Residual Activity Research Articles

Comparative efficacy of alternative insecticides for Rhyzopertha dominica management in Colombia: Beyond organophosphates and pyrethroids

Here, we directly compared the insecticide efficacy of diamides (chlorantraniliprole), neonicotinoids (thiamethoxam), and spinosyns (spinosad and spinetoram) with that of organophosphates (pirimiphos-methyl) and pyrethroids (deltamethrin) in Colombian populations (i.e., Ibagué; El Espinal; Granada; Pore, and Sahagún) of R. dominica. Furthermore, we established the lethal concentrations for spinetoram in our insecticide-susceptible pattern population (Ibagué) and compared this insecticide residual activity against individuals of Ibagué and El Espinal populations. The Ibagué population was most susceptible to deltamethrin and chlorantraniliprole, while Ibagué and El Espinal populations showed higher susceptibility to pirimiphos-methyl and spinetoram. Thiamethoxam exhibited similar toxicity to the Ibagué, Granada, and Sahagún populations, and the susceptibility to spinosad was similar across all populations. The spinetoram exhibited high toxicity (LC50 = 0.221 (0.174–0.287) mg a.i./Kg of grains) and residual activity against individuals from Ibagué and El Espinal populations, reinforcing its potential as alternative molecule for the management of R. dominica. Our findings expanded the insecticide options for controlling R. dominica in Colombia, contributing to avoid undesired effects of pyrethroids and organophosphates.

Enhancing Solidification in One-Part Geopolymer Systems through Alkali-Thermal Activation of Bauxite residue and Silica fume Integration

This work describes the synthesis of geopolymers, a family of amorphous alumino-silicates, employing Silica fume and bauxite waste as precursor materials in an alkali-activated polycondensation reaction and the investigation of their properties. The purpose of the study is to determine the feasibility of producing one-part alkali-activated geopolymer for use in cast-in-place construction. Following calcination of the bauxite residue at 800 °C, different amounts of solid activator Na2O (10%, 15%, and 20%) were added. Furthermore, Silica fume content ranging between 10% and 40% was utilised at intervals of 10%, in lieu of alkali-thermally treated bauxite residue. The primary objective of the study is to evaluate the fresh properties throughout the first 28 days of geopolymer formation, including consistency, flowability, heat evolution, initial setting time (IST), and final setting time (FST). Scanning electron microscopy (SEM) images are used in conjunction with the 28-day average compressive strength to demonstrate the solidification of one-part geopolymers. Furthermore, these properties are affected by adding Silica fume at regular intervals between 10% and 40% as a replacement of the alkali-thermally treated Bauxite residue. The 28-day average compressive strength, with a maximum value of 19MPa indicating successful geopolymer formation, supports the solidification of one-part geopolymers.

Nano-chitosan as a bio-enhancer for improving insecticide formulations: A study on indoxacarb and methomyl against cotton leafworm

This study was designed to improve the effectiveness and safety of two commercial insecticides, Camvaal EC 15 % (indoxacarb) and Nudrin SP 90 % (methomyl), against Spodoptera littoralis (Boisd.) through nanotechnology. Insecticide nanoemulsions were enhanced using nano-chitosan as a natural biopolymer. Nanoformulations of Camvaal and Nudrin were synthesized and characterized by Transmission Electron Microscopy (TEM) and Zetasizer Nano instruments. The insecticidal efficacy against the 4th instar of S. littoralis larvae revealed that the nano-formulations were more effective than the commercial formulations. LC50 values decreased from 0.049 to 0.009 mg/L for Camvaal and from 20.82 to 4.97 mg/L for Nudrin, indicating 81.63 and 76.09 % increased efficiency, respectively. Semi-field persistence studies established higher residual activity of the nano-formulations with LT50 values extending from 16.75 days (commercial) to 40.66 days (nano-formulation with chitosan). Cytotoxicity tests on human skin cells showed that nano-chitosan effectively lowered the toxicity of the insecticides with mortality percentages of 10 % for Camvaal and 12.30 % for Nudrin nano-formulations at 1000 μg/mL, compared to 100 and 73.20 % for the commercial products, respectively. These results reveal that nano-chitosan can be used as a bio-enhancer for the formulation of safer and more effective insecticides for sustainable pest control in agriculture.

Comprehensive analysis of genotypic and phenotypic characteristics of biotinidase deficiency patients in the eastern region of Türkiye

Background. Biotin is a water-soluble vitamin that plays a key role in carboxylation. The formation of free biotin is impaired in biotinidase deficiency (BD), resulting in impaired biotin-dependent carboxylase functions. Based on the percentage of residual serum enzyme activity, BD is classified as partial and profound. Methods. Retrospective data including gender, age, parental consanguinity, family history, biotinidase activity analyses, type of deficiency (partial-profound), physical examination, treatment, and genotypes were evaluated in patients diagnosed with biotinidase deficiency in a single center in the eastern region of Türkiye. Patients whose biotinidase enzyme activity was below 30% with biallelic variants in the BTD gene were diagnosed as BD. Results. A total of 302 patients were included in the study. Parental consanguinity was present in 135 (44.7%) of them. Two hundred eighty-six (94.7%) were diagnosed by neonatal screening, 14 (4.6%) by family screening and two (0.06%) by clinical symptoms. Ninety-two (30.5%) of the patients were followed-up with profound deficiency and 210 (69.5%) with partial deficiency. A total of 306 variants were detected. Twenty different variants (3 novel - 3 rare) and 31 different genotypes were detected. The 3 most frequently detected variants were c.410G>A (p.Arg137His; 47.3%), c.1270G>C (p.Asp424His; 29.7%), and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36; 15.3%). The 3 most frequently identified genotypes were c.410G>A (p.Arg137His) / c.1270G>C (p.Asp424His) compound heterozygous (32.4%), c.410G>A (p.Arg137His) homozygous (24.8%), and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) / c.1270G>C (p.Asp424His) compound heterozygous (12.2%). Patients with c.410G>A (p.Arg137His) homozygous variant, c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) homozygous variant and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) / c.410G>A (p.Arg137His) compound heterozygous variant were statistically significantly associated with profound deficiency. Compound heterozygosity of c.410G>A (p.Arg137His) / c.1270G>C (p.Asp424His) variants were significantly associated with partial deficiency. Conclusions. The association between the BTD genotype and biochemical phenotype is not always consistent. Our study provides valuable data by adding variants with genotype-phenotype correlations to the literature and three novel variants, which can provide significant guidance in clinical follow-up.

Complementary activity of Trifludimoxazin and Saflufenacil when used in combination for postemergence and residual weed control

Abstract Trifludimoxazin is a new herbicide that inhibits protoporphyrinogen oxidase and is targeted for commercial market introduction in North America, South America, and Asia. It will be available both as a standalone product and in a 1:2 mixture with saflufenacil. The herbicide is intended for use in preplant burndown (PPBD) and preemergence applications in cereal, corn (Zea mays L.), soybean [Glycine max (L.) Merr.], and pulse crops to control a variety of annual broadleaf and grass weed species. Additionally, it is planned to be used in tree crops, oil-palm (Elaeis quineensis Jacq.), and non-crop areas. In this study, we meticulously evaluated the performance and effectiveness of both the standalone herbicide and the innovative mixture concept in combating prevalent weeds commonly encountered in corn and soybean fields. Our findings revealed that both products exhibited exceptional efficacy, significantly reducing the presence of these troublesome weeds. Furthermore, the mixture concept not only demonstrated commendable soil mobility but also showcased impressive residual activity, positioning it as a powerful tool for sustainable weed control. These promising effects are further substantiated by our comprehensive ADME (Adsorption-Distribution-Metabolism-Extraction) studies, which provide insight into the behavior and longevity of the herbicides in the agricultural ecosystem.

Planting Strategy Influences Vegetable Response to Glyphosate and Glufosinate Applied Preplant in Plasticulture

Abstract In Georgia plasticulture vegetable production, a single installation of plastic mulch is utilized for up to five cropping cycles over an 18-month period. Preplant applications of glyphosate and glufosinate ensure fields are weed-free before transplanting, but recent data suggest residual activity of these herbicides may pose a risk to transplanted vegetables. Glyphosate and glufosinate were applied preplant in combination with three different planting configurations, including 1) a new plant hole into new mulch, 2) a pre-existing plant hole, 3) or a new plant hole spaced 15 cm from a pre-existing plant hole (adjacent). Following herbicide application, overhead irrigation was utilized to remove residues from the mulch before punching transplanting holes for tomato, cucumber, or squash. Visible injury, widths, biomass, yield of tomato, cucumber, or squash were not influenced by herbicide in the new mulch or adjacent planting configurations. When glyphosate was applied at 5.0 kg ae ha-1 and the new crop was planted into pre-existing holes, tomato was injured 45%, with reduced heights, biomass, and yields; at 2.5 kg ae ha-1 injury of 8% and a biomass reduction was observed. Cucumber and squash were injured 23% to 32% by glyphosate at 5.0 kg ae ha-1, with reductions in growth and early-season yield; lower rates did not influence crop growth or production when the crop was placed into a pre-existing plant hole. Glufosinate at the same rates did not affect tomato growth or yield when planted into pre-existing plant holes. Cucumber, when planted into pre-existing plant holes, was injured 43 to 75% from glufosinate, with reductions in height and biomass, and yield losses at 1.3 and 2.6 kg ai ha-1; similar results from glufosinate were observed in squash. In multi-crop plasticulture production, growers should ensure vegetable transplants are placed a minimum of 15 cm away from soil exposed to these herbicides.

Stability of immobilized L-arginine deiminase from Penicillium chrysogenum and evaluation of its anticancer activity

The aim of the present work was to immobilize L-arginine deiminase on suitable supports such as chitosan, alginate, and silica gel to study its stability. Additionally, the study aims to investigate the anticancer effects of the free purified enzyme on hepatocellular carcinoma (Hep-G2) and breast cancer (MCF-7) cell lines. L-arginine deiminase (ADI: EC 3.5.3.6) was immobilized on chitosan, Ca-alginate, and silica gel, with immobilization efficiencies of 89.0%, 72.8%, and 66.5%, respectively. The optimal immobilization time for the highest efficiency was 4 h. Increasing the concentration of glutaraldehyde improved the immobilization efficiency of ADI on chitosan. The chitosan-immobilized ADI retained about 45% of its activity after 8 cycles. The optimal pH values were 6 for the free purified ADI and 7 for the chitosan-immobilized ADI. The optimal temperature increased from 40 °C for the free enzyme to 45 °C after immobilization. The activation energies for the free and chitosan-immobilized enzymes were 71.335 kJ/mol and 64.011 kJ/mol, respectively. The Km values for the free and chitosan-immobilized ADI were 0.76 mM and 0.77 mM, respectively, while the Vmax values were 80.0 U/mg protein for the free ADI and 71.4 U/mg protein for the chitosan-immobilized ADI. After 30 days of storage at 4 °C, the residual activities were 40% for the free purified ADI and 84% for the chitosan-immobilized ADI. At 25 °C, the residual activities were 10% for the free ADI and 75% for the chitosan-immobilized ADI. The chitosan-immobilized ADI exhibited significantly higher stability against proteases such as pepsin and trypsin compared to the free enzyme. The purified ADI also demonstrated enhanced potential anticancer effects and significant cytotoxicity against the Hep-G2 and MCF-7 tumor cell lines compared to doxorubicin. These findings suggest that purified ADI has potential as an anticancer agent, though further in-depth studies are required.

The molecular reactive pathway between lipoxygenase and lipase and reactive species generated in dielectric barrier discharge atmospheric cold plasma: An investigation using molecular docking

The molecular docking was explored to study the interactions between reactive species generated by cold plasma and the enzymes lipoxygenase (LOX) and lipase (LPS), with the aim of elucidating the molecular mechanisms governing these interactions. Molecular docking results suggest that both LOX and LPS are primarily involved in hydrogen bonding interactions with the seven reactive species. The key binding sites for LOX and LPS were identified as Ile 663 and Glu 188, respectively. Notably, the lowest docking energy was observed between LOX and NO (−13.75 kcal/mol), whereas for LPS, it is between LPS and NO3 (−12.08 kcal/mol). Increased treatment voltage and time resulted in higher inactivation levels, with LPS exhibiting higher residual activity compared to LOX. When the voltage was 75 kV and the time was 120 s, the residual activities of LOX and LPS were 42.88% and 56.77%, respectively. Consequently, the results enhance our understanding of the mechanisms underlying the inhibition of enzyme activity by reactive species generated by cold plasma. Moreover, cold plasma may serve as a novel preservation technology for inhibiting lipid oxidation of food by controlling enzyme activity.

Aeruginosin 525 (AER525) from Cyanobacterium Aphanizomenon Sp. (KUCC C2): A New Serine Proteases Inhibitor

Aeruginosins (AERs) are one of the most common classes of cyanobacterial peptides synthesised through a hybrid non-ribosomal peptide synthase/polyketide synthase pathway. They have been found in Microcystis, Nodularia spumigena, Oscillatoria/Plantothrix, and Nostoc. The presence of AER in Aphanizomenon isolated from the Curonian Lagoon was reported for the first time in our previous work. Here, the structure of aeruginosin 525 (AER525), isolated from Aphanizomenon sp. KUCC C2, was characterised based on high-resolution mass spectrometry. This new AER variant shows potent activity against thrombin. It also inhibits trypsin and carboxypeptidase A but has no effect on elastase and chymotrypsin. In terms of the N-terminal residue and biological activity, AER525 displaces some similarity to dysinosins, which belongs to the most potent inhibitors of thrombin among AERs. The findings underline the potential of AER525 as a new anticoagulant agent.

Toxicity and residual activity of insecticides to Grapholita molesta (Lepidoptera: Tortricidae) in apple orchards: implications for insecticide resistance management.

Organophosphate (OP) insecticides have been used to control Grapholita molesta (Busck, 1916) in Brazil since 1990. However, the evolution of pest insecticide resistance and toxicological reassessments has led to the replacement of OPs with other chemical groups. This study evaluated the toxicity of the insecticides abamectin + cyantraniliprole (1.1 + 3.7g a.i./100L-1), acetamiprid + etofenproxi (9.1 + 16.5g a.i./100L-1), emamectin benzoate (0.5g a.i./100L-1), Sophora flavescens (Fabales: Fabaceae) (22.8g a.i./100L-1), phosmet (87.5g a.i./100L-1), and indoxacarb (11.2g a.i./100L-1) to eggs, larvae, and adults of G. molesta. Additionally, the residual activity of these insecticides in apple orchards was assessed, and the diagnostic concentration for resistance monitoring was estimated using LC99 values (Lethal Concentration that causes 99% mortality in a susceptible population). In larval studies, all insecticides resulted in mortality rates of more than 90%. The insecticide acetamiprid + etofenproxi reduced larval hatching by 99%. This insecticide with phosmet and Sophora flavescens caused over 80% adult mortality and had a 14-day residual activity in apple orchards. The estimated LC99 (μg.ml-1) for resistance monitoring were 1.75 (indoxacarb), 6.45 (metaflumizone), 9.10 (acetamiprid + etofenproxi), 9.67 (Sophora flavescens), 36.13 (phosmet), and 45.61 (abamectin + cyantraniliprole). The insecticides evaluated have high toxicity for G. molesta, being new tools for integrated pest management and for insecticide resistance management in apple orchards.

Identification and Application of Novel Patulin-Degrading Enzymes from Bacillus subtilis 168.

Patulin (PAT), a toxic secondary metabolite produced mainly by Penicillium species that frequently contaminates fruit and fruit-derived products, poses serious health risks to humans and animals. In the present study, three short-chain dehydrogenases/reductases (SDRs) with PAT-degrading ability, designated BsSDR1, BsSDR2, and BsSDR3, were identified from the genome of Bacillus subtilis 168. BsSDR1 and BsSDR2 showed powerful PAT elimination abilities, which can completely convert PAT to nontoxic E-ascladiol. Moreover, BsSDR1, BsSDR2, and BsSDR3 shared the highest sequence identity of 36.03% with the reported PAT-degrading enzymes, indicating that they are novel PAT-degrading enzymes. BsSDR1, BsSDR2, and BsSDR3 exhibited the highest activity against PAT at 40, 40, and 35 °C, respectively. Additionally, BsSDR1, BsSDR2, and BsSDR3 displayed remarkable thermostability, retaining 32.50, 24.63, and 46.74% residual activity, respectively, after incubation at 50 °C for 1 h. Three-dimensional (3D) simulation and site-directed mutagenesis indicated that the catalytic triad formed by the residues (Ser, Tyr, and Lys) was the key for SDR activity, and this conserved catalytic mechanism was followed in the catalytic process of novel PAT-degrading enzymes BsSDR1, BsSDR2, and BsSDR3. More importantly, BsSDR1, BsSDR2, and BsSDR3 can degrade PAT in apple juice at rates of 86.90, 90.17, and 61.57%, respectively. The identification of BsSDR1, BsSDR2, and BsSDR3 enriched the PAT-degrading enzyme libraries, providing promising candidates for PAT decontamination in the food industry.

New evidence on the spread in Catalonia of the invasive longhorn beetle, Xylotrechus chinensis, and the efficacy of abamectin control.

The invasive wasp-mimicking Tiger Longicorn Beetle, Xylotrechus chinensis, a potentially lethal pest of mulberry trees (Moraceae: Morus spp.), was first reported in Europe in 2018. In Catalonia its spread has been impressive: one district, four towns, 44km2 in February 2018; four districts, 12 towns, 378km2 in July 2020; seven districts, 65 towns, 1134km2 in December 2023. It is likely the infestation will spread further in the future, in particular along the Mediterranean coast from Spain to Greece. The flight period of this beetle in Mediterranean countries is expanding on both ends (from the end of April to mid-October), probably due to increasing temperatures because of climate change, although this should be further proved. The adult beetle life span is 18-23 days in insectaria, likely somewhat shorter in the wild since they do not feed and spend more energy flying outside. The single treatment with abamectin carried out in a Catalonian town in June 2022 on a sample of 24 trees (12 treated, 12 untreated) has been effective in reducing the infestation of this beetle in relation to untreated trees. But the reduction was not complete since new exit holes appeared during the summer of the following year. Therefore, an additional treatment carried out during the first ten days of October should be tested to achieve a better control of this pest. It would also be worthwhile trying a single treatment of abamectin in mid-July since it should be able to kill larvae hatching before the treatment and still have sufficient residual activity for larvae hatching after the treatment, and thus cover the entire activity period. Also, to get satisfactory mulberry tree protection, it is likely that treatments might have to be repeated for a few consecutive years. Yearly re-infestations from untreated public and/or private mulberry trees are also a concern and make short-term control of this pest difficult, no doubt accounting for the rapid spread of this beetle.

Lipoxygenase activity in tropical fish: Changes during surimi processing and some biochemical characterization in lizardfish (Saurida tumbil).

The lipoxygenase (LOX) activity of major tropical fish used for surimi production, including threadfin bream (TB), lizardfish (LZ), and goatfish (GF), was measured in the gills, skin, and muscle. The highest LOX activity was observed in the LZ samples (p<0.05), with the gills exhibiting the greatest activity at 376.56U/mg (p<0.05). The highest peroxide value was detected in the TB samples, particularly in the gills (p<0.05). Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were the main polyunsaturated fatty acids in all the tissues and surimi. The total lipid and DHA contents of washed mince reduced considerably after the screw press process. Although LOX activity decreased during surimi production, a residual activity of 21.33U/g was observed in the finished surimi. LOX was partially purified and characterized from LZ gills. The purification was conducted using two successive chromatographic steps, Sephacryl S-200 and diethylaminoethyl (DEAE)-sepharose, resulting in a 3.52% yield and a 22.43-fold increase in purity. The optimum activity was found at 25°C and pH 7.5, with pH stability between 6.0 and 8.5. The relatively high thermal stability at 4°C-10°C suggested that LOX might contribute to fish lipid oxidation during cold storage. The enzyme was thermally inactivated at 60°C. The preferred substrate was EPA. LOX from the LZ gills was inhibited by 1mM ethylenediaminetetraacetic acid and activated by 1mM Fe2+, Na+, and Ca2+. PRACTICAL APPLICATION: Elucidating lipoxygenase activity and lipid oxidation in various tropical fish tissues, as well as understanding the characteristics of LOX, can help take appropriate postharvest actions to afford high-quality surimi.

Zebrafish are resilient to the loss of major diacylglycerol acyltransferase enzymes

In zebrafish, maternally deposited yolk is the source of nutrients for embryogenesis prior to digestive system maturation. Yolk nutrients are processed and secreted to the growing organism by an extra-embryonic tissue, the yolk syncytial layer (YSL). Export of lipid from the YSL occurs through the production of triacylglycerol-rich lipoproteins. Here we report that mutations in the triacylglycerol synthesis enzyme, diacylglycerol acyltransferase-2 (Dgat2), cause yolk sac opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. Though triacylglycerol synthesis continues, it is not properly coupled to lipoprotein production as dgat2 mutants produce fewer, smaller, ApoB-containing lipoproteins. Unlike DGAT2-null mice, which are lipopenic and die soon after birth, zebrafish dgat2 mutants are viable, fertile and exhibit normal mass and adiposity. Residual Dgat activity cannot be explained by the activity of other known Dgat isoenzymes, as dgat1a;dgat1b;dgat2 triple mutants continue to produce YSL lipid droplets and remain viable as adults. Further, the newly identified diacylglycerol acyltransferase, Tmem68, is also not responsible for the residual triacylglycerol synthesis activity. Unlike overexpression of Dgat1a and Dgat1b, monoacylglycerol acyltransferase-3 (Mogat3b) overexpression does not rescue yolk opacity, suggesting it does not possess Dgat activity in the YSL. However, mogat3b;dgat2 double mutants exhibit increased yolk opacity and often have structural alterations of the yolk extension. Quadruple mogat3b;dgat1a;dgat1b;dgat2 mutants either have severely reduced viability and stunted growth, or do not survive past 3 days post fertilization, depending on the dgat2 mutant allele present. Our study highlights the remarkable ability of vertebrates to synthesize triacylglycerol through multiple biosynthetic pathways.

Developing Strategies to Mitigate Squash Injury from the Soil Residual Activity of Glyphosate in Bareground Systems

Fresh market vegetables are an essential component of the human diet. Maximizing yield is critical, and to achieve this goal, fields must be weed-free when vegetable crops are planted. Historically, removing emerged weeds just before planting has been accomplished using the herbicide glyphosate. However, recent research has indicated that glyphosate applied to sandy, low-organic-matter soils just before transplanting vegetables can be injurious. Two field experiments investigated 1) the response of transplanted squash to the residual activity of glyphosate, and 2) the effects of implementing tillage, irrigation, or extending the plant-back interval after application and before planting to mitigate injury from glyphosate. Glyphosate applied at 1.3, 2.5, or 3.8 kg ae/ha 1 day before transplanting injured squash 13%, 29%, and 53%, respectively; extending the interval between application and planting to 7 days reduced injury to 1%, 11%, and 28% at the same rates. An interaction between application rate and planting interval was also observed on squash plant widths and biomass, as well as early-season and total marketable fruit numbers and weights. Total marketable fruit number was reduced 29% and 52% by glyphosate at 2.5 or 3.8 kg ae/ha, respectively, and a reduction in fruit production of 36%, 28%, and 23% was observed when glyphosate was applied 1, 4, or 7 days before transplanting, respectively. In a separate study, light tillage (5 cm deep) was the most effective cultural practice evaluated because it eliminated damage by glyphosate. Overhead irrigation of 0.6 cm was not beneficial in mitigating injury by glyphosate. Recommendations from this research will help vegetable growers avoid injury from the residual activity of glyphosate through a FIFRA 2(ee) recommendation label.

Cold-Active Lipase from the Ice Cave Psychrobacter SC65A.3 Strain, a Promising Biocatalyst for Silybin Acylation.

Cold-active lipase from the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania) was cloned and characterized as an extremophilic biocatalyst for silybin acylation. Structural analyses highlighted conserved motifs confirming a functional lipase and the presence of primary structure elements for catalysis at low temperatures. The recombinant enzyme (PSL2) heterologously expressed in Escherichia coli was purified in one step by affinity chromatography with a yield of 12.08 ± 1.72 µg L-1 of culture and a specific activity of 20.1 ± 3.2 U mg-1 at 25 °C. Functional characterization of PSL2 showed a neutral (7.2) optimal pH and a high thermal stability up to 90 °C. Also, this lipase was stable in the presence of different organic solvents, with 60% residual activity when using 20% DMSO. Kinetic measurements indicated performant catalytic efficiency of PSL2 for different short and long chain fatty acids, with Km in the mM range. The catalytic activity of PSL2 was assessed for silybin acylation with various fatty acids and fatty acid methyl esters, demonstrating a 90% silybin conversion when methyl decanoate ester was used. This result clearly highlights the biocatalytic capability of this new cold-active lipase.

The efflux system CdfX exports zinc that cannot be transported by ZntA in Cupriavidus metallidurans.

Cupriavidus metallidurans is able to survive exposure to high concentrations of transition metals, but is also able to grow under metal starvation conditions. A prerequisite of cellular zinc homeostasis is a flow equilibrium combining zinc uptake and efflux processes. The mutant strain ∆e4 of the parental plasmid-free strain AE104 with a deletion of all four chromosomally encoded genes of previously known efflux systems ZntA, CadA, DmeF, and FieF was still able to efflux zinc in a pulse-chase experiment, indicating the existence of a fifth efflux system. The gene cdfX, encoding a protein of the cation diffusion facilitator (CDF) family, is located in proximity to the cadA gene, encoding a P-type ATPase. Deletion of cdfX in the ∆e4 mutant resulted in a further decrease in zinc resistance. Pulse-chase experiments with radioactive 65Zn(II) and stable-isotope-enriched 67Zn(II) provided evidence that CdfX was responsible for the residual zinc efflux activity of the mutant strain ∆e4. Reporter gene fusions with cdfX-lacZ indicated that the MerR-type regulator ZntR, the main regulator of zntA expression, was responsible for zinc- and cadmium-dependent upregulation of cdfX expression, especially in mutant cells lacking one or both of the previously characterized efflux systems, ZntA and CadA. Expression of zntR also proved to be controlled by ZntR itself as well as by zinc and cadmium availability. These data indicate that the cdfX-cadA region provides C. metallidurans with a backup system for the zinc-cadmium-exporting P-type ATPase ZntA, with CdfX exporting zinc and CadA cadmium.IMPORTANCEBacteria have evolved the ability to supply the important trace element zinc to zinc-dependent proteins, despite external zinc concentrations varying over a wide range. Zinc homeostasis can be understood as adaptive layering of homeostatic systems, allowing coverage from extreme starvation to extreme resistance. Central to zinc homeostasis is a flow equilibrium of zinc comprising uptake and efflux reactions, which adjusts the cytoplasmic zinc content. This report describes what happens when an imbalance in zinc and cadmium concentrations impairs the central inner-membrane zinc efflux system for zinc by competitive inhibition for this exporter. The problem is solved by activation of Cd-exporting CadA or Zn-exporting CdfX as additional efflux systems.

Immobilization of Phospholipase D on Magnetic Graphene Oxide for Efficient Phosphatidylserine Production

Phosphatidylserine (PS) has significant applications in various sectors, such as the medical and food industries. However, its production relies heavily on phospholipase D (PLD), a crucial tool that is hindered by issues like poor stability and irrecoverability. Immobilization presents itself as an effective solution to overcome these limitations. In this study, magnetic graphene oxide (MGO) modified with an amino (NH2) group was synthesized and utilized for PLD immobilization. The activity of the immobilized PLD (MGO-PLD) reached 3062 U/gMGO, with a specific activity of 33.9 U/mgPLD, virtually identical to that of the free PLD. MGO-PLD was utilized to synthesize PS efficiently in a biphasic system. Under optimal conditions, the PS yield reached 18.66 g/L, with a conversion rate of 92.8% and a productivity of 3.11 g/L/h. Notably, MGO-PLD retained an impressive PS conversion rate of 77.4% even after seven repetitive usages. Moreover, MGO-PLD displayed enhanced thermal and pH resistance properties compared to free PLD, alongside augmented storage stability. After an 8-week preservation at 4 °C, its residual activity was maintained at 76.3%. This study provides a sustainable and highly efficient pathway for the biocatalytic synthesis of PS.

Concurrent SOS1 and MEK suppression inhibits signaling and growth of NF1-null melanoma.

Neurofibromin (NF1) is a negative regulator of RAS signaling, frequently mutated in cancer. NF1-mutant melanoma is a highly malignant tumor for which targeted therapies are lacking. Here, we use biochemical and pharmacological assays on patient-derived models and isogenic cell lines to identify potential pharmacologic targets, revealing that NF1-null melanomas are dependent on RAS activation and that MEK inhibition relieves ERK-dependent negative feedback, increasing RAS signaling. MEK inhibition with avutometinib abrogates the adaptive rebound in ERK signaling, but the antitumor effects are limited. However, concurrent inhibition of MEK and SOS1 abrogates ERK activation, induces cell death, and suppresses tumor growth. In contrast to the NF1-deficient setting, concurrent SOS1 and SOS2 depletion is required to completely inhibit RAS signaling in NF1 wild-type cells. In sum, our data provide a mechanistic rationale for enhancing the therapeutic efficacy of MEK inhibitors by exploiting the lower residual SOS activity in NF1-null tumor cells.

A novel alkali and thermotolerant protease from Aeromonas spp. retrieved from wastewater

Enzymes are integral to numerous industrial processes, with a growing global demand for various enzyme types. Protease enzymes, in particular, have proven to be cost-effective, stable, and compatible alternatives to traditional chemical processes in both industrial and environmental applications. In this study, an alkaline protease-producing strain of Aeromonas spp. was isolated from a wastewater treatment plant in Iran. The protease production was confirmed by culturing the strain on casein agar medium. The bacterium was identified through morphological, biochemical, and 16 S rRNA sequencing analyses. The optimal culture medium for bacterial growth and enzyme production was obtained using peptone, salt, yeast extract, galactose, and CaCl₂ at an initial pH of 8. Maximum protease production was achieved after 20 h of incubation at 40 °C. To partially purify the enzyme, the supernatant of the bacterial culture medium was first centrifuged, and the enzyme was precipitated using ammonium sulfate, followed by dialysis. Zymography revealed the production of one type of protease during bacterial growth. The partially purified protease exhibited optimal activity at pH 8.5 and maximum stability at pH 9. The optimum temperature for maximum enzyme activity was observed at 50 °C, with 100% residual activity retained for 1 h at 0 °C. The effect of metal ions on enzyme activity was assessed, revealing that KCl induced the most significant effects (p < 0.0001) on enzyme activity. Chemical amino acid modifiers and inhibitors, such as EDTA, DEPSI, and IAA, did not exhibit significant inhibition. In contrast, PMSF and HNBB significantly (p < 0.0001) reduced enzyme activity, suggesting that the enzyme could be classified as a serine protease. The protease also demonstrated high stability in the presence of 2% SDS, showing no signs inactivation. The alkaline pH optimum, thermal stability, and resistance to SDS exhibited by the protease produced by the Aeromonas strain are particularly promising characteristics that warrant further investigation. Based on preliminary tests and the enzyme’s characteristics, this protease can be recommended for various applications, pending further studies.