Natural Substrate Research Articles

Season-long comparison of trap lures for integrated management of the navel orangeworm (Lepidoptera: Pyralidae) in almond and pistachio

Monitoring navel orangeworm, Amyelois transitella (Lepidoptera: Pyralidae) using pheromone lures is now complicated by the use of mating disruption. Two commercially available lures that are minimally affected by mating disruption use either natural ovipositional substrates (Bait lures) or a combination of phenyl propionate (PPO), a volatile chemical that attracts both sexes and pheromone lures (Combo lures). We compared the season-long trap capture for these two lures in a sterile insect technique (SIT) demonstration project in Fresno County, California, 2020–2023. A total of 194,233 males (96.52%) were caught using Combo lures and 7006 (3.48%) caught using Bait lures, while 95,947 females (48.83%) were caught using Combo lures and 100,532 (51.17%) caught using Bait lures. Capture of A. transitella was 2.3-fold higher in pistachio (278,492) than in almond (119, 316), and within a commodity, there was a moderate correlation in female capture between the two lures; correlation was stronger during the period leading up to July 21 (Julian date 202). For Bait lures, 48.7% of the trap catch in almond occurred by July 21 and 87.1% of the trap catch occurred in pistachio by July 21. For Combo lures, 38.9% of the trap catch in almond occurred by July 21 while 54.6% of the catch occurred in pistachio. Bait lures became less effective after July 21, likely due to diminished attractiveness as new crop nuts split in August through October. Our data indicate that Combo lures are preferable when the goal is consistent season-long capture of A. transitella in almond and pistachio.

Abstract 4777: A small-molecule SMUG1 activator enhances repair of 5-hydroxymethyl-2’-deoxyuridine-mediated pyrimidine lesions in DNA

Abstract Background: Pharmacological upregulation of DNA repair pathways presents a potentially promising approach for cancer prevention in populations that are genetically high-risk. SMUG1, an enzyme involved in base excision repair, removes uracil and certain oxidized bases from DNA to mitigate adverse genotoxic and mutagenic effects and represents a promising target for cancer therapy. A growing body of evidence suggests that SMUG1 is involved in tumorigenesis, and deficiency of SMUG1 correlates with poor prognosis in several cancers. We hypothesize that upregulation of SMUG1 by a small molecule activator may be a viable strategy for reducing tumorigenesis in at-risk populations. We report on the properties of SU0547 - a SMUG1 activator that was derived from the EGFR inhibitor drug gefitinib - which shows reduced kinase inhibition and potent activation of SMUG1 both in vitro and in human cell lines. Methods: MTT proliferation assays were used to screen for toxicity and to determine the effect of SU0547 on 5-hydroxymethyl-2’-deoxyuridine (5-hmdU)-treated cell lines. The effect of the compound on SMUG1 expression was determined by Western blot and RT-qPCR while its effect on DSBs via γH2AX expression in 5-hmdU-treated cells was determined by immunofluorescence and Western blot. Results: SU0547 activates SMUG1 by 347 ± 50% in vitro at 100 nM, with an AC50 of 2.55 ± 0.62 µM. We examined whether it can modulate SMUG1 activity in a panel of human cell lines by artificially inducing DNA damage with the native SMUG1 substrate 5-hmdU and treating cells with the activator. In all five human cancer cell lines tested, SU0547 caused the cells to become significantly more resistant to 5-hmdU by factors of 2.1 to 5.3-fold. A series of CRISPR-Cas9 generated SMUG1 KO cell lines showed resistance to 5-hmdU and SU0547 had no effect on these treated cells, confirming that this activator acts on endogenous SMUG1. To determine the effect of the activator on double-strand breaks (DSBs), cells were treated with 5-hmdU with or without the compound and DSBs were visualized and quantitated by examining γH2AX foci. 5-hmdU treatment induced γH2AX foci while SU0547 treatment significantly reduced these foci to near or below baseline levels. These results were confirmed using Western blot quantitation of γH2AX. Conclusions: We envision that this activator will serve as a useful tool for further studying the role of SMUG1 in mediating 5-hmdU toxicity and will enable the testing of our hypothesis that enhancing DNA repair processes may serve to inhibit mutagenesis and slow progression of pre-neoplastic lesions to invasive cancers associated with genomic instability. Citation Format: Lisa A. McPherson, Yixuan Gao, Shanthi Adimoolam, Samyuktha Suresh, David L. Wilson, Ishani Das, Elizabeth R. Park, Christine S. Ng, Yong Woong Jun, James M. Ford, Eric T. Kool. A small-molecule SMUG1 activator enhances repair of 5-hydroxymethyl-2’-deoxyuridine-mediated pyrimidine lesions in DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4777.

Abstract 3256: Novel SOX2-targeting bioPROTACs for the treatment of cancers

Abstract Targeted protein degradation (TPD) technology has been developed showing the therapeutic potential to modulate historically ‘undruggable’ disease-associated proteins. Currently, proteolysis targeting chimeras (PROTACs) using small-molecule binders represent the TPD field, and PROTAC-induced proximity between target proteins and E3 ligases enables specific degradation of target proteins. However, substantial amounts of clinically relevant cancer targets such as transcription factors lack active sites or allosteric binding pockets for small molecules which challenges the development of classical PROTACs for these targets. SOX2 is one of the pluripotency transcription factors and has been well-known for its crucial role in tumor development through promoting cancer cell survival, metastasis, stemness, and drug resistance. Numerous studies have also reported SOX2 amplification and overexpression in various tumor tissues. Thus, SOX2 has been validated as an attractive anticancer target, but directly targeting SOX2 has been proved to be difficult since SOX2 is an “undruggable” transcription factor. EPD Biotherapeutics develops EPDegTM, a next-generation engineered protein degrader platform in order to overcome the limitations of small molecule-based PROTAC. Here we report a series of SOX2-targeting bioPROTACs, being composed of a nanobody against SOX2 with high affinity and several ubiquitin E3 ligases with deletion of natural substrate binding domain to allow highly selective degradation of SOX2 protein. To evaluate the in vitro efficacy, SOX2 bioPROTACs were generated by mRNA IVT with CleanCap ® and modified uridine. First, robust degradation of SOX2 with a half-maximal degradation concentration (DC50) of the picomolar range was observed with the treatment of SOX2 bioPROTACs in multiple SOX2-overexpressing cancer cell lines. The degradation was ubiquitin- and proteosome-dependent manner, and neither nanobody nor ubiquitin E3 ligase alone affected SOX2 protein level. Then, SOX2 bioPROTACs led to significant cancer cell growth inhibition caused by apoptotic cell death with a half-maximal growth inhibition concentration (GI50) of the picomolar range. Notably, chemo-resistant cancer cell lines expressing SOX2 protein in high levels were sensitized to chemotherapy in combination with our SOX2 bioPROTACs. Furthermore, i.v. injection of one of our lead candidates formulated with LNP significantly inhibited tumor growth in the A549 xenograft model, proving the in vivo anti-tumor efficacy of SOX2 degraders for the first time in the world. Taken together, EPD’s SOX2 bioPROTACs show potent in vitro and in vivo anti-tumor efficacy as first-in-class SOX2 degraders and provide new insights for the therapeutic potential of transcription factor-degrading strategies. Citation Format: Jaehyun Choi, Saeyi Lim, Xuelian Bai, Hyerin Song, Yeonji Oh, Yeryoung Yong, Seunghyun Lee. Novel SOX2-targeting bioPROTACs for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3256.

Functional analysis of CYP71AV1 reveals the evolutionary landscape of artemisinin biosynthesis.

Artemisinin biosynthesis, unique to Artemisia annua, is suggested to have evolved from the ancestral costunolide biosynthetic pathway commonly found in the Asteraceae family. However, the evolutionary landscape of this process is not fully understood. The first oxidase in artemisinin biosynthesis, CYP71AV1, also known as amorpha-4,11-diene oxidase (AMO), has specialized from ancestral germacrene A oxidases (GAOs). Unlike GAO, which exhibits catalytic promiscuity toward amorpha-4,11-diene, the natural substrate of AMO, AMO has lost its ancestral activity on germacrene A. Previous studies have suggested that the loss of the GAO copy in A. annua is responsible for the abolishment of the costunolide pathway. In the genome of A. annua, there are two copies of AMO, each of which has been reported to be responsible for the different product profiles of high- and low-artemisinin production chemotypes. Through analysis of their tissue-specific expression and comparison of their sequences with those of other GAOs, it was discovered that one copy of AMO (AMOHAP) exhibits a different transcript compared to the reported artemisinin biosynthetic genes and shows more sequence similarity to other GAOs in the catalytic regions. Furthermore, in a subsequent in vitro enzymatic assay, the recombinant protein of AMOHAP unequivocally demonstrated GAO activity. This result clearly indicates that AMOHAP is a GAO rather than an AMO and that its promiscuous activity on amorpha-4,11-diene has led to its misidentification as an AMO in previous studies. In addition, the divergent expression pattern of AMOHAP compared to that of the upstream germacrene A synthase may have contributed to the abolishment of costunolide biosynthesis in A. annua. Our findings reveal a complex evolutionary landscape in which the emergence of a new metabolic pathway replaces an ancestral one.

Artificial language creation as a tool for assessing children’s metalinguistic abilities

The author aims to analyse two projects of the artificial language Uniling. They were created by students of years 5 to 11 within the linguistic framework of the "Formula de Integreco" training camp in June 2017 and January 2018. The research methods included structural analysis of the artificial language, as well as its creation process in comparison with Russian, i. e. the native language for all participants in the experiment. Research results: the specific conditions for artificial language development enable us to trace the stages of its accelerated "evolution" and complexity buildup at all levels. The latter becomes possible by shifting the initial focus away from observing the economy principle in language and raising randomly occurring exceptions to the status of a rule. Such rules are further extrapolated to other same-level units of the artificial language. This indicates that children attach importance to the concept of linguistic norm, as well as the possibility of clear grammatical differentiation when the grammatical meaning is secondary relative to the lexical-conceptual one. Children give no less significance to the inner form of words, which makes it possible to create lexemes employing the means of the artificial language itself without relying on the material of natural substrate languages. Moreover, the inner form helps to outline the systemic connections within the thematic groups. However, the hypernym/hyponym relations are obviously weak as generic concepts are not found in the Uniling lexis. At the same time, the aesthetics and euphony notions often come to the fore, which is reflected in the expression plane of artificial language lexemes and in the idea of the pragmatic potential of the language. This is most clearly seen at the syntax level due to the introduction of particular semantic role markers not typical of the Russian language (benefactives and malefactives). Conclusions: artificial language analysis enables us to identify the strengths and weaknesses of children’s metalinguistic representations, which can be considered in the process of teaching Russian at school.

Palladium (II)-Catalyzed C-H Activation with Bifunctional Ligands: From Curiosity to Industrialization.

In 2001, our curiosity to understand the stereochemistry of C-H metalation with Pd prompted our first studies in Pd(II)-catalyzed asymmetric C-H activation (RSC Research appointment: 020 7451 2545, Grant: RG 36873, Dec. 2002). We identified four central challenges: 1. poor reactivity of simple Pd salts with native substrates; 2. few strategies to control site selectivity for remote C-H bonds; 3. the lack of chiral catalysts to achieve enantioselectivity via asymmetric C-H metalation, and 4. low practicality due to limited coupling partner scope and the use of specialized oxidants. These challenges necessitated new strategies in catalyst and reaction development. For reactivity, we developed approaches to enhance substrate-catalyst affinity together with novel bifunctional ligands which participate in and accelerate the C-H cleavage step. For site-selectivity, we introduced the concept of systematically modulating the distance and geometry between a directing template, catalyst, and substrate to selectively access remote C-H bonds. For enantioselectivity, we devised predictable stereomodels for catalyst-controlled enantioselective C-H activation based on the participation of bifunctional ligands. Finally, for practicality, we have developed varied catalytic manifolds for Pd(II) to accommodate diverse coupling partners while employing practical oxidants such as simple peroxides. These advances have culminated in numerous C-H activation reactions, setting the stage for broad industrial applications.

Palladium (II)‐Catalyzed C−H Activation with Bifunctional Ligands: From Curiosity to Industrialization

AbstractIn 2001, our curiosity to understand the stereochemistry of C−H metalation with Pd prompted our first studies in Pd(II)‐catalyzed asymmetric C−H activation (RSC Research appointment: 020 7451 2545, Grant: RG 36873, Dec. 2002). We identified four central challenges: 1. poor reactivity of simple Pd salts with native substrates; 2. few strategies to control site selectivity for remote C−H bonds; 3. the lack of chiral catalysts to achieve enantioselectivity via asymmetric C−H metalation, and 4. low practicality due to limited coupling partner scope and the use of specialized oxidants. These challenges necessitated new strategies in catalyst and reaction development. For reactivity, we developed approaches to enhance substrate–catalyst affinity together with novel bifunctional ligands which participate in and accelerate the C−H cleavage step. For site‐selectivity, we introduced the concept of systematically modulating the distance and geometry between a directing template, catalyst, and substrate to selectively access remote C−H bonds. For enantioselectivity, we devised predictable stereomodels for catalyst‐controlled enantioselective C−H activation based on the participation of bifunctional ligands. Finally, for practicality, we have developed varied catalytic manifolds for Pd(II) to accommodate diverse coupling partners while employing practical oxidants such as simple peroxides. These advances have culminated in numerous C−H activation reactions, setting the stage for broad industrial applications.

Computational analyses of drug resistance mutations in katG and emb complexes in Mycobacterium tuberculosis.

The number of antibiotic resistant pathogens is increasing rapidly, and with this comes a substantial socioeconomic cost that threatens much of the world. To alleviate this problem, we must use antibiotics in a more responsible and informed way, further our understanding of the molecular basis of drug resistance, and design new antibiotics. Here, we focus on a key drug-resistant pathogen, Mycobacterium tuberculosis, and computationally analyze trends in drug-resistant mutations in genes of the proteins embA, embB, embC, and katG, which play essential roles in the action of the first-line drugs ethambutol and isoniazid. We use docking to predict binding modes of isoniazid to katG that agree with suggested binding sites found in our laboratory using cryo-EM. Using mutant stability predictions, we recapitulate the idea that resistance occurs when katG's heme cofactor is destabilized rather than due to a decrease in affinity to isoniazid. Conversely, we have identified resistance mutations that affect the affinity of ethambutol more drastically than the affinity of the natural substrate of embB. With this, we illustrate that we can distinguish between the two types of drug resistance-cofactor destabilization and drug affinity reduction-suggesting potential uses in the prediction of novel drug-resistant mutations.

Control of three-carbon amino acid homeostasis by promiscuous importers and exporters in Bacillus subtilis: role of the "sleeping beauty" amino acid exporters.

The Gram-positive model bacterium Bacillus subtilis can acquire amino acids by import, de novo biosynthesis, or degradation of proteins and peptides. The accumulation of several amino acids inhibits the growth of B. subtilis, probably due to misincorporation into cellular macromolecules such as proteins or peptidoglycan or due to interference with other amino acid biosynthetic pathways. Here, we studied the adaptation of B. subtilis to toxic concentrations of the three-carbon amino acids L-alanine, β-alanine, and 2,3-diaminopropionic acid, as well as the two-carbon amino acid glycine. Resistance to the non-proteinogenic amino acid β-alanine, which is a precursor for coenzyme A biosynthesis, is achieved by mutations that either activate a cryptic amino acid exporter, AexA (previously YdeD), or inactivate the amino acid importers AimA, AimB (previously YbxG), and BcaP. The aexA gene is very poorly expressed under most conditions studied. However, mutations affecting the transcription factor AerA (previously YdeC) can result in strong constitutive aexA expression. AexA is the first characterized member of a group of amino acid exporters in B. subtilis, which are all very poorly expressed. Therefore, we suggest to call this group "sleeping beauty amino acid exporters." 2,3-Diaminopropionic acid can also be exported by AexA, and this amino acid also seems to be a natural substrate of AerA/AexA, as it can cause a slight but significant induction of aexA expression, and AexA also provides some natural resistance toward 2,3-diaminopropionic acid. Moreover, our work shows how low-specificity amino acid transporters contribute to amino acid homeostasis in B. subtilis.IMPORTANCEEven though Bacillus subtilis is one of the most-studied bacteria, amino acid homeostasis in this organism is not fully understood. We have identified import and export systems for the C2 and C3 amino acids. Our work demonstrates that the responsible amino acid permeases contribute in a rather promiscuitive way to amino acid uptake. In addition, we have discovered AexA, the first member of a group of very poorly expressed amino acid exporters in B. subtilis that we call "sleeping beauty amino acid exporters." The expression of these transporters is typically triggered by mutations in corresponding regulator genes that are acquired upon exposure to toxic amino acids. These exporters are ubiquitous in all domains of life. It is tempting to speculate that many of them are not expressed until the cells experience selective pressure by toxic compounds, and they protect the cells from rare but potentially dangerous encounters with such compounds.

Oxygen Activation in Aromatic Ring Cleaving Salicylate Dioxygenase: Detection of Reaction Intermediates with a Nitro-substituted Substrate Analog.

Cupin dioxygenases such as salicylate 1,2-dioxygense (SDO) perform aromatic C-C bond scission via a 3-His motif tethered iron cofactor. Here, transient kinetics measurements are used to monitor the catalytic cycle of SDO by using a nitro-substituted substrate analog, 3-nitrogentisate. Compared to the natural substrate, the nitro group reduces the enzymatic kcat by 500-fold, thereby facilitating the detection and kinetic characterization of reaction intermediates. Sums and products of reciprocal relaxation times derived from kinetic measurements were found to be linearly dependent on O2 concentration, suggesting reversible formation of two distinct intermediates. Dioxygen binding to the metal cofactor takes place with a forward rate of 5.9×103 M-1 s-1: two orders of magnitude slower than other comparable ring-cleaving dioxygenses. Optical chromophore of the first intermediate is distinct from the in situ generated SDO Fe(III)-O2⋅- complex but closer to the enzyme-substrate precursor.

The role of macroinvertebrate taxonomic resolution in bioassessment of urban streams: A case study from the City of Lodz, Poland

Benthic macroinvertebrates were sampled at 28 sites situated on ten urban streams (Łódź, Poland) representative of a wide range of anthropogenic modifications of water chemistry and bed structure. A total of 5,043 individuals, comprising 121 species belonging to 37 families and 15 orders, were collected. Diptera (35.8 %) and Ephemeroptera (31.8 %) were dominant.The studied sites were grouped into clusters based on separately-analyzed environmental parameters, various levels of taxonomic diversity, and bioassessment variables. All analyses demonstrated similar trends of anthropogenic modification, intensifying concentrically from the suburbs towards the city centre. The first cluster included the upper section of streams; less polluted, richer in ecotone zones and natural stream bed substrate. The second cluster comprised streams demonstrating the influence of moderate human pressure. The third cluster included streams flowing through the city center in concreted beds and which were connected to a combined sewage system in which sewage and rain water are transported through a single canal system and enters the nearest stream. Modification of stream beds was significantly negatively correlated with the number of species, families and orders, but not with total abundance.The number of species, the number of families and the number of orders were highly mutually correlated. Moreover, they were similarly correlated with biodiversity and biotic indices, which are used to assess the ecological status of the aquatic environment. Hence, different levels of taxonomic identification demonstrate similar effectiveness in assessment, i.e. higher taxa of aquatic macroinvertebrates may be suitable for simplified ecological monitoring of urban watercourses.

Optimization of bacterial cellulose production by Komagataeibacter sucrofermentans in synthetic media and agrifood side streams supplemented with organic acids and vitamins

The effect of thiamine (TA), ascorbic acid (AA), citric acid, and gallic acid (GA) on bacterial cellulose (BC) production by Komagataeibacter sucrofermentans, in synthetic (Hestrin and Schramm, HS) and natural substrates (industrial raisins finishing side stream extract, FSSE; orange juice, OJ; green tea extract, GTE), was investigated. The Response Surface Methodology was found reliable for BC yield prediction and optimization. Higher yields were achieved in the FSSE substrates, especially those supplemented with AA, TA, and GA (up to 19.4 g BC/L). The yield in the non-fortified substrates was 1.1–5.4 and 11.6–15.7 g/L, in HS and FSSE, respectively. The best yield in the natural non-fortified substrate FSSE-OJ-GTE (50–20-30 %), was 5.9 g/L. The porosity, crystallinity, and antioxidant properties of the produced BC films were affected by both the substrate and the drying method (freeze- or oven-drying). The natural substrates and the process wastewaters can be further exploited towards added value and sustainability. Take Home Message Sentence: Raisin and citrus side-streams can be efficiently combined for bacterial cellulose production, enhanced by other vitamin- and phenolic-rich substrates such as green tea.

Effects of Xylanase A double mutation on substrate specificity and structural dynamics

While protein activity is traditionally studied with a major focus on the active site, the activity of enzymes has been hypothesized to be linked to the flexibility of adjacent regions, warranting more exploration into how the dynamics in these regions affects catalytic turnover. One such enzyme is Xylanase A (XylA), which cleaves hemicellulose xylan polymers by hydrolysis at internal β-1,4-xylosidic linkages. It contains a “thumb” region whose flexibility has been suggested to affect the activity. The double mutation D11F/R122D was previously found to affect activity and potentially bias the thumb region to a more open conformation. We find that the D11F/R122D double mutation shows substrate-dependent effects, increasing activity on the non-native substrate ONPX2 but decreasing activity on its native xylan substrate. To characterize how the double mutant causes these kinetics changes, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were used to probe structural and flexibility changes. NMR chemical shift perturbations revealed structural changes in the double mutant relative to the wild-type, specifically in the thumb and fingers regions. Increased slow-timescale dynamics in the fingers region was observed as intermediate-exchange line broadening. Lipari-Szabo order parameters show negligible changes in flexibility in the thumb region in the presence of the double mutation. To help understand if there is increased energetic accessibility to the open state upon mutation, alchemical free energy simulations were employed that indicated thumb opening is more favorable in the double mutant. These studies aid in further characterizing how flexibility in adjacent regions affects the function of XylA.

Zeolite-Y-catalyst production from locally sourced Meta-kaolin: Computer-Aided scale-up process design and economic analysis with Monte-Carlo-Simulation

Production of zeolite-Y catalyst from natural substrate has been a research trend in the scientific community. Published articles revealed that zeolite-Y recovery from locally sourced metakaolin is confined to laboratory practice. Scale-up process design and its economic feasibility for zeolite-Y catalyst recovery are rarely found in the scientific bibliography. Therefore, this study presented conceptual scale-up process design, base-case techno-economics and Monte-Carlo simulation of zeolite Y recovery from Nigerian metakaoline. ASPEN Base Case Simulation (ABCS), scale-up design and economics were accomplished using inherent design and costing algorithms in ASPEN Batch Process Developer (ABPD) V10. Process economic parameters: Net Present Value (NPV), Internal Rate of Return (IRR), Return on Investment (ROI) and Payback Time (PBT), were modelled and optimized using Design Expert V13 software; while zeolite Unit Production Cost (UPC), Annual Production Cost (APC), Total Capital Investment (TCI) and interest/discount rate were considered as model inputs. Monte-Carlo Simulation (MCS) in Crystal Ball Oracle software was used to perform the sensitivity and uncertainty analyses. The base-case techno-economic results of process design of 600,000 kg/year zeolite production gave batch size 5000 kg/batch with 104 batches/year, batch time (4149 min), TCI ($15,930,306), APC ($147,145), NPV ($41,983,375), ROI (38.13 %) and PBT (2.14 years). The coefficient of determination (R2) of the economic models were 0.9978, 0.9989 and 0.9986 for NPV, ROI and IRR respectively. The optimum economic variables that maximized synthesis of 5000 kg/batch zeolite Y are UPC ($11.68), APC ($100,033) and TPC ($15,930,200). MCS uncertainty for NPV, IRR and ROI are negligible. Therefore, this study demonstrated that scale-up zeolite-Y production from the local substrate is economically feasible.

Sessile Biofouling on Electrolytic Carbonated Structures: Stages of Colonization and Succession

The ecological succession has been widely studied by means of biofouling assemblages among different substrates, and mainly targeted in early stages on artificial ones. The present study focuses on biofouling that colonizes carbonated structures, a material similar to the natural substrate produced by the electrolysis of seawater, which is relatively very little studied. We have observed the colonization of sessile macrofouling of the port of Alicante (SE Spain, Western Mediterranean) on two types of substrates (electrolytic carbonated and steel) over 12 months of succession. The assemblages of both substrates have been analyzed by means of diversity indexes and multivariate analysis (PERMANOVA and SIMPER) in order to see the differences over time. The carbonated substrate has presented a community with higher values of biological diversity, structure and complexity, although the differences in species composition between substrates are not evident during all immersion periods. Thus, these results seem to indicate that, even after 12 months of immersion, communities are still in a dynamic successional stage.

Characterization and optimization of a quantitative colorimetric acetylcholine esterase inhibition assay for biochip integration demonstrated by neurotoxicity evaluation of malathion

Organophosphates (OPs) and carbamates as insecticides, nematicides, fungicides, and herbicides are constantly increasing. Their neurotoxic nature requires careful usage, and misuse can lead to fatalities. OPs classified as 'Class 1′ toxic compounds irreversibly inhibit cholinesterases due to their molecular structure resembling the natural acetylcholine substrate, leading to toxic events in the human brain. Monitoring such chemicals is relevant for agricultural applications and essential for the military sector to ensure the safety of personnel and civilian populations. State-of-the-art analytical detection methods require time-consuming pre-treatments and costly reagents and face challenges associated with pesticide properties like thermal lability, low volatility, and high polarity, which can compromise analysis performance. Advanced systems like electrophoresis or liquid chromatography are used to address these, but these are not well suited for field analysis. Miniaturized colorimetric assays are becoming more popular for various portable devices and kits (i.e., metabolic or blood cell assays) due to their ease of use and practicality. Here, we aimed to establish and optimize a straight-forward paper-based microfluidic acetylcholine esterase inhibition assay for mobile organophosphate detection, laying the groundwork for future microdevice modules to be used in environmental monitoring, public health, and CBRN applications.

A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.

Partial purification and characterization of protease extracted from kinema

Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30–70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5–9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens’ plots showed the protease had Vmax of 1.001 U/ml with corresponding Km value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them.

High crystalline quality homoepitaxial Si-doped β-Ga2O3(010) layers with reduced structural anisotropy grown by hot-wall MOCVD

A new growth approach, based on the hot-wall metalorganic chemical vapor deposition concept, is developed for high-quality homoepitaxial growth of Si-doped single-crystalline β-Ga2O3 layers on (010)-oriented native substrates. Substrate annealing in argon atmosphere for 1 min at temperatures below 600 °C is proposed for the formation of epi-ready surfaces as a cost-effective alternative to the traditionally employed annealing process in oxygen-containing atmosphere with a time duration of 1 h at about 1000 °C. It is shown that the on-axis rocking curve widths exhibit anisotropic dependence on the azimuth angle with minima for in-plane direction parallel to the [001] and maximum for the [100] for both substrate and layer. The homoepitaxial layers are demonstrated to have excellent structural properties with a β-Ga2O3(020) rocking curve full-widths at half-maximum as low as 11 arc sec, which is lower than the corresponding one for the substrates (19 arc sec), even for highly Si-doped (low 1019 cm−3 range) layers. Furthermore, the structural anisotropy in the layer is substantially reduced with respect to the substrate. Very smooth surface morphology of the epilayers with a root mean square roughness value of 0.6 nm over a 5 × 5 μm2 area is achieved along with a high electron mobility of 69 cm2 V−1 s−1 at a free carrier concentration n=1.9×1019 cm−3. These values compare well with state-of-the-art parameters reported in the literature for β-Ga2O3(010) homoepitaxial layers with respective Si doping levels. Thermal conductivity of 17.4 Wm−1K−1 is determined along the [010] direction for the homoepitaxial layers at 300 K, which approaches the respective value of bulk crystal (20.6 Wm−1K−1). This result is explained by a weak boundary effect and a low dislocation density in the homoepitaxial layers.

Enhancing ecological complexity in soft‐bottom coastal ecosystems: the impact of introducing hard substrates

Coastal ecosystems globally face pressures, with natural coastal habitats being replaced by engineered structures. While hard structures for navigation‐purposes and coastal defense can negatively impact native communities, they can also be applied in ecological restoration as artificial reefs. This way substrates may facilitate establishment of biogenic (shellfish) reefs and provide habitat heterogeneity in soft‐sediment ecosystems. In a 1.5‐year experiment, we introduced six different types of natural or biodegradable hard substrates in a subtidal soft‐sediment system. We compared the substrates with surrounding soft sediment and evaluated differences among substrate types using biodiversity indices, community composition analyses, and food web indicators. This offers a comprehensive understanding of how the introduced hard substrates affect ecosystem complexity. Overall ecological complexity indicators were higher in the second year of the experiment. We found a significant increase in species richness, community composition, and link density (number of feeding interactions per species) on hard substrates compared to the surrounding sediment. However, Shannon diversity index and food web connectance (measure of food web saturation) did not differ between substrates and the surrounding sediment. Although differences among types of hard substrates were small, certain species were uniquely associated with specific substrates with wood and granite performing best. While there was no establishment of epibenthic shellfish reefs within 1.5 years, we conclude that introducing hard substrates effectively enhances ecological complexity in soft‐bottom coastal ecosystems within that timeframe. We recommend the use of substrates with a historical ecological relevance in the system for scaling up introduction of substrates for ecological restoration.