The Secreted Aminopeptidase of Pseudomonas aeruginosa (PaAP).

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in compromised hosts. P. aeruginosa infections are difficult to treat because of the inherent ability of the bacteria to develop antibiotic resistance, secrete a variety of virulence factors, and form biofilms. The secreted aminopeptidase (PaAP) is an emerging virulence factor, key in providing essential low molecular weight nutrients and a cardinal modulator of biofilm development. PaAP is therefore a new potential target for therapy of P. aeruginosa infections. The present review summarizes the current knowledge of PaAP, with special emphasis on its biochemical and enzymatic properties, activation mechanism, biological roles, regulation, and structure. Recently developed specific inhibitors and their potential as adjuncts in the treatment of P. aeruginosa infections are also described.

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.

Silence of Aminopeptidase N 2 gene reveals the trade-offs for acquiring Cry1Ac resistance in Plutella xylostella.

Bacillus thuringiensis (Bt) is widely used as a biopesticide worldwide. To date, at least eight pest species have been found to be resistant to Bt in the field. As the first pest that was reported having resistance to Bt in the field, considerable research has been done on the mechanisms of Bt resistance in Plutella xylostella. However, whether the acquisition of Bt resistance by P. xylostella comes at a fitness cost is also a valuable question. In this study, Aminopeptidase-N 2 (APN2), a Cry toxin receptor gene of P. xylostella, was knocked down by RNA interference, resulting in improved resistance to Cry1Ac. It was also found that larval mortality of APN2 knockdown P. xylostella was significantly higher than that of the control, while the pupation rate, pupal weight, eclosion rate, fecundity (egg/female), hatchability, and female adult longevity were significantly lower in APN2 knockdown P. xylostella than in the control. These results illustrate that if Cry1Ac resistance was obtained only through the reduction of APN2 expression, P. xylostella would need to incur some fitness costs for it.

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.

Effective inhibition of PDCoV infection in chimeric APN gene-edited neonatal pigs.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, is a serious threat to piglets and has zoonotic potential. Here, we aimed to further explore the role of aminopeptidase N (APN) as a receptor for PDCoV and test the inhibitory effect of a chimeric APN protein strategy on PDCoV infection. PK-15 cells and LLC-PK1 cells expressing chimeric APN were selected and infected with PDCoV. Viral replication was significantly decreased in these chimeric APN cells compared with that in control group cells. To further characterize the effect of the chimeric APN strategy on PDCoV infection in vitro, primary intestinal epithelial cells isolated from chimeric APN pigs were inoculated with PDCoV. Viral challenge of these cells led to decreased PDCoV infection. More importantly, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. Taken together, these findings deepen our understanding of the mechanism of PDCoV infection and provide a valuable model for the production of disease-resistant animals. IMPORTANCE: Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are currently no effective measures for the prevention or control of PDCoV infection. Here, we have developed PK-15 cells, LLC-PK1 cells, and primary intestinal epithelial cells expressing chimeric APN, and viral challenge of these cells led to decreased PDCoV infection. Furthermore, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. These data show that chimeric APN is a promising strategy to combat PDCoV infection.

Substrate Characterization for Hydrolysis of Multiple Types of Aromatic Esters by Promiscuous Aminopeptidases.

Synthetic aromatic esters, widely employed in agriculture, food, and chemical industries, have become emerging environmental pollutants due to their strong hydrophobicity and poor bioavailability. This study attempted to address this issue by extracellularly expressing the promiscuous aminopeptidase (Aps) from Pseudomonas aeruginosa GF31 in B. subtilis, achieving an impressive enzyme activity of 13.7 U/mg. Notably, we have demonstrated, for the first time, the Aps-mediated degradation of diverse aromatic esters, including but not limited to pyrethroids, phthalates, and parabens. A biochemical characterization of Aps reveals its esterase properties and a broader spectrum of substrate profiles. The degradation rates of p-nitrobenzene esters (p-NB) with different side chain structures vary under the action of Aps, showing a preference for substrates with relatively longer alkyl side chains. The structure-dependent degradability aligns well with the binding energies between Aps and p-NB. Molecular docking and enzyme-substrate interaction elucidate that hydrogen bonding, hydrophobic interactions, and π-π stacking collectively stabilize the enzyme-substrate conformation, promoting substrate hydrolysis. These findings provide new insights into the enzymatic degradation of aromatic ester pollutants, laying a foundation for the further development and modification of promiscuous enzymes.

Research progress of porcine epidemic diarrhea virus S protein.

Porcine epidemic diarrhea virus (PEDV) is a single-stranded RNA virus with a capsid membrane that causes acute infectious gastrointestinal disease characterized by vomiting, diarrhea, and dehydration in swine. Piglets are more susceptible to PEDV than adults, with an infection rate reaching 90% and a fatality rate as high as 100%. Moreover, PEDV has a rapid transmission rate and broad transmission range. Consequently, PEDV has caused considerable economic losses and negatively impacted the sustainability of the pig industry. The surface spike (S) glycoprotein is the largest structural protein in PEDV virions and is closely associated with host cell fusion and virus invasion. As such, the S protein is an important target for vaccine development. In this article, we review the genetic variation, immunity, apoptosis-induction function, virulence, vaccine potential, and other aspects of the PEDV S protein. This review provides a theoretical foundation for preventing and controlling PEDV infection and serves as a valuable resource for further research and development of PEDV vaccines.

Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S.

Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H2S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H2S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits.

Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination.

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

The DNA repair kinase ATM regulates CD13 expression and cell migration.

Classically, ATM is known for its role in sensing double-strand DNA breaks, and subsequently signaling for their repair. Non-canonical roles of ATM include transcriptional silencing, ferroptosis, autophagy and angiogenesis. Angiogenesis mediated by ATM signaling has been shown to be VEGF-independent via p38 signaling. Independently, p38 signaling has been shown to upregulate metalloproteinase expression, including MMP-2 and MMP-9, though it is unclear if this is linked to ATM. Here, we demonstrate ATM regulates aminopeptidase-N (CD13/APN/ANPEP) at the protein level. Positive correlation was seen between ATM activity and CD13 protein expression using both "wildtype" (WT) and knockout (KO) ataxia telangiectasia (AT) cells through western blotting; with the same effect shown when treating neuroblastoma cancer cell line SH-SY5Y, as well as AT-WT cells, with ATM inhibitor (ATMi; KU55933). However, qPCR along with publically available RNAseq data from Hu et al. (J. Clin. Invest., 2021, 131, e139333), demonstrated no change in mRNA levels of CD13, suggesting that ATM regulates CD13 levels via controlling protein degradation. This is further supported by the observation that incubation with proteasome inhibitors led to restoration of CD13 protein levels in cells treated with ATMi. Migration assays showed ATM and CD13 inhibition impairs migration, with no additional effect observed when combined. This suggests an epistatic effect, and that both proteins may be acting in the same signaling pathway that influences cell migration. This work indicates a novel functional interaction between ATM and CD13, suggesting ATM may negatively regulate the degradation of CD13, and subsequently cell migration.

Modulation of endogenous opioid signaling by inhibitors of puromycin-sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

A study of the association between single nucleotide polymorphisms of the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene and the risk of ankylosing spondylitis in Egyptians.

BACKGROUND: Ankylosing spondylitis (AS) is often regarded as the prototypical manifestation of spondylo-arthropathies that prevalently involves the axial skeleton with the potential attribution of ERAP2 polymorphisms to AS predisposition. The purpose of this study was to determine the genetic association between ERAP2 gene rs2910686, and rs2248374 single nucleotide polymorphisms (SNPs) and the risk of ankylosing spondylitis in the Egyptian population. METHODS AND RESULTS: A cross-sectional work involved 200 individuals: 100 AS individuals diagnosed based on modified New York criteria in 1984 with 100 healthy controls matched in age and gender. The study included a comprehensive evaluation of historical data, clinical examinations, and evaluation of the activity of the disease using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). A comprehensive laboratory and radiological evaluation were conducted, accompanied by an assessment and genotyping of the ERAP2 gene variants rs2248374 and rs2910686. This genotyping was performed utilizing a real-time allelic discrimination methodology.Highly statistically substantial variations existed among the AS patients and the healthy control group regarding rs2910686 and rs2248374 alleles. There was a statistically significant difference between rs2910686 and rs2248374 regarding BASDAI, BASFI, mSASSS, ASQoL, V.A.S, E.S.R, and BASMI in the active AS group. CONCLUSIONS: ERAP2 gene SNPs have been identified as valuable diagnostic biomarkers for AS patients in the Egyptian population being a sensitive and non-invasive approach for AS diagnosis especially rs2910686. Highly statistically significant variations existed among the AS patients and the healthy control group regarding rs2910686 alleles and genotypes.Further research is recommended to explore the potential therapeutic implications of these SNPs.

Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17.

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

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.

Bestatin attenuates breast cancer stemness by targeting puromycin-sensitive aminopeptidase.

Breast cancer is a prevalent malignant tumor among women with an increasing incidence rate annually. Breast cancer stem cells (BCSCs) are integral in impeding tumor advancement and addressing drug resistance. Bestatin serves as an adjuvant chemotherapy, triggering apoptosis in cancer cells. In this study, the effects of bestatin on sorted BCSCs from breast cancer cell lines have been studied. Our results indicated that bestatin inhibits the migration and proliferation of breast cancer cells by reducing the stemness of BCSCs both in vitro and in vivo. Puromycin-sensitive aminopeptidase is implicated in the process through the regulation of cell cycle, resulting in heightened cell apoptosis and diminished cell proliferation of BCSCs. Our study suggest that targeting cancer stem cell may offer a promising approach in breast cancer treatment, presenting noval therapeutic strategies for patients with breast cancer.

On-target, dual aminopeptidase inhibition provides cross-species antimalarial activity.

To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum, is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817-resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817, and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial.

The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds.

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

In Vivo Imaging of Acute Hindlimb Ischaemia in Rat Model: A Pre-Clinical PET Study.

BACKGROUND: To better understand ischaemia-related molecular alterations, temporal changes in angiogenic Aminopeptidase N (APN/CD13) expression and glucose metabolism were assessed with PET using a rat model of peripheral arterial disease (PAD). METHODS: The mechanical occlusion of the base of the left hindlimb triggered using a tourniquet was applied to establish the ischaemia/reperfusion injury model in Fischer-344 rats. 2-[18F]FDG and [68Ga]Ga-NOTA-c(NGR) PET imaging performed 1, 3, 5, 7, and 10 days post-ischaemia induction was followed by Western blotting and immunohistochemical staining for APN/CD13 in ischaemic and control muscle tissue extracts. RESULTS: Due to a cellular adaptation to hypoxia, a gradual increase in [68Ga]Ga-NOTA-c(NGR) and 2-[18F]FDG uptake was observed from post-intervention day 1 to 7 in the ischaemic hindlimbs, which was followed by a drop on day 10. Conforming pronounced angiogenic recovery, the NGR accretion of the ischaemic extremities differed significantly from the controls 5, 7, and 10 days after ischaemia induction (p ≤ 0.05), which correlated with the Western blot and immunohistochemical results. No remarkable radioactivity was depicted between the normally perfused hindlimbs of either the ischaemic or the control groups. CONCLUSIONS: The PET-based longitudinal assessment of angiogenesis-associated APN/CD13 expression and glucose metabolism during ischaemia may continue to broaden our knowledge on the pathophysiology of PAD.

Safety evaluation of the food enzyme leucyl aminopeptidase from the genetically modified Aspergillus oryzae strain NZYM-BU.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the genetically modified Aspergillus oryzae strain NZYM-BU by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes. Dietary exposure to the food enzyme TOS was estimated to be up to 1.508 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 4,928 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 3,268. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.