Contrary to the popular belief, we show that the Open, non-equilibrium systems with balanced gain and loss, known as parity-time ( The second category has systems that are far removed from equilibrium2, making perturbative methods inapplicable. Open systems with balanced gain and loss, called parity-time 〉24. In a sharp contrast with the nature-given lattices in condensed matter systems, waveguide lattices can be fabricated with a wide range of site-to-site tunneling amplitudes and on-site potentials; local or long-ranged “impurity” potentials; and on-site or tunneling disorder. This versatility has permitted the observation of disorder-induced localization, its insensitivity to the source of the disorder, as well as the signatures of the disorder-source in Hanbury-Brown Twiss correlations in disordered waveguide lattices31 and fibers32.Discrete 20. It does so because a random disorder does not preserve the symmetries of the underlying Hamiltonian. A straightforward way to salvage the fragile 33. However, this approach imposes highly non-local correlations on the randomness and is therefore difficult to implement, even with an engineered disorder. Thus questions about localization and 35.What is the fate of a disordered In this report, we show that the N-site tight-binding lattice with gain and loss potentials ±iγ located at parity symmetric sites m0 ≤ N/2 and Nth site has only one neighbor each. The distance between the gain and the loss sites, N − 1 to one (two) when N is even (odd). The non-Hermitian, HPT = H0 + Γ whereConsider an J > 0 is the constant tunneling amplitude that sets the energy-scale for the Hermitian Hamiltonian H0 and |k〉 is a single-particle state localized at lattice site k. Since the Hamiltonian HPT commutes with the antilinear operator 37. The spectrum is real when γ ≤ γPT(m0) where the γPT(m0) denotes the gain-location dependent N is even, the threshold is maximum when the gain and loss potentials are nearest to each other or farthest away from each other, i.e., γPT = J when d = 1 and d = N − 1. When N is odd, γPT → J/2 when d = 2 and γPT → J when d = N − 1. This unexpected robustness of the 39. In the presence of a random, uncorrelated disorder, the threshold is suppressed to zero. In the following subsection, we show that introducing a periodic disorder alleviates this problem.p,We consider two classes of Hermitian disorders, one in the tunneling amplitude and the second in the on-site potential, each with lattice period λ ≥ 0 and Δ ≥ 0 represent the strength of tunneling and on-site disorder respectively, {r1, …, rp} are independent, identically distributed (i.i.d.) random numbers with zero mean and unit variance, and the periodic nature of disorder implies that rk′ = rk if k′ − k = 0 mod p. We remind the reader that although the randomness of disorder is only confined to a unit-cell of size p, the lattice with N sites may or may not contain integer number of such unit cells. The existence of a finite threshold depends critically on these details and, thus, cannot be obtained via the Bloch-theorem approach. Figure N = 11 sites and gain potential iγ at site m0 = 3. The tunneling disorder VT has period p = 3, and the three independent, random tunnelings within a unit cell are given by Jk = J(1 + λrk). Figure N = 15 sites, gain potential at site m0 = 4, and disorder period p = 4; the four independent, random potentials within a unit cell are given by Vk = JΔrk. Note that the periodic disorder potential in each case is not The dimensionless numbers γPT for an N = 17 lattice with tunneling disorder strength λ = 1. Its key features are as follows. The threshold γPT is nonzero only when N + 1 and m0 are multiples of disorder period p. Thus, when p = 2 the threshold is nonzero only when m0 is even, for p = 3 it is nonzero for m0 = {3, 6}, and for p = 6, it is nonzero only when m0 = 6. It is identically zero for periods p = {4, 5, 7, 8} for any gain-site location m0. These results, obtained for a particular realization of the tunneling disorder, are generic. They show that a tunneling disorder with appropriate period p and gain locations m0 leads to a positive Figure γPT > 0 when N + 1 = 0 mod p and m0 = 0 mod p. Thus, periodicities p = {2, 3, 6} have a positive threshold for appropriate gain locations, while γPT = 0 for all other disorder periods. In addition, when p = 2 , the symmetry breaking threshold is nonzero for odd values of gain location as well. This is the only qualitative difference between the threshold results for tunneling vs. on-site disorders. It arises because for an odd N and p = 2, the on-site disorder is always γPT > 0 only when the gain potential site is even.Figure they show that the symmetry breaking threshold is robust against disorders that are not reflection symmetric40. They hint at the existence of another antilinear operator that commutes with the disordered Hamiltonian37. In the next subsection, we uncover this symmetry and discuss its signatures.Results in Fig. H0 = UDU† where Dαβ = εαδαβ = −2J cos pαδαβ is the eigenvalues matrix, the unitary matrix has entries pα = πα/(N + 1) are the quasimomenta consistent with open boundary conditions. The spectrum of H0 is particle-hole symmetric, α. For a given H0, one can generate a family of operators P = USU† where S = diag is a diagonal matrix with randomly chosen entries ±1; there are 2N−1 such distinct operators. When S = 1N the result is the identity and when N−1disorder-dependent operators P. It is easy to show that P = P† = P−1 and P commutes with H0. However, in general, the operator The tunneling Hamiltonian of a uniform lattice can be expressed as k, k′ are both multiples of the disorder period p. When k ≠ 0 mod p, the unit weight is distributed to other elements in the same column. These results are generic and apply for on-site potential disorder, panel (b); tunneling disorder, panel (c); or a combination of the two, panel (d). In all cases, the H0. A positive symmetry-breaking threshold, then, is possible if and only if the antilinear operator N + 1 and m0 are integer multiples of the disorder period.Figure entclass1pt{minimaN = 5 site lattice and tunneling disorder with period p = 2. Without loss of generality, this system is mapped onto a dimer model with alternating tunneling amplitudes given by Jr(1 ± λ) where Jr is the average tunneling amplitude for a dimer and λ is proportional to the variance of the disorder. It is straightforward to obtain the Π operator in this case,What does the Π operator represent? An insight into its structure is offered by the simplest example where an explicit, analytical expression for the Π operator can be obtained. Let us consider an λ = 0. For λ > 0 and an odd k, the unit weight at We see that Eq.  reduces Umα are symmetric or antisymmetric, i.e., iγ vanish, and leads to a positive 20. Are the eigenfunctions of the disordered Hamiltonian H0 also reflection symmetric? To address this question, we define a disorder- and site-dependent asymmetry functionAn complementary insight into the vanishing commutator, A ≥ 0 in general and for a uniform lattice, A(k) ≡ 0. The asymmetry functions A(k) for M = 100 different disorder realizations on an N = 23 site lattice are shown in Fig. p = 8, A(k) = 0 only at sites k = {8, 16} (top panel), whereas when p = 3 the function vanishes exactly when k = 0 mod 3 (bottom panel). Figure p = 6 (top panel) and p = 4 (bottom panel). Once again, we see that A(k) = 0 if and only if the site index is a multiple of p. The asymmetry function is nonzero everywhere when the disorder period and lattice size do not satisfy N + 1 = 0 mod p. Results in Fig. Umα are neither symmetric nor antisymmetric, but, when restricted to specific sites, they show these symmetries40. Thus, although the Hamiltonian HPT = H0 + Γ is not H0 gives rise to the positive It follows that PDF(γPT) depend on the strength of the disorder? Does it depend on the distribution of the disorder? Is it different for on-site and tunneling disorders? What is the fate of localization in Disordered models with positive PDF(γPT) in the presence of on-site potential disorder, panel (a), and tunneling disorder, panel (b). The results are for the m0 = 3 in an N = 17 lattice, obtained by using M = 5 × 104 realizations of disorder with period p = 3. We remind the reader that when N + 1 is not a multiple of the disorder period p, the γPT(m0) = 0 for all locations that are not multiples of the period p. Thus, in the following, we only focus on configurations that lead to a positive γPT/J. Panel (a) shows that as the on-site disorder strength Δ increases, the threshold distribution PDF(γPT) becomes broader, and skewed towards values smaller than its clean-limit value. In addition, PDF(γPT) is independent of the disorder distribution, i.e., it is the same whether the random, periodic potential is drawn from a Guassian distribution with zero mean and variance Δ  or a uniform distribution with the same mean and variance . Qualitatively similar results are obtained for other lattice sizes N, disorder periods p, and gain potential locations m0 as long as they have a positive threshold. These results are consistent with what we would expect. Introducing disorder suppresses the PDF(γPT) - a single particle property - is independent of the underlying disorder distribution41.Figure PDF(γPT) is a bell shaped distribution centered about its clean-limit value. It becomes broader when the tunneling disorder strength λ is increased, and its center shifts towards the origin. For a uniform disorder , we find that PDF(γPT) is now a flat-top distribution approximately centered about its clean-limit value. These results are remarkable because for a tunneling disorder, the threshold distribution PDF(γPT) mimics the disorder distribution and is not universal.Figure 30. In optical-waveguide realizations of a Hermitian disordered lattice, it is manifest by a disorder-averaged intensity profile that, after an initial ballistic expansion, develops a steady-state value31. For an initial state on site k0, the disorder-averaged intensity profile d denotes averaging over different disorder realizations, |ψ(t)〉 = G(t)|ψ(0)〉, and the time-evolution operator is G(t) = exp(−iHt) where we have used ħ = 1. In the Hermitian case the time-evolution operator is unitary and the total intensity at each time is constant, In one-dimensional Hermitian systems, a random disorder exponentially localizes all states. In transport experiments, this localization is inferred from a scaling analysis of the resistivity in the presence of disorderγ < γmin = minγ{PDF(γPT) > 0}, the system is in the Id(k) with constant total intensity 33. When γ > γmin the system is in the m0. As a result, the disorder-averaged intensity Id develops a peak at the gain site m0 whose weight increases with time. We note that in this regime, the intensity Id does not reach a steady state value35.In the Id. The results are for an N = 39 site lattice with on-site disorder, p = 10, number of disorder realizations M = 103, and an initial state localized at the center of the lattice, |ψ(0)〉 = |k0 = 20〉. Panel (c) shows the disorder-averaged intensity Id for the Hermitian case, γ = 0. A periodic disorder leads to a steady-state profile Id(k) that is exponentially localized about the initial site k0 = 20, along with satellite peaks at sites k = 20 ± 10 = {10, 30}. These satellite peaks are signatures of extended states that exist in one-dimensional systems with periodic disorder43. As the disorder strength Δ is increased, the peak intensity of the satellites decreases. We remind the reader that when the disorder is purely random, these satellite peaks are absent.Figure γ/J = 0.05 at site m0 = p = 10 (red filled circle); the corresponding loss potential −iγ at site k = k0 mod p, a new peak emerges at the gain location. It arises because a disordered system with γ/J = 0.05 is, sometimes, in the broken Id at time Jt = 100. In the Hermitian case, the steady-state intensity profile Id(k) shows localization peaks at the initial site k0 = 20 and satellite peaks at sites k = {10, 30} (blue filled circles). In the m0 in the Hermitian limit.Panel (d) shows corresponding results for a disordered N. It is straightforward to see, via perturbative arguments, that if the randomly generated potentials iγk are uncorrelated, the N random potentials {iγ1, …, iγN}, one restricts to random, periodic entries {iγ1, …, iγp} that are then repeated, the threshold is again zero. Indeed, for a nonzero k. We remind the reader that implementing a single pair of balanced gain and loss potentials is experimentally challenging at present, and therefore, implementation of such a disorder, with N/2 balanced pairs, is exceedingly difficult in the near future.Until now, we have confined our attention to Hermitian, periodic, on-site or tunneling disorders that are easily implementable. In this subsection, we will consider the effects of purely imaginary (gain-loss) disorder on the H(σ) = H0 + Γd(σ) wherex] stands for the integer part of x and iγk are [N/2] i.i.d. random numbers drawn from distribution with zero mean and variance σ. We note that the zero mean ensures that the potentials iγk in the first half of the lattice approximately average out, i.e. the lattice is locally γk ≥ 0, which corresponds to random gain-only disorder in the first half of the lattice, with a counterpart loss-only disorder in the second part of the lattice. In this case, we find that 44. For a locally σ is small, the spectrum of the disordered Hamiltonian H(σ) is purely real. It transitions to a complex-conjugate spectrum when the strength exceeds a threshold σPT. This threshold is disorder-realization dependent, and therefore, we obtain a distribution of the The Hamiltonian for a disordered system is given by PDF(σPT) for different lattice sizes N, obtained with M = 5 × 104 realizations drawn from a uniform distribution (a) and Guassian distribution (b). Note that the horizontal axis has a logarithmic scale. We see that the distribution for a Gaussian disorder is wider than that for the uniform disorder. In both cases, as the lattice size N is doubled, the threshold probability distribution shifts to smaller values, but its width on the log-scale is essentially unchanged for large lattices, PDF(γPT) depended sensitively on the underlying distribution of Hermitian disorder, these threshold distribution functions PDF(σPT) are qualitatively similar for uniform (a) and Guassian (b) disorders. Thus, the non-Hermitian gain-loss disorder is “traditional”, in the sense that its statistical effects over a large number Figure ψ in a waveguide array with realistic parameters45 via the beam propagation method (BPM)47. The continuum Schödinger equation is given by n0 is the cladding index of refraction, c is the speed of light in vacuum, n(x) is the position-dependent index of refraction in the waveguide array, and k0 = 2π/λ is the wave number of the rapidly varying part of the electric field The results for the mentclass2pt{minimmentclass2pt{minimn(x) differs from that of the cladding only within each waveguide. In the limit of small contrast, n(x) = n0 + Δn with V(x) = 2ck0Δn/n0, and we implement the gain and loss potentials by adding appropriate imaginary parts to the index contrast. Figure N = 8 waveguide-lattice in the presence of an on-site disorder with period p = 3. The initial state, marked by a white semicircle, is a normalized Gaussian with width σ = W/2 in the 5th waveguide, where W is the width of each waveguide. Each panel shows the time- and space-dependent intensity I where we have switched to the distance along the waveguide z = ct/n0 as a measure of time for an easier comparison with experiments. The bar-chart at the top of each panel shows a randomly generated index contrast Δn(x) with period p = 3. The gain-potential waveguide is shown by a red bar, the reflection-symmetric lossy waveguide is shown by a blue bar, and the linear scale on the vertical axis in each bar-chart ranges from Δn = 4.8 × 10−4 to Δn = 5.2 × 10−4.The index of refraction I in Fig. γ = 0.7 cm−1 in the first waveguide, m0 = 1. It shows that at long times, z ≥ 10 cm, the intensity is largely confined to the gain waveguide and the system is in the broken m0 = 2; it also shows intensity localized in the gain waveguide and thus indicates that the system is in the I is larger than the average intensity I with the same gain in the third waveguide, m0 = 3. It is clear from the intensity plot that the system is in the γ = 1.4 cm−1, the system enters a The intensity plot 40. Therefore, any phase-insensitive observable will reflect the signatures of this symmetry. Experimentally, the models studied here can be realized in coupled waveguide arrays with one gain waveguide and one lossy waveguide. Ideally, if the on-site potentials or tunneling amplitudes are tunable - for example, via voltage-controlled top-gate heaters - it will permit experimental investigations of interplay between localization due to a periodic disorder and the In this paper we have introduced non-Hermitian lattice models with balanced gain and loss that are robust against random, periodic disorder. We have uncovered a veiled symmetry that is exhibited by eigenfunctions of such disordered, Hermitian lattices. This symmetry is phase-sensitive, and it ensures equal weights at specific reflections-symmetric sites, but not equal wave functionsPDF(γPT) on the source and the distribution of disorder is, at this point, poorly understood. A generalization of these models to non-sparse matrices with a positive 48, will provide an approach to investigate the spectral properties random, Mathematically, the lattice models considered here correspond to tridiagonal matrices with Hermitian, random, periodic entries, in addition to non-Hermitian, fixed, gain-loss potential entries along the main diagonal. The statistical properties of eigenvalues of such matrices are essentially unexplored. In particular, the dependence of the threshold distribution


Further we demonstrate, using time resolved two-site amplified FRET (A-FRET) analysis of xenograft tumours, that inhibition of tumour growth correlates with the degree of mTORC2 uncoupling from its downstream targets, as demonstrated for Akt. These data add weight to the body of evidence that mTORC2 represents a pharmacological target in cancer independently of mTORC1.Mammalian target of rapamycin (mTOR) is a central regulator of growth and metabolism. mTOR resides in two distinct multi-protein complexes – mTORC1 and mTORC2 – with distinct upstream regulators and downstream targets. While it is possible to specifically inhibit mTORC1 with rapamycin, or inhibit both mTOR complexes together with ATP pocket directed mTOR kinase inhibitors, it is not possible to assess the specific roles for mTORC2 pharmacologically. To overcome this, we have developed a novel, inducible, dominant negative system for disrupting substrate recruitment to mTORC2. Previously we identified the mTORC2 specific subunit Sin1 as a direct binding partner for AGC kinases Akt and PKC. Sin1 mutants, which retain the ability to bind Rictor and mTOR, but fail to recruit their AGC client kinases, inhibit AKT and PKC priming and block cell growth. In this study, we demonstrate that uncoupling mTORC2 from AGC kinases in DLD1 colon cancer cells inhibits Akt activation and blocks tumour growth  The mammalian target of rapamycin (mTOR) is a key regulator of eukaryotic cell growth and represents a major drug target in numerous cancers -3. ThereAmong the key targets for the mTOR complexes are AGC family kinases, including Akt, p70S6 kinase, SGK and PKC. AGC kinase activity is typically regulated by PDK1 mediated phosphorylation of the generic kinase domain activation loop and mTOR mediated phosphorylation of conserved hydrophobic- and turn-motifs within a C-terminal kinase domain extension. These phosphorylations cooperate to fully activate the AGC kinase domain . mTORC1 The emergence and early promise of mTOR inhibitors has provided evidence that targeting of mTORC1 with rapalogues, or both mTORC1 and mTORC2 with active site directed inhibitors, may be beneficial therapeutically in a number of malignancies. Dose limiting toxicities  and potemTORC2 specific inhibitors remain to be described and in their absence alternative means are required to specifically assess this pathway as an independent target . We prevWe previously identified that Sin1 interacts directly with AGC client kinases in yeast-2-hybrid and HEK293 pull-down assays, and mapped the interaction to the CRIM domain of Sin1 . In thisn = 3) with no change in levels of associated mTOR. Together these data indicate that the ∆Sin1 construct incorporates into >80% of the endogenous mTORC2 complex without affecting the net expression levels of the complex. Levels of endogenous Sin1 immunoprecipitated by the Sin1 polyclonal were also reduced to the same degree (Relative Intensity 0.16 ± 0.14) indicating that displaced endogenous Sin1 is unstable and degraded [In order to examine and quantify the integrity of the mTORC2 complex, and the degree to which endogenous Sin1 has been displaced, we immunoprecipitated mTORC2 using either Rictor or Sin1 polyclonal antibodies Figure . Inductidegraded . InductiConsistent with our previous findings in HEK293 cells, ∆Sin1 expression in DLD1 cells suppressed Akt Ser473 phosphorylation but had no effect on phosphorylation of the mTORC1 target p70S6K Thr389 Figure  and 2B. Together these data demonstrate that inducible expression of Sin1 constructs can be used to modulate mTORC2 complex functionality while maintaining complex integrity. In contrast, Sin1 or Rictor ablation results in complex disruption with unknown adaptive consequences , 23.In order to assess a role for mTORC2 in tumour growth we conducted subcutaneous xenograft studies in NOD/SCID mice. Mice were injected with DLD1 ∆Sin1 tumour cells on both flanks and mice were randomly assigned to two groups: control and doxycycline. For doxycycline treatment, to induce ∆Sin1 expression, drinking water was supplemented with 1% sucrose (w/v) and 2mg/ml doxycycline; control mice were maintained on 1% sucrose alone as a vehicle control. Tumour growth was monitored three times weekly by caliper measurements and the experiment was terminated before tumour burden reached the maximum permitted volume. Doxycycline treatment significantly inhibited tumour incidence and average tumour burden Figure  and 3B. 3. Average tumour growth was modestly reduced by ∆Sin1 induction and individual tumour growth rate was also suppressed. In contrast to xenograft growth, ∆Sin1 expression did not affect growth of DLD1 cells under standard 2D cell culture conditions as previously described for HEK293 cells (data not shown). This concurs with the observation that Sin1 (unlike Rictor) knockout fibroblasts do not show a growth deficit in cell culture [in vivo can suppress mTORC2 target phosphorylation and impede tumour development.To mimic therapeutic targeting we also assessed the effect of ∆Sin1 induction on the growth of established tumours Figure  and 3F.  culture , 24. We in vivo we conducted a time resolved amplified FRET (A-FRET) analysis of Akt activation status in tumour sections [To determine the whether ∆Sin1 was able to target mTORC2 function sections . Here, wThe first generation of mTOR targeting drugs stem from the eponymous allosteric inhibitor of mTORC1, rapamycin. Rapamycin and its analogues  have been approved for the treatment of a number of malignancies, including Renal Cell Carcinoma, HER2 negative breast cancer and various neuroendocrine tumours . DespiteSecond generation active site directed mTOR inhibitors, that target both mTORC1 and mTORC2, demonstrate significantly improved anti-cancer activity in preclinical models when compared with rapalogues -30; a va3 downstream of PI3 kinase both releases inhibition and co-localises mTORC2 with its substrate Akt. Interestingly, in a previous study, we found that incorporation of PH domain disrupted Sin1 into mTORC2 did not significantly modify basal Akt phosphorylation in HEK293 cells [In order to model the effects of targeting mTORC2 alone, strategies which perturb the function of the mTORC2 specific subunits Sin1 or Rictor are necessary. Rictor and Sin1 null cells from knockout mice have thus been instrumental in defining mTORC2 complex regulation and targets. Deletion of either Sin1 or Rictor results in complete loss of the mTORC2 complex , 35. As 93 cells . This so93 cells , 37.in vivo response to mTORC2 blockade. Encouragingly, suppression of mTORC2 significantly impeded tumour development in our xenograft model. Two-site amplified FRET analysis revealed target suppression in vivo with significant reduction in phosphorylation of Akt on both mTORC2 targeted Ser473 and PDK1 targeted Thr308 (a weaker inhibitory effect was observed on this latter site). Associated with the direct effect of mTORC2 inhibition, loss of Thr308 was also observed in cell culture. This likely results from reduced stability of Thr308 phosphorylation in the absence of Ser473 mediated kinase domain stabilisation. An alternative mechanism, where disruption of PIF pocket dependent recruitment of PDK1 in the absence of Akt Ser473 phosphorylation is unlikely to explain Thr308 reduction, as blocking this interaction is not sufficient to block Akt activation by PDK1 [Despite regulation through a genetic mechanism, we have generated a system where we can inducibly ablate approximately 90% of cellular TORC2 activity following doxycycline administration; this pharmacomimetic system provides unique insight into the potential  by PDK1 . Interes by PDK1 , potenti by PDK1 . Alterna2. Tetracycline-inducible Sin1 lines were generated using the pcDNA5.0 FRT T-Rex™ system (Invitrogen) according to the manufacturer’s instructions. Sin1 expression was induced with 100 ng/ml tetracycline. Animal studies were compliant with UK Home Office regulations and carried out under license PPL 70/8066. Female NOD/SCID mice were inoculated subcutaneously on the hind flank with 1x106 DLD1 cells before being assigned randomly to control and doxycycline cohorts. Drinking water was supplemented with 1 % sucrose (w/v) and 2mg/ml doxycycline. Tumour growth was monitored by caliper measurements twice weekly and volume calculated as (w2 x l)/2. Excised tumours were fixed in formalin and paraffin embedded for pathology and FRET analysis.FRT-DLD1 cells were cultured in DMEM, 10% foetal bovine serum, penicillin (50 units/ml) and streptomycin (0.05 mg/ml) in 10% COMouse anti-Akt/PKB (A-FRET) mAb (SKB1) was from Millipore (#: 05-591), anti-pAkt (Thr308) (D25E6) rabbit (#: 13038S), anti-pAkt (Ser473) (D9E) rabbit (#: 4060S), and anti-Akt (immunoblot) mAb (#:40D4) were obtained from Cell Signaling Technology. Affinity-purified F(ab’)2 fragments Perox-Apure, Fab-Frag Anti-Rabbit -HRP (#: 711-036-152) were purchased from Jackson ImmunoResearch, Suffolk, UK. Peroxidase Suppressor (#35000) and Tyramide Signal Amplification (TSA) kit with Alexa Fluor 594 tyramide (#: T-20925) was purchased from Thermo Fisher Scientific, UK. ATTO488 NHS-ester dye (#: 41698-1MG-F) from Sigma. Sin1 (NB110-40424), Rictor (NB100-612) and mTOR (NBP-19855) polyclonal antibodies were purchased from Novus Biologicals. Anti-myc (9E10) was prepared by the In-House antibody facility. Sin1 mAb was from R&D Systems (MAB8168).For immunoprecipitation of mTORC2, cells were lysed in 0.3% CHAPS, 40 mM Tris pH 7.4, 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM Pyrophosphate, 10 mM β-glycerophosphate, 2 mM Na orthovanadate supplemented with cOmplete™ protease inhibitor cocktail (Roche). 2 µg of antibody was conjugated to protein G DynaBeads (Invitrogen) and incubated overnight with 1-2mg centrifuged lysate and washed four times with lysis buffer. Immunoprecipitates or cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose and incubated with designated antibodies overnight. Bands were visualised and quantified using an AI600 imaging system , using HRP conjugated secondary antibodies and enhanced chemiluminescence.ATTO488-NHS conjugation to the anti-mouse-specific Fab secondary antibody (1mg/ml) was performed. Briefly, the ATTO488-NHS-ester dye was reconstituted in anhydrous DMF to a concentration of 1mg/ml and added (125µg of dye/1mg of protein) to the Fab fragment antibody solution. The reaction mixture was incubated with constant agitation at room temperature for 1 hour. Purification of labelled antibodies was performed using pre-equilibrated  gravity flow PD-10 dye removal columns. The dye: protein ratio required for these experiments should not exceed a 4:1 ratio.® Gold anti-fade. The donor lifetimes of ATTO488 were determined from at least 3 regions from each section with elevated donor intensity. Lifetime data was analysed using a purpose-built algorithm and the FRET efficiency was calculated using the following equation: Ef = 1-(tDA/tD)*100%; where tD is donor lifetime and tDA is donor plus acceptor lifetime [For the acquisition of donor (ATTO488) fluorescent lifetime an automated multiple frequency lifetime imaging microscope (Lambert Instruments) was used. Two identical tumour sections (4 μm) were de-waxed, rehydrated and subjected to heat antigen retrieval by microwaving in Tris-EDTA (pH 9.0) buffer, for 10 minutes. Sections were then incubated in freshly prepared sodium borohydride (1mg/ml in PBS) buffer for 10min at RT, followed by blocking with 1% BSA/PBS. Tissue sections were incubated with peroxidase suppressor for 15min. For the A-FRET assay, the first slide was incubated with mouse anti-panAkt (1:50), and the second slide with mouse anti-panAkt (1:50) plus rabbit anti-pT308 1:200) primary antibodies, overnight at 4°C. The first slide was further immunolabeled with (donor fluorophore) ATTO488-conjugated anti-mouse Fab fragment secondary antibody (20µg/ml). The second slide was immunolabeled with ATTO488-conjugated anti-mouse Fab fragment (20µg/ml) and HRP-conjugated anti-rabbit Fab fragment secondary antibody (10µg/ml), which was detected by using (acceptor fluorophore) Alexa-594-TSA assay. These slides were mounted with ProLong0 primary


Carnobacterium maltaromaticum is frequently associated with foods having extended shelf-life due to its inhibitory activity to other bacteria. The quantification of such inhibition interactions affected by various environmental factors is limited. This study investigated the effect of environmental factors relevant to vacuum-packaged beef on inhibition between two model isolates of C. maltaromaticum, D0h and D8c, specifically D8c sensitivity to D0h inhibition and D0h inhibitor production. The effects of temperature , atmosphere (aerobic and anaerobic), pH , lactic acid  and glucose  on D8c sensitivity (diameter of an inhibition zone) were measured. The effects of pH, glucose, lactic acid and atmosphere on D0h inhibitor production were measured at 25 °C. Sensitivity of D8c was the highest at 15 °C, under aerobic atmosphere, at higher concentrations of undissociated lactic acid and glucose, and at pH 5.5 (p < 0.001). pH significantly affected D0h inhibitor production (p < 0.001), which was the highest at pH 6.5. The effect of lactic acid depended upon pH level; at relatively low pH (5.5), lactic acid decreased the production rate (arbitrary inhibition unit (AU)/mL/h). This study provides a quantitative description of intra-species interactions, studied in in vitro environments that are relevant to vacuum-packaged beef. Pseudomonas aeruginosa to deliver bacteriolytic effectors to other Gram-negative bacterial cells [Enterobacteriaceae [Bifidobacterium bifidum, a probiotic organism that is often found in human intestines, and Listeria monocytogenes, an opportunistic pathogen, mutually promote each other’s growth [Bacteria interact in any given niche, and these interactions may have a positive or negative impact on the involved species . Bacterial cells . Furthereriaceae . Converseriaceae ,7,8,9. Fs growth . Accordis growth , previouSerratia marcescens and Pseudomonas fluorescens, the main spoilage organisms in pork, were increased by quorum sensing compounds in vitro. A large number of studies regarding interactions among food-sourced bacterial species mainly focus on the inhibitory activity of lactic acid bacteria (LAB) on pathogenic or spoilage bacteria in vitro or in situ within food [Bacterial interactions have been frequently reported in food-relevant environments. A few researchers have observed the inhibitory effect of natural microbiota of meat on the growth of pathogens, which has been defined as the ‘Jameson effect’ ,13,14. Nhin food ,17,18. Shin food ,20,21. TCarnobacterium maltaromaticum is a dominant, and often preferred, LAB species. Numerous reports demonstrate that its presence is associated with foods having an extended shelf-life, including vacuum-packaged (VP) meat [C. maltaromaticum may be represented by a single strain, and the effect is likely attributed to bacteriocins—inhibitory compounds that suppress other bacteria within the spoilage community [Among the numerous microorganisms that contaminate food, VP) meat ,22,23,24C. maltaromaticum  inhibited a wide spectrum of species, including Serratia spp., Pseudomonas spp., Leuconostoc spp. and other Carnobacterium spp. [Pseudomonas spp. and two Bacillus spp. showed relatively large inhibitory activities. Interestingly, the CFS of a few Gram-negative bacteria including Pseudomonas spp. and Enterobacteriaceae also showed growth-promoting activity. That study has helped to understand bacterial interactions among a large variety of species related to VP beef. However, a more complete understanding of how populations of species and strains interact and compete requires a quantitative description of the effect(s) of environmental factors.Examining a broad spectrum of bacterial species isolated from VP beef produced at different abattoirs, we showed that cell-free supernatant (CFS) of ium spp. . Under aC. maltaromaticum D0h and D8c, specifically the effects of environmental factors relevant to VP beef. Firstly, a preliminary test was performed to investigate the production kinetics of inhibitory compounds by D0h, and then, D0h CFS with the highest level of inhibition was obtained to study the effects of pH, atmosphere, glucose, temperature and lactic acid on D8c sensitivity under an agar model system with the diameter of an inhibition zone measured. To better understand the inhibition between C. maltaromaticum D0h and D8c, the production of D0h inhibition in broth medium with various combinations of pH, atmosphere, glucose and lactic acid was also tested at 25 °C.The present study describes intra-specific inhibitory activity between two model VP beef bacterial isolates, C. maltaromaticum, D0h and D8c, respectively, were isolated from VP beef [Effector and target strains of  VP beef ,27 and sCarnobacterium maltaromaticum D0h was inoculated in BHI at an initial cell density of 105 CFU/mL and then incubated at 25 °C. Broth was sampled every 3 h for the first 9 h and then at 1.5-h intervals, and the optical density was measured at 600 nm (OD600) by a plate reader . D0h CFS was prepared by centrifuging cultures at 10,000× g for 5 min, followed by filtration through a 0.2 µm pore-sized membrane . The inhibitory activity of CFS was determined by an agar overlay assay reported by Aween et al. [8 CFU/mL , and then 10 mL of melted TSA  were mixed with 107 CFU D8c and poured into Petri dishes. After the agar had solidified, 10 µL of filter-sterilized CFS of D0h were spotted on the agar in triplicate using a micropipette. After incubation at 25 °C for 24 h, the diameter (mm) of inhibition (DI) was measured using ImageJ software (v1.49 (http://rsb.info.nih.gov/ij/index.html)).n et al. , with slThe sensitivity of the target isolate D8c to inhibitory activity of D0h CFS was tested under environmental conditions relevant to VP beef: temperature: −1, 7, 15 and 25 °C; atmosphere: aerobic and anaerobic; pH: 5.5, 6 and 6.5; lactic acid: 0, 25 and 50 mM; glucose: 0, 0.56 and 5.55 mM. A full factorial design (4 × 2 × 3 × 3 × 3) was used.The agar overlay method was used with a basal medium of modified brain heart infusion (mBHI) broth, without glucose . L (+) lactic acid  and D (+) glucose  were added into 27 variations of mBHI . pH was 7 CFU D8c and added to Petri dishes; the dishes were previously prepared with a bottom layer containing 15 mL of the same mBHI agar used in the overlay.After cooling to 50 °C, 10 mL mBHI agar were mixed with 10Results showed that the highest levels of inhibitory compounds  in D0h broth cultures occurred between 19.5 and 22.5 h of incubation . TherefoAfter the CFS was absorbed into agar, Petri dishes were incubated at different temperatures both aerobically and anaerobically until inhibition zones were observed .2, ≥13% CO2) were produced using a GasPak EZ anaerobic pouch system  in sealed jars. Incubation temperature was recorded during the experiment with data loggers . At the end of the experiments, DI was measured and defined as D8c sensitivity.Anaerobic conditions  of atmosphere, lactic acid, glucose and pH was used in 54 combinations of the four environmental factors. Using BHI (without glucose) as the basal medium, 27 formulations of modified BHI broth medium (mBHI) were prepared, based on the factorial design . C. maltaromaticum D0h 24-h culture, for an initial density of 105 CFU/mL. Two replicate wells were used for each test condition. Plates were incubated at 25 °C, aerobically and anaerobically, respectively. When culture OD600 reached 0.05, samples were collected at 2-h intervals until the bacteria reached the stationary phase. CFS was obtained by filtering the two replicate samples through 0.2-µm filters and serially diluted using BHI.The 1.8 mL of mBHI broth were added to individual wells of a 24-well plate and then inoculated with 200 µL of C. maltaromaticum D8c, was reduced to 104 CFU. A mixture of molten medium and target bacteria was poured over the solidified agar made from the same medium. After air-drying in a hood, 10 µL of each CFS dilution were spotted onto the solidified agar surface and incubated at 25 °C for 48 h. The highest dilution of CFS showing inhibition was defined as one arbitrary inhibition unit (AU) [® .The level of inhibition in CFS was measured by the agar overlay method described above, with modification to increase detection sensitivity. Instead of TSA, agar medium (pH 6.5 ± 0.1) was made with 37 g/L BHI, 5.06 g/L L(+)-lactic acid and 15 g/L agar. Furthermore, the cell density of target bacteria, nit (AU) . Each co10 CFU/mL) of each sample was calculated according to a regression equation between cell density and log OD600: cell density = optical density*1.1168 + 9.4726 (R2 = 0.996), which was developed in a preliminary experiment  pairwise differences. The correlation coefficient between undissociated lactic acid and DI and undissociated lactic acid and inhibitor production (AU/mL/h) was calculated via linear regression in Excel®.The overall effect of environmental factors on D8c sensitivity and D0h inhibitor production (per hour and per log CFU) was evaluated using analysis of variance (ANOVA), employing the GLM  procedure in SAS . If the Inhibitory activity (DI) of D0h CFS was detected between 6 and 9 h of incubation with a blurred inhibition zone; hence, DI was not determined for this time point . InhibitpH had a significant effect both on inhibitor production per hour and per biomass unit (CFU) . The ratp = 0.0098 for the interaction of lactic acid with pH  was heavily dependent on the pH level of a culture condition, with  with pH . At pH 5 with pH B. Conver with pH . Hence, p = 0.404) (p = 0.0017)  was not significant (= 0.404) . At pH 6 0.0017) . Further 0.0017) , which a 0.0017) . Hence, p = 0.236 for production per hour and p = 0.731 for production per CFU, respectively)  , the proctively) .p < 0.0001, F-test) on the sensitivity of D8c to D0h CFS  compared to anaerobic conditions (mean DI = 15.7 mm) , but thep = 0.0545).Significant interactions were observed between temperature and atmosphere, temperature and glucose, temperature and lactic acid, temperature and pH, glucose and pH and pH and lactic acid . A slighAtmosphere produced a strong discriminating effect on D8c sensitivity at 7 °C, compared with other temperatures A, as the2 = 0.965) (The effects of atmosphere and pH on D8c sensitivity were greater in the presence of 5.55 mM glucose G. These = 0.965) H.Brochothrix thermosphacta was inhibited by the presence of LAB in agar media [Pseudomonas spp. was found to enhance the survival of the pathogenic species Campylobacter jejuni in vitro and in poultry [C. maltaromaticum isolates, i.e., D8c sensitivity and D0h inhibitor production.Bacterial interactions have long been investigated in culture media and food model systems. For example, ar media . The pre poultry ,32. In tC. maltaromaticum D8C sensitivity to D0h CFS, interpreted by DI, increased at lower pH, agreeing with Ganzle et al. [+ affected bacteriocin activity via changes in the surface charge of target bacteria, thereby causing changes in conformation/oligomerization of bacteriocin peptides. Nisin is believed to have greater activity in acidic foods, due to increased solubility and stability [C. maltaromaticum D0h is undissociated at low pH (5.5), which increased the overall inhibitory activity of D0h CFS. We found that D8c cell density in agar was reduced at lower pH  (Inhibition by D0h CFS was greater under aerobic versus anaerobic conditions B. Accord= 0.055) E, with tC. maltaromaticum had no effect at 4 and 15 °C. Due to lower growth rates at −1, 7 and 15 °C, mBHI agar was incubated for longer time periods  comparedC. maltaromaticum D0h and D8c better, the effects of pH, glucose, atmosphere and lactic acid on inhibitor production by D0h were investigated at 25 °C. C. maltaromaticum D0h produced small or no detectable levels of inhibitory compounds in media with initial pH 5.5. This may result from a bacterial strategy to shift energy from biosynthesizing metabolites and translocating them to the external medium, to maintaining internal pH in high H+ environments [C. maltaromaticum [To understand inhibition between ronments ,57. Relaomaticum ,58. As momaticum ,59; we fC. maltaromaticum strains. Under aerobic conditions, the D8c sensitivity and D0h inhibitor production rate were both slightly higher  compared to an anaerobic environment (2. It is also possible that residual O2, especially at the beginning of the storage of VP beef, may benefit inhibitor strains of C. maltaromaticum to compete against sensitive strains.In our study, the inhibitory effect produced by D0h and the sensitivity of D8c to inhibition were studied separately. The effects of environmental factors were complicated, not only affecting inhibitor production, but also sensitivity and/or growth of D8c. Nevertheless, it is possible to estimate the effects of environmental factors on the net outcome of the bacterial interaction under a few conditions. For example, at 25 °C and pH 6, glucose increased D8c sensitivity, but did not significantly affect D0h inhibitor production ; hence, ironment . TherefoC. maltaromaticum strains, D0h vs. D8c. Experimental data were generated in vitro to define the effects of environmental factors more clearly, without the potential complicating factors of a complex meat matrix. These findings are an important first step to help to explain future research that will focus on monitoring the growth of a microbial community including different bacterial strains and/or species. Such bacteriological medium-based studies have been extensively used to understand how environments influence bacterial growth, resulting in predictive models [This study focused on the effects of environmental conditions on the intra-specific inhibitory activity between two model bacteria, e models ,61,62,63


October 15, 2017, marks the 10th annual Global Handwashing Day. This observance helps to increase awareness and understanding of handwashing with soap as an effective and affordable way to prevent disease around the world.Handwashing with soap has an important role in child survival and health. Approximately 1.4 million children aged <5 years die each year from diarrheal diseases and pneumonia, the top two causes of death among young children globally (https://www.cdc.gov/handwashing. Information on water-related hygiene is available at https://www.cdc.gov/healthywater/hygiene.Although persons around the world clean their hands with water, few use soap to wash their hands, because soap and water might be less accessible in developing countries. Even when soap is available, it might be reserved primarily for laundry and bathing instead of for handwashing. Washing hands with soap removes germs more effectively than water alone (


Infrared control is a new technique that uses pulsed infrared lasers to thermally alter electrical activity. Originally developed for nerves, we have applied this technology to embryonic hearts using a quail model, previously demonstrating infrared stimulation and, here, infrared inhibition. Infrared inhibition enables repeatable and reversible block, stopping cardiac contractions for several seconds. Normal beating resumes after the laser is turned off. The block can be spatially specific, affecting propagation on the ventricle or initiation on the atrium. Optical mapping showed that the block affects action potentials and not just calcium or contraction. Increased resting intracellular calcium was observed after a 30-s exposure to the inhibition laser, which likely resulted in reduced mechanical function. Further optimization of the laser illumination should reduce potential damage. Stopping cardiac contractions by disrupting electrical activity with infrared inhibition has the potential to be a powerful tool for studying the developing heart. Infrared control is a powerful new technology for controlling excitable cells, enabling new studies in small cardiac samples. It has several advantages over conventional electrical techniques, including being contact-free and having higher spatial precision. This is critical for studies of cardiac development in tiny embryonic hearts as electrical techniques interfere with electrical recordings, are prone to cause damage, and may be difficult to position.,,–,Infrared control was first developed as infrared neural stimulation and uses pulsed infrared laser light to elicit firing of an action potential,,,We have recently shown that infrared control can also be used to inhibit electrical activity.Here, we demonstrate reversible infrared inhibition of early embryonic avian hearts, both in intact embryos and excised hearts, with spatial selectivity, and we further investigate the target of inhibition and potential damage.Coturnix coturnix communis; Boyd’s Bird Company, Pullman, Washington) were incubated for 2 days to Hamburger–HamiltonQuail eggs . No excitation–contraction uncoupler was used to abolish motion, so recordings show motion artifacts, but the presence or absence of calcium transients remains clear. Hearts were imaged before, during, and after inhibition on the ventricle . CalciumOptical recordings of membrane voltage were acquired from excised hearts stained with di-4-ANEPPS. Hearts were imaged before, during, and after inhibition on the ventricle . Membran,–,–Both infrared stimulation and inhibition work through the mechanism of the infrared laser light being absorbed by water, which converts the energy into heat.To test for thermally induced necrosis, hearts were stained with propidium iodide, a membrane impermeant dye, for 10 min after 30 s of exposure to the inhibition laser. No increase in staining was detected, indicating that infrared inhibition did not result in compromised cell membrane via necrosis.However, using Fluo-4 staining, we did detect a large increase in calcium concentration during the resting phase of the action potential  on the sThe damage tests were done with a longer single exposure (30 s) than used for any of the other experiments included in this study . Since some hearts were inhibited up to three times without any detectable effect on mechanical function, it is possible that shorter exposures avoid damage. Additional studies will be conducted to determine the thresholds for mechanical dysfunction and apoptosis after optimization of the inhibition laser parameters.To reduce possible damage to the heart, the power of the inhibition laser should be minimized. In this study, the power was not optimized to avoid damage. Rather, the power was selected to provide a high success rate on the first attempt, so in many cases, the laser power could be decreased to reduce potential damage.Additionally, in the tubular embryonic heart, it is necessary to inhibit conduction of an entire cross-section; otherwise, action potentials would continue to propagate through any gap in the block. Changing the illumination profile on the heart could also reduce potential damage by avoiding over illumination of tissue proximal to the laser in an effort to reach the block threshold distally. Radiant exposure on the proximal side of the heart could be reduced by use of multiple illumination points so that the light would not have to penetrate across the entire heart tube. Radiant exposure in the center of the illumination spot could also be reduced by reshaping the beam to a more uniform profile to deliver energy more evenly to the entire illumination spot, rather than the Gaussian beam profile used here, where the majority of the delivered energy is concentrated at the center of the illumination spot. This would also likely allow inhibition with a smaller spot size, reducing the chance of off-target effects from heating other tissues.Furthermore, the temporal profile of the laser could be altered to reduce damage. It may be desirable to use a variable-power illumination protocol with a higher initial and lower steady-state laser powers. Having a higher initial power would allow the temperature to more quickly rise to the block threshold, reducing the exposure time before inhibition is established. Also, a lower steady-state temperature may be sufficient to maintain block after inhibition has already been established, reducing the long-term radiant exposure.In conclusion, we demonstrate here that infrared light can be used to inhibit cardiac activity in early embryos. This inhibition can be achieved with spatial selectivity, stopping the whole heart by targeting the atrium or part of the heart by targeting the ventricle. With optical mapping, we show that infrared inhibition stops contractions by inhibiting action potentials, which results in the abolition of intracellular calcium transients and contraction. Increased resting intracellular calcium was detected, which may indicate temporary mechanical attenuation at low levels or potential tissue damage at high levels, but short exposures or further optimization of the laser parameters will likely decrease any chances of damage during inhibition. Infrared inhibition has many potential applications in the embryonic heart, including enabling cardiac imaging without gating, allowing injections or tissue collection in a temporarily still heart, and producing disease models by disrupting normal function. Further in the future, the ability to inhibit cardiac electrical activity with high spatial specificity may enable novel experimental studies or more targeted treatments of arrhythmias.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen. Aspergillus flavus is associated with disease in plants, animals and humans and is the common causal agent of aflatoxins contamination. Aflatoxins are polyketide-derived secondary metabolites that are produced by both A. flavus and A. parasiticus, which are highly toxic, mutagenic, and carcinogenic to animals and humansSaccharomyces cerevisiae, Synechococcus spp., Drosophila melanogaster, mouse, and human891011A. nidulans and Candida albicans13Reversible protein phosphorylation at serine (S), threonine (T), and tyrosine (Y) residues is the most widespread type of post-translational modification (PTM) in cells, regulating crucial cellular functions, including signal transduction, cell proliferation, and development4A. parasiticus NRRL 2999estA) is regulated by aflR1A. flavus NRRL 3357, despite the fact that, according to genome analysis, this pathogenic fungus contains at least 119 kinases and 54 phosphatases. Global analysis of phosphorylated proteins in this pathogen would facilitate understanding of the role of phosphorylation in aflatoxins biosynthesis and signal transduction pathways, and this analysis could provide a firm basis for future research. However, to our knowledge, only a few A. flavus phosphoproteins have been identified to date, and little progress has been made in ascertaining precisely which A. flavus proteins are phosphorylated, which kinases/phosphatases are involved in this process, and which cellular functions are targeted by this important PTM.In recent years, major advances have been made in elucidating the mechanism underlying aflatoxins production. For example, dephosphorylation of calcineurin-mediated enzymes ensures production of aflatoxins in the pathogenic fungus 2 enrichment and LC-MS/MS analyses. In total, we identified 598 phosphorylation sites on a total of 283 phosphoproteins from A. flavus. Consistent with previous reports, unbiased bioinformatics analyses indicated that a large proportion of these phosphorylation sites are present on proteins involved in signal transduction pathways. Furthermore, we also identified phosphorylated proteins involved in metabolic pathways, related to aflatoxins biosynthesis. Immunoblotting analysis indicated that phosphorylation could play a key role in responses to environmental stimuli and aflatoxins generation. Further functional studies revealed that phosphorylation of Ste11 affected colonial morphology, conidial generation, sclerotial formation and aflatoxins biosynthesis in A. flavus. These results provide novel insights into the range of functions regulated by phosphorylation in A. flavus. The findings of this study may help improve the general understanding of the mechanism underlying aflatoxins biosynthesis and may lead to the development of innovative strategies to control aflatoxins production.To fill this gap, we performed a global phosphoproteomic analysis of this pathogen, using a combination of TiOA. flavus, western blotting analysis was performed using a polyclonal anti-phosphotyrosine antibody. As shown in A. flavus was cultivated for only 1 d, whereas high levels of aflatoxins were generated after cultivation for 6 d , followed by pT (16.4%), and pY (2.5%)  . We reasA. flavus, we performed the Gene Ontology (GO) functional annotation analysis of all identified phosphoproteins according to the biological process, molecular function and cellular compartment. Our results showed that the phosphoproteins were distributed throughout a variety of biological processes, including metabolic processes, cellular processes, and biological regulation , nucleocytoplasmic transport (p = 7.85E-03) and so on, suggesting that protein phosphorylation may play a role in the regulation of protein transport, as previously describedp = 7.94E-03, p = 1.49E-03) and phosphotransferase activity (p = 1.32E-02) within the enzymatic activity cluster, it was easy to associate that phosphoproteins may play a role in signal transduction. Furthermore, phosphoproteins were enriched in the nuclear pore (p = 1.36E-02), pore complex (p = 1.36E-02) and nuclear envelope (p = 1.59E-02) in the cellular component category. Taken together, these results implied that the identified phosphoproteins had a wide distribution of cellular localizations and functions, and phosphorylation may play an important role in the regulation of diverse cellular processes in A. flavus.To gain further insight into the potential functional implications of phosphorylation, we carried out GO term enrichment using DAVID tool . The majA. flavus proteome  with the entire set extracted from the proteome . Interesproteome , such asA. flavus phosphoproteome allowed us to infer kinase–substrate relationships in this fungus and to explore the extent of their phosphorylation pathways. Possible kinase groups targeting each of the 598 phosphorylation sites were predicted by NetworKIN (version 2)  is the most intensively studied PTM in eukaryotic cells and ePKs belong to one of the largest protein familiesrsion 2) . After aA. flavus phosphoproteins using NetSurfPp = 5.161E-06) and less frequently in ordered helical regions  than all S/T/Y, which is quite different from those of lysine PTM eventsp = 9.073E-04).We next predicted the local secondary structures and solvent accessibilities of pS/pT/pY and all S/T/Y of via oxidative decarboxylation of pyruvate and β-oxidation of fatty acidsWe next sought to establish protein interaction networks to identify the physical and functional interactions among the identified phosphoproteins, using protein interaction information from the String database. In total, 89 phosphoproteins and 236 edges were mapped to the protein interaction database . Then, fin vivo, the site-specific anti-phosphopeptide antibodies and anti-non-phosphopeptide antibodies were used to validate the phosphorylation status of five phosphoproteins, viz., acetyl-CoA carboxylase ACC (S1138), pyruvate decarboxylase PdcA (S355), MAP kinase kinase kinase Ste11 (S187), phosphotransmitter protein Ypd1 (T14) and serine/threonine protein kinase (S153). As expected, the relative abundance of phosphoproteins differed depending on the different physiological/developmental stage of A. flavus (2O2) and during aflatoxins formation (6 d culture), as shown in 31To validate our phosphoproteomics findings and to determine whether environmental stimuli affect phosphorylation . flavus . The oriA. flavus. Therefore, we assigned all identified phosphoproteins to KEGG pathways to evaluate whether the phosphoproteins played a role in any biological/signaling pathways. As shown in A. flavus as previously describedvia oxidative decarboxylation of pyruvate, β-oxidation of fatty acids or from ketogenic amino acids, in primary metabolism13437The large group of phosphoproteins observed in this study could yield new insights into metabolic pathways of the model pathogenic fungus Aspergillus nidulans, deletion of the MAPKK gene, steC, a homologue of A. flavus ste11, results in a slower growth rate, the formation of more branched hyphae, altered conidiophore morphology, an inhibition of heterokaryon formation and a block of cleistothecium developmentste11 gene mutagenesis (Δste11), complemental strain (COM) and site-directed mutagenesis (S187A and S187D) of phosphorylation site of Ste11. The identified phosphorylation site 187th S was converted to a non-phosphorylatable alanine (A) and to aspartic acid (D) to mimic a constitutively phosphorylation state. All mutations were verified by PCR or DNA sequencing  Ste11, it is conceivable that phosphorylation plays a vital role in the signal transductionquencing  and then for 4 d . AccordiES media . It was for 10 d . Consequ1 level would increase when strain was grown into the stationary phase that led to nutritional deficiency. Recent study has also revealed the stress response linkage to the production of natural products, including mycotoxinsAspergillus fumigatusA. flavus by regulating the MAP kinase cascade system. To test this hypothesis, we performed the TLC analysis of the WT, Δste11, COM, S187A and S187D. Compared with the WT strain, the production of aflatoxin B1 in Δste11 strain and S187A mutant were decreased memorably, whereas COM strain and S187D mutant had similar yield of aflatoxin B1  in A. nidulans∆ste11. Without phosphorylation, we speculated that Ste11 could not transmit phosphate signal. Hence, downstream Fus3 and VeA could be not activated. Finaly, aflatoxins biosynthesis was suppressed. It is convincible that phosphorylation may play an important role in regulating the aflatoxins biosynthesis in A. flavus.To our knowledge, the MAPK cascade module was well conserved in all eukaryotes, playing a fundamental role in the regulation of various environmental responses Taken together, it is safe to say that phosphorylation is involved in aflatoxins biosynthesis.A. flavus. Phosphorylated proteins in A. flavus were involved in a variety of biological processes. Further functional studies demonstrated that phosphorylation may be a mechanism involved in aflatoxin biosynthesis in A. flavus. Therefore, our results provided a foundation for functional analyses of the phosphorylated proteins and opened an avenue toward better understanding of signaling pathways in this pathogen.In conclusion, we presented a comprehensive phosphoproteome analysis of the model pathogenic fungus A. flavus NRRL 3357, A. fumigatus Af293 and A. flavus CA14Δku70ΔpyrGΔniaD were obtained from Prof. Zhumei He , Dr. Yang Liu  and Dr. Ana M. Calvo , respectively. Conidia of A. flavus NRRL 3357 were cultivated in 150 mL liquid YES media6 conidia/mL liquid media)A. fumigatus Af293 was cultivated with YES agar statically in the dark at 28 °C to extract DNA and amplify pyrG. A. flavus CA14Δku70ΔpyrGΔniaD was cultivated with YGTUU agarvia centrifugation and separated using a separatory funnel. All samples were dried and then resuspended in chloroform. Then, 5 μL of each extract was loaded onto a silica gel TLC plate , and the extracts were separated using acetone: chloroform . Photographs were captured following exposure to UV radiation at 312 nm wavelength. The intensity of the samples was analyzed using a JD-801 Computer-aided Image Analysis System (JEDA Co.) by visual comparison with standard aflatoxin B1.Cultures were added with equal volume of chloroform and agitated for 30 min. Aflatoxins dissolved in the chloroform were collected A. flavus NRRL 3357 were inoculated into liquid YES media and cultivated for 1 d or 6 d. Subsequently, a phosphatase inhibitor solution g for 20 min at 4 °C, and the supernatant was pipetted into Millipore Amicon Ultra-15 Centrifugal Filters  to remove pigment and other small molecules with centrifugation at 4,000 × g for 30 min at 4 °C; this purification process was repeated twice until the lower liquid was colorless. Finally, the entire retentate fraction was transferred into a new centrifuge tube for further processing. Protein concentrations were determined with a BCA Protein Assay Kit (Tiangen).Conidia of 18 material . The columns were washed three times with 15 mL of 100% acetonitrile (ACN) and then equilibrated three times with 15 mL of 20 mM ammonium formate. After the digested solution was loaded sequentially onto the self-packed C18 columns three times, the columns were washed with 5 mL of 20 mM ammonium formate, for sample desalting, and eluted with a series of elution buffers (2 mL) composed of 20 mM ammonium formate and different concentrations of ACN . Fractions were collected in 2 mL centrifuge tubes, concentrated to less than 50 μL, and stored at −80 °C for further use.Protein extracts (20 mg) were subjected to disulfide reduction, alkylation and digestion as described previously122 Kit  with slight modifications of the manufacturer’s protocol. Specifically, the fractions were redissolved in 200 μL of TiO2 phosphobind buffer containing 50 g/L 2,5-dihydroxybenzoic acid. Then, 50 μL of TiO2 phosphobind resin was added to the mixtures, and then incubated on an oscillator. After 1 h incubation, mixtures were centrifuged, and the supernatant was discarded. Subsequently, the TiO2 phosphobind resin was washed three times with wash buffers I and II. Thereafter, phosphopeptides bound to the resin were eluted twice with 0.5% ammonium solution (pH 10.5) in 50% ACN. The eluates were combined and concentrated. Then, the combined eluates were concentrated to dryness and redissolved in 25 μL of an equal volume of 0.1% TFA and 5% ACN for future processing.Phosphopeptides were enriched using a Phosphopeptide Enrichment TiO® binary pump system with tray cooling and analyzed online using a Triple TOF™ 5600 Mass Spectrometer (AB SCIEX). The samples were delivered to a Nano cHiPLC Trap Column  and then trapped on a Nano cHiPLC Column . Peptides were eluted with a linear solvent gradient (described in Phosphopeptides were separated on an Eksigent NanoLC-UltraTM 3000 nano-LC System (Thermo Scientific™ Dionex™) in tandem with an electro-spray ion-trap mass spectrometer HCT Ultra . The methods used for sample loaded, peptide elution from the column and the settings for the mass spectrometer were as described by Yang et al.Enriched phosphopeptides were also separated on an Ultimatehttp://abi.inf.uni-tuebingen.de/Services/YLoc/webloc.cgi)http://david.abcc. ncifcrf.gov/tools.jsp) was used to perform interpro and GO term enrichment analyseshttp://motif-x.med.harvard.edu/motif-x.html)http://www.cbs.dtu.dk/services/NetSurfP/)p-values of the identified phosphorylated residues were calculated as described by Wagner et al.http://networkin.info/version_2_0/newPrediction.php)http://string-db.org/)The identified phosphoproteins were group into biological process, molecular function and cellular compartment based on gene ontology (GO) term by Blast2GO softwareA. flavus NRRL 3357 were denatured in protein-loading buffer and separated by 12% SDS-PAGE. After electrophoresis, gels were stained with Coomassie Brilliant Blue or transferred to PVDF membranes . Immunoblotting was performed with the generated anti-phospho-site-specific antibodies (1:1000 dilution) or anti-non-phosphopeptide antibodies (1:1000 dilution), followed by incubation of the blots with a peroxidase-conjugated secondary antibody . As a loading control, monoclonal mouse anti-actin antibodies were used. Membranes were washed three times with TBST, and proteins were detected by chemiluminescence using SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The results of western blotting were recorded with ImageQuant TL .Proteins from A. flavus ste11 gene orthologs and A. fumigatus Af293 pyrG gene ortholog were identified, searched and downloaded from the Aspergillus Comparative Database (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html). The full-length replacement mutation of ste11 gene was introduced by a previous descriptionste11 gene (including upstream promoter element and downstream 3′-non-translated region) was amplified and digested  and attached to digested pPTR I with ClonExpress II One Step Cloning Kit . Then complementation vector was transformed into protoplasts  to construct complementation strain (COM), filtered by pyrithiamine resistance gene. The complementation vector was also used as the template of point mutant vectors (S187A and S187D). DpnI was used to eliminate template (complementation vector), and then point mutant vectors were transformed into protoplasts (∆ste11), filtered by pyrithiamine resistance gene. A. fumigatus Af293 pyrG gene was amplified and transformed into protoplasts (A. flavus CA14Δku70ΔpyrGΔniaD), used as wild type (WT).The 4 spores of WT, ∆ste11, COM, S187A and S187D were point inoculated onto YES agar, cultivated for 4 d. Colonial phenotypes were recorded. Conidia were collected with 7% DMSO and 0.5% Tween-20 and counted using a hemocytometer. Conidial formation was observed as described previously4 conidia of each strain were spread on Wickerham media plates and statically incubated at 37 °C for 10 d in the dark. Plates were sprayed with 70% ethanol to kill and wash away conidia to aid in enumeration of sclerotia. Each experiment was performed with four replicates for three times.10How to cite this article: Ren, S. et al. Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus. Sci. Rep.6, 34078; doi: 10.1038/srep34078 (2016).


Dear Editor:1. APAF1, CASP9, and CASP3 knockout mice display typical NTD phenotypes including severe craniofacial malformations and exencephaly5. Genes identified using mouse models have been explored as candidates in human NTDs and corresponding analyses of risk association. However, examinations of human populations have not provided persuasive genetic evidence to support a role for the apoptosis pathway in human NTDs.Neural tube defects (NTDs) are common congenital anomalies that occur because of the failure of neural tube closure in early embryogenesis. More than 250 NTD causative genes have been reported in mouse models. This suggests the involvement of distinct molecular mechanisms in NTD etiology, including the apoptosis pathwayCASP9, APAF1, and CASP3. All these variants were case-specific variants that only exist in 352 NTD samples but not in any of 224 controls . Subsequent sequence alignment of 14 candidate variants in NTDs suggested that CASP9 R180C (p.Arg180Cys), CASP3 Q217H (Gln217His), and APAF1 P335R (Pro335Arg) were extremely conserved in different species and caspase sub-family members  in three apoptosis related genes ols Fig. . 10 of 1ers Fig. . Notablyers Fig. .Fig. 1RaCASP9 C287A (active site) and D315A (cleavage site) mutations as positive controls6, we found that rare mutation in CASP9, especially R180C totally abolished spontaneous cleavage of CASP9 whereas CASP9 K292E identified from controls has no effect on procaspase9 cleavage . Overall, 81 and 67 DEGs were identified in HEK cells transfected with WT CASP9 and CASP9 (Y251C), respectively, when compared to the control sample Fig. . Unexpecp-value = 2.17e-5). Furthermore, the distribution of 57 LoF variants identified from ExAC, especailly 41 singletion LoF variants, were markedly varying in different ethnicities. No singletion high-confidence LoF variants was derived from East Asian population. In this study, we identified a new singleton LoF variant in APAF1 in NTDs patients. Although the etiology of NTDs in human is multifactorial7, our results strongly suggest that rare mutations in apoptosis-related genes including CASP9, APAF1, and CASP3 contribute to etiology of neural tube defects in Han Chinese population.In the ExAC database, a total 615 non-synonymous variants (MAF < 1%) including 57 LoF variants and 558 missense variants were identified in 60,706 unrelated individuals for three genes. Despite all this, the frequencies of non-synonymous mutations identified in three genes in NTD patients (14/352) were significantly higher than that in ExAC database (615/60706) by using fisher’s exact test (Supplementary filesSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5


The corrosion inhibitory effects of HCB on mild steel in 1.0 M HCl were investigated using potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS). The results showed that HCB inhibited mild steel corrosion in acidic solution and inhibition efficiency increased with an increase in the concentration of the inhibitor. The inhibition efficiency was up to 96.5% at 5.0 mM. Changes in the impedance parameters suggested that HCB adsorbed on the surface of mild steel, leading to the formation of a protective film. The novel corrosion inhibitor synthesized in the present study was characterized using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectral data. The specimens were cleaned according to the ASTM standard, G1-03 [−4 to 5 × 10−4 M. Solutions were freshly prepared from analytical grade chemical reagents using distilled water. All of the measurements were performed in triplicate, and the average values are reported. The measurements were performed using a Gamry Instrument Potentiostat/Galvanostat/ZRA. DC105 and EIS300 software produced by Gamry were used for potentiodynamic scans and electrochemical impedance spectroscopy (EIS). The potentiodynamic current-potential curves were swept from −0.25 to +0.25 Vsce at a scan rate of 0.5 mVs−1. All of the impedance data were fitted to appropriate equivalent circuits (EC) using Gamry Echem Analyst software. To stabilize the steady state potential, electrochemical measurements were initiated approximately 30 min after the working electrode was immersed in the solution. Mild steel specimens obtained from the Metal Samples Company were used as working electrodes throughout the study. The composition (wt.%) of the mild steel can be described as follows: Fe = 99.21, C = 0.21, Si = 0.38, P = 0.09, S = 0.05 and Al = 0.01 [sample area = 4.5 cmd, G1-03 . The meaIn the present study, 2-(1-methyl-4-((E)-(2-methylbenzylidene)amino)-2-phenyl-1H-pyrazol-3(2H)-ylidene)hydrazinecarbothio-amide (HCB), a novel hydrazinecarbothioamide, was synthesized and characterized using various spectroscopic methods. Changes in the electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization data were used to study the inhibition of mild steel corrosion in 1.0 M HCl solution at 30 °C, and HCB was used as an inhibitor at various concentrations. HCB exhibited excellent inhibition performance as a mixed-type inhibitor. In general, the acidic corrosion of mild steel was reduced upon the addition of an appropriate concentration of HCB. The inhibition efficiencies obtained from EIS data were comparable to those obtained from polarization measurements performed on the inhibitory solution, which were greater than those displayed by the non-inhibitory solution. HCB acted as an efficient corrosion inhibitor in 1.0 M HCl and exhibited a maximum inhibition efficiency of 95%–97%. The adsorption of HCB on mild steel surface obeys the Langmuir adsorption isotherm.