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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.

Abstract

Root-knot nematodes (RKNs) are a vital pest that causes significant yield losses and economic damage to potato plants. The use of chemical pesticides to control these nematodes has led to environmental concerns and the development of resistance in the nematode populations. Endophytic fungi offer an eco-friendly alternative to control these pests and produce secondary metabolites that have nematicidal activity against RKNs. The objective of this study is to assess the efficacy of Aspergillus flavus (), an entophyte fungus isolated from Trigonella foenum-graecum seeds, against Meloidogyne incognita in filtered culture broth using GC–MS analysis. Among them, various nematicidal secondary metabolites were produced: Gadoleic acid, Oleic acid di-ethanolamide, Oleic acid, and Palmitic acid. In addition, biochemical compounds such as Gallic acid, Catechin, Protocatechuic acid, Esculatin, Vanillic acid, Pyrocatechol, Coumarine, Cinnamic acid, 4, 3-indol butyl acetic acid and Naphthyl acetic acid by HPLC. The fungus was identified through morphological and molecular analysis, including ITS 1–4 regions of ribosomal DNA. In vitro experiments showed that culture filtrate of A. flavus had a variable effect on reducing the number of egg hatchings and larval mortality, with higher concentrations showing greater efficacy than Abamectin. The fungus inhibited the development and multiplication of M. incognita in potato plants, reducing the number of galls and eggs by 90% and 89%, respectively. A. flavus increased the activity of defense-related enzymes Chitinas, Catalyse, and Peroxidase after 15, 45, and 60 days. Leaching of the concentrated culture significantly reduced the second juveniles’ stage to 97% /250 g soil and decreased the penetration of nematodes into the roots. A. flavus cultural filtrates via soil spraying improved seedling growth and reduced nematode propagation, resulting in systemic resistance to nematode infection. Therefore, A. flavus can be an effective biological control agent for root-knot nematodes in potato plants. This approach provides a sustainable solution for farmers and minimizes the environmental impact.

Abstract

Background/Objectives: Adaptation can reduce or completely eliminate the effectiveness of antibiotics and antiseptics at clinical concentrations. To our knowledge, no studies have examined fungal adaptation to antiseptics. This study aimed to preliminarily investigate the potential for Candida albicans adaptation to eight antiseptics. Methods: The minimal inhibitory concentration (MIC), drug susceptibility, adaptation to antiseptics, and Karpinski Adaptation Index (KAI) of C. albicans strains were assessed. Results: The antiseptics with the most effective MICs activity against C. albicans were octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHX), and polyhexamethylene biguanide (polyhexanide, PHMB). Sodium hypochlorite (NaOCl) and ethacridine lactate (ET) demonstrated moderate activity, while boric acid (BA), povidone–iodine (PVI), and potassium permanganate (KMnO4) showed the weakest activity. The MIC values for NaOCl and KMnO4 were close to or equal to the clinical concentrations used in commercial products. The studied strains were susceptible to econazole, miconazole, and voriconazole. Resistance to other drugs occurred in 10–30% of the strains. Antifungal resistance remained unchanged after antiseptic adaptation testing. The lowest KAI values, indicating very low resistance risk, were observed for CHX, OCT, and PHMB. PVI and BA presented a low risk, ET a moderate risk. KMnO4 and NaOCl had the highest KAI values, indicating high and very high resistance risk in Candida yeasts. Conclusions: C. albicans strains can adapt to antiseptics to varying extents. For most antiseptics, adaptation does not significantly affect their clinical efficacy. However, due to adaptation, NaOCl and KMnO4 may become ineffective against C. albicans strains even at clinical concentrations.

2.3. Adaptation of Candida albicans Strains to Antiseptics

The studies demonstrated that C. albicans yeasts undergo adaptation when exposed to increasing concentrations of antiseptics. The strains showed weak adaptation to OCT, CHX, PVI, and ET, managing to grow at concentrations several times higher than their respective MIC values. For NaOCl and KMnO₄, adaptation levels were also low but were close to or equal to the initial commercial concentration. This finding suggests that the development of adaptation may lead to the ineffectiveness of commercial products containing NaOCl and KMnO4 against C. albicans. The highest increase in adaptation relative to MIC was observed for BA and PHMB; however, these values were still much lower than the commercial concentrations.

2.3. Adaptation of Candida albicans Strains to Antiseptics

Based on the Karpinski Adaptation Index, the lowest KAI values were observed for CHX, OCT, and PHMB, indicating a very low risk of clinical resistance development in the studied C. albicans strains. PVI and BA had a low risk, while ET posed a moderate risk for clinical resistance development. The highest KAI values were observed for KMnO₄ and NaOCl, indicating a high and very high risk, respectively, of clinical resistance development in Candida yeasts. The results of C. albicans adaptation to antiseptics are provided in . Detailed raw data on the C. albicans adaptation are presented in the .

4.1. Antiseptics

Eight antiseptics were examined in the study: octenidine dihydrochloride (OCT; Schülke & Mayr GmbH, Norderstedt, Germany), polyhexamethylene biguanide (PHMB, Cosmocil PG; Arxada AG, Basel, Switzerland), sodium hypochlorite (NaOCl; Cerkamed, Stalowa Wola, Poland), potassium permanganate (KMnO4; Hasco-Lek S.A., Wrocław, Poland), chlorhexidine digluconate (CHX; Sigma-Aldrich, Poznań, Poland), povidone–iodine (PVI, poly(vinylpyrrolidone)–iodine complex; Sigma-Aldrich, Poznań, Poland), boric acid (BA, Borasol; Herbapol, Poznań, Poland), and ethacridine lactate (ET, Rivanol; Herbapol, Poznań, Poland). The initial concentrations of active substances matched the clinical concentrations found in commercial products used for wound and oral antiseptics: 0.05% (500 µg/mL) OCT, 0.1% (1000 µg/mL) PHMB, 0.01% (100 µg/mL) NaOCl, 1% (10 mg/mL) KMnO4, 0.1% (1000 µg/mL) CHX, 7.5% (75 mg/mL) PVI, 3% (30 mg/mL) BA, and 0.1% (1000 µg/mL) ET.

Abstract

Gene therapy offers potentially transformative strategies for major human diseases. However, one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue. Here, we report a novel virus-like nanoparticle, the bioorthgonal engineered virus-like recombinant biosome (reBiosome), for efficient gene therapies of cancer and inflammatory diseases. The mutant virus-like biosome (mBiosome) is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site, and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry. The results show that the reBiosome exhibits reduced virus-like immunogenicity, prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci (like tumor and arthritic tissue). Furthermore, reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems, respectively. In conclusion, this study develops a universal, safe and efficient platform for gene therapies for cancer and inflammatory diseases.

Site-Specific Codon Mutated Virus-Like mBiosome and Its Gene Transfection Efficiency

To prepare the mutated VSV-G enveloped biosome packaging the GFP mRNA (mBiosomeGFP), a series of steps were undertaken. Firstly, HEK 293T cells were co-transfected with the mutated pCMV-VSV-G plasmid along with other three plasmids, pIRE4-Azi, pCMV-capsid10-flag (encoding the capsid10 protein), and pCMV-cargoGFP (encoding the GFP mRNA). This transfection process was carried out in the presence of 1 mM Azi, as depicted in Fig. c. After the concentration of mBiosomes, the membrane proteins were extracted and analyzed by Western blot assay. The results clearly demonstrated a successful incorporation of mutated VSV-G proteins into the envelope of mBiosomes (Fig. d). In order to gain further insights, the morphologies, particle sizes and Zeta potentials of the mBiosomes were examined using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The TEM images showed that eBiosome had a spherical shape and was encapsulated with spike proteins, closely resembling general enveloped virus. Similarly, three different types of mBiosomes displayed similar morphological characteristics to the eBiosome (Fig. e). Moreover, the particle sizes and Zeta potential values of the three different types of mBiosomesGFP were evaluated and compared to the eBiosomeGFP. The results obtained from DLS analysis demonstrated that all three types of mBiosomesGFP shared comparable particle sizes and Zeta potential values with the eBiosomeGFP (Figs. f and S3).

Expression of plant EFE-like proteins causes guanidine production in E. coli

The detection of free guanidine indicated that 2-ODD-C23 might be homoarginine-6-hydroxylases, as 6-hydroxyhomoarginine is expected to decay spontaneously to yield guanidine and AASA. The latter is in spontaneous equilibrium with P6C, which can be converted to pipecolate by P5CR (proC in E. coli, ). Indeed, we detected pipecolate in the E. coli cultures in quantities proportional to the amount of guanidine (). Only in the cells expressing Din11s, the proportion of pipecolate was lower, suggesting that Din11s used both arginine and homoarginine as substrates. In contrast to guanidine, pipecolate was predominantly retained inside the cells (). To analyze if the plant enzymes can convert 2-oxoglutarate to ethylene, we compared bacterial cultures expressing either PsEFE or one of the Arabidopsis enzymes (). By GC-FID analysis of the headspace of bacterial cultures expressing EFE, Din11s, At3g49630, or At3g50210 and incubated with 1 mM arginine or homoarginine for 18 hr in air-tight tubes, we observed the formation of up to 0.18% (v/v) ethylene by cultures expressing EFE. In contrast, none of the plant enzymes produced even traces of ethylene [detection limit 0.0002% (v/v)]. As expected, the cultures expressing EFE or Din11s contained guanidine, irrespective of the supplement. The cultures expressing At3g49630 or At3g50210 only contained guanidine, when they were fed with homoarginine.

Introduction

Only two enzymes are known to release guanidine from arginine in side reactions. The Ethylene Forming Enzyme (EFE) from Pseudomonas savastanoi converts 2-oxoglutarate (2-OG) into ethylene and this activity depends on structural changes induced by binding of arginine to the active site (; ). In a second reaction with roughly 50% turnover rate, EFE also catalyzes the hydroxylation of arginine at the C5 atom, yielding unstable 5-hydroxyarginine, which spontaneously decays to glutamate-5-semialdehyde (GSA) and guanidine. The arginine-4,5-desaturase NapI predominantly catalyzes desaturation of the C4-C5 bond but also produces a small proportion unstable 5-hydroxyarginine (). Both enzymes belong to the class of Fe2+ and 2-OG-dependent dioxygenases (2-ODD), which use molecular oxygen to catalyze a broad range of reactions typically involving an FeIV-oxo-intermediate formed after the initial conversion of 2-OG to succinate (; ; ). Other enzymes from this family, like VioC or OrfP, catalyze the hydroxylation of arginine at C3 or C3 and C4, respectively, without initiating guanidine release (; ). A third guanidine-producing enzyme, the guanylurea hydrolase GuuH from Pseudomonas mendocina strain GU, is from a different protein family and may have evolved recently to utilize guanylurea generated by metformin degradation ().