Type Enzyme Research Articles

Simultaneous enhancement of multiple functional properties using evolution-informed protein design

A major challenge in protein design is to augment existing functional proteins with multiple property enhancements. Altering several properties likely necessitates numerous primary sequence changes, and novel methods are needed to accurately predict combinations of mutations that maintain or enhance function. Models of sequence co-variation (e.g., EVcouplings), which leverage extensive information about various protein properties and activities from homologous protein sequences, have proven effective for many applications including structure determination and mutation effect prediction. We apply EVcouplings to computationally design variants of the model protein TEM-1 β-lactamase. Nearly all the 14 experimentally characterized designs were functional, including one with 84 mutations from the nearest natural homolog. The designs also had large increases in thermostability, increased activity on multiple substrates, and nearly identical structure to the wild type enzyme. This study highlights the efficacy of evolutionary models in guiding large sequence alterations to generate functional diversity for protein design applications.

Occurrence and types of carbapenamase enzymes amongst enterobacterales and Pseudomonas spp. using automated phenotypic method

Occurrence and types of carbapenamase enzymes amongst enterobacterales and Pseudomonas spp. using automated phenotypic method

Enzymatic Synthesis of Austroeupatol Esters with Enhanced Antiprotozoal Activity.

Austroeupatol, the principal diterpene isolated from the invasive shrub Austroeupatorium inulifolium, holds promise for structural diversification and biological assessment of its derivatives due to its abundant availability and high yield isolation. We propose an efficient enzymatic synthesis of a series of austroeupatol esters derived from aliphatic and heterocyclic carboxylic acids. Systematic optimization of reaction parameters, including enzyme type and quantity, acylating agent amount, solvent, and temperature, was conducted. Thermomyces lanuginosus lipase in cyclohexane at 55 °C, yielded esters with favorable conversion rates. Through enzymatic catalysis, mono- and diacylated derivatives were obtained, with a diacylation-monoacylation ratio influenced by temperature and acylating agent amount. The antiprotozoal activity of austroeupatol and all synthesized derivatives was evaluated, observing that acylation improved it. The 19-valeroyl, 19-indolylpropyl, and 19-octyl derivatives were the most potent compounds against Trypanosoma cruzi and Leishmania infantum, highlighting this approach as a valuable method for synthesizing austroeupatol derivatives as potential antiparasitic agents.

Pattern of Liver Enzymes in Type -2 Diabetes Mellitus Subjects with and without Dyslipidemia

Pattern of Liver Enzymes in Type -2 Diabetes Mellitus Subjects with and without Dyslipidemia

Performance of broiler chickens fed palm kernel cake with enzyme supplementation

Abstract The purpose of this study was examined the performance of broiler chickens fed palm kernel cake (PKC) with enzyme supplementation. This research used 1,260 Indian River strain male broiler chickens divided into seven treatments and six replications, with 30 chickens in each replication. This research used two types of cocktail enzymes, enzyme one and enzyme two. Enzyme one consists of mannanase 182 g/ton (182×103 U), NSPase 200 g/ton (α – amylase 54×103 U, selulase 500 ×103 U, xylanase 375×103 U, and protease 180×103 U) and protease 130 g/ton (240.5 U), while enzyme two consists of mannanase 182 g/ton (182×103 U), NSPase 400 g/ton (α – amylase 108×103 U, selulase 106 U, xylanase 750×103 U, and protease 360×103 U), and protease 260 g/ton (481 Unit enzyme). This study’s treatment was: P0 - basal feed (control), P1 – P0 with 10% PKC, P2 – P0 with 20% PKC, P3 – P0 with 10% PKC and enzyme one, P4 – P0 with 10% PKC and enzyme one, P5 – P0 with PKC 20% and enzyme two, and P6 – P0 with PKC 20% and enzyme two. Chickens were kept for 35 days. The variables observed included feed intake (FI), average daily gain (ADG), Feed conversion Ratio (FCR), Performance Index (PI), and carcass percentage. The data were analyzed using Analysis of Variance, followed by Duncan’s Multiple Range Test (DMRT). Enzyme addition did not affect feed consumption. The P3 had the largest ADG (2,219.75 g), the lowest FCR (1.59), and had the best FCR compared to the other treatments. The P4 and P6 could increase ADG compared P0, but did not increase compared to P3. The addition of 10% PKC could reduce FCR compared to the control, while the addition of 20% PKC did not reduce FCR. The addition of 10% PKC could increase PI by 19.07% (P1) and 28.58% (P3) compared to the control. The P4 and P6 did not increase PI. The P3 could increase carcass percentage by 6.15% and 6.19% (P5) compared to the control. Carcass percentage in P3 and P5 was highest than the control which was 71.53% and 71.56%. This research concluded that the P3 treatment consisting of basal feed with 10% PKC and the addition of enzymes mannanase 182 g/ton, NSPase 200 g/ton, and protease 130 g/ton could increase ADG, PI, and carcass percentage, and reduced FCR.

Selenocysteine variant of human Peroxiredoxin 2 is more active than the wild type enzyme

Selenocysteine variant of human Peroxiredoxin 2 is more active than the wild type enzyme

Comparing the Efficacy of Homemade Bio-enzymes to Store-Bought Enzymes

The cleaning industry has seen a surge in the use of manufactured enzymes for their effectiveness in breaking down organic stains and residues. Although commercial, enzyme-based cleaning products have amassed popularity among the general public, concerns over their environmental consequences persist. This research study intends to comprehensively compare the efficacy of store-bought enzymes and homemade bio-enzymes in antimicrobial applications. Bio-enzymes are synthesized through fermentation of jaggery and complex polyphenols (sourced from orange rinds) in water. Both types of enzymes are assessed on their antimicrobial effectiveness by observing activity in gelatin-based spread bacteria plates. Finally, this study intends to address future research into the possibilities of manufacturing recyclable cleaning agents through enzyme immobilization in polymer composites.

The two-step strategy for enhancing the specific activity and thermostability of alginate lyase AlyG2 with mechanism for improved thermostability

To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Ya mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Ya mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Ya system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.

Recent progress in enzymatic degradation and recycling of polyurethanes

The widespread production of polymeric materials has precipitated severe environmental challenges, prompting a pressing need for effective solutions. Among these materials, polyurethanes (PUs) stand out significantly due to their versatility in synthetic approaches, manufacturing, properties, and applications. This versatility has led to a high demand for PUs, resulting in significant waste accumulation at the end of their lifecycle. Along with that, the PU’s inherent chemical stability prevents the occurrence of natural hydrolytic processes. Consequently, there is a critical need for sustainable recycling methods, particularly those capable of recovering reusable monomers. Recycling is a leading way to manage waste by reducing plastic pollution and its environmental impact, which can be performed through mechanical, chemical, and biological processes. Biological degradation occurs through the action of enzymes and microbes that can potentially convert the polymers into monomers or other valuable chemicals. This approach presents an opportunity to establish a sustainable and circular economy within the PUs industry while considerably reducing the environmental impact. This review highlights enzymatic degradation as a promising solution for PU recycling by discussing the types of enzymes utilized in the process, potential biochemical pathways for PU degradation, recent achievements, and future remarks for this process.

Lipidomics of phospholipase A2 reveals exquisite specificity in macrophages

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze phospholipids at their sn-2 fatty acyl position. Our laboratory has demonstrated that PLA2 enzymes regulate membrane remodeling and cell signaling by their specificity toward their phospholipid substrates at the molecular level. Recent in vitro studies show that each type of PLA2, including Group IVA cytosolic PLA2 (cPLA2), Group V secreted PLA2 (sPLA2), Group VIA calcium independent PLA2 (iPLA2) and Group VIIA lipoprotein-associated PLA2, also known as platelet-activating factor acetyl hydrolase, can discriminate exquisitely between fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse PUFA precursors of inflammatory metabolites. We now determined PLA2 specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a role in which specific phospholipids are hydrolyzed by each enzyme type. We used PLA2 stereospecific inhibitors in tandem with a novel UPLC-MS/MS-based lipidomics platform to quantify more than a thousand unique phospholipid molecular species demonstrating cPLA2, sPLA2, and iPLA2 activity and specificity toward the phospholipids in living cells. The observed specificity follows the in vitro capability of the enzymes and can reflect the enrichment of certain phospholipid species in specific membrane locations where particular PLA2’s associate. For assaying, we target 20:4-PI for cPLA2, 22:6-PG for sPLA2, and 18:2-PC for iPLA2. These new results provide great insight into the physiological role of PLA2 enzymes in cell membrane remodeling and could shed light on how PLA2 enzymes underpin inflammation and other lipid-related diseases.

Endophytic fungal isolates from apple tissue: Latent pathogens lurking within?

Fungal endophytes inhabit a similar ecological niche to that occupied by many phytopathogens, with several pathogens isolated from healthy tissues in their latent phase. This study aimed to evaluate the pathogenicity, the colonisation ability, and the enzyme activity of 37 endophytic fungal isolates recovered from apparently healthy apple shoot and leaf tissues. The pathogenicity of the isolates was assessed on ‘Royal Gala’ and ‘Braeburn’ fruit and detached ‘Royal Gala’ shoots. For the non-pathogenic isolates, their ability to endophytically colonise detached ‘Royal Gala’ shoots was evaluated. Enzyme activity assays were undertaken to determine whether the pathogenicity of the endophytes was related to the production of the extracellular enzymes, amylase, cellulase, pectinase, protease, and xylanase. Of the 37 isolates studied, eight isolates, representing the genera Colletotrichum, Diaporthe, Fusarium, and Penicillium, were shown to be pathogenic on both apple shoots and fruit. Two isolates identified as Trichoderma atroviride, were pathogenic only on shoots, and three isolates, representing the genus Diaporthe, were pathogenic only on fruit. Of the remaining 24 isolates, 22 (Biscogniauxia (n = 8), Chaetomium (n = 4), Trichoderma (n = 3), Epicoccum (n = 2), Neosetophoma (n = 2), Xylaria (n = 1), Daldinia (n = 1), and Paraphaeosphaeria (n = 1)) were recovered from the inoculated apple shoots but two failed to colonise the shoot tissues. Of the isolates tested, 20 produced amylase, 15 cellulase, 25 pectinase, 26 protease, and 13 xylanase. There was no correlation between the range and type of enzymes produced by the isolates and their pathogenicity or ability to endophytically colonise the shoot tissue. The study showed that approximately one-third (13/37) of the isolates recovered from the apparently healthy apple shoot tissues were observed as latent pathogens. The isolates that did not cause disease symptoms may have the ability to reduce colonisation of apple tissues by pathogens including Neonectria ditissima associated with European canker of apple.

Stress-mediated polysorbate 20 degradation and its potential impact on therapeutic proteins

PurposePolysorbates are the most commonly used surfactants in formulations to stabilize therapeutic proteins against interfacial stresses. Polysorbates can undergo oxidative or enzyme-mediated hydrolytic degradation to produce free fatty acids (FFAs) and subvisible particles in formulations. To determine which product related variables contribute to PS20 degradation, we investigated the effects of storage temperature, formulation, pH, presence of hydrolytic enzymes, and specific fatty acid composition on different grades of PS20 in relation to their PS20 degradation profile and consequently the quality of protein drug products.MethodsBevacizumab and T-DM1 were reformulated in the freshly prepared therapeutic protein formulations containing either compendial PS20 or non-compendial PS20 with high % lauric acid and spiked with exogenous esterase or lipase. The release of FFAs and formation of particles were monitored at 4°C and 37°C. Protein quality was assessed for secondary structures, purity, and biological activity.ResultsHydrolytic release of FFAs and formation of subvisible particles were found to be dependent on grades of PS20, types of enzymes used, incubation temperature, and pH. Esterase- or lipase-mediated degradation of PS20 and formation of subvisible particles in drug formulation showed no significant impact on the biological activity and stability of therapeutic proteins against degradation or aggregation.ConclusionsOur study suggests that degradation of PS20 and formation of FFA particles depend on the fatty acid composition of PS20, types of hydrolytic enzymes, pH, and temperature. The presence of FFA subvisible particles showed no significant impact on the purity and biological activity of the therapeutic proteins under the tested conditions.

From laboratory to industrial use: Understanding process variation during enzymatic protein hydrolysis with dry film fourier-transform infrared spectroscopy

Industries are continuously looking for dedicated sensor solutions for evaluating the chemical composition of products that can provide valuable insights into process control, process optimization, and product quality. A rapid and robust analytical method, Fourier-Transform Infrared (FTIR) spectroscopy, holds significant potential in this regard as it provides detailed compositional values of food nutrients. The main aim of the present study was to use dry film FTIR spectroscopy as an analytical tool to characterize products from an industrial enzymatic protein hydrolysis process and link this to understand industrial process variations. For this purpose, 463 protein hydrolysate samples were obtained from an industrial enzymatic protein hydrolysis plant. In the same period, systematic variations in process parameters such as raw material composition, enzyme type, and water addition, were performed. All samples were analyzed using dry film FTIR spectroscopy. Two subsets containing 200 and 68 hydrolysate samples were chosen for measuring average molecular weight (AMW) and collagen content respectively, providing reference data for constructing calibration models based on partial least squares regression (PLSR). The percentage of low molecular weight constituents was derived from the molecular weight distribution of size exclusion chromatograms of protein hydrolysates and also used in the modeling. Calibration models for the prediction of AMW, low molecular weight constituents, and collagen content were obtained with a good fit and lower estimation errors. Principal component analysis (PCA) of protein hydrolysates' FTIR spectra effectively differentiated process variations (enzyme types and raw materials) without extensive reference analysis. One-factor analysis of variance (ANOVA) tests was used to observe the impact of process variation on product quality. FTIR proved to be a sensitive method for liquid protein analysis and process control with a significant potential in process optimization approaches. To the authors’ knowledge, this is the first time dry film FTIR spectroscopy has been used to evaluate an industrial bioprocess with designed process variations.

Genetic and functional diversity of β-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis

β-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. β-N-Acetylgalactosaminidases hydrolyze β-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-β-N-acetylgalactosaminidases that specifically act on β-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight β-N-acetylgalactosaminidases and β-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse β-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

Effect of enzymatic hydrolysis with Alcalase or Protamex on technological and antioxidant properties of whey protein hydrolysates

The aim of this study was to evaluate the effect of the enzymatic hydrolysis, performed using Alcalase and Protamex enzymes, on the technological functionalities and the antioxidant capacity of whey protein hydrolysates (WPHs) to identify the conditions allowing to obtain target functionality/ies. Samples were characterized for hydrolysis degree (DH), molecular weight distribution, structural properties, and food-related functionalities. Free sulfhydryl groups and surface hydrophobicity significantly decreased with the increase in DH, regardless of the used enzyme. The foaming and antioxidant properties of Alcalase WPHs were higher as compared to those of WPI, reaching the maximum value at DH = 18–20 %, while higher DH resulted in impaired functionality. Gelling properties were guaranteed when WPI was hydrolysed by Protamex at DH < 15 % while foaming and antioxidant abilities were fostered at 15 < DH < 21 %. These results were well correlated with MW distribution and were rationalized into a road map which represents a useful tool in the selection of proper hydrolysis conditions (time, DH, enzyme type) to obtain WPHs with tailored functionalities. Research outcomes highlighted the possibility to drive protein hydrolysis to optimize the desired functionality/ies.

Nanodiamonds and natural deep eutectic solvents as potential carriers for lipase

This study investigates the use of nanodiamonds (ND) as a promising carrier for enzyme immobilization and compares the effectiveness of immobilized and native enzymes. Three different enzyme types were tested, of which Rhizopus niveus lipase (RNL) exhibited the highest relative activity, up to 350 %. Under optimized conditions (1 h, pH 7.0, 40 °C), the immobilized ND-RNL showed a maximum specific activity of 0.765 U mg−1, significantly higher than native RNL (0.505 U mg−1). This study highlights a notable enhancement in immobilized lipase; furthermore, the enzyme can be recycled in the presence of a natural deep eutectic solvent (NADES), retaining 76 % of its initial activity. This aids in preserving the native conformation of the protein throughout the reusability process. A test on brine shrimp revealed that even at low concentrations, ND-RNL had minimal toxicity, indicating its low cytotoxicity. The in silico molecular dynamics simulations performed in this study offer valuable insights into the mechanism of interactions between RNL and ND, demonstrating that RNL immobilization onto NDs enhances its efficiency and stability. All told, these findings highlight the immense potential of ND-immobilized RNL as an excellent candidate for biological applications and showcase the promise of further research in this field.

Abstract PO2-18-04: STX-478 is a potentially best-in-class mutant-selective PI3Kα inhibitor that demonstrates robust efficacy in ER+ breast cancer models as monotherapy and in combination with standard of care agents

Abstract Activating mutations in PI3Kα are among the most prevalent genetic aberrations in breast cancer. Constitutive signaling of PI3Kα is an established oncogenic driver in these cancers, as these mutations are predictors of response to alpelisib, an approved PI3Kα inhibitor, which inhibits both wild type and mutant PI3Kα. While inhibiting PI3Kα has proven to be efficacious, the concomitant inhibition of wild type enzyme in normal tissue results in metabolic dysfunction and ultimately dose-limiting toxicities. Selectively targeting the mutant PI3Kα enzyme is expected to inhibit tumor growth while sparing normal tissues, thus increasing the therapeutic index compared to non-mutant selective PI3Kα inhibitors. Therefore, we have developed STX-478, an allosteric, CNS penetrant, mutant-selective PI3Kα inhibitor. In a high-throughput viability screen, STX-478 selectively inhibited growth of cell lines with kinase domain and helical domain mutations in PI3Kα. In a panel of PI3Kα mutant CDX and PDX models, STX-478 provided efficacy that was similar or superior to higher than clinically achievable doses of alpelisib. Robust suppression of pharmacodynamic (PD) markers, pAKT, was observed in tumors, but not in skeletal muscle. Importantly, the efficacy achieved with STX-478 occurred at doses that did not cause metabolic dysfunction, in contrast to alpelisib which caused insulin release and suppressed glucose uptake in skeletal muscle in a 13C glucose tolerance test. Collectively, these data indicate that STX-478 has an expanded therapeutic window to selectively target mutant PI3Kα in vivo. In efficacy studies using human tumor xenograft models, STX-478 caused tumor regressions as a single agent. In multiple advanced ER+ breast cancer PDX models including models that are insensitive to the approved CDK4/6 inhibitor palbociclib, STX-478 combined with standards of care was highly efficacious and well tolerated. In a representative PDX model, STX-478 combined with the ER degrader fulvestrant and palbociclib was well tolerated for &amp;gt; 90 days of dosing in mice and resulted in durable tumor regressions that were superior to STX-478 alone. In conclusion, these data demonstrate STX-478 is a highly efficacious, well tolerated, mutant-specific PI3Kα inhibitor that can be combined safely with standards of care in breast cancer and provide the therapeutic benefits of PI3Kα inhibition without the side effects associated with inhibiting WT PI3Kα. These properties combined with the potential for CNS penetration and excellent pharmacokinetic profile in higher species make STX-478 a potentially best-in-class mutant-selective PI3Kα inhibitor. STX-478 is currently being evaluated in a Phase I clinical trial (NCT05768139). Citation Format: Trang Tieu, Leonard Buckbinder, David St. Jean, Samantha Manimala, Gregory Dowdell, Michael Huff, Jacob Alltucker, Erica Jackson, Angel Guzman-Perez, Darrin Stuart. STX-478 is a potentially best-in-class mutant-selective PI3Kα inhibitor that demonstrates robust efficacy in ER+ breast cancer models as monotherapy and in combination with standard of care agents [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-04.

Characterization of Yeast Protein Hydrolysate for Potential Application as a Feed Additive.

Yeast protein can be a nutritionally suitable auxiliary protein source in livestock food. The breakdown of proteins and thereby generating high-quality peptide, typically provides nutritional benefits. Enzyme hydrolysis has been effectively uesed to generate peptides; however, studies on the potential applications of different types of enzymes to produce yeast protein hydrolysates remain limited. This study investigated the effects of endo- (alcalase and neutrase) and exotype (flavourzyme and prozyme 2000P) enzyme treatments on yeast protein. Endotype enzymes facilitate a higher hydrolysis efficiency in yeast proteins than exotype enzymes. The highest degree of hydrolysis was observed for the protein treated with neutrase, which was followed by alcalase, prozyme 2000P, and flavourzyme. Furthermore, endotype enzyme treated proteins exhibited higher solubility than their exotype counterparts. Notably, the more uniform particle size distribution was observed in endotype treated yeast protein. Moreover, compared with the original yeast protein, the enzymatic protein hydrolysates possessed a higher content of β-sheets structures, indicating their higher structural stability. Regardless of enzyme type, enzyme treated protein possessed a higher total free amino acid content including essential amino acids. Therefore, this study provides significant insights into the production of protein hydrolysates as an alternative protein material.

Systematic evaluation of the influence of Taq enzyme choice and amplification conditions on targeted environmental DNA assay performance and detection in field samples

AbstractTargeted environmental DNA (eDNA) studies mainly rely on quantitative real‐time polymerase chain reaction (qPCR) to amplify extremely low concentrations of DNA present in environmental samples. Understanding factors that influence targeted eDNA assay performance and detection in field samples, such as Taq DNA polymerase enzyme type and thermocycle protocol, is critical for the interpretation of qPCR results. We completed a systematic performance evaluation of five distinct targeted eDNA assays (eANFI6, eFISH1, eANBO5, eGLIN1, and eLICA3 targeting sablefish, general fish, Boreal toad, wood turtle, and the American bullfrog) by subjecting the same samples to analysis by five different Taq enzyme reagent mixes (Immolase, Environmental Master Mix (EMM), Amplitaq, QIAcuity, and QuantiNova) and the commonly used 2‐step (95°C, 60°C) and 3‐step (95°C, 64°C, 72°C) thermocycle protocols. We evaluated assay performance using a standardized dilution series of synthetic dsDNA target sequences and calculated limits of detection (LOD) and quantification (LOQ) and 95% confidence intervals for each combination of Taq enzyme and thermocycle protocol. All assays performed within acceptable performance criteria as defined by the Canadian national standard for targeted eDNA assays. Based on data generated by synthetic dsDNA fragments, the eDNA assays performed comparably regardless of the enzyme reagent mix and protocol used, except for eANFI6 and eGLIN1 using EMM and the 3‐step protocol, where no amplification was observed. On freshwater field samples, EMM and Immolase performed best. On marine field samples, Immolase, EMM, Qiacuity, and QuantiNova performed equally well, although EMM failed to amplify some samples. The work reveals that an enzyme reaction mix or thermocycle protocol can affect the result of an eDNA assay, but the appropriate choice also depends on the nature of the field sample. It is therefore imperative that these are considered when selecting appropriate reaction conditions and that they are clearly reported.

ACE2 and a Traditional Chinese Medicine Formula NRICM101 Could Alleviate the Inflammation and Pathogenic Process of Acute Lung Injury

The coronavirus disease 2019 (COVID-19) is a highly transmittable respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and acute lung injury (ALI) is the major complication of COVID-19. The challenge in studying SARS-CoV-2 pathogenicity is the limited availability of animal model. Therefore, it is necessary to establish animal models that can reproduce multiple characteristics of ALI to study therapeutic application. The present study established a mouse model that has features of ALI that are similar to COVID-19 syndrome to investigate the role of human angiotensin-converting enzyme type II (ACE2) and the administration of the Chinese herbal prescription Chingguan Yihau (NRICM101) in ALI. The mice with ACE2 genetic modifications, including the overexpression of human ACE2 (K18-hACE2 TG) and the absence of ACE2 (mACE2 KO), were intratracheally instillated with hydrochloric acid to induce ALI. The acid intratracheal instillation induced the imbalance between angiotensin-converting enzyme (ACE) and ACE2 (i.e., ACE/ACE2), severe immune cell infiltration, cytokine storms, inflammatory effect, and pulmonary disease in the mice. Compared with K18-hACE2 TG mice, mACE2 KO mice exhibited dramatically increased levels of multiple inflammatory cytokines Interleukin-6 (IL-6) and Tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid, histological evidence of lung injury, the production of the ACE expression, and the activation of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression. The results indicate that overproduction of ACE2 and induction of the ACE2-Ang-(1-7)/Mas axis could counteract the proinflammatory effect of acid-induced ALI and effectively alleviate the pathogenic process of acid-induced ALI by regulating MAPK, p-ERK1/2, and p-STAT3 pathways and dysregulation of MMP-2/MMP-9 activation. In addition, NRICM101, which is an effective TCM that has both antiviral and anti-inflammatory effects against COVID-19, shows potential for treating acid-induced ALI. NRICM101 could alleviate the disease progression of acid-induced ALI by suppressing IL-6 and TNF-α production in alveoli. In conclusion, the established mouse model provided an effective platform for researchers to investigate the role of ACE2 on pathological mechanisms and develop therapeutic strategies for ALI, including COVID-19-related pulmonary diseases. The effects of NRICM101 administration is a potential strategy for curing acid-induced ALI evaluated in the mouse model in this study. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.