Läs källbundna sammanställningar.

Abstract

The success and longevity of a resin composite restoration is determined by its good bonding to the tooth structure, with the adhesion being a challenge to dentin due to its complexity and structural heterogeneity. The present study aimed to compare the adhesive strength of dentin conditioned with 18% ethylenediaminetetraacetic acid (EDTA) versus 35% phosphoric acid (H3PO4) in human premolars. Materials and Methods: This in vitro experimental study was performed on 40 human premolars. The occlusal thirds were sectioned and randomly placed into four groups according to the type of dentin conditioning: Group 1 (control), Group 2 (18% EDTA), Group 3 (35% H3PO4) and Group 4 (18% EDTA plus 35% H3PO4). Then, 10,000 thermocycles between 5 +/− 2 °C and 55 +/− 2 °C were applied. Adhesive strength was tested by shearing with a digital universal testing machine at a crosshead speed of 0.75 mm/min. The values obtained were analyzed in megapascals (MPa). The mean and standard deviation were used as measures of central tendency and dispersion. In addition, a one-factor intergroup ANOVA test was applied with Tukey’s post hoc test considering a significance level of p < 0.05. Results: The 18% EDTA and 18% EDTA plus 35% H3PO4 showed significantly higher adhesive strength compared to the control group that did not receive dentin conditioning (p = 0.047 and p < 0.001, respectively). However, the group conditioned with 18% EDTA did not present significant differences compared to the group conditioned with 35% H3PO4 (p = 0.997). In addition, the group conditioned with 18% EDTA plus 35% H3PO4 showed significantly higher adhesive strength compared to the groups conditioned with 18% EDTA (p = 0.002) and 35% H3PO4 (p = 0.001). Conclusion: The adhesion of bulk fill resin composite to dentin was favorable when preconditioning was performed using 18% EDTA followed by 35% H3PO4. In contrast, when both etchants were used separately, the bulk fill resin composite showed similar bond strength values in both cases, but significantly lower compared to their sequential application.

2.4. Dentin Conditioning and Resin Composite Bonding

The procedure for each group was as follows ():Group 1: No dentin etching. Only rinsed in water for 5 s, and excess moisture was dried with sterile gauze. Then, a layer of Tetric® N-Bond adhesive was placed with a microbrush, and air was gently applied for 5 s. Finally, a block of Tetric® N-Ceram Bulk-Fill resin composite color A2 was placed and light cured perpendicularly for 10 s at a maximum distance of 1 mm to the upper surface with a Bluephase N LED (Ivoclar Vivadent AG, Schaan, Liechtenstein) at an intensity of 1200 mW/cm2 for 10 s. (). The light intensity of curing unit was previously verified using a radiometer (Bluephase Meter II, Ivoclar Vivadent AG, Schaan, Liechtenstein).Group 2: Dentin etching was performed with 18% EDTA gel for 90 s. Then it was washed with water for 10 s, and the excess moisture was dried with sterile gauze. Then, the adhesive was applied, and the resin composite was placed using the same procedure as group 1 [].Group 3: Dentin etching was performed with 35% H3PO4 gel for 15 s. Then, it was washed with water for 10 s, and the excess moisture was dried with sterile gauze. Then, the adhesive was applied, and the resin composite was placed using the same procedure as groups 1 and 2.Group 4: Dentin etching was performed with 18% EDTA gel for 90 s. Then, it was washed with water for 10 s, and the excess moisture was dried with sterile gauze. Then, 35% H3PO4 gel was applied for 15 s. Then, it was again washed with water, and excess moisture was dried with sterile gauze. Then, the adhesive was applied, and the resin composite was placed using the same procedure as in groups 1, 2 and 3.

Abstract

Methods. Eighty-nine nonvital premolar and molar teeth with periapical lesions (PAI: 3‒4) without any clinical symptoms were included. The patients were randomly assigned to three groups according to the final irrigation solutions used: G1: 2 mL of Qmix (n=29), G2: 2 mL of 17% ethylenediaminetetraacetic acid (EDTA) (n=30), and G3: 2 mL of 2% chlorhexidine gluconate (CHX) (n=30). All the patients were prescribed 100 mg of flurbiprofen to use as needed for pain. The patients were asked to rate their pain status according to the verbal rating scale at 12, 24, 48, and 72 hours, and one week. The data were analyzed using Pearson’s chi-squared test, Fisher’s exact test, and chi-squared analysis with Monte Carlo simulation. The significance level was set at P≤0.05.

Abstract

Results. No significant differences were observed in postoperative pain rates at 12, 48, and 72 hours and one week (P>0.05). However, in the Qmix group, a significantly lower pain level was observed at 24 hours with EDTA and CHX (P=0.019). The rate of mild pain in the EDTA group at 72 hours (18.8%) was significantly higher in premolar teeth than in molar teeth (P=0.012). The moderate pain level in the EDTA group at 12 hours was significantly higher in those>60 years of age (P=0.008).

Patient selection

The method and design of this prospective, randomized, double-blind clinical trial were approved by the Ethics Board of the University (No: 929). The study protocol was published and registered at www.clinicaltrials.gov (identifier: ). Before inclusion, all the patients were informed of the study protocol, aims, complications, and possible risks, and written informed consent was obtained from each patient. Patients who met the inclusion criteria were treated by a single endodontist (SIY) from the Faculty of Dentistry, Department of Endodontics, between October 2019 and February 2020.

Root canal treatment procedures

Group 2 (EDTA, n = 30): After applying 2 mL of saline solution to each root canal for 30 sec, 2 mL of 17% EDTA was applied for 60 seconds.

Abstract

This study aimed to determine the effects of dietary lanthanum chloride on the growth and health performance of juvenile Clarias gariepinus when fed diets experimentally contaminated with mixtures of aflatoxin B1 and fumonisin B1. A control diet, (mycotoxin free, diet A), mycotoxin contaminated (diet B), and two mycotoxin-contaminated diets amended with lanthanum chloride (200 mg/kg, diet C; and 400 mg/kg, diet D), were fed to 450 fish divided equally into five groups (each with three replicates) for 56 days. The fish were randomly sampled at the time points: day 7, 28 and day 56 for the zootechnical, hematological and serum biochemical evaluations. The fish fed the diets amended with lanthanum chloride exhibited significantly (p < 0.05) better performance indices compared with the fish fed only the mycotoxin-contaminated diet. Lanthanum chloride elicited significant (p < 0.05) increases in erythrocytes and leucocytes count and significant (p < 0.05) reduction in serum transaminase, alkaline phosphatase, lactate dehydrogenase activities, urea and uric acid concentrations in the fish fed the diets contaminated with mixtures of aflatoxin B1 and fumonisin B1. The study indicates that juvenile Clarias gariepinus may be beneficially cultured with mycotoxin-contaminated grains amended with 200 to 400 mg/kg lanthanum chloride.

2.1. Fiber Extraction and Treatment

Butea parviflora (BP) is a woody climber belonging to the Fabaceae family. It is found in the regions of Kanniyakumari, Tamil Nadu. The whole plant is fibrous, but the strength and texture vary from the outer to inner space. Due to its strength, bark fibers of Butea plant are used by locals for tying, knotting, and binding requirements. Bark contains 1% rotenone, which is used as an insecticide. In order to separate mature fibers from the plant, a metal teeth tool is used. Collected fibers are treated in distilled water and potassium permanganate; treatment involves soaking of fibers in 0.1 M NaOH solution for 20 min, followed by 15 min in the same molarity of potassium permanganate at 27 °C. Treated fibers are rinsed in distilled water. Fibers are first drained in shade, then dried completely using a vacuum desiccator. Variation in soaking time and concentration of KMnO4 will bring varying results. Different treatments at 0.025% and 0.05% of KMnO4 on the Musa plant eliminated the non-cellulosic debris and manifested the crystalline and tensile property of fiber. The delignification process that takes place during permanganate action facilitates the rearrangement of microfibrils, resulting in an improvement in crystallinity. Moreover, the wetting of fibers in 0.1% KMnO4 induces capillary forces that increase their resistance to water []. . presents water and KMnO4 treated BP fibers.

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking of Orai1 TM domains can lead to CRAC channel-like activation.Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the absence of STIM1. Stars indicate sites for UAA incorporation. Ca2+ imaging measurements of Orai1 A137Bpa (a), Orai1 L174Bpa (b) and Orai1 A254Azi (c) using R-GECO1.2. Intracellular Ca2+ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with the above mentioned UAA-containing Orai1 mutants in HEK 293 cells, were monitored initially in 0 mM Ca2+ solution followed by a 2 mM Ca2+ solution. Under 2 mM Ca2+ solution conditions, UV light was applied for 10 s and 30 s, respectively. d–f Time courses of Ca2+ current-densities after whole-cell break-in of above mentioned UAA-containing Orai1 mutants. UV light was applied for 15 s during the recording leading to UV-induced activation of Ca2+ currents. g–i Corresponding current/voltage (I/V) relationships were taken after 100 s of measurement. Inlet represents reversal potential (Vrev) of wild-type CRAC channel (Orai1 + STIM1) versus light-sensitive Orai1 mutant currents. Single values are indicated in gray. Data represent mean values ± SEM of indicated number (n) of experiments. Mann–Whitney test was applied to show that Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking of Orai1 TM domains can lead to CRAC channel-like activation.Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the absence of STIM1. Stars indicate sites for UAA incorporation. Ca2+ imaging measurements of Orai1 A137Bpa (a), Orai1 L174Bpa (b) and Orai1 A254Azi (c) using R-GECO1.2. Intracellular Ca2+ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with the above mentioned UAA-containing Orai1 mutants in HEK 293 cells, were monitored initially in 0 mM Ca2+ solution followed by a 2 mM Ca2+ solution. Under 2 mM Ca2+ solution conditions, UV light was applied for 10 s and 30 s, respectively. d–f Time courses of Ca2+ current-densities after whole-cell break-in of above mentioned UAA-containing Orai1 mutants. UV light was applied for 15 s during the recording leading to UV-induced activation of Ca2+ currents. g–i Corresponding current/voltage (I/V) relationships were taken after 100 s of measurement. Inlet represents reversal potential (Vrev) of wild-type CRAC channel (Orai1 + STIM1) versus light-sensitive Orai1 mutant currents. Single values are indicated in gray. Data represent mean values ± SEM of indicated number (n) of experiments. Mann–Whitney test was applied to show that Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

In control experiments, we ensured that an intact plasma membrane expression of the mutants could only be achieved in the presence of the UAA and the expression of the respective tRNA/aaRS pair. In the absence of the latter, only weak background fluorescence was detectable in the cytosol, but not in the plasma membrane (Supplementary Fig. ) in line with abolished UV-mediated Ca2+ influx comparable to untransfected cells (Supplementary Fig. ). Furthermore, examination of the UV-evoked Ca2+ currents of cells co-expressing Orai1 A137Bpa-YFP and Orai1-CFP wild-type as a function of the Orai1 A137Bpa:Orai1 fluorescence intensity ratio showed a tendency for higher maximum currents with increasing ratio and highest UV-induced activation when only Orai1 A137Bpa was expressed (Supplementary Fig. ), indicating efficient UAA incorporation. Moreover, rapid UV-mediated activation occured independent of endogenous STIM and Orai isoforms, as shown in CRISPR/Cas9 STIM1/STIM2 double-knockout (DKO), CRISPR/Cas9 STIM1/Orai1 DKO and CRISPR/Cas9 Orai1/Orai2/Orai3 triple KO (TKO) cells (Supplementary Fig. ), although to slightly lower maximum current levels, likely owing to lower expression levels in the KO compared to wild-type HEK293 cells (Supplementary Fig. ). Moreover, we showed that ER Ca2+ levels detected by LAR-GECO1 in HEK293 cells expressing either Orai1 or Orai1 A137Bpa compared to untransfected cells remained comparable before and after UV light irradiation and subsequent thapsigargin (TG)-induced store-depletion led to a similar decrease in Ca2+ levels (Supplementary Fig. ), thus excluding that UV light-triggered Ca2+ entry leads to Ca2+ overload in the ER.

Abstract

Nitrosative stress promotes protein glycoxidation, and both processes can occur during an infection with the SARS-CoV-2 virus. Therefore, the aim of this study was to assess selected nitrosative stress parameters and protein glycoxidation products in COVID-19 patients and convalescents relative to healthy subjects, including in reference to the severity of COVID-19 symptoms. The diagnostic utility of nitrosative stress and protein glycoxidation biomarkers was also evaluated in COVID-19 patients. The study involved 218 patients with COVID-19, 69 convalescents, and 48 healthy subjects. Nitrosative stress parameters (NO, S-nitrosothiols, nitrotyrosine) and protein glycoxidation products (tryptophan, kynurenine, N-formylkynurenine, dityrosine, AGEs) were measured in the blood plasma or serum with the use of colorimetric/fluorometric methods. The levels of NO (p = 0.0480), S-nitrosothiols (p = 0.0004), nitrotyrosine (p = 0.0175), kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan fluorescence was significantly (p < 0.0001) lower in COVID-19 patients than in the control group. Significant differences in the analyzed parameters were observed in different stages of COVID-19. In turn, the concentrations of kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan levels were significantly (p < 0.0001) lower in convalescents than in healthy controls. The ROC analysis revealed that protein glycoxidation products can be useful for diagnosing infections with the SARS-CoV-2 virus because they differentiate COVID-19 patients (KN: sensitivity—91.20%, specificity—92.00%; NFK: sensitivity—92.37%, specificity—92.00%; AGEs: sensitivity—99,02%, specificity—100%) and convalescents (KN: sensitivity—82.22%, specificity—84.00%; NFK: sensitivity—82,86%, specificity—86,00%; DT: sensitivity—100%, specificity—100%; AGE: sensitivity—100%, specificity—100%) from healthy subjects with high sensitivity and specificity. Nitrosative stress and protein glycoxidation are intensified both during and after an infection with the SARS-CoV-2 virus. The levels of redox biomarkers fluctuate in different stages of the disease. Circulating biomarkers of nitrosative stress/protein glycoxidation have potential diagnostic utility in both COVID-19 patients and convalescents.

A comparison of the levels protein glycoxidation products in patients infected with the SAR-CoV-2 virus, convalescents, and healthy controls, including in reference to the severity of COVID-19

Tryptophan concentration was significantly lower in COVID-19 patients than in the control group (p < 0.0001) and convalescents (p = 0.0498), and significantly lower in convalescents than in the control group (p < 0.0001). In turn, KN, NFK, DT, and AGE levels were significantly higher in COVID-19 patients than in healthy controls (p < 0.0001), and significantly higher in convalescents than in the control group (p < 0.0001). In addition, KN and NFK levels were higher in patients infected with the SARS-CoV-2 virus than in the control group (p = 0.0009, p = 0.0394) (Fig. A–E). An analysis of protein glycoxidation products revealed no significant differences in COVID-19 patients with different severity of symptoms evaluated based on the MEWS (Table ). However, tryptophan concentration decreased, whereas KN, NFK, and DT concentrations increased with the aggravation of disease symptoms (Table ). In turn, a comparison of patients with mild/moderate symptoms and moderately severe/severe symptoms revealed that the concentrations of all protein glycoxidation products were higher in MEWS 3 + 4 subgroups than in MEWS 1 + 2 subgroups (tryptophan p = 0.0021, KN p = 0.0051, NFK = 0.0187, dityrosine p = 0.0213, AGEs p = 0.0065) (Fig. E,G–J).Figure 2Comparison of the levels protein glycoxidation products: in COVID-19 patients, convalescents, and the control group (A, B, C, D, E); in COVID-19 patients with mild/moderate (1 + 2) and moderately severe/severe (3 + 4) symptoms (F, G, H, I, J). Key: AGEs advanced glycation end products.

Discussion

According to the literature, oxidative and nitrosative stress influences the pathogenesis and severity of infections with the SARS-CoV-2 virus,,, and the present study was undertaken to evaluate nitrosative stress parameters (NO, S-nitrosothiols, and NT) and protein glycoxidation products (tryptophan, KN, NFK, DT, and AGEs) in COVID-19 patients and convalescents, and to examine the above parameters in COVID-19 patients with different severity of disease symptoms evaluated based on the MEWS. The concentrations of NO, NT, S-nitrosothiols, KN, NFK, DT, and AGEs were higher, whereas tryptophan concentration was lower in COVID-19 patients than in the control group and convalescents. The study demonstrated that in patients with a history of COVID-19, the levels of KN, NFK, DT, and AGEs were significantly higher, whereas tryptophan concentration was significantly lower than in persons who had never been infected with the SARS-CoV-2 virus. Significant differences in nitrosative stress parameters and protein glycoxidation products were observed among COVID-19 patients with different disease severity. In addition, protein glycoxidation products were useful diagnostic biomarkers for differentiating COVID-19 patients and convalescents from healthy subjects. This is the first study to analyze changes in the levels of nitrosative stress and protein glycoxidation products in convalescents, and it provides valuable information for predicting long-term consequences of COVID-19.

Introduction

Recently, we discovered the biosynthetic pathway of guanitoxin, a potent freshwater neurotoxin from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 which possesses a cyclic guanidine moiety in its highly polar structure (B). Previously named anatoxin-a(s), guanitoxin exerts its neurotoxicity through the irreversible inhibition of acetylcholinesterase within the peripheral nervous system. If consumed via contaminated water, guanitoxin can lead to an overexcitation of cholinergic neurons and downstream muscle spasms, respiratory arrest, and potentially death. Within the nine-enzyme biosynthetic pathway that transforms l-arginine into the mature neurotoxin, the pyridoxal-5′-phosphate (PLP)-dependent enzyme GntC constructs the key cyclic guanidine moiety upon which the anticholinesterase O-methylphosphate pharmacophore is assembled. Specifically, GntC performs a cyclodehydration of substrate (S)-γ-hydroxy-l-arginine (1) to l-enduracididine (2), cleanly inverting the stereochemistry at the γ-position. This reaction is highly diastereoselective under in vitro enzyme assay conditions following 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (l-FDAA, Marfey’s reagent) derivatization and ultra performance liquid chromatography mass spectrometry (UPLC-MS) analysis. In our initial characterization of GntC, we failed to observe any enzymatic production of allo-l-enduracididine diastereomer (2′) nor did we observe any conversion of substrate epimer (R)-γ-hydroxy-l-arginine (1′) within our limits of detection. While the GntC reaction occurs relatively early in the guanitoxin biogenesis, the biochemical validation was foundational for establishing the biosynthetic pathway. Both 1 and 2 were previously isolated from guanitoxin-producing Anabaena flos-aquae cyanobacteria, and the incorporation of backbone-deuterated 1 into the mature toxin was supported via in vivo feeding experiments., Although these stable isotope experiments directly implicated 1 in guanitoxin biogenesis, an intriguing reintroduction of hydrogen atoms at the former β-position implied that additional deprotonation/reprotonation chemistry was involved in toxin production (). Following full pathway characterization, one of these β-position reprotonations could be justified using the retro-aldol (GntG) and transamination (GntE) reactions identified in this study. However, we propose that a mechanistic interrogation into GntC-mediated cyclization of 1 could help further rationalize this in vivo result.

GntC PLP Dependency

To fuel our investigation, substrate (1, 1′) and product (2, 2′) diastereomers were synthesized following previously established synthetic protocols.,Sphaerospermopsis torques-reginae ITEP-024 GntC was heterologously expressed as an Escherichia coli codon-optimized N-terminal His6-tagged construct and purified to near homogeneity using Ni-NTA affinity chromatography.In vitro GntC assays were setup in potassium phosphate (KPi) buffer, amine-containing components were derivatized with l-FDAA, and reaction mixtures were analyzed by UPLC-MS as previously reported. Because GntC co-purifies with its requisite PLP cofactor, we had yet to firmly confirm its dependency in 2 production. Treatment of purified GntC with PLP inhibitor hydroxylamine followed by buffer exchange abolished any catalytic activity with 1 in the apo-GntC form (). Reintroduction of two molar equivalents of PLP to the apoenzyme restored 2 production, validating the cofactor dependence of the intramolecular cyclase GntC.

GntC In Vitro Assay Optimization

We initially investigated a range of pHs in KPi buffer to identify ideal conditions for 2 production. Under a 20:1 substrate/enzyme ratio, relative product formation was compared to an l-FDAA-derivatized glycine standard to correct for any derivatization discrepancies, and we determined that GntC behaved optimally at pH 8.0 (). Previous reports identified that divalent metal cations can aid PLP-dependent enzyme catalysis through the promotion of electron displacement, and we wanted to determine if a similar observation occurred with GntC. Counterintuitively, 2 production was not enhanced in the presence of any 1 mM divalent metal salts; instead, incubation with chelator EDTA showed comparable activity to wild-type GntC. Most other ionic additives (Mg2+, Ca2+, Mn2+, Fe2+, Zn2+, Co2+) showed similar or reduced in vitro2 formation, with the addition of Cu2+ completely abolishing production (). We finally investigated the impact of the His6 tag on in vitro2 production, given the significance of the N-terminus on homodimerization, shaping the active site pocket and establishing catalysis in other PLP-dependent enzymes., We recloned GntC as a C-terminal His6 construct and found a slight reduction in catalytic activity compared to the original N-terminal His6 construct (). In spite of additional optimization efforts, in vitro GntC assays were done under identical reaction conditions to those previously described. Following the development of an l-FDAA-derivatized 2 standard curve (), we determined the steady-state kinetic parameters of GntC toward substrate 1 (kcat = 0.0061 ± 0.008 min–1, KM = 150 ± 80 μM ()).

Abstract

Humans are the only species who generate waste materials that cannot be broken down by natural processes. The ideal solution to this waste problem would be to employ only compostable materials. Biodegradable materials play a key role in creating a safer and greener world. Biodegradability is the gift that keeps on giving, in the sense of creating an Earth worth living. The future is thus best served by green energy, sustainability, and renewable resources. To realize such goals, waste should be considered as a valuable resource. In this context, Zea mays (Zm) root fibres, which are normally considered as agricultural waste, can be used as reinforcing substances in polymer matrices to produce structural composite materials. Before being used in composites, such fibres must be analysed for their physical properties. Chemical treatments can be employed to improve the structural quality of fibres, and the changes due to such modification can be analysed. Therefore, the current work examines the effect of permanganate treatment on the surface properties of Zm fibres. The raw and potassium permanganate-treated samples were assayed for various properties. Physical analysis of the fibre samples yielded details concerning the physical aspects of the fibres. The thermal conductivity and moisture absorption behaviour of the samples were analysed. Chemical analysis was employed to characterize the composition of both treated and untreated samples. p-XRD was employed to examine the crystalline nature of the Zm fibres. Numerous functional groups present in each sample were analysed by FTIR. Thermogravimetric analysis was used to determine the thermal stability of Zm fibres. Elemental analysis (CHNS and EDS) was used to determine the elemental concentrations of both raw and treated samples. The surface alterations of Zm fibres brought on by treatment were described using SEM analysis. The characteristics of Zm roots and the changes in quality due to treatment were reviewed, and there were noticeable effects due to the treatment. Both samples would have applications in various fields, and each could be used as a potential reinforcing material in the production of efficient bio-composites.

Potassium permanganate treatment on natural fibres

Zea mays (Zm) root fibres were chemically pre-treated with alkali solution and then with permanganate solution in the present work. They were then tested for the changes in their qualities related to being better reinforcement materials for polymer composite-based products.