Epilepsy is a common neurological disorder worldwide and anti-epileptic drugs (AEDs) are always the first choice for treatment. However, more than 50% of patients with epilepsy who take AEDs have reported bone abnormalities. Cytochrome P450 (CYP450) isoenzymes are induced by AEDs, especially the classical AEDs, such as benzodiazepines (BZDs), carbamazepine (CBZ), phenytoin (PT), phenobarbital (PB), and valproic acid (VPA). The induction of CYP450 isoenzymes may cause vitamin D deficiency, hypocalcemia, increased fracture risks, and altered bone turnover, leading to impaired bone mineral density (BMD). Newer AEDs, such as levetiracetam (LEV), oxcarbazepine (OXC), lamotrigine (LTG), topiramate (TPM), gabapentin (GP), and vigabatrin (VB) have broader spectra, and are safer and better tolerated than the classical AEDs. The effects of AEDs on bone health are controversial. This review focuses on the impact of AEDs on growth and bone metabolism and emphasizes the need for caution and timely withdrawal of these medications to avoid serious disabilities. Epilepsy, a common neurological disorder, affects about 50 million people around the world. The prevalence of epilepsy is approximately 6.8 per 1000 in the US , and 3.32+, and alkaline phosphatase. Routine X-rays can identify bone fractures, but cannot detect these bone diseases if the reduction of bone mass density (BMD) is less than 30%  (accounts for 80%) and R enantiomers of MHD [(R)-MHD] (accounts for 20%) [R)-MHD and (S)-MHD is similar [OXC azepine-5-carboxamide) has been designed via structural variation of CBZ . After othan 1%) . Orally-than 1%) . Therefofor 20%) . The ant similar .OXC, like CBZ, specifically inhibits voltage-dependent sodium , potassiN-2 glucuronide, N-5 glucuronide, N-2 methyl, and N-2 oxide are the main metabolites of LTG [LTG -1,2,4-triazine-3,5-diamine) is rapidly and completely absorbed after oral administration. The oral bioavailability of LTG is 98%. The blood level of LTG is 1.4 to 4.8 h . Metabols of LTG . Most ofs of LTG . LTG gens of LTG .LTG acts on pre-synaptical voltage-sensitive sodium channels. LTG blocks N- and P/Q/R-type calcium channels. These blocking effects and others may stabilize neuronal membrane potential . LTG canO-(1-methylethylidene)-β-d-fructopyranose sulfamate) is a derivative of monosaccharide d-fructose. TPM is rapidly and completely absorbed after oral administration and concomitant food intake does not affect the metabolism of TPM. The peak blood level of TPM is 1.4–4.3 h [t1/2 of TPM is 19–25 h and is decreased by co-administration of EIAEDs such as CBZ and PT [TPM -type glutamate receptors, and blocks kinases to activate these channels . All of 2δ subunit to attenuate their activities [A or GABAB receptors, nor does it disturb GABA uptake or metabolism, but can increase the concentration of GABA to reduce firing inputs [GP (1-(aminomethyl) cyclohexane acetic acid), structurally-related to GABA, was originally developed to treat spasticity . GP is ativities ,180. GB g inputs  and enhag inputs . For itsg inputs . GB inhig inputs , causingg inputs . Due to g inputs , low levg inputs , and reng inputs , GB is lg inputs . GB is eg inputs .Sexual dysfunction, weight gain, dizziness, somnolene, and fatigue, but no serious idiosyncratic reactions or toxicities, have been reported ,190. LonVB (4-amino-5-hexenoic acid) is a GABA-aminotransferase inhibitor to antagonize the GABA degradation in synapses . VB is rAEDs are widely used for seizure treatment. However, abnormalities in bone and mineral metabolism have been frequently reported in individuals receiving EIAEDs because EIAEDs may cause hypocalcemia through triggering the catabolism of vitamin D. In vitro studies demonstrated that PB induces cultured human hepatocytes to increase the mRNA of CYP2C9, CYP2C19 , CYP2B6,


R) and it is overexpressed in insulin-resistant subjects. In keeping with this, we stimulated human umbilical vein endothelial cells (HUVECs) with insulin and UA to investigate the effects of UA on insulin signaling pathway, testing the hypothesis that UA can interfere with insulin signaling by the activation of ENPP1. Cultures of HUVECs were stimulated with insulin, UA and the urate transporter SLC22A12 (URAT1) inhibitor probenecid. Akt and endothelial nitric oxide synthase (eNOS) phosphorylation levels were investigated by immunoblotting. ENPP1 binding to IR and its tyrosine phosphorylation levels were tested by immunoprecipitation and immunoblotting. UA inhibited insulin-induced Akt/eNOS axis. Moreover, UA induced ENPP1 binding to IR that resulted in an impairment of insulin signaling cascade. Probenecid reverted UA effects, suggesting that UA intracellular uptake is required for its action. In endothelial cells, UA directly interferes with insulin signaling pathway at receptor level, through ENPP1 recruitment. This evidence suggests a new molecular model of UA-induced insulin resistance and vascular damage.High levels of uric acid (UA) are associated with type-2 diabetes and cardiovascular disease. Recent pieces of evidence attributed to UA a causative role in the appearance of diabetes and vascular damage. However, the molecular mechanisms by which UA induces these alterations have not been completely elucidated so far. Among the mechanisms underlying insulin resistance, it was reported the role of a transmembrane glycoprotein, named either ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) or plasma cell antigen 1, which is able to inhibit the function of insulin receptor (I Uric acid (UA) is the end-product of purine metabolism, with both anti-oxidant and pro-oxidant properties; it has been demonstrated to have a key role in the redox process related to oxidative stress  that is In keeping with this, we previously demonstrated that serum UA levels, independently from classical cardiovascular risk factors, are associated with endothelial dysfunction  and the R), whereas others may impair one or more of the downstream signaling steps (R function and having high expression levels in cells of insulin-resistant subjects (R (IRα), impairs IR signaling by inhibiting its autophosphorylation, and consequently insulin receptor substrate-1 phosphorylation and glucose transport . In particular, we tested the hypothesis that UA can interfere with insulin signaling throughout the activation of ENPP1.Human umbilical vein endothelial cells were obtained from ScienCell Research Laboratories  and maintained in endothelial cell medium according to the manufacturer’s instructions. Growth medium was replaced every 48/72 h and cells were grown until 95–100% confluent. We used the same batch of HUVECs from a single donor for all the experiments, at different passages ranging from 2 to 6.−7M) for the last 7 min of UA or UA+Probenecid incubation.Human umbilical vein endothelial cells, seeded in 100 or 150 mm tissue culture dishes, were cultured until 80% confluent. UA  was dissolved in pre-warmed (37°C) medium; the mixture was then warmed again  and sterile filtered. Probenecid, a urate transporter  inhibitor, was added from a concentrated stock, prepared in NaOH at the maximal allowed concentration (50 mg/dl), to 1 mM treatment concentration . HUVECs Rα antibody or directly loaded to SDS-PAGE gels. For immunoprecipitation experiments, immune complexes were collected by incubation with protein A-Sepharose and resuspended in Laemmli buffer before loading to SDS-PAGE gels. Gels were then transferred to nitrocellulose membranes and immunoblotted with the appropriate primary antibodies, according to standard protocols. To evaluate ENPP1 binding to IR and IR tyrosine phosphorylation, the IRα immunoprecipitated proteins were immunoblotted, with an anti-ENPP1 and or an anti-tyrosine phosphorylated proteins antibody. To normalize for protein levels, the blots were stripped and reprobed with primary antibodies against the total unphosphorylated form of the appropriate protein. Primary antibodies used for this study were purchased from Cell Signaling Technology, Beverly, MA, USA  or from Merck Millipore (clone 4G10). Blots were visualized using appropriate peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence detection, and band densities were quantified by densitometry using an ImageJ software.Cells were lysed in buffer containing 1.5% NP-40 and analyzed by immunoprecipitation and Western blotting as previously described –31. Briet-test. Values of P < 0.05 were considered statistically significant. Analyses were performed with GraphPad Prism version 6 software.All results are given as mean fold variation ± SE over control. Statistical differences were assessed by Student’s R is able to activate a dual signaling pathway: the PI3K/Akt signaling pathway, promoting metabolic effects, and the MAPK-related signaling pathway, promoting cellular proliferation, differentiation, and gene expression. In endothelial cells, the metabolic and hemodynamic effects of insulin are mediated by the activation of PI3K/Akt axis that induces a phosphorylation cascade leading to glycogen synthesis and glucose uptake and, above all, eNOS activation that increases NO production promoting in turn vasodilation and improving endothelial function.As well-known, insulin binding to I473, Figure 1177, Figure As shown in Figure R tyrosine phosphorylation. As shown in Figure R tyrosine phosphorylation.Given the inhibitory effect of UA on Akt/eNOS signaling pathway, we hypothesized that UA could exert a direct action on insulin signaling at membrane level. Since preliminary results demonstrated that the maximal inhibitory effect of UA is observed at 30′ exposure, we used this time point for subsequent experiments. Initially, we performed an immunoprecipitation test to evaluate the molecular effect of UA on IR to ENPP1, a well-known inhibitor of IR auto-phosphorylation that has been shown to be overactive in condition of insulin resistance —mediated entry of UA in endothelial cells .R association. As shown in Figure R in presence of insulin and UA.HUVECs exposure to Probenecid restored Akt insulin-induced phosphorylation levels in presence of UA and the increased Akt phosphorylation resulted, in turn, in an enhanced eNOS activation Figure . Given tR, inhibiting its activation. This evidence clearly shows that UA directly interferes with the insulin signaling pathway in a way totally independent of its ability to increase the oxidative stress and the inflammatory burden at cellular level. Thus, data obtained from this study demonstrate the hypothesis that UA has a primary role in the appearance and progression of insulin resistance and, then, in the development of diabetes. In fact, the demonstration that UA is directly able to impair the insulin signaling pathway, by inhibiting the cellular trigger of insulin signal at receptor level, attributes to hyperuricemia a causal independent role in the pathogenetic mechanisms of insulin resistance beyond its mediator role in the oxidative stress and inflammation.In this study, we demonstrated for the first time that UA promotes the binding of ENPP1 to Iin vivo study it was seen that hyperuricemia caused by fructose plays a role in the pathogenesis of metabolic syndrome (Identification of hyperuricemia as a risk factor for diabetes has been uncertain for many decades because it has been considered a consequence of insulin resistance rather than a cause , 33. In syndrome . Thus, oNotably in the present study, we were also able to show that UA has a key role in reducing Akt–eNOS axis activity that is involved in the normal vascular function; it is clearly demonstrated that its impairment induces endothelial dysfunction that represents the first step in the atherosclerotic process . We prevUric acid also stimulates vascular smooth muscle cells through a specific organic anion transport pathway, platelet-derived growth factor-dependent proliferation, monocyte chemoattractant protein-1 and cyclooxygenase-2-dependent thromboxane synthesis, and through the activation of renin–angiotensin system , 44, allFinally, UA crystals have been reported to be able to activate the NLRP3 inflammasome, which in turns promotes the cleavage of caspase-1 and the consequent increased production of pro-inflammatory cytokines .R in endothelial cells could affect the vascular integrity ensured by the protective action of insulin. Thus, in all clinical conditions associated with an insulin resistance status, such as diabetes, obesity, hypertension, and metabolic syndrome, the presence of hyperuricemia represents an important factor capable to induce and sustain endothelial damage.In this study, we demonstrated that UA is able to exert a direct effect on insulin signaling inducing NO synthesis, as observed in condition of insulin resistance. In fact, inhibitory effect of UA on IIn conclusion, data obtained from this study clearly demonstrate that UA directly interfere with insulin signaling pathway, being able to inhibit the trigger of insulin signaling at receptor level through an ENPP1 recruitment. This evidence proposes a new molecular model of UA-induced insulin resistance that goes beyond the increase of oxidative stress and the promotion of inflammation. This finding attribute to UA the role of leading player in the pathogenesis of insulin resistance and endothelial dysfunction, suggesting that hyperuricemia can significantly contribute to the pathophysiological mechanisms of atherosclerosis and to the appearance of new diabetes. This has several important clinical implications because the reduction of UA levels might represent an innovative treatment goal in the prevention of both diabetes and vascular damage, two conditions that significantly worsen cardiovascular risk profile. This is particularly important, especially in view of the fact that fructose is largely used in industrial food. Importantly, since fructose is a precursor of UA , it woulET, AC, and MH gave substantial contributions to acquisition, analysis, and interpretation of data for the work. MP, AS, FA, GS, and FP gave substantial contributions to conception, design, and interpretation of data of the work. All the authors contributed to the manuscript, provided critical revisions, approved the final version, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bruton's tyrosine kinase (BTK) mediated signaling is critical for B cell development and activation. The physiological role of BTK is evident since its functional loss results in a differentiation block of B cells beyond the pro-B cell stage causing X-linked agammaglobulinemia. Inhibitors of BTK, such as ibrutinib and acalabrutinib profoundly impair BCR signaling. They are highly effective in the treatment of many lymphoid tumors, including chronic lymphocytic leukemia, mantle cell lymphoma, Waldenström's macroglobulinemia and marginal zone lymphoma. Most patients respond, but escape mutants of different origins may appear –3. The mcis, thereby impeding enzyme activity. This phosphorylation is reversible and the CD45 transmembrane phosphatase is directly involved [In light of the potent effect of BTK inhibitors, it seems important to consider the endogenous/physiological control of the BCR signaling pathway, including how activated BTK is turned off. Immediately upstream of BTK are SRC-family kinases (SFKs). They phosphorylate tyrosine 551 in BTK, which changes the conformation leading to an activated form of the kinase. In B lymphocytes LYN is the predominant member of this family  are involved in the down-regulation of BCR signaling, often with 14-3-3 proteins being crucial contributors to this process. Examples of the BCR downstream proteins involved in this process are BTK, Spleen tyrosine kinase (SYK) and B cell linker/adapter protein (BLNK) . Thus, wIn further support of the idea that S/T kinases down-regulate BCR signaling is the fact that Protein kinase C (PKC) β abates the activity of BTK by phosphorylating serine 180 in the Tec homology (TH) domain . The undHowever, other molecules in the BCR signaling cascade are affected by S/T kinases too, such as the cytoplasmic linker/adaptor protein BLNK, also known as SLP-65. BLNK interacts with BTK and defects of either molecule cause related forms of primary immunodeficiency, both confined to the B-cell lineage. AKT is also implicated in the 14-3-3 mediated degradation of BLNK . HoweverFinally, the SYK kinase, which is another key player for BCR signaling initiation, is also affected by AKT phosphorylation Fig. . However


We propose and experimentally demonstrate that the intensity noise correlation and the noise level of intensity-difference and intensity-sum in Stokes and anti-Stokes channel can be well controlled by the relative nonlinear phase in spontaneous parametric four-wave mixing process. By modulating the relative nonlinear phase, including self-phase modulation and cross-phase modulation, switching the correlation into anti-correlation and the relative intensity noise level between the intensity-difference and intensity-sum are realized. We also show that the variation tendencies of the relative intensity noise level and the corresponding intensity fluctuations correlation are in accordance with each other. Recent research progresses related to atomic coherence in such solid-state materials, including electromagnetically induced transparency (EIT)The experimental preparation, manipulation, and detection of correlated and entangled photon pairs are of great interest for research in fundamental physics and application of quantum information processing12673+:Y2SiO3 crystal. We demonstrated that the switching the correlation into anti-correlation as well as the relative noise about the intensity-difference noise level (IDNL) and intensity-sum noise level (ISNL) can be realized by modulating the relative nonlinear phase, including self-phase modulation and cross-phase modulation. Especially, the correlation and relative noise level can be manipulated by power and polarization of the dressing field participating in the SP-FWM process.In this paper, the intensity noise correlation and relative noise level of intensity-difference and intensity-sum in Stokes and anti-Stokes signals are investigated in Pr3+:Y2SiO3 crystal) is held at 77 K in a cryostat (CFM-102). two tunable dye lasers (with a 0.04 cm−1 linewidth) pumped by an injection locked single-mode Nd:YAG laser  are used to generate the pumping fields E1  and E2  with the frequency detuning of Δi = ωmn − ωi , respectively, where Ωmn is the transition frequency between level E1 drives the transition E2 couples to the transition E1 with peak power of about 6 kW and beam waist of 280 μm is combined with pumping beam E2 (about 12 kW) at an angle of 3 mrad at the center of Pr3+:Y2SiO3 crystal, where E1 crosses with E2 and amplified with a gain of G≈3.5. Based on these settings, two narrow band correlated photons pairs (Stokes ES and anti-Stokes EAS) are generated via SP-FWM process, and the corresponding photon counting rate for Stokes and anti-Stokes channel are 1 × 106 s−1 and 6 × 107 s−1, respectively.E1 and E2 are switched on, in the V-type three-level ES and anti-Stokes field EAS) are generated via SP-FWM process, satisfying the phase-matching condition k1 + k2 = kAS + kS. ES and anti-Stokes signal EAS are reflected by polarized beam splitters (PBS) and detected by photomultiplier tubes, and recorded by D1 and D2. To study fluctuations in stokes and anti-stokes channel transmitted through Pr3+:YSO, the dependence of the intensities on time and frequency are registered by digital oscilloscope and photomultiplier tubes, respectively, as shown in δIi(t)  recorded by digital oscilloscope, the temporal waveform correlations between stokes and anti-stokes channel are investigated by When two pumping field In the interaction picture, the coupled Stokes channel and anti-Stokes channel can be described byκ is called the coupling parameter of the SP-FWM, depending on the nonlinearity coefficient χ(3) and intensity of the pump field, and EAS (ES) channel, GSi = μkESiE/ħ Rabi frequency of Si., μijelectric dipole moment between energy stateand, and N averaged atomic density. According to the Liouville pathways EAS) and ES), the density-matrix element for ES and EAS can be expressed as Gi = μiEi/ħ  is the Rabi frequency of the field Ei, and Γij is the decay rate between the energy levels d20 = Γ20 + iΔ2, d10 = Γ10 + iΔ1, d00 = Γ00 + i(Δ2 − Δ1), where E1 and E2 into account, the revised density-matrix element for ES and EAS can be rewritten asNext, taking the dressing effects of d21 = Γ21 − iΔ1, d01 = Γ01 − iΔ1.where E2 is modulated by inserting a quarter-wave plate (QW) in E2 beam  of Stokes and anti-Stokes signals reflected by PBS are detected. Therefore, the effective density matrix elements for ES and EAS are given byIn addition, the polarization of beam see , while pG2|2 in cx,y is the anisotropic factor in different directions of crystal, θ is the rotated angle of the QWP’s axis from the x axis.In this case, the Rabi frequency |Based on the measurements of the second-order coherence functions, See Methods for theoretical derivations of the intensity distribution at each output channel, the intensity fluctuations correlation between the Stokes and anti-Stokes signals1415S − tAS is the selected time delay between Stokes and anti-Stokes signal, and T is the time of integration, in our case T = 10 μs. Δφ = φS − φAS Is relative nonlinear phase between Stokes and anti-Stokes signal based on XPM and SPM.where averaging over the time is defined as Different from the intensity-difference squeezing171819τ) is two photon envelop function.where Ii〉 + δIi(t)  are registered. Here 〈Ii〉 is the average intensity of anti-Stokes/Stokes beams, and δIi(t) are corresponding intensity fluctuations. After the intensity fluctuations is averaged by a fast gated integrator over 10 pulses, and then using the averaged data, the correlation curve is calculated via cross correlation of the intensity fluctuation in stokes and anti-stokes channel.To study the role of the relative nonlinear phase played on intensity fluctuations correlation as well as the intensity noise level between the IDNL and ISNL of anti-Stokes and Stokes beams, the dependence of the intensities on time 〈EAS and ES under different dressing powers, where the curve is calculated by substituting the recorded intensity fluctuations δIAS,S(t) into 1 = Δ2 = 0 and P1 = 4 mW. When P2 = 6 mW, as shown in τ = 0 has amplitude of 0.83 ± 0.01 and corresponding full width at half maximum (FWHM) is 2.2 μs. If we set the power of E2 at P2 = 8 mW, as shown in τ = 0 is switched from positive (0.83 ± 0.01) to negative (−0.82 ± 0.01), therefore, the intensity fluctuations is changed from correlated to anti-correlated. The FWHM of the correlation peaks has increased to 1.8 μs. In contrast, at P2 = 10 mW, the correlation peak τ = 0 is adjusted back to correlated with amplitude 0.83 ± 0.01 and corresponding FWHM is further increased to 1.5 μs, as shown in E2) can modulate the nonlinear refractive index of Kerr medium and lead to XPM, the relative nonlinear phase between Stokes and anti-Stokes signal φ = φS − φAS is the relative nonlinear phase between Stokes and anti-Stokes signal based on XPM, whereas φS (φAS) is nonlinear phases induced on Stokes (anti-Stokes) signal, respectively and r is the beam radium of Gaussian beam incident from laser and z is length of YSO crystal’s lens (z = 3 mm). Specifically, by changing the power of E2 at 6, 8, and 10 mW, the relative nonlinear phase between Stokes and anti-Stokes are modulated to Δφ = 0, π, and 2π, respectively. Therefore, P2 = 6 mW and 10 mW have positive correlation peak, and at P2 = 8 mW have negative correlation peak.δIAS,S(t) into φ = 0, π, and 2π, respectively. we can see that the noise level of intensity-difference signal is lower than the intensity-sum signal when Δφ = 0 and 2π, as in φ = π, see On the other hand, by substituting E2, but changing the polarization of E2 as well. As stated above, the polarization of the dressing field E2 can be controlled by inserting a QWP, and the effective Rabi frequency of E2 for matrix element The modulation of the nonlinear phase can be realized not only by changing the power of E2. θ = 0), elliptical polarization (θ = π/6), and circular polarization (θ = π/4), respectively, with setting Δ1 = Δ2 = 0, P1 = 5 mW and P2 = 10 mW. We can see from EAS and ES depends crucially on the correlation functions, therefore, the corresponding relative noise level of intensity-difference is switched from lower than the intensity-sum to higher than the intensity-sum as polarization modulated from linear to circular. This phenomenon can be interpreted from a = 0, +, and − are corresponding to linear, left circular, and right circular polarization, respectively.) induced by XPM at circular polarization is about π, and at linear (θ = 0) and elliptical (θ = π/6) polarization is about 0 and 2π. Therefore, relative intensity noise level between IDNL and ISNL and correlation can also be switched by polarization of pumping field.Now, we focus on the polarization dependences of the intensity fluctuations correlation and the relative intensity noise level between IDNL and ISNL. Modulated polarization can be achieved by inserting QWP in EAS and ES are demonstrated, as shown in 1 = Δ2 = 0 and setting P1 = 5 mW and P2 = 10 mW. At the high intensity time position (2nd), τ = 0 has amplitude −0.88 ± 0.01 and corresponding FWHM is 1.6 μs. It is interesting that the correlation is switched to correlation with amplitude 0.86 ± 0.01 and 0.89 ± 0.01 at the lower intensity time position (1st) and (3rd), respectively. This phenomenon are tightly related to the intensity of generated field EAS and ES, and can be explained by relative nonlinear phase induced by SPM ζ = |G2/GS/AS|2 is intensity ratio of strong field G2 and obtained weak fields nd for intensity of EAS and ES are the largest at this timing point, therefore, the relative phase induced by SPM at 2nd is about Δφ ≅ π. On the other hand, at 1st and 3rd, the SPM can be ignored for faint intensity of EAS and ES, therefore, the relative phase between Stokes and anti-Stokes is same Δφ ≅ 0, which is the reason that nd  crystal, and the triplet energy-level (3H4) and singlet energy-level (1D2) of Pr3+ are selected which can be distinguished by investigating the optical spectrum of Pr3+. The degeneracy of the energy levels of the Pr3+ ion is removed by the crystal field of YSO, where the terms in 3H4 and 1D2 states are split into nine and five Stark components, respectively. The Pr3+ impurity ions occupy two nonequivalent cation sites  in the YSO crystal lattice. The energy levels are labeled by a Greek letters with and without asterisk for site II and I sites, respectively. The coupling between Pr3+ ions localized at different cation vacancies can happen due to induced dipole-dipole interactions, so one can treat the two ions as a hetero-nuclear-like molecule. Therefore, we can construct a V-type three-level subsystem  is nonlinear phases induced on Stokes (anti-Stokes) signal, respectively, and the relative nonlinear phase between Stokes and anti-Stokes signal can be defined as Δφ = φS − φAS. Here, the induced nonlinear phase shift Δφ can be defined as n1 is the linear refractive index. Therefore, from where nonlinear susceptibility coefficients where the gain coefficient EAS) and ES), the non-steady state solutions of density-matrix element for ES and EAS can be expressed asAccording to the Liouville pathways where τ) is obtained asIn addition, taking the population distributions in levels |1〉 and |2〉 into account, where the Boltzmann distribution with finite temperature is considered in the deduction, the two-photon envelope function Θ.


Bull fertility is the degree of sperm’s ability to fertilize and activate the egg and support embryo development, and this is critical for herd reproductive performance. We used the bull as a unique model organism for the study of male fertility because cattle genetics and physiology is similar to those of other mammals including humans. Moreover, reliable fertility data along with well-established in vitro systems are available for bovine. The objective of this original study was to ascertain evolutionary diversification and expression dynamics of Testis Specific Histone 2B (TH2B) in sperm from Holstein bulls with different fertility scores.The intensity of TH2B was determined by using flow cytometry in sperm from 13 high and 13 low fertility bulls. Expression levels of TH2B were measured using immunofluorescence and Western blotting in sperm from five high and five low fertility bulls. Sequence identity, evolutionary distance and interactome of TH2B were evaluated by dotmatcher, STRING and Cytoscape. Data were analyzed using linear mixed effects model and regression plots were drawn.P = 0.0182). The intensity of TH2B in sperm from the high fertility group decreased (P = 0.0055) as fertility increased. TH2B was constantly detectable in sperm and expression levels of TH2B decreased in relation to fertility in sperm from the high fertility group (P = 0.018). TH2B biological functions include male gamete generation, chromosome organization, DNA packaging, DNA conformation change, chromatin organization, nucleosome organization, chromatin disassembly, spermatid nucleus elongation, spermatid nucleus differentiation, sperm motility, chromatin organization, chromatin condensation, chromatin silencing, nucleus organization, and chromatin remodeling (P < 0.05).The intensity of TH2B as measured by flow cytometry was significantly affected by an interaction between fertility group and fertility score (We elucidated the cellular localization and molecular physiology of TH2B using both computational and cell biology approaches. In addition to advancing the fundamental science of mammalian male gamete, the present findings can be potentially used to evaluate semen quality and predict male fertility in the future.This study did not involve any live animals. We did not perform any anesthesia, euthanasia, or any kind of animal sacrifice. The cryopreserved semen samples were obtained from Alta Genetics, Inc., Watertown, WI, USA. All samples were preserved in liquid nitrogen. Spermatogenesis is the development of mature male germ cells in the testes capable of forming highly differentiated sex cells called spermatozoa. During mammalian spermatogenesis, complex biochemical and morphological transformations occur in the sperm head. The nucleus of a sperm progressively differentiates, undergoing chromosomal condensation, synapsis, genetic recombination, and extensive chromatin reorganization , 2. Duri.Transition of H2B to TH2B sets nucleosome stability, ensuring a genome-wide change of nucleohistone to intermediate structural entities, which in turn are required for the assembly of TNPs (TNP1 & TNP2) and protamines (PRM1) . TH2B in/or after fertilization. Defects in sperm chromatin are linked to spontaneous abortion and failures of assisted reproductive techniques in human [The octamer-shaped nucleohistone complex consists of histones H2A, H2B, histone 3 (H3) and histone (H4), all involved in sperm DNA packaging. Along with protamines, they form a tightly coiled and compacted structure known as the toroidal model . Retainein human , 13. Thein human , 15. Sucin human , 17. Abnin human . Therefoin human . Functioin human , 19.p = 0.0055) and intensity (p = 0.018) were lower in sperm from the high fertility bulls as compared to low fertility ones. Protein sequence of TH2B is conserved among human, rat, mouse and bovine. Further, TH2B has significant gene ontology terms for molecular functions in chromatin and DNA binding and biological functions in sperm development differentiation and motility, nucleosome assembly, chromatin remodeling and spermatid nucleus differentiation (P < 0.05). Because of the genetic and physiological similarities among mammals [Abnormal retention of histones in the sperm is an indication of immature sperm, and abnormal ratios of protamine to histone cause infertility . Therefo mammals –23, the Cryopreserved semen straws from Holstein bulls  were used in the present study. Straws from 26 bulls were used for the flow cytometry experiment Table.  and 10 bg at 4 °C for 5 min. And pellets were washed twice in washing buffer (WB: PBS with 0.1% Bovine Serum Albumin BSA) and again centrifuged at 2000 x g at 4 °C for 5 min. The pellets were then fixed in 1 ml of 4% formaldehyde at RT for 1 h in separate centrifuge tubes. The samples were then centrifuged at 3000 x g at 4 °C for 5 min and pellets were resuspended in 250 μl of PBS and immediately permeabilized in 250 μl of 0.1% Triton X-100 in 0.1% sodium citrate in PBS on ice for 2 min. The pellets were resuspended in 500 μl of PBS, filtered through a flow cytometric tube using a cell strainer cap , and then incubated with the primary antibody at 4 °C overnight. Primary antibody was TH2B . Next day, samples were centrifuged at 3000 x g at 4 °C for 5 min, washed once in 500 μl of washing buffer, centrifuged at 3000 x g at 4 °C for 5 min and incubated with secondary antibodies for 2 h at RT. The secondary antibody was donkey anti-rabbit IgG-FITC . Following the incubation, the samples were washed twice in WB (3000 g at 4 °C for 5 min). Sperm samples were then analyzed using the BD-FACSCalibur flow cytometer .Flow cytometry experiments were performed according to methods described by Dogan et al. (2015)  and Odhig at room temperature (RT) for 5 min (min). In addition, the sperm pellets were incubated with 20 mM CHAPS at RT for 20 min. Sperm chromatin was then decondensed in 10 mM DTT and 1 mg/ml of heparin at RT for 30 min [Immunofluorescence was performed according to the methods described by Li et al. (2008)  and de Og at 4 °C for 5 min. An aliquot containing 25–40 × 106 sperm was used to extract the nuclear proteins. In order to lyse the cells, sperm samples were washed twice with 400 μL of 1 mM Phenyl methylsulfonyl fluoride (PMSF) in ddH2O, centrifuged each time at 700×g at 4 °C for 5 min. Then, 100 μL of 20 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 100 mM Tris (pH 8.0) were added to the pellets followed by the addition of 100 μL of 6 M guanidine hydrochloride, 575 mM dithiothreitol (DTT), and 200 μL of 552 mM iodoacetamide. The samples were protected from light and incubated at 20 °C for 30 min. In addition, the samples were supplemented with 1 mL of cold ethanol (−20 °C), and each sample was then incubated at −20 °C for 1 min and centrifuged at 12000×g at 4 °C for 10 min. The ethanol wash was repeated once more and the pellet was resuspended in 1 mL of 0.5 M HCl and incubated at 37 °C for 15 min and centrifuged at 10,000×g at 25 °C for 10 min. The supernatant was kept and the nuclear proteins were precipitated by the addition of 300 μL of 100% trichloroacetic acid (TCA) to a final concentration of 20% TCA. The solution was incubated at 4 °C for 5 min and centrifuged at 12000×g for 10 min. The pellet was washed twice in 500 μL of 1% 2-mercaptoethanol in acetone. The final pellet was dried out and stored at −30 °C.Sperm TH2B was extracted according to the methods of Aoki et al. (2005)  and de Og at 4 °C for 10 min. The supernatants were discarded and the pellets were resuspended in 50 μL of 1× Laemmli sample buffer  with 5% 2-mercaptoethanol, vortexed (10 s), boiled for 10 min, and stored −30 °C. An aliquot of sperm nuclear proteins (10 μg) was reduced, denatured and separated with a vertical polyacrylamide gel electrophoresis . Protein bands were transferred from the gels to an Immobilon®-P polyvinylidene difluoride (PVDF) membrane using HEP-1 semi-dry electro blotting  set at 46 mA for 2.5 h. Binding sites were blocked with 5% BSA in PBS-0.1% Tween 20 (PBS-T) at RT under mild agitation for 1 h, followed by an incubation with primary antibodies against bovine TH2B . Lamin B1 was used as internal loading control  in PBS-T with 1% of BSA at RT for 1 h. Because, the expression levels of Lamin B1 were not stable, therefore another protein band (~25 KDa) served as second internal control. Membranes were then washed three times for 10 min in PBS-T and incubated with secondary antibodies  and Precision Protein™ StrepTactin-HRP Conjugate  in PBS-T with 1% of BSA at RT for 1 h. Membranes were washed with PBS-T three times for 5 min each. The bands were revealed using a chemoluminescence reagent  and Image Laboratory software (Bio-Rad®) for 30 s. The band intensity of TH2B histone protein was analyzed using ImageJ 1.x [Protein concentration in samples containing sperm protein extracts was determined in triplicates using Quick Start™ Bradford Protein Assay Kit 2 . In addition, sperm nuclear proteins were precipitated with cold acetone at −20 °C for 3 h, followed by centrifugation at 10,000×ageJ 1.x .http://www.bioinformatics.nl/cgi-bin/emboss) to ensure the sequence similarity of bovine TH2B for downstream computational biology analysis. Protein sequences of predicted TH2B and H2B of bovine were compared using computational tools. Protein sequences of TH2B and H2B were first extensively searched and aligned using UniProt for the bovine . The sequence alignment was performed using Clustal Omega method and the percent identity matrixes between the sequences were obtained .Emboss dotmatcher was used to compare protein sequences of predicted bovine TH2B with human TH2B, bovine H2B with human H2B, and bovine TH2B with bovine H2B (http://string-db.org/) [TH2B using the biological networks gene ontology tool (BiNGO) within Cytoscape 3.3 (http://www.cytoscape.org) was elucidated as well [HIST1H2B (TH2B), a merged-network was generated in Cytoscape by entering the query keywords “HIST1H2B for TH2B”, “H2A”, “H3”, “H4”, “TNP1”, “TNP2”, “PRM1”, “PRM2”, “H2B”, “BRD4” and “CHD5” into the search bar and results were retrieved from the BiNGO plugin to assess overrepresentation of GO categories.Predicted protein-protein interactions among TH2B, H2B, H2A, H3, H4 TNP1, TNP2, PRM1, BRD4 and CHD5 of bovine, human and mouse were obtained using STRING database  . The tes as well . To analThe associations between in vivo fertility scores of bulls with sperm TH2B intensity (measured by flow cytometry) and expression levels of TH2B (estimated by Western blotting) were assessed using mixed model analysis with PROC MIXED from SAS for Windows 9.4. For this mixed model, in vivo fertility group was the fixed effect. The random statement included replicate within repeat, repeat within bull, and bull as random effects. The relationship between fertility score and TH2B intensity was assessed through mixed model linear regression using PROC MIXED with SAS for Windows 9.4. In an initial model, fertility score, fertility group, and their interaction term were included as fixed effects. The random statement included replicate within repeat, repeat within bull, and bull as random effects. Fitting this model indicated no significant interactions between fertility score and fertility group, but the fertility scores were widely separated between the two groups. Accordingly, separate models with fertility score as the fixed effect were fit for both the high and low fertility groups to better understand the relationships between fertility and TH2B intensity. Regression line plots were developed using SGPLOT with SAS for Windows 9.4. The distribution of the conditional residuals was assessed to ensure that assumptions for the statistical models have been met. An alpha level of 0.05 was used to determine statistical significance.P = 0.0182). The expression levels of TH2B was highly significant compared to fertility score in the high fertility group  but no significance was found in the low fertility group .Total of 150,000 sperm per each bull were analyzed to investigate the expression of testis specific histone 2B (TH2B) in sperm from 13 high fertility and 13 low fertility bulls . In high fertility bulls, as fertility decreased, TH2B expression increased  signifying that the bulls with the highest fertility had the least TH2B retention in their spermatozoa. In low fertility bulls, as fertility decreased, TH2B expression decreased  has been used in our study, because bovine TH2B gene as well as TH2B protein have not been sequenced yet. Therefore, the predicted gene HIST1H2BA (TH2B) had 85.7% similarity with known bovine H2B, using multiple sequence alignment method Clustal Omega  [Histone variant of TH2B was identified as the testis specific H2B , 36. TH2HIST1H2B . Mouse Tequenced , and a pustalo/) . Compariustalo/) .Fig. 6ThWe used the STRING database to depict the interactions of sperm proteins in human, mice, and bovines respectively. Analyses of the interactions of nuclear proteins of developed using STRING for human, mouse, and bovine Fig.  showed tP < 0.05; Table. The interactome networking of bovine TH2B with nuclear proteins  showed the significant gene ontology overexpression terms . The expression levels of TH2B decreased in sperm from high fertility bulls as fertility score increased (P = 0.0055) and increased in low fertility bulls but was not significant (P = 0.0737). Bovine TH2B and H2B sequences were highly similar (85.71%), as revealed by percent identity matrix (PIM) score. The H2B Emboss dotmatcher showed that the predicted bovine TH2B sequence is similar to TH2B of human, which has few possible frame shift mutation sites. Bovine H2B and human H2B have a remarkably perfect diagonal line match, revealing that H2B is highly conserved. STRING database [Our study presents evidence that bovine TH2B was localized in the sperm head. The intensity of TH2B in sperm from high fertility bulls decreased as fertility increased (database ) was empth2b gene in the mouse resulted in absence of TH2B protein, causing sterility in mouse [Since TH2B plays an important role in eviction of canonical histones and replacement of TNPs with protamines in the mouse , disruptin mouse . HoweverOur results shed light on the role of TH2B in shaping bovine sperm chromatin and interacting with chromatin remodeling proteins BRD4 and CHD5. The intensity as well as expression levels of TH2B were significantly lower in sperm from high fertility group, paving the way for increased incorporation of protamines into the matured sperm chromatin resulting into increased number of healthy sperm. However, this trend was completely reversed in low fertility group; higher TH2B retention resulting in less protamine incorporation into chromatin suggesting loosening of sperm chromatin and higher numbers of immature sperm. Another possibility is that eviction of the canonical histones by TH2B might be hindering the protamine incorporation to a certain level because of abnormal retention TH2B. This is consistent with the findings of Dogan et al. (2015) , who shohttp://www.ncbi.nlm.nih.gov/nuccore/XM_010825421.2) because the actual bovine sequences have not yet been reported to NCBI. However, an 85% homology between human sperm TH2B and somatic H2B has been clearly demonstrated through laboratory experiments [The sample size in our immunocytochemistry analysis limited the statistical power needed to detect significant differences between the groups of bulls with high and low fertility. In addition, TH2B fluorescence signal was faint in the sperm tail, probably because TH2B was not involved with sperm chromatin . In thiseriments . MoreoveIn the bull, sperm chromatin dynamics was thought to be controlled by replacement of histones by protamines. This is a multi-step process involving chromatin remodelers BRD4, CHD5 and TH2B with a scope of many additional nuclear chromatin associated proteins. Therefore, the mechanisms of histone to protamine replacement are multi-cascade reactions, in which different chromatin remodelers such as BRD and CHD family proteins are involved in loosening the DNA nuclear protein binding in sperm chromatin. The results presented in our study advance the understanding of the role of TH2B in remodeling sperm chromatin dynamics.Presence of TH2B variant in bull sperm initiates replacement of histones by TNPs and then by protamines, all of which are in accordance with the prior findings about the function of TH2B in the mouse , 8, 19.


To determine the influence of serum uric acid (UA) levels on renal impairment in patients with UA stone.We retrospectively analyzed 463 patients with calcium oxalate and/or calcium phosphate stones (CaOx/CaP), and 139 patients with UA stones. The subjects were divided into the serum UA-high (UA ≥ 7.0 mg/dL) or the UA-low group (UA < 7.0 mg/dL). The control group comprised 3082 community-dwelling individuals that were pair-matched according to age, sex, body mass index, comorbidities, hemoglobin, serum albumin, and serum UA using propensity score matching. We compared renal function between controls and patients with UA stone , and between patients with CaOx/CaP and with UA stone . Logistic regression analysis was used to evaluate the impact of the hyperuricemia on the development of stage 3 and 3B chronic kidney disease (CKD) .The renal function was significantly associated with serum UA levels in the controls and patients with CaOx/CaP and UA stones. In pair-matched subgroups, patients with UA stone had significantly lower renal function than the control subjects  and patients with CaOx/CaP stones  regardless of hyperuricemia. Multivariate logistic regression analysis revealed that patients with UA stone, CaOx/CaP, hyperuricemia, presence of cardiovascular disease, higher body mass index, older age and lower hemoglobin had significantly higher risk of stage 3 and 3B CKD .Patients with UA stones had significantly worse renal function than controls and CaOx/CaP patients regardless of hyperuricemia. Urolithiasis (CaOx/CaP and UA stone) and hyperuricemia had an association with impaired renal function. Our findings encourage clinicians to initiate intensive treatment and education approaches in patients with urolithiasis and/or hyperuricemia in order to prevent the progression of renal impairment. The prevalence of urolithiasis has been increasing in Japan, similar to other developed countries . UrinaryUrolithiasis is reported to be associated with metabolic syndrome (MetS) . MetS isThe study was conducted in accordance with the ethical standards of the Declaration of Helsinki and was approved by the Ethics Committee of Hirosaki University Graduate School of Medicine (authorization number 2016–225). For this type of retrospective study, formal patient consent is not required. The cross-sectional data collection from the Iwaki Health Promotion Project was approved by the Ethics Committee of Hirosaki University School of Medicine (authorization number 2014–015), and all of the subjects provided written informed consent before participating in the study. This study was registered as a clinical trial UMIN000022962.Between January 2010 and September 2015, 1319 patients with urolithiasis were treated at the Oyokyo Kidney Research Institute and Hirosaki University Hospital. Patients with infection stones, and patients lacking sufficient clinical data regarding urinary stones and blood examinations were excluded from the study. Ultimately, 602 patients who underwent laboratory testing to evaluate renal function, serum UA levels, and lipid metabolism were included in this retrospective study. Among them, 463 patients with CaOx/CaP and 139 patients with UA stones were identified. The patients were divided into two groups according to serum UA levels: the UA-high group with hyperuricemia (serum UA ≥ 7.0 mg/dL) or the UA-low group with normal UA levels (serum UA < 7.0 mg/dL) groups. The 3082 community-dwelling control subjects (Ctrl) were selected from the Iwaki Health Promotion Project. The Iwaki Health Promotion project was a comprehensive study for clarification of etiology for lifestyle-related diseases such as hypertension, cardiovascular diseases, atherosclerosis, chronic renal failure, osteoporosis, arthritis, asthma, cancer, chronic liver disease or cirrhosis, chronic obstructive pulmonary disease, type 2 diabetes, obesity, metabolic syndrome, dementia, Alzheimer’s disease and depression. It was planned to prevent lifestyle-related diseases and promote health to extend the life span of residents of Hirosaki city (Iwaki district), a northern part of Japan in collaboration with Hirosaki University, Hirosaki City, and Aomori Prefecture general screening center. This project conducted comprehensive screening of general health status including comprehensive biological and physical examinations.4MgPO4], and cystin). CVD was defined as a positive history of cardiac surgery, angina, myocardial infarction, or stroke or taking any cardiotonic agents or coronary vasodilators. Estimated glomerular filtration rate (eGFR) was used as an index of renal function, and it was calculated using the Modification of Diet in Renal Disease equation for Japanese patients [2, respectively.The analyzed pre-treatment variables were age, sex, body mass index, history of hypertension (HTN), blood pressure, diabetes mellitus (DM), cardiovascular disease (CVD), renal function, hemoglobin, serum albumin (Alb), serum UA, dyslipidemia  and type of stones  or Mann-Whitney U-test . The correlation between two parameters was analyzed using Spearman’s correlation coefficient. Probability (P) values < 0.05 were considered statistically significant.Statistical analyses were conducted using SPSS v. 24.0  and GraphPad Prism v. 5.03 . Categorical variables were presented as percentages and compared using Fisher’s exact test or Chi-square test. Quantitative data were expressed as the mean ± standard deviation. Differences between groups were compared using Student’s To select the appropriate control subjects from the 3082 community-dwelling individuals, we compared renal function between control subjects and patients with UA stones , and between CaOx/CaP and patients with UA stones  using propensity score matching as previously described . Propens2 values showed higher correlation in UA stone patients  than that in the Ctrl  or in the CaOx/CaP or   .The number of CaOx/CaP patients with stage 3 CKD were not significantly different between the UA-low and UA-high . The num2) compared with control subjects (71 ± 17 mL/min/1.73m2) (P = 0.007) (P = 0.005)The UA-low group comprised 92 control subjects and pair-matched 46 patients with UA stones. There were no statistically significant differences in background between UA stone patients and control subjects . However= 0.007) . The pre2) compared with control subjects (66 ± 19 mL/min/1.73m2) (P < 0.001) (P = 0.020).The UA-high group comprised 21 patients with UA stones and 42 pair-matched control subjects. There were no statistically significant differences in background between UA stone patients and control subjects . However< 0.001) . The pre2) compared with CaOx/CaP (66 ± 20 mL/min/1.73m2) (P = 0.034) (P = 0.414).The UA-low group comprised pair-matched 75 patients with CaOx/CaP and 75 patients with UA stones. There were no statistically significant differences in background between UA stone patients and CaOx/CaP patients . The eGF= 0.034) . However2) compared with CaOx/CaP (60 ± 25 mL/min/1.73m2) (P = 0.032) (P = 0.028).The UA-high group comprised 21 patients with CaOx/CaP and pair-matched 21 patients with UA stones. There were no statistically significant differences in background between UA stone patients and control subjects . The eGF= 0.032) . The preIndependent risk factors for development of stage 3 and 3B CKD in the 3082 control subjects and 602 stone patients was evaluated using uni- and multivariate logistic regression analyses including 12 variables. In univariate analysis, all variables were selected as independent risk factors for development of stage 3 and 3B CKD. UA stone , CaOx/CaP stone , and hyperuricemia  persisted as an independent risk factors for stage 3 CKD after multivariate logistic regression analysis accounting for the strong confounders such as age, body mass index, CVD, and hemoglobin , Table 4Hyperuricemia is a significant risk factor for urolithiasis and CKD , but it Although hyperuricemia is a well-known risk factor for urolithiasis and renal impairment , 18, theIn the present study, we found that an UA stone patient have impaired renal function even with a normal serum UA level . AlthougChronic asymptomatic hyperuricemia  may promote development of CKD and formation of UA stones. However, the lack of biomarkers for UA accumulation within the body remains an unmet need in patients with hyperuricemia. As efforts to improve chronic asymptomatic hyperuricemia are applied to patients with gout, this might be also a valuable therapeutic approach for patients with a combination of UA stones, insulin resistance, and MetS . Optimal2 to define CKD was also the limitation of the present study. Because the definition of CKD is the presence of GFR < 60 mL/min/1.73m2 for 3 months, eGFR evaluation at one point in time does not allow for conclusions regarding whether there is CKD or not. Even though the OR for UA stones was higher than hyperuricemia, it also had the widest 95%CI (2.93–4.74). Therefore, the reason why UA stone patients have impaired renal function remains unsolved from the present study. Despite these limitations, this is the first report to assess the implication of serum UA levels on renal impairment in patients with UA stones. In addition, the current evidences suggested the influence of MetS on UA stones and CKD. Therefore, it is necessary to improve MetS in patients with UA stones in order to prevent the progression of renal impairment.Several limitations in this study should be noted. The limited sample size and retrospective study design prevented us from making definitive conclusions regarding the effect of serum UA levels on renal impairment in patients with UA stones. Furthermore, we were unable to control selection bias and other potentially confounding factors in UA stone patients or control subjects, despite the use of controlled matching methods. Our results did not account for some other factors known to influence renal function, including purine diet, the use of niacin, the amount of alcohol consumed or the amount of high fructose containing beverages, cigarette smoking, blood pressure control, background medications, proteinuria, and hydronephrosis. A history of urinary tract infections and/or administration of agents that are potentially renoprotective, or may alter plasma or urine uric acid excretion also may have an influence on renal function. The use of the Modification of Diet in Renal Disease equation for Japanese patients and using a cut-off of < 60 mL/min/1.73mPatients with UA stones had significantly worse renal function than controls and CaOx/CaP patients regardless of hyperuricemia. Urolithiasis (CaOx/CaP and UA stone) and hyperuricemia had an association with impaired renal function. Our findings encourage clinicians to initiate intensive treatment and education approaches in patients with urolithiasis and/or hyperuricemia in order to prevent the progression of renal impairment.S1 Dataset(XLSX)Click here for additional data file.


Mycobacterium llatzerense was previously described as frequently associated with FLA in a drinking water network. The present study aimed to characterize the interactions between M. llatzerense and FLA. M. llatzerense was internalised by phagocytosis and featured lipid inclusions, suggesting a subversion of host resources. Moreover, M. llatzerense survived and even multiplied in presence of A. castellanii. Using a genomic-based comparative approach, twelve genes involved in phagocytosis interference, described in M. tuberculosis, were identified in the M. llatzerense genome sequenced in this study. Transcriptomic analyses showed that ten genes were significantly upregulated during the first hours of the infection, which could partly explain M. llatzerense resistance. Additionally, M. llatzerense was shown to actively inhibit phagosome acidification. In conclusion, M. llatzerense presents a high degree of resistance to phagocytosis, likely explaining its frequent occurrence within FLA in drinking water networks. It underscores that NTM should be carefully monitored in water networks to prevent human health concerns.Nontuberculous mycobacteria (NTM) are environmental bacteria increasingly associated to public health problems. In water systems, free-living amoebae (FLA) feed on bacteria by phagocytosis, but several bacteria, including many NTM, are resistant to this predation. Thus, FLA can be seen as a training ground for pathogenic bacteria.  Mycobacterium is a genus that is composed of at least 170 species, among which are found highly pathogenic mycobacteria, such as M. tuberculosis and M. leprae, causing tuberculosis and leprosy, respectively. Besides these pathogens causing major health concerns for humans and cattle, are nontuberculous mycobacteria (NTM), a vast group encompassing all other species from this genus. NTM are ubiquitously found in environments such as soil and water4579Legionella pneumophila1213in vitro, as well as some NTM species, being thus considered as ARB16171819in vitro within Acanthamoeba spp.Mycobacterium species were studied for deciphering cellular processes involved in phagocytosis resistance, although most of the existing literature is focused on the highly pathogenic M. tuberculosis, as recently reviewedM. tuberculosis222324Mycobacterium marinum as a surrogate for the pathogenic M. tuberculosis bacilli. In this context, it was shown that the type 7 secretion system variant ESX-1 was required for intracellular thriving, when infecting Acanthamoeba26Mycobacterium avium, the major slowly-growing species involved in NTM diseasesM. aviumMycobacterium abscessus, the major rapidly growing mycobacteria involved in human diseases30M. abscessus whole genome highlighted the presence of a horizontally acquired phospholipase C (PLC), that was involved in intracellular proliferation within A. castellaniiSeveral M. llatzerense as a predominant NTM co-occurring with FLAM. mucogenicum group, has been repeatedly identified within drinking water networks34353637M. llatzerense was recently reported in immunocompromised patients, raising the possibility that it may be an opportunistic pathogen as well3940We recently reported the frequent occurrence of FLA-NTM association in the drinking water network of Paris, France, and identified M. llatzerense and FLA, and hypothesized that this species was able to resist amoebal phagocytosis. Thereby, our work aimed to understand how M. llatzerense is coping with the amoeba model Acanthamoeba castellanii. This FLA species was chosen in this study because it is among the most frequently encountered FLA in the drinking water systems, as well as in association with mycobacteria9M. llatzerense to persist and multiply in presence of A. castellanii. The subcellular localisation of M. llatzerense within the amoeba host was characterised. A comparative genomics approach, using newly sequenced M. llatzerense genome, was used to identify conserved genes based on their role in mycobacterial resistance to phagocytosis. The involvement of these genes was subsequently monitored, by quantifying transcription levels during the infection process. Finally, an investigation at the phenotypic level allowed us to quantitatively evaluate the presence and survival of M. llatzerense within intracellular acidic compartments.In order to explain why and how this NTM co-exists with FLA in water networks, we investigated the interactions of Mycobacterium llatzerense (80% to 90% of identified NTM sequences) in association with FLA isolated from a drinking water networkM. llatzerense interactions with Acanthamoeba castellanii. In this study, environmental isolates of M. llatzerense were collected from endpoint sites of the above-mentioned drinking water network, provided by treated groundwater. Their resistance to A. castellanii was determined through a droplet test, for comparing the ability of bacterial isolates to grow in presence or absence of A. castellanii seeded on the agar medium. This ability was assessed for five M. llatzerense environmental isolates, as well as a strain of Mycobacterium septicum, moderately resistant to A. castellanii predation, and isolated from the same drinking water network. E. coli was used as a control, as it is a commonly used food source for FLA  (To confirm ter 16 h . This inocytosis . A signi t-test) .M. llatzerense and A. castellanii, infected amoeba cultures were observed using transmission electron microscopy (TEM). M. llatzerense bacilli were mostly found intracellularly in vacuoles, which suggested an active internalisation process , while harbouring a similarly high GC content of 66.6%, versus 66.3% for M. llatzerense CLUC14. Further analyses between the two assemblies indicated an average nucleotide identity of 98.12%, hence confirming they belong to the same species ; mutant deleted for these genes were significantly enriched in acidified phagosomeM. tuberculosis was shown to block phagosome maturation in murine macrophages through disruption of Rab5 and Rab7 GTPases, thus blocking phagosomes fusion with endosomal and lysosomal compartmentsM. tuberculosis infecting macrophages. PhoP and FbpA were both indirectly involved in blocking phagosomal maturation, as they regulate independently other virulence factors such as type 7 secretion systems (PhoP) or the cell wall lipid composition (FbpA)23M. llatzerense genome, whose heterodimeric effector EsxG-EsxH was recently involved in disruption of M. tuberculosis-containing phagosomes addressing to the lysosomeAmong an initial set of thirteen coding genes, eleven highly similar, as well as one moderately similar genes were identified in ed below . Compara4 genome . Rv3707cM. llatzerense resistance to A. castellanii, their expressions were quantified by qRT-PCR at 0 h, 2 h, 4 h and 8 h post infection, corresponding to the early stage of M. llatzerense internalisation . After 2 h, only three of the selected genes were significantly upregulated, corresponding to secA2 (8.23 folds), fbpA (7.28 folds) and phoP (6.23 folds). After 4 h, significant upregulations of transcription levels were observed for 8 other genes, namely cut2, esxG, fbpA, phoP, ptpA, ptpB, rv3707c and secA2. After 8 h, all genes except glyA1 and ppe10 were significantly upregulated. Only genes that were upregulated after 2 h reached more than 10-times fold changes . These results, coupled with previous observations at the transcription level, strongly suggest that M. llatzerense is actively blocking phagosomal maturation, ultimately resulting in M. llatzerense persistence in poorly acidified compartments.In addition, the characterisation of abelling . In contMycobacterium genus have been repeatedly reported to develop resistance to FLA predation1619M. llatzerense was recurrently found in drinking water, and in association with FLA. Therefore, we undertook the characterisation of resistance mechanisms employed by M. llatzerense to A. castellanii phagocytosis.NTM are frequently found in aqueous environments along with FLA, facing therefore a potentially strong predatory pressure, which suggests that NTM resistance stand as an evolutionary advantage to persist in such environments71048M. llatzerense to persist and even grow in presence of FLA was described. As for other mycobacteria, A. castellanii internalised M. llatzerense through phagocytosis. Once internalised, M. llatzerense were found in vacuoles corresponding to phagosomes. The observed infectivity of M. llatzerense was comparable to those of M. marinum and M. avium infecting Acanthamoeba spp., as reported previously26M. llatzerense growth in presence of A. castellanii was comparable to this of M. avium infecting the same FLA host, while higher than the growth observed for M. fortuitum and M. marinum52In the present study, the ability of M. llatzerense harboured many intracytoplasmic lipid inclusions. Lipid bodies can represent a form of energy storage, but are also thought to be involved in host inflammatory and immune responsesM. marinum was able to actively subvert host lipid bodies when infecting D. discoideumMycobacterium.Interestingly, intracellular M. llatzerense resistance to A. castellanii, a comparative genomic approach was undertaken. M. llatzerense whole genome sequencing revealed a large-sized 6.7 Mb genome, which is in accordance with its environmental and generalist lifestyleM. tuberculosis H37rv  synthesis, an important surface glycolipid also designated as “cord factor”fbpA might lead to an enrichment of TDM, which was correlated with M. tuberculosis intracellular survivalphoP gene in M. llatzerense could suggest a role in blocking phagosome acidification. To support this hypothesis, M. tuberculosis mutants deficient for PhoP expression failed at blocking phagosome acidification, while still able to prevent phagolysosomal fusionsecA2 gene showed one of the earliest and strongest overexpression following internalisation. This gene, as part of the sec system, was recently suggested to be involved in secretion of acid phosphatase PtpA and PtpB 22M. llatzerense the upregulation of SecA2 transcription was parallel to those of PtpA and PtpB, which is in accordance with the proposed hypothesis. Indeed, a thorough investigation will be required to confirm the role of the Sec system in PtpA and PtpB secretion. Transcription analysis of ndk highlighted a late uperegulation. In M. tuberculosis, NdK was shown to inhibit phagosomal fusion with early endosomes and lysosomesEsxG and EsxH transcription, which were significantly upregulated at 4 h and 8 h post infection, respectively. These genes, encoding an ESX3 heterodimeric effector, were linked to the inhibition of phagolysosome fusion5859Cut2 and Rv3707c transcription was also significantly upregulated at 4 h post infection, however their roles are not yet clearly elucidatedIn order to explore the genomic basis of glyA1 and ppe10 were not differentially transcribed throughout the experiment. The roles for these genes have been identified by the characterisation of mutants unable to prevent phagosomal acidificationMycobacterium pathogenicityM. llatzerense seemed surprising. It is however worth noting that only a moderate sequence similarity (40% identity) was identified between PPE10 from M. tuberculosis and the corresponding protein in M. llatzerense. Furthermore, M. llatzerense EDP_4 was shown to harbour a total of 27 PE-PPE genes in its genome. It would be thus relevant to analyse the expression of these other genes during infection, in order to evaluate their importance in FLA phagocytosis resistance. So far the secreted hydroxymethyltransferase GlyA1 has only been described in M. bovis BCG for blocking phagosome maturation. As no significant difference in expression of GlyA1 was observed in our study, we cannot confidently conclude on its role in the survival of M. llatzerense infecting A. castellanii. Transcription analyses indeed provide strong arguments in favour of the involvement of significantly modulated genes. However, it has to be kept in mind that functional analyses are required to confirm the importance of such genes in M. llatzerense resistance to A. castellanii phagocytosis, such as phenotypic characterisation of M. llatzerense survival within FLA when impaired for selected gene’s expression, through mutagenesis or interference experiments.In contrast to the significant changes observed in transcription levels of the other selected genes, both M. llatzerense – infected A. castellanii resulted in a significant decrease of bacterial survival. However, efficient M. llatzerense killing appeared nuanced, as 50% of intracellular mycobacteria were still viable after autophagy induction. The partial survival of M. llatzerense following autophagy activation might reflect its inherent resistance to advert conditions such as acidic and oxidative stresses. This finding is in accordance with a previous study describing M. avium ability to multiply within phagolysosomes, while M. tuberculosis was only able to persist in such conditionM. llatzerense to actively block A. castellanii phagocytosis process, and corroborate the transcription profiles of the analysed genes.To complete transcription analyses, an investigation at the phenotypic level was performed. Most notably, a particular focus was given on the potential role of the autophagic machinery in modulating the infection. Autophagy, firstly described as a process maintaining cellular homeostasis through nutrients and organelles recycling, is also characterised as an antimicrobial mechanism, allowing the degradation of intracellular pathogensM. llatzerense, an environmental NTM, to resist A. castellanii predation. M. llatzerense was shown to infect efficiently A. castellanii through phagocytosis, remained viable and was able to grow for up to 96 h. M. llatzerense intracellular localisation within A. castellanii indicated persistence in vacuoles, as well as a potential subversion of host lipid metabolism. A comparative genomic approach identified twelve conserved virulence factors, shared between M. llatzerense and M. tuberculosis. Analyses of transcriptional profiles highlighted that all selected genes except glyA1 and ppe10 were significantly upregulated during the early stage of A. castellanii infection. This led to suggest that M. llatzerense is very likely able to actively hamper phagosomal acidification, while resisting to autophagy-induced advert conditions. Taken together, this study clearly defined M. llatzerense as an amoeba resistant bacteria. M. llatzerense was shown to establish an elaborate and efficient strategy not only to survive but to take advantage as well of the presence of FLA for its growth. This capability might very well explain the frequent recovery of this NTM in several drinking water networks.In conclusion, the work presented here provides insights into mechanisms employed by Escherichia coli strain K-12 strain was grown and maintained on LB-agar  plates at 37 °C. Acanthamoeba castellanii strain ATCC 30234 was cultured axenically in PYG medium 2(SO4)2, pH 6.5) at 30 °C.NTM were isolated from Paris drinking water network. Briefly, water samples (1 L) were filtered through a 0.45 μm cellulose membrane, sonicated for 10 minutes, and decontaminated using 3% lauryl sulfate and 1% NaOH, as described previouslyA. castellanii monolayers, grown in PYG medium for 3 days, were collected, washed twice using PAS buffer , adjusted to a final concentration of 2.5 × 105 cells/mL and 1 mL was distributed in each well of a 12-wells plate. Mycobacteria grown for 4 days (i.e. early stationary phase) were used to infect A. castellanii at a multiplicity of infection (MOI) of 1 or 10 at 30 °C.6A. castellanii trophozoïtes on Middlebrook 7H10 agar supplemented with 10% (v/v) OADC (120X120 mm Petri dishes). Mycobacteria suspensions were prepared in PAS buffer, supplemented with 0.005% triton X-100 to avoid clumping, and homogenised by three passages using a 27 gauges needle. The suspension was spotted (from 1 × 108 to 1 × 103 bacteria/mL) on agar medium in presence or absence of A. castellanii as prepared previously, and incubated at 30 °C or 37 °C for optimal bacterial growth.Droplet tests were performed by seeding homogeneously 4.10A. castellanii monolayers in 12-wells plates were infected at a MOI of 1 for 16 h. Supernatant was discarded, cells washed twice and resuspended in 1 mL of fresh PAS buffer at 0, 24, 72 and 96 hours. Trophozoites were lysed and mycobacteria enumeration was performed as described above.For the assessment of mycobacteria viability, A. castellanii monolayers in 12-wells plates were infected at a MOI of 1. Trophozoites were washed three times using PAS buffer before processing. Cells were centrifuged at 800 g for 5 minutes, supernatant was discarded, and the pellet resuspended in 50 μL of PAS, spread on a glass slide, heat-fixed and processed for Ziehl-Neelsen staining. Percentages of infected amoebae were calculated on the basis of at least 200 amoebae cells par slides, in a minimum of five different fields. In order to determine internalisation mechanisms of M. llatzerense, A. castellanii monolayers in 12-wells plates were infected at a MOI 10 for 4 h, in co-incubation with cytochalasin D at 100 μM in PAS buffer. For induction of autophagy A. castellanii trophozoïtes were infected at a MOI of 10 for 4 h, and treated for 1 h using 1 μM or 10 μM rapamycin in PAS.For evaluating the number of infected cells, M. llatzerense suspension prepared as described previously were pre-labelled using pHrodoTM Red succinimidyl ester (ThermoFischer Scientific) following manufacturer recommendations, but excluding the methanol washing step. Briefly, bacterial suspensions were incubated for 1 h in 0.1 M sodium bicarbonate buffer containing 20 μM pHrodoTM Red succinimidyl ester, in the dark. Bacteria were then pelleted by centrifugation at 12000 g for 5 min, and washed twice in PAS buffer supplemented with 0.005% triton X-100. In one condition, mycobacteria were killed before the initial incubation by heating the suspension at 85 °C for 15 min, in order to assess the importance of bacterial viability in subsequent analyses. A. castellanii monolayers were infected by either alive of heat-killed labelled mycobacteria at a MOI of 10, in 6 wells plates. After 2 h of incubation, trophozoites were detached, fixed with 2% paraformaldehyde for 15 min in the dark. Cells were pelleted by centrifugation at 800 g for 10 min, and resuspended in a 30 μL of SlowFade Diamonds antifade mountant (ThermoFischer Scientific) with Hoechst at a concentration of 400 ng/mL. Samples were examined using a confocal laser scanning microscope . To estimate the fraction of M. llatzerense residing in acidified phagosomes, a ratio was calculated based on the total number of bacteria (as detected by Hoechst staining) divided by the number of bacteria being labelled as well by pHrodo, which fluoresces only in acidic environment.M. llatzerense colony was suspended in phosphate buffer saline, subsequently bead-beaten in tubes containing 500 mg of small diameter glass beads (100 μm) and 4 glass beads of 2 mm diameter (Sigma) using Fastprep apparatus for 30 s, put on ice for 1 min, and bead-beaten again using the same parameters. The suspension was then processed for DNA extraction using NucleoSpin Tissue kit (Macherey-Nagel), following manufacturer recommendations for bacterial DNA extraction.Isolated ® reagent (Life Technology) was added to each sample, and incubated at room temperature for 5 min. 100 μL of chloroform/isoamyl alcohol (24:1 v/v) was added to each sample, shaken vigorously, incubated for 1 min and centrifuged for 5 min at 14000 g. Aqueous phase was transferred into a new tube, treated again with 200 μL of chloroform/isoamyl alcohol as described above. The collected aqueous phase was washed with 500 μL of isopropanol for 1 h on ice and centrifuged at 16000 g for 30 min. Supernatants were discarded and pellets washed with 1 mL of 70% ethanol, centrifuged 15 min at 16000 g. Pellets were air dried and suspended 50 μL of ultrapure water, 7.5 mM sodium acetate, and 150 μL of absolute ethanol. Samples were incubated at −20 °C overnight and centrifuged for 30 min at 16000 g. Pellets were rinsed as described above, let air dried, and suspended in 30 μL of ultrapure water. RNA samples were subsequently treated using TURBOTM DNase (Life Technologies) according to manufacturer recommendations, purified once again using phenol/chloroform and quantified by spectrophotometry. RNA were aliquoted, stored at −80 °C, and one aliquot of each condition was used immediately for reverse transcription, using GoScriptTM Reverse transcription System (Promega). The resulting cDNA were used for downstream quantification by qPCR.For RNA extraction cells were collected, suspended in 400 μL of resuspension solution  and transferred to a tube containing 400 mg of small diameter glass beads (100 μm) and 500 μL of acid phenol. Samples were bead-beaten as described above and centrifuged for 5 min at 14000 g, 4 °C. Supernatants were kept on ice and transferred into clean tubes. 1 mL of TRIzolloci from total DNA were done by PCR. Mixtures were prepared in a final volume of 25 μL, containing 5 μL of 5x GC buffer, 400 nM of each deoxynucleotide triphosphate, 1,5 mM MgCl2, 500 nM of each primer, 0.5 U of Phusion polymerase (Thermo Scientific), 2.5 μL of template DNA and PCR grade water in sufficient quantity for 25 μL. Primers U341F (5′-CCTACGGGAGGCAGCAG-3′) U926R (5′-CCGTCAATTCMTTTRAGT-3′) and Myco F (5′-GGCAAGGTCACCCCGAAGGG-3′) Myco R (5′-AGCGGCTGCTGGGTGATC-3′) were used for partial amplification of 16 S and rpoB genes, respectively® Green PCR Master Mix (Life Technologies), 250 nM of each primer, 2 ng of cDNA, and molecular grade water in sufficient quantity for 20 μL. Experiments were carried out in a Viia7 real time thermocycler (Life Technologies), consisting in an initial denaturation step at 95 °C for 10 min, 45 cycles of denaturation at 95 °C for 15 sec, annealing for 15 sec at 63 °C to 65 °C, and elongation at 72 °C for 20 sec. Fusion curves were collected for each experiments. Quantification of transcripts were performed by calculating fold changes using 2−ΔΔCt method, corrected by qPCR efficiency for each gene, and normalised to the control condition at 0 h, as well as the control gene expression of 16S rRNAAmplifications of targeted rpoB sequencing, total DNA were processed for whole genome sequencing, using GS junior 454 pyrosequencer (Roche). Library preparation, emulsion PCR and sequencing were performed following manufacturer’s protocols. Raw sequences were then assembled using GS denovo Assembler software (Roche). The assembly was uploaded for annotation using RAST online annotation tool68Once identification and purity of mycobacterial isolates were confirmed by both 16 S and M. llatzerense inside A. castellanii was investigated using transmission electron microscopy following the protocol described inM. llatzerense at a MOI of 1, and fixed in 3% glutaraldehyde and osmium tetroxide. Cells were dehydrated in successive baths of 50%, 70%, 90% and 100% acetone. Cells were embedded in araldite resin, and ultrathin section of 60 nm were cut and stained with uranylacetat and lead salts according to Reynolds. Ultrathin sections were observed using JEOL 1010 transmission electron microscope operating at 75 kV.The localisation of How to cite this article: Delafont, V. et al. Mycobacterium llatzerense, a waterborne mycobacterium, that resists phagocytosis by Acanthamoeba castellanii. Sci. Rep.7, 46270; doi: 10.1038/srep46270 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Gynecologic malignancies account for 1.09 million new cancer cases worldwide and forabout 12% of the tumors affecting females. Approximately 10% of all female cancersurvivors are aged 40 years or younger. Since cancers affecting female genital organsare usually treated with radical surgery, chemotherapy or chemoradiotherapy - approachesthat induce permanent reproductive function impairment - the development of strategiesto preserve fertility is one of the most important goals in gynecologic oncology , human oocytes can be cryopreserved withexcellent survival and pregnancy rates. Research has proven that oocyte vitrification issafe and offers women a new way to preserve fertility. In 2012, the American Society forReproductive Medicine (ASRM) stated that egg vitrification was no longer considered anexperimental procedure .Oocyte vitrification is an elective medical procedure used to successfully cryopreservehuman oocytes. The age at which patients usually undergo oocyte vitrification rangesfrom 21 to 39 years of age. In fertility preservation, the younger a woman is when shehas her eggs vitrified, the better are her chances of having a successful pregnancy at alater time. Aging negatively impacts a woman's fertility. The decrease in fertility islargely explained by increased levels of aneuploidy  in humanoocytes, which contributes to infertility, miscarriage and genetic anomalies in liveborn infants.Fertility preservation processes are very similar to in-vitro fertilization. Femalepatints are screened prior to treatment to ensure they are good candidates for oocytevitrification. Follicle-stimulating hormone (FSH) levels, antral follicle counts (AFC),and antimullerian hormone (AMH) levels are measured on day three of the menstrual cycle.Medical and special needs are addressed. The ovaries are stimulated with gonadotropininjections to support the development of multiple follicles, and ovulation is blockedwith either a GnRH agonist or antagonist. The ovaries are usually stimulated for about10 days, and follicular development is monitored with regular transvaginal ovarianultrasound examination and blood work. Mature oocytes are then removed from thefollicles by transvaginal ultrasound-guided oocyte aspiration. Good quality oocytes arevitrified and stored in liquid nitrogen (at -196 °C) for future use. Vitrified oocytescan be stored safely for many years.Interestingly, a close friend told me that she offered to pay for the cryopreservation ofthe eggs of her daughters as a gift for their thirtieth birthdays. As a parent of ateenage daughter myself, this idea intrigued me and I thought long and hard about whatnew technology will be available when my daughter turns thirty. I feel confident thatthe field of fertility preservation is going to keep advancing in new and exciting ways,resulting in new procedures to help successfully beat the pressures of the biologicalclock.


A most important property of biochemical systems is robustness. Static robustness, e.g., homeostasis, is the insensitivity of a state against perturbations, whereas dynamics robustness, e.g., homeorhesis, is the insensitivity of a dynamic process. In contrast to the extensively studied static robustness, dynamics robustness, i.e., how a system creates an invariant temporal profile against perturbations, is little explored despite transient dynamics being crucial for cellular fates and are reported to be robust experimentally. For example, the duration of a stimulus elicits different phenotypic responses, and signaling networks process and encode temporal information. Hence, robustness in time courses will be necessary for functional biochemical networks. Based on dynamical systems theory, we uncovered a general mechanism to achieve dynamics robustness. Using a three-stage linear signaling cascade as an example, we found that the temporal profiles and response duration post-stimulus is robust to perturbations against certain parameters. Then analyzing the linearized model, we elucidated the criteria of when signaling cascades will display dynamics robustness. We found that changes in the upstream modules are masked in the cascade, and that the response duration is mainly controlled by the rate-limiting module and organization of the cascade’s kinetics. Specifically, we found two necessary conditions for dynamics robustness in signaling cascades: 1) Constraint on the rate-limiting process: The phosphatase activity in the perturbed module is not the slowest. 2) Constraints on the initial conditions: The kinase activity needs to be fast enough such that each module is saturated even with fast phosphatase activity and upstream changes are attenuated. We discussed the relevance of such robustness to several biological examples and the validity of the above conditions therein. Given the applicability of dynamics robustness to a variety of systems, it will provide a general basis for how biological systems function dynamically. Cells use signaling pathways to transmit information received on its membrane to DNA, and many important cellular processes are tied to signaling networks. Past experiments have shown that cells’ internal signaling networks are sophisticated enough to process and encode temporal information such as the length of time a ligand is bound to a receptor. However, little research has been done to verify whether information encoded onto temporal profiles can be made robust. We examined mathematical models of linear signaling networks and found that the relaxation of the response to a transient stimuli can be made robust to certain parameter fluctuations. Robustness is a key concept in biological systems—it would be disastrous if a cell could not operate if there was a slight change in its environment or physiology. Our research shows that such dynamics robustness is a property of linear signaling cascades, and we outline the design principles needed to generate such robustness. We discovered that two conditions regarding the speed of the internal chemical reactions and concentration levels are needed to generate dynamics robustness. In general, it is the ability of an organism to maintain a state or behavior against external or internal perturbations, and many frameworks of robustness have emerged –7. Homeodynamics robustness, i.e., robustness in the temporal course, is little understood as compared with extensive studies on static robustness. Here we study dynamics robustness, the insensitivity of transients to initial conditions or parameters. We adopt the term dynamics robustness as opposed to dynamic or dynamical robustness since those terms have been defined elsewhere in a different context. For example in . If this measure is less than 0.3, then we consider the temporal profiles to be robust against perturbations. We stress again that 0.3 was chosen arbitrarily, but any reasonable threshold will work. We performed a similar analysis for β1 and β2 in β0 and β2. . However, the second module is sensitive to perturbations in the phosphatase activity.In ith layer is too low, then the logarithmic gain of the duration against βi is roughly −1, i.e., the duration of the response is strongly dependent on the rate-limiting module in the cascade. In βi becomes less than all other β values, and the logarithmic gain indeed becomes −1 near this point. However, the upper bound for the phosphatase concentration that exhibits duration robustness cannot be described by the rate-limiting effect only.In all three cases, there are common features in the plots of the duration. As mentioned earlier, if the phosphatase activity in any layer is too high, then the system is in an inactivated state, which is colored in gray in ϑ vs. βi) for different values of αi  > 0.5. gi, the initial steady-state phosphorylation level at each stage, can be written as a sequence of iterations:αk will proportionally increase only for i ≤ k. For i > k, increasing αk has a negligible effect on βi. This is somewhat counterintuitive because one usually considers alterations propagating downstream in a linear signaling cascade, whereas alterations in the kinase activity affects the range of duration robustness only in upstream modules. This is because the constraint for initial conditions back-propagates. In general, having fast kinase activity in the downstream modules is ideal if one wishes to generate a region of duration robustness against the upstream phosphatase activities.Here, the upper limit of duration robustness is roughly given by the largest value of g2 as a function of βi does play a crucial role.To better understand how duration robustness is generated and the criteria needed, we analyzed the linearization of the Heinrich model about the origin, the only equilibrium point once the stimulus is removed. Duration robustness is also a property of the linear model as can be seen in β0 ≠ β1 ≠ β2, the normalized solution  model of a linear signaling cascade . This model is a complete mass action description of a MAPK signaling pathway, which is a linear cascade with three layers. The middle and last layers represent double phosphorylation events, which lead to ultrasensitivity. The HF model also explicitly assumes that a phosphatase at each layer removes the active phosphate groups, and thus, the phosphatase activity is directly proportional to the total phosphatase concentration, The same numerical analysis as for the Heinrich model was performed for the HF model and the results are displayed in dynamics robustness, i.e., the insensitivity of the time courses against changes in certain parameters, is observed in the relaxation process of signaling cascades. By using a general linear cascading system, the time course of the output layer downstream is shown to be almost insensitive to changes in upstream parameters. As a consequence of dynamics robustness, the duration in which the activated state lasts is also robust to parameter changes, a property we termed duration robustness. By analyzing the cascading process, the conditions for duration robustness are given by the constraints on the rate-limiting process and on the initial conditions. Since multiple layers are needed to generate duration robustness, this suggests that this property is a byproduct of how temporal information is processed downstream.In the present paper, we have demonstrated that The robustness of flux at steady state has been extensively studied since the pioneering study of Kacser-Burns , but a tith stage, and that two main conditions are responsible for this phenomenon:We have shown that linear signaling cascades of varying complexity display duration robustness against perturbations in the phosphatase activity in the 1) The constraint on the rate-limiting process. The phosphatase activity in the ith stage, βi, should not be the minimum β value. This unfortunately means that the slowest module in a linear cascade will not display duration robustness. This constraint determines the lower limit for the range of duration robustness, i.e., β < βi. If βi is the minimum β value in the cascade, then the duration time is inversely proportional to βi as described by usual relaxation processes. In general linear signaling cascades, this means that the phosphatase activity in module i should not be the slowest. For certain parameter regions, our results are contrary to the idea that upstream phosphatase activity controls the duration of the system more than downstream in a strongly activated cascade [ cascade .2) The constraint on the initial conditions. To achieve duration robustness, the initial phosphorylation level of the output layer also has to be robust. For the Heinrich model, the initial steady-state phosphorylation level, gi, is given as a sequence of iterations as gi will be robust against changes in the upstream phosphatase activity. In other words, changes in upstream layers are shielded by the strong kinase activity. This constraint determines the upper limit for duration robustness.βi can be is largely determined by the kinase activity, αi. A stronger kinase activity allows the phosphatase to be at a higher level and the system to remain in an activated state. Although too low kinase activity changes the initial phosphorylation level, too high kinase activity has little effect, due to the saturation of the phosphorylation. This determines the upper limit of βi for the region of duration robustness.Intuitively, if the kinase activity is low, the phosphatase activity should be low enough to allow the cascade to be active. How large ith layer could be given asfi is a decreasing function of the kinase activity, αi, and an increasing function of the phosphatase activity, βi. In this case, increasing the kinase activity downstream can shield upstream changes, and then lead to duration robustness against changes in the phosphatase activity in upstream modules. This is interesting as changes appear to be propagated upstream. This type of downstream-to-upstream perturbation transference was reported in the steady-state concentration of linear cascades as retroactivity [In general, linear signaling cascades do display such saturation, as a result of conservation of the substrate at each layer, and as for the Heinrich model, the initial steady-state phosphorylation level in activity , whereasOur results showed that within the range of a biologically relevant parameter set of a MAPK signaling pathway reported in , the durWhereas our results show that the response duration and profile can be robust to some parameters, sensitivity to other external control parameters is necessary. Pathways need to be sensitive to certain parameters to function in the signaling process. Compatibility between robustness and plasticity (or sensitivity) to external changes is a basic characteristic of a biological system. In biological clocks, it is represented as reciprocity between robustness of period and plasticity in phase of oscillation , and it The fast-slow-fast organization of the kinetics in the three-stage cascade adopted in the present model is not necessary for dynamics robustness since it is observed in two-stage cascades as well. We looked at other parameter setups in linear cascades and their results intuitively agreed with the results in this paper; the rate-limiting module tends to control the duration and the other modules display robustness under the constraints discussed. Whether the fast-slow-fast organization is a byproduct of another selected property or is selected for a beneficial trait regarding dynamics robustness is unknown. However, one possible benefit is the emergence of a plateauing response as observed in Figs As a design principle, a signaling cascade with the conditions discussed previously are ideal for robust transients and this parameter organization is reflected in . Since rThe present paper focused on linear signaling cascades because of the recent interest in their temporal dynamics, but our idea of dynamics robustness can be generalized to any biochemical network. Two common properties of the cascade architectures we examined were mass conservation and an active molecule working as a kinase downstream. This suggests that such robustness can be achieved by similar designs such as the two-component signaling network in bacteria, and may be a universal feature in biological signaling cascades via protein modifications. There are some biochemical processes which are known to be reliably timed, such as lysis of bacteria and chromosome segregation during mitosis , 30, anddynamics robustness we have presented here should be timely and of importance. In many biological phenomena, the time course, such as the response against external stimuli or the developmental process, is crucially important, and must be sufficiently robust to perturbations. This point has been noted before, but so far there is no theory for such dynamics robustness. For example, the scale invariance of time course has gathered much attention as fold-change detection [The concept and explicit results of etection . DynamicFuture work will be done to incorporate more complicated design elements, such as the addition of feedback loops. It will be important to examine other cascading networks such as transcription regulatory cascades. For example, robustness of sensory response is discussed in transcription networks where feedback loop network motfis work as a rate limiting process . It willWe demonstrated dynamics robustness in standard models of signal transduction. As these models are based on experimental data, and agree rather well with them, our dynamics robustness can be straightforwardly confirmed in cell-signaling experiments. Also considering the generality of our results, many other experimental topics will benefit from dynamics robustness.We looked at different models of varying complexity. Although we use the nomenclature of kinases and phosphatases to represent the activating enzymes and deactivating enzymes, our model can be applied generally to any linear signaling cascade. We used mass action kinetics to simulate the chemical reactions, and all of our equations were solved using MATLAB’s (version R2009a) built-in numerical integrator ode15s.E0, converts M0 to M1 to M2 to Mi is activated is βi. We assume that after an initial, constant stimulus and equilibration of the system, the receptor is immediately shut off and the system relaxes.In the Heinrich model described in  and diagMi. Although these assumptions ignore some details, they enable us to analyze the models mathematically while still capturing the overall behavior of a signaling cascade. The corresponding set of equations post-stimulus is:There are a few major simplifying assumptions in this model that make it useful for examining the qualitative behavior of linear cascades. The assumptions are that the intermediate complexes formed by each kinase-substrate pair is negligible, that the backward reaction from the complexes is insignificant, and that the active phosphatase concentration is nearly constant. This means that the phosphatases and the intermediate complexes can be ignored, the desphosphorylation rate can be expressed as a first-order reaction rate, and that the sum of the inactive and active forms of each substrate is constant, i.e., y) against a parameter (say x) on a log-log scale, then the logarithmic gain at a point is the slope of the tangent at that point. In other words, the logarithmic gain at a point x0 is x0. If y is inversely proportional to x, then the logarithmic gain will be −1. This concept has been used in systems biology to measure the robustness of steady-state concentration levels and transition times [How robust a system is to a perturbation in a parameter is quantitatively measured by logarithmic gain. If one plots the dependent variable Click here for additional data file.