Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin β2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin β2 translocation are specifically or predominantly dependent on coronin 1. Vascular injury leads to exposure of prothrombotic extracellular matrix proteins, which facilitates the entrapment and activation of platelets through specialized receptors. These interactions contribute to stable adhesion of platelets by generating intracellular signals that lead to shape change, secretion of granules, and activation of integrins. Activation of integrins facilitates the binding of the plasma protein fibrinogen, which subsequently supports platelet aggregation and clot formation, rapidly consolidated by secreted soluble agonists . While tPlatelet activation is the result of multiple integrated signaling cascades that ultimately drive remodeling of the platelet cytoskeleton and sustain the morphological changes required for adhesion, spreading, aggregation, and secretion at the sites of vascular damage . The cytCoronin 1 )  participCoro1 is abundantly expressed in cells of the hematopoietic lineage, where it is essential for the survival of naïve T cells ,17,18,19Coro1a knockout (KO) model [p = 0.8164, Student’s t-test)  model . We confO) model ,20. The  t-test) B.We assessed the expression of characteristic surface platelet receptors  by flow cytometry both in unstimulated and in thrombin-stimulated platelets. Thrombin stimulation caused a significant increase in the expression of GPVI, CD41 (integrin αIIb), and CD49b (integrin α2) (20–40%) and a significant decrease in the expression of CD42b (GP1b) (32–43%), the latter due to cleavage and internalization of the GP1b/IX/V complex . Both bap = 0.1016). However, upon thrombin stimulation expression increased significantly in WT platelets to 1562 ± 158  but only modestly in KO platelets  (Coro1 interacts with the cytoplasmic tail of integrin β2 and regulates its function in neutrophils . Althouge to WT) A,B. The e to WT) C.2) (2. No obvious differences were apparent in cell area between WT and KO platelets (2) to those observed with 0.1 U/mL thrombin. This indicates that low thrombin doses (0.025 U/mL) are sufficient to elicit full spreading on native BSA and Coro1 is dispensable for this response. Integrin β2 main ligand is intercellular adhesion molecule-1 (ICAM-1), a glycoprotein expressed in endothelial cells and leukocytes. We used fluorescence microscopy to investigate the effect of Coro1 absence on platelet adhesion and spreading on surfaces coated with 5 mg/mL native BSA, a surrogate method of assessing binding to ICAM-1, both basally and upon stimulation with 0.1 U/mL thrombin ,28. On a2) D–F. Throlatelets D–F. To iWe assessed the potential effects of Coro1 deficiency on integrin αIIbβ3 activation with the activation state-specific antibody JON/A by flow cytometry. Stimulation with a wide range of agonists , as well as adenosine diphosphate (ADP) and the thromboxane analog U46619 alone or in combination) caused activation of αIIbβ3, in the case of thrombin and CRP in a dose-dependent manner A. HoweveWe next explored whether Coro1 KO platelets have a defect in granule secretion. To monitor alpha and dense granule secretion, we induced P-selectin and CD63 expression, respectively, by the same agonists as in the αIIbβ3 activation experiment. In both cases, thrombin produced a clear dose–response effect, CRP had little effect and ADP and U46619 had a synergistic effect in both WT and KO platelets B,C. Nonep = 0.0420) and a moderately higher velocity  compared to WT platelets. We did not observe any statistically significant difference between WT and KO platelets at any dose of collagen or CRP.A functioning actin cytoskeleton remodeling is critical for platelet aggregation and for adhesion and spreading on extracellular matrix proteins. We next investigated the implications of Coro1 deficiency for those processes. Stocker et al. reported subtle defects in aggregation induced by low doses of collagen using impedance-based aggregometry on whole blood . We appl2 in the WT vs. 15.83 ± 0.85 µm2 in the KO) than on fibrinogen (10.75 ± 0.74 µm2 in the WT vs. 11.89 ± 0.71 µm2 in the KO) (The effect of Coro1 absence on platelet adhesion and spreading was further investigated on surfaces coated with collagen (100 μg/mL) or fibrinogen (100 μg/mL) by fluorescence microscopy. On average, slightly more platelets per observation field adhered on fibrinogen; however, there were no statistically significant differences in the numbers of platelets adhering to either surface between the WT and the KO platelets (60.4 ± 4.5 vs. 61.4 ± 5.1 on fibrinogen and 48.3 ± 4.6 vs. 52.3 ± 7.4 on collagen) A, B. Irr the KO) C. Charac2 in the WT vs. 11.43 ± 0.81 µm2 in the KO) and higher on CRP (22.92 ± 2.24 µm2 in the WT vs. KO 21.96 ± 0.77 µm2 in the KO) compared to collagen  (for α2β1 integrin), or CRP (for GPVI). Approximately 50% less platelets adhered on GFOGER and CRP compared to collagen and the trend was similar in both WT and KO platelets B. Surfaccollagen C. On CRPWe have shown that in platelets Coro1 co-immunoprecipitates and colocalizes with components of the Arp2/3 complex and that the activity of the complex is necessary for extension of lamellipodia and accumulation of Coro1 at the cell cortex of spreading platelets . We usedJayachandran et al. have shown that Coro1 interacts with Gαs and modulates the cAMP signaling pathway in neurons and T cells ,29. In pCoro1a deletion on hemostasis, we examined tail bleeding  when associated with integrin αL, is one of the 6 integrins expressed in mouse platelets and the fourth most abundant . β2 inteDictyostelium discoideum regulates cAMP-dependent initiation of multicellular aggregation [Magnaporthe oryzae interacts with a Gαs subunit to regulate cAMP production and pathogenicity [Jayachandran et al. have uncovered the role of Coro1 in modulating the cAMP signaling pathway in excitatory neurons, where deficiency of the protein resulted in the loss of excitatory synapses and a range of neurobehavioral disabilities . Coro1 iregation  and the genicity . In platgenicity , absencegenicity . Furthergenicity  but CDK5genicity ,39, althgenicity , suggestgenicity .In summary, we propose that class I coronins display a large extent of functional overlap in platelets. This would explain the absence of a strong phenotype in most platelet functional assays while aspects like integrin β2 translocation reported by us and the formation of F-actin and cofilin dephosphorylation in response to agonists reported by others  are specPrimary antibodies against following proteins were used: Coro1 (ab56820 and ab72212), β-actin (ab20272) from Abcam ; Coro3 (K6-444 hybridoma supernatant) ; β3-inteCoro1a gene have been previously described [C57Bl/6 mice with a homozygous targeting of the escribed  and are g for 5 min. The platelet-rich plasma (PRP) was collected in a separate tube, modified Tyrode’s buffer was added to the pellet, and the procedure repeated to increase the platelet yield. For washed platelet preparation, the PRP was pelleted at 800× g for 6 min and platelets resuspended in modified Tyrode’s buffer and allowed to rest for 30 min at 37 °C prior to experiments.Blood was taken by cardiac puncture into acid citrate dextrose (ACD)  or sodium citrate (109 mM tri-sodium citrate pH 7.4) and centrifuged at 100× Lysates were prepared from washed platelet suspensions by mixing with one volume of 2× Laemmli buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and blotted onto polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with the relevant primary antibody and the corresponding fluorochrome-labeled secondary antibody and visualized and quantified with an LI-COR Odyssey CLx Imaging System (LI-COR Biosciences).2), CRP, ADP, or U46619 for 20 min at 37 °C in the presence of FITC-conjugated anti-P-selectin (BD Biosciences), PE-conjugated JON/A  and APC/Cy7-conjugated anti-CD63 (Biolegend) antibodies. Platelets were subsequently fixed and analyzed by fluorescence-activated cell sorting (FACS) using an LSRFortessa cell analyzer (BD Biosciences) and FlowJo software.PRP was prepared in sodium citrate and stimulated with thrombin , GPVI, integrin α2 (CD49b) , integrin αIIb (CD41) , or PE-conjugated antibodies against integrin β2 (CD18) (Biolegend). Receptor expression was also studied upon stimulation with 0.1 U/mL thrombin for 20 min at 37 °C in the presence of 10 µM Gly-Pro-Arg-Pro-NHg for 10 min on poly-l-lysine (0.01% in PBS) coated coverslips. Platelets were stained for 1 h at room temperature with the indicated primary antibodies followed by the corresponding secondary antibodies and fluorescently labeled phalloidin diluted in PBG , 0.05% fish gelatin in PBS). For adhesion studies, coverslips were coated overnight at 4 °C with fibrinogen, collagen, CRP, or GFOGER in PBS at the concentrations indicated and blocked with 5 mg/mL heat-denatured fatty acid free BSA for 1 h before the experiment. For adhesion on native BSA, coverslips were coated overnight at 4 °C with 5 mg/mL fatty acid free BSA in 0.05 M sodium bicarbonate buffer pH 9 [Platelet aggregation in response to agonists was recorded in washed platelets under constant stirring conditions (1000 rpm) for 7 min at 37 °C using light transmission aggregometry with a CHRONO-LOG 490 aggregometer . Washed platelets in suspension were fixed with an equal volume of ice-cold 4% paraformaldehyde (PFA) and spun at 350× fer pH 9 . Washed Mice were anesthetized with 50 mg/kg ketamine and 1 mg/kg medetomidine. The tail was cut off at 2 mm from the tip and immediately immersed in 37 °C PBS. Bleeding time between the cut and cessation of bleeding for at least one minute was monitored by visual inspection until hemostasis for up to 10 min.p ≤ 0.05. Experimental data were analyzed by GraphPad Prism v6.0 . Data are presented as mean ± standard error of the mean (SEM) of at least 4 independent experiments. Normality was assessed by the Shapiro–Wilk test. Differences between groups were assessed using the appropriate parametric or nonparametric test and statistical significance was taken at


Our optical measurements show a ln(I525/I545) vs. 1/T dependence for both 800 nm and 980 nm excitations. Despite widespread evidence promoting the benefits of 800 nm over 980 nm CW excitation in avoiding thermal heating in biological imaging, in contrary, we find that given the pulsed laser intensities appropriate for single particle imaging, at both 800 and 980 nm, that there is no significant local heating in air and in water. Finally, in order to confirm the applicability of infrared imaging at excitation intensities compatible with single nanoparticle tracking, DNA tightropes were exposed to pulsed infrared excitations at 800 and 980 nm. Our results show no appreciable change in the viability of DNA over time when exposed to either wavelengths. Our studies provide evidence for the feasibility of exploring protein-DNA interactions at the single molecule scale, using UCNPs as a reporter.Upconversion Nanoparticles (UCNPs) enable direct measurement of the local temperature with high temporal and thermal resolution and sensitivity. Current studies focusing on small animals and cellular systems, based on continuous wave (CW) infrared excitation sources, typically lead to localized thermal heating. However, the effects of upconversion bioimaging at the molecular scale, where higher infrared intensities under a tightly focused excitation beam, coupled with pulsed excitation to provide higher peak powers, is not well understood. We report on the feasibility of 800 and 980 nm excited UCNPs in thermal sensing under pulsed excitation. The UCNPs report temperature ratiometrically with sensitivities in the 1 × 10 In contrast to other researchers, where both I525 and I545 emission lines are derived from a spectral scan over the appropriate wavelength range, we collect both lines simultaneously, in order to avoid heat dissipation effects. Our optical measurements show a ln(I525/I545) vs. 1/T dependence for both 800 nm and 980 nm excitations, in the Yb3+/Er3+ codoped and triply doped Yb3+/Er3+@Yb3+/Nd3+ samples. We also observe a strong influence of the laser intensity on the relative spectroscopic ratio of I525/I545. Additionally, we find that given the pulsed laser intensities appropriate for single particle imaging, at both 800 and 980 nm, that there are no significant differences in the local heating effects. This result is in contrast with that obtained when comparing excitation at both wavelengths under CW excitation. We further demonstrate our observations by comparing the differences in a DNA tightrope denaturation experiment at 800 and 980 nm pulsed irradiation respectively, and find no significant change in denaturation at excitation fluences that have previously been shown to support upconversion imaging utilizing 976 nm excitation at the single particle level at diameters of 10 nm with an intensity of 3e4 W/cm2 and diameter of 25 nm at an intensity of 4e4 W/cm2 as demonstrated by Gargas et al. , Y(CF3COO)3 (0.29 mmol), Yb(CF3COO)3 (0.20 mmol) and Er(CF3COO)3 (0.01 mmol) precursors were mixed with oleic acid (5 mmol), oleyamine (5 mmol), and 1-octadecene (10 mmol) in a two-neck round bottom flask. The mixture was heated to 110°C to form a transparent solution followed by 10 min of degassing. Then the mixture was heated to 300°C at a rate of 15°C/min under dry argon flow, and maintained at 300°C for 30 min to form the α-NaYF4:40%Yb, 2%Er intermediate UCNPs. After the mixture cooled to room temperature, the α-NaYF4:40%Yb, 2%Er intermediate UCNPs were collected by centrifugal washing with excessive ethanol . In the second step, the α-NaYF4:40%Yb, 2%Er intermediate UCNPs were redispersed into oleic acid (10 mmol) and 1-octadecene (10 mmol) together with CF3COONa (0.5 mmol) in a new two-neck round bottom flask. After degassing at 110°C for 10 min, this flask was heated to 325°C at a rate of 15°C/min under dry argon flow, and maintained at 325°C for 30 min to complete the phase transfer from α to β. After the mixture cooled to room temperature, the β-NaYF4:40%Yb, 2%Er UCNPs were collected by precipitated with equal volume of ethanol and centrifugation afterwards . The β-NaYF4:40%Yb, 2%Er UCNPs were stored in hexane (10 mL).The β-NaYF4:40%Yb, 2%Er core UCNPs served as cores for the epitaxial growth of core-shell UCNPs. A hexane stock solution of β-NaYF4:40%Yb, 2% Er core UCNPs was transferred into a two-neck round bottom flask, and the hexane was sequentially evaporated by heating. CF3COONa (0.50 mmol), Y(CF3COO)3 (0.40 mmol) and Yb(CF3COO)3 (0.10 mmol) were introduced as UCNP shell precursors with oleic acid (10 mmol) and 1-octadecene (10 mmol). After 10 min of degassing at 110°C, the flask was heated to 325°C at a rate of 15°C/min under dry argon flow and maintained at 325°C for 30 min to complete the shell crystal growth. After the mixture cooled to room temperature, the β-NaYF4:40%Yb, 2%Er@NaYF4:20%Yb core-shell UCNPs were collected by precipitated with equal volume of ethanol and centrifugation afterwards . The β-NaYF4:40%Yb, 2%Er@NaYF4:20%Yb core-shell UCNPs were stored in hexane (10 mL).Next, the as-synthesized β-NaYF4:40%Yb, 2%Er@NaYF4:20%Yb core-shell UCNPs served as cores for the epitaxial growth of shell crystal. A hexane stock solution of β-NaYF4:40%Yb, 2%Er@NaYF4:20%Yb core-shell UCNPs was transferred into a two-neck round bottom flask, and the hexane was sequentially evaporated by heating. CF3COONa (0.50 mmol), Nd(CF3COO)3 (0.45 mmol) and Yb(CF3COO)3 (0.05 mmol) were introduced as UCNP shell precursors with oleic acid (10 mmol) and 1-octadecene (10 mmol). After 10 min of degassing at 110°C, the flask was heated to 325°C at a rate of 15°C/min under dry argon flow and maintained at 325°C for 30 min to complete the shell crystal growth. After the mixture cooled to room temperature, the β-NaYF4:40%Yb, 2%Er@NaYF4:20%Yb@NaNdF4:10%Yb core-shell-shell UCNPs were collected by precipitated with equal volume of ethanol and centrifugation afterwards . The β-NaYF4:40%Yb, 2%Er@NaYF4:20%Yb@NaNdF4:10%Yb core-shell-shell UCNPs were stored in hexane (10 mL).Afterwards, the as-synthesized β-NaYF4:20%Yb, 2%Er  to determine average diameter. The β-NaYFl Figure  UCNPs weObjects resolved on an optical microscope  imaged by a Andor NEO sCMOS camera with excitation from a 1,000 hz, tunable Nd: YAG laser with a 4.5 ns pulse width were selected by size, ensuring that imaged objects were less than the size of the diffraction limited spot of the wavelength of collection. Spectral distributions were separated using a half-meter monochromator with a custom exit with two slits. The gap between the slits correspond to a 10 nm wavelength difference centered at the monochromator's single slit location. By setting the monochromator to 535 nm, the 525 nm peak and 545 nm peak were effectively separated at the exit slits and further separated by a prism and coupled into separate SPCM-AQRH avalanche photodiodes. Controlled heating was performed using a peltier heater fixed to the back of the sample slide by epoxy. A thermistor attached to a PTC2.5K-CH temperature controller was embedded in the epoxy to ensure a constant set temperature during operation, and a K-type thermocouple was used to monitor the temperature at the sample surface.2 was injected into the flow cell and pushed back and forth for 10 min using a syringe pump at a flow rate of 300 uL/min  were ligated using the Quick Ligation Kit (New England BioLabs) at room temperature for overnight and then purified by phenol-chloroform extraction to remove the ligase  vs. 1/T measured for the both the β-NaYF4:20%Yb, 2%Er UCNPs and β-NaYF4:40%Yb, 2%Er@NaYF4:20%Yb@NaNdF4:10%Yb core-shell-shell UCNPs at 976 and 806 nm respectively. Since the population of the Er: 2H11/2 and Er: 4S3/2 levels fluctuates as a function of the Boltzmann's distribution  vs. 1/T plot, a ΔE of 887.170 cm−1  and 966.176 cm−1 (976 nm core only) is obtained. The calculated difference between 545 and 525 nm peaks is 700 cm−1. The sensitivity S is defined as,where by taking the slope of the ln level, at high pump power intensities. As the pump power is increased, the increase in phonon coupling to the lattice, and subsequent non-radiative energy transfer from the 2H9/2 to 2H11/2 and the 4S3/2 level to the 4F9/2 level occurs. Since the energy gap for the 4S3/2 to 4F9/2 transition of 3117 cm−1 coincides with the typical value of 3,000–3,600 cm−1 for OH vibrations level population significantly and the ratio decrease should be accounted for.Figure s Figure , R is ob4:40%Yb, 2%Er@NaYF4:20%Yb@NaNdF4:10%Yb core-shell-shell UCNPs, at 976 nm excitation, shows a drop in R, but to a smaller degree, with increasing pump power intensity. This is attributed similarly to the β-NaYF4:20%Yb, 2%Er UCNPs as shown earlier. The smaller drop may be due to the protective nature of the core-shell-shell configuration, which limits the effect of lattice vibration effects. In contrast, at 806 nm excitation, an increase in R is seen with increasing pump power. The immediate assumption is that of an increase in local temperature, but results shown later will demonstrate that this is not the case. The increase in R appears to be an optical effect only. Under 806 nm excitation, the Nd3+: 4F5/2 and Nd3+: 2H9/2 levels are populated due to transitions from the Nd3+:4I9/2 ground state. Subsequent non-radiative relaxation from these two states occur, leading to population of the Nd3+: 4F3/2 state. The resonant energy levels of Nd3+: 4F3/2 and Yb3+: 2F5/2 ensures a very high efficiency energy transfer (ET) between Nd3+ ions and Yb3+ ions, 2H11/2(green), 4S3/2(green), and 4F9/2(red) emissions at two different excitation intensities. It can be seen that at a higher excitation intensity (5.6 × 104 W/cm2) in Figure 4F9/2 and 4S3/2 states excitation pathways are more strongly coupled as evidenced by the narrowing of the gap between their rise times  and 976 nm (7 × 104 W/cm2) excitation for 2-min durations only cause dsDNA breaks 10% of the time. For a control DNA tightrope experiment where there was no exposure to near infrared excitation, the rate of occurrence of dsDNA breaks were also around 10%. Therefore, any damage observed is consistent with 10 s of UV or blue light nicking of the DNA, from the Xenon Arc Lamp source used to detect YOYO-1 emission. Clearly, from our measurements, the lack of DNA damage under infrared exposure leads us to infer that thermal heating is not significant at the current pump power intensities used in our measurements especially when considered alongside the lack of heating in water, as shown in Figure As a demonstration of the effects of 976 vs. 806 nm irradiation at the single-molecule level, we compared DNA tightropes exposed to both wavelengths at intensities shown previously to support single particle imaging (Gargas et al., 3+ sensitization may be good candidates for application as nanothermometers in single biological molecule experimental situations. It was demonstrated that each type of UCNP was able to report temperature ratiometrically with sensitivities in the 1 × 10−4 K−1 range. In addition, it was determined that excitation intensity is a parameter of nanothermometry that must be strongly controlled in the single particle level to avoid ratio suppression due to alteration of the optical pathways involved in ratio reporting. Finally, the excitation intensities previously used to image single nanoparticles were shown to be applicable to DNA tightrope experiments without appreciable change in the viability of DNA over time windows appropriate for the study of protein-DNA interactions.UCNPs with and without NdThe study aims were proposed by SL. KH synthesized the UCNPs. KG designed and performed the spectroscopic experiments. HP prepared the biologicals for the DNA tightrope experiments. Data interpretation and analysis were performed by KG, KH, GH, and SL.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.


The hypoxic tumor microenvironment is the major contributor of chemotherapy resistance in solid tumors. One of the key regulators of hypoxic responses within the cell is the hypoxia inducible factor-1α (HIF-1α) that is involved in transcription of genes promoting cell survival and chemotherapy resistance. Multidrug resistance gene-1 (MDR1) and Lysosome-associated protein transmembrane 4B-35 (LAPTM4B-35) are among those notable players which augment their responses to cellular hypoxia. MDR1 is the hypoxia responsive gene involved in multidrug resistance phenotype while LAPTM4B-35 is involved in chemotherapy resistance by stabilizing HIF-1α and overexpressing MDR1. Overexpression of HIF-1α, MDR1 and LAPTM4B has been associated with poor disease outcome in many cancers when studied individually at tissue level. However, accessibility of the tissues following the course of chemotherapy for ascertaining chemotherapy resistance is difficult and sometimes not clinically feasible. Therefore, indication of hypoxic biomarkers in patient’s blood can significantly alter the clinical outcome. Hence there is a need to identify a blood based marker to understand the disease progression. In the current study the expression of hypoxia associated chemotherapy resistance genes were studied in the peripheral blood lymphocytes of solid tumor patients and any potential correlation with disease progression were explored. The expression of HIF-1α, MDR1 and LAPTM4B was studied in blood of 72 breast, 42 ovarian, 32 colon and 21 prostate cancer patients through real time PCR analysis using delta cycle threshold method. The statistical scrutiny was executed through Fisher’s Exact test and the Spearman correlation method. There was 12–13 fold increased in expression of HIF-1α, two fold increased in MDR1 and 13–14 fold increased in LAPTM4B mRNA level in peripheral blood of breast, ovarian, prostate and colon cancer patients. In the current study there was an association of HIF-1α, MDR1 and LAPTM4B expression with advanced tumor stage, metastasis and chemotherapy treated group in breast, ovarian, prostate and colon cancer patients. The Spearman analysis also revealed a positive linear association among HIF-1α, MDR1 and LAPTM4B in all the studied cancer patients. The elevated expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumor patients can be a predictor of metastasis, disease progression and treatment response in these cancers. However, larger studies are needed to further strengthen their role as a potential biomarker for cancer prognosis. Solid tumors are characteristically known to harbor a hypoxic microenvironment which serves as a major impediment to cure by conventional radiotherapy and chemotherapeutic regimens . In receDespite the fact that hypoxia is lethal to both normal as well as tumor cells, cancer cells undergo adaptive and genetic changes for their survival and even proliferation in hypoxic conditions, outpacing their normal counterparts. These changes involve transcription of many genes under the direction of transcription factor-hypoxia inducible factor-1 (HIF-1) . HIF-1 iMultidrug resistance gene-1 is one of the oldest and well reported genes associated with chemotherapy resistance . IncreasLysosomal-associated protein transmembrane-4 beta is an oncogene, initially identified in liver cancer. LAPTM4B associated with mammalian 4-tetratransmembrane spanning protein superfamily, and is largely located on plasma membrane and on membranous organelles such as endosomes and lysosomes . Hence, The expression of HIF-1α, MDR1 and LAPTM4B in individual cancers has been well reported and related to cancer progression and metastasis at tissue level however in isolated studies. However, the expression of these genes in serum of cancer patients and their potential relation with clinico-pathological features are scarcely reported. Hence, these finding necessitate investigation regarding whether there will be co-expression of these genes in peripheral blood of solid tumor patients and whether they will be associated with clinico-pathological characteristics. The applicability of such an investigation may lead to novel approach for studying cancer prognosis utilizing serum rather than actual tissues.The study subjects were identified as patients who were having either biopsy proven breast, ovarian, prostate or colon cancer, aging between 25 and 70, having no other comorbidity such as diabetes, hypertension or congenital heart disease. The patients were asked for written informed consent for their potential participation in the study. Ethical approval of the study was obtained from institutional review board of Atta-ur Rahman School of Applied Biosciences NUST (IRB-15) and Shifa International Hospital, Islamabad (shifa-ref-183-2015).Blood samples (five mL) from 72 breast, 42 ovarian, 21 prostate and 32 colon cancer patients were collected. The samples were collected in HTS sterile vacuum collection tubes and immediately put on ice to avoid RNA degradation. The clinical characteristics of patients including stage and metastasis were obtained from pathological reports . The staExtraction of total RNA from whole blood was conducted using TriZol reagent . All the reactions were performed on ice in order to avoid degradation. The concentration and purity of RNA was determined through NanoDrop 2000  and the samples with ratio A260/A280 > 1.6 were used for cDNA synthesis. For cDNA synthesis 20 µL of reaction volume was prepared by adding 100ng of RNA, 1.5 mM dNTPs, 100 µM oilgodT, 200 U reverse transcriptase, 10 U RNase inhibitor and DEPC water up-to 20 µL. The reverse transcription reaction was started at 42 °C for 60 min and terminated at 70 °C for 10 min. The cDNA was then stored at −20 °C.The expression analysis of HIF-1α, MDR1 and LAPTM4B genes was carried out using real time PCR analysis. Primers used for expression analysis of HIF-1α forward 5′- CGCATCTTGATAAGGCCTCT-3′, Reverse 5′- TACCTTCCATGTTGCAGACT-3′, MDR1 forward 5′- AACGGAAGCCAGAACATTCC-3′, Reverse 5′- AGGCTTCCTGTGGCAAAGAG-3′, LAPTM4B forward 5′- CCTCACTGCCAGATC-3′, reverse 5′- CTATCTGTGGCATACCT-3′ and GAPDH  forward 5′- CCCCTTCATTGACCTCAACTACA-3′, reverse 5′- CGCTCCTGGAGGATGGTGAT-3′. No template/negative controls was included for all the primers in each run. The reaction mixture comprised of 200 ng of template cDNA, 0.5 µM of each forward and reverse primer, 12.5 µL of SYBER Green master mix and nuclease free water to make up 20 µL of final volume. The thermal cycler conditions were as follows; initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 60 s and extension at 72 °C for 45 s. All the experiments were run in triplicates and their average was accounted for analysis. Data was analyzed using comparative cycle threshold method (ΔCt) and normalized according to GAPDH expression in each sample.p < 0.05.Statistical inquiry was performed using Statistical software package SPSS 21 . The Fisher’s Exact test was used to show the relationship between different clinico-pathological variables and expression of HIF-1α, MDR1 and LAPTM4B. Spearman correlation was performed to find the association between HIF-1α, MDR1 and LAPTM4B. The significance is defined as There was a high expression of HIF-1α observed in peripheral blood of breast (2.389 ± 0.1597), ovarian (2.647 ± 0.1541), prostate (2.689 ± 0.2272) and colon (2.369 ± 0.1810) cancer patients as compared to healthy controls (0.1806 ± 0.1236). In general, there was 12–13 fold increase in expression of HIF-1α in all the studied cancer patients. There was also an elevated expression of MDR1 observed in the blood of breast (2.717 ± 0.1145), ovarian (2.498 ± 0.1191), prostate (2.391 ± 0.2224) and colon (2.634 ± 0.1782) cancer patients compared to healthy controls (1.245 ± 0.08544) . Hence tp > 0.05). Instead, they showed an association with tumor stage and metastasis (p < 0.05). In the advanced stage group, there were >80% of patients with higher expression of all the three genes. In the lower stage group, the high expression of HIF-1α, MDR1 and LAPTM4B was found in 31%, 37% and 56% of patients, respectively. A similar scenario was observed in the metastatic and non-metastatic groups, where in the metastatic group, >80% of patients with elevated expression while in the non-metastatic group 38%, 71% and 66% of patients were having elevated expression of HIF-1α, MDR1 and LAPTM4B, respectively. It was also observed that increased expression in patients who underwent the chemotherapy treatment at the time of sample collection as compared to the treatment naïve group . Increased expression of HIF-1α, MDR1 and LAPTM4B was observed with higher tumor stage and distant metastasis (p < 0.05). Among advanced stage group, 80% of patients were presenting high expression while in the lower stage group 40–60% of patients were exhibiting high expression of three genes. In the non-metastatic group, 60% of patients with high expression of three genes while among metastatic group, 85%, 67% and 75% of patients were having elevated expression of HIF-1α, MDR1 and LAPTM4B, respectively. We observed an elevated expression of these genes in pre-treated group (p < 0.05)  . In pre-p < 0.05). 50% of patients in lower stage group while 66% and 80% of patients in the advanced stage group exhibiting high expression of HIF-1α and LAPTM4B, respectively. Among metastatic and localized disease group, high expression of HIF-1α and LAPTM4B was observed in patients with distant metastasis (p < 0.05). As there were 69% and 76% of patients among metastatic group were having high expression of HIF-1α and LAPTM4B, respectively. No correlation of MDR1 expression was observed with tumor stage and metastasis (p > 0.05). High expression of HIF-1α and MDR1 expression was observed in the patients who were under the chemotherapy treatment (p < 0.05). In treatment naïve group 42% of patients while in pre-treated group, 71% and 64% of patients were having elevated expression of HIF-1α and LAPTM4B, respectively.The relationship of different clinical parameters of prostate cancer with HIF-1α, MDR1 and LAPTM4B expression is shown in p < 0.05). In the advanced tumor stage, 84% and 57% of patients, while in lower stage group 30% and 46% of patients, were having elevated expression of HIF-1α, MDR1 and LAPTM4B. There was elevated expression of all the three genes was observed in metastatic group compared to patients with non-metastatic disease as there was 85%, 62% and 52% of patients in the metastatic group were exhibiting high expression of HIF-1α, MDR1 and LAPTM4B, respectively. The elevated expression was also observed in patients who were under the chemotherapy treatment at the time of sample collection (p < 0.05). 78%, 68% and 58% of patients in the pre-treated group were showing elevated expression of HIF-1α, MDR1 and LAPTM4B, respectively.In colon cancer, high expression of HIF-1α, MDR1 and LAPTM4B was correlated to clinical features of patients ; Fig. 5.p < 0.001; R ≥ 0.8) in the blood of breast . A similar pattern of linear correlation was also found among HIF-1α, MDR1 and LAPTM4B in case of prostate cancer  with 12 patients having high expression of these genes while six patients having low expression of these genes. In case of colon cancer 12 patients were those having high expression of HIF-1α and MDR1 while 15 were those with high expression of LAPTM4B also harboring high expression of MDR1 and HIF-1α. Hence a positive linear correlation was observed with R = 0.825 and p < 0.001. The positive correlation among all the studied cancers suggest similar upregulation of LAPTM4B leading to increased HIF-1α and MDR1 genes.The Spearman analysis revealed a linear positive correlation between HIF-1α and MDR1 expression  ranges from between 40 and 65 mm Hg; however dropping to 10 mm of Hg or less occurs in 60% of the solid tumors  metastasis (B) tumor stage; (C) treatment response. The scatter plot of expression of three genes in ovarian cancer with (D) metastasis (E) tumor stage; (F) treatment response. The scatter plot of expression of three genes in prostate cancer with (G) metastasis (H) tumor stage; (I) treatment response. The scatter plot of expression of three genes in colon cancer with (J) metastasis (K) tumor stage; (L) treatment response.Click here for additional data file.10.7717/peerj.6309/supp-3Supplemental Information 3Click here for additional data file.


Softwood is a promising feedstock for lignocellulosic biorefineries, but as it contains galactoglucomannan efficient mannan-degrading enzymes are required to unlock its full potential.β(1→4)-mannanases (endomannanases) from GH5 and GH26, including 6 novel GH26 enzymes. The endomannanases from Trichoderma reesei (TresMan5A) and Podospora anserina (PansMan26) were investigated with and without their carbohydrate-binding module (CBM). The pH optimum and initial rates of enzyme catalysed hydrolysis were determined on pure β-mannans, including acetylated and deacetylated spruce galactoglucomannan. Melting temperature (Tm) and stability of the endomannanases during prolonged incubations were also assessed. The highest initial rates on the pure mannans were attained by GH26 endomannanases. Acetylation tended to decrease the enzymatic rates to different extents depending on the enzyme. Despite exhibiting low rates on the pure mannan substrates, TresMan5A with CBM1 catalysed highest release among the endomannanases of both mannose and glucose during softwood saccharification. The presence of the CBM1 as well as the catalytic capability of the TresMan5A core module itself seemed to allow fast and more profound degradation of portions of the mannan that led to better cellulose degradation. In contrast, the presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification.Boosting of the saccharification of pretreated softwood (Canadian lodgepole pine) was investigated for 10 fungal endo-TresMan5A as the best endomannanase for increasing cellulase catalysed glucose release from softwood. Except for the superior performance of TresMan5A, the fungal GH5 and GH26 endomannanases generally performed on par on the lignocellulosic matrix. The work also illustrated the importance of using genuine lignocellulosic substrates rather than simple model substrates when selecting enzymes for industrial biomass applications.This study identified The online version of this article (10.1186/s13068-018-1184-y) contains supplementary material, which is available to authorized users. Enzymatic degradation of softwood to fermentable monomeric sugars is, however, still challenging due to its complex composition and inhomogeneous architecture . Not onl enzymes , 3. The  enzymes . The mai and C-3 . The typ 0.2–0.3 –11. Vari, 1.6:1) , 12.Fig.O-acetylated galactoglucomannan found in softwood -mannanases , β-mannosidases (EC 3.2.1.25), β-glucosidases (EC 3.2.1.21), α-galactosidases (EC 3.2.1.22) and acetyl mannan or glucomannan esterases (EC 3.1.1.-) [β(1→4)-glucanases (EC 3.2.1.4), primarily attacking the cellulose fraction of the softwood, have been shown to have activity on glucomannans [It requires a coordinated interplay of different types of enzymes to degrade the ood Fig. . A varie3.1.1.-) , 15. Alsomannans .β/α)8-TIM barrel fold in their structure, and catalyse the cleavage of the O-glycosidic bonds with retention of the anomeric configuration [Cellvibrio mannanases, it has been proposed that GH26 enzymes may primarily attack soluble mannans, whilst the GH5 counterparts primarily attack insoluble mannans [Podospora anserina endomannanases, PansMan26A and PansMan5A [PansMan26A together with the GH26 endomannanase from Aspergillus nidulans, AnidMan26A, were also found to accommodate more galactopyranosyl residues in the active site pocket than their GH5 counterparts [Endomannanases are important enzymes for facilitating the solubilization and release of mannan from the substrate matrix , 17. Endguration –21. Base mannans , 23. How mannans . RegardiansMan5A , 25. Theβ-mannans, uronic acids and α-d-galactopyranosyl residues on carbohydrate polymers [Many fungal GH5 endomannanases are modular, typically having a carbohydrate-binding module from family 1 (CBM1) as part of their structure. CBM1 is known to confer cellulose binding and increase the mannan hydrolysis of complex substrates such as softwood and ivory nut extractions containing both mannan and cellulose , 28. Funpolymers , 31.Trichoderma reesei GH5 endomannanase, (TresMan5A) [The capacity of endomannanases to boost saccharification of softwood to fermentable monomers has been demonstrated and studied mostly with selected fungal GH5 endomannanases. The available literature in particular includes several studies with the esMan5A) , 17, 32,esMan5A) . A few sesMan5A) , 34, butesMan5A) ., Pinus contorta) with 12% mannan left after pretreatment. The saccharification studies were performed at low temperature (30 °C) to focus on comparing activity of the enzymes, and at 50 °C to mimic industrial saccharification conditions. A subsidiary aim was to address the importance of the CBM35 in softwood saccharification by testing the Podospora anserina GH26 endomannanase with and without its CBM35. To touch upon any possible differences in the biological role of the fungal GH5 and GH26 endomannanases, and to assess if any of the substrate preferences on pure mannans could help explain performance differences on the softwood substrate, the initial rate of the studied endomannanases on soluble mannans, including acetylated and deacetylated spruce galactoglucomannan were also determined.Based on the hypothesis that fungal endomannanases differ in their capacity to catalyse removal of galactoglucomannans from cellulose fibrils, and thus in turn may have different effects on enzymatic cellulose saccharification, this study compares 10 fungal endomannanases and their boosting effect on enzymatic cellulose degradation from softwood  or CBM35 (PansMan26A)  Table . When coβ-glucanase cross-activity was found for any of the tested endomannanases, CvirMan26A, PansMan26A, PansMan26A core, AnigMan5A, TresMan5A and TresMan5A core (assessed on barley β-glucan and carboxymethyl cellulose).The initial rates of endomannanase catalysed hydrolysis of locust bean gum, guar gum, konjac glucomannan, as well as acetylated and deacetylated spruce galactoglucomannan were determined at pH 5 and at 37 °C, to assess the activity without confounding effects of differential thermostability of the enzymes Fig. . No β-glThe endomannanases showed different activity levels on the pure mannan substrates.YpenMan26A had a significantly higher initial rate than all the other enzymes on the two galactomannans: locust bean gum  and guar gum . In contrast, WspMan26A had the highest initial rates of all tested endomannanases on the glucomannans: konjac glucomannan , acetylated galactoglucomannan (8650 U/µmole), and deacetylated galactoglucomannan (9150 U/µmole) , but did not generally affect rates or caused rate increase. The lowest initial rates were observed for TresMan5A on the acetylated spruce galactoglucomannan and on konjac glucomannan (on both substrates 600 U/µmole). In general, TresMan5A had lowest initial rates of all the endomannanases on all the tested mannans, irrespective of the presence of the CBM1. The lack of significance of the CBM1 presence is in accordance with TresMan5A CBM1 being known to bind to cellulose and not to mannan [PansMan26A containing a CBM35 tended to be higher than those for the PansMan26A core, especially on locust bean gum and konjac glucomannan. A positive effect of the CBM35 may be related to the reported interaction of CBM35 with β-mannans or α-d-galactopyranosyl residues [PansMan26A CBM35 interacts with the β-mannan backbone than with the α-d-galactopyranosyl substitutions, since the positive effect of the CBM was not found on the highly substituted guar gum.GH5 endomannanases tended to exhibit lower initial rates than the GH26, but no consistent discrimination between the enzymes’ substrate preferences was evident  compared to on konjac glucomannan . All other tested GH26 endomannanases had higher (or similar) initial rates on konjac glucomannan than on locust bean gum, including PansMan26A core for which the CBM35 were removed artificially. YpenMan26A and AnidMan26A do not have a CBM, hence the substrate preferences exhibited by these enzymes as compared to those with a CBM35 must be tied to the enzyme core properties rather than to presence of the CBM35 domain. Interestingly, Katsimpouras et al. [Myceliophthora thermophila had similar substrate preference trends, i.e., showing higher activity on konjac glucomannan compared to locust bean gum.The wild type core GH26 endomannanases, s et al.  reportedTresMan5A and AnigMan5A, had significantly higher initial rates on locust bean gum (1150 and 5450 U/µmole) compared to on konjac glucomannan (600 and 2650 U/µmole). The data for the GH5 enzymes correspond with the substrate preferences reported for A. nidulans GH5 endomannanases [Like the wild type core GH26 endomannanases, nnanases .® CTec3, where in each case the Cellic® CTec3 had been supplied with a pure GH2 β-mannosidase from Aspergillus niger (BM2). When assessed on locust bean gum Cellic® CTec3 itself exerted weak mannan-degrading activity. The endomannanase addition levels were ten times higher than this background activity. The release of glucose, mannose and xylose, respectively, was quantified at 24, 48 and 144 h  and the release of glucose from 3.06 to 3.18 g/l (0.12 g/l increase)  galactoglucomannans [® CTec3 preparation and/or by weak glucomannan degrading capacity by some endoglucanases of this enzyme cocktail  and 15% (increase in 0.23 g/l mannose) higher than that of the control  is in agreement with data published by Westh et al. [KM) cancels thermoactivation (increase in kcat). In the present study, the substrate concentration was low (2% DM ~ 1% cellulose). The effective accessible substrate concentration may have been lower, because not all cellulose is equally accessible. Since industrial lignocellulose conversion is usually executed at 50 °C, the data strongly indicate that addition of TresMan5A to commercial cellulase preparations can efficiently boost glucose yields in industrial softwood saccharification reactions.When the two endomannanases, ent Fig. , and theh et al. , 40 who ® CTec3 and endomannanase doses used  is not correlated to high boosting effect. Despite its high boosting capacity, TresMan5A was found to have the lowest initial rate on the pure mannans . This is likely an overestimated rate for the endomannanase since it attacks the mannan backbone, while it is the surplus of BM2 that causes the release of mannose. On the other hand, the rate is probably not constant throughout the first 24 h, but higher during the initial reaction period. Even if the initial reaction rate for TresMan5A on the softwood substrate was 20 U/µmole, this rate is still 45 times lower than the initial rates obtained for TresMan5A on extracted deacetylated spruce galactoglucomannan at 900 U/µmole (the acetyl moieties are expected to be lost during pretreatment of the softwood). On this insoluble lignocellulosic matrix, the hydrolysis rate of the endomannanases is comparable with the rate of cellulases working on insoluble cellulose [In general, the hydrolysis rate of mannan during softwood saccharification was much lower than the initial hydrolysis rates measured on the pure mannans. During the first 24 h, the mannose release from the softwood substrate by ellulose . NeitherTresMan5A with its CBM1 was the most efficient for softwood saccharification among the tested endomannanases (when added to Cellic ® CTec3). It has previously been observed that hemicellulases from T. reesei reduce hemicellulose exposed at the cellulose surface of wood materials to a greater extent than hemicellulases from Aspergillus sp. [TresMan5A catalyses hydrolysis of softwood galactoglucomannan [TresMan5A catalyses a faster or more profound degradation of a certain type of mannan that is not immediately accessible for the other endomannanases and which moreover, when degraded, allows for a more profound cellulose degradation. Since at least part of softwood mannan is closely associated with cellulose [TresMan5A helps target the cellulose-associated mannan more efficiently than its truncated counterpart lacking CBM1. This view is supported by a previous study of TresMan5A action on cellulose-mannan complexes and the CBM1 cellulose binding capacity [Our results clearly show that llus sp. , and thacomannan . Our hypellulose , it canncapacity . When plcapacity .TresMan5A core without the CBM1 was among the top performers in the softwood saccharification, the core module itself also played a role for the efficient degradation of the insoluble mannans. The reason that the other GH26 endomannanases with a CBM1, i.e., CvirMan26A and AstMan26, were not releasing the same levels of mannose and glucose as TresMan5A, could be because their core modules are not as optimal as TresMan5A for degradation of mannan associated to cellulose. Another reason might be that their CBM1s have slightly different specificities than the TresMan5A CBM1.Since the TresMan5A was superior to all other tested GH5 and GH26 endomannanses  in genuine softwood saccharification, despite being among the slowest on purified mannan substrates. Two GH5 and eight GH26 endomannanases (including six novel endomannanases) were successfully recombinantly expressed, purified, and characterized with focus on their performance in softwood saccharification. The fungal GH26 endomannanases from Yunnania penicillata and Westerdykella sp. were found to have highest initial rates among the tested enzymes on pure soluble galactomannans and glucomannans, respectively. The acetyl groups on extracted spruce galactoglucomannan tended to decrease the initial enzymatic rates when compared to the initial rates on the deacetylated substrate. However, these initial rates on the pure mannans did not correlate with the results obtained in the extended softwood saccharification reactions. All tested endomannanases caused increased glucose release during softwood saccharification when compared to the glucose release catalysed by Cellic ® CTec3 plus the A. niger β-mannosidase alone. However, the GH5 endomannanase from T. reesei with a CBM1 produced a markedly higher mannose and glucose release at all time points than all the other tested endomannanases. Based on the data, our hypothesis is that TresMan5A is able to attack an additional portion of mannan in the lignocellulosic matrix, allowing for better cellulose degradation. Both the catalytic efficiency of the core module and the presence of the CBM1 play important roles in the superior performance of this enzyme on softwood. The other nine GH5 and GH26 endomannanases performed on par with the softwood lignocellulosic matrix, giving no clear signs of different biological roles for these fungal endomannanases. Presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification.This study strongly confirms that fungal endomannanases differ in their capacity to degrade galactoglucomannan in softwood, and that this degradation contributes significantly to obtain increased enzymatic cellulose saccharification of softwood. Apart from this main finding, the key novel result was that the well-studied The data obtained highlight the problematic strategy of selecting enzymes for industrial applications based on basic characterisation on pure and well-defined substrates. Neither rates nor substrate preferences observed in the basic characterisation correlated with efficient softwood saccharification. Evidently, the data of this study have implications for the selection and use of endomannanases in industrial softwood saccharification applications, especially as new pretreatment methods leaves more hemicellulose in the lignocellulosic matrix after pretreatment.β-glucan , and carboxymethyl cellulose, were purchased from Megazyme (Ireland). Spruce galactoglucomannan  was prepared as described previously [O-acetyl moieties of the O-acetylated spruce galactoglucomannan were removed by alkaline hydrolysis in the presence of ammonium hydroxide as described by Jacobs et al. [Locust bean gum , guar gum (high viscosity), konjac glucomannan (high viscosity), eviously . The O-as et al. . All othCollariella virescens (CvirMan26A), Mycothermus thermophiles (MtheMan26A), Neoascochyta desmazieri (NdesMan26A), Ascobolus stictoideus (AstiMan26A), Westerdykella sp. (Wsp.Man26A), Aspergillus nidulans (AnidMan26A), Yunnania penicillata (YpenMan26A), Podospora anserina (PansMan26A and PansMan26A core), the fungal GH5 endomannanases from Aspergillus niger (AnigMan5A), and Trichoderma reesei (TresMan5A and TresMan5A core) were recombinantly expressed in Aspergillus oryzae MT3568an amdS [PansMan26 core and TresMan5A core were expressed without the linker and the N-terminal CBM35 and the C-terminal CBM1, respectively. The enzymes were purified to electrophoretic purity using hydrophobic interaction and ion exchange chromatography . ε at 280 nm of all proteins were estimated by GPMAW 9.20 (Lighthouse Data), and were based on mature proteins without modifications.The fungal GH26 endomannanases from 8an amdS . PansManThe hydrolytic activity was determined at 37 °C, after 15 min, over a pH range from 2.0 to 12.0, with 1 pH unit intervals. The hydrolysis volume was 200 µl, with 2.5 mg/ml locust bean gum in a buffer with 50 mM acetic acid, 50 mM HEPES, 50 mM glycine, 0.01% Trition X-100, 50 mM potassium chloride and 1 mM calcium chloride. The buffer pH was adjusted with sodium hydroxide from pH 2.0–12.0. Released reducing sugars were measured with the 4-hydroxybenzoic acid hydrazide (PAHBAH) method described by Lever , with maβ-glucan, and carboxymethyl cellulose by the endomannanases were determined with 2.5 mg/ml substrate in 50 mM sodium acetate pH 5 at 37 °C. All substrates, except the deacetylated spruce galactoglucomannan and the carboxymethyl cellulose, were soluble at the concentration employed. The deacetylated spruce galactoglucomannan and the carboxymethyl cellulose were only partly soluble, but these substrates were kept in suspension during reaction and sampling via vigorous mixing. Released reducing sugars were measured with the PAHBAH method as described above, except that glucose was used as standard for measurements on β-glucan and carboxymethyl cellulose. All hydrolysis assays were carried out at seven different endomannanase doses as described elsewhere [The initial rate on locust bean gum, guar gum, konjac glucomannan, acetylated and deacetylated spruce galactoglucomannan, lsewhere . InitialThe thermal stability at pH 5 was investigated with differential scanning calorimetry (DSC) as described elsewhere . The thePinus contorta) were pretreated, and the chemical composition was analyzed . Wood chips were screened and a size fraction between 2.5 × 2.5 and 5.0 × 5.0 cm was collected and used as feedstock for pretreatment. The pretreatment was performed in a similar manner to the procedure developed by Chandra et al. [Commercially available grey-stage beetle killed lodgepole pinewood (LPP) chips , and a commercially available cellulase- and xylanase-rich enzyme cocktail (Cellic® CTec3). Except for the endomannanases, which were purified in this study, the enzymes were kindly provided by Novozymes. The applied enzyme doses can be seen in Table ® CTec3 is a Trichoderma-based product with different recombinant enzymes. One can assume that the mannan-degrading activity from Cellic® CTec3 is equivalent to TresMan5A. Cellic® CTec3 supplies approximately 1.3 × 10 −9 mol/g dry matter (DM) endomannanase (TresMan5A) to the softwood hydrolysis, when added in a concentration of 10 mg Cellic® CTec3/g DM. The lowest addition of purified endomannanase was 1.26 × 10−8 mol/g DM.The applied enzymes were the purified endomannanases  for separation and quantification of glucose amount and by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization followed by reverse phase ultra-performance liquid chromatography (UPLC) for separation and quantification of mannose and xylose amounts.Enzymatic hydrolysis of the pretreated softwood was performed in 50 ml falcon tubes with two metal balls (9 mm). Softwood was added to give 0.4 g dry matter per tube (resulting in 2% dry matter) along with sodium acetate buffer, pH 5, and proxel to give concentrations of 50 mM and 0.2%, respectively, in the final mixture. Milli-Q water was added to give a total reaction mass of 20 g after addition of the required amount of enzyme. The tubes were incubated in a heated (30 or 50 °C) 20 cm diameter drum, rotating at 20 rpm. All experiments were performed in triplicates and run for 144 h. At sampling, 2 ml representative whole slurry sample was transferred to Eppendorf tubes and centrifuged 18,2132SO4. Calibration curves for glucose were plotted (Empower) and used to estimate the amount of glucose released.The amount of glucose was analysed by a Waters HPLC system coupled with a refractive index detector and equipped with a Aminex HPX 87H column (300 by 7.8 mm). 10 µl sample was injected and separation was performed at 65 °C with a flow rate of 0.6 ml/min 5 mM Hd-glucose as internal standard, 20 µl 4 M NaOH, and 200 µl 0.5 M PMP in aqueous methanol. The mixtures were mixed well and incubated at 70 °C for 30 min, cooled to room temperature and mixed with 20 µl 4 M HCL and 400 µl methanol. The separation and quantification of mannose and xylose was analysed by a Waters Acquity UPLC system coupled with a UV (245 nm) detector and equipped with a Waters Acquity CSH C18 column . 3 µl sample was injected and separation was performed at 65 °C with a flow rate of 0.5 ml/min. A two-eluent system was used, (A) 0.15% formic acid in MiliQ water and (B) 0.15% formic acid in ACN with the following gradient: 0 min, 83:17 (% A:B); 1 min, 83:17 (% A:B); 10 min, 77.2:22.8 (% A:B); 10.5 min, 5:95 (% A:B); 11 min, 83:17 (% A:B). The total run time per injection was 13 min. Calibration curves for mannose and xylose were plotted (Empower3) and used to estimate the amount of released mannose and xylose. All results are expressed in g/l.The derivatization reaction, for analysing the amount of mannose and xylose, was performed with 200 µl sample in an appropriate concentration, 20 µl 6-deoxy-Additional file 1: Table S1 and S2. Ratings from one-way ANOVA analyses of initial reaction rates by endomannanases on pure mannans  was determined on locust bean gum at 30 °C, pH 5 and are given as mean values ± SD (n=3). Please consult the manuscript for enzyme abbreviations. Figure S2. Softwood saccharification. Endomannanases or BSA were added in equal molar amounts on top of Cellic ® CTec3 plus an A. niger GH2 β-mannosidase (BM2). Samples were taken after 24, 48 and 144 h saccharification at 30 °C. Glucose , mannose (dark grey) and xylose (black) yields are given as mean values ± SD (n=3). One-way ANOVA analyses can be seen in Additional file Figure S3. SDS-PAGE gels of the purified enzymes. The protein concentration in the samples was 0.5 mg/ml. Prior to gel loading, samples were diluted 1:1 with loading mix. Loading mix was prepared as a 9:1 mix of Novex ® Tris-Glycine SDS Sample Buffer (2X) (Life Technologies) and Nupage ® Sample Reducing Agent (10X) (Life Technologies). Please consult the manuscript for enzyme abbreviations. Samples with NdesMan26A and MtheMan26A both contain molecules with and without the CBM35.


Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the s2C containing tRNA can be methylated to carry ms2C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms2C as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach.RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (ms Bacterial tRNA is modified by thiolation of nucleosides. Here the authors identify 2-methylthiocytidine in bacterial tRNA using nucleic acid isotope labeling coupled mass spectrometry. Exposure to methylating agents converts 2-thiocytidine to 2-methylthiocytidine, which is repaired by demethylase AlkB in vivo. In addition, both DNA and RNA are target of modifying enzymes which introduce methylations or other functionalities. To this day, over 160 modified nucleosides have been identified in RNA from organisms in all domains of life2. In addition, bacteria use various electrophiles as chemical warheads  to harm other bacteria by methylation and alkylation of their macromolecules6. Thus, bacteria are constantly forced to overcome these alkylating reagents and various adaptive mechanisms can be found. To study the impact of direct methylation, electrophiles such as methyl-methanesulfonate (MMS) or dimethyl sulfate (DMS) are used. Methylation damage of both RNA and DNA is repaired by direct demethylation through the alpha-ketoglutarate dependent dioxygenase AlkB in bacteria8. So far the methylated aromatic nitrogens of 3-methylcytidine (m3C), 1-methyladenosine (m1A) and 1-methylguanosine (m1G) are reported substrates of AlkB. In addition, the exocyclic amines of N2,N2-dimethylguanosine  can be demethylated by AlkB in vitro, which finds application in the RNA modification sequencing technique ARM-Seq9. So far, no non-nitrogen methylations have been described as substrates of AlkB in RNA.Due to the nucleophilic character of the canonical nucleobases, both RNA and DNA are prone to direct methylation. Inside cells these electrophiles comprise molecules such as S-adenosylmethionine (SAM), which is the common methyl-donor for enzymatic methylation reactions but it is also known to directly alkylate nucleic acids3-labeled methionine in a nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) assay, this discrimination can be achieved and, e.g., enzymatic 7-methylguanosine (m7G) and MMS-damage derived m7G can be quantified independently10. With NAIL-MS, the analysis of alkylation damage of canonical nucleosides was possible in the presence of natural methylated nucleosides. A key finding in this study was that m7G damage is as common as m1A, which was believed to be the only dominant RNA damage product upon direct methylation in vivo11. In addition, NAIL-MS was used in a pulse-chase setup to follow the in vivo demethylation kinetics of m1A and m3C during MMS recovery10. Especially m3C is quickly and efficiently (~80%) repaired within nine hours of recovery whereas only 20% of m1A damage sites are repaired within the same timeframe12. For m3C, the demethylation by AlkB is reported in vitro, but so far not in vivo13.In addition to the nucleobase methylations introduced by direct alkylation, cells in all domains of life use SAM for the enzymatic methylation of RNA. The occurrence of these natural methylated nucleosides makes the detection of damage-derived methylated nucleosides challenging. With the application of CD2C). The thiolated nucleosides at position 32 and 37 of tRNA flank the anticodon (position 34–36) allowing for a more flexible anticodon loop and thus improve the binding of the codon during translation. s2C is incorporated into bacterial tRNAArgCCG, tRNAArgICG, tRNAArgUCU and tRNASerGCU by the thiotransferase TtcA at position 32. Its effects on tRNA structure and bacterial translation has been addressed in several studies15.While methylation is a major modification of RNA, especially tRNA contains additional and more complex modifications. One group of unique tRNA modifications is the enzymatic thiolation. For example, bacteria thiolate cytidine at position 32 of the tRNA to form 2-thiocytidine   and s2C (m/z 260) is 14 Dalton, which is a strong indicator of methylation of s2C.With the goal of discovering novel modified nucleosides in bacterial tRNA, 84 from 1C-labeled2C) is feasible, and thus the methylation reaction at position 2 of s2C was performed. Interestingly, the synthetic route was already described in 2010, when the Suzuki lab19 used 2-methylthiocytidine as an intermediate of agmatidine synthesis. We followed their synthetic route under milder conditions and received pure ms2C in 28% yield . The precursor of ms2C, s2C is reported in three of the five tRNAArg isoacceptors and one of the five tRNASer isoacceptors21. Therefore, we designed DNA probes , a tRNA with 2’-O-methylcytidine at position 32  and tRNAs without a C32, tRNAGlyCCC and tRNAAlaUGC. As expected, we found s2C in tRNAArgCCG, tRNAArgICG, tRNAArgUCU and tRNASerGCU . We observed decreased ms2C formation in various knockout strains grown in M9 medium but not in LB medium. To exclude an involvement of these enzymes in ms2C formation, we analyzed E. coli strains that overexpress tRNA modifying enzymes , non-lethal MMS doses (3 mM and 0.5 mM) and the LD50 dose of the natural antibiotic streptozotocin  in M9 medium  and s2C (0.008 ms2C/tRNA). Overall, ms2C formed readily during alkylation stress and in comparable extent to m1A.Intrigued by the possibility that s1A is known to be a direct methylation product, the increase in ms2C might be caused by either direct methylation or SAM-derived enzymatic methylation. To elucidate the origin of the methylation in ms2C under MMS stress, we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to discriminate direct and SAM-derived methylation during MMS stress in vivo. Adapted from our recently established assay10, we cultured E. coli WT bacteria in the presence of CD3-methionine to allow complete CD3-labeling of all SAM dependent methylated nucleosides. One aliquot of these bacteria was exposed to the LD50 dose of MMS, while the other remained unstressed. After 1 h of MMS exposure the tRNA was purified and analyzed by LC-MS/MS. Enzymatically methylated nucleosides carry a + 3 label whereas the directly MMS methylated nucleosides are unlabeled and thus the two species are distinguishable by mass spectrometry. Absolute quantification of the enzymatically and directly methylated nucleosides in the unstressed and stressed cells revealed the abundance of methylated nucleosides in the samples. The fold changes between the stressed and unstressed samples were calculated and plotted in Fig. 2C was again found to be labeled through CD3-methionine dependent pathways, with a slightly higher abundance in the stressed samples. This small increase in CD3-methylated ms2C does not reflect its substantial abundance in Fig. 3U (5 fold), m6A (47 fold), m7G (64 fold) m1A (64 fold) and m3C (74 fold)10. However, the highest increase was for the product of direct s2C methylation, ms2C, which was over 500 fold increased in the stressed bacteria compared to the unstressed  in unlabeled medium. After cell harvesting, the unstressed and stressed bacteria were mixed to avoid purification biases, as recently suggested12. From the resulting co-purified total tRNA, the tRNA isoacceptors SerGCU, ArgCCG, ArgICG and ArgUCU were isolated, digested and subjected to LC-MS/MS analysis. The mass spectrometer was set to distinguish the 13C-labeled nucleosides (unstressed bacteria) from the unlabeled nucleosides (stressed bacteria) and thus the abundance of each nucleoside can be determined , AGA (tRNAArgUCU) and CGC, CGU and CGA (tRNAArgICG)14. The methylation of s2C and formation of ms2C might result in electronic and electrostatic shifts of the nucleobase, which potentially changes the properties of the whole anticodon loop of the affected tRNAs. Thus translation of the respective serine and arginine codons might be influenced by the methylation of s2C. To address this question, the effect of MMS on the synthesis of superfolder green fluorescent protein (sfGFP) in E. coli WT was determined. We decided to study sfGFP  cells of the mid-log growth phase were induced by arabinose. Fluorescence intensity of single E. coli cells of strain BW25113 was determined after 1 and 5 h , e.g. m/z (s2C) 260. All methylated nucleosides had a CH3 mark . In this medium, the cells were exposed to MMS (LD50) and s2C was converted to ms2C and, e.g., A to m1A. After exposure, MMS was removed by exchanging the media with heavy-isotope media. During the following recovery period, newly transcribed RNA were [15N] labeled, newly methylated nucleosides were [CD]3 labeled and new s2C had a [34S] label .The ms1A if referenced to the sum of all tRNAs . The fast decrease is caused by dilution due to ongoing tRNA transcription. In the past, this decrease could be misinterpreted as active tRNA demethylation, but with NAIL-MS we can follow the fate of modified nucleosides inside living cells by excluding transcription processes. By referencing m1A abundance to the original tRNAs, we saw a slow but steady decrease of m1A in the damaged tRNAs over time which reflects true demethylation  but five units . In case of the imagined scenario of spontaneous dethiomethylation of ms2C into cytidine followed by rethiolation into s2C, only sulfur-34 is available and thus the original [32S]-s2C would turn into [34S]-s2C with an m/z of 262. The original [32S]-s2C produced by AlkB demethylation and the hypothetical turnover [34S]-s2C formed after dethiomethylation/rethiolation can be distinguished by mass spectrometry  nucleoside was confirmed after synthesis by various analytical tools including fragmentation patterns in high resolution mass spectrometry. Methylated derivatives of other thiolated nucleosides like s4U or mnm5s2U might also form endogenously, but were not detected in our nucleoside discovery experiments.In this study, we identified s2C was found at position 32 of tRNAArgICG, tRNAArgCCG, tRNAArgUCU and tRNASerGCU. Only 1% of the s2C modified tRNA isoacceptors carry ms2C under unstressed growth conditions. The endogenous formation of ms2C was not found to be connected to common tRNA modifying enzymes. At this stage we cannot determine whether the endogenous, but low abundant, ms2C modification of bacterial tRNA is introduced enzymatically or by direct methylation through, e.g., SAM. For low abundant nucleoside modifications it is often an open question whether they are lesions or functional entities24.ms2C forms naturally in the presence of the methyl donor SAM, we were wondering whether ms2C is formed by direct methylation during alkylation stress after MMS exposure. MMS is a direct mono-methylating agent, which is known to methylate RNA nucleosides and DNA nucleosides. From our systematic study, we know that the main damage products of canonical nucleosides are m1A and m7G10. In the current study, we also focused on the quantities of ms2C formed during an LD50 MMS exposure of E. coli. We find 0.1 m1A damage sites per average tRNA and surprisingly 0.05 ms2C per average tRNA. At lower MMS doses (3 mM), the number of m1A damage sites is reduced to 0.014 per average tRNA and 0.018 ms2C per average tRNA. It is now clear that ms2C is an equally prominent damage product as m1A in total tRNA. If one considers that the average tRNA in bacteria is composed of 15 adenosines while only 0.1 s2C are found in the average tRNA, our results are rather intriguing. Although adenosine is 150 fold more abundant than s2C in total tRNA, the methylation products m1A and ms2C are of comparable quantities. From our data, we conclude that s2C is an at least equal or even better substrate of direct methylation than adenosine in the tRNA of the bacterium E. coli.Intrigued by the observation that ms2C influences the translation in vivo. Serine can be translated by tRNASerGGA (no s2C) reading the TCC codon or tRNASerGCU reading the AGC or AGU codons. We found that sfgfp with the AGC or AGU codon was less efficiently translated than sfgfp with the TCC codon under MMS stress. This finding indicates that the translation of the AGC and AGT codon is disturbed after MMS stress, potentially due to the presence of ms2C.Although the abundance of modified nucleosides is low in comparison to the amount of canonical nucleosides in the total RNA pool, modified nucleosides fulfil a variety of important functions within the cell. The damage of a modified nucleoside will most likely interrupt the modification’s function and might be disadvantageous to the organism. We have tested how the methylation of s1A and m3C by oxidative demethylation by the enzyme AlkB has been shown7. In our NAIL-MS studies, we use stable isotopes such as carbon-13, nitrogen-15 or sulfur-34 to distinguish RNA that was present during the stress event  and new RNA transcribed during the recovery period. We can thus distinguish repair by RNA degradation and demethylation. With NAIL-MS, we observe AlkB dependent demethylation of ms2C, m3C and potentially m1A.The repair of damaged ribonucleosides might occur by two potential mechanisms. RNA is a transient molecule and constantly transcribed from DNA within the cell. Upon damage, it is possible to remove the damage by controlled degradation of the RNA. Due to the fact that tRNA maturation and modification is energy consuming, the second repair scenario of direct damage repair by, e.g., demethylation, is more attractive. In bacterial RNA, the repair of m3C had been suggested in previous studies but has never been shown in vivo. Surprisingly, m1A repair was similar in the WT and ΔalkB mutant. Only for the 3 mM MMS dose, 3 h after stress exposure, the WT strain showed less m1A per tRNA compared to the ΔalkB strain. This is in accordance with the only other in vivo study, where m1A repair by AlkB was observed in radioactive tRNA 3 h after stress at a low dose of alkylating agent23.The AlkB dependent repair of malkB bacteria, we did not observe an accumulation of ms2C . Only after exposure to alkylating agents, alkB is induced and six hours after 3 mM MMS exposure 6000 AlkB copies/cell have been found11.In unstressed Δ2C repair, we found an increase of original [32S]-s2C in vivo, which reflects direct sulfur demethylation by AlkB. With our sophisticated NAIL-MS approach we could additionally observe an AlkB independent formation of s2C which originated from dethiomethylation of s2C to cytidine and subsequent rethiolation. The initial dethiolation step might be enzymatically catalyzed, but due to the electrophilic nature of the C2 in ms2C, a direct nucleophilic attack by water is also possible.Concerning ms1A was thought to be the main methylation damage product in bacterial RNA and thus to be the main substrate of AlkB. Our data shows that ms2C is a methylation damage in bacterial tRNAs of comparable extent after MMS exposure. In addition, AlkB is faster during the repair of ms2C damaged tRNAs compared to m1A damaged tRNAs.So far, m2C.Since its discovery, AlkB has been an enzyme full of surprises. In this work, we present the next surprise, which is AlkBs extended substrate repertoire and its preference for the sulfur methylated nucleoside ms15NH4Cl (>98 atom %) and [D3]-L-methionine (98 atom %) were obtained from Sigma-Aldrich. Isotopically labeled 13C6- glucose (≥99 atom %) and isotopically labeled Na234SO4 (99.11 atom %) were obtained from Eurisotope . All solutions and buffers were made with water from a Millipore device . The nucleosides adenosine, cytidine, guanosine, uridine, and N2-methylguanosine (m2G) were obtained from Sigma-Aldrich. 1-Methyladenosine (m1A), 2-methyladenosine (m2A), N3-methylcytidine (m3C), N6-methyladenosine (m6A), 7-methylguanosine (m7G), 5-methylcytidine (m5C), 5-methyluridine (m5U), 2’-O-methylcytidine (Cm), 2′-O-methylguanosine (Gm), 1-methylguanosine (m1G), and 3-methyluridine (m3U) were obtained from Carbosynth .All salts were obtained from Sigma-Aldrich  at molecular biology grade unless stated otherwise. The isotopically labeled compounds 2C were stirred in 1 mL anhydrous ethanol under nitrogen atmosphere.7.4 mg of NaHCO3 and 24 µL of methyl iodide were added and the reaction was stirred overnight at room temperature. The clear yellow solution was evaporated and two times purified by silica gel chromatography in dichloromethane with 10 % methanol. The Rf value (TLC) in DCM/MeOH (10:1) was 0.46.Twenty milligrams of s1H NMR :δ = 8.39 , 6.60 , 5.98 , 4.38 , 4.23-4.18 , 3.99 , 3.85 , 2.67 . 13C NMR :δ = 166.4 (C-2), 162.1 (C-4), 141.5 (C-6), 102.1 (C-5), 92.7 (C-1′), 84.6 (C-3′), 74.9 (C-2′), 68.5 (C-4′), 59.8 (C-5′), 14.4 (C-7).E. coli wild-type strain BW25113 and the isogenic knockout strains were purchased from the Keio database26. Pseudomonas aeruginosa PA14 was a generous gift of Prof. Peter Dedon. Knockout strains with kanamycin resistance were grown on kanamycin LB agar plates (50 µg/mL). The E. coli strain AG1 (ME5305) transformed with the indicated pCA24N-based vectors was used for gene overexpression and ordered from the ASKA library database27. Strains with chloramphenicol resistance were grown in the presence of chloramphenicol (30 µg/mL). All cultures were grown in a shaking incubator at 37 °C at 250 rpm (Orbit = 10 mm). Overnight cultures were grown in 5 mL of the media used for the respective experiment. The cells were grown starting with an OD600 of 0.1, 0.5 or 1 (as specified in the respective section) and grown until reaching stationary phase (OD600 ~ 4). The used For LB media, LB Broth (Luria Miller) from Roth  was used. 1.5% (wt/vol) agar plates were prepared with LB-broth and Agar-Agar (Kobe I from Roth) according to manufacturer’s protocol.2, Na2SO4, and CaCl2 (as detailed below). For unlabeled 10 × M9 stock solution, Na2HPO4 (68 g/L), KH2PO4 (30 g/L), NaCl (2.5 g/L), and NH4Cl (10 g/L) were mixed and autoclaved. For 15N-labeled 10 × M9 stock solution, 15NH4Cl (10 g/L) was used. MgCl2 (0.1 M), CaCl2 (0.1 M), Na2SO4 (0.1 M), and 20% (wt%) glucose were prepared by sterile filtration. A 20% (wt%) 13C6-labeled glucose solution was prepared for 13C-labeled M9 media. For 34S-labeled M9 media a 0.1 M Na234SO4 solution was prepared. Final M9 media was prepared by mixing, e.g., 500 μL M9 stock solution with 100 μL glucose, 100 μL MgCl2, 100 μL Na2SO4, 5 μL CaCl2 and water to a final volume of 5 mL. For 15N-labeled cultures, the 15N-10 × M9 stock solution was used. For 13C-labeled cultures, the 20% (wt%) 13C6-labeled glucose solution and for 34S-labeled cultures, the 0.1 M Na234SO4 solution was used. For CD3-methylome labeling, 200 μL CD3-methionine (stock 5 g/L) was added to 5 mL of culture volume.M9 minimal medium was used with and without the indicated isotopes. Unlabeled M9 was prepared by mixing a 10 × M9 stock solution with glucose, MgCl600 = 0.5. After 3 h the cells were harvested and the RNA was isolated and purified. The overexpression strains were cultured in LB media starting with an OD600 = 0.5 and an IPTG concentration of 0.1 mM. After 1 h incubation at 37 °C and 250 rpm (Orbit = 10 mm) the IPTG concentration was increased to 1 mM to induce full overexpression. The bacteria were incubated for further 2 h before the cells were harvested and RNA was isolated and purifiedThe knockout strains were cultured in LB or M9 media, starting from OD600 1.0  were prepared from an E. coli overnight culture. After 60 min incubation (30 min with Streptozotocin), 100 µL of the culture were diluted 10−5 or 10−6 with sterile water. From this dilution 70 µL were plated on a prewarmed LB agar plate. The colony number of this plate represents 100% bacterial survival. MMS  or Streptozotocin  was added to the remaining bacteria in defined concentrations. After 60 min (30 min) exposure, 100 µL of the culture were diluted and plated. The LB plates were incubated at 37 °C overnight and the colonies counted for determination of the survival at the respective MMS or STZ concentration.Unlabeled M9 media was used throughout the survival assay. A 5 mL culture with ODE. coli strain BW25113 (WT) the OD600 was brought to 1.0 and the culture was grown for 1 h. To 5 mL bacterial culture 125 µL of a 10 mM STZ solution was added  and the culture was incubated for 30 min. Afterwards, the bacteria were centrifuged and the resulting pellet was used for RNA isolation and purification.From an overnight culture of g for 5 min. The supernatant was discarded and the cell pellet was resuspended in 1 mL TRI reagent (Sigma-Aldrich) per 5 mL bacteria culture. The total RNA was isolated according to the supplier’s manual. tRNA was purified by size exclusion chromatography (SEC) according to published procedures10. The tRNA was resuspended in water (30 μL).The bacteria culture was centrifuged at 1200 × 22. One microgram of total tRNA was incubated with 100 pmol of the biotinylated DNA probe and purified using Dynabeads T1  according to the manusfacturer’s protocol. For tRNA isoacceptor purification, pre-purified total tRNA was used. The sequences of the biotinylated 2′-deoxyoligonucleotide probes are listed in Supplementary Table The procedure was adapted from Hauenschild et al.E. coli culture was grown overnight in an unlabeled M9 medium and another in a 13C labeled M9 medium. From these cultures, a labeled and unlabeled exposure culture with an OD600 of 1.0 and a volume of 5 mL were prepared. After 60 min growth, 8.5 μL MMS  were added to the unlabeled culture and 8.5 μL of water to the 13C-labeled culture (MOCK). After 60 min of exposure both cultures were mixed and the RNA was purified immediately. Total tRNA and tRNA isoacceptors were isolated as described above. For validation of the comparative NAIL-MS assay, the experiment was repeated by mixing and co-purifying RNA  from an unlabeled MOCK treated culture and a 13C-labeled MOCK treated culture.One 3-labeled M9 medium was used at all times. A 5 mL bacterial solution with an OD600 of 0.1 was prepared from an overnight culture. After 60 min growth, 8.5 μL MMS  were added. As a control, 8.5 µL water was added to a second culture. After 60 min of exposure, the RNA was isolated and total tRNA purified by SEC.For this assay, CDE. coli strains BW25113, BW25113 ΔalkB and BW25113 ΔttcA were transformed with pBAD24 containing a copy of sfgfp under the control of the arabinose-inducible PBAD promoter. To test the effects of different serine codons  the only serine codon of the used sfgfp sequence (corresponds to amino acid position 2) was exchanged by using the respective primers during the cloning process. To enhance the number of serines, the sfgfp sequence was extended at the 5′-end with multiple motifs coding for proline-alanine-serine (ASPAAPSASAPSAASAAPSAA)-sequence, modified from previous studies28. We used the primers PAS-gfp_for/ PAS-AGT_gfp_for / PAS-TCC_gfp_for plus gfp_rev to introduce different types of codons for serine  in the sfGFP variants extended with the proline-alanine-serine (ASPAAPSASAPSAASAAPSAA)-sequence or respectively gfp_for / AGT_gfp_for / TCC_gfp_for plus gfp_rev to produce the sfGFP variants with different types of codons for serine . The restrictions sites EcoRI and XbaI were used for integration of the sfgfp variants into the pBAD24 plasmid.For in vivo quantification of the effects of tRNA modifications after MMS stress on translation, the 29.For each experiment cells of an overnight culture were inoculated into fresh M9 minimal medium supplemented with 0.5 % (vol/vol) glycerol as a sole carbon source and incubated under vigorous shaking at 37 °C. Cells were grown to mid-log phase, and the culture was divided. One half was stressed by the addition of MMS to final concentrations of 3 and 20 mM, respectively, the other half remained untreated. After 1 h of incubation in presence of MMS the medium was exchanged with fresh, pre-tempered M9 minimal media supplemented with 0.5% (vol/vol) glycerol and 0.2% (vol/vol) arabinose to induce sfGFP expression. As a control, cells were cultivated without addition of arabinose. To measure sfGFP fluorescence, cells were fixed on an agarose pad  placed on a microscope slide with coverslip. Micrographs were taken on a Leica microscope DMI 6000B equipped with a Leica DFC 365Fx camera . sfGFP fluorescence was visualized using an excitation wavelength of 460 nm and a 512 nm emission filter with a 75-nm bandwidth. Fluorescence intensities of a minimum of 300 cells per transformant were collected and quantified using FijiE. coli BW25113 or E. coli JW2200-KC  was picked and grown in unlabeled M9 medium (5 mL) overnight. From the first overnight culture, a 50 mL culture was prepared in unlabeled M9 medium and grown overnight. From the second overnight culture, 120 mL culture (OD600 of 1.0) was prepared in unlabeled M9 medium. After 60 min growth, the first aliquot (7 mL) was taken for RNA isolation. The remaining culture was split into two flasks of 56.5 mL each. One was exposed to MMS  the other to water (MOCK) and inverted before both cultures were cultivated for 60 min. An aliquot (7 mL) was drawn from each culture, and the RNA was isolated. The remaining bacteria were centrifuged , and the MMS/MOCK-containing supernatants were discarded. The bacteria pellets were washed with 15N, 34S and CD3-methionine labeled M9 medium (5 mL), and each bacterial pellet was suspended in fresh 15N/34S/CD3 M9 medium (50 mL). The bacteria were allowed to grow and recover from the MMS/MOCK treatment. Seven millilitres of each bacterial culture were harvested after 1, 2, 3, 4, and 23 h. The RNA was isolated and the tRNA purified by SEC. The experiment was also done with lower volumes and different concentrations of MMS.A single colony of 2C synthetic standard solution (0.4 nmol) were mixed with 0, 500, 1000, 1500 or 2000 equivalents of SAM (32 mM stock) or MMS (20 mM stock) in a final volume of 100 μL containing 50 mM Na2HPO4/NaH2PO4 buffer (pH 7.0). The mixture was incubated at 37 °C for 60 min in a shaking heat block. After incubation, 900 µL water were added (1:10 dilution) and spiked with SILIS to be analyzed by quantitative LC-MS/MS.4.3 μL of a 93 μM sE. coli was mixed with 45 μL of AlkB. (45 µL AlkB was prepared with 7.5 μL KCl (100 mM), 1.5 μL freshly mixed sodium α-ketoglutarate (10 mM), 2.5 μL Tris buffer , 5 μL freshly mixed sodium L-ascorbate (20 mM), 1.5 μL freshly mixed Fe(II)(NH4)2(SO4)2 (10 mM) and 1.5 μL of AlkB enzyme  and water.) The reaction mixture was incubated at 37 °C for 60 min. After incubation, 500 μL of LiClO4 in acetone (2 vol%) was added for tRNA precipitation and mixed thoroughly. After 10 min at room temperature, the sample was centrifuged at 5000 × g for 10 min. The RNA pellet was washed with 70 vol% ethanol and resuspended in 20 µL water before digestion for LC-MS/MS analysis.Five microlitres of purified tRNA (30 ng/µL) from 20 mM MMS exposed 2 (1 mM) containing buffer. Furthermore, 0.5 µg tetrahydrouridine , 1 µM butylated hydroxytoluene, and 0.1 µg pentostatin were added to avoid deamination and oxidation of the nucleosides30. The mixture was incubated for 2 h at 37 °C and then filtered through 96-well filter plates  at 3000 × g and 4 °C for 30 min. 1/10 Vol. of SILIS  as prepared in31 was added to each filtrate before analysis by QQQ mass spectrometry.tRNA (100 ng) in aqueous digestion mix (30 μL) was digested to single nucleosides by using 0.2 U alkaline phosphatase, 0.02 U phosphodiesterase I , and 0.2 U benzonase in Tris  and MgCl18, 100 Å, 100 × 2 mm . Mobile phase A was 2 mM ammonium acetate and mobile phase B was 80% acetonitrile containing 2 mM ammonium acetate. Gradient elution started with 0% B and increased to 12% B after 10 min and to 80% after 12 min. After 4 min elution at 80% B and subsequently regeneration of starting conditions to 100% A after 5 min, the column was equilibrated at 100% A for 8 min. The flow rate was 0.2 mL/min and the column temperature 30 °C. High-resolution mass spectra of precursor and product ions were recorded by a ThermoFinnigan LTQ Orbitrap XL. The parameters of the mass spectrometer were tuned with a freshly mixed solution of adenosine (5 μM). The parameters were sheath gas flow rate, 16 arb; auxiliary gas flow rate, 11 arb; sweep gas flow rate, 4 arb; spray voltage, 5.0 kV; capillary temperature, 200 °C; capillary voltage, 20 V, tube lens 65 V.The ribonucleosides were separated using a Dionex Ultimate 3000 HPLC system on an Interchim Uptisphere120-3HDO C18 or a Synergi, 2.5 μm Fusion-RP C2 gas temperature of 230 °C and N2 gas flow of 6 L/min, sheath gas (N2) temperature of 400 °C with a flow of 12 L/min, capillary voltage of 2500 V, nozzle voltage of 0 V, and nebulizer at 40 psi. The instrument was operated in dynamic MRM mode.For quantitative mass spectrometry an Agilent 1290 Infinity II equipped with a diode-array detector (DAD) combined with an Agilent Technologies G6470A Triple Quad system and electrospray ionization  was used. Operating parameters: positive-ion mode, skimmer voltage of 15 V, cell accelerator voltage of 5 V, N18, 100 Å, 150 × 2.1 mm) at 35 °C and a flow rate of 0.35 mL/min were used in combination with a binary mobile phase of 5 mM NH4OAc aqueous buffer A, brought to pH 5.6 with glacial acetic acid (65 μL), and an organic buffer B of pure acetonitrile . The gradient started at 100% solvent A, followed by an increase to 10% over 10 min. From 10 to 15 min, solvent B was increased to 45% and was maintained for 3 min before returning to 10% solvent A and a 3 min re-equilibration period.For separation a Core-Shell Technology column  and (2) for st-test analysis. p-values of student t-test  were calculated using Excel or Graphpad Prism.All experiments were performed at least three times  to allow student Further information on research design is available in the Supplementary InformationPeer ReviewReporting Summary


For all of the Brønsted acids investigated, the catalytic performances do not depend on the catalyst loading (mol ratio of Brønsted acid to substrate), but rather depend on the pH value of the aqueous reaction solution. All of them can give more than 98% conversion and selectivity in their aqueous solution at pH 1.30, no matter how much the catalyst loading is and what the Brønsted acid is. This pH-dependence principle is a very novel perspective to understand the Brønsted-acid catalysis system compared with our common understanding of the subject.A pH-dependence of the Brønsted acid-catalyzed oxidation of sulfides to the corresponding sulfoxides with H Among tperoxide ,36. Howeperoxide ,39,40,41peroxide ,43,44,45peroxide ,53,54,55peroxide ,57,58, hperoxide ,59,60,61peroxide ,64,65,662SO4) [3) [3BO3) [3COOH) [p-toluenesulfonic acid (H3CC6H4SO3H) [2NSO3H) [Several Brønsted acids have been employed in the Brønsted-acid catalysis system to catalyze the chemoselective oxidation of sulfides to corresponding sulfoxides, such as sulfuric acid (H2SO4) , hydroch2SO4) , nitric SO4) [3) , boric a) [3BO3) , chiral ) [3BO3) , acetic  [3COOH) , oxalic  [3COOH) , p-tolue6H4SO3H) , sulfami[2NSO3H) , dodecyl[2NSO3H) , immobil[2NSO3H) , immobil[2NSO3H) , immobil[2NSO3H) , and imm[2NSO3H) .Excellen2O2, and finally a pH-dependence principle was obtained. In this paper, we disclose the pH-dependence principle of the Brønsted acid-catalyzed chemoselective oxidation of sulfides with H2O2 based on our systematic investigation of the catalytic performance of a series of Brønsted acids, in which the conversion, yield and chemoselectivity depended only on the pH of the aqueous reaction solution, no matter how much the catalyst loading is and what the Brønsted acid is. To the best of our knowledge, our study reveals a commonness in the Brønsted acid-catalyzed oxidation of sulfides with H2O2 for the first time, so these attempts to explore the commonness in the different Brønsted acid-catalyzed chemoselective oxidation of sulfides with H2O2 will not only provide a summary of the abundant previous studies, but also an important reference for further studies of the Brønsted acid-catalyzed oxidation of sulfides, and even other Brønsted-acid catalysis systems.In our screening of modifying groups for β-cyclodextrin to construct artificial enzymes ,73,74, eCamphor-10-sulfonic acid derived from the natural product camphor was the first Brønsted acid investigated in our research. In order to optimize the reaction conditions, we chose the catalytic oxidation of methyl phenyl sulfide as a probe reaction and systematically evaluated the effect of catalyst loading on the probe reaction. As shown in p-toluenesulfonic acid, methanoic acid and acetic acid. The experimental data is presented in the p-toluenesulfonic acid, methanoic acid and acetic acid, respectively, as shown in In order to verify the generality of our findings, the scope of Brønsted acids were extended from camphor-10-sulfonic acid to methanesulfonic acid, In order to further confirm this commonness in the Brønsted acid-catalyzed oxidation of sulfides to the corresponding sulfoxides, some shared features in the aqueous solution reactions in p-toluenesulfonic acid, methanoic acid, sulfuric acid, hydrochloric acid, trifluoroacetic acid and boric acid were selected to prepare aqueous solutions with a series of pH values, and their catalytic performance in the probe reaction were investigated systematically. As shown in To verify our speculation, several typical Brønsted acids, such as camphor-10-sulfonic acid, methanesulfonic acid, A further decrease in the pH value led to no increase in all of the conversion, yield and selectivity, but rather a slight decrease. Similar catalytic performance profiles with pH of the aqueous solution reaction were obtained for all of the Brønsted-acid catalysis systems employed herein. The experimental data is presented in the In order to verify the generality of the pH-dependence principle, both the scope of Brønsted acids and sulfides were extended. It is clear from 2O and CH3CN in the volume ratio of 1:1 was employed as solvent to enhance the substrate dispersion, and satisfactory catalytic performance was achieved too.When the scope of sulfides was extended to a series of structurally diverse sulfides, a good substrate tolerance was observed when employing camphor-10-sulfonic acid as a typical Brønsted acid catalyst in pH 1.30 aqueous solution. As shown in 1H-NMR and 13C-NMR spectra were measured on a AVANCE III NMR spectrometer  at 400 MHz in CDCl3. Tetramethylsilane was used as the internal standard (0.00 ppm) in CDCl3. The ESI-MS spectra were measured on an Agilent 6210 TOF LC/MS mass spectrometer . GC analyses were performed on a GC-14B instrument  using a SPB-5 column (0.25 mm × 30 m). HPLC analyses were performed on a Waters 1525 chromatograph  equipped with a Waters 2996 Photodiode Array Detector using a Hypersil ODS2-5 column (4.6 mm × 250 mm). All the catalytic reaction were carried out in the Wattecs parallel synthesis system .1H-NMR, 13C-NMR and ESI-MS. Camphor-10-sulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, methanoic acid, sulfuric acid, hydrochloric acid, trifluoroacetic acid, boric acid, taurine (2-aminoethanesulfonic acid), homotaurine (3-aminopropanesulfonic acid) were purchased from Shanghai Energy Chemical Co. Ltd., Aladdin Chemistry Co. Ltd., and TCI Co. Ltd. 30% H2O2 were purchased from Shanghai Lingfeng Chemical Reagent Co. Ltd. , All the other common reagents were of an analytical grade. All of the reagents were used as received without further purification unless otherwise noted.Sulfides used were purchased from Shanghai Energy Chemical Co. Ltd. , Aladdin Chemistry Co. Ltd. , TCI Co. Ltd. , Aldrich Co. Ltd. , or synthesized from thiophenol and corresponding alkyl bromide. Methyl phenyl sulfoxide was purchased from Aldrich Co. Ltd. . Other sulfoxides were synthesized following the literature procedure  and char2O2 (1.15 mmol) was added. Then, the resultant mixture was kept stirring at 25 °C for 24.0 h. At the end of the reaction, saturated aqueous Na2SO3 (2.0 mL) was added to stop deep oxidation. The obtained solution was extracted with ethyl acetate (3 × 2.0 mL), and the combined organic phase was washed with brine (2.0 mL) and dried over anhydrous Na2SO4. The obtained solution was analyzed by GC or HPLC to determine the conversion and yield with p-xylene as internal standard.Sulfide (1.0 mmol) was added to a solution of Brønsted acid in water (2.0 mL) in corresponding amount, after stirring at 25 °C for 15 min, 30% H2O2 (1.15 mmol) was added. Then, the resultant mixture was kept stirring at 25 °C for 24.0 h. At the end of the reaction, saturated aqueous Na2SO3 (2.0 mL) was added to stop deep oxidation. The obtained solution was extracted with ethyl acetate (3 × 2.0 mL), and the combined organic phase was washed with brine (2.0 mL) and dried over anhydrous Na2SO4. The obtained solution was analyzed by GC or HPLC to determine the conversion and yield with p-xylene as internal standard.Sulfide (1.0 mmol) was added to a aqueous solution of Brønsted acid in certain pH (2.0 mL), after stirring at 25 °C for 15 min, 30% H2O2 is reported for the first time. For all the Brønsted acids investigated, their catalytic performances depend on the pH value of the aqueous solution reaction, and do not depend on the Brønsted acid loading (mol ratio of Brønsted acid to substrate). All of the Brønsted acids investigated can give more than 98% conversion and selectivity in their aqueous solution of pH 1.30 with an excellent substrate tolerance, no matter what the Brønsted acids are. To the best of our knowledge, this pH-dependence principle is the first common feature reported in the Brønsted acid-catalyzed chemoselective oxidation of sulfides and represents a very novel perspective to understand the Brønsted-acid catalysis system compared with our common understanding of these reactions. Thus, our finding will be an important reference not only to further studies of Brønsted acid-catalyzed oxidations of sulfides, but also to other Brønsted-acid catalysis systems. Further investigation on the catalytic mechanism and attempts to apply the pH-dependence principle to other Brønsted acid-catalyzed systems are under way.In conclusion, a pH-dependence principle in Brønsted acid-catalyzed chemoselective oxidation of sulfides to the corresponding sulfoxides with H