Total chromatography time for each run was 9 min. Time required for the sample pretreatment procedures was approximately 4 h. The coefficients of variation (CVs) of intra-assay were 10.4%, 3.2 % and 2.3% for vitamin K1 in three levels of quality control samples; were 14.3%, 3.2% and 6.7% for menaquinone-4; and were 11.1%, 6.0% and 7.0% for menaquinone-7. The inter-assay CVs were 12.8%, 11.3% and 7.4% for vitamin K1; were 15.2%, 9.2% and 8.7% for menaquinone-4; and were 13.2%,11.1% and 7.2% for menaquinone-7. No interference was found between K1, menaquinone-4 and menaquinone-7, nor any deuterated internal standards. This method was then used to determine reference values for Caucasian populations of central European origin. Samples were measured from 191 healthy volunteers (51.2 ± 16.2 years (mean ± SD)) and the values concerning K1 were 0.044–1.357 ng/mL for women and 0.030–1.214 ng/mL for men. The values for menaquinone-4 and menaquinone-7 did not exhibit any differences between women and men, and were 0.050–1.598 and 0.074–0.759 ng/mL, respectively.A novel application of the liquid chromatography method combined with the triple quadrupole tandem mass spectrometry method was developed for the quantification of vitamin K A synthetic form of vitamin K menadione (vitamin K3) can be found as an additive in some animal feeds. Recently, new synthetic vitamin K forms have been described: vitamin K4 (menadiol sodium phosphate), which is the water-soluble form derived from menadione by reduction; and vitamin K5 (4-amino-2-methyl-1-naphtol). Vitamin K1 is the main form of vitamin K in the Western diet (about 90%), whereas menaquinones make up about 10%. This form of vitamin K is important mainly for blood coagulation. The main functions of vitamin K2 were discovered to be bone metabolism, vascular calcification and cell proliferation. In some studies, vitamins K2, K3, K4 and K5 have been shown to have anticancer effects (Vitamin K is a cofactor for enzymatic modification of glutamic acid residues (Glu) to gamma-carboxyglutamic acid residues (Gla) in vitamin K-dependent Gla proteins. These Gla proteins are necessary for hemostasis, bone metabolism, vascular calcification and cell proliferation. Vitamin K is found in nature as phylloquinone . Before it can be reused, this vitamin K epoxide has to be converted back to reduced vitamin K (hydroquinone) by vitamin K epoxide reductase (VKOR) in a two-step reaction. At first, vitamin KO is converted to vitamin K by the VKOR. The second step is catalyzed by either VKOR or possibly another yet-to-be-defined reductase. Those two steps are sensitive to vitamin K antagonists such as warfarin. There is one warfarin-resistant pathway where the reduction of vitamin K to hydroquinone is catalyzed by a NAD(P)H-dependent reductase . Tie et Recommended daily intakes (RDIs) of vitamin K are very variable. In the US, the recommended adequate intake (AI) of vitamin K is 120 µg/day for men and 90 µg/day for women. The Italian Society of Human Nutrition suggested that the AI of vitamin K should be 140 µg/day for people 18–59 years old and 170 µg/day for people >60 years old. In the UK, the AI of vitamin K has not been precisely established. Previous data calculated AI using one µg/kg body weight. This amount might be adequate for blood coagulation, but it is insufficient for bone metabolism and vascular calcification . In the The most commonly used methods for determination of vitamin K levels are high performance liquid chromatography (HPLC) with post-column reduction and fluorescent or electrochemical detection, but these methods require extensive sample pre-purification due to interference of lipids. The newest methods are based on the principle liquid chromatography method used with tandem mass spectrometry (LC–MS/MS) methods, which should be more sensitive and specific with a lower limit of detection .1) were obtained from Toronto Research Chemicals Inc. .Liquid chromatography–mass spectrometry-grade methanol, HPLC-grade ethanol, LC–MS-grade isopropanol, n-hexane, diethyl ether, ammonium fluoride, and analytical standards of: phylloquinone, MK-4 and MK-7, were obtained from Supelco and Honeywell . Deuterium-labelled internal standards of the vitamins . Other necessary equipment were MS 40+ Vacuum Products  and a nitrogen generator NM32LA . The sample separation was carried out on a reversed phase column SB-C8, 1.8 µm, 2.1 × 100 mm .The column was operated at 30 °C. A chromatographic separation was achieved under the gradient flow of eluents, initially at 15/85 mix of mobile phase [A] water and methanol  and [B] methanol (100% with 0.1% ammonium fluoride). The gradient set up is shown in The Agilent Jet Stream with electrospray ionization (ESI) ion source operated in positive ion mode. The scan type used dynamic multiple reaction monitoring (MRM). The measurements used were: gas temperature at 340 °C, gas flow eight L/min, nebulizer at 28 psi, sheath gas temperature 380 °C, and sheath gas flow 12 L/min. An overview of the MRM transitions, collision energies and retention times for the analytes are given in 1, d7-MK-4, d7-MK-7) were prepared by dissolving of each vitamin to the final concentration of 200 ng/mL and to the final concentration of 500 ng/mL for each deuterated IS in ethanol. Calibration standards were prepared from stock solutions and pooled serum from healthy volunteers. The pooled serum was exposed to UV light for at least 24 h due to photodegradation of endogenousvitamin K. After 24 h, the pooled serum was measured to demonstrate the elimination of endogenous vitamin K. The calibration range was from 0.03 to 10 ng/mL: 0.03; 0.05; 0.1; 0.3; 0.6; 1.0; 1.5; 2.0; 4.0; 10.0 ng/mL, respectively. Stock and working standards of analytes and deuterated IS were stored at −20 °C. The pooled serum was spiked with the stock solution of the analytes to obtain low- and high-quality control (QC) samples in concentrations of 0.5 and 1.5 ng/mL. Prepared QC samples were stored at −80 °C.Stock solutions of the analytes  and deuterated IS ), with 149 participants being female and 42 male. Ethical approval for this study was obtained from Ethics committee of University Hospital Motol (Ref.No.: EK-1422/13). Written informed consent was obtained from the participants at recruitment prior to their inclusion in the study.g, 10 min), the serum was transferred to plastic tubes, protected from light and stored at −80 °C until analysis. Before analysis, 500 μL aliquots of serum were transferred into glass tubes and spiked with 10 μL of each ISs. Two mL of ethanol were added due to the denature of proteins. After briefly mixing, four mL of n-hexane were added. After mixing for 5 min, the solution was centrifuged at 2,147×g for 10 min. The supernatant was transferred to a new covered glass tube, and the bottom layer was re-extracted by another four mL of n-hexane. Mixing and centrifugation conditions were the same as the first extraction. Both supernatants were collected and evaporated under a stream of nitrogen at 50 °C. The lipid extract was dissolved by two mL of n-hexane, and then a solid phase extraction (SPE) was required. The sample was applied to the silica Sep-Pak extraction cartridge  connected to a Visiprep SPE Vacuum Manifold , which was washed prior with three mL diethyl ether:n-hexane (1:1) and then 3 × 3 mL of n-hexane. After the sample application, the cartridge was washed with two mL of n-hexane and then was washed again with 3 × 3 mL of n-hexane. The sample was eluted with 3 × 3 mL diethyl ether:n-hexane (3:97). The eluate was evaporated under nitrogen. The residue was reconstituted in 65 μL isopropanol. The prepared sample was transferred to amber micro-vials, capped and placed into the cooled autosampler rack. The aliquot of four μL was automatically injected into the system. The samples had to be protected from light during their preparation.The samples were collected in five mL Greiner Tubes (Vacuette) containing a clot activator. After centrifugation  corresponds to the lower limit of quantification (LLOQ). Limits of detection (LoD) were determined by a further dilution of the first calibration point (LLOQ). Concentrations of 0.0019 ng/mL for K1 and MK-7 and 0.00375 ng/mL for MK-4 were obtained.Our LC–MS/MS method for analysis of K1; were 14.3%, 3.2% and 6.7% for MK-4; and were 11.1%, 6.0% and 7.0% for MK-7. The inter-assay CVs were 12.8%, 11.3% and 7.4% for K1; were 15.2%, 9.2% and 8.7% for MK-4; and were 13.2%, 11.1% and 7.2% for MK-7.The intra- and inter-assay variation was determined using three concentration levels of QC samples: 0.05, 0.5 and 2.5 ng/mL. Intra-assay precision was obtained from 10 replicates measured in a single series and inter-assay imprecision from 25 different assays over a period of 6 weeks. The intra-assay coefficients of variation (CVs) were 10.4%, 3.2% and 2.3% for K1 were 10.2%, 3.5% and 4.9%; for MK-4 were 10.7%, 0.5% and 6.0%; and for MK-7 were 6.7%, 2.0% and 6.6%. The recoveries for K1 were 102.6–108.3%, for MK-4 were 94.0–108.7, and for MK-7 were 100.6–106.7%. Chromatograms of K1, MK-4 and MK-7 obtained by LC–MS/MS are shown in 1, and 23.0% for MK-7 after 7 days storage of serum in the refrigerator. The vitamins stored in the freezer were stable throughout the storage. Mean decreases of all vitamins were lower than 1%. The matrix effect was evaluated from vitamin free serum and water. The pretreatment procedure was performed, and after the SPE extraction the standards of each vitamin were added. The matrix factor was calculated in a range from −2.8% to +13% for all analytes. We performed a stability test of prepared sample stored in cooled autosampler for 168 h. There was a small decrease in vitamin K levels. The decreases did not exceed 12% against the original value in each form of vitamin K. We also tested an atmospheric pressure chemical ionization (ACPI) mode, but this type of ionization reported a lower sensitivity than the method with ESI. Our method with ESI is more convenient due to a routine laboratory mode.The within-day accuracy was expressed by calculating the bias between observed and theoretical concentrations, and for K1, MK-4 and MK-7 from the healthy Central European population were: 0.03–2.19 (0.20) ng/mL, 0.05–2.79 (0.19) ng/mL, and 0.05–1.39 (0.09) ng/mL, respectively (min—max (median)).We measured the vitamin K levels in a healthy Central European population. The group of patients consisted of 191 volunteers, 22% of them male, ages 51 ± 16 years (mean ± SD). Serum levels of K2 has been published in the literature. What can be found in the literature are different reference ranges for vitamin K1. For example, 1:0.044–1.357 ng/mL for women and 0.030–1.214 ng/mL for men. We performed Wilcoxon rank sum test with continuity correction, the p-value = 5.387e-06.The reference range calculated was 0.050–1.598 ng/mL for MK-4 for both genders, and 0.074–0.759 ng/mL for MK-7 for both women and men. The overview of our reference values is shown in No reference range for vitamin K1, MK-4 and MK-7 with low limits of quantification, which is suitable for the routine measurement of these vitamers in serum. This method enables us to find out the status of vitamin K. Vitamin K belongs to a group of liposoluble vitamins and is essential for bone metabolism and vascular health. Its key role was described in blood coagulation. Low vitamin K concentrations  are associated with increased risks of osteoporotic fractures in the elderly, and vascular calcification  or by measuring undercarboxylated proteins . Patients treated with warfarin are routinely monitored for INR in order to minimize the risk of bleeding. This test provides no information about the levels of vitamin Kitamin K .It has been published that it is much easier to find out the status of vitamin K by measuring under-carboxylated vitamin K dependent proteins, where the analytical methods are less complicated. However, ucOC is also affected by vitamin D, therefore the levels of ucOC may not reflect the actual concentration of vitamin K properly. We also performed another research showing that proteins induced in the absence of vitamin K (PIVKA II) did not reflect the levels of MK-7 in patients treated with 45 µg/day of MK-7 for at least for 3 months. PIVKA could be a sensitive marker of mild vitamin K deficiency, but with a slow response. The next indirect assays were measurements of urinary vitamin K metabolites such as 7C-aglycone and 5C-aglycone which belong to non-invasive assays. Results indicated a good relationship between urinary metabolites with dietary vitamin K intake. Urinary vitamin K metabolites were mainly tested in pediatric populations. The main disadvantages of the indirect assays of vitamin K status were slow response to vitamin K deficiency and non-differentiation of vitamin K forms .In recent years, research on LC–MS/MS methods have found them to be more sensitive and specific than HPLC methods . Quantif1, MK-4 and MK-7. The method has particular demands for the preparation of samples, but this is necessary, especially for the detection of low levels of MK . We developed and described the LC–MS/MS method for quantitation of K1, and it is from Immundiagnostik  with an LoD 0.15 ng/mL. The kit is for HPLC system using post-column zinc reduction and fluorescence detection. We performed some experiments using this kit, but we were not able to separate peaks of K1 and IS sufficiently.Presently, there is only one commercially available kit used only for the direct determination of K1, MK-4, and MK-7 using liquid chromatography with ACPI and tandem mass spectrometry (LC–APCI–MS). Their subjects were divided into three groups by age; the first group was premenopausal women 30–49 years old, the second group was postmenopausal women 50–69 years old, and the third group was postmenopausal women older than 70 years old. All women came from the region of Nagano, Japan, where the traditional meal “natto” is rich in MK-7. All groups had higher levels of MK-7 compared to our subjects: they had 4.96 ± 6.96 ng/mL in the first group, 8.42 ± 11.44 ng/mL in the second group and 4.21 ± 6.81 ng/mL in the third group. However, our subjects achieved a higher level of MK-4 compared to the subjects of 1 and up to 10 forms of vitamin K2 (MK-4 to MK-13) in faeces, serum and food. The serum levels of K1 were 0.45 ± 0.27 ng/mL in the group consuming refined grain and 0.54 ± 0.32 ng/mL in the group consuming whole grain. Serum levels of MK-4 to MK-13 were under LoD. 1 with LoD 0.3 ng/mL. This LoD is quite high considering the fact they used the LC–ACPI–MS method. An LC–MS/MS method was published for determination of K1, MK-4 and MK-7 using isotopically labeled vitamins as IS. 1 and MK-7; and 0.04 ng/mL for MK-4. The serum levels of PK, MK-4 and MK-7 in postmenopausal women with osteoporosis and without osteoporosis were published. Serum levels of K1, MK-4 and MK-7 in women with osteoporosis were: 0.43 ± 0.39, 0.89 ± 0.29 and 1.00 ± 1.02 ng/mL, respectively. Serum levels of K1, MK-4 and MK-7 in women without osteoporosis were: 0.49 ± 0.40, 0.83 ± 0.27 and 1.19 ± 1.08 ng/mL, respectively.1 and MK-7 levels in concentrations of 0.51 ± 0.37 and 0.29 ± 0.18 ng/mL, respectively (mean ± SD). In elderly normal subjects (n = 17), the levels of PK and MK-7 were 0.60 ± 0.28 and 0.33 ± 0.17 ng/mL, respectively (mean ± SD). Both of the MK-7 subject levels are very similar to our results 0.25 ± 0.21 ng/mL (mean ± SD), but there are high levels of vitamin K1. 1 in older men (n = 86) and women (n = 92) in China. In this paper, they did not determine the levels of menaquinones. The levels of PK were 0.85 ± 0.99 and 1.12 ± 1.45 ng/mL, respectively, in men and women (mean ± SD). They also had higher levels of K1 than our results. n = 741) for the determination of K1. The level of K1 was 0.69 ± 0.90 ng/mL. 1, MK-4 and MK-7 in 20 healthy subjects. Levels of vitamins K were 1.81 ± 1.11, 0.15 ± 0.17 and 16.27 ± 20.58 ng/mL for K1, MK-4 and MK-7 (mean ± SD), respectively. In this case, there were levels of MK-4 lower than our results, while levels of MK-7 were several times higher than our results. This could be present in the traditional Japanese meal nattō, which is rich in MK-7 as shown in the Tsugawa article (2006). n = 20) were 1.22 ± 0.57, 0.39 ± 0.46 and 6.37 ± 7.45 ng/mL for K1, MK-4 and MK-7 (mean ± SD), respectively. The serum levels of vitamin K reported in previous studies are shown in At this time, HPLC and LC–MS/MS methods are used for determination of vitamin K levels. Both types of methods can be used with sufficient pre-purification of the samples. The sufficient pre-purification of the sample is necessary for achievement of low LoD. No immunoassay for determination of vitamin K has been published.1, MK-4 and MK-7) determination. Due to the performance of solid-phase extraction, the LoD are low, and the method is sensitive and robust enough to quantify phylloquinone and two types of menaquinones in human serum. In order to improve the outcomes of some medical treatments, especially those related to bone metabolism and inhibition of vascular calcification, it is recommended to measure the levels of phylloquinone and menaquinones in serum or plasma.Our LC–MS method presented here is applicable for three forms of vitamin K . Determination of vitamin K levels is more suitable due to the early response of the organism. So far, the biggest problem is the lack of data of RDIs and reference ranges for all forms of vitamin K, which makes assessing the status of vitamin K more difficult. The difficult pre-purification sample process is time-consuming and requires high-tech devices. Therefore the measurement of this vitamin is only possible in a specialized laboratory. The low toxicity and beneficial effects of MK-7 on both bone metabolism and vascular calcification should be strongly considered as a therapeutic agent in the treatment of several diseases. Vitamin K could additionally play a role in the treatment of cancer or in the prevention of diabetes mellitus. Determination of vitamin K levels would be beneficial for patients undergoing warfarin therapy.10.7717/peerj.7695/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.7695/supp-2Supplemental Information 2Click here for additional data file.


The results showed a high stability of amorphous imatinib mesylate adequate to enable its use in solid dosage form.The proposed study examined the characterization and stability of solid-state amorphous imatinib mesylate (IM) after 15 months under controlled relative humidity (60 ± 5%) and temperature (25 ± 2 °C) conditions. After 2 weeks, and 1, 3, 6, and 15 months, the samples were characterized using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray powder diffractometry (XRPD), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM). Additionally, the amorphous form of imatinib mesylate was obtained via supercooling of the melt in a DSC apparatus, and aged at various temperatures  and time periods (1–16 h). Glass transition and enthalpy relaxation were used to calculate molecular-relaxation-time parameters. The Kohlrausch–Williams–Watts (KWW) equation was applied to fit the experimental enthalpy-relaxation data. The mean molecular-relaxation-time constant ( N-u[4-methyl-3-(4-pyridin-3-yl)pyrimidine-2-ylamino)phenyl]benzamide methanesulfonate, is a tyrosine-kinase inhibitor commonly used in the treatment of chronic myeloid leukemia [α- and β-form. Grillo et al. showed that these forms transform into amorphous IM upon grinding, followed by conversion to the crystalline phase during aging or thermal treatment. Furthermore, recent studies have found that α and β-forms are enantiotropically related. Detailed reports have confirmed these solid forms based on crystallographic and spectroscopic techniques [Amorphous substances form a separate class of solids, which are distinct from the more common and well-known crystalline solid. Crystalline materials consist of a three-dimensional long-range structure, which is not present in the amorphous state. Furthermore, compared to crystalline state, the positioning of amorphous state molecules are more irregular, as well as possessing high internal energy and specific volume, which improves dissolution and bioavailability ,2,3. Theleukemia ,21,22 anleukemia ,24,25. Ileukemia ,27, throleukemia ,29, and leukemia ,31. Imatleukemia ,35,36,37leukemia ,42,43,44chniques ,46.Tg prevents crystallization and ensures a physically stable drug during its shelf-life [τ” and relaxation-time distribution parameter β using the Kohlrausch–Williams–Watts (KWW) equation. Moreover, the physicochemical characteristics of IM at time ageing were determined by thermal techniques X-ray powder diffractometry (XRPD), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM). This is the first time such a study of amorphous IM was presented.To the best of our knowledge, information related to the stability and characterization of IM amorphous form in the literature is non-excitant. However, there is a strong relationship between molecular mobility and stability of amorphous form in pharmaceutical substances. To predict the life expectancy of pure amorphous compounds in the study, we searched for correlations between crystallization tendency of glass formers with molecular mobility and thermodynamic properties of the amorphous state. It was recognized that storage well below elf-life ,47,48. Iα form used in this study was synthetized in a new continuous-flow microwave reactor, described in [Crystalline imatinib mesylate ribed in  and donaα form [Our previous studies confirmed that both commercial and synthetized IM occur in crystalline α form .Preparation of the amorphous form of a drug for physical stability studies was achieved by melting the drug in a stainless-steel beaker on a hot plate, followed by subsequent quenching by cooling the melt over crushed ice (method A). The quench-cooled product was ground, sieved using 315 μm sieve, and examined after 2 weeks, 1 month, 3 months, 6 months, and 15 months, respectively.Additionally, the amorphous form of IM was obtained by supercooling the melt in a DSC apparatus (method B), and aged at various temperatures  for periods of time between 1 and 16 h.The physical stability of amorphous IM was monitored for fifteen months under controlled relative humidity (60 ± 5%) and temperature (25 ± 2 °C), which was the long-term stability condition, selected based on Guideline for Industry Q1A (R2) Stability Testing of new Drug Substances and Products . Periodi® 7.1.0 analysis software . The apparatus was calibrated using standard samples from calibration set 6.239.2-91.3 supplied by Netzsch . All samples were weighed on a Sartorius CPA225D-0CE analytical balance  with a resolution of 0.01 mg.Differential scanning calorimeter DSC 214 Polyma instrument  equipped with an Intracooler IC70  was used. Measurements of the amorphous samples were performed using the heat-flow measurement method at a heating rate of 10 °C per minute over a temperature range of 10–250 °C in a nitrogen dynamic atmosphere (25 mL/min). The samples of approx. 8.5 mg were placed in an aluminium pan (25 µL) with a pierced lid, and subsequently sealed. An empty pan of the same type was employed as reference. DSC peak area and transition temperatures were determined using Netzsch ProteusaT)  for a predetermined period of ageing time (at) . Following the specified aging time, the glassy materials were heated at 10 °C per minute to 185 °C. A cycle for unaged sample was processed. This second heating determined the change heat capacity (pΔC), the extrapolated onset glass transition temperature (gT) and the enthalpy recovery (tΔH).In this study, the ageing experiments of IM crystals were also performed. The schematic representation of the entire temperature program is illustrated in ® 7.1.0 analysis software  was used to determine the mass change of the samples and DTG curves.The thermal stability of the samples was investigated by thermogravimetric analysis using TG 209 F1 Libra  thermobalance. The experiments were carried out in alumina crucibles (150 μL), under nitrogen atmosphere (25 mL/min) at a heating rate of 10 °C per minute. TGA/DTG curves were obtained in the temperature range of 25–600 °C. Netzsch Proteusθ/2θ) horizontal geometry between 7° and 40° (2θ) (step size 0.02° and 0.1 s/step) at 295 K. XRD patterns were analyzed using the software Diffrac.EVA v.2.1 Crystalline/amorphous nature of IM was determined using Bruker D2 PHASER diffractometer  with a LynxEYe detector. Cu Kα radiation (1.5418 Å) was used at 30 kV and 10 mA. Diffractograms were obtained in the Bragg-Brentano  analyzer equipped with OMNIC analysis software was used for FTIR spectra registration. Spectra were collected in attenuated total reflectance (ATR) mode. A small amount of each sample was placed on the diamond crystal and scanned over a wavelength of 400 cmScanning electron microscope (SEM) images were taken on Zeiss EVO MA25  apparatus at an accelerating voltage of 20 kV. The samples were covered with gold and palladium  using a Quorum machine .DSC curves of amorphous IM samples stored at 25 °C under controlled relative humidity at different ageing times are presented in tΔH) data for amorphous IM were obtained. Using the KWW equation, the average relaxation time constant τ and dimensionless relaxation time distribution parameter β were estimated , enthalpy recovery . The quality of matching the KWW functions to the experimental data was determined by coefficients of determination R2 (R-squared). The correctness of the entire model was additionally checked by residual analysis (50%t listed in t∅ = 0.50. By comparing τΦ(t) = 50% values of each glass at each aging temperature, it was found that as aging temperature decreased, τΦ(t) = 50% values increased dramatically, hence, the rate of the aging process dramatically decreased at lower aging temperatures. A rapid decrease in 50%t values was observed with increasing aging temperatures from 4.91695 × 1033 h at 3 °C to 8.21992 × 1016 h at 30 °C, which again slowed the aging process at lower temperatures. Calculated mean molecular relaxation time constants for amorphous IM range from 3.609324 × 1039 to 6.393168 × 1021 s, which are comparable with values obtained for griseofulvin, tolbutamide, troglitazone, and simvastatin [The statistical significance of analysis . For eacvastatin .β ranging from 0 to 1 were characterized. The smaller the β value, a greater distribution of molecular motion deviates from a single exponential behavior was obtained. Estimated values of β parameters were comparable at different ageing temperatures but vary significantly from the unit. Therefore, this indicates a distribution of time scale rather than one single relaxation time. Furthermore, with an increase in ageing temperature the mean relaxation-time constants τ decreased.The width of relaxation-time distribution parameter ® ver. 13.1 from Dell.The statistical analysis presented in mT); extrapolated onset temperature of decomposition (OnsetT); rate of mass loss, which corresponds to mT; extrapolated temperature at which the degradation process ends (EndsetT); temperature at which 0.5 wt.% and 1.0 wt.% loss occurs and the amount of residue (Res) at 600 °C, are presented in Thermogravimetry and derivative thermogravimetry (DTG) was used to identify the thermal stability of amorphous IM. TGA and DTG curve of the substance at 0 time and after a specified time are presented in −1, the rate of mass loss. As shown in OnsetT and T0.5 wt.% should be taken into consideration during thermal stability assessment.From the above results it can be extrapolated that the thermal degradation of the analyzed samples occurs in one step. The average temperature of maximum weight loss rate for the main effect was 371.57 °C, which corresponds to 5.42% minmT = 376.0 °C, rate mass loss = 5.61 %·min−1, OnsetT = 325.6 °C, EndsetT = 424.1 °C, 0.5 wt.%T = 278.6 °C, 1.0 wt.%T = 291.7 °C, and Res = 40.29% were determined. In the temperatures ranging between 25–140 °C no mass loss was observed related to moisture.According to TGA/DTG curve registered for the crystalline form of the substance, decomposition occurs in one step. The thermal decomposition parameters: θ values of 10.38°, 11.17°, 11.89°, 12.11°, 12.85°, 13.78°, 14.82°, 16.42°, 17.62°, 18.02°, 18.53°, 19.03°, 21.19°, 19.75°, 21.19°, 21.55°, 22.56°, 23.12°, 23.66°, 24.83°, 26.24°, 27.33°, 27.93°, 28.46°, and 31.92. The obtained results correspond to α form and literature data [XRPD pattern of crystalline IM confirms its crystalline nature a. The sture data . The amo−1 region are presented in −1 (N–H stretching vibration), 1657 cm−1 (C=O band), 1570, 1525, and 1444 cm−1 , 1158 cm−1 (C–N stretching vibration), 1036 cm−1 (C–O stretching vibration), and 807 cm−1 (aromatic C–H deformations out of plane). ATR-FTIR spectra for crystalline and amorphous IM samples were qualitatively similar. The spectra of amorphous forms showed peak broadening attributed to the relative disorder of the molecules in the amorphous form, resulting in a broader distribution of bond lengths and energies with respect their crystalline counterparts.ATR-FTIR spectra of IM samples in the 4000–400 cmIn order to estimate the morphology of IM samples, scanning electron microscopy (SEM) analysis was performed. SEM images of crystalline and amorphous IM are shown in A delicate balance exists among the chemical and physical properties of amorphous materials. Slight alterations or disturbances can have a major influence on stability, as well as future production and dissolution characteristics of the final product . UnfortuHerein, we showed efficient methodology for the preparation of amorphous IM via its crystalline form. The process eliminated unwanted chemical degradation and the presence of amorphous IM was confirmed using analytical techniques: DSC, ATR-FTIR, and XRPD. Glass transition and enthalpy relaxation were experimentally determined and used to calculate molecular relaxation-time parameters. The KWW equation fitted the experimental enthalpy relaxation data well. Two non-linear optimization algorithms: Levenberg–Marquardt and Quasi–Newton were applied to predict the long-term amorphous stability of IM. It was determined that the mean molecular relaxation time constant (τ) increased with decreasing ageing temperature.By calculating the relaxation time of amorphous IM, it was possible to estimate the timescale of undesirable solid-state degradation or crystallization as a function of temperature. The calculated mean molecular-relaxation time constants for amorphous IM confirmed the long-term stability of the examined samples. This amorphous substance could be a potential candidate for the development of novel drug products.


The dose-response PDI results confirm the relevance of the number of positive charges on the porphyrin molecule in the PDI of E. coli. The difference between the Hill coefficients of cationic porphyrins with 1–3 positive charges and the tetra-cationic porphyrin (4) revealed potential variations in their mechanism of inactivation. Fluorescent live-cell microscopy studies showed that cationic porphyrins with 1–3 positive charges bind to the cell membrane of E. coli, but are not internalized. On the contrary, the tetra-cationic porphyrin (4) permeates through the membrane of the cells. The contrast in the interaction of cationic porphyrins with E. coli confirmed that they followed different mechanisms of inactivation. This work helps to have a better understanding of the structure-activity relationship in the efficiency of the PDI process of cationic porphyrins against Gram-negative bacteria.Photodynamic inactivation (PDI) is a non-antibiotic option for the treatment of infectious diseases. Although Gram-positive bacteria have been shown to be highly susceptible to PDI, the inactivation of Gram-negative bacteria has been more challenging due to the impermeability properties of the outer membrane. In the present study, a series of photosensitizers which contain one to four positive charges ( A mixtu2Cl2 (30 mL) and flushed with N2 at room temperature. NO2BF4  was added dropwise to the reaction mixture and stirred for 60 min. The nitration of TPP was monitored by thin layer chromatography (TLC). Four aliquots of NO2BF4 (0.625 mL) were added dropwise every 60 min for a total of 2.5 mL of NO2BF4 solution. After the final addition, TLC analysis showed a small amount of unreacted TPP and other red spots representing the mono- and di-nitrated porphyrins. To produce more di-nitrated porphyrin, the reaction was stirred under N2 for an additional 24 h. Next, the reaction mixture was extracted with water. The organic layer was collected, and the volatile removed under vacuum. To remove sulfolane, a minimal amount of acetone (<5 mL) was added, followed by cold water (50 mL) which caused the porphyrin mixture to precipitate. The porphyrin mixture was collected by vacuum filtration. MALDI-TOF analysis of the mixture showed that the reaction produced a mixture of TPP and mono- and di- nitrated porphyrins. Separation of the mixture was performed using silica chromatography and eluting with CH2Cl2: Hexanes (1:1). The desired di-nitrated porphyrin was collected as the third red band. Yield: 67% wt. 1H NMR : δ 8.52–8.66 , 8.34–8.37 , 8.10–8.13 , 7.93–7.95 , 7.52–7.59 , −3.07 . FTIR : 3316 (N–H stretch), 2851–3102 (C–H stretch), 1594 (Aromatic), 1342, 1513 (NO2 stretch), 964 (C–N stretch). MALDI-TOF (m/z): [M]+ = 704.50. Calculated for [M]+ = 704.22.TPP  was dissolved in CH2∙H2O  was added to this solution, and the reaction mixture was heated to 65 °C. The reaction was stirred for 24 h at 65 °C. D.I. H2O (50 mL) was added to dilute the solution. Then 0.1 M NH4OH was added until pH = 8 was reached. The solution was washed with chloroform (100 mL). The aqueous and organic layers were separated using a separatory funnel. The organic layer was collected, and the solvent was removed using rotary evaporation to dryness. Silica chromatography and chloroform were used to obtain the products. The desired di-amine porphyrin mixture eluted as the second band. Yield: 87% wt. 1H NMR : δ 8.80–8.92 , 8.19–8.22 , 7.96–7.99 , 7.72–7.74 , 7.01–7.04 , −2.74 . FTIR : 3451, 3362, 3321 (N–H stretch), 2851–3026 (C–H stretch), 1468 (C–H bend), 1616 (aromatic). MALDI-TOF (m/z): [M]+ = 644.61; Calculated for [M]+ = 644.27.5,10-(4′-nitrophenyl)-15,20-triphenylporphyrin and 5,15-(4′-nitrophenyl)-10,20-triphenyl porphyrin mixture  was dissolved in 37% HCl (42.5 mL) and sonicated to assist dissolution. SnCl2 at room temperature. CH3I  was added and the reaction stirred for 24 h. After 24 h, CH2Cl2 (25 mL) and water (25 mL) were added to the reaction mixture. The aqueous and organic layers were separated using a separatory funnel. The desired porphyrin product and starting porphyrin resided in the organic layer. Therefore, the organic layer was collected, and the solvent was removed using rotary evaporation to dryness. CH2Cl2 (1 mL) was added back to the round bottom flask and an excess of diethyl ether was added which caused the alkylated porphyrin to precipitate down. The precipitate was collected by vacuum filtration. Yield: 75% wt. 1H NMR : δ 8.77–8.87 , 8.47–8.50 , 8.41–8.44 , 8.19–8.22 , 7.83–7.85 , −2.95 , 3.91 . FTIR : 3317, 3422 (N–H stretch), 2851–2988 (C–H stretch), 1596 (aromatic). ESI-MS (m/z): [M–2(I−)-2H]+ = 364.49; Calculated for [M–2(I−)-2H]+ = 364.19.5,10-(4′-aminophenyl)-15,20-triphenylporphyrin and 5,15-(4′-aminophenyl)-10,20-triphenylporphyrin mixture  was dissolved in anhydrous DMF (5 mL) and flushed with N2Cl2 (30 mL) and flushed with N2 at room temperature. One aliquot of NO2BF4  was added dropwise to the reaction vessel. The reaction mixture stirred at room temperature for 60 min. Additional aliquots of NO2BF4  was added every 60 min until TLC analysis indicated a complete consumption of TPP. A total of ten aliquot of NO2BF4 were added . After the final addition, the reaction stirred under N2 for an additional 24 h. The reaction mixture was extracted with water. The organic layer was collected, and the volatile removed under vacuum. To remove sulfolane, a minimal amount of acetone (<5 mL) was added, followed by cold water (25 mL) which caused the porphyrin mixture to precipitate. The porphyrin mixture was collected by vacuum filtration. MALDI-TOF analysis of the mixture showed that the reaction produced a mixture of mono-, di-, and tri-nitrated porphyrins. Separation of the mixture was performed using silica chromatography and eluting with CH2Cl2:Hexanes (1:1). The desired tri-nitrated porphyrin was collected as the third red band. Yield: 44% wt. 1H NMR : δ 8.83–8.84 , 8.73 , 8.68–8.70 , 8.56–8.59 , 8.31–8.33 , 8.10–8.12 , 7.69–7.71 , −2.91 . FTIR : 3315 (N–H stretch), 2857–3046 (C–H stretch), 1593 (aromatic), 1341–1512 (NO2). MALDI-TOF (m/z): [M+1]+ = 750.65. Calculated for [M+1]+ = 750.20.TPP  was dissolved in CH2∙H2O  was added to this solution, and the reaction mixture was heated to 65 °C. The reaction was stirred for 24 h. D.I. H2O (25 mL) was added to dilute the solution. Then 0.1 M NH4OH to diluted solution until a pH = 8 was reached. The solution was washed with chloroform (50 mL). The aqueous and organic layers were separated using a separatory funnel. The organic layer was collected, and the solvent was removed using rotary evaporation to dryness. Silica chromatography was used to purify the product. The mixture was eluted with chloroform and methanol (1%) and the second band was collected giving the desired tri-amine porphyrin. Yield: 40% wt. 1H NMR : δ 8.92–8.93 , 8.81–8.83 , 8.22 , 7.95–7.98 , 7.73 , 6.97–6.99 , −2.68 . FTIR : 3316 (N–H stretch), 2853–2922 (C–H stretch), 1598 (aromatic), 1473 (C–H stretch), 1035 (C–N stretch). MALDI-TOF (m/z): [M–1]+ = 658.51, [M]+ = 659.58. Calculated for [M]+ = 659.28.5,10,15-tris(4′-nitrophenyl)-20-phenylporphyrin  was dissolved in 37% HCl (25 mL) and sonicated to assist dissolution. SnCl2 at room temperature. CH3I  was added and the reaction stirred for 24 h at 45 °C. After 24 h, an excess of acetone (25 mL) was added which caused the alkylated porphyrin to precipitate down. The precipitate was collected by vacuum filtration. Yield: 28% wt. 1H NMR : δ 8.85–8.91 , 8.79–8.80 , 8.49–8.52 , 8.42–8.45 , 8.20–8.23 , 7.78–7.87 , 3.93 , −2.96 . FTIR : 3397, 3316 (N–H stretch), 2849–3005 (C–H stretch), 1597 (aromatic). ESI-MS (m/z): [M–3(I−)]+ = 262.58; Calculated for [M–3(I−)]+ = 262.81.5,10,15-tris(4′-aminophenyl)-20-phenylporphyrin  was dissolved in anhydrous DMF (5 mL) and flushed with N3I  in anhydrous DMF (10 mL) was heated to 45 °C and stirred for 24 h under N2. After 24 h, excess acetone (25 mL) was added to precipitate down the product. The precipitate was collected by vacuum filtration. Yield 68% wt. 1H NMR : δ 8.89 , 8.49–8.51 , 8.46–8.48 , 3.96 , −2.94 . FTIR : 3420 (N–H stretch), 2752–3019 (C–H stretch), 1460 (C–H bend), 1577 (Aromatic). ESI-MS (m/z): [M–4(I−)]+ = 211.80. Calculated for [M–4(I−)]+ = 211.63.A mixture of 5,10,15,20-tetrakis(4′-aminophenyl) porphyrin  and CHStock solutions (1 mM) of each porphyrin were prepared in DMSO and DMF. For biological assays, the stock solutions of photosensitizers prepared in DMSO were diluted to the final concentrations in phosphate buffer solution  of 1% DMSO.F) in DMSO were determined using the comparative method, as shown in Equation (2). TPP with a quantum yield of 0.12 benzene was used as the reference [www.graphpad.com.Absorption and emission measurements for the cationic porphyrins were collected on solutions of 5 µM in DMSO. The fluorescence quantum yields for air-saturated solutions (Φeference . The porΔ = 0.62) [5 (ΦΔ = 0.54) [www.graphpad.com.The quantum yields of singlet oxygen were determined through the absorbance decay of DMA in DMF using the comparative method, as shown in Equation (1). Solutions containing DMA (50 µM) and the PS  were prepared in DMF saturated with oxygen. After preparation, the solutions were protected from light. A volume of 2 mL of the solution was filled in a quartz cuvette (1 cm × 1 cm), set into a fluorometer  and irradiated at 515 nm. The irradiation period, controlled by a shutter, was maintained for time intervals (0–600 s). The decay of DMA was monitored at 379 nm. A reference spectrum of the PS in DMF was taken before each experiment and subtracted from the final data. The experiments were performed at least two times for each PS. The experimental design was confirmed by comparison with two references, TPP (Φ = 0.62)  and 5 (Φ = 0.54)  in DMF. E. coli (427) was inoculated in liquid Luria Broth (LB) containing 50 µg/mL kanamycin and grew at 37 °C for approximately 6–8 h until the optical density at 600 nm (OD600nm) reached approximately 0.8 (a.u.). Cell suspension solutions were prepared using PBS . Bacterial cells were harvested through centrifuging  and washed with PBS. After discarding the supernatant, the remaining bacterial cell pellet was resuspended in PBS, 1% DMSO solution (1.0 mL) to an OD600nm ~0.6–0.8.−2 for 20 min without stirring. The light power density was measured with OPHIR VEGA power/energy meter.The effect of the cationic porphyrin derivatives was evaluated by exposing bacterial suspensions to white light (400–700 nm) with a distance of 10 cm from the surface of the cell culture to the light source, and at an irradiance of 44 J cm600nm ~0.6–0.8; 3–4 108 CFU/mL) in PBS (1% DMSO) solution (1.0 mL) was incubated at 37 °C for 30 min in the dark in the presence of each porphyrin (concentration = 1 nM to 10 µM). After 30 min, an aliquot of 100 µL representing the “dark toxicity” was removed for each experiment and stored in a 1.5 mL Eppendorf centrifuge tube. The original cell suspension was centrifuged  to remove unbound PS. The obtained bacterial cell pellets were resuspended in PBS (1% DMSO) solution (900 µL) and exposed to white light (44 J cm−2) for 20 min. After white light exposure, another aliquot of 100 µL representing the “light toxicity” sample was removed and stored in a new 1.5 mL Eppendorf centrifuge tube.A bacterial suspension  and the dilution factor. The cell survival percentage of the dark and light samples were calculated as a percent of the control using Equation (3).The survival percentage was determined by counting the colony-forming units (CFU). The control, dark toxicity and light toxicity solutions were serially diluted 10600nm ~0.6–0.8; 3–4 108 CFU/mL) were incubated with each porphyrin (concentration = 1 µM) at 37 °C for 30 min in the dark. After discarding the unbound PS by centrifugation , the cell pellet and tightly-bound porphyrin was resuspended in PBS . Aliquots of 40 µL cell suspensions were pipetted to glass slides containing agarose pads (0.7%).To visualize the interaction between bacterial cells and cationic porphyrins, fluorescence live-cell deconvolution microscopy was used. Bacterial suspensions (ODSamples were excited with 390 nm and emission captured at 679 nm using Delta Vision’s Standard DAPI/FITC/TRITC/Cy5 filter set. Z-stack images were collected at 0.2 µm z-increments on a DeltaVision Elite Workstation (GE-Helathcare) based on an inverted microscope  using a 100×/1.4 NA oil immersion lens and Nomarski optics. Images were captured at room temperature with a 12-bit CCD camera  and deconvoluted using the interactive-constrained algorithm and the measured point spread function. All image analyses were performed using ImageJ v1.51n .4 into bacterial cells occurred via the self-promoted uptake pathway, the uptake of compounds 4 and 3 into E. coli cells was studies by incubating the tetracationic porphyrin in bacterial culture solutions pre-exposed to increasing concentrations of Mg+2, source MgCl2. A stock solution of MgCl2 (1 M in autoclaved DI water) was prepared. Next, different volumes of MgCl2 (1M in autoclaved DI water) were added to bacterial suspensions (OD600nm ~0.6–0.8) in PBS solution (1.0 mL) to obtain final concentrations of 0, 10, 25, and 50 mM MgCl2. The bacterial solutions were incubated at 37 °C for 30 min. The bacterial solutions were centrifuged  to remove unbound Mg+2. The obtained bacterial pellets were dispersed in a pre-made PS solution . Then the PS/bacterial mixture was incubated at 37 °C and protected from light for 30 min. After incubation, unbound PS was removed from the suspension by centrifuging . To extract the cell-bound PS from the bacterial cells, the obtained pellet was dispersed in 2% sodium dodecyl sulfate (SDS) at room temperature for 14 h. The concentration of PS in solution was analyzed by fluorescence. The samples were excited at 415 nm and the emission of the PS was monitored at the fluorescence maximum . The PS concentration was found by interpolation with a calibration plot constructed with known concentrations of each PS in 2% SDS . The significance of charge number and porphyrin concentration on bacterial inactivation was assessed by one-way univariate analysis of variance (ANOVA) model with the Tukey’s post hoc test. A value of E. coli. The dose-response graph showed that the PDI efficiency of the porphyrins against E. coli was directly correlated with the number of positive charges following the order: 5 < 1 < 2 < 3; nevertheless, this trend is not followed by compounds 4 and 6. Hill coefficients obtained from the dose-response data indicate that compounds 1–3 may follow a different mechanism of inactivation than compounds 4 and 6. Fluorescent live-cell microscopy revealed that indeed the interaction of compounds 1–3 with E. coli is completely different than the one for compounds 4 and 6. Under the conditions used for our experiments, compounds 1–3 are localized in the cell membrane; however, compounds 4 and 6 have already been internalized by E coli cells. The contrast in the cellular localization of the cationic porphyrin derivatives leads to different molecular targets for the PDI. These results shed light to explain the difference in PDI efficacy between cationic porphyrin derivatives against E. coli. Nevertheless, the mechanisms of cellular death after PDI treatment with these cationic porphyrins derivatives at biomolecular level needs to be further investigated.The results in this study confirmed that the number of cationic groups on porphyrins has a major impact on the PDI of


Collagen is widely used in the pharmaceutical, tissue engineering, nutraceutical, and cosmetic industries. In this study, acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the skin of red stingray, and its physicochemical and functional properties were investigated. The yields of ASC and PSC were 33.95 ± 0.7% and 37.18 ± 0.71% (on a dry weight basis), respectively. ASC and PSC were identified as type I collagen by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis, possessing a complete triple helix structure as determined by UV absorption, Fourier transform infrared, circular dichroism, and X-ray diffraction spectroscopy. Contact angle experiments indicated that PSC was more hydrophobic than ASC. Thermal stability tests revealed that the melting temperature of PSC from red stingray skin was higher than that of PSC from duck skin, and the difference in the melting temperature between these two PSCs was 9.24 °C. Additionally, both ASC and PSC were functionally superior to some other proteins from terrestrial sources, such as scallop gonad protein, whey protein, and goose liver protein. These results suggest that PSC from red stingray skin could be used instead of terrestrial animal collagen in drugs, foods, cosmetics, and biological functional materials, and as scaffolds for bone regeneration. Using the first diffraction peaks of ASC and PSC (peak A1) at 8.05° and 7.94°, respectively, the distances between the molecular chains of ASC and PSC were 1.10 and 1.11 by Bragg’s Law. The distance between PSC molecular chains was greater than that between ASC molecular chains, indicating that pepsin selectively cleaves the terminal peptide sequences of collagen fibers, weakens the interaction between collagen molecules, and increases the distance between collagen molecular chains . Using tBoth collagens carried positive charges at pH 3–6 and negative charges at pH 7–10. At zero zeta potential, the isoelectric points (pIs) of ASC and PSC were 6.71 and 6.41, respectively. At pH levels above or below the pI of collagen, the repulsion between collagen peptide chains increased, thereby increasing its net solubility . The pI The static WCA of ASC was 100.3° ± 2.31°, and that of PSC was 94.96° ± 0.59°. With values greater than 90°, the contact angles indicate hydrophobicity in both ASC and PSC . The lard) and melting temperature (Tm). Td is the temperature at which the collagen triple helix structure dissociates into random coils in a solution [d = 24.7 °C, ASC; 26.7 °C, PSC) [d = 28.0 °C, ASC; 25.5 °C, PSC) [d = 24.3 °C) [d ≈ 34.5 °C, ASC; 34.5 °C, PSC) [d = 29.5 °C, ASC; 27.5 °C, PSC) [d = 14.5 °C, ASC; 16.8 °C, PSC) [d = 15.12 °C, ASC; 14.66 °C, PSC) [m is defined as the temperature at which the physical form of collagen changes from solid to liquid [m, collagen loses its primary structure. DSC performed on the ASC and PSC from red stingray skin provided melting temperatures of 85.25 °C and 95.46 °C, respectively  , the ski°C, PSC) , and of °C, PSC)  and of r°C, PSC) . Further°C, PSC) . Tm is do liquid . At tempectively B. The diss-links . Collagess-links . ConsideAs the concentration of ASC and PSC increased from 5 to 25 mg/mL, complex viscosity (η*) increased, indicating that concentration affects the stability of the network structure in ASC and PSC solutions . AdditioThe η* of the PSC solution decreased gradually when the temperature increased to 20–45 °C and the solution exhibited shear-thinning flow behavior A,C. The p <0.05). The collagens from red stingray skin had a higher WAC than scallop gonad protein (6.5 mL/g) [p <0.05). As demonstrated by Jitngarmkusol et al., the OAC of a protein is related to its non-polar amino acid residues. The hydrophobic interaction between the non-polar amino acids of protein molecules and the hydrocarbon chains of oil determines the OAC of the protein [The WACs of ASC and PSC were 20.76 ± 0.55 and 28.48 ± 0.69 mL/g, respectively, and were significantly different (.5 mL/g) , gluteli.5 mL/g) , and gre.5 mL/g) . These d protein . The OAC protein , gluteli protein , and gre protein . As repo protein ; thus, tThe foaming and emulsifying properties of collagen samples were characterized by foaming capacity (FC), foam stability (FS), emulsifying activity index (EAI), and emulsifying stability index (ESI). The FC of ASC and PSC varied between 47.62 ± 3.50 and 146.67 ± 2.89 and between 71.43 ± 3.20 and 151.67 ± 5.77%, respectively A. The FC2/g and between 80.19 ± 0.04 and 105.53 ± 0.41 m2/g, respectively (2/g) [2/g) [2/g) [The EAI values of ASC and PSC varied between 61.06 ± 0.83 and 117.11 ± 0.25 mectively C. Meanwhectively D. The EAg) [2/g) , and goog) [2/g) . These rp < 0.05). Additionally, there was a significant difference in OD value between the negative control and collagen samples on day 1 , suggesting the potential use of ASC and PSC in drugs, foods, cosmetics, and as biomedical materials [Cell proliferation experiments were performed to determine the biocompatibility of NIH3T3 fibroblasts. A higher optical density (OD) value indicated a greater cell proliferation rate . As showaterials . HoweverDasyatis akajei) skin was purchased from an aquatic product processing plant located in Zhangzhou, China. All red stingray experiments were executed according to the protocol approved by Institutional Animal Care and Use Committee of Third Institute of Oceanography, Ministry of Natural Resources. . Meat and fat layers were removed from the skin by hand. After washing with water, the skin was stored in polyethylene bags and kept at −20 °C for future analyses. N,N,N´,N´-tetramethylethylenediamime (TEMED), sodium dodecyl sulphate (SDS), and Coomassie blue R-250 were obtained from Bio-Rad Laboratories . High-molecular-weight markers, pepsin, trifluoroacetic acid (TFA), and formic acid were obtained from Sigma Chemical Co. . All other chemicals and reagents used in this study were analytical grade.Red stingray (3 for 6 h to remove non-collagenous proteins and pigment. The skin was then stirred and extracted with 0.5 mol/L acetic acid at a 1:10 (w/v) ratio for 24 h. Extracted ASC was centrifuged at 20,000× g for 15 min. The supernatant of the ASC extract underwent salt-induced precipitation with 2–5% (w/v) NaCl. The precipitate was collected after centrifugation at 10,000× g for 20 min and re-dispersed at a 1:9 (w/v) ratio in 0.5 mol/L acetic acid. The solution was dialyzed against a 20-fold volume of 0.1 mol/L acetic acid for 24 h, followed by 24 h of dialysis against distilled water. All processes were carried out below 5 °C. ASC was lyophilized and then stored at −20 °C until use.ASC from stingray skin was extracted following the methods previously described by Zhao et al.  with modw/w) at a 1:10 (w/v) ratio. After extraction, the PSC was centrifuged, salt-precipitated, dialyzed, and freeze-dried using the same methods as used for ASC.PSC from the stingray skin was extracted following the methods previously described by Zhao et al.  with mod1 is the weight of lyophilized collagen and m2 that of initial dry fish skin.The extraction yields of ASC and PSC were calculated using Equation (1)w/v) and 20% glycerol (v/v) at a 1:1 (v/v) ratio. Samples were loaded onto polyacrylamide gels  and electrophoretically separated under a constant current of 20 mA/gel. The gel was then treated with 25% methanol (v/v) and 5% acetic acid (v/v) for 30 min and stained for 30 min with 0.1% Coomassie Brilliant Blue R-250 dye (w/v)  in 30% methanol (v/v) and 10% acetic acid (v/v), followed by 30 min of de-staining in 30% (v/v) methanol and 10% (v/v) acetic acid. The staining ratio of α1 to α2 were analyzed using Quantity One 4.6.0 . High-molecular-weight markers were used to estimate the molecular weight of the samples. SDS-PAGE was performed following the methods previously described by Laemmli (1970)  with modUV spectroscopy was performed following the methods previously described by Veeruraj et al.  with mod−1 at a resolution of 4 cm−1. Analyses of the spectral data were carried out using the OPUS v6.5 data-collection software .FTIR spectroscopy was performed following the methods previously described by Chen et al.  with modCD spectroscopy was performed following the methods previously described by Zhang et al. . SamplesKα with a tube voltage of 40 kV and tube current of 25 mA, scanning the 2θ interval at 10° and 50° with an angular speed of 0.06°/s.XRD spectroscopy was performed following the methods previously described by Chen et al.  with modThe zeta potential of the samples was analyzed following the methods previously described by Chen et al.  with modAnalyses of the static water contact angle were conducted following the methods previously described by Deng et al.  with modAnalyses of the dynamic water contact angle were conducted following the methods previously described by Deng et al.  with modd values of the samples were measured following the methods previously described by Zhao et al. [d of the samples. Td was calculated using Equation (2).The To et al.  with modm was assessed following the methods previously described by Huang et al. [w/v) ratio and stored at 4 °C for 48 h. Roughly 5–10 mg of swollen collagen samples were added to an empty aluminum pan for differential-scanning-calorimetry (DSC) measurements using the aluminum pan as the blank . The settings of the differential scanning calorimeter were as follows: temperature range, 20–140 °C; heating rate, 1 °C/min; nitrogen flow in the sample chamber, 50 mL/min. The peak on the DSC spectrum is the Ts of collagen.Tg et al. . SamplesThe rheological behavior was analyzed following the methods previously described by Wang et al.  with modV1 is the volume of water added during the experiment (mL), V2 the volume of water left after centrifugation (mL), and m the sample mass (g);V1 is the volume of oil added during the experiment (mL), V2 the volume of oil left after centrifugation (mL), and m the sample mass (g).WAC and OAC were evaluated following the methods previously described by Wani et al.  with modw/v) collagen solution. The pH values of 20 mL aliquots of the solution were adjusted to 2, 4, 6, 8, or 10 with 1 M HCl or 1 M NaOH. Each solution was homogenized with a homogenizer at 25 °C at 16,000 rpm/min for 120 s. Then, the sample was quickly transferred to a 250-mL measuring cup. The total volume of the sample was recorded after 30 s. FC and FS were calculated using the following equations:V1 (mL) is the volume of the sample after homogenization, and V2 (mL).is the volume before homogenization. V3 (mL) is the foam volume of the sample after standing at room temperature for 2 h. FC and FS were assessed following the methods previously described by Celik et al.  with modw/v) collagen solution. The pH values of 6-mL aliquots of the prepared solution were adjusted to 2, 4, 6, 8, or 10 with 1 M HCl or 1 M NaOH. Corn oil (2 mL) was added to each aliquot and homogenized in a homogenizer for 60 s at 16,000 rpm/min. At 0 and 10 min after homogenization, the emulsion was transferred to a 2-mL centrifugal tube and diluted 100 times with 0.1% (w/v) sodium dodecyl sulfate (50-µL sample in 5 mL of 0.1% sodium dodecyl sulfate) with thorough mixing. Absorptions were measured at 500 nm using 0.1% sodium dodecyl sulfate as the blank. EAI and ESI were calculated using the following equations:A0 and A10 are the absorptions of the emulsions measured at 0 and 10 min, respectively, and Δt = 10 min.The EAI and ESI of the samples were determined following the methods previously described by Celik et al. . The samCell proliferation experiments were performed following the methods previously described by Chen et al. . Collagep < 0.05.Statistical analyses were conducted using SPSS software . One-way ANOVA analyses followed by Dunnett’s test for multiple comparisons of treatment means with a control were used. Statistical significance was defined as In this study, ASC and PSC were isolated from the skin of red stingray and the yield of PSC was found to be higher than ASC. SDS-PAGE analyses confirmed that both collagens belonged to type I collagen. UV, FTIR, CD, and XRD spectroscopy revealed the intact triple helix structure of ASC and PSC, which native collagens possess. The pI of ASC and PSC were 6.71 and 6.41, respectively, as per the zeta-potential analyzer results. Rheological tests showed that both ASC and PSC solutions exhibited shear-thinning flow behavior. In the thermal stability tests, PSC from red stingray skin was found to have a higher melting temperature than duck skin PSC. Contact angle experiments demonstrated the superior hydrophobicity of PSC to ASC. Functional property analyses indicated better functional properties of ASC and PSC than other proteins from terrestrial sources in terms of WAC, OAC, foaming properties, and emulsifying properties. Collectively, these results support the potential use of collagens from red stingray skin as an alternative for terrestrial collagen sources, as well as their potential use in drugs, foods, and cosmetics, and as biological functional materials.


Recent studies found that levels of serum uric acid (SUA) were positively associated with serum 1,5-anhydroglucitol  in subjects with type 2 diabetes mellitus (T2DM). In the current study, we investigated the association between 1,5-AG and UA in T2DM patients with different renal functions.  A total of 405 T2DM patients, 213 men and 192 women, participated in the study. Patients' clinical information was collected, and serum 1,5-AG, SUA, and other clinical characteristics were measured. Correlation analyses were carried out to analyze their correlation with serum 1,5-AG and SUA. μmol/L, respectively, P < 0.01). Pearson's correlation coefficients determine that SUA was positively associated with 1,5-AG in both men  and women , and such relationship can be influenced by the renal function. The positive association still existed with moderate impaired renal function. Moreover, 1,5-AG had a negative association with haemoglobin A1c (HbA1c) in T2DM subjects with eGFR ≥ 30 mL/min/1.73 m2 (P < 0.01).  The male group showed higher levels of SUA than the female group (282.1 ± 91.2 and 244.7 ± 71.89  The positive association between SUA and 1,5-AG still exists in T2DM with moderate renal failure. 1,5-AG can still reflect the glucose levels in patients with CKD stages 1-3. DietarySerum uric acid (SUA), a weak organic acid, is the end product of purine metabolism in humans. SUA levels tend to rise with increasing blood glucose levels in the healthy and prediabetes population, while SUA levels decline in a patient with type 2 mellitus (T2DM) . SimilarIn the current study, therefore, we investigated the correlation between SUA and 1,5-AG using classification by a CKD disease stage in T2DM subjects.In this retrospective study, we enrolled 405 patients that were diagnosed with T2DM [according to guidelines of the World Health Organization (WHO) in1999] and treated from January 2013 to December 2015 in the Endocrinology and Metabolism Department of Southeast University-Affiliated Zhongda Hospital. Patients with cancer, liver dysfunction, or other illnesses affecting renal function such as renal artery stenosis were excluded, and patients using drugs like UA-lowering agents, diuretics, or fructose that might influence the level of UA were excluded. Type 1 diabetes and mitochondrial diabetes were excluded by clinical and immunological criteria.−0.999 × age−0.176 × BUN−0.170 × Alb+0.318× 0.762  [2 were categorized under stage 1 , 60-89 mL/min/1.73 m2 under stage 2 , 30-59 mL/min/1.73 m2 under stage 3 , and 15-29 mL/min/1.73 m2 under stage 4 (severely reduced kidney function). Nearly a quarter of the subjects in our study were found to have mild to moderate renal impairment including 62 patients with mild renal failure and 29 patients with moderate renal failure. Moreover, urine albumin-to-creatinine ratio (ACR) ≥ 30.0 mg/gCre is a risk factor for GFR decline and is included in the definition of CKD if it persists for ≥3 months [We collected patients' information: medication use, duration of diabetes, body mass index (BMI), blood pressure, and so on. Moreover, we sampled blood samples from all subjects between 7:00 and 9:00 a.m. and tested the serum concentrations of UA, creatinine, TC, TG, LDL, and HDL . Fasting blood glucose (FBG) and HbA1c were measured with an automated analyzer . 1,5-AG was estimated by using the GlycoMark assay . Estimated glomerular filtration rate (eGFR) was calculated by the Modified Diet in Renal Disease (MDRD) study equation for the Chinese diabetic population as follows: eGFR = 1.211 × 170 sCr , 13. Sub3 months , 15. Thet-test, bivariate analysis, and multivariate regression were conducted to calculate the data. Continuous data are given as the mean ± SD, and categorical data are expressed as the percentage. P value < 0.05 was considered statistically significant.SPSS Software  was utilized for the statistical analyses. Independent P < 0.01). The male group showed higher levels of SUA than the female group , while eGFR, ACR, and serum 1,5-AG levels had no significant difference. Moreover, SUA levels among women with menopause were higher than those among premenopausal women   .P = 0.016 in female and P = 0.039 in male), UA , and HbA1c (P < 0.01 in both genders). In addition, as shown in r = 0.213, P < 0.05; female: r = 0.223, P < 0.05), and the positive correlation also exists in premenopausal women  and postmenopausal women . Nevertheless, in both groups, there was no significant relationship between 1,5-AG and BMI or FPG.With an increase of 1,5-AG concentration, the glycemic levels like FPG and HbA1c significantly decreased and SUA levels were significantly elevated . Therefo2 (P < 0.05) or with ACR < 300 mg/gCre (P < 0.01).Next, we investigated the factors correlated with the positive association between serum 1,5-AG and SUA. The positive relationship still existed after adjusting possible influencing factors, such as age, BMI, blood pressure, lipids, HbA1c, and FBG, by multivariate regression analysis in both genders . Interes2 (P < 0.01).Finally, regression analyses were conducted to investigate the correlation between 1,5-AG and HbA1c in T2DM with different renal functions. As shown in 2, and 1,5-AG remains a reliable glycemic control marker.The current study provides the evidence that SUA in relation to 1,5-AG in T2DM subjects with CKD stages 1-2. The relation was noted in both men and women. Furthermore, the negative association between HbA1c and 1,5-AG still exists in T2DM patients with eGFR ≥ 30 mL/min/1.73 mUA is a product of the metabolic breakdown of purine nucleotides and is excreted and absorbed via the kidney. When the concentration of blood glucose reaches a kidney's reabsorptive threshold, glucose will be excreted in the urine causing the decline of reabsorption of UA, and any impairment of kidney function can result in hyperuricemia –18. In t2 or with ACR < 300 mg/gCre. However, this positive association no longer existed when eGFR declined under 60 mL/min or ACR ≥ 300 mg/gCre. Consequently, we suggest that the renal function can affect the association between SUA and 1,5-AG and some explanations may illustrate the results. Firstly, both 1,5-AG and UA were competitively reabsorbed with renal glucose in the proximal tubules [Although the positive relation between SUA and 1,5-AG has been found in a large number of studies, little is known about the association in patients with chronic renal disorder. In our present study, multivariate analyses found a positive association between SUA and 1,5-AG, independent of HbA1c and FBG. Interestingly, when taking eGFR and ACR into account, the significant association mentioned previously disappeared. Therefore, the subjects were classified into different groups based on eGFR or ACR. We found that 1,5-AG and SUA still associated positively in T2DM patients with eGFR ≥ 60 mL/min/1.73 m tubules , 26, and tubules , 28. Sec tubules . Moreove tubules . Further2. Considering these results, we conclude that 1,5-AG remain a reliable glycemic control marker even if patients suffer mild or moderate renal dysfunction, which is consistent with a previous observation [Currently, HbA1C is the “gold standard” for identifying the overall blood glucose control; however, 1,5-AG which can reflect rapid changes in glycemia is more accurate than HbA1C . Therefoervation . In addiervation .The limitations of the present study include the following: our study is a cross-sectional design in a single center with a high risk of selection bias; the consumption of meat or alcohol was not examined, and urate oxidase was not assessed.Collectively, the present study provides the evidence that 1,5-AG was positively associated with SUA in T2DM patients with mild and moderate renal failure. In addition, 1,5-AG can reflect the glucose metabolic situation in patients with CKD stages 1-3.


Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.  Organisms from all domains of life display circadian ~24 h) rhythms in their metabolism, physiology, and behavior that arose as an adaptation to daily cycles of ambient light and temperature . These e4 h rhythJust as a watchmaker’s apprentice learns the mechanism of a watch by studying its gears as they move, it is informative to observe components of the cyanobacterial clock as they move. Typically, time points of in vitro cyanobacterial clock reactions are analyzed ex post facto using denaturing polyacrylamide gel electrophoresis (SDS PAGE) to resolve different states of KaiC phosphorylation. It was used to resolve the ordered temporal pattern of KaiC phosphorylation: S/T→S/pT→pS/pT→pS/T→S/T→…, where S and T represent residues S431 and T432, the two phosphorylation sites of KaiC, and pS and pT denote their phosphorylated states ,7,8,9,10, and the plasmid construct for expressing KaiB-FLAG-K25C  .Quikchange PCR primers.Synechococcus elongatus KaiB-FLAG-K25C:Critical step: Quikchange PCR primers for Forward-CCAAACTCAGTCCGTGCCCTCTGCACGCTCAAGAACATTCTCGReverse-CGAGAATGTTCTTGAGCGTGCAGAGGGCACGGACTGAGTTTGG10× PfuTurbo Cx reaction buffer (1.0 units/50 µL PCR) .PfuTurbo DNA polymerase (1.0 units/50 µL PCR).10 mM deoxynucleotide triphosphates (dNTPs) solution mix .1.5% agarose gel in Tris base , acetic acid , and EDTA , referred to as (TAE) buffer.E.coli cells.Competent BL21-DE3 Luria Broth (LB) agar plates with 50 µg/mL kanamycin .Critical step: Autoclave 1 L of Luria broth (LB) media broth in 2 L Erlenmeyer flask on liquid cycle. Allow to cool to 50 °C and add 1 mL (50mg/mL) kanamycin to the sterilized 2 L LB flask. Pour into sterile plastic plates and immediately cover with lids. Let sit at room temperature for 20 min before wrapping in aluminum foil and storing at 4 °C until use. We find that these plates are good for up to one month.500 µL of 2× YT bacterial culture media broth per sample.+ media broth.LB kan1.5% agarose gel in Tris base, acetic acid, and EDTA (TAE) buffer.Freshly transformed cells harboring the expression construct.LB media to grow cells.50 mg/mL kanamycin antibiotic.1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) .2PO4 , 500mM NaCl , pH 8.0).Lysis buffer phosphine (TCEP)  was used to prevent unwanted disulfide bond formation between proteins.Critical step: Proteins can crosslink through cysteinyl –SH groups. To minimize this unwanted reaction, a final concentration 20 mM TCEP was added to the elution volume following Ni-NTA column purification. More TCEP can be added after the 2nd Ni-NTA column purification step, preceding concentration of the sample for FPLC (see steps 55–62). No additional TCEP is added following size-exclusion chromatography as to not interfere with 6IAF labeling efficiency.Fluorescent dye compatible with thiol (-SH) click chemistry attachment (6-iodoacetamidofluorescein) .Critical step: Here, for each fluorescently labeled protein preparation, 1 mg of 6IAF was suspended with 80 µL of methanol and then mixed in a 5:1 ratio with KaiB-FLAG-K25C at pH 7.0 to facilitate conjugation (see step 72).Reduced KaiB-K25C mutant construct with cysteinyl residue available for fluorophore labeling .Labeling buffer .Critical step: After labeling, a desalting column is used to both adjust pH to 8.0 for oscillation reactions while also removing free dye from the sample.Desalting column buffer for protein storage .Fluorescently labeled clock protein see .2 , 1 mM ATP , 0.5 mM EDTA, 50 µg/mL kanamycin, pH 8.0) for each reaction sample.600 µL reaction buffer  to KaiC and KaiB proteins, respectively, to increase solubility and stability.Low protein-binding syringe filter (13 mm with 0.2 um membrane).Micropipettes  and pipette tips.DNA molecular size ladder .0.2 mL individual PCR tubes with snap shut caps to hold PCR reactions.Thermocycler.Gel electrophoresis apparatus .1.5 mL sterile centrifuge tubes.Ice bath .Water bath (set to 42 °C).Temperature controlled shaker (set at 37 °C).Glass rod for spreading BL21-DE3 transformed cells on LB agar plates.Alcohol lamp or Bunsen burner for sterile technique.Incubator (set at 37 °C) for cultured LB agar plates.Micropipettes  and pipette tips.15 mL sterile Falcon tubes.DNA molecular size ladder.Thermo Scientific GeneJET Plasmid Miniprep Kit .Benchtop centrifuge.Gel electrophoresis apparatus .Thermo Scientific Sorvall Legend RT+ Centrifuge.Thermo Scientific NanoDrop UV-Vis 2000 Spectrophotometer.Temperature-controlled shaker at 37 °C and capable of 220 rpm.Inoculating loop.Autoclave.UV-Vis spectrometer .Temperature controlled shaker (set at 37 °C).g.Centrifuge to harvest cells, and to separate protein supernatant from insoluble debris after cracking cells. We use a Thermo Scientific Sorvall RC 6 Plus Superspeed centrifuge. It has 4 L of capacity, speeds of up to 22,000 rpm, and RCF’s to 55,200× Centrifuge rotors for 50 mL and 300 mL centrifuge bottles.Sterile volumetric pipet.Transfer bulb for volumetric pipet.Homogenizer for cell disruption.Critical step: An Avestin C3 homogenizer was used to produce homogenous cell-disrupted samples. Allowing the samples to cycle through the instrument for ~5 min was sufficient to consistently achieve homogenous products. Subsequent centrifugation yielded clear separation of supernatant containing soluble proteins and cell debris pellet.Nickel-nitrilotriacetic acid (Ni-NTA) gravity-flow chromatography columns for protein purification (10 mL polypropylene columns with QIAGEN Ni-NTA agarose beads) .Amicon stirred cell and a 10 kDa cut-off ultrafiltration disc .Benchtop centrifuge and 1.5 mL centrifuge tubes.Liquid chromatography 5 mL injection syringe, needle, and adaptor for injection into instrument.Fast protein liquid chromatography (FPLC) to collect protein eluates.GE Healthcare Life Sciences size-exclusion HiLoad 16/600 Superdex 75.Nanodrop to measure protein concentrations by Bradford Assay.Critical step: Protein concentrations were determined with bovine serum albumin (BSA) standard curve Bradford Assay. A standard curve was made fresh each time a protein was measured.Covered sterile Corning tube to hold protein sample during labeling reaction without light exposure.Critical step: Simply wrapping the corning tubes with aluminum foil, fixed with tape or parafilm is sufficient to protect the fluorescent dye from light exposure during labeling.FPLC to separate labeled KaiB construct from free dyes.Stirred cell concentrator.Critical step: The concentrator employed here was Amicon stirred cell 50 mL concentrator with a 10 kDa molecular weight cutoff regenerated cellulose membrane. This apparatus was connected to a nitrogen gas line to provide pressure, and magnetic stir plate allowed for stirring while concentrating. Concentration occurred in a 4 °C refrigerator.Liquid chromatography 2 mL injection syringe, needle, and adaptor for injection into instrument.GE Healthcare Life Sciences HiPrep 26/10 desalting column.Nanodrop to measure protein concentrations by Bradford Assay.Photon counting spectrofluorimeter.Critical step: ISS Photon Counting Spectrofluorimeter (PC1) shown in Three-cuvette sample compartment for fluorimeter.Circulating water bath at 30 °C and tubing compatible with fluorimeter for sample temperature control.5 mm quartz cuvettes to hold reaction samples.Low protein-binding 0.2 µm membrane filter for each reaction sample .1 mL syringe for sample filtration.2 and ATP solutions.Stock MgCl2 stocks at concentrations of 300 mM and 200 mM, respectively, were used. These should be prepared in 20 mM Tris with pH 8.0. Note that KaiA and KaiB protein stocks lack ATP and MgCl2.Critical step: ATP and MgCl1.5 mL sterile centrifuge tubes, three for each reaction sample (see steps 80–88).1.Prepare Quikchange PCR solutions in PCR tubes see .2.Setup thermo cycler to run program see .Critical step: The PCR products should be run on 1.5% agarose gels before performing DpnI digestion. Load 3 µL PCR product and 3 µL 6× orange loading dye. Mix well with micropipette and heat to 100 °C for 1 min before loading agarose gel. Only add DpnI to the products that show bright bands at the correct size of the plasmid.3.Add 1 µL of DpnI enzyme to each Quikchange PCR product, and spin down in benchtop centrifuge at 13,000 rpm for 1 min.4.Incubate Quikchange PCR products with DpnI for 1 h at 37 °C.5.PCR purification kit, optional (QUIAGEN).6.Measure Quikchange PCR product concentration with Nanodrop. Ensure the 260 nm/280 nm ratio is near 1.8 for pure DNA product.260/280). Highly pure DNA in should be ~1.8, while highly pure RNA should be ~2.0. Proteins absorb light at 280 nm. Therefore, lower ratios are indicative of protein contamination in the sample [Critical step: Sample purity can be obtained by comparing the absorbance at 260 nm against that of 280 nm .11.Heat shock the cells at 42 °C in a water bath for 45 s.12.Incubate them on ice for 2 min.13.Add 500 µL of warmed 2× YT broth to each sample.14.Incubate cells in temperature-controlled shaker at 37 °C and 220 rpm for 1 h.15.Spin down cells at 5000 rpm for 1 min in benchtop centrifuge (at room temperature).16.Discard 350 µL of supernatant and resuspend the transformed cell pellet with the remaining supernatant (150 µL).17.Pipet 150 µL of resuspended cells per LB agar plate containing kanamycin (50 µg/mL).18.Spread cells on agar surface using sterile glass spreader.+ agar plates. Flame the glass rod for 3–5 s, dip into ethanol, flame again for 3–5 s, and then allow cooling for 10–12 s. Repeat this process between streaking samples.Critical step: Glass rods should be sterilized prior to spreading cells on LB-kan19.Incubate plates for 16–18 h at 37 °C.+ media broth. After overnight incubation at 37 °C with shaking at 220 rpm, use a miniprep kit to isolate DNA. DNA sequencing of this sample will determine whether the Quikchange PCR and transformation steps were successful  with 5 mL LB-kan22.With an inoculating loop, and aseptic technique, pick a colony from the plate, and dip & swirl loop into a Falcon tube containing media for each. Do this step for three colonies.23.Incubate samples for 4–6 h at 37 °C with shaking at 220 rpm.+ broth media may be stored at 4 °C. This culture will be utilized following gel electrophoresis analysis to ensure proper DNA length (see step 29).Critical step: Following incubation, 1 µL incubated transformation product will be used in step 24, and the remained of the cultured 5 mL LB-kan24.Prepare PCR centrifuge tubes with 1 µL incubated transformation product, 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 10 µL dNTP (10 mM), 1 µL Pfu Turbo DNA Polymerase (1 unit/50 µL), 5 µL 10× Pfu Turbo Cx Reaction Buffer, and dilute to 50 µL with deionized water see .25.Run PCR thermo cycle to amplify transformed DNA sequence see .26.Prepare a 1.5% agarose gel.27.Load 30 µL of PCR product into 1.5% agarose gel, along with 1 kbp DNA ladder, and run at 60 V for 20 min.28.Analyze PCR product bands to confirm that the DNA sequence is both present at a reasonable concentration and the correct length.29.Spin down remainder of cultures for 10 min at 4 °C and 4000 rpm using Thermo Scientific Sorvall Legend RT+ Centrifuge.30.Discard supernatant and isolate DNA plasmid from pelleted cells using Thermo Scientific GeneJET Plasmid Miniprep Kit.31.Measure DNA miniprep product with Nanodrop. Expect concentration of 100–300 ng/µL.32.Prepare 500 ng of miniprep product in deionized water .33.Each sample requires two tubes, one with forward primer (0.5 µL at 10 µM) and the other with reverse primer (0.5 µL at 10 µM) added.34.Send samples to DNA sequencing facility.35.Analyze results to ensure desired mutations are present before moving forward with protein expression.36.Add 25 g LB to 1 L of deionized water in 2 L Pyrex flask.2HPO4, 3 g KH2PO4, 0.5 g NaCl, and 1 g NH4Cl, then autoclave on liquid cycle. Following sterilization, add 10 mL (0.2 µm filtered) 20% D-glucose, 2 mL 1 M MgSO4, 100 µL 1 M CaCl2, and 1 mL 50 µg/mL kanamycin.Critical step: LB media should be used for KaiA and KaiB-FLAG expression. For FLAG-KaiC, M9 minimal media is highly recommended. To produce 1 L of M9 media, mix 6 g Na37.Autoclave on a liquid cycle to sterilize media (121 °C for 20 min).38.Allow to cool to 50 °C, and add 1 mL (50mg/mL) kanamycin to the sterilized 1 L LB media.39.Pipet 5 mL LB kanamycin into sterilized 15 mL Falcon tubes for each variant protein that will be expressed.40.Add 2 µL of bacterial glycerol stock into corresponding falcon tubes.Critical step: All glycerol stocks consist of transformed cells in 16% glycerol. These solutions are prepared by adding 100 µL of 80% glycerol (autoclaved) to 400 µL of transformed cell media. These samples are stored at −80 °C until use.41.Incubate overnight in shaker for 16 h at 37 °C and 220 rpm.42.Add 5 mL overnight culture to autoclaved 1 L LB kanamycin media.43.600), measured with a UV-Vis Spectrometer, is reached.Incubate at 37 °C and shake at 220 rpm until the optical density of 0.6 at 600 nm  and Sorvall Instruments SS-34 (50 mL bottles) rotors are used. All samples must be within 0.1 g of one another to ensure the rotors are balanced (steps 47 & 51).48.2PO4, 500mM NaCl, pH 8.0).Discard supernatant and resuspend cell pellet with 30 mL lysis buffer  on the same day is preferred.50.Lyse cells in homogenizer.51.Transfer the cell lysate into 50 mL centrifuge tubes and spin down at 15,000 rpm for 45 min.52.Prepare Ni-NTA columns for purification by running two full column volumes of deionized water through the nickel-chelated resin, followed by equilibration with a full column volume of lysis buffer.53.Load supernatant onto Ni-NTA columns.2+ ions, trapping the proteins of interest on the Ni-NTA column.Critical step: Preceding the SUMO tag is a 6-His-tag that binds the Ni54.2PO4, 500 mM NaCl, 20 mM imidazole, pH 8.0) to remove non-His tagged proteins.Wash column with 50 mL wash buffer .Elute His tagged protein with 5 mL elution buffer  or wash (undesirable).56.Add 150 µL of 100 µM ubiquitin-like-specific-protease 1 (Ulp1).6-SUMO tag from the fusion protein, allowing for purification by subsequent passes through Ni-NTA columns in which the His6-SUMO tag binds while the protein does not.Critical step: The Ulp1 enzyme specifically cleaves His57.6-SUMO fusion proteins at 4 °C.Allow 8–14 h for Ulp1 to cleave His58.Dilute the solution to 25 mM imidazole by adding lysis buffer to the sample.59.Clean Ni-NTA columns with two column volumes of DI water, followed by two bed volumes of 0.2 µm-filtered solution of 6 M guanidine hydrochloride (GdHCl) in 0.2 M acetic acid. Then wash with another two column volumes of DI water.60.Pass Ulp1-treated protein samples through clean Ni-NTA columns, and collect the flow-through in a clean, labeled corning tube.6-SUMO fusion tag can be captured on the Ni-NTA column, while proteins of interest pass through. This was performed by attaching a 1–2” section of soft rubber tubing to the bottom of each Ni-NTA column, which can then be partially clamped to allow flow rates of ~50–60 drop/min.Critical step: When passing Ulp1-treated protein samples through Ni-NTA columns, it is important to regulate flow rates to ensure that the free His61.6-SUMO tags.Repeat steps 59 & 60 to ensure that protein sample has been sufficiently separated from His6-SUMO by Ulp1 and purification by Ni-NTA columns, a confirmation step can be performed to verify that the protein of interest is present and pure. Transfer 3–5 µL of nickel column eluate to a centrifuge tube and add an equal volume of 2× SDS-dye. Load the sample alongside a protein ladder on SDS polyacrylamide gel (17% for KaiB) and run for 30 min at 60 V, followed by 100 min at 120 V. A comparison of stained bands against molecular weight markers allow for the estimation of protein purity and yield.Critical step: After cleavage of His62.Concentrate purified samples to 6 mL for subsequent FPLC injection (see 3.4.5) in an Amicon Stirred Cell concentrator using a 10 kDa molecular weight cut-off (MWCO) membrane.63.Prepare mobile phase buffer in which the purified protein will be stored .Critical step: The preferred ratio of 6IAF to protein is 10–20:1, although we have had success at 5:1 fluorescently labeling KaiB. During the conjugation reaction the recommended pH is 7.0–7.5, which is accomplished by preparing the mobile phase for FPLC at this pH. All labeling was performed at 4 °C overnight in the dark.64.Divide the 6 mL of concentrated protein sample into 1 mL aliquots in six 1.5 mL centrifuge tubes and spin down at 13,000 rpm for 2 min on a benchtop centrifuge. This will allow for removal of any precipitate and particles from the sample before FPLC.65.Draw the supernatant from the centrifuge tubes into a 10 mL syringe using a long blunt-end needle.66.Exchange the needle on the syringe with an adaptor for connecting to the FPLC sample injection port.67.Remove all bubbles from the syringe.68.Attach the syringe to the sample injection port on the FPLC. Do so without introducing air into the instrument by filling the injection port with DI water as the syringe adaptor is inserted.69.Run the FPLC with HiLoad 16/600 Superdex 75 size-exclusion column, at 0.8 mL/min flow rate, and 0.5 MPa maximum pressure.70.Collect eluate peak fractions for proteins of interest.71.Following purification, concentrate eluate to desired concentration with an Amicon Stirred Cell concentrator using a 10 kDa molecular weight cut-off (MWCO) membrane.72.Mix thiol-reactive fluorescent dye (1 mg 6IAF resuspended in 80 uL methanol) with protein at the ratio recommended for the particular dye being used.Critical step: 6IAF was chosen because it is inexpensive with high quantum yield, photostability, and moderate excitation/emission spectrum such that it may be paired as a FRET-acceptor or -donor in the future. As stated in step 63, a 5:1 ratio of 6IAF to protein is sufficient for ~99% labeling efficiency .73.Incubate at 4 °C for 14–16 h, covered to avoid light contamination.74.Use Amicon Stirred Cell concentrator with 10 kDa MWCO membrane to concentrate sample to 2 mL.75.Split the 2 mL sample into two 1.5 mL centrifuge tubes and spin down at 13,000 rpm for 2 min using a benchtop centrifuge.76.Repeat steps 65–68.77.Run the FPLC with HiPrep 26/10 Desalting Column, 3 mL/min flow rate, and 0.3 MPa max pressure to separate labeled protein from free fluorophore.78.Collect eluate from 11–19 mL.79.Following purification, concentrate eluate to desired molarity with the Amicon Stirred Cell concentrator and 10 kDa MWCO membrane.80.2, 1 mM ATP, 0.5 mM EDTA, 50 µg/mL kanamycin, pH 8.0) to 1.5 mL centrifuge tubes.Add reaction buffer  and 520 nm (slit width 1.0 nm), respectively, were read every 15 min. Fluorescence anisotropy measurements are achieved on the PC1 with an emission polarizer isolating parallel emission detection followed by subsequent perpendicular emission detection with the same photomultiplier tube (PMT). G-factor determination is set to the “once” option to adjust parallel and perpendicular gain values in order to eliminate instrumental bias in fluorescence anisotropy measurements.87.Load clock reaction samples into 1 mL syringe, attach 0.2 µm membrane filter, and filter samples into fresh 1.5 mL centrifuge tubes.Critical step: Filtering the solutions with low protein-binding 0.2 µm membrane ensures bacteria and/or large contaminants are not present in the final reaction mixtures. This step may be omitted if long term oscillations, many days/weeks, are not planned.88.Transfer into a fresh 1.5 mL centrifuge tube the volume of sample necessary to achieve a final volume of 600 µL upon adding fluorescently labeled protein in step 89.89.In the dark, add KaiB-FLAG-K25C-fluorophore to a final concentration of 0.05 µM and total volume of 600 µL.90.In the dark, mix the sample and transfer it to a fluorimeter cuvette.91.Place the cuvette in the fluorimeter and start data collection once the 30-min timer (see step 86) expires. Record the time representing t = 0.


Semiliquidambar cathayensis Chang was a traditional medicinal plant and used to treat rheumatism arthritis and rheumatic arthritis for centuries in China with no scientific validation, while only 15 components were reported. Thus, a rapid, efficient, and precise method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) was applied in both positive- and negative-ion modes to rapidly analysis the main chemical compositions in S. cathayensis for the first time. Finally, a total of 85 chemical compositions, including 35 alkaloids, 12 flavonoids, 7 terpenoids, 5 phenylpropanoids, 9 fatty acids, 7 cyclic peptides, and 10 others were identified or tentatively characterized in the roots of S. cathayensis based on the accurate mass within 5 ppm error. Moreover, alkaloid, flavonoid, phenylpropanoid, and cyclic peptide were reported from S. cathayensis for the first time. This rapid and sensitive method was highly useful to comprehend the chemical compositions and will provide scientific basis for further study on the material basis, mechanism and clinical application of S. cathayensis roots. Semiliquidambar cathayensis Chang, is an epiphyllum tree belonging to the Hamamelidaceae family, native only to China, and grows in Jiangxi, Guangxi, Guangdong, Hainan and Guizhou + ion at m/z 632.3032, 616.3089 and 646.3199. They have similar fragmentation pathways, including [M + H − HAc]+, [M + H − HAc − CH3OH]+, [M + H − HAc − CH3OH − CO]+, [M + H − HAc − 2CH3OH − CO]+, [M + H − HAc − 2CH3OH]+, and [M + H − HAc − 3CH3OH − C6H5COOH]+, which were identified as mesaconitine, hypaconitine, and aconitine, respectively. The possible fragmentation mechanism of mesaconitine is depicted in Compounds 59 and 64 gave [M + H]+ ion at m/z 648.3002 and 662.3168, 16 Da greater than that of mesaconitine and aconitine, respectively. As C10 position was commonly substitued by hydroxyl in aconitum alkaloids, they were presumed as 10-OH mesaconitine and 10-OH aconitine, respectively. However, compounds 70 and 72 gave [M + H]+ ion at m/z 630.3256 and 614.3302, 16 Da and 32 Da less than aconitine, respectively. Their tandem mass spectra were smililar to aconitine, and some fragment ions of compounds 70 and 72 were 16 Da and 32 Da less than the fragment ions of aconitine, respectively. They were reported as deoxyaconitine and 3,13-dideoxyaconitine, respectively. Compound 71 showed [M + H]+ ion at m/z 660.3396, 14 Da greater than that of aconitine. Moreover, as C14 position was commonly substitued by methoxybenzoyl in aconitum alkaloids, 71 was presumed as yunaconitine + and [M + H − H2O]+, respectively, and it was identified as 14-acetyl-karakoline by comparing with the literature +, [M + H − H2O]+, [M + H − H2O − CH3OH]+, and [M + H − H2O − 2CH3OH]+, respectively, and it was tentatively identified as bullatine C  and 10 flavonoid glycosides in the roots of S. cathayensis extract were indentified in negative ion mode (3 (15 Da), H2O (18 Da) and CO (28 Da) were feature loss. The main MS/MS behavior of aglycones described previously was retro Diels-Alder (RDA) fragmentation pathway. RDA fragments of m/z 135 and 119 were the feature fragments in negative ion mode − ion at m/z 289.0715. It yielded fragments at m/z 205.0532, 203.0700, 187.0372, and 125.0280 by loss of 2(C2H2O), 2(C2H3O), 2(C2H2O)-H2O, and C9H8O3 moieties. It was consistent with literature and identified as catechin + ion at m/z 231.13795, and its fragments were at m/z 163.0778 [M + H − C5H8]+, 155.0848 [M + H − HCOOH − C2H6]+, 143.0931 [M + H − HCOOH − C3H6]+, and 105.0712 [M + H − HCOOH − C3H6 − C4H2]+. Its fragmentation process was the same as the literature − ion at m/z 455.3495, and its fragments were at m/z 409.2443 [M − H − CH2O2]− and 391.2341 [M − H − CH2O2 − H2O]−. Its fragmentation process was the same as the literature − ion at m/z 623.0455, and its fragments were at m/z 461.1451 [M − H − C9H6O3]– and 315.1067 [M −H − C9H6O3 − C6H10O4]−. Its fragmentation process was the same as the literature [Taking compound 5, 69, 74, 77, 78, 80, 83, 84, and 85) were identified based on the existing literatures [Fatty acids found in medicinal plants vary in length chains from 12 to 22 carbon atoms, of which 16–20 carbon atoms are the most common fatty acids in nature. It was reported that these compositions have a wide range of biological activities, including stabilizing cell membranes, maintaining the balance of cytokines and lipoproteins, and fighting cardiovascular diseases. In this study, nine fatty acids with long aliphatic hydrocarbon chains and a carboxyl group at one end  were purchased from Fisher Scientific . Deionized water was purified by a Milli-Q ultrapure water system . All other regents used of at least analytical grade.S. cathayensis were collected in the town of Longsheng, Guilin City, Guangxi, China, in October 2016. A botanical voucher specimen of this plant (no. SC20161022) was deposited at authors’ laboratory and was identified by one of the authors Ronghua Liu. 1.0 g aliquots of the roots of S. cathayensis powders were weighed and transferred into a 100-mL conical flask. 50 mL of 50% aqueous ethanol solution was added, and then extracted with a reflux twice for 60 min. Then, the fluid was filtrated and concentrated under reduced pressure in a rotary evaporator. Subsequently, the concentrated extract was lyophilized and dissolved in ACN. The solution was filtered through a 0.22-μm PTFE membrane before submitting for instrumental analysis.The roots of ®BEH C18  keeping at 35 °C. 0.1% aqueous formic acid  and acetonitrile (B) were used as the mobile phase. The gradient elution with the flow rate of 0.3 mL/min was performed as follows: 0–5 min 5–15% B; 5–15 min 15–18% B; 15–25 min 18–35% B; 25–35 min 35–95% B; 35–37 min 95–95% B; 37–37.1 min 95–5% B; 37.1–40.0 min 5–5% B. The sample inject volume was 3 μL.The UHPLC analysis were carried out on a Shimadzu System , equipped with a LC-3AD solvent delivery system, a SIL-30ACXR auto-sampler, a CTO-30AC column oven, a DGU-20A3 degasser and a CBM-20A controller. Chromatographic separation was conducted on a ACQUITY UPLCTM 5600+ system with a Duo Spray source in both positive and negative electrospray ion mode . The MS analysis was carried out by the ESI source in both positive- and negative-ion modes. The parameters were set as follows: ion spray voltage, −5500 V; ion source temperature, 500 °C; curtain gas, 40 psi; nebulizer gas (GS1), 50 psi; heater gas (GS2), 50 psi; and decluster potential (DP), -100 V. Mass ranges were set at 100–1500 Da for the TOF-MS scan and 100–1500 Da for the TOF MS/MS experiments. In the IDA-MS/MS experiment, the collision energy (CE) was set at 45 eV, and the collision energy spread (CES) was (±) 15 eV for the UHPLC-Q-TOF-MS/MS detection. The most intensive five ions from each TOF-MS scan were selected as MS/MS fragmentation. LC-MS/MS data were analyzed using PeakView®1.2 software .UHPLC-Q-TOF-MS/MS detection was conducted on a Triple TOFS. cathayensis for the first time. By the virtue of high resolution and high separation speed of UHPLC, and accurate MS data of Q-TOF-MS, a total of 85 components, including 35 alkaloids, 12 flavonoids, 7 terpenoids, 5 phenylpropanoids, 9 fatty acids, 7 cyclic peptides, and 10 others were identified by comparisons of their retention times, accurate masses, fragment ions, related literatures. Moreover, alkaloid, flavonoid, phenylpropanoid, and cyclic peptide were reported from S. cathayensis for the first time. This rapid and sensitive method was highly useful to comprehend the chemical compositions and will provide a scientific basis for further study on the material basis, mechanism and clinical application of S. cathayensis roots.In this study, a rapid, efficient, and precise UHPLC-Q-TOF-MS/MS approach was developed for the separation and identification of the main compositions in the roots of