This scalability relies on fast gates: entangling gates faster than the trap period.Large-scale digital quantum simulations require thousands of fundamental entangling gates to construct the simulated dynamics. Despite success in a variety of small-scale simulations, quantum information processing platforms have hitherto failed to demonstrate the combination of precise control and scalability required to systematically outmatch classical simulators. We analyse how fast gates could enable trapped-ion quantum processors to achieve the requisite scalability to outperform classical computers without error correction. We analyze the performance of a large-scale digital simulator, and find that fidelity of around 70% is realizable for  Quantum simulation promises the ability to study the dynamics of highly complex quantum systems using more easily accessible and controllable systems12Many systems are considered as potential quantum simulators: cold atoms in optical lattices7912131415161819In this work, we show that a simpler gate, equally capable of efficiently implementing the fermionic simulation, may be constructed using faster resonant two-qubit gatesWe investigate a digital quantum simulation of a fermionic lattice system using an ion trapmth ion in the trap. The MS gate couples every pair of ions in the trap evenly,Here, x operator on ion k, and L the total number of ions. Although MS gates can be implemented with high fidelity in ion traps22where It was also shown in the simulation proposalcoupling only pairs involving the locally-rotated ion. It was suggested that these UMQ gates could be constructed using fast two-qubit phase gates24252628293031In this paper, we perform numerical simulations of fast gates in a full 40-qubit crystal to calculate the fidelity of a digital quantum simulation scheme under typical noise parameters. We benchmark the laser repetition rates which will allow us to overcome the time barrier imposed by the MS gate and significantly reduce the operation time of digital quantum simulations, as well as greatly increasing the final fidelity. The latter implies that large-scale quantum simulations could be performed without error correction, bringing nearer the solutions for problems in condensed matter, quantum chemistry, and high-energy physics, which are infeasible to classical computers.Fast two-qubit gates can be performed beyond the trap-period limit and implement the ideal unitary for a geometric phase gate,z operator on the kth ion. These gates are implemented using counter-propagating laser π-pulse pairs, which act as state-dependent momentum kicks on the motional modes while preserving the internal states of the ions. This behaviour is described in the optical rotating-wave approximation by the kick unitarywhere xc is the position operator on ion c, ζ is the number of π-pulse pairs used for the momentum kick and k is the laser wavenumber. These kicks are nearly instantaneous compared to the trap period, so the evolution of a real gate operation can be expressed as the product of the kick unitaries interspersed with free motional evolution,Here, Uckick is the cth kick unitary, δtc is the time between kicks c and c + 1 (0 if c = N) and N is the total number of kicks. An analytic solution for this evolution to implement the ideal unitary in where The structure of the kicks is chosen to be antisymmetric, which approximately restores the motional state - the dominant motional error term is cubic with respect to gate timez is the set of kick magnitudes associated with the times t and n is an integer. Negative and positive kick amplitudes represent kicks in opposite directions. The originally proposed FRAG scheme restricts the order of the kicks, so τ1 > τ2 > τ3 > 0. Removing this restriction preserves the antisymmetric structure of the sequence whilst allowing numerical optimisations to search a larger parameter space. This often results in higher-fidelity gate solutions, so we ignore the kick ordering restriction in this work.where π pulse  and thus errors will contribute negligibly compared with the infidelity of the fast gates, which use hundreds of π pulses.We address the axial phonon modes of the trap - it is possible to perform quantum gates using transverse phonon modesL starting at one end, this is done by performing a fast gate on ions 1 and 2, a SWAP gate on these ions, then a fast gate on ions 2 and 3 and so on until we reach ions L − 1 and L. The SWAP gate is given in the computational basis byWe can use fast geometric phase gates to implement the UMQ gates required in digital quantum simulations. If we label the ions in a Paul trap from 1 to and is equivalent to ‘swapping’ the internal states of the two ions.This process gives the UMQ unitary required up to local rotations, not preserving the position of the internal states in the trap. The reversed UMQ gate in the simulation step restores the position of the internal states. It should be noted that this method of constructing a UMQ gate requires fast SWAP gates, which can be implemented with three CNOT gatesL − 2 SWAP gates and an additional L − 1 fast gates, for a total of 4L − 7 fast gates. Due to the relative timescales and fidelities of local rotations, which require only single laser pulses that take negligible time and can be performed with very high fidelityThis allows us to implement a non-position-preserving UMQ gate using F for a UMQ gate by first calculating the fidelities of the two-qubit fast gates used to construct the UMQ operation. This calculation was performed with a state-averaged fidelity measure, assuming an initial thermal product state for the motional modes of the trap. This fidelity measure is given byWe estimated fidelity Ure and Uid are the real and ideal gate operations, respectively. The assumption of a thermal product state simplifies expectation values of motional displacement operators that appear upon expanding this expression, allowing the calculation of an analytic expression for the state-averaged fidelity given a set of kick times. This expression simplifies numerical optimisation by removing the need to calculate the full unitary Ure. We optimised the component gate fidelities for every adjacent pair of ions in Paul traps of various sizes. The longitudinal trap frequency was chosen as ν = 2.1856 MHz × L−0.865, where the power of L represents a no-buckling limit for the trap derived from molecular dynamics simulations37νx = 5 MHz. The Lamb-Dicke parameter was η = 0.16 for the two-ion trap, and scaled appropriately with trapping frequency for larger trapswhere et al.L/2 where L is the ion number. We see that the achieved fidelities decay with the size of the trap, which is expected because larger traps have a more complex motional phase space, and because more two-qubit gates are needed to construct a UMQ gate in a larger trap. However, we do see very high fidelity overall, even for larger traps. Assuming perfect laser control and no trap heating or dephasing, the n = 50 fast gate, corresponding to a minimum laser repetition rate of around 10 GHz, achieves a UMQ gate fidelity of 99.998% in a 40-ion trap. This would correspond to fidelity of around 95%, without including error correction, for 10 Trotter steps of a 20-site fermionic lattice simulation, requiring 2680 UMQ gates. UMQ gates with n = 50 or more are capable, as shown in The results of this optimisation are shown in n = 50 fast gates, but work is in progress on a pulsed laser system for 40Ca+ control with a 5 GHz repetition rateCurrent repetition rates for applicable pulsed laser systems are up to 300 MHz, with small modifications required to increase repetition rates to approximately 1 GHz32n = 50 case, the time required for a 40-ion UMQ gate is 17.9 μs, comparable with the trap period of 10 μs for this trap. Furthermore, the trap period does not place any fundamental limit on the speed of fast gates as it does with MS gates. In general, an MS gate for N ions will take a time t1 being the time it takes for 1 ion. This can be seen from the expression of the gate’s phase in terms of experimental parameters, η is the Lamb-Dicke parameter, which for N ions is given by η1 is the single-ion Lamb-Dicke parameter, when the COM vibrational mode is considered. The detuning δ sets the gate time to t = 1/δ. To compensate for the 1/N decay rate of θ with the number of ions, the detuning has to be decreased accordingly, n = 50, as shown in the figure. Higher available repetition rates would lead to even more significant gains, as seen in the n = 100 case. The lowest heating rates in current traps are on the order of 10 s−1, which limits the total simulation time to around 100 ms. This places a corresponding maximal threshold on gate time, shown in In N) gates, it can be seen that UMQ gates would perform faster for a number of ions on the order of 1000 or below. This is far beyond the expected capacity of linear traps, and for this number of ions more creative ion arrays will be required, most likely composed of linear traps where the UMQ gates prove to be the most practical.A large scale quantum simulation with MS gates sits on the verge of current motional coherence times, and should be possible with coherence times expected in the near future. However, from the time scaling of MS  and gate time, we numerically optimised fast gates using the FRAG scheme with n = 1 to n = 100 for our two-ion trap. The resulting gate times are shown in Finally, to achieve a better understanding of the interplay between laser repetition rate, momentum (i.e. fast gate n (and thus momentum) approximately as n−2/3. This agrees very well with theoretical results for the GZC pulse schemeA simple least-squares fit to this data reveals that gate time scales with fr and gate time, we can use the approximation n = TGfr, which works very well for fast gates in this regimen−2/3 scaling of gate time with the number of pulse pairs to arrive atTo understand the relationship between laser repetition rate The time taken for the entire simulation should scale according to this power law as achievable laser repetition rates increase.t) is the Rabi frequency and τ is the pulse length) have been shown to affect gate fidelity significantly - it is expected that pulse energy fluctuations will be the most significant source of error for a fast gateThere is a clear trend towards higher fidelity and lower gate time with higher momentum in the results shown in π pulse area on a gate applied to an adjacent pair of ions in a large trap, we employed a method relying on the assumption that the motional mode dynamics are separable during the gate, a valid assumption for small π pulse errors in harmonic trapping potentials. This enables computationally feasible simulation of the gate evolution, allowing simple calculation of a representative-state fidelity for various levels of π pulse errorTo explore the effect of an error in where τ is the pulse time such that ξ = 1 gives a perfect π pulse. Results are plotted against rotational infidelity 1 − Fπ, where Uπre,id are the real and ideal unitaries for the π pulse. For this square pulse this rotational infidelity is approximately π - this means there is no residual population transfer between the 0 and 1 states, even with imperfect pulse area, helping to reduce gate error from pulse imperfections32We simulated two-qubit fast gates on the outermost adjacent pair of ions (ions 1 and 2) in traps of varying size. The results of these simulations are shown in n = 2) fast gates and imperfect π pulses. Comparing the infidelity of the entire UMQ operation to that of a single fast gate with the same π pulse imperfection, it was determined that errors due to π pulse imperfections in successive fast gates are uncorrelated, and thus this type of error scales with the number of gates applied as π pulses, shown in −6, the gate infidelity is too high to implement a 40-ion simulation with an estimated fidelity above 70%. Thus, we can place this limit on π pulse errors for fast gates to be useful for a quantum simulation outperforming current classical computers without error correction. Proposals exist for composite pulse schemes which can produce extremely high fidelities, robust to the type of intensity instability or miscalibration that would damage a square pulse42−6 with microwave pulses36We also simulated a full UMQ gate on a three-ion trap with low-momentum (n = 2) fast gate on a two-ion trap. The full evolution of this gate was simulated, assuming the momentum kicks to be approximately instantaneous and thus unitary, and solving a master equation using a Monte Carlo trajectory approach during the free evolution periods of the gate.We also considered the effect of decoherence during fast gate evolution, in order to place a limit on the rate of decoherence processes to maintain the high fidelity of fast gates. In this case, a full simulation is not feasible for a 40-ion trap, so our results are inferred from simulations of decoherence effects on a low-momentum . This confirms that anharmonicity of the interaction has no appreciable effect on fast gates in the momentum ranges suggested in this work.To confirm the usefulness of gate schemes found using the harmonic approximation, we performed a full simulation of the dynamics of a two-ion trap during an −6 and decoherence rates below 10 s−1. If these requirements are satisfied, it appears that fast gates can significantly outperform MS gates in terms of gate time, and can be implemented with very high fidelity if high-repetition-rate lasers are available3239515253Fast gates have been proposed as a superior two-qubit gate for trapped ion quantum information processing, being much faster than existing gates and robust to many sources of error. In this work, we have analysed the potential for fast gates to implement useful digital quantum simulations, enough to outperform a classical computer. We have also studied the limits on experimental setups to successfully implement high-fidelity operations using fast gates. High-performing gates require rotational fidelities on the order of 10How to cite this article: Taylor, R. L. et al. A Study on Fast Gates for Large-Scale Quantum Simulation with Trapped Ions. Sci. Rep.7, 46197; doi: 10.1038/srep46197 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Transplantation of cardiosphere-derived cells (CDCs) has been shown to exert a therapeutic effect in patients with myocardial infarction (MI). However, poor survival of transplanted CDCs limits their beneficial effect. Metformin (MET) activates AMP-activated protein kinase (AMPK) which is associated with cell survival. The aim of this study is to determine whether MET improves CDC survival in the transplantation microenvironment and enhances the therapeutic effect of CDC transplantation against MI.CDCs were isolated and expanded from transgenic β-actin-GFP mice. CDCs were pretreated with MET and intramyocardially injected into wild-type C57 mouse heart with MI injury. The survival of CDCs was quantified, and the infarct size and cardiac function of treated hearts were evaluated.CDC transplantation modestly reduced infarct size and improved cardiac function in the post-MI heart, which was further improved by MET treatment. MET pretreatment significantly increased the survival of CDCs transplanted into the myocardium. MET also reduced CDC apoptosis induced by oxidative stress in vitro. The anti-apoptotic effect of MET was blocked by the AMPK inhibitor compound C. MET increased AMPK phosphorylation and upregulated endothelial nitric oxide synthase (eNOS) in CDCs under oxidative stress, which might be associated with the anti-apoptotic effect of MET.MET improves the survival of transplanted CDCs in the myocardium, thereby enhancing their therapeutic effect against MI injury. The pro-survival function of MET on CDCs might be associated with an AMPK-eNOS-dependent mechanism.The online version of this article (doi:10.1186/s13287-017-0476-7) contains supplementary material, which is available to authorized users. Myocardial infarction (MI) is a major severe heart disease that threatens human health worldwide. The substantial loss of functional cardiomyocytes contributes to the pathogenesis of the disease, leading to cardiac failure. Stem cell therapy offers possibilities for restoring lost myocardium and cardiac function. Multiple cell types, including bone marrow-derived cells and adipose tissue-derived cells, have shown modest therapeutic effect on post-MI patients . AnotherIrrespective of the cell types used for transplantation, effective cardioprotection and cardiac muscle regeneration are hindered by the poor survival of transplanted cells in the infarcted myocardium, which is high in inflammatory factors and free radicals . GeneticMetformin (MET), a first-line antidiabetic medication for the treatment of type 2 diabetes, has been reported to inhibit the mitochondrial respiratory chain and activate AMP-activated protein kinase (AMPK). AMPK can stimulate fatty acid oxidation, promote glucose transport, accelerate glycolysis, and inhibit both triglyceride and protein synthesis. AMPK stimulation promotes cardiomyocyte cell survival through a mitochondrial apoptotic mechanism . MET attMale wild-type C57 mice were purchased from the Laboratory Animal Center of Daping Hospital, and transgenic β-actin-GFP mice (C57BL/6-Tg (CAG-EGFP) C14-Y01-FM131Osb) were purchased from the Jackson Laboratory, USA. All experimental procedures using the animals were carried out in accordance with the regulations of the Animal Care and Use Committee of the Third Military Medical University, China. All animal protocols were approved by the Medical Ethics Committee of the Third Military Medical University (Permit Number: 2012[13]). All efforts were made to minimize animal suffering.3, washed, and partially digested with trypsin . These tissue fragments were cultured as cardiac explants on fibronectin-coated  dishes in 2 mL cardiac explant media  supplemented with 20% fetal bovine serum (FBS) and 0.1 mmol/L 2-mercaptoethanol). After a variable period of growth, the loosely adherent cells surrounding the explant (cardiac outgrowth) were harvested using mild enzymatic digestion (0.05% trypsin/EDTA) and dispensed to poly-d-lysine-coated dishes. Several days later, cardiospheres were harvested, plated on fibronectin-coated flasks, and cultured in cardiosphere growth medium /F12 supplemented with 35% IMDM, 7% FBS, 29.2 mg/mL l-glutamine, 0.1 mmol/L 2-mercaptoethanol, 2% B27 supplement, 25 ng/mL cardiotrophin, 20 ng/mL basic fibroblast growth factor (bFGF), 10 ng/mL epidermal growth factor (EGF), and 5 U thrombin) to generate CDCs. The characterization of CDCs was investigated by flow cytometry. CDCs were incubated with antibodies against CD29, CD105, CD90, CD31, CD45, and CD34 for 30 min. Isotype-identical antibodies served as a negative control. Quantitative analysis was performed using a BD Accuri™ C6 Cytometer and FlowJo software.Mouse CDCs with transgenic green fluorescent protein (GFP) labeling were obtained and expanded with a protocol similar to that previously reported . GFP lab5 CDCs. MET was used to pretreat CDCs (dissolved in PBS at 100 μmol/L containing the CDCs before transplantation) and intraperitoneally injected into the recipient animal.Animals were randomly allocated into groups with different treatments. The mouse MI model was performed in 4-month-old wild-type C57 mice according to the methods we described previously . Under aEchocardiography was performed (GE vivid 7 dimension) to determine cardiac structure and function in anesthetized mice. Mice underwent echocardiography at 4 weeks after surgery. After the induction of anesthesia by inhalation of isoflurane, the hearts were imaged two dimensionally (2D) in long-axis views at the level of the greatest left ventricular (LV) diameter. Left ventricular ejection fraction (LVEF) was automatically calculated by the echocardiography software using Simpson’s method. EF% was calculated as ((LVVd – LVVs)/LVVd) × 100, where LVVd is left ventricular end-diastolic volume (μL) and LVVs is left ventricular end-systolic volume (μL). Left ventricular fractional shortening (FS) was calculated as ((LVDd – LVDs)/LVDd) × 100, where LVDd is left ventricular end-diastolic dimension (mm) and LVDs is left ventricular end-systolic dimension (mm). Echocardiographic data were conducted and analyzed by blinded investigators.2+-free normal Tyrode’s solution containing 1 mg/mL collagenase II (Roche) and 0.1 mg/mL protease (Sigma) for about 10 min, depending on digesting conditions. The ventricles were cut off and minced in solution, followed by pipetting to disassociate the cells, and then filtered through a nylon mesh to remove big pieces of undigested tissues. The GFP+ cells represented CDCs or their derived cells, which were carefully counted. The survival rate = 100% × (number of survived GFP+ cells/1 × 105).To quantify the survival rate of post-transplanted CDCs, heart cells were isolated from the mouse heart at different time points after CDC transplantation using a method of enzymatic digestion . Brieflyhigh and CD11b+. Data were analyzed by FlowJo software.Similar to the method previously described , mouse h+ CDCs in hearts, anti-GFP, anti-sarcomeric tropomyosin (Sigma), and DAPI for nuclei were used to stain the heart tissue. To trace CDC differentiation, immunostaining against GFP, sarcomeric tropomyosin (a cardiomyocyte marker), von Willebrand factor , and α-smooth muscle actin  were performed.Hearts were fixed with 4% paraformaldehyde, subsequently embedded in paraffin, and cut to 5-μm thick sections. Similar to the method previously described , quantit2O2 (30 μmol/L for 24 h) was used to induce CDC apoptosis. CDCs were pretreated with MET at different dosages  and for different time periods  for inhibiting H2O2-induced CDC apoptosis. Administration of MET at 100 μmol/L for 60 min was selected for the experiment. AICAR (500 μmol/L for 60 min) was used to activate AMPK, and compound C  was used for inhibiting AMPK [CDCs were cultured in serum-free media for 24 h and HTotal cellular extracts were obtained by cell lysis, as described previously . Briefly2O2 were purchased from Sigma-Aldrich (USA). Antibodies against phospho-AMPK-Thr-172 and AMPK were purchased from Cell Signaling Technology. The antibodies against endothelial nitric oxide synthase (eNOS) and phospho-eNOS-Ser-1177 were purchased from Santa Cruz Biotechnology.1-Dimethylbiguanide hydrochloride (metformin hydrochloride), 5-aminoimidazole-4-carboxamide ribonucleoside , 6-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-3-pyridin -4-yl-pyyrazolo pyrimidine  and Ht test was performed. For multiple groups, one-way analysis of variance (ANOVA) with post-hoc comparisons by the Tukey’s test was used. Data were analyzed using SPSS 13.0 for Windows . A P value <0.05 was considered significant.Data are reported as the mean ± SEM. For analysis of differences between two groups, a student’s CDCs were isolated and expanded from transgenic β-actin-GFP mice with a protocol similar to that reported previously . GFP labTo identify the multi-differentiation potential of the CDCs, permanent ligation of the left anterior descending coronary artery was performed to induce MI injury, and CDCs were intramyocardially injected into the infarct border zone. Immunostaining was performed 4 weeks post-transplantation. As shown in Additional file 5 CDCs were intramyocardially injected into the infarct border zone immediately after MI. MET treatment was induced by intraperitoneal injection of MET at a dose of 125 mg/kg/day for a week. The initial areas at risk  of the infarcted hearts in the different groups with MI injury were comparable   Fig.  and c.Fi+/Ly6Chigh cells was robustly upregulated by MI injury, which was reduced by MET treatment or CDC transplantation. However, the combination of MET and CDC transplantation did not exert stronger anti-inflammatory effects  assay. As shown in Fig. 2O2 induced CDC apoptosis and decreased CDC viability. MET pretreatment (100 μmol/L for 60 min) significantly decreased the TUNEL+ CDCs and annexin V+/PI– CDCs subjected to H2O2 insult. The decreased CDC viability induced by H2O2 was also largely restored by MET (Fig. 2O2-induced CDC apoptosis, which was blocked by CC pretreatment (Fig. Oxidative stress is known to be a major factor causing stem cell apoptosis in the transplantation microenvironment of the post-MI heart. CDCs were treated with 30 μmol/L HMET Fig. . When thent Fig.  and b. T2O2 treatment did not change the expression of phosphorylated α-AMPK at Thr-172 in CDCs. The level of phosphorylated α-AMPK significantly increased when CDCs were treated with MET or AICAR, which was blocked by pretreatment with the AMPK inhibitor CC (Fig. 2O2 treatment. MET and AICAR significantly increased the eNOS phosphorylation, which was also blocked by pretreatment with CC (Fig. To further determine how MET resulted in AMPK activation, α-AMPK phosphorylation in CDCs was evaluated with Western blotting analysis. The total AMPK protein expression was comparable among each experimental group. Compared with control, H CC Fig. . As the  CC Fig. . These dMuch progress has been made in the field of stem cell transplantation for treating MI, but challenges such as poor cell survival in the harsh transplantation microenvironment greatly limit its therapeutic effect. In this study, we combined MET treatment and CDC transplantation for treating MI injury in mice, and found that MET improved CDC survival thereby enhancing its therapeutic effect after transplantation. Our data showed that the anti-apoptotic function of MET on CDCs was associated with AMPK activation and eNOS phosphorylation. Since MET is a safe medication in clinical practice, combination of MET treatment with stem cell therapy is of clinical significance to treat MI patients.The fact that CDC transplantation elicits cardioprotective effects has been shown in small and large animals, and in patients with MI injury. Aside from autologous CDC transplantation, ‘off-the-shelf’ allogeneic human CDCs are being tested clinically in the ALLSTAR and DYNAMIC trials . CDCs haDespite the progress in recent years, the survival of transplanted cells in infarcted myocardium is still poor , 22. PreMET is recognized to activate AMPK, which is essential for keeping the balance of energy production and metabolism in various tissues, thereby modulating cell apoptosis. MET can also exert cardioprotective effects independently of anti-hyperglycemic effects. MET-induced AMPK activation exerts protective effects against acute oxidative stress-induced injury in cardiomyocytes . AMPK woOur study reveals that MET treatment significantly enhances the therapeutic effect of CDC transplantation against MI injury. The pro-survival effect of MET on CDCs might be through an AMPK-eNOS-dependent mechanism. Since MET is a safe and ready-to-use medication, combination of MET treatment and stem cell therapy may be a promising strategy in clinical practice.


China has experienced unprecedented economic growth since the 1980s. Despite this impressive economic development, this growth exists side by side with the human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) and severe acute respiratory syndrome (SARS) crises and the persisting deficiencies in public health provision in China. Acknowledging the prevailing health problems, the Chinese government has encouraged the development of health non-governmental organizations (NGOs) to respond to the health challenges and address the gaps in public health provision of the government. HIV/AIDS-focused NGOs have been perceived as the most outstanding civil society group developed in China. Considering the low priority of health policies since the economic reform, the limitation of the “third sector” activity permitted in authoritarian China, together with the political sensitivity of the HIV/AIDS problem in the country, this article aims to explain the proliferation of HIV/AIDS-focused NGOs in China with the usage of the securitization framework in the field of international relations (IR).The research that underpins this article is based on a desk-based literature review as well as in-depth field interviews with individuals working in HIV/AIDS-focused NGOs in China. Face-to-face interviews for this research were conducted between January and May in 2011, and between December 2016 and January 2017, in China. Discourse analysis was in particular employed in the study of the security-threat framing process (securitization) of HIV/AIDS in China.This article argues that the proliferation of HIV/AIDS-related NGOs in China is largely attributed to the normative and technical effects of HIV/AIDS securitization ushered in by the United Nations Security Council (UNSC) and supported by the Global Fund to Fight AIDS, Tuberculosis, and Malaria  observed in China. Despite depicting a positive scenario, the development of HIV/AIDS-focused NGOs in China generated by the international securitization efforts is largely limited. An internal and external factor was identified to verify the argument, namely (1) the reduction of international financial commitments, as well as (2) the fragmentation of HIV/AIDS-focused NGO community in China.This article shows that international securitization weakened with the rise of Chinese commitment on HIV/AIDS interventions. In other words, HIV/AIDS-related responses delivered by the national government are no longer checked by the global mechanism of HIV/AIDS; thus it is unclear whether these NGOs would remain of interest as partners for the government. The fragmentation of the HIV/AIDS community would further hinder the development, preventing from NGOs with the same interest forming alliances to call for changes in current political environment. Such restriction on the concerted efforts of HIV/AIDS-related NGOs in China would make achievement of the Sustainable Development Goals (SDGs) to foster stronger partnerships between the government and civil society difficult, which in turn hindering the realization of ending HIV/AIDS in the world by 2030. China has experienced remarkable economic growth since the initiation of the economic reform in the 1980s. The average GDP growth in China was 9.6% annually during the first two decades of the economic reform . JakovljAizibing cun) in Henan and its outlying provinces, where a large percentage of rural residents were infected with HIV/AIDS  during this past 15 years,” as claimed by Michel Sidibe, executive director of the UNAIDS , 43. TheIn China, many of the first cases of HIV/AIDS since 1985 occurred among foreigners and homosexuals. Therefore, the official discourses at that time promoted the idea that HIV/AIDS only affected non-local Chinese people and foreigners, asserting “drug taking, alcoholism, robbery, homicide, suicide, divorce, prostitution, homosexuality, syphilis, AIDS…These kinds of Western social ills come from their ideology”, and also claiming “China had no sources of HIV/AIDS” , 45. TheGaige kaifang) of China to the outside world. The thirst for economic growth and performance preceded the urgency of disease management: local authorities feared their respected jurisdictions would lose external or foreign investment if the full extent of the problem were known, or that they would be punished by superiors for failing to prevent the HIV/AIDS spread. The “cover-up” practice also occurred during the early outbreak of SARS in China in 2003, for the infectious disease clearly posed a negative impact on the Chinese flourishing trade and tourist industry.One probable reason for the resistance to HIV/AIDS securitization is economic: the period of early HIV/AIDS outbreak coincided with the initial years of Open Door Policy and economic reform .common enemy of the whole mankind as it seriously threatens public health and safety. The Chinese government has attached great importance to HIV/AIDS prevention and treatment and has treated it as a strategic issue for social stability, economic development, national prosperity and security, making it a first priority of the government work” [emphasis added]     . The Chis added] . The ides added] . China’ss added] . Based oSuch an alteration in perception toward the epidemic brought about the political recognition of the HIV/AIDS-focused NGOs, which has also been highlighted by multiple statements delivered by various top-level government officials since 2003. In 2004, Vice Premier Wu Yi stated, “We should mobilize all the partners in society to participate in the fight against HIV/AIDS. We need to improve our policies and strategies to build a better environment for the society to participate in the response, and try our best to facilitate the involvement of all sectors” . In 2011The previous section illustrates the change in the perception of the Chinese government toward the HIV/AIDS problems: from denial to the acceptance of the threats that could be posed by the disease on the country and its people. Such threat recognition also brought about the acknowledgement that the government alone could not resolve the HIV/AIDS problems because of the complicated nature of the epidemic; the involvement of civil society groups was a globally recognized recommendation leading to the successful containment of the epidemic. Nevertheless, verbal recognition is not sufficient to enhance the engagement and development of HIV/AIDS-focused NGOs, especially in the preexisting authoritarian government structure; the governance and funding mechanism for HIV/AIDS responses requires alternation in China. As illustrated in the following section, this was the mechanism of the Global Fund that enhanced the funding for HIV/AIDS-focused NGOs, facilitating the involvement of civil society groups in translating the rhetorical acts into emergency responses of HIV/AIDS in China.Among the international funding agencies emerging after the HIV/AIDS securitization launched by the UNSC, the Global Fund was the most renowned major contributor to the HIV/AIDS interventions in China. The Global Fund finally accepted the first proposal submitted by the Chinese government on HIV/AIDS interventions in late 2003, after the previous proposals being turned down twice in 2002 . BetweenIntervention of the Global Fund’s funding mechanism was deemed to transform the primary HIV/AIDS governance in China . The GloThe involvement of NGOs in national HIV/AIDS programs was notably highlighted in Round 6 of the Global Fund in China, since the theme of the grant proposal submitted by the CCM was directly linked to the development of civil society groups  of the country, the Chinese government  was required to disburse a designated portion of the grants to HIV/AIDS-related NGOs (in the form of sub-recipients) operating in the country . FollowiFirst, the Global Fund required at least 25 percent to 30 percent of the grant should be given to grassroots NGOs. However, the government failed to achieve this requirement. Second, the management fee in some provinces was too high. Third, one of the elements in the RCC program was related to budget aggregation . However, the Chinese government did not allocate enough money for the aggregated budget (CCDC/NHFPC-1).The suspension of the Global Fund in 2011 demonstrated that the funding mechanism to some extent held the Chinese government accountable for the financial supports of HIV/AIDS-focused NGOs. The Global Fund once suspended its funding to Chinese HIV/AIDS programs in early 2011 because the Chinese CCM failed to allocate 35% of the US$283 million HIV/AIDS grant to grassroots NGOs as pledged; less than 11% was distributed to HIV/AIDS-focused NGOs . A progrThe determination of the Chinese government to resolve the suspension of the Global Fund was observed in the course of fieldwork research. During a face-to-face interview inside the office building of the Ministry of Health  in May 2011, the respondent told the investigator “the representatives of the Chinese government and the Global Fund representatives are meeting upstairs to negotiate for the resumption of the 2011 grants” (CCDC/NHFPC-2). The dispute was eventually resolved; the Chinese government promised to allocate at least 25% of the Global Fund grant to NGO work . In thisConsidering HIV/AIDS as a health threat to the country and its people, the recognition and involvement by the central government and the NHFPC and other related government agencies is of particular vital to the proliferation of HIV/AIDS-focused NGOs in a state-dominated society in the current political regime. Despite depicting a positive scenario, this article further illustrates that the development of HIV/AIDS-focused NGOs in China generated by the international securitization efforts is largely limited. In other words, the growth of the number of NGOs working on HIV/AIDS in China would be ceased or even decreased in long term, and also the political space for grassroots to involve in HIV/AIDS policy making and implementation would be further restricted. An internal and external factor was identified to verify the argument, namely (1) the reduction of international financial commitment, as well as (2) the fragmentation of HIV/AIDS-focused NGO community in China.The Global Fund stopped the phase two of the consolidated program suddenly . As a result, the number of NGOs working on HIV/AIDS decreased drastically from 1,000 to 500 between 2013 and 2014 (INGO-1).Owing to a lack of international and national HIV/AIDS funding, the number of NGOs working on HIV/AIDS was drastically reduced; many NGOs and CBOs were simply closed down, only a few of them could barely survive with the support by other funding sources .China resembles some developing countries in that it has faced the problem of decreasing international HIV/AIDS financial commitments in supporting its national HIV/AIDS responses . Given tshehui zuzhi canyu aizibing fangzhi jijin), providing financial resources for HIV/AIDS-related NGOs via the submission of grant proposal starting from 2017 [The retreat of the Global Fund has subsequently created a policy vacuum for the Chinese government in taking the lead in managing the development of HIV/AIDS-related NGOs in China. Mimicking the funding mechanism of the Global Fund, the State Council launched the China AIDS Fund for Non-Governmental Organizations (CAFNGO)  and registered NGOs working on HIV/AIDS are included in HIV/AIDS national policy implementation processes, whereas excluding unregistered grassroots NGOs that are perceived as illegal or “anti-government” organizations. Based on the nature of services that individual HIV/AIDS-focused NGOs provided, service providers are preferred by the state, whereas NGOs serving as advocates of human rights and agencies providing legal services for HIV/AIDS-infected people would be subjected to prosecution and coercion. A HIV/AIDS specialist working in an international NGO in Beijing stated, “During a 2011 HIV/AIDS NGO meeting, the Chinese government clearly claimed that the authorities would like to work with NGOs/CBOs conducting service delivery, but not with those working on HIV/AIDS-related human rights or gender issues” (INGO-1). To further illustrate this point, the Chinese political leaders publicly praised a Guangxi-based NGO named AIDS Care China, recognizing the work the organization had been done in HIV/AIDS prevention, meanwhile stifling numbers of HIV/AIDS activists through imprisonment, house arrest, or assault. Dr. Wang Yanhai and Dr. Gao Yaojie were two of the prominent Chinese HIV/AIDS activists that fled to the United States in 2009 and 2010, respectively, due to the recurrent pressure and disturbance exerted by Chinese authorities owing to their advocacy work. In June 2015, a HIV/AIDS-related legal aid NGO, Beijing n regime , while pFew NGOs have cooperative relationship. There are lots of “fake” NGOs in China. The CDC found some patients to set up grassroots NGOs and then through the name of such NGO to apply for the Global Fund. After they received the money, CDC would become the one who controlled the funding (self-help group-2).Eighty-five percent of the HIV/AIDS-focused NGOs in Yunnan have government background…for those NGOs that are funded by the Global Fund or China-Bill Gates Projects, these are organizations with the government background…or if you read their introduction saying “with the help and support of government”, you will know these are NGOs with the government background (self-help group-3).While a formal local network for HIV/AIDS-related NGOs or civil society groups has been established in many countries in Asia, such coordination mechanism between non-state actors is largely absent in China.A “we-they” distinction has apparently prevailed amongst the HIV/AIDS NGOs community in China, reinforcing the atomization of HIV/AIDS-focused NGOs, discouraging the proliferation of civil society in China. Horizontal relationship amongst NGOs working on HIV/AIDS is relatively weak and ill-organized, thereby avoiding these organizations joining hand to call for favorable political environment or monetary supports to continue their operation in China. Owing to the reduction of international financial commitments, as well as the fragmentation of HIV/AIDS-focused NGO community in China, the proliferation of HIV/AIDS-focused NGOs in China generated by the international securitization efforts is nevertheless largely limited in long run. Such restriction on the concerted efforts of HIV/AIDS-related NGOs will inevitably pose challenges to China to fulfill the pledge of the 2030 Agenda to end HIV/AIDS as one of the targets of the Sustainable Development Goals (SDGs) .HIV/AIDS was officially announced as a security issue in 2000 by the UNSC Resolution 1308, which claimed that the pandemic could pose security threats if left unchecked. The rhetorical security-threat claim resulted in the burgeoning number of GHIs organized by the international and bilateral organizations operating at the state level. This article illustrates the ways in which the UNSC Resolution 1308 and the Global Fund influencing the HIV/AIDS governance in China. Rhetorically acknowledged the HIV/AIDS problems and the irreplaceable role of HIV/AIDS-focused NGOs in combating the disease by the Chinese government, the monetary and technical supports by the Global Fund enabled HIV/AIDS-focused NGOs to participate in the national policymaking process, facilitating the proliferation and development of civil society in China.This success nevertheless does come with concerns regarding sustainability. This article shows that international securitization weakened with the rise of Chinese commitment on HIV/AIDS interventions. In other words, HIV/AIDS-related responses delivered by the national government are no longer checked by the global mechanism of HIV/AIDS; thus it is unclear whether these NGOs would remain of interest as partners for the government . The fraThis research has several limitations. Firstly, there is a lack of available information regarding the operation of local or grassroots HIV/AIDS-specific NGOs that are in particular helping MARPs in China, owing to the political sensitivity of HIV/AIDS, the unregistered status of NGOs working on HIV/AIDS, the increasing restrictive environment for NGOs operating in China especially under the Xi administration, together with the social stigma attached to their service objects as well as the NGOs themselves as the service providers. Secondly, it is difficult to conduct longitudinal study on the development of HIV/AIDS-specific NGOs in China. The researcher realized that many of the NGOs that had been interviewed in 2011 were no longer under operation in the follow-up fieldwork in 2017, either because these NGOs could not secure sustainable funding, or they were cracked down by local authorities for advocating HIV/AIDS-related human rights or gender equality, or having very close links to foreign (western) organizations. Further research therefore needs to be conducted to remedy these technical weaknesses. It is suggested that comparative research could be conducted on the effects (if any) of the regime types, such as China and Thailand, and the level of socio-economic development of the country, such as China and Singapore, on the effects of international HIV/AIDS securitization in the national levels.


Inappropriate activation of c-mesenchymal-epithelial transition (MET), the receptor tyrosine kinase (RTK) for hepatocyte growth factor (HGF), has been implicated in tumorigenesis and represented a promising therapeutic target for developing anticancer agents. In contrast to other solid tumors, there are limited data describing the functional role of HGF/c-MET signaling pathway in lymphoma. In the current review, we summarize recent findings about the expression, cellular mechanisms/functions, and therapeutic application of HGF/c-MET in different types of lymphoma, especially B cell lymphoma, T and NK cell lymphoma, and Hodgkin lymphoma. We also discuss the existing problems and future directions about studying the HGF/c-MET pathway in lymphoma cells. MET proto-oncogene encodes for the receptor tyrosine kinase (RTK) of growth factor tyrosine kinase [. The structure of MET protein comprises a highly glycosylated 45-kDa extracellular α-subunit and a 145-kDa transmembrane β-subunit, which are linked together by a disulfide bridge  can be utilized as a signaling receptor for hepatocyte growth factor (HGF), and it has been reported that in humans, the e kinase , 2. The Weimar et al. reported that in several B cell-derived lymphoma cell lines , two of them  were c-MET positive . Within In one of the most common B cell-derived lymphomas, DLBCL, c-MET was overexpressed in 26–73.2% of cases and significantly associated with other signaling molecules such as p-AKT, p-GSK3, and Ki-67 , 19. IntKaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL), which comprises transformed B cells harboring the viral episome and arises preferentially within the pleural or peritoneal cavities of patients infected with HIV . PEL is Among Burkitt’s lymphoma cell lines, EB4 and Raji expressed high levels of c-MET, while Namalwa cell line was negative . HGF proc-MET was found in the lymphocytes composing mucosa-associated lymphoid tissue (MALT) lymphoma, and HGF was recognized mostly in the endothelial cells and macrophages in the MALT lymphoma . HGF actHodgkin lymphoma is a B cell neoplasm characterized by a minority of Reed-Sternberg (RS) cells in the infiltrating reactive cells. In classical Hodgkin lymphoma (cHL) , the expIn anaplastic large cell lymphoma (ALCL), a type of T cell lymphoma, c-MET is expressed in Karpas299 cells and some other ALCL cell lines . HoweverIn addition, the expression of c-MET was found in PBMCs from adult T cell leukemia/lymphoma (ATLL) patients and HTLV-1-infected T cell lines. HGF secreted in paracrine rather than in autocrine mechanism seems to be involved in the pathogenesis of ATLL . ATLL ceBecause of its pleiotropic role in oncogenesis and cancer progression, HGF/c-MET is considered to be an important target in anticancer therapy. However, currently, there are no HGF inhibitors that have been tried in lymphoma treatment, so here, we will focus on c-MET inhibitors, their application in lymphoma treatment Table . c-MET iSU11274, another ATP-competitive c-MET selective inhibitor, blocked the constitutive phosphorylation of c-MET and downstream kinases and induced G2/M cell cycle arrest and significantly suppressed cell growth in a dose-dependent manner . TreatmeMET gene mutation and amplification represent one of the major mechanisms causing constitutive active HGF/c-MET signaling in cancer cells. However, no amplification of the MET gene was found in B cell lymphoma cell lines and patient samples [There are only limited data studying the role of HGF/c-MET signaling pathway in lymphomas, when compared to those from other solid tumors. For example,  samples . Current samples . Therefo samples , 63. How


High expression of c-Met strongly correlated with the expression of two CSC markers, ALDH1A3 and CD133 in breast cancers. In addition, breast cancers at tumor stage III-IV expressing both c-Methigh and ALDH1A3high had poor prognosis. Furthermore, treatment with c-Met inhibitors  in MDA-MB157 cells with high c-Met protein expression resulted in significant suppression in cell viability, contrary to MDA-MB468 cells with low c-Met protein expression. These c-Met inhibitors also suppressed cell viability and tumor-sphere formation of ALDH1high breast cancer cells with high c-Met expression. These results suggest that c-Met in ALDH1 positive CSCs seems to play an important role in breast cancer repopulation. Therefore, we conclude that c-Met is a potential therapeutic target in ALDH1 positive breast CSCs.c-Met is a receptor-type tyrosine kinase, which is involved in a wide range of cellular responses such as proliferation, motility, migration and invasion. It has been reported to be overexpressed in various cancers. However, the role of c-Met in breast cancer stem cells (CSCs) still remains unclear. We herein, show that  Breast cancer is one of the most common cancers occurring in women worldwide with 1.7 million new cases  and 0.5 million-cancer deaths  according to an estimate from the International Agency for Research on Cancer (IARC) , 4, of wTumors are comprised of population of cancer cells and distinct cancer stem cells (CSCs), which are largely undifferentiated tumorigenic cells with stem-like properties such as self-renewal and multipotency , 8. MostALDH1A1 and ALDH1A3 are also known as CSCs markers in several cancers [ALDH1A3 has been reported to contribute significantly to ALDH1 activity in breast cancer cells and its expression significantly correlates with cancer type, tumor grade and metastasis in breast tumor patients [CSCs in breast tumor patients can be identified based on the expression of aldehyde dehydrogenase (ALDH) isoforms. ALDH1 has been reported to be enriched in CSCs of several cancer types, including breast cancer and is a potential CSC marker , 10, 11. cancers , 13, 14.patients . On the patients , 17, 18.c-Met is a receptor-type tyrosine kinase, which is involved in wide range of cellular responses such as proliferation, motility, migration, invasion and tumor angiogenesis , 20. c-MALDH1A3 in breast cancer. Patients with co-expression of c-Met and ALDH1A3 at tumor stage III-IV showed poor clinical outcome. Furthermore, c-Met inhibitors suppressed the viability and tumor-sphere formation of ALDH1high cells. These results suggest that c-Met is essential for the viability and tumor formation of ALDH1 positive breast CSCs. Therefore, c-Met protein is a promising therapeutic target for ALDH1 positive breast CSCs.In this study, we show that high expression of c-Met correlates with the expression of c-Met with CSC markers such as ALDH1A1, ALDH1A3, CD44, and CD133 at gene expression levels in human breast cancers, we analyzed mRNA data and the clinical information of 1904 patients of breast cancers from cBioPortal for Cancer Genomics [c-Met (MET+) correlated with expression of CSC markers, ALDH1A1 (p < 0.001), ALDH1A3 (p < 0.001), and CD133 (p < 0.001) in breast cancers. In addition, scatter plots analysis also indicated that c-Met expression correlated with ALDH1A1 (p = 0.0077), ALDH1A3 (p < 0.001) and CD133 expression (p < 0.001) , 45% were c-Met+ (n = 573), contrary to 59% of c-Met+ (n = 74) at tumor late stage lesions . As c-Met+ tumor lesions were higher in tumor stage III-IV, in contrast with stage 0, I, and II, we next focused to analyze the relationship between c-Met gene expression and CSC markers in breast cancer subtypes at tumor stage III-IV. c-Met mRNA was found to be enriched in Basal-like type in comparison with other subtypes at stage III and IV . c-Met+ also weakly associated with CD133+ (p = 0.0025). Scatter plots analysis also indicated that c-Met expression correlated with ALDH1A3 (p < 0.001) and CD133 expression (p < 0.001)   and CD133high(p = 0.0088) showed poor prognosis , and with CD133high (p = 0.0023) indicated poor prognosis  Figure , whereass Figure . Interess Figure . These rhigh cells derived from both MDA-MB157 and MDA-MB468 cell lines showed CSCs properties such as self-renewal, multi-differentiation, and tumorigenesis  of Crizotinib, Foretinib, and PHA-665752 were found to be lower in MDA-MB157 cells expressing higher c-Met protein than that in MDA-MB468 cells expressing lower c-Met protein , 0.21 μM (Foretinib), 3.4μM (PHA-665752), and 0.18 μM (Tivantinib)  Figure . These rTaken together with aforementioned results, it can be inferred that c-Met is specifically essential for cell viability and tumor-sphere formation of ALDH1 positive human breast CSCs.c-Met correlated with the expression of ALDH1A3 in Basal-like type of breast cancer . In spite of no correlation between c-Met and ALDH1A3 expression, major population of Basal-like type patients expressed high expression of c-Met and ALDH1A3 (n = 93 in 199). Therefore, c-Met may play an important role in cancerous progression in Basal-like type. On the other hands, at tumor stage III-IV, of total analyzed patient samples (n = 124), each subtypes were distributed as follows; Luminal A , Luminal B , HER2-enriched , Claudin-low , Normal-like , and Basal-like . Therefore, co-expression of both c-Met and ALDH1A3 at late tumor stages may contribute to poor clinical outcome not only in Basal-like but also in other subtypes. Since efficacy of chemotherapy at cancer spreading stage III-IV is extremely crucial, targeting c-Met in ALDH1 positive breast CSC may possibly decrease the severity of metastatic breast cancer and hence may lead to the survival of breast cancer patients. In addition, previous studies reported that ALDH1 is required for maintaining a drug-resistant cell subpopulation of stomach and breast cancer cells [c-Met and ALDH1A3 should be analyzed in detail in the future for targeted cancer therapy.Several studies have reported that patients with higher expression of ALDH1 have poor prognosis in several cancers , 35. In s Figure . Howeverer cells , 38, 39.high cells  were obtained from American Type Culture Collection . Cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS)  and penicillin/streptomycin. Cells were cultured at 37°C in a humidified atmosphere with 95% air/5% COc-Met inhibitors  were purchased from Namiki Inc. (Japan). All compounds dissolved in DMSO. Rabbit polyclonal c-Met antibody was purchased from Santa Cruz Inc. (USA). Rabbit monoclonal phospho-Met (Tyr1234/1235) antibody, HRP-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Cell Signaling Technology (USA). Mouse monoclonal β-actin antibody was obtained from Wako Inc. (Japan).high cells were isolated from MDA-MB157 and MDA-MB468 cells by ALDEFLUOR assay kit (Stem Cell Technology) or AldeRed ALDH detection assay kit (MERCK) according to the manufacturer's instructions. Briefly, cells (2×106) were incubated with the substrate for ALDH1 (5μL substrate/mL medium) for 30 min at 37°C. As a negative control for the ALDEFLUOR assay and AldeRed assay, cells were incubated with ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB). The ALDH1high cells were sorted by cell sorter  by taking the negative control into consideration. The analysis of CD10/EpCAM positive cells from MDA-MB157 and MDA-MB468 cells. Suspended MDA-MB468 cells (1×106) were incubated with anti-CD10 (APC) (BD Bioscience) and anti-EpCAM (PE) (BD Bioscience) for 1hr on ice, after which the sample was washed with fresh FACS buffer (2%FBS in 1×PBS (-)). For this experiments, cells were analyzed using a FACS Calibur (BD Bioscience).Cells were exfoliated from culture dish by accutase (Innovative Cell Technology) and filtered through 40μm cell strainers (Greiner) to obtain single cells. The ALDH15/well) were seeded into 96 well culture plate (Sigma). One day post seeding, cells were treated with c-Met inhibitors for 3, 5, and 7 days. Cell viability was detected by WST-8 assay (Cell Counting Kit-8 (DOJINDO)). The formazan dye formed was measured by ARVOTM MX (PerkinElmer) at 450 nm. Numerical values of test groups are shown with respect to 0.02% DMSO treated group.Cells (3×10Cells were dissolved in RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.5 w/v% sodium deoxycholate, 0.1 w/v% SDS, 1.0 w/v% Nonidet P-40 and protease inhibitor cocktail (Thermo Fisher)). Eight μg of whole cell lysate proteins was electrophoresed by SDS-PAGE (8% gel) and transferred to Immobilon-P Transfer Membrane (Millipore) or Immobilon-FL Transfer Membrane (Millipore). The transferred membranes were then blocked with 5% BSA in TTBS (25 mM Tris (pH 7.5), 140 mM NaCl, 2.5 mM KCl and 0.1% Tween 20) and incubated with the primary antibodies. The membranes were then probed with the horseradish peroxidase-conjugated secondary antibody. Specific signals were detected by chemiluminescence reagent, such as Immunostar LD/Immunostar Basic (Wako) using ChemiDoc MP (Bio-Rad).2. ALDH1high cells (1x103/well) were seeded and cultured in ultra low attachment 96-well plate (Greiner) for 6 days with or without inhibitory compounds. CellTiter-Glo® luminescence assay (Promega) was performed by TR717 Micro plate Luminometer (TROPIX) using 96 well Micro-assay-plate (Greiner). Numerical values of test groups are shown with respect to 0.02% DMSO treated group.Tumor-spheres were grown in DMEM culture medium containing 10% FBS, penicillin and streptomycin, 0.6% methyl cellulose (Wako), and 0.05 mM 2-mercaptoethanol (Sigma) at 37°C in a humidified atmosphere with 95% air/5% COn = 140; Luminal A, n = 679; Luminal B, n = 461; HER2-enriched, n = 220; Claudin-low, n = 199; Basal-like, n = 199; Not classified, n = 6). We retrieved the mRNA expression (Z-scores) of genes and evaluated co-expression of c-Met and several stem cell markers in either all or each of the tumor stage groups. We defined the c-Met expression as follows; all stage patients were divided into c-Met+ and c-Met- in Figure + and c-Met- in Figure c-Met expression in all or stage III-IV groups. Quantitative variables were analyzed by Tukey's test. Data with p value less than 0.05 were considered significant. Survival curves were plotted by the Kaplan-Meier method and compared by the Gehan-Breslow generalized Wilcoxon test using BellCurve for Excel ver2.11. “High” and “low” were defined as the upper top 40% and the lower 60% of Z-score respectively, in several genes at stages III-IV breast cancer patients. Follow-up period after diagnosis ranged from 5.8 to 274.3 months stages III-IV breast cancer patients.Gene expression data was analyzed using METABRIC, Nature 2012 & Nat. Commun. 2016 dataset deposited in cBioPortal , 30, 31.


MAX2 ubiquitin ligase, but the universality of this mechanism is not clear. Multiple proteins have been suggested as targets for strigolactone signalling, including both direct proteolytic targets of SCFMAX2, and downstream targets. However, the relevance and importance of these proteins to strigolactone signalling in many cases has not been fully established. Here we assess the contribution of these targets to strigolactone signalling in adult shoot developmental responses. We find that all examined strigolactone responses are regulated by SCFMAX2 and D14, and not by other D14-like proteins. We further show that all examined strigolactone responses likely depend on degradation of SMXL proteins in the SMXL6 clade, and not on the other proposed proteolytic targets BES1 or DELLAs. Taken together, our results suggest that in the adult shoot, the dominant mode of strigolactone signalling is D14-initiated, MAX2-mediated degradation of SMXL6-related proteins. We confirm that the BRANCHED1 transcription factor and the PIN-FORMED1 auxin efflux carrier are plausible downstream targets of this pathway in the regulation of shoot branching, and show that BRC1 likely acts in parallel to PIN1.Strigolactones are a recently identified class of hormone that regulate multiple aspects of plant development. The DWARF14 (D14) α/β fold protein has been identified as a strigolactone receptor, which can act through the SCF Summary: Strigolactones signal through D14 to regulate shoot development by targeting SMXL6-clade proteins, but not BES1 or DELLA proteins, for degradation. BRC1 and PIN1 plausibly act downstream to regulate branching. Striga spp.) , a group of carotenoid-derived terpenoid lactones. SLs were first identified as a component of root exudates that cause seed germination in parasitic witchweeds -class cytochrome P450s , encoding a HEAT SHOCK PROTEIN101-like protein, suppress aspects of the max2 phenotype that are associated with karrikin responses, but not those related to SL responses, supporting the idea of separate target proteins downstream of MAX2 for KL and SL signalling  TCP-domain transcription factor has been implicated as a transcriptional target of SL, since ranching , and SL n in pea . Howevern in pea . This abn in pea , and racma cells .rac-GR24 does not specifically activate the SL signalling pathway . It also has shorter petioles and leaf blades, but no reduction in blade width, leading to an altered leaf shape .The most intensively studied aspect of SL developmental responses has been shoot branching, but the phenotypes of SL synthesis mutants include other aspects of adult shoot development. For example, in Arabidopsis SL has been implicated in the control of leaf blade and petiole length, leaf senescence, internode elongation and final height, branch angle, stem diameter, and cambial development . To provmax4-5 . Under omax4-5 phenotype, and gain-of-function mutants should suppress the phenotype of SL-deficient/insensitive mutants. For negative regulators these expectations are inverted. Mutants in downstream effectors should display changes in the SL-sensitivity of relevant phenotypes. In practice, the genetic materials do not exist to assess all these aspects for each candidate gene, and genetic analysis is often complicated by problems of pleiotropy, redundancy and epistasis. Nevertheless, we were able to gather sufficient materials for each candidate to assess their role in SL signalling.Having established a phenotypic platform for understanding the effects of SL deficiency in adult shoots, we tested whether mutations in proposed or potential SL signalling genes confer the expected phenotypic profile. For positive regulators of SL signalling, loss-of-function mutants should phenocopy the d14-1 are essentially indistinguishable from max4-5 . Consistent with previous reports  . By contrast, we did not observe any clear phenotypic similarities between max4-5 or the kai2-2 or dlk2-3 mutants . The kai2 mutant has distinct phenotypic effects in the shoot that are not seen in max4-5, including strongly accelerated flowering time  and increased leaf blade width . We conclude that D14-dependent signalling is fully responsible for SL effects on shoot branching.As discussed above, two proteins are known to be required for SL signalling, MAX2 and D14. The leaf dimensions and leaf senescence, branching level, branch angle, height and stem thickness phenotypes of m max4-5 ; Fig. S1rts e.g. , we also mutants ; Fig. S1de width A. In conhis line , Fig. S1d14-1, the max2-1 mutant is not a simple phenocopy of max4-5 . However, leaf blade length is not reduced in max2, and there are additional phenotypes, including wider leaf blades. Since MAX2 has been implicated in signalling downstream of KAI2, we reasoned that the max2-1 phenotype may represent combined loss-of-function of signalling downstream of these two receptors, which we confirmed by analysing a d14-1 kai2-2 double mutant, which closely phenocopies max2-1 . Given the similarity of the d14-1 and max4-5 phenotypes, and the lack of obvious redundancy with KAI2 and DLK2, we conclude that for all the phenotypes we examined, SL signalling is mediated by D14 acting through MAX2.We reasoned that if DLK2 acted redundantly with D14 or KAI2, the effect of losing DLK2 would be more obvious in the sensitised 1 kai2-2 ; Fig. S1MAX2 show the expected phenotypes of negatively regulated targets. We first examined the DELLA proteins, constitutive repressors of growth that are degraded in the presence of gibberellins (GA). DELLA proteins have been identified as SL signalling targets based on their physical interactions with D14 (gai mutant in which the GIBERRELIC ACID INSENSITIVE (GAI) DELLA protein is stabilised, phenocopying severe GA deficiency  mutant, in which all DELLA protein activity is lost , only the absolute dimensions of the leaf . The effect of DELLA activity on leaves is thus qualitatively different from the effect of SL signalling. There was also no alteration in leaf senescence in della relative to Ler, but there may be a delay in the gai-1 mutant . As anticipated, height was increased in della, and reduced in gai relative to Ler . With respect to height, the effect of gai is thus qualitatively similar to SL mutants, but is quantitatively much more extreme. Stem diameter follows the same pattern, being increased in della, and reduced in gai . We observed no difference in branch angle between Ler and gai, but branch angle was increased in della . Finally, we examined whether either mutant had a branching phenotype under standard long-day growth conditions, but did not observe any statistically significant difference from the Ler wild type in terms of total primary branches in della or gai   H. The di P>0.05) H, but thranching , but fougai and della mutants share some phenotypic characteristics with reduced and increased SL signalling mutants, respectively, their phenotypic syndromes and the correlations within them are both qualitatively and quantitatively different. It is therefore plausible, if unlikely, that SL could regulate some aspects of shoot phenotype by targeting DELLA proteins for degradation. To assess more directly the effects of SL on DELLA stability, we treated roots expressing a GFP-RGA fusion protein (rac-GR24 for 45 min (a relevant timeframe for SL action), but observed no decrease in the level of fluorescence of the fusion protein relative to mock-treated plants  . We thus conclude that SL is unlikely to control development through targeting DELLA proteins for degradation. Consistent with this idea, SL acts independently of GA and DELLAs in the control of internode elongation in pea  F-H. We ttability I-K (t-ten in pea .bes1-D mutant (in which BES1 is stabilised) was reported to have increased branching, while BES1-RNAi lines were reported to have reduced branching . There is no difference in any leaf dimension between bes1-1 and Col-0  , and leaf senescence is not delayed in bes1-D or bes1-1 relative to Col-0 . We observed no significant difference in height between Col-0, bes1-1 and bes1-D  , and no difference in stem diameter between Col-0 and bes1-1, though there is a significant reduction in bes1-D relative to Col-0  . There is also a significant increase in branch angle in bes1-D relative to Col-0, but branch angle in bes1-1 is not different from Col-0  .BES1, a transcription factor which regulates brassinosteroid (BR) responses along with its homologues BZR1 and BEH1-BEH4, has been proposed as a direct target of SL signalling, based primarily on biochemical approaches . Consistranching . However1-D line  from theild type , as well  although bes1-D (but not bes1-1) shows a slight increase in branching in the more sensitive decapitation-based assay   . This result contrasts to previous reports that BES1-RNAi lines suppress the branching phenotype of max2-1. The BES1-RNAi lines have highly pleiotropic phenotypes and are generally lacking in vigour, making the results difficult to interpret  A,B,D. Wenterpret .bes1-D alters sensitivity to SL, because the SL analogue rac-GR24 does not reduce hypocotyl length in the bes1-D background . Indeed the kinetics of bud outgrowth in response to either NAA or NAA+rac-GR24 treatment are slightly retarded relative to wild type, rather than accelerated as would be predicted if BES1 is a target for SL signalling in this response , and transformed it into Arabidopsis. As with SMXL7, we observed a clear nuclear localization for SMXL6 in cells of the Arabidopsis root meristem . As anticipated, SMXL6Δpl-YFP was resistant to rac-GR24-induced degradation  . The buds of Δpl-YFP35S:SMXL6 plants are insensitive to the application of rac-GR24 when tested in an excised node assay  . They have the same blade length:width ratio as max2-1  , but are not as large as max2-1 leaves  C,E; ther P=0.39) E,F. The  mutants A,D. 35S:1 leaves D. Whilstpromoter . This mar 7 days B. Converhenotype B. Thus SBRC1 has been suggested as a transcriptional target of SL signalling, based on the SL-resistant increased shoot branching phenotype observed in brc1 loss-of-function mutants, and the lack of genetic additivity in some, but not all, brc1 max double mutants    . However, plant height is reduced in brc1-2 brc2-1, although not to the same extent as seen in d14-1  . These data suggest that BRC1 is a plausible target of SL signalling, although only in the contexts of shoot branching and stature. This is consistent with the reported expression pattern of BRC1 and BRC2  B,C. We fig. S5A) . The resild type . We founand BRC2 . HoweverOsSPL14 and the rice BRC1 orthologue have been described, leading to the hypothesis that BRC1 transcription is regulated by OsSPL14     B,C. We t P<0.05) F. We als P<0.05) E. We alse leaves A,D. Thus in rice .max1-1, max3-9 and max4-1 have increased auxin transport in the primary inflorescence stem, and that max1-1 and max3-9 have increased levels of the PIN1 auxin efflux carrier at the basal plasma membrane of cambial and xylem parenchyma cells in the stem , while there is no change in auxin transport in kai2-1 (here in the Ler background) or dlk2-1 relative to wild type   E-G.Fig.Δp-loop-YFP35:SMXL6, further demonstrating the equivalence in function of SMXL6 and SMXL7 . Furthermore, we have shown that loss of smxl6, smxl7 and smxl8 causes a dramatic reduction in auxin transport and PIN1 levels in inflorescence stems  . These data demonstrate that if BRC1 is involved in SL signalling, it does not act upstream of the regulation of PIN1 accumulation. We next tested whether BRC1 expression is modulated by changes in PIN1 accumulation and/or auxin transport, i.e. whether BRC1 is downstream of PIN1. The max2 mutant has increased PIN1 accumulation and auxin transport, and reduced BRC1 expression. Thus, we hypothesised that, if BRC1 is downstream of PIN1, the tir3 mutant, which has decreased PIN1 accumulation and auxin transport, ought to have increased levels of BRC1 expression. However, we found that BRC1 expression in tir3 is strongly reduced, as in max2  and used several different assays, including direct tests of SL sensitivity. Our results suggest that, contrary to previous suggestions, neither BES1 nor DELLA proteins fit the profile of an SL target in the regulation of shoot development. DELLA proteins had only been implicated as SL targets on the basis of biochemical interaction with D14 , and preorphisms . Our anagnalling , 2014. Msmxl6 smlx7 smxl8 triple mutant completely suppresses relevant aspects of the max2 phenotype  is not downregulated in SL mutant buds and does not respond to SL treatment  independently of BRC1. SL mutants can have stronger and different branching phenotypes than brc mutants  . Data presented for kai2-1 are in the Landsberg erecta background, except for kai2-1 allele has been backcrossed into Col-0 background. Double mutants between lines were constructed using visible, fluorescent and selectable markers or by PCR genotyping as previously described  , gai-1   (primers: ATGCCGACGCCGGTGACTACG and CCATATCACATCCACCTTCGCCG). The SMXL6ΔP-loop variant, lacking amino acids 705-712 (FRGKTVVD), was made with the Q5 Site-Directed Mutagenesis Kit (NEB) (primers TACGTAACCGGTGAGTTATC and TTTGTCATCAAGGGAACAATG). SMXL6 and SMXL6ΔP-loop entry clones were sub-cloned into a pEarlyGate101 destination vector, between the 35S promoter and a C-terminal YFP tag. The resultant constructs were transformed into the Col-0 or max2-1 genetic background using the Agrobacterium floral dip method . Three technical replicates were run for each of three biological replicates. Expression levels were calculated using the Light Cycler 480 II software and the 2nd derivative maximum method assuming equal primer efficiencies. Primers: BRC1-F CTTAGTCAACTACAAACCGAACTCAT; BRC1-R GATCCGTAAACTGATGCTGCT; UBQ10-F CCACTTGGTCTTGCGTCTGC; UBQ10-R TCCGGTGAGAGTCTTCACGA.For analysis J, active−2 s−1). For axenic growth, seeds were sterilised, and stratified at 4°C for several days. Seedlings were grown using ATS media  in controlled environment rooms with light provided by white fluorescent tubes, , and then measured again at 37 dpg to confirm that growth of these leaves was arrested. The maximum length and width of the leaf blade were measured, in addition to the length of the petiole (the petiole was not included in the blade length). Leaf senescence assays were performed as described by Seeds were sterilised and stratified at 4°C for several days. The seeds were sown into 500 ml jars  containing 60 ml ATS with 1% sucrose, solidified with 0.8% agar. For intact plant assays, plants were grown on ATS agar containing 5 µM GR24 or an equivalent volume of acetone (solvent control) for 6 weeks, and branching was then measured. For excised nodal assays, plants were grown on plain ATS agar for ∼3 weeks, until bolting. Young nodes with buds <1.5 mm in length were excised and placed between two agar blocks, to which hormones could be added independently . The grorac-GR24 or an equivalent volume of solvent control for 45 min before imaging. Images were taken using laser-scanning confocal microscopy using a Zeiss LSM700 imaging system with 20× water immersion lenses. Excitation was performed using 488 nm (15% laser power) and 555 nm (6%) lasers. Chloroplast autofluorescence was detected above 600 nm, and GFP/YFP fluorescence below 555 nm. The same settings for GFP/YFP detection were used within experiments for each line, except where stated. GFP quantification was performed on non-saturated images, using Zeiss ZEN software. For PIN1-GFP, fluorescence intensity in the GFP channel was measured in four or five basal plasma membranes per sample, in at least eight independent samples, except where stated. For RGA-GFP, fluorescence intensity in the GFP channel was measured in five nuclei per sample, in eight independent samples per treatment.For PIN1-GFP, GFP-RGA and SMXL6-YFP imaging in the shoot, hand sections were made through the vascular bundles of basal internodal stem segments of 6-week-old plants, and the slices were then embedded in agar plates. For GFP-RGA GR24 treatments, stems were covered in ATS solution containing 5 µM rac-GR24 or an equivalent volume of solvent control in the mounting solution, then imaged after 45 min using a Zeiss LSM700 imaging system with a 20× lens. Excitation was performed using a 488 nm laser, and GFP fluorescence was detected below 555 nm. GFP quantification was performed on non-saturated images, using Zeiss ZEN software. Fluorescence intensity in the GFP channel was measured in five nuclei per sample , in 12 independent samples per treatment.For GFP-RGA imaging in the root, 7-day-old seedlings were mounted on glass slides with 5 µM rac-GR24, 5 µM KAR1 or an equivalent volume of solvent control in the mounting solution, then imaged after 20 min using a Zeiss LSM780 imaging system with 20× lenses. For MG132 treatments, seedlings were pre-treated for 1 h with 50 µM MG132, then mounted as above. Excitation was performed using a 514 nm laser. YFP fluorescence was detected below 555 nm. The same settings for YFP detection were used within experiments for each line.For SMXL6-YFP imaging in the root, 3-5-day-old seedlings were mounted on glass slides with 5 µM


Aconiti Lateralis Radix Praeparata (Fuzi) is obtained from processed daughter roots of Aconitum carmichaeli, a toxic plant with a high medical value well known in Chinese medicine. In addition to the known toxic alkaloids  and bioactive alkaloids , three rarely found alkaloids have been previously reported in Fuzi, i.e., yunaconitine, 8-deacetyl-yunaconitine, and crassicauline A, and they were reported in recent years to cause potential risk to patients who took Fuzi or related products. To better control the quality of this herb and its related products and ensure safe use, developing a method to simultaneously determine these 9 alkaloids is important. In this research, sensitive and accurate ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method was established and used to examine 51 Fuzi and 27 Fuzi-containing products. Unexpectedly, 8-deacetyl-yunaconitine was detected in 17 Fuzi samples (33.3%) and 3 Fuzi-containing products (11.1%); yunaconitine in 10 Fuzi samples (19.6%) and 10 Fuzi-containing products (37.0%); and crassicauline A in 3 Fuzi samples (5.8%). Industry and clinics should be aware of the unusually high detection rate of these three toxic alkaloids in the Fuzi herb and its related products and take the necessary precautions to protect patients from any potential risk. Aconiti Lateralis Radix Praeparata (Fuzi in Pinyin), the processed daughter root of Aconitum carmichaeli Debx. (Family Ranunculaceae), is a well-known Chinese medical material recorded in the Chinese Pharmacopoeia (CHP)1 and other traditional Chinese medicine (TCM) texts. Fuzi has been used for over 2000 years as an analgesic and anti-inflammatory agent in clinics2. According to TCM theories, Fuzi is very toxic2. Modern experimental studies have shown that diester-diterpenoid alkaloids (DDAs) are the active toxic ingredients in Fuzi. These toxic DDAs have cardiac, analgesic, and anti-inflammatory activities5. To reduce the toxicity, crude Fuzi must be processed by heating or steaming, and processed Fuzi must be boiled before oral administration according to TCM theories and practice. During these processes, aconitine (AC), mesaconitine (MA), and hypaconitine (HA), which are the main toxic DDAs in Fuzi, degrade into less toxic but still active monoester-diterpenoid alkaloids (MDAs), such as benzoylaconine (BAC), benzoylmesaconine (BMA), and benzoylhypaconine (BHA), respectively6. Therefore, 6 aconitum alkaloids have been selected in the CHP as the target elements for the quality control of aconitum herbs. According to the CHP (2015 Edition), the total amount of AC, MA and HA in Fuzi must be less than 0.020%, and the total amount of BAC, BMA and BHA must be higher than 0.010%. Nevertheless, poisoning cases are still occasionally reported. From 1989 to 2010, 140 cases of aconitum poisoning, including one fatal case, were reported in Hong Kong7. Additionally, 17 cases were reported in Taiwan from 1990–1999, 2017 cases in China from 1989–2008, and 121 cases in Korea from 1995–20078. Multiple reasons for aconitum poisoning exist and include over doses, inadequate processing, aconitum contamination in other herbs, dispensing and management errors, and hidden risk factors7. In the 17 cases reported in Hong Kong, yunaconitine (YAC), crassicauline A (CCA), and 8-deacetyl-yunaconitine (DYA) were detected instead of AC, MA and HA in the urine samples of the aconitum poisoning patients9. Although YAC and CCA are two DDAs with toxicities similar to AC10, they are mainly isolated from A. forrestii, A. crassicaule, and other aconitum species9, not from A. carmichaeli. Therefore, the surveillance of toxic alkaloids from aconitum herbs always neglects YAC and CCA. DYA is the hydrolytic product of yunaconitine. Although DYA is less toxic than YAC, it still presents a safety risk because of the conversion between the two compounds11. Because YAC, DYA and CCA were detected in the urine of the aconitum poisoning patients, these alkaloids are considered to be hidden risk factors and should be covered in laboratory screenings for toxic compounds9. Therefore, a method to simultaneously determine the levels of these 9 alkaloids is needed for quality control of the herb and its products and to ensure the safe use of these medical materials.18, the maximum number of aconitum alkaloids simultaneously determined using these methods is only 7, i.e., AC, MA, HA, BAC, BMA, BHA, and YAC. The challenge in simultaneously determining the 9 aconitum alkaloids is that the level of some these alkaloids are significantly lower than the detection limits of the current methods. In the present study, ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC–QQQ–MS/MS) was used to simultaneously determine the contents of 9 aconitum alkaloids in Fuzi and Fuzi-containing products. The method was rapid, sensitive, accurate and fully validated and was used to analyze 51 Fuzi samples and 27 Fuzi-containing product samples Table Although the high-performance liquid chromatography (HPLC) and liquid chromatography hyphenated with mass spectrometry (LC/MS) methods have been broadly applied for the quantification of aconitum alkaloids in herb or herbal products and body fluids over the past few yearsDifferent solvents and gradient profiles of the mobile phase were compared to achieve a good resolution within 15 min. Acetonitrile and an acidic aqueous solution significantly improved the resolution and the symmetry of the target compounds. Acetonitrile-0.1% formic acid was used as the mobile phase with the gradient mentioned in the Materials and methods section at a flow rate of 0.35 mL/min.The negative and positive ion modes were compared for the MS analysis. The positive mode resulted in a higher sensitivity and cleaner mass spectral background than the negative mode. The collision energy and fragmentor voltage parameters were optimized to obtain the highest relative abundance of the exclusive ions and production in the multiple reaction monitoring (MRM) optimization conditions. The final conditions for the collision energy and fragmentor voltage are shown in the Instruments and UHPLC-MS conditions section. The MS/MS ion spectra of the 9 alkaloids are shown in Fig. The extraction method, including the extraction solvent and extraction times, was optimized to effectively extract the 9 alkaloids with a large difference in the content levels for the UHPLC-MS analysis. A 0.1 M hydrochloric acid solution (50 mL), 50 mL of methanol with 3 mL of the ammonia test solution , and 50 mL of isopropanol (IPA) and ethyl acetate (EA) (1:1) with 3 mL of the ammonia TS were compared as the extraction solvents. Ethyl acetate and isopropanol (1:1) with 3 mL of ammonia TS were the most efficient for extracting the alkaloids  of a Fuzi sample with a known amount of the mixed standards (n = 6). The recoveries were calculated using the equation below:Recovery (%) = /amount spiked × 100%As shown in Table The established method was successfully used for the simultaneous determination of 9 alkaloids in 51 Fuzi samples and 27 Fuzi-containing products. The representative MRM chromatograms of the reference standard mixture and the Fuzi sample are shown in Fig. An optimized method for quantifying the main constituents in pharmaceutical preparations is important for quality control standards to ensure the safety, quality and effectiveness of Chinese herbal products. In this study, 3 active alkaloids and 6 toxic alkaloids in Fuzi were selected as chemical markers for quality control. A total of 51 samples, 49 processed Fuzi and 2 unprocessed Fuzi samples, were collected from 10 areas in China, including 8 provinces and 2 special administrative regions, namely, Guangdong , Hunan (Changsha), Anhui (Bozhou), Jiangxi, Gansu, Liaoning , Yunnan, and Taiwan Provinces, as well as Hong Kong and Macao, providing a good geographical distribution of the samples.The total concentrations of BAC, BMA, and BHA in the 49 processed Fuzi samples complied with the CHP criteria except for one sample (no. FZ49) that was below the CHP limits. In these 49 samples, the contents of AC, MA and HA were beyond the CHP limits in 10 batches; i.e., approximately 20.4% of the processed Fuzi samples did not meet the requirements of the national pharmacopoeia of China, which implies a quality control matter for processed Fuzi. The contents of the six aconitum alkaloids  in the two unprocessed aconitum roots (No. FZ46 and FZ47) were above the CHP limits, showing that aconitum roots need to be processed to ensure safety. The unqualified Fuzi samples were collected from Hong Kong , Changsha , Gansu Province , Guangzhou , Yunnan Province  and Dalian  Fig. .Figure 5YAC was detected in 10 of the 51 Fuzi samples at concentrations ranging from 0.00711 to 2.25 μg/g. These samples were collected from five places, including Hong Kong, Changsha, Guangzhou, Dalian and Macao. Meanwhile, DYA was detected in 17 Fuzi samples at concentrations ranging from 0.00483 to 3.57 μg/g. Figures In addition, 27 samples of Fuzi-containing products were also analyzed. YAC was found in 10 samples Fig. , and a t18. The detection of the three alkaloids at such a high rate implies that there may be unknown risks for patients who consume Fuzi or Fuzi-containing products. One of the major alkaloids in A. vilmorinianum, A. hemsleyanum and A. nagarum is YAC19. The roots of the above species are occasionally misrepresented as Radix Aconiti Kusnezoffii and used as “Caowu” (in Chinese) in Chinese herbal medicine. In addition, the detection of YAC was reported in some herbs that are closely related to Fuzi, such as Aconiti Radix Preparata, Aconiti Kusnezoffii Radix, and Aconiti Kusnezoffii Radix Preparata, and products containing these herbs, but not in Fuzi14. The previously published literature also implies that the determination of YAC, DYA and CCA contents in Fuzi and Fuzi-containing products is a safety issue when Fuzi and its products are used in clinics.YAC, DYA and CCA were detected in one third of the Fuzi samples (17/51) and 37% of the Fuzi-containing products (10/27). DYA was the most frequently detected alkaloid in the Fuzi samples, and YAC was the most frequently detected alkaloid in the Fuzi-containing products. Both compounds have a high detection rate, which has not been reported before. YAC, DYA and CCA are toxic aconitum alkaloids, and the acute toxicity of YAC is comparable to that of aconitine21. However, the Fuzi source was not clearly presented in the reports, and the evidence was insufficient to draw a conclusion. Therefore, a possible reason for the detection of YAC, DYA and CCA in Fuzi could be contamination. YAC and CCA, which are mainly found in certain aconitum roots from Southwest China, were most commonly detected22. Fuzi may be contaminated by aconitum roots via accidental contamination, misidentification or even intentional adulteration. In the production, storage, packaging and transport of herbal medicines, unwanted introduction of foreign substances, including toxic plants or weeds, can occur . Determining how non-toxic herbs are contaminated with aconitum roots is not easy. Thus, strengthening the source control through good agricultural and supply practices and appropriate quality assurance is important.In recent years, the frequency of aconitum poisoning has increased, and this could be a result of the YAC, DYA and CCA contents in processed Fuzi, as discovered by the current study. Currently, the YAC or CCA contents in aconitum are unknown, and only two studies have reported the isolation of YAC and DYA from FuziIn this study, a method for determining 9 aconitum alkaloids in Fuzi samples was established and validated, providing an important reference for the quality control of Fuzi and research on related toxic components, such as YAC. Does Fuzi contain YAC and other ingredients, and what content levels should be set for safe use? These questions should be further investigated in future pharmacological and toxicological studies. The existing information on the quality control of YAC, DYA and CCA in Fuzi and Fuzi-containing products is insufficient. In this work, these 3 alkaloids were all detected in commercial Fuzi samples and Fuzi-containing products, implying that YAC, DYA and CCA might be hidden toxic ingredients and should be used as quality control and toxicological monitoring indicators. In addition, limit levels for the three alkaloids should be set after careful and thorough studies on their safety and pharmacological effects. The current findings should indicate to manufacturers of Fuzi and Fuzi-containing products that strictly controlling the quality of these medicinal materials is important.Fuzi samples were purchased from local drug stores or herb markets in different places in P.R. China, including Guangdong, Liaoning, Hunan, Yunan, Jianxi, Anhui, Sichuan Provinces and Hong Kong, Macao, and Taiwan. All these materials were authenticated by Dr. Zhi-Feng Zhang . Voucher specimens were deposited in the State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology). The Fuzi-containing products used in this study were Heishunpian granule (HSP), Danfupian granule (DFP), Baifuzi granule (BFZ), Paofupian granule (PFP), You-gui-wan (YGW), Si-ni-tang (SNT), Ma-huang-fu-zi-xi-xin-tang (MFXT), Zhen-wu-tang (ZWT), Fu-zi-li-zhong-tang (FLT), and Jin-gui-shen-qi-wan (JGSQ) and were purchased from local pharmacies in Shenzhen and Hong Kong P.R. China. Xiang-sha-liu-jun-zi-tang (XSLJZT) that is free of Fuzi was used as a negative control. The sample information, including the sample number, sample name and sample source, is shown in Table The reference standard compounds (purity ≥ 98%), aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, and crassicauline A, were purchased from the Chengdu Must Bio-technology Co., Ltd. . Yunaconitine (purity ≥ 98%) was purchased from the Testing Laboratory for Chinese Medicine . 8-Deacetyl-yunaconitine (purity ≥ 98%) was kindly provided by Prof. Xiao-Xia Ma of Yunnan University of TCM (P.R. China). The structures are given in Fig. 18 column  at 30 °C. The mobile phase consisted of 0.1% acetic acid (A) and acetonitrile (B), and the gradient elution was as follows: 20–25% B from 0-4 min, 25% B from 4–10 min, 90% B from 10.01–12 min, and 20% B from 12.01–15 min. An aliquot of 2 μL was injected, and the flow rate was 350 μL/min. The detection of the 9 alkaloids was performed using MRM and an ESI source in a positive ion mode. The transitions of the 9 compounds were m/z 646.4 → 586.3  for AC, m/z 632.0 → 105.1  for MA, m/z 616.3 → 556.3  for HA, m/z 604.3 → 105.1  for BAC, m/z 590.3 → 105.1  for BMA, m/z 574.3 → 105.1  for BHA, m/z 660.2 → 135.0  for YCA, m/z 618.4 → 135.1  for DYA, and m/z 644.4 → 135.0  for CCA. The other parameters were as follows: drying gas (N2) flow rate, 11.0 L/min; drying gas temperature, 300 °C; nebulizer, 15 psig; and capillary voltage, 4000 V.An UHPLC system  coupled with an Agilent 6460 Triple Quadrupole Mass Spectrometer  was used to quantitatively detect the 9 aconitum alkaloids. Chromatographic separations were performed on a Waters Acquity UPLC CAn appropriate amount of the 9 reference standards was dissolved with methanol to prepare stock solutions, which were stored at 4 °C. The standard mixture solution was obtained by accurately mixing the 9 stock solutions and diluting them with 50% methanol. The final concentrations of AC, MA, HA, BAC, BMA, BHA, YAC, DYA, and CCA in the mixture solution were 6.56, 5.46, 7.89, 5.65, 16.5, 5.91, 4.95, 5.08 and 2.53 μg/mL, respectively. The different concentrations of the standard solutions in Table 1 and the US Food and Drug Administration bioanalytical method validation 24.The method was validated in accordance with the Guidelines for the Validation of Quality Standard of TCM The Fuzi powder (2.0 g) was accurately weighed and ultrasonically extracted for 30 min using 3 mL of ammonia TS and 50 mL of ethyl acetate-isopropanol  in a conical flask that was covered after the solution cooled to room temperature. The loss in weight was supplemented via the addition of more extraction solvent. The extraction was filtered, and 25 mL of the successive filtrate was evaporated to dryness below 40 °C. The residue was precisely dissolved in 3 mL of isopropanol- dichloromethane . The solution was then diluted with methanol in a 1:10 ratio and filtered with a 0.22 μm microporous membrane filter. Two microliters of the successive filtrate was injected into the UHPLC-MS system for the analysis, according to the method mentioned in the Instruments and UHPLC-MS conditions section.


Cochlospermum regium, popularly known as “algodãozinho-do-cerrado,” are used for the treatment of genitourinary infections. However, the removal of their subterranean structures results in the death of the plant, and the use of the leaves becomes a viable alternative. Therefore, the antimicrobial activity of Cochlospermum regium leaf's ethanolic extract and its action on the biofilm formation of microorganisms associated with urinary infection were evaluated. The total phenolic compounds, flavoids, and tannins were quantified using the reagents Folin-Ciocalteu, aluminum chloride, and vanillin, respectively. The antimicrobial activity was evaluated by the broth microdilution method and the effect of the extract in the biofilm treatment was measured by the drop plate method. Cytotoxicity was evaluated by the method based on the reduction of MTS and the mutagenicity by the Ames test. The ethanolic extract of C. regium leaves presented 87.4 mg/EQ of flavonoids, 167.2 mg/EAG of total phenolic compounds, and 21.7 mg/ECA of condensed tannins. It presented reduction of the biofilm formation for E. coli and C. tropicalis and antimicrobial action of 1 mg/mL and 0.5 mg/mL, respectively. The extract showed no cytotoxicity and mutagenicity at the concentrations tested. This study demonstrated that C. regium leaves are a viable option for the treatment of genitourinary infections and for the species preservation.The roots of Escherichia coli being the most predominant microorganism  was added to the solution and the tubes were homogenized and poured into glycated minimum agar (GMA) plates . The number of revertant colonies perμg/plate) for TA97a and TA98 and sodium azide  (2.5 μg/plate) for TA100 and mitomycin C  (0.5 μg/plate) for TA102. In the metabolic activation assays the 2-aminoanthracene compound  (0.625 μg/plate) was used for all strains. Negative controls were performed with the sample solvent, DMSO (5 μL/plate).The positive controls used in the assays without metabolic activation were 4-nitro-o-phenylenediamine  . Ames test data were analyzed without the statistical program Salanal . C. regium indicated the presence of organic acids, steroids and triterpenes, flavonoids, total phenolic compounds and condensed tannins, and absence of alkaloids. A total of 87.4 ± 0.9 mg/EQ of flavonoids, 167.2 ± 2.7 mg/EAG of total phenolic compounds, and 21.7 ± 0.1 mg/ECA of condensed tannins were quantified.The ethanolic extract of the leaves (EEF) of E. coli and a moderate activity (0.5 mg/mL) C. tropicalis  foropicalis . C. regium EEF reduced the preformed E. coli biofilm and at the concentration of 16 mg/mL the C. tropicalis biofilm was also reduced , at all tested concentrations and for all evaluated strains of Salmonella Typhimurium (Tables The EEF ofm Tables  and 3. C. regium presented organic acids, steroids and triterpenes, flavonoids, total phenolic compounds, condensed tannins, and absence of alkaloids. These results corroborate with Vasconcelos Filho et al. [ C. regium and report that phenolic compounds are among the most commonly found compounds in these parts of the plant, among them tannins and absence of alkaloids in their leaves.The EEF ofo et al.  in a stu C. regium have activity against fungi and bacteria [ C. regium also presents as major compounds the phenolic compounds and flavonoids, which have been reported in the literature as antimicrobials, being responsible for the rupture of the cell membrane of microorganisms and inhibition of nucleic acid synthesis and energetic metabolism [ C. regium presented an antimicrobial action against E. coli, establishing a correlation with the popular use, since this bacterium is responsible for 80–90% of cases of urinary infection [ Cochlospermum [ E. coli, reiterating that this genus has antimicrobial properties.According to Goulas et al. , phytochbacteria , 40. In tabolism , 42. Thenfection . Anotherospermum  has also Candida tropicalis, the leaf extract presented antifungal action at the concentration of 0.5 mg/mL; this data is in agreement with the results of Inácio et al. [ C. regium root, this way, indicating that the leaves can be used in substitution to the popular use of the roots helping in the preservation of this species. Rodrigues and de Carvalho [Foro et al. , who demCarvalho  report tCandida is a genus of yeast fungi, which are present in the mucosa, skin, and gastrointestinal tract and make up the vaginal and urethra microbiota. They are considered commensal and opportunistic, and if there is an imbalance due to low immunity, they may prevail the microbiota and become pathogenic [ Candida tropicalis is among the most isolated microorganisms in patients with urinary tract infection [thogenic . Authorsnfection . C. regium EEF reduced the biofilm formed by E. coli and C. tropicalis. Adhesion and biofilm formation are virulence factors that promote the increase of antimicrobial resistance and the permanence of these microorganisms in the genitourinary tract. The reduction of the formed biofilm is relevant data, since, when adhered, these microorganisms become less susceptible to the antimicrobial activity.The C. regium EEF did not show cytotoxicity against VERO cells and it evidenced a potential antiproliferative activity against HeLa cells. Another study has also shown absence of cytotoxicity in C. regium [The use of medicinal plants in the treatment of diseases requires information that ensures absence of toxicity . The C. . regium . C. regium EEF did not show mutagenicity by the Ames test. The assay was performed in the presence and absence of S9 microsomal fraction which simulates an exogenous metabolization process similar to the hepatic metabolism. The sample is considered mutagenic when at least one of the strains has a mutagenicity index greater than or equal to 2 and a dose response relationship between the concentrations tested [ C. regium root extract with Drosophila melanogaster germ cells and found that the extract also does not present mutagenicity by this model.Thes tested . Nunes as tested  evaluate Cochlospermum regium EEF showed antimicrobial activity and reduction of biofilm formation in relation to uropathogens, such as Escherichia coli and Candida tropicalis; in addition, it does not present cytotoxicity and mutagenicity by the evaluated tests and concentrations. In this way, the present study enables the use of the leaves, since its use minimizes the impacts caused by the removal of roots and Xylopodia.The study showed that the