute to the relative effectiveness of A1R antagonists to potentiate EPSCs in slices from young rats. Regional differences in the expression of A1Rs may be sufficient to explain the differential effects of CCPA on synaptic responses in adult tissue, but they would likely be in addition to any regional differences in intracellular signaling molecules described above. For example, both of the calcium-activated adenylyl cyclases, AC1 and AC8, increase with age in mouse hippocampus (Nicol et al., Although the developmentally-regulated pattern of immunolabeling for the Ai et al.  has not i et al. , our finmpus see . Interesmpus see . Neverthnses see , and robnses see  indicate CA2 see , may be 1R-mediated synaptic potentiation in area CA2 induced by caffeine or DPCPX (Simons et al., 1R agonist CPA on glutamate receptor phosphorylation and internalization indicates a key role for protein phosphatase 2A (Stockwell et al., 1R agonist for a much shorter incubation period than in the previous report, we found no effect of the protein phosphatase 2A inhibitor, fostriecin, on the CCPA-mediated depression of EPSCs in either CA2 or CA1 (see 1R-mediated depression (Atwood et al., The precise mechanism underlying the induction of CCPA-mediated synaptic depression in Schaffer collateral inputs to CA2 and CA1 remains unknown, but it is clear that NMDA receptors or constant synaptic stimulation are not required see 1,2,B1,2. CA1 see 1,2. We a CA1 see , was sim CA1 see 1,2. Thus CA1 see , many of1R antagonists in area CA2 in slices obtained from young animals despite the lack of any obvious enrichment of A1Rs.A difficult result to interpret is the observation that internal application of the different protein kinase A inhibitors elicited different effects across the various hippocampal subfields (KT5720 and PKI; see via genetic manipulations to prevent glutamate release results in deficits in social recognition memory (Hitti and Siegelbaum, Avpr1b, reduces aggressive behavior in mice (Wersinger et al., 1Rs are expressed in neurons throughout the brain, if their function is greatest in area CA2 where the receptor is highly concentrated then it is not surprising that an A1R agonist impairs social recognition memory and that caffeine enhances it (Prediger and Takahashi, 1R knockout mice display no deficits in spatial learning, but they do exhibit pronounced anxiety-like behaviors and aggression (Giménez-Llort et al., 1Rs is what distinguishes CA2 from CA2 early in postnatal development. Further, they suggest that the modulation of key neurotransmitter systems in the hippocampus, like adenosine, may differ depending on the stage of development.Hippocampal area CA2 has only recently been appreciated for its role in various forms of cognition. Indeed, evidence has been amassed linking CA2 to a wide array of social behaviors. Specifically, lesioning area CA2 or silencing CA2 neurons The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee.SD and DC conceived and designed the studies and wrote the manuscript. DC conducted experiments and analyzed data. SD supervised the project.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.


Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumor stroma, marking them for surgical removal. pHLIP ICG is non-toxic, marks blood flow for hours after injection, and effectively delineates tumors for improved resection on the day after administration. The molecular mechanism of action of acidity targeting by peptides of the pHLIP family has been well-investigated and has been the subject of many publications (see review3 and references within it). pHLIP is a water-soluble, moderately hydrophobic peptide containing several carboxyl groups in a sequence of hydrophobic and polar residues  promotes the protonation of Asp and Glu residues and the C-terminal, membrane-inserting end of pHLIP peptide. Protonation increases the peptide’s hydrophobicity and triggers its partition into a membrane, which is accompanied by a coil-helix transition to form a stable helix across the membrane (transmembrane helix). pHLIP peptides target tumors with high precision, since high cell surface acidity or low pH (pHsurf) is a byproduct of the elevated glycolytic metabolism and of overactivated carbonic anhydrases (CAIX) at cancer and activated immune cells7. A variety of imaging and therapeutic agents are successfully delivered to tumors by pHLIP peptides (see review8 and references within it). pHLIP peptides tumor targeting has been demonstrated in human cancerous tissues and more than 20 different human and murine cancer models including transgenic breast, prostate, pancreatic and skin models. pHLIP peptides tumor uptake was correlated with tumor extracellular pH11. pHLIP peptides tumor targeting has been shown to be enhanced in tumors that are further acidified by co-injection of glucose12 or by overexpression of CAIX at the surface of HCT116 cancer cells10. Alternatively, pHLIP peptides tumor targeting was shown to be reduced by alkalization of tumors in mice fed with bicarbonate drinking water13. Micro-metastases near primary tumors and in distant organs were shown to be very well targeted by pHLIP peptides16. In multiple studies, it has been found that non-protonatable (at low pH) pHLIP variants, where some or all Asp/Glu residues were replaced by Lys, did not target tumors and did not exhibit pH-dependent membrane insertion. pHLIP peptides distribution within tumors, cellular localization and correlation with a variety of markers have been intensively investigated18.More than a decade ago, the first pH-Low Insertion Peptide (pHLIP) was introduced as an agent for targeting acidity at the surfaces of cellular membranes in vitro and in vivoues Fig. a. The di14, we chose a widely used clinical fluorescent dye, indocyanine green (ICG). In addition to its long record of safety, ICG was chosen to couple to pHLIP peptide as a clinical imaging candidate for two main reasons: first, the fluorescence of pHLIP ICG is enhanced about 20 times when pHLIP tethers ICG to the membrane, as compared to the emission of pHLIP ICG in aqueous solution20, thus reducing the probability of false positive signal and contrast-to-noise ratio12. Second, many clinical instruments for imaging ICG fluorescence have already been developed and are clinically available21, which streamlines pHLIP ICG’s clinical adoption. Here we present the results of the translational studies, including in vitro and in vivo efficacy, pharmacology and toxicology, that reveal important properties of a pHLIP agent and form the basis for the clinical trials that will soon be initiated.Since pHLIP peptides offer a unique approach to the targeting of a spectrum of tumor subtypes, we were motivated to develop a fluorescent pHLIP candidate in order to improve the surgical resection of tumors guided by fluorescence. After testing a variety of fluorescent dyes with pHLIP peptidesAll manufacturing procedures were completed in accordance with good laboratory practice (GLP) quality control specifications. pHLIP ICG was synthesized and the agent was produced by Iris Biotech, GmbH (Germany) in partnership with Chemical and Biopharmaceutical Laboratories (CBL) (Greece) and then further optimized by CordenPharma. GLP pHLIP ICG verification batch #1912127 was manufactured in Frankfurt by CordenPharma; the acceptance criteria are presented in Supplementary Table The 28-amino-acid pHLIP Var3 peptide with free N- and C-terminus was synthesized on solid support followed by purification and conjugation of its single Cys residue with ICG-maleimide to obtain the pHLIP ICG imaging agent Fig. a. Furthe−1 cm−1 and was used to calculate the concentration of pHLIP ICG. The absorption and emission spectra of pHLIP ICG in DMF (to mimic the hydrophobic environment of the membrane) are shown in Fig. 22. The midpoint of the transition for pHLIP ICG insertion in liposomes is at pH 6.3 as calculated from graph presented on Fig. 24, about 70% of pHLIP ICG molecules are expected to be inserted across plasma membrane at equilibrium, while at pH 7.4  < 1% of pHLIP ICG molecules are expected to be inserted into the membranes. Since the rate constant of pHLIP ICG insertion into a membrane is high (approximately 5 s−1), the insertion will occur even under conditions of fast blood flow and limited exposure time of pHLIP ICG to acidic cancer cells.The molar attenuation coefficient (i.e. extinction coefficient) of pHLIP ICG at 810 nm measured in methanol  was performed in vitro using 86 enzyme and receptor targets. No inhibitory effect was observed, except a mild inhibitory effect of pHLIP ICG on progesterone receptor B: the established The cytotoxicity of pHLIP ICG was assessed by treatment of human mammary epithelial cells (HMEpC) with increasing concentrations of pHLIP ICG (up to 16 µM) for 72 h, followed by assessment of cell viability. pHLIP ICG did not show any cytotoxic effect at any tested concentration.Hemolysis assays were performed on single donor human whole blood samples treated with increasing concentrations (up to 8 nmol/mL) of pHLIP ICG. Hemolysis was assessed by the release of hemoglobin into the supernatant as monitored by measuring absorbance at 450 nm after centrifugation to remove red blood cells (RBC). The amount of RBC lysis was less than 2% in all samples compared to 100% release upon lysis by water or Triton X-100.The genotoxicity of pHLIP ICG was evaluated using in vitro bacterial reverse mutation and mammalian cell micronucleus tests in human peripheral blood lymphocytes (see details in Cl), volume of distribution (Vz) and elimination half-time (T1/2) values obtained for mice and dogs were very similar: Cl = 9.53 mL/h/kg (mice) and 11.6 mL/h/kg (dogs); Vz = 53.51 mL/kg (mice) and 61.6 mL/kg (dogs); and T1/2 = 3.9 h (mice) and 3.7 h (dogs).Pharmacokinetic studies were performed in mice and dogs. Blood samples were collected at 5, 15, 30 min and 1, 2, 4, 6, 8, 12 or 16, 24 and 48 h after a single i.v. dose of pHLIP ICG (12.3 mg/kg to mice and 0.064 mg/kg to dogs). Evidence of systemic exposure to pHLIP ICG was observed in all animals and was quantifiable up to 8–16 h post administration  and pHLIP ICG (2.5 nmol of each). As expected, ICG-Cys was cleared from the blood within the first 5 min Fig. . To furtBiodistribution and tumor targeting were assessed after a single tail vein injection of pHLIP ICG in mice bearing murine and human tumors. Biodistribution studies were carried out in female and male mice bearing a triple-negative 4T1 tumor, which closely mimics stage IV human breast cancer. Targeting of 4T1 tumors in BALB/c mice by pHLIP ICG is shown in Supplementary Figure 25, so the observed targeting is likely to be reporting cancerous LNs. Removal of the tumor and imaging of the tumor bed is shown in Supplementary Figure 13. The ability of pHLIP ICG to stain both tumor mass and tumor stroma is expected to improve margin resection during surgery.pHLIP ICG tumor targeting was observed in several breast tumor models , lung (A549 human and LLC murine), epithelial (melanoma) (M4A4 human), cervical (HeLa human), urinary bladder (UM-UC3 human) and prostate (LNCaP human) cancers. NIR fluorescence imaging on live animals using the Stryker clinical imaging system was performed at 24 h after a single i.v. administration of pHLIP ICG  per Body Surface Area (BSA)pHLIP ICG was administered by single i.v. bolus injection at increasing doses such as 0.063 mg/kg , 1.08 mg/kg (15 × h.d.), 2.16 mg/kg (30 × h.d.) or once daily for 7 days at 2.16 mg/kg/day (30 × h.d. per day) to dogs, and did not adversely affect the overall health or condition of the animals, any of the measured clinical pathology parameters, organ weights, macroscopic or microscopic pathology. Based on these results, the no-observed-adverse-effect level (NOAEL) was above 2.16 mg/kg/day (30 × h.d. per day) for 7 days repeated administration of pHLIP ICG in males and females.pHLIP ICG was administered once daily for 7 days at 4.92 mg/kg/day , 7.38 mg/kg/day (15 × h.d. per day) and 12.3 mg/kg/day (25 × h.d. per day). The NOAEL was above 12.3 mg/kg/day (25 × h.d. per day) for 7 days repeated administration of pHLIP ICG in male and female mice.A single administration of pHLIP ICG at dose levels of 0.072 mg/kg , 0.36 mg/kg (5 × h.d.), and 1.44 mg/kg (20 × h.d.) to male dogs registered no effect on systemic blood pressures, heart rate, body temperature, electrocardiographic intervals, or qualitative ECG parameters up to 24 h post-dose.A single administration of pHLIP ICG at dose levels of 0.124 mg/kg  and 1.86 mg/kg (15 × h.d.) through all routes of administration was well-tolerated and did not result in any pHLIP ICG-related changes.A single administration of pHLIP ICG at dose levels of 0.218 mg/kg , 0.248 mg/kg (1 × h.d.), 3.72 mg/kg (15 × h.d.) and 7.44 mg/kg (30 × h.d.) had no effect on the central nervous system up to 24 h post-dose.A single administration of pHLIP ICG at dose levels of 6.25 mg/kg , 12.5 mg/kg (50 × h.d.) and 25 mg/kg (100 × h.d.) showed no evidence of genotoxic activity.31. Emerging uses in fluorescence-guided surgery promise to improve surgical outcomes by revealing tumor margins and, even more importantly, by marking flat lesions and micro-metastases adjacent to primary tumors, which are difficult to identify35.Fluorescence imaging, which has long been used to image blood flow, is finding a new range of applications in image-guided surgery. For decades, fluorescence angiography has been applied to assess blood flow and tissue perfusion in preoperative, intraoperative and postoperative settings41. ICG circulates in blood for a few minutes 43 and is cleared through the liver, appearing in the bile about 8 min after injection37. If administered intraperitoneally, ICG is found in the nearest lymph nodes within 15 min, and in the regional lymph nodes within 1–2 h45. The IV dose of ICG varies from 0.5 to 2.0 mg/mL/kg of body weight. At a high dose of 5 mg/kg, no acute toxicity has been observed37 and chronic toxicity has not been reported over decades of many clinical uses of ICG.Several FDA-approved fluorescent dyes and compounds are now in clinical use. The most widely used is the near-infrared emitting ICG, first approved for angiography and subsequently used in ophthalmology and elsewhere for imaging blood vessels46. However, ICG does not have any inherent tumor-targeting properties based on selective binding. Also, the use of ICG for steady-state blood vessels visualization is limited by its very fast blood clearance profile, which can necessitate multiple injections in a single surgical procedure. pHLIP ICG resolves these deficiencies.Notwithstanding the utility that fluorescence-guidance using ICG has afforded to surgical procedures, there remain several important limitations associated with its use. ICG  does not strongly accumulate in tumors except for a very weak signal from enhanced permeation retention (EPR) in vascularized tumors. ICG may be taken up by some cancerous lesions only if blood flow uptake is enhanced for the particular lesion. In other cases, ICG has been used to visualize a lesion via a lack of signal, where the uptake/signal of ICG was much lower within the lesion compared to the surrounding tissue19 and the human upper urinary tract47. The agent was applied ex vivo after the surgical removal of organs, followed by washing and NIR fluorescent imaging using clinical imaging instruments. Malignant bladder lesions were targeted by pHLIP ICG and identified by NIR imaging; pathology showed a sensitivity of 97% and specificity of 100%. Carcinoma in situ was accurately diagnosed in 11 cases, whereas only four of these cases were seen using white light. All malignant upper tract lesions were targeted by pHLIP ICG and visualized on NIR imaging with a sensitivity of 100% (compared to 78.9% white light imaging) and a specificity of 100%. Benign collecting systems and ureters did not show uptake of the pHLIP construct.Tumor targeting by pHLIP peptides has been well-documented in a large variety of animal tumor models and human tissues. pHLIP ICG was used to image cancerous lesions in human bladdersTaken together, the published work on the mechanism of pHLIP peptides action, on tumor targeting and visualization, as well as in the preclinical studies with pHLIP ICG presented here, including pharmacology and toxicology assessments, motivate the clinical translation of pHLIP ICG for systemic administration for (i) real-time blood vessels visualization and (ii) targeting and identification of cancerous lesions during surgical procedures. Further, real-time blood vessels imaging with pHLIP ICG is expected to significantly extend the imaging time window from several minutes to several hours, allowing imaging to be performed at any time during a surgical procedure after a single injection of the agent. Potentially, multi-color imaging could be developed: pHLIP ICG could be used to visualize blood vessels, and pHLIP peptides with a fluorescent dye emitting in the range of 600–700 nm could be used for tumor imaging, allowing for the imaging of cancerous lesions and blood vessels during the same surgical procedure. It is expected that pHLIP ICG will allow accurate identification of tumor margins by staining of tumor mass and tumor stroma, targeting and visualization of flat lesions and, potentially, identification of micro-metastasis nearby primary tumor masses.49. Two factors are known to have an important role: the process of identification of tumor negative margins and breast cancer biology. The tumor margin is first established intra-operatively (by eye), marked by ink, the tumor is resected, and, later, the “no ink on tumor margin” is confirmed by a pathologist. Difficulties in the marking of margins, specimen handling and pathology evaluation remain limitations to be overcome52. Pathology assessment takes time and the margin status is known only after surgery. Even when the margin is negative (as accurately as can be established), the local recurrence is still high, especially in the cases of triple-negative breast cancer, which test negative for estrogen receptor (ER), progesterone receptor (PR) and hormone epidermal growth factor receptor 2 (HER2), significantly limiting targeting and evaluations of this tumor type. In our study, we show targeting of triple-negative breast cancer in murine 4T1 and human MDA-MB-231 mouse models, including targeting of cancerous LNs. If our findings are successfully translated to the clinic, significant improvements of surgical outcomes should be expected.pHLIP ICG currently is in clinical translation for the fluorescence-guided surgical resection of breast tumors. According to the American Cancer Society, ~ 276,480 and ~ 48,530 new cases of invasive and non-invasive breast cancer will be diagnosed in US in 2020, and ~ 42,170 women will die from breast cancer. Surgery remains a dominant first approach to treatment, and there has been a significant shift in surgical practice toward breast-conserving surgery since 1980. The goal is to remove the tumor mass and, at the same time, preserve as much healthy tissue as possible. Unfortunately, ~ 25% of patients with invasive carcinoma and one-third of those with ductal carcinoma in situ require re-excision with about half of the re-excisions done in patients with negative margins, defined as “no ink on tumor”Supplementary Information.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.


Tripterygium wilfordii (TW) in acute myeloid leukemia (AML) based on network pharmacology.This is a study on the potential therapeutic targets and pharmacological mechanism of Active components of TW were obtained by network pharmacology through oral bioavailability, drug-likeness filtration. Comparative analysis was used to investigate the overlapping genes between active ingredient's targets and AML treatment-related targets. Using STRING database to analyze interactions among overlapping genes. Both KEGG pathway analysis and Gene Ontology enrichment analysis were conducted in DAVID. These genes were analyzed for survival in OncoLnc database.We screened 53 active ingredients; the results of comparative analysis showed that 8 active ingredients had an effect on AML treatment. On the basis of the active ingredients and overlapping genes, we constructed the Drug-Compounds-Genes-Disease Network. Survival analysis of overlapping genes indicated that some targets possessed a significant influence on patients’ survival and prognosis. The enrichment analysis showed that the main pathways of targets were Toll-like receptor signaling pathway, NF-kappa B signaling pathway, and HIF-1 signaling pathway.This study, using a network pharmacologic approach, provides another strategy that can help us to understand the mechanisms by which TW treats AML comprehensively. Although advances in AML treatment have significantly improved the prognosis of young patients, a mass of the new cases are elderly, and the prognosis of the elderly is still very poor. Even with current treatments, the prognosis is poor, with a 5-year relative survival rate of only 30% to 50%.Acute myeloid leukemia (AML) is one of the common tumors in the blood system and is caused by the cloning and amplification of undifferentiated myeloid progenitor cells. Some TCMs have been proven to have therapeutic effects on AML, such as arsenic trioxide. And some other experiments also proved that TCM has a therapeutic effect on AML.Tripterygium wilfordii (TW), as one of the TCMs, there is evidence to show that the TCMs have anti-tumor ability. Proved by research, through mitochondrial-mediated pathway, triptolide can cause apoptosis of AML cells. Although the active ingredients in TW have been identified, its relevant mechanism of anti-AML pharmacological effects remains to be studied. In recent years, not only the continuous development of system biology, but also network pharmacology and computer technology are booming. Network pharmacology is a new discipline based on the theory of systems biology, which analyzes the network of biological systems and selects specific signal nodes (Nodes) for multitarget drug molecular design. Network pharmacology emphasizes the multichannel regulation of signal pathways, improves the therapeutic effect of drugs, reduces toxic side effects, thereby increasing the success rate of clinical trials of new drugs and saving drug development costs. Therefore as a useful tool, it could assist us to further understand what the drugs can do, and help us to deepen our insights on how to improve the discovery of drugs in diseases.,13Traditional Chinese medicine (TCM) doctors in China have been using TCM clinically for more than 2500 years.In this study, the potential mechanism of TW treatment AML was analyzed by the method of network pharmacology. The target protein of TW active ingredient and the target of AML treatment were intersected. With TW and AML as the center, Drug-Compounds-Genes-Disease Network was constructed. Construct a protein-protein interaction (PPI) network based on overlapping genes, and conduct enrichment analysis of GO and KEGG pathways. We hope to explore TW's potential therapeutic targets in anti-AML, and provide a basis for the study of TW pharmacological mechanism Fig. .22.1http://tcmspw.com). As a special database that contains a lot of herbs, active constituents, and their targets, TCMSP is established by using the pharmacology of TCM systems. It also includes some pharmacokinetic properties of active compounds, such as oral bioavailability (OB), drug-likeness (DL), and others. OB, as one of the most important pharmacokinetic parameters, indicates the ratio of the drug taken to the blood circulation. The larger the OB value of compounds, the more likely it is to become a clinical drug. The similarity between a compound and a drug which is already known can be expressed in terms of DL. It represents that although the compounds is not a drug, it has the possibility of becoming a drug. Therefore, the greater the value of DL, the greater the possibility that this ingredient will become a potential therapeutic drug for AML. The larger the value of OB, the higher the effectiveness of the ingredient in treating AML.The active compounds of TW and their target proteins were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , as a database that contains a lot of genes, such as genome, transcriptome, proteome and genetics, as well as clinical and functional information from 150 web sources. The other database is the Online Mendelian Inheritance in Man (OMIM) database (http://www.omim.org/).The target genes for AML in this study were collected from 2 databases. The first database is Gene Cards (2.3https://cytoscape.org/) to visually analyze these data to construct D-C-G-D.,24At the beginning, we took the target genes of TW's active component and the therapeutic targets for AML obtained from the above 2 databases to get the overlapping genes. Subsequently, we integrated the information of the component, drug, genetic, diseases. Finally, we used Cytoscape software , which is a database that can predict known protein interactions.PPI data were attained from the STRING database (2.5http://www.oncolnc.org/). It contains RNA-SEQ expression and survival data of 8647 patients’ mRNAs and miRNAs in 21 cancer studies, which are conducted by the Cancer Genome Atlas (TCGA).The information for these gene survival analyses comes from the OncoLnc database (2.6https://david.ncifcrf.gov/), every genes’ GO as well as KEGG enrichment analysis were obtained. Three aspects are contained in GO enrichment analysis: biological process (BP), molecular function (MF), and cellular component (CC).,28 All the GO and KEGG enrichment analysis results were selected by P value less than .05 as the critical criterion.In this study, processed by DAVID6.8 . Then, we downloaded the data about 53 active compounds’ target. After deleting the useless items, 64 known targets were finally obtained. The details of these target are shown in Supplementary 2.3.2From Gene Card database, total 11,619 Therapeutic targets, which are already known for AML, were acquired. In order to improve the accuracy of our data, Gifts greater than 10 were used as a standard to streamline the data. Then, 1093 Known therapeutic targets were collected. Finally, we downloaded 348 known therapeutic targets from OMIM to enrich our data.3.3By taking the intersection of drug targets and disease targets, we got 37 genes Fig. A and 8 a3.4For the purpose of figuring out how overlapping genes interact, we uploaded the information of them to the STRING database and then structure a PPI network, which contained 37 nodes and 324 edges (Fig. 3.5P < .05 are shown in Figure To clarify the impact of these genes on survival and prognosis of AML, survival analysis was performed. The results of 3.6P value from small to large as shown in Figure For the purpose of figuring out what the relationships between 37 genes and AML are, we carried out GO enrichment analysis on these genes in DAVID database and the result of GO enrichment analysis included 3 different levels: BP, MF, and CC. We selected the top 10 according to the 3.7P value from small to large as shown in Figure To further explore how TW affects AML through these target genes, we uploaded these 37 genes to the DAVID database and performed KEGG enrichment analysis. And we selected the top 10 according to the 4 The interaction network of drugs, active ingredients, diseases, and target proteins is an important way to express the mechanism of drugs and their active ingredients in the treatment of a certain disease, which helps to deepen the understanding of the therapeutic effects of drugs and offer a theoretical basis for new drugs development. As an important pharmacological research method, network pharmacology can better demonstrate the relationship between drugs, components, and targets. The application of this idea in the research of TCM can help to understand the interaction mechanism of multicomponent multitarget model of TCM.It is well-known that Chinese medicine has been utilized in various illnesses treatment for long times in China. In the research of active pharmaceutical ingredients, the method based on network interactions is of great help in deepening the understanding of the role of drugs in multilayer information.,10 Triptolide can inhibit the expression of MDM2 and lead to the p53-dependent pathway inhibition and thus achieve the curative effect on AML. Other active ingredients in TW have also been shown to have therapeutic effects on other cancers. Such as Hederagenin, it can inhibit the proliferation of cervical cancer CaSki cells and promote their apoptosis by blocking the STAT3 pathway. And Isoxanthohumol can inhibit the formation of lung metastasis in B16-F10 murine melanoma model. Tripterygium also exhibited significant inhibitory effects on many cancer cells.In this research, by taking the intersection of the active ingredient target of TW and the target of AML treatment, we obtained 37 target genes and 8 active compounds. In these 8 active compounds, Triptolide has been shown to have a therapeutic effect on AML.–37 This means that multiple active ingredients in TCM can act on a single target to take effect in the treatment of diseases. As for the active component, Triptolide is associated with 25 target genes, such as PTGS2, CD40, CASP3, JUN, etc, which is consistent with the fact that a single active compound can act on multiple targets.On the contrary, it can be seen from the D-C-G-D Network that these active ingredients have a common target, which shows that they have some synergistic effects in the treatment of AML. As one of the predicted targets of TW for the treatment of AML, PTGS2 can be acted by 7 active compounds in TW. It has been reported that it can selectively inhibit cox-2 and play a role in the treatment of AML.–40 This means that these genes may be the crucial target of TW in the treatment of leukemia.In order to clarify the impact of these genes on the survival and prognosis of patients, we conducted a survival analysis of these 37 genes. The results show that some of these genes have a significant impact on the survival and prognosis of patients. And some genes have been experimentally proven to be the target of TW treatment of diseases such as AR, CD14, BCL2. Due to the indirect anti-leukemia effect caused by the activation/stimulation of normal immuneocompetent cells, such as eradicating residual leukemia cells after induction therapy, TLR is considered a possible drug target in AML. In addition, the release of pro-inflammatory cytokines due to TLR stimulation can enhance the immunogenicity of AML blasts, making it easier to treat. It is well known that NF-κB is constitutively activated in most patients with AML, leading to apoptosis resistant. Studies have shown that inhibiting NF-κB can induce apoptosis in AML cells. Studies have shown that HIF1α promotes the proliferation of AML cells through a pro-tumoral chemokine factor signaling pathway.Next, we conducted GO and KEGG enrichment analysis on these target genes. The major signaling pathways related to AML were found to include Pathways in cancer, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, Transcriptional misregulation in cancer, HIF-1 signaling pathway, and other pathways closely connected to the occurrence and development of AML. This indicates that TW may have multiple active ingredients through multiple pathways to prevent and treat AML. Such as Toll-like receptor signaling pathway, as a part of the innate immune system, studies have shown that various hematological malignant cells, including AML cells, express TLRs, and TLR agonists may directly effect on the leukemic cells. In-depth analysis of the anti-AML pharmacological actions of TW, as well as the targets and pathways of the active ingredients are necessary to be further verified. And a large number of experimental verifications in vivo or in vitro are required.Due to the complexity of TCM ingredients and the limitations of experimental research methods, there are no reports on the effective ingredients, targets, and mechanisms of TW for AML treatment. This study shows that using network pharmacology to study the effective ingredients and therapeutic targets of TW in the treatment of AML is of great significance for deepening the understanding of its related mechanism of action and discovering new effective ingredients. For the first time, the multitargets and multipathways that play a role in AML are used to explain the molecular mechanism of TW's therapeutic effect. However, the pathogenesis of AML is more complicated. Signaling pathways cross and regulate each other, presenting a pattern of multiple pathways and multiple targets. There is a dose--effect relationship between drugs and diseases, and the current network pharmacology technology is difficult to achieve the purpose of quantification. Finally, most of the research based on network pharmacology is still in static network analysis, and body function is a continuous dynamic process; the occurrence of diseases, drug development, and efficacy process are also dynamic changes.5In this study, 8 active compounds with TW between AML and 37 overlapping genes were selected by the method of network pharmacology. After that, we constructed a PPI network of these genes, and conducted a survival analysis on the top 20 nodes to identify the effect of these genes on patients’ survival and prognosis. The enrichment analysis of GO and KEGG was conducted to find the potential mechanism of action of TW in AML treatment. In conclusion, this study provides another strategy to understand the mechanisms comprehensively by which TW treats AML.Wenjun Liu and Tingting Fang conceived and designed the studies. Lanqin Liu participated in this work. All authors participated in drafting of the manuscript and revising it before final submission.Conceptualization: Wenjun Liu.Funding acquisition: Wenjun Liu.Investigation: Tingting Fang, Lanqin Liu, Wenjun Liu.Methodology: Tingting Fang.Resources: Tingting Fang, Lanqin Liu.Software: Tingting Fang, Lanqin Liu.Supervision: Wenjun Liu.Validation: Tingting Fang, Lanqin Liu, Wenjun Liu.Writing – original draft: Tingting Fang.Writing – review & editing: Wenjun Liu.


Dipeptides [glycyl-L-phenylalanine 2-naphthylamide (GPN) and L-leucyl-L-leucine methyl ester] that are inducers of lysosomal membrane permeabilization (LMP) uncoupled endoplasmic reticulum Ca2+-store depletion from SOCE by interfering with Stim1 oligomerization and/or Stim1 activation of Orai. Similarly, the K+/H+ ionophore, nigericin, that rapidly elevates lysosomal pH, also inhibited SOCE in a Stim1-dependent manner. In contrast, other strategies for manipulating lysosomes  had no effect upon SOCE. Finally, the effects of GPN on SOCE and Stim1 was reversed by a dynamin inhibitor, dynasore. Our data show that lysosomal agents not only release Ca2+ from stores but also uncouple this release from the normal recruitment of Ca2+ influx.Pharmacological manipulation of lysosome membrane integrity or ionic movements is a key strategy for probing lysosomal involvement in cellular processes. However, we have found an unexpected inhibition of store-operated Ca Summary: Lysosomal agents uncouple ER Ca2+-release from store-operated Ca2+ entry, predominantly by inhibiting Stim1 oligomerization and its activation of Orai. Lysosom release . LMP is  release .2+ release from lysosomes (2+ release from the endoplasmic reticulum (ER) Ca2+ stores (2+ release can act as a ‘trigger’ to recruit the ER Ca2+ store ‘amplifier’ via Ca2+-induced Ca2+ release (CICR)  , or elsechanisms .2+ store by directly monitoring the ER luminal [Ca2+]. This unexpectedly revealed that lysosomal agents substantially emptied the ER but, surprisingly, without recruiting store-operated Ca2+ entry (SOCE). We demonstrate that lysosomal agents uncouple ER Ca2+-store depletion from SOCE by inhibiting the influx pathway in multiple ways.We have extended these studies to investigate how these agents may interact with the ER Ca2+ changes in the cytosolic and ER luminal compartments using genetically encoded reporters, GCaMP6s (Kd 148 nM) and R-CEPIA1er (Kd 565 µM). Under resting conditions, the ER is Ca2+-replete, but R-CEPIA1er was not saturated because the R-CEPIA1er signal could be further increased by overloading the cell with Ca2+ using ionomycin/10 mM extracellular Ca2+ to determine the Fmax . This confirms that the response range of the reporter is appropriate for monitoring even initial changes of the ER Ca2+ concentration ([Ca2+]ER).In Cos-7 cells, we simultaneously recorded Ca2+-ATPase (SERCA) inhibitor, cyclopiazonic acid (CPA), ionomycin and the IP3-linked purinergic receptor agonist, ATP. Unsurprisingly, R-CEPIA1er showed antiparallel changes in [Ca2+]ER compared with the cytosolic Ca2+ concentration ([Ca2+]cyt) as simultaneously monitored with GCaMP6s: CPA and ionomycin induced a complete emptying of the ER and an accompanying high cytosolic Ca2+ plateau   . In spit[Ca2+]ER D,F, and + gradient drives H+ uptake and an acidification of the cytosol, as we confirmed using the cytosolic pH probe, 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein (BCECF) (4Cl (note that the time-to-peak of Ca2+ release is faster than of acidification). For comparison, this contrasts with the smaller transient alkalinization induced by GPN  showed a significant (∼70%) reduction in the nigericin-induced acidification BCECF signal . These results imply two things: (1) that these Ca2+ responses are mainly driven by nigericin acting on internal membranes; (2) the cytosolic Ca2+ spikes are predominantly due to release from internal stores (including the ER) because Ca2+ influx is inhibited in high K+ due to membrane depolarization  G, which d by GPN . We thenF signal G, withouormal K+ E,F; indetion see D.Fig. 4+-ATPase inhibitor, bafilomycin A1 which stimulates lysosomal Ca2+ release in some cell types , but poorly stimulate Ca2+ entry.Finally, we applied acutely the lysosomal V-Hll types . In contf the ER H,I. Becaa2+ leak , the data2+ leak . Either 2+ spiking that depletes the ER stepwise (indicated by R-CEPIA1er and the poor subsequent ATP response). In contrast, CPA evokes a slow release from stores that resulted in a sustained cytosolic Ca2+ response due to SOCE . To ensure that the inhibition by nigericin was not simply due to cytosolic acidification, we repeated the protocol in high-K+ medium . Broadly speaking, the effect of GPN and LLOMe on lysosomes occurred over similar concentration ranges as the effect on SOCE, although whether it was the magnitude of inhibition  that showed closer correspondence differed between GPN and LLOMe. Unfortunately, with nigericin, we could not systematically quantify the Lysotracker fluorescence changes: although nigericin reassuringly eliminates lysosomal labelling, the Lysotracker translocates and is retained by other endomembranes so that the overall mean fluorescence is not reduced . Overall, the potency of the effect of the agents on SOCE overlaps with the potency at lysosomes.Thus far, we have described the inhibition of SOCE by these agents but have not resolved a mechanism or target(s). Until recently, GPN and LLOMe were thought to act by inducing LMP , althoughibition E,F or it+ pump (V-ATPase) with bafilomycin A1, to increase the lysosomal pHL and, possibly, to deplete the lysosomes of Ca2+ indirectly  . These data suggest that lysosomal pHL does not play a major role in regulating SOCE: chronically increasing lysosomal pHL does not per se inhibit SOCE, nor does it affect the ability of GPN to block SOCE (i.e. the GPN effect is pHL-independent).Probing lysosomes in another manner, we specifically inhibited the lysosomal Hrsially) . As Cos- release H,I, we p release H,I, whic release J is an u plateau K,L, and mycin A1 . In cont1 P>0.2; M,N. MoreFig. S2). Monitoring Ca2+, we found that the CPA-evoked SOCE phase was itself unaffected by lysosomal repositioning  contacts and activates the plasma membrane Orai (herein not referring to a specific isoform unless otherwise mentioned) channel family (We probed further which aspect(s) of the SOCE pathway was inhibited by the lysosomal agents. In the simplest model, the depletion of the ER Cal family . We therFig. S3A,B), or an increase in shape circularity . Remarkably, GPN addition rapidly reversed this aggregation and restored the resting ER/microtubular morphology . Similarly, nigericin also dispersed Stim1 puncta . In contrast to the effects of nigericin and GPN on Stim1 oligomerization, LLOMe failed to disperse Stim1 puncta . As with the wild-type Stim1, GPN reversed the CPA-induced aggregation of the EYFP-Stim1ΔK variant , even though the morphological changes were clear  evokes a redistribution of plasma membrane proteins, including Orai1, via changes to the inner leaflet, which is likely to reduce the interaction interface area with Stim1.In addition to implicating Stim1, we tested whether lysosomal agents also affected Orai. For example, could these lysosomal agents reduce Orai interactions with Stim1 by promoting a redistribution of Orai away from Stim1 puncta? First, we imaged Orai1-EYFP. To our surprise, the addition of GPN led to a substantial redistribution of Orai1 in the plane of the membrane such that dark patches appeared, presumably due to occlusion of Orai1-EYFP from these areas A,C. GPN ngements H. We con2+ levels nor prevented GPN-induced Ca2+ release  lysosomal Ca2+ release is a ‘trigger’ that secondarily recruits the ‘amplifier’, the major ER Ca2+ store  the SOCE plateau phase induced by the SERCA inhibitor, CPA, is acutely inhibited by these agents; and (3) circumstantially, when we compare GPN- and ATP-induced Ca2+ oscillations, the pattern of ER Ca2+ oscillations with GPN more closely resembles that with ATP in Ca2+-free medium, i.e. ER Ca2+ does not recover well per spike, but proceeds as a stepwise depletion. This is consistent with SOCE normally contributing to the replenishment of ER Ca2+ stores during spiking, but this occurs poorly with lysosomal agents. Furthermore, we consider it unlikely that the repression of the SOCE phase by lysosomal agents is merely due to enhanced Ca2+ removal: the agents inhibit the SOCE phase promptly and persistently, and yet these agents do not repress their own Ca2+ release when applied alone.Multiple lines of evidence suggest that lysosomal agents inhibit SOCE: (1) they deplete ER Ca2+-store refilling; (2) Stim1 Ca2+-sensing; (3) Stim1 oligomerization; (4) Stim1 activation of Orai; and (5) Orai-pore block. First, we exclude ER Ca2+ store refilling because R-CEPIA1er recordings demonstrate that the ER remains depleted when GPN inhibits SOCE  ER Caits SOCE , 3; it i2+-sensor of Stim1? For GPN to reverse SOCE by this mechanism, it would need to enhance (or mimic) Ca2+-binding to the Stim1 luminal Ca2+-binding sites, even when the Ca2+ store is depleted. Given that the R-CEPIA1er Ca2+ affinity is 565 µM  form of Stim1 that does not bind Ca2+, even up to 10 mM 2+-sensor2+ entry. This reversal of Stim1 puncta can readily explain the inhibition of SOCE and has been seen previously with other agents, such as 2-APB and its analogues .Stim1 puncta formation is a hallmark of the SOCE pathway and occurred as expected with all EYFP-Stim1 variants tested. Consistent with their inhibition of SOCE, GPN and nigericin dispersed Stim1 puncta . Most renalogues  or the knalogues . Indeed,2+ entry  can reverse puncta and thereby inhibit SOCE. However, our data are somewhat paradoxical because we also show that the expression of EYFP-Stim1 variants renders SOCE less sensitive to inhibition by GPN . Can we 2+ entry . We sugg2+-release and Stim1 oligomerization, we activated Orai using a cytosolic fragment of Stim1 that contains most of the SOAR . LLOMe is not such a consistent stimulus of Ca2+ release, which is why its inhibition kinetics are not so masked. We hypothesise that lysosomotropic agents inhibit SOAR-Orai interactions, but it will require Stim-Orai intermolecular fluorescence resonance energy transfer studies , we also investigated whether the downstream activation of Orai might also be affected. To activate Orai independently of ER Cathe SOAR ; we usedthe SOAR . Both GP hBACCS2 . This su hBACCS2 M, implyi hBACCS2 M. Howeve studies  to furtha values 5.8 and 4.6, respectively); (2) the loss of fluorescence is so spatially restricted, as well as sustained; and (3) GPN acts as a weak base, not an acid (GPN (but not the other agents) may also unexpectedly affect Orai1 activation by inducing its redistribution in the plasma membrane. As revealed with Orai1-EYFP, GPN promoted the formation of large dark patches within the Orai1 labelling . The drop in fluorescence in these patches is either going to be due to quenching of the tag or to redistribution of the proteins. We consider it unlikely to be due to quenching by acidification when (1) the tags EYFP and TagRFP-T exhibit different sensitivities to pH  and by interfering with Orai interactions with SOAR (GPN and LLOMe). Are these effects dependent on their shared action at lysosomes or are these merely off-site effects? Unfortunately, our data do not definitively answer this key question. In favour of a primary lysosomal target, the three chemically unrelated lysosomal agents inhibit SOCE at concentrations that manifestly affect lysosomes. It is intriguing that the so-called Stim1 inhibitor ML-9 has also emerged as a lysosomotropic agent , with ef2+-buffering effects  regulates SOCE? There is indeed a correlation between LMP and the inhibition of SOCE since LLOMe, GPN and nigericin all evoke LMP . Application of these lysosomal agents should take into account these other potential consequences for the ER and SOCE.In summary, we have shown that Ca2. Cells were periodically treated with mycoplasma removal agent. For imaging, cells were either subcultured onto 25-mm diameter no. 1 glass coverslips or into ten-well Cellview Dishes (Greiner).Cos-7 cells were a generous gift from Prof. Colin Akerman , cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO2, 1.8 mM CaCl2, 6 mM NaHCO3, 25 mM HEPES and 10 mM Glucose, pH 7.4) at room temperature. To examine the effect of ECM with high K+, immediately before imaging, cells were washed three times and maintained in High-K+ ECM . Cells were imaged using a Nikon A1R laser-scanning confocal microscope equipped with 20×, 40× and 60× objectives, and used in Galvano mode to collect an image collected every 3-5 s. Multi-channel images were collected in channel-series mode (to reduce bleed through). The following standard blue, green, red and far-red spectral configurations were used unless otherwise stated (excitation/emission): 405 nm/450 nm, 488 nm/525 nm, 561 nm/595 nm and 647 nm/700 nm; all emission bandwidths are 50 nm except the 700 nm (75 nm). All images are single confocal sections.Unless otherwise stated, cells were loaded, maintained and used in extracellular medium . For ratiometric cytosolic Ca2+ recordings, GEM-GECO1 was used, with cells excited at 405 nm and dual emissions recorded at 450/525 nm, the emission ratio being directly proportional to Ca2+. Luminal ER [Ca2+] was monitored using R-CEPIA1er. Single-cell fluorescence data were analysed using custom-written Magipix software .Cytosolic CaTo monitor cytosolic pH semi-quantitatively, cells were loaded with 2 µM BCECF/AM plus 0.03% Pluronic F127 for 50 min at room temperature and imaged on a Nikon A1R confocal microscope using dual excitation (405 and 488 nm) and single emission (525±25 nm). Data are expressed as the 488/405 ratio, which is directly proportional to pH. Lysosomal pH was qualitatively assessed by loading cells with 300 nM Lysotracker Red for 5 min at room temperature.After 1-3 days, when cells were at 50-70% confluency, they were transfected for 4-6 h with various plasmids using the transfection reagent JetPEI (Polyplus Transfection) in a ratio of 1 µg DNA to 2.0-2.5 µl of JetPEI. Cells were washed and used 16-24 h later.The following plasmids were obtained as gifts from these authors via Addgene: GCaMP6s  ; R-CEPIAGPN was obtained from Santa Cruz Biotechnology or Abcam. LLOMe was purchased from Cayman Chemical or Merck. CPA was from obtained from Merck. Dynasore was purchased from Abcam. AP21967  was obtained from Takara Bio. Lysotracker Red, Lysotracker Deep Red and BCECF/AM were obtained from Life Technologies. JetPEI was sourced from Polyplus Transfection. Mycoplasma removal agent was purchased from Bio-Rad. All other reagents were obtained from Sigma-Aldrich.All morphological analyses were conducted using Nikon NIS-Elements software (version 4).t-tests in GraphPad Prism 6 using the Holm–Sidak method .The rapalog-induced repositioning of lysosomes was assessed by quantifying lysosomal fluorescence along a line (5-pixel width) drawn between the nucleus and plasma membrane, i.e. a profile intensity plot. Fluorescence at each point was then expressed as a percentage of the total mean fluorescence along the line. Because cells are different sizes, the nuclear-plasmalemmal distance was normalized to 100% and the mean fluorescence binned from each 5%-distance increment to allow data to be collated from different cells. Significance compared to the control was determined by paired To quantify Stim1-puncta formation and reversal, the size and shape of YFP-Stim1 structures were determined using threshold masking. Images were first smoothed to remove noise, and the threshold-mask settings of the single cell were defined using an image of stable CPA-induced puncta. These settings were then propagated through every frame of the time series. The binary-mask properties were exported from single images for each condition: basal, CPA plateau and 2-5 min after addition of the lysosomal agent. Multiple structures were detected per cell, and these were averaged to give single-cell means for each parameter .To quantify patch size in the plasma membrane marker, images were first smoothed to remove noise and the single-cell boundary was drawn to first determine the total cell area. To determine the patch size, a threshold mask selected the non-patch plasmalemmal fluorescence and this mask was inverted to select non-fluorescent regions; by generating the intersection with the single-cell boundary, the area of the patches was quantified and expressed as a percentage of the total cell area.2+ signals (GEM-GECO1), multiple regions of interest (ROI) were drawn manually, one per contiguous plasma membrane patch, and fluorescence monitored over time. For each ROI, the amplitude (ΔF/F0) and lag time were determined (lag=the time between agent addition and the first point of deviation from F0). These ROI values were then averaged per cell to give a single-cell mean.When simultaneously monitoring plasma membrane remodelling (inner- and outer-leaflet) and Cat-test, whereas a one-way ANOVA and Tukey–Kramer (or Bonferroni) post-hoc test were used for three or more conditions. For concentration-response curves, Dunn's multiple comparisons test compared different concentrations with the vehicle control. Data were paired where appropriate. Normality was determined using the Kolmogorov and Smirnov test, and non-parametric tests were applied when data failed normality. Experiments were conducted on at least three separate cell preparations on different days, with multiple transfection replicates per condition. Data throughout are expressed as the mean±s.e.m. of n cells (from N different experiments).Statistics were determined either using GraphPad Prism 6 or GraphPad Instat 3.1. Two data sets were compared using a two-tailed Student's


B cell lymphoma 2 (BCL-2) family proteins play an important role in intrinsic apoptosis. Overexpression of BCL-2 proteins in acute myeloid leukemia can circumvent resistance to apoptosis and chemotherapy. Considering this effect, the exploration of anti-apoptotic BCL-2 inhibitors is considered to have tremendous potential for the discovery of novel pharmacological modulators in cancer. This review outlines the impact of BCL-2 family proteins on intrinsic apoptosis and the development of acute myeloid leukemia (AML). Furthermore, we will also review the new combination therapy with venetoclax that overcomes resistance to venetoclax and discuss biomarkers of treatment response identified in early-phase clinical trials. Acute myeloid leukemia (AML) is one of the most common hematological malignancies in adults, with a median age at diagnosis of 68 . For thevia apoptosis  chromosomal translocation in follicular lymphoma and diffuse large B cell lymphoma , 14. It ;18 chromOwing to subtle differences in their BH3 domains and in the grooves of the anti-apoptotic proteins, the various BCL-2 family proteins have differential specificity of binding to one another. For example, BAX and BAK have high affinities for BCL-XL, and BAK has higher affinities for MCL-1, as do BAX and BCL-2 . Interesin vitro   and thenin vitro, and the same activity was demonstrated in a murine xenograft model in vivo -based screening . In a pr in vivo –44. High in vivo –47. HoweNavitoclax, which is a derivative of ABT-737, is an improved orally bioavailable BH3 mimetic with high affinity for BCL-2, BCL-XL, and BCL-w and substantially lower affinity for MCL-1 than ABT-737 . Navitocin vitro and in a mouse xenograft model in vivo  for BCL-2 and nanomolar affinity (Ki<245 nM) for BCL-XL. Based on this feature, it circumvents significant thrombocytopenia due to concomitant inhibition of BCL-XL, making it clinically available for the treatment of AML, a disease typically associated with thrombocytopenia , 58. In To date, many studies on BCL-XL inhibitors have focused on solid tumors  because solid tumors rely on BCL-XL for their survival. Selective BCL-XL inhibitors include WEHI-539, A-1155463, and A-1331852 –63, and MCL-1 has been shown to play a significant role in promoting cell survival in AML cell lines , and itsin vitro and potent efficacy in vivo in preclinical mouse models of diverse hematological malignancies  has been disclosed, with two clinical trials under investigation in patients with AML or myelodysplastic syndrome    , 74.AMG176 is a first-in-class selective MCL-1 inhibitor that is being studied in humans. It targets the BH3-binding groove of MCL-1 and thus frees BAX, resulting in activation of the intrinsic apoptotic pathway. In preclinical studies, it showed potent effects on AML cell lines, xenograft models, and primary patient samples. Based on this background, AMG176 is under several clinical investigations for AML   patients achieving CR/CRi. Moreover, another 2 patients with IDH mutations demonstrated anti-leukemic activity that did not meet formal criteria for response due to a lack of hematological recovery. This is consistent with preclinical studies, in which IDH1/2 mutant AML suppresses the activity of cytochrome c oxidase and lowers the mitochondrial threshold to trigger apoptosis upon BCL-2 inhibition . AnotherPreclinical models have proven a synergistic effect upon combining venetoclax and hypomethylating agents (HMAs) in AML cell lines and primary AML patient samples , 80. Morin vitro by decreasing amino acid uptake, as confirmed by decreased α-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. These metabolic perturbations suppress oxidative phosphorylation, which in turn efficiently and selectively targets LSCs  that recruited 212 patients over the age of 60 years with treatment-naïve AML. They conducted a dose escalation and expansion study of venetoclax , 84. At ets LSCs , 87.Similarly, preclinical studies demonstrated that cytarabine can boost venetoclax activity in AML by reducing MCL-1 levels , 89. An In addition, a “real-world” report of venetoclax combined with azacitidine for the same patient populations at the same institution has been published . Thirty-In summary, venetoclax in combination with HMAs or LDAC seemed to achieve a positive outcome in similar patient populations. On the basis of the above-described clinical data, two phase 3 trials comparing venetoclax + azacitidine/LDAC with azacitidine/LDAC alone are ongoing . The promising responses observed in these trials led to the FDA approval of venetoclax in combination with HMAs or LDAC for treatment-naïve AML patients who were at least 75 years old or ineligible for intensive induction chemotherapy because of comorbidities.Less than 10% of patients with relapsed/refractory AML are cured with current standard chemotherapy. Allogeneic HSCT is the only realistic hope of a cure for these patients, but only a minority of patients consider this option . There iAlthough numerous reports have shown that venetoclax-based therapies exert a promising effect on AML, they still remain partly resistant to venetoclax. It is necessary to explore how resistance evolves and how this can be overcome. The upregulation of MCL-1 protein in AML cells is one of the most well-known reasons for resistance to treatment with venetoclax . TherefoHere, we introduce several combinations that can reduce the MCL-1 level indirectly to circumvent the resistance. Indirect MCL-1 inhibitors include the following compounds: bromodomain extra-terminal protein inhibitors (BETis), which reduce MCL-1 and BCL-XL levels while increasing BIM levels and enhance the lethal effects of venetoclax on AML ; cyclin-The specific population that is most suitable for venetoclax remains unknown. However, recent studies have shown that mutations in NPM1, RAD21, MLL, or IDH1/IDH2 may predict venetoclax sensitivity , 111. InTargeting the BCL-2 protein has shown compelling clinical promise in AML. Venetoclax, a selective BCL-2 inhibitor that was approved by the FDA for treatment-naïve elderly AML patients, has shown potential to be further explored. According to a recent study, previously untreated patients aged 75 years or older who were ineligible for intensive chemotherapy experienced a longer overall survival and a higher incidence of remission after treatment with azacitidine plus venetoclax than patients who received azacitidine alone . In addiChimeric antigen receptor T cells, which have shown exciting results in B cell lymphocytic leukemia, are another a novel strategy for AML treatment. What outcomes would occur if this or other immune therapies was combined with venetoclax? Moreover, venetoclax-based therapies have shown effects on some high-risk AML subtypes. Could these therapies be combined with allogeneic HSCT or be used as a consolidation therapy after HSCT? Will these therapies affect graft versus-host disease or graft anti-leukemia? All of these questions remain unanswered. Furthermore, preclinical studies with MCL-1 inhibitors have demonstrated effective antitumor activity in AML. Will MCL-1 inhibitors be another promising strategy to cure AML? In summary, continued efforts to explore BCL-2 family proteins are necessary.MZ designed the research. YW, YC, RS, LC, XX, XJ, XH and WL performed the research and analyzed the data. YW wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Sciences Foundation of China , the National Natural Sciences Foundation of China , and Tianjin First Central Hospital.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.


N-acetyllactosamine (bi-SiaLN) oligosaccharide and 3-O-sulfated Lewis X trisaccharide (suLeX). Here, we assessed the ability of these glycans to regulate sperm Ca2+ influx, capacitation and affect sperm lifespan. After 24 h, the viability of sperm bound to immobilized bi-SiaLN and suLeX was higher (46% and 41% respectively) compared to viability of free-swimming sperm (10–12%). Ca2+ is a central regulator of sperm function so we assessed whether oviduct glycans could affect the Ca2+ influx that occurs during capacitation. Using a fluorescent intracellular Ca2+ probe, we observed that both oviduct glycans suppressed the Ca2+ increase that occurs during capacitation. Thus, specific oviduct glycans can regulate intracellular Ca2+. Because the increase in intracellular Ca2+ was suppressed by oviduct glycans, we examined whether glycans affected capacitation, as determined by protein tyrosine phosphorylation and the ability to undergo a Ca2+ ionophore-induced acrosome reaction. We found no discernable suppression of capacitation in sperm bound to oviduct glycans. We also detected no effect of oviduct glycans on sperm motility during capacitation. In summary, LeX and bi-SiaLN glycan motifs found on oviduct oligosaccharides suppress the Ca2+ influx that occurs during capacitation and extend sperm lifespan but do not affect sperm capacitation or motility.Before fertilization, sperm bind to epithelial cells of the oviduct isthmus to form a reservoir that regulates sperm viability and capacitation. The sperm reservoir maintains optimum fertility in species, like swine, in which semen deposition and ovulation may not be well synchronized. We demonstrated previously that porcine sperm bind to two oviductal glycan motifs, a biantennary 6-sialylated  In a variety of mammals –7, birds2+ is central to sperm function in preparation for fertilization , sterile filtered) as described [Several media were used for these experiments. The medium used when sperm capacitation was desired was dmTALP  as described . The medescribed .Sus scrofa boars . Approval from the Institutional Animal Care and Use Committee was not necessary because semen was obtained from a commercial facility. Semen was extended in BSA-free Preserv Xtra , cooled to 17°C, transported to the laboratory, and processed within 24 h. The extended semen was pooled and 3 ml were washed through a Percoll cushion containing 4 ml of NC-TALP, 0.6 ml of 10X HBS , and 5.4 ml of Percoll for 10 min at 800 x g. The resulting pellet was re-suspended in 5 ml of NC-TALP and centrifuged for 3 min at 600 x g. Sperm concentration was estimated by hemocytometer and only samples with greater than 80% motile sperm were used for experiments.For each replicate, semen was collected by applying pressure to the glans penis from 3 to 5 mature X glycan residues to extend the lifespan of non-capacitated porcine sperm. To link glycans to beads, approximately 60 μg of glycans [Glycan-coated streptavidin-Sepharose High-Performance beads  were used to test the ability of bi-SiaLN and Le glycans  covalent5 sperm/ml was prepared to receive 2 μl of glycan-coated beads. Non-capacitated sperm and beads were co-incubated at 39°C and collected for evaluation of viability at 0.5, 4, 8, 12, and 24 h.To prepare fibronectin-coated beads, fibronectin  was first biotinylated by incubating 45 μl of 10 mM biotin with 1 ml of a 1 mg/ml solution of FN, both in PBS, pH 7.35. After incubation for 2 h at 5°C, free biotin was removed using a desalting spin column . The biotinylated FN (60 μg) was incubated with 20 μl of streptavidin-Sepharose beads for 90 min at room temperature as above for biotinylated glycans. Beads incubated with biotinylated FN or glycans were washed twice in dmTALP and re-suspended in 100 μl of dmTALP. Once the glycan-coupled beads were ready for use, a 100 μl-droplet containing 2 x 10N-acetyllactosamine and suLeA beads to about 200 bound to the suLeX and bi-SialLN beads. At least 100 free sperm for each treatment replicate were also counted. Sperm that were self-agglutinated were not included in the counts. The experiment was documented using AxioVision 4.5 software .The viability of bound sperm was determined using the LIVE/DEAD Sperm Viability Kit . Live-cell stain SYBR14 at 100 nM and dead-cell stain propidium iodide at 12 μM were incubated with sperm for 5 min at 39°C. Subsequently, sperm were observed by fluorescence microscopy using a 20X dry objective on a Zeiss Axioskop equipped with an Axiocam  using Zeiss filters 09  for SYBR14 and filter 15  for PI. For each treatment, 10 beads were randomly selected in triplicate droplets and the total number of bound live and dead sperm was enumerated. Some sperm aggregated together and could not be evaluated. For each experiment, sperm bound to 10 beads in triplicate droplets were counted (triplicate droplets in one experiment). So the total number of sperm evaluated in each replicated experiment ranged from about 30 bound to the 2+ in sperm populations at a final concentration of 5 x 106 sperm/ml was assessed by a spectrofluorometric assay using the Ca2+ probe Fluo-4 as used before [2+ just before each measurement. Some samples were treated with 5 μM ionomycin as a positive control [2+ binding to Fluo-4 was detected by argon-ion laser excitation at 494 nm and emission at 516 nm in a QuantaMaster 4CW fluorescence spectrophotometer .Intracellular Cad before ,47. Fluo control . Ca2+ biX, LN and suLeA; Motility of sperm bound to soluble glycans was assessed using the Hamilton Thorne Semen Analysis CASA system . Sperm were incubated with 40 μg of each soluble glycan  was assessed by the ability to undergo a Ca2+ ionophore (A23187)-induced acrosome reaction. Sperm were incubated with 40 μg of soluble glycans per ml at 39°C in either dmTALP or NC-TALP. Acrosome status was assessed on aliquots at 0, 2 and 4 h of incubation after addition of 5 or 10 μM A23187 and, 10 min later, Coomassie staining [Capacitation status of sperm incubated with soluble oviduct glycans , dmTALP (positive control for tyrosine phosphorylation), and dmTALP containing 40 μg/ml of either bi-SiaLN, suLeX, LeX, LN, suLeA or LeA , and loaded into a 4–20% gradient gel . After electrophoresis, proteins were transferred to a nitrocellulose membrane. The membranes were blocked with 5% BSA and incubated with primary antibody. Phosphotyrosine antibody  was used at 1:1000 dilution in TBST  with 5% BSA. Membranes were washed in TBST and incubated with a polyclonal anti-mouse IgG conjugated to HRP  diluted 1:2000. After washing, the membranes were incubated with a chemiluminescent peroxidase substrate . Chemiluminescent signals were documented using an ImageQuant LAS 4000 . The primary antibody was replaced with normal IgG as a control. Three independent biological replicates were done for each treatment and time point.Changes in sperm protein tyrosine phosphorylation during capacitation were assessed using SDS-PAGE and immunoblotting. After washing, sperm concentration was adjusted to 5 x 10A or LeA . Sperm wP < 0.05 using Tukey’s test for multiple comparisons.Replicates were performed independently using semen pooled from different boars. At least 3 replicates were performed for each condition. Differences among means were determined using a one-way analysis of variance in SAS . The results are shown as means ± SEM and the means were considered to belong to distinct populations if X structures, and related controls . We used glycans attached to a 30-kDa polyacrylamide chain, so-called neoglycoproteins, to mimic the multivalency common in glycoproteins. These glycans were attached to agarose beads to make them insoluble, resembling their attachment to oviduct epithelial cells. Following incubation of sperm with beads and gentle washing, some sperm remained bound to bi-SiaLN, suLeX and fibronectin (FN) bound to beads but not to LN-beads, a disaccharide found in both suLeX and bi-SiaLN and used as a control (P<0.05). Although not as potent as bi-SiaLN, a biantennary oligosaccharide with N-acetyllactosamine termini , which binds fewer sperm [P<0.05). Sulfated suLeX also extended sperm lifespan during 4–24 h incubation in vitro . The increase in viability was not due only to tethering of a sperm because when fibronectin was linked to the agarose beads and then incubated with sperm, although a high number of sperm bound to the beads, fibronectin did not increase sperm lifespan to the same degree as glycan-bound beads (P<0.05). The increase in viability was not because moribund sperm were released from the glyco-beads. The number of sperm bound to any of the glyco-beads did not change significantly over 24 h, demonstrating that the percentage of viable sperm was not affected by the release of moribund sperm  for this experiment. Although there was no difference in the first 60 min, control sperm showed a gradual, albeit non-significant, increase in intracellular free Ca2+ (P>0.10). By 90 min of incubation, glycans that bound sperm, bi-SiaLN, LeX, and suLeX were able to suppress completely the increase in intracellular free Ca2+ in sperm (P<0.05). In fact, in the presence of bi-SiaLN, LeX, and suLeX, there was less intracellular Ca2+ at 90 min than at 0 min. The presence of biantennary structure and a sialyl residue attached to the 6-position of each galactosyl residue in bi-SiaLN, which are required for maximum sperm binding [2+ increase because the disaccharide, N-acetyllactosamine, did not affect Ca2+ (P>0.10). Furthermore, the positional isomers of LeX and suLeX, Lewis A GlcNAc) and sulfated Lewis A (3’-(O-SO3)Galβ1-3(Fucα1–4)GlcNAc) had no effect on intracellular Ca2+ (P>0.10). Thus, very specific structures were required for glycans to affect intracellular Ca2+.Previous studies indicated that binding to the oviduct regulated sperm intracellular Ca2+ ,31. Limicitation . Using sin sperm ; P<0.05. binding , were al2+ influences many sperm behaviors such as the development of hyperactivated motility, sperm protein tyrosine phosphorylation and the ability to response to acrosome reaction inducers [Intracellular free Cainducers . CASA ob3- that does not capacitate sperm [3-  decreased the number of sperm with intact acrosomes when added at either 2 h or 4 h  would follow, as previously published in porcine sperm . Additioh or 4 h .X), C with biantennary 6-sialylated N-acetyllactosamine oligosaccharide (bi-SiaLN), C with 3-O-sulfated Lewis A trisaccharide (suLeA), and C with N-acetyllactosamine (LN). Path velocity (VAP) μm/sec, progressive velocity (VSL) μm/sec, track speed (VCL) μm/sec, amplitude of lateral head displacement (ALH) μm, beat cross frequency (BCF) Hz. Results are means and SEM from 3–5 experiments.Sperm were incubated in capacitating dmTALP (C), NC-TALP (NC), C with 3-O-sulfated Lewis X trisaccharide  not only blocked the normal Ca2+ influx associated with capacitation but actually reduced intracellular Ca2+ below the concentration found at the beginning of capacitation. In contrast, LeA, a positional isomer of LeX, and LN, a disaccharide that lacks N-acetylneuraminic acid and is not multivalent, did not affect Ca2+ entry. These glycans that did not affect intracellular Ca2+ also do not bind sperm [2+ by the oviduct is key to fertility, particularly in species in which fertilization and ovulation are poorly synchronized [2+, which we propose allows proper timing of sperm capacitation for maximal fertility [The effect of glycan binding on the suppression of Careaction . Here, wnd sperm ,43. Reguhronized ,19. Bindhronized ,51,54. Oertility ,31,55. H2+ is present [3 and BSA [3 and BSA but with 2 mM Ca2+ Click here for additional data file.S1 Data(XLSX)Click here for additional data file.