Aminopeptidase Research Articles

Enzyme Activity-Based Optical Probe: Imaging Aminopeptidases N in Vulnerable Plaque and Serum.

Cardiovascular disease, a chronic and progressive arterial wall disease, is increasingly recognized for its clinical significance. Aminopeptidases N (APN), crucial in the pathophysiological processes of vulnerable plaque, have been linked to endothelial dysfunction, oxidative stress, and plaque formation, thus highlighting their potential as biomarkers for disease progression. However, current detection methods for APN in body fluids and in vivo have limitations, including insufficient sensitivity and specificity, time delays, and the inability to directly reflect enzyme activity in plaques. To address these challenges, we developed an optical probe, HD-APN, for in vivo imaging of aminopeptidases, providing a potential implementation in cardiovascular disease. Our work demonstrated the applicability of HD-APN for specific monitoring of aminopeptidase levels in plaques and serum, shedding light on its potential for further research in cardiovascular disease.

Benzylhydroxamic acids as inhibitors of insulin regulated aminopeptidase (IRAP)

With the objective of finding new classes of cognitive enhancers with potential for the treatment of neurodegenerative disorders, such as Alzheimer's disease, small molecule inhibitors of insulin-regulated aminopeptidase (IRAP) were designed and synthesized. IRAP is a member of the M1 family of zinc aminopeptidases and is abundantly expressed in areas of the brain associated with cognition, such as the amygdala, hippocampus and cerebral cortex. IRAP inhibitors were previously shown to enhance memory and learning in animal models. A comprehensive high throughput screening of 400,000 small molecules from the European Lead Factory library provided a series of 50 promising compounds in a qualified hit list (QHL). More than 30 IRAP inhibitors with an IC50 below 3.5 μM were identified. Herein, selected compounds from this QHL were assayed for solubility and permeability. Most of the selected compounds displayed good solubility, but further permeability studies on the best compounds revealed low blood brain barrier (BBB) permeability and high efflux in cells overexpressing P-gp pumps, rendering them less promising as starting points in drug discovery processes. Two compounds from the QHL were prioritized for further structural optimization; the pyridyl-substituted isoxazole 1a (QHL27) and the benzylhydroxamic acid derivative 1b (QHL1), both demonstrating fair BBB permeability and no indication of efflux. While our attempts to improve the isoxazole derivative 1a were not fruitful, a structural modification of 1b to provide the chloro-substituted benzylhydroxamic acid 14b resulted in a ten-fold improvement of the IRAP inhibition with an IC50 value of 60 nM. The binding modes of 1b and 14b were determined by free energy perturbation (FEP) analysis performed on candidate docking poses, determining a binding mode that accurately explained the experimental SAR. Further FEP studies of compound 14b suggested that it exhibits selectivity towards IRAP over Aminopeptidase N (APN), indicating its potential for targeted therapeutic applications.

Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

Genome-wide CRISPR/Cas9 library screen identifies C16orf62 as a host dependency factor for porcine deltacoronavirus infection

ABSTRACT Porcine deltacoronavirus (PDCoV) is an emerging pathogen that can cause severe diarrhoea and high mortality in suckling piglets. Moreover, evidence of PDCoV infection in humans has raised concerns regarding potential public health risks. To identify potential therapeutic targets for PDCoV, we performed a genome-wide CRISPR/Cas9 library screening to find key host factors important to PDCoV infection. Several host genes in this screen were enriched, including ANPEP, which encodes the PDCoV receptor aminopeptidase N (APN). Furthermore, we discovered C16orf62, also known as the VPS35 endosomal protein sorting factor like (VPS35L), as an important host factor required for PDCoV infection. C16orf62 is an important component of the multiprotein retriever complex involved in protein recycling in the endosomal compartment and its gene knockout led to a remarkable decrease in the binding and internalization of PDCoV into host cells. While we did not find evidence for direct interaction between C16orf62 and the viral s (spike) protein, C16orf62 gene knockout was shown to downregulate APN expression at the cell surface. This study marks the first instance of a genome-wide CRISPR/Cas9-based screen tailored for PDCoV, revealing C16orf62 as a host factor required for PDCoV replication. These insights may provide promising avenues for the development of antiviral drugs against PDCoV infection.

Biomarkers of oxidative stress, biochemical changes, and the activity of lysosomal enzymes in the livers of rainbow trout (Oncorhynchus mykiss Walbaum) vaccinated against yersiniosis before a Yersinia ruckeri challenge

Abstract Introduction This study aimed to evaluate biomarkers of oxidative stress (2-thiobarbituric acid reactive substances, aldehyde and ketone derivatives of oxidatively modified proteins and total antioxidant capacity), the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), that of lysosomal enzymes (alanyl aminopeptidase, leucyl aminopeptidase, β-N-acetylglucosaminidase and acid phosphatase) and changes in biochemical parameters (alanine aminotransferase, aspartate aminotransferase, de Ritis ratio, lactate dehydrogenase activity, lactate and pyruvate levels and their ratio) in the liver tissue of fish that were vaccinated against enteric redmouth disease and challenged with its causative agent, the bacterium Yersinia ruckeri. Material and Methods The vaccine was administered orally to trout, some of which were challenged with Y. ruckeri 61 days later. For comparison, unvaccinated and unchallenged trout and unvaccinated and challenged trout were also evaluated. Results In the unvaccinated fish, infection with Y. ruckeri disrupted the pro-oxidant/antioxidant balance, led to a significant increase in lipid peroxidation and oxidative modification of proteins, decreased total antioxidant capacity and significantly increased the activity of lysosomal enzymes. In vaccinated fish, the Y. ruckeri challenge increased the activity of glutathione-related enzymes and decreased lipid peroxidation, anaerobic metabolism and the activity of lysosomal enzymes in fish livers relative to the unvaccinated and challenged group. In contrast, these parameters increased after the Y. ruckeri challenge in unvaccinated trout relative to those in the untreated group. Conclusion Vaccination exerted a protective effect during the Y. ruckeri challenge and had no adverse effect on fish livers.

Differential regulation of viral entry-associated genes modulated by inflammatory cytokines in the nasal epithelium.

This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry-associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), and beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) were selected as key entry-associated genes for SARS-CoV-2, HCoV-229E, MERS-CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air-liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real-time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry-associated genes showed distinct regulation patterns in response to each interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL-4 and IL-13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF-α and IFN-γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN-γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS-CoV-2 and HCoV-229E infections but higher susceptibility to MERS-CoV and influenza infections.

Macrogenome-based study on the mechanism of Bacillus velezensis SX13 in regulating rhizophere environment and cucumber growth under different cultivation environments

Microbial activities are the dynamic core of nutrient cycling in organic substrates, and the exploitation of plant growth-promoting rhizobacteria strains contributes to sustainable agricultural development. This study aimed to investigate the effect and mechanism of Bacillus velezensis SX13 in nutrient cycling and plant promotion under different substrate supply conditions. The effects of reduced substrate amount (sCK) and inoculation of SX13 strain under both substrate supply conditions (Bv and sBv) on rhizosphere microenvironment and plant growth were investigated using conventional substrate amount (CK) as a control. Results showed no significant difference in the α-diversity indexes (Chao1 and Shannon) of the rhizospheric microbial community among the four treatments. However, nonmetric multidimensional scaling analysis and principal coordinate analysis revealed that compared with CK treatment, the inoculation of SX13 strain and reduced substrate supply reshaped the β-diversity structure of microbial communities. Furthermore, inoculation with B. velezensis SX13 under both substrate supply conditions increased the abundance of Proteobacteria (1.64%–2.46 %), Acidobacteria (14.09%–43.07 %), and Firmicutes (179.29%–861.29 %). The results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the metabolic pathway with the highest abundance of enriched genes was also the pathway with the most enriched differential genes caused by reducing substrate supply or inoculation of B. velezensis SX13. The rhizosphere inoculation of B. velezensis SX13 significantly up-regulated the top genes related to carbohydrate esterases, carbohydrate binding modules, glycoside hydrolases, glycoside transferases, and polysaccharide lyases. As a result, the activities of carbon and nitrogen cycle-related enzymes such as cellobiohydrolase, β-glucosidase, urease, l-leucine amino peptidase, and β-1 4-N-acetylglucosaminidase were increased, which in turn accelerated nutrient cycling. B. velezensis SX13 and its mediated improvement of the rhizospheric microenvironment resulted in the up-regulation of root CsNRT family genes (such as CsNRT1.1, CsNRT1.4a, CsNRT1.4b, CsNRT1.5a, CsNRT1.5b, CsNRT1.5c, and CsNRT1.8), which accelerated nitrogen uptake, accumulation, and utilization efficiency and ultimately improved the yield and quality of cucumber. The effect of SX13 strain was more stable and efficient under conventional substrate supply conditions than under reduced substrate supply conditions.

A Nanofluorescent Probe for Evaluating the Fluctuation of Aminopeptidase N in Nonalcoholic Fatty Liver Disease and Hepatic Fibrosis.

Aminopeptidase N (APN/CD13) is a widely expressed transmembrane ectoenzyme that is crucial for maintaining normal physiological activities. It exhibits abnormal activity closely associated with hepatic fibrosis and nonalcoholic fatty liver disease (NAFLD). Therefore, there is a high demand for noninvasive detection of aminopeptidase N (APN) in the diagnosis and research of related diseases. Here, we developed a small molecule fluorescent probe, Hcy-APN, which is a fluorescent probe with high sensitivity and selectivity for the detection of APN. Furthermore, we synthesized the fluorescent nanoprobe Hcy-APN@MSN by self-assembling Hcy-APN and mesoporous silica nanoparticles in solution using a combination of molecular probe design and nanofunctionalization strategies. The detection limit of this probe was 1.5 ng/mL. Hcy-APN@MSN exhibits more stable spectral characteristics compared to Hcy-APN and is suitable for detecting APN activity in live cells and mice. Hcy-APN@MSN was utilized for in vivo and intracellular imaging of NAFLD and hepatic fibrosis at different stages, as well as for a systematic assessment of APN levels in the liver. The results confirm an elevation in the expression levels of APN in NAFLD and hepatic fibrosis models. Furthermore, we investigated the inhibitory effect of the APN inhibitor bestatin in nonalcoholic fatty liver and hepatic fibrosis disease models, confirming its regulatory effect on APN levels in cells and in vivo in both disease models. Therefore, this study may offer diagnostic possibilities for detecting NAFLD and hepatic fibrosis.

Aminopeptidasic Enzymes as Early Biomarkers of Cardiac Surgery-Associated Acute Kidney Injury and Long-Term Events.

Diagnosis of acute kidney injury (AKI) relies on serum creatinine (SCr) changes. This study investigated if urinary aminopeptidases are early and predictive biomarkers of cardiac surgery-associated AKI (CSA-AKI). Glutamyl aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), proteinuria, albuminuria, N-acetyl-β-D-glucosaminidase (NAG), and neutrophile gelatinase-associated lipocalin (NGAL) were measured in urine samples from 44 patients at arrival in the intensive care unit (ICU) after cardiac surgery. Sensitivity, specificity, and positive and negative predictive value for diagnosis of stages 1, 2, and 3 of AKI were analyzed for the highest quartile of each marker. We also studied the relationship with SCr after surgery, 6- and 12-month glomerular filtration rates (GFRs), and other long-term events over the next 5 years. GluAp diagnosed the maximal number of patients that developed stage 2 or 3 of AKI, increasing diagnostic sensitivity from 0% to 75%. In addition, GluAp and DPP4 were related to the decrease in GFR at 6 or 12 months after surgery. Urinary aminopeptidases are a potential tool for the early diagnosis of CSA-AKI, with GluAp being the most effective marker for diagnosing stage 2 or 3 of AKI at ICU admission. GluAp and DPP4 serve as predictive biomarkers for a decrease in GFR.

Low temperature exposure influences nitrogen metabolism resulting in decreased Cry1Ac insecticidal endotoxin content in cotton seeds

BackgroundSudden temperature drops, resulting from extreme weather events, often occur during the boll-setting period of cotton in Xinjiang, China, causing decreased expression of Bacillus thuringiensis (Bt) insecticidal proteins in cotton bolls. The precise threshold temperatures and durations that lead to significant changes in Cry1Ac endotoxin levels under low temperatures remain unclear. To address this, we investigated the effects of different temperatures and stress durations on Cry1Ac endotoxin levels in cotton bolls. In 2020–2021, two Bt transgenic cotton varieties, conventional Sikang1 and hybrid Sikang3, were selected as experimental materials. Various low temperatures (ranging from 16 to 20 °C) with different durations (12 h, 24 h and 48 h) were applied during the peak boll-setting period.ResultsAs the temperature decreased, the Cry1Ac endotoxin content in the boll shell, fiber, and seed exhibited a declining trend. Moreover, the threshold temperature which caused a significant reduction in Cry1Ac endotoxin content increased with the prolonged duration of low-temperature stress. Among the components of cotton bolls, seeds were most affected by low-temperature stress, with the threshold temperature for a significant reduction in Cry1Ac endotoxin content ranging from 17 °C to 19 °C. Correlation analysis indicated that low temperatures led to a decrease in protein synthesis capacity and an increase in degradation ability, resulting in reduced Cry1Ac endotoxin content. Pathway analysis revealed that both free amino acid and peptidase had significant negative effects on Cry1Ac endotoxin content.ConclusionIn summary, when the daily average temperature was ≤ 19 °C, implementing cultural practices to reduce free amino acid content and peptidase activity could serve as effective cold defense strategies for Bt cotton production.

Cell-surface d-glucuronyl C5-epimerase binds to porcine deltacoronavirus spike protein facilitating viral entry.

The identification of viral receptors is of great significance, potentially extending our understanding of viral infection and pathogenesis. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus with the potential for cross-species transmission. However, the receptors or coreceptors of PDCoV are still poorly understood. The present study confirms that d-glucuronyl C5-epimerase (GLCE) is a positive regulator of PDCoV infection, promoting viral attachment and internalization. The anti-GLCE antibody suppressed PDCoV infection. Mechanically, GLCE interacts with PDCoV S and promotes the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. This work identifies GLCE as a novel cell-surface factor facilitating PDCoV entry and paves the way for further insights into the mechanisms of PDCoV infection.

Effects of exogenous copper on microbial metabolic function and carbon use efficiency of Panax notoginseng planting soil.

Soil copper (Cu) pollution is a serious environmental risk in the Panax notoginseng planting area. However, the effect of Cu on soil microbial metabolism and nutrient cycling in this area remains unknown. Therefore, Biolog ECO-plate and enzyme stoichiometry methods were utilized in this study to investigate the impact of exogenous Cu (control: 0 mg·kg-1; Cu100: 100 mg·kg-1; Cu400: 400 mg·kg-1; and Cu600: 600 mg·kg-1) on the metabolic function of soil microbial and nutrient limitation in the P. notoginseng soil. The results indicated that Cu100 significantly increased soil organic carbon (SOC), total phosphorus (TP), soil C:N, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) 9.89%, 15.65%, 17.91%, 61.87%, and 90.56% higher than the control, respectively. Moreover, the carbon source utilization ratio of carbohydrates, amino acids, and amphiphilic compounds of Cu100 also increased by 7.16%, 25.47%, and 84.68%, respectively, compared with the control. The activities of β-1,4-glucosidase, cellobiohyrolase, leucine amino peptidase, β-1,4-N-acetylglucosaminidase, and phosphatase significantly decreased with increasing Cu concentration. Soil enzyme stoichiometry showed that all treatments were limited by nitrogen (vector angle < 45°; 19.045-22.081). Cu600 led to the lowest carbon limitation (1.798) and highest carbon use efficiency (CUE:0.267). The PLS-SEM model also showed that MBC, MBN, MBP, and microbial diversity positively affected carbon and nitrogen limitation (0.654 and 0.424). Soil carbon, nitrogen, phosphorus, stoichiometric ratio, MBC, MBN, and MBP positively affected CUE (0.527 and 0.589). The microbial diversity index significantly negatively affected CUE (-1.490). Multiple linear stepwise regression analyses showed that CUE was mainly influenced by MBC, AP, C:P, and LAP. Thus, P. notoginseng soil can benefit soil microbial carbon and nitrogen limitations at low Cu concentrations. Clarifying the metabolic activity and nutritional status of microorganisms under Cu stress can provide some theoretical basis for realizing China's comprehensive and effective management and control policies for environmental risks from metals by 2035.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Association of Perinatal Cardiovascular Features with Angiotensin System Expressions in Maternal Preeclampsia.

We hypothesized and investigated whether prenatal exposure to preeclampsia (PE) would simultaneously affect perinatal cardiovascular features and angiotensin system expressions. This prospective study was composed of mother-neonate dyads with (n = 49) and without maternal preeclampsia (n = 48) in a single tertiary medical center. The neonates exposed to PE had significantly larger relative sizes for the left and right coronary arteries and a higher cord plasma level of aminopeptidase-N, which positively correlated with the maternal diastolic blood pressures and determined the relative sizes of the left and right coronary arteries, whereas the encoding aminopeptidase-N (ANPEP) mRNA level in the PE cord blood leukocytes was significantly decreased, positively correlated with the neonatal systolic blood pressures (SBPs), and negatively correlated with the cord plasma-induced endothelial vascular cell adhesion molecule-1 mRNA levels. The PE cord plasma significantly induced higher endothelial mRNA levels of angiotensin II type 1 receptor (AT1R) and AT4R, whereas in the umbilical arteries, the protein expressions of AT2R and AT4R were significantly decreased in the PE group. The endothelial AT1R mRNA level positively determined the maternal SBPs, and the AT4R mRNA level positively determined the neonatal chamber size and cardiac output. In conclusion, PE may influence perinatal angiotensin system and cardiovascular manifestations of neonates across placentae. Intriguing correlations between these two warrant further mechanistic investigation.

Malaria transmission blocking activity of Anopheles stephensi alanyl aminopeptidase N antigen formulated with MPL, CpG, and QS21 adjuvants.

Malaria, a preventive and treatable disease, is still responsible for annual deaths reported in most tropical regions, principally in sub-Saharan Africa. Subunit recombinant transmission-blocking vaccines (TBVs) have been proposed as promising vaccines to succeed in malaria elimination and eradication. Here, a provisional study was designed to assess the immunogenicity and functional activity of alanyl aminopeptidase N (APN1) of Anopheles stephensi, as a TBV candidate, administered with MPL, CpG, and QS21 adjuvants in the murine model. The mouse groups were immunized with recombinant APN1 (rAPN1) alone or formulated with CpG, MPL, QS-21, or a combination of adjuvants (CMQ), and the elicited immune responses were evaluated after the third immunization. The standard membrane feeding assay (SMFA) measured the functional activity of antibodies against bacterial-expressed APN1 protein in adjuvanted vaccine groups on transmission of P. falciparum (NF54) to An. stephensi mosquitoes. Evaluation of mice vaccinated with rAPN1 formulated with distinct adjuvants manifested a significant increase in the high-avidity level of anti-APN1 IgG and IgG subclasses; however, rAPN1 induced the highest level of high-avidity anti-APN1 IgG1, IgG2a, and IgG2b antibodies in the immunized vaccine group 5 (APN1/CMQ). In addition, vaccine group 5 (receiving APN1/CMQ), had still the highest level of anti-APN1 IgG antibodies relative to other immunized groups after six months, on day 180. The SMFA data indicates a trend towards higher transmission-reducing activity in groups 2 and 5, which received the antigen formulated with CpG or a combination of three adjuvants. The results have shown the capability of admixture to stimulate high-affinity and long-lasting antibodies against the target antigen to hinder Plasmodium parasite development in the mid-gut of An. stephensi. The attained results authenticated APN1/CMQ and APN1/CpG as a potent APN1-based TBV formulation which will be helpful in designing a vaccine in the future.

Neutralizing antibodies reveal cryptic vulnerabilities and interdomain crosstalk in the porcine deltacoronavirus spike protein

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of PDCoV remains unknown. The virus relies on its spike (S) protein for cell entry, making it a prime target for neutralizing antibodies. Here, we generate and characterize a set of neutralizing antibodies targeting the S protein, shedding light on PDCoV S interdomain crosstalk and its vulnerable sites. Among the four identified antibodies, one targets the S1A domain, causing local and long-range conformational changes, resulting in partial exposure of the S1B domain. The other antibodies bind the S1B domain, disrupting binding to aminopeptidase N (APN), the entry receptor for PDCoV. Notably, the epitopes of these S1B-targeting antibodies are concealed in the prefusion S trimer conformation, highlighting the necessity for conformational changes for effective antibody binding. The binding footprint of one S1B binder entirely overlaps with APN-interacting residues and thus targets a highly conserved epitope. These findings provide structural insights into the humoral immune response against the PDCoV S protein, potentially guiding vaccine and therapeutic development for this zoonotic pathogen.

A transformable and self-oxygenated smart probe for enhanced tumor sonodynamic therapy

Sonodynamic therapy (SDT) is emerging as a promising modality for cancer treatment. However, improving the tumor bioavailability and anti-hypoxia capability of sonosensitizers faces a big challenge. In this work, we present a tumor microenvironment (TME)-mediated nanomorphology transformation and oxygen (O2) self-production strategy to enhance the sonodynamic therapeutic efficacy of tumors. A smart probe Ce6-Leu@Mn2+ that consists of a glutathione (GSH) and leucine amino peptidase (LAP) dual-responsive unit, a 2-cyanobenzothiazole (CBT) group, and a Mn2+-chelated Ce6 as sonosensitizer for tumor SDT was synthesized, and its SDT potential for liver tumor HepG2 in living mice was systematically studied. It was found that the probes could self-assemble into large nanoparticles in physiological condition and spontaneously transformed into small particles under the dual stimulation of GSH and LAP in TME resulting in enhanced tumor accumulation and deep penetration. More notably, Ce6-Leu@Mn2+ could convert endogenous hydrogen peroxide to O2, thereby alleviating the hypoxia and achieving effective SDT against hypoxic tumors under the excitation of ultrasound. We thus believe this smart TME-responsive probe may provide a noninvasive and efficient means for malignant tumor treatment. Statement of significanceSonodynamic therapy (SDT) is emerging as a promising therapeutic modality for cancer treatment. However, how to improve the tumor bioavailability and anti-hypoxia capability of sonosensitizers remains a huge challenge. Herein, we rationally developed a theranostic probe Ce6-Leu@Mn2+ that can transform into small-size nanoparticles from initial large particles under the dual stimulation of LAP and GSH in tumor microenvironment (TME) resulting in enhanced tumor accumulation, deep tissue penetration as well as remarkable O2 self-production for enhanced sonodynamic therapy of human liver HepG2 tumor in living mice. This smart TME-responsive probe may provide a noninvasive and efficient means for hypoxic tumor treatment.

Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 μM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

Anti-Coronavirus Potential of Polyether Ionophores: The New Application of Veterinary Antibiotics in Livestock.

Coronaviruses have consistently posed a major global concern in the field of livestock industry and public health. However, there is currently a lack of efficient drugs with broad-spectrum antiviral activity to address the challenges presented by emerging mutated strains or drug resistance. Additionally, the method for identifying multitarget drugs is also insufficient. Aminopeptidase N (APN) and 3C-like proteinase (3CLpro) represent promising targets for host-directed and virus-directed strategies, respectively, in the development of effective drugs against various coronaviruses. In this study, maduramycin ammonium demonstrated a broad-spectrum antiviral effect by targeting both of the proteins. The binding domains 4 Å from the ligand of both target proteins shared a structural similarity, suggesting that screening and designing drugs based on these domains might exhibit broad-spectrum and highly effective antiviral activity. Furthermore, it was identified that the polyether ionophores' ability to carry zinc ion might be one of the reasons why they were able to target APN and exhibit antiviral effect. The findings of this experiment provide novel perspectives for future drug screening and design, while also offering valuable references for the utilization of polyether ionophores in the management of livestock health.