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Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in suckling piglets and has the potential for cross-species transmission, posing a threat to animal and human health. However, the susceptibility profile of different species of mice to PDCoV infection and its evolutionary characteristics are still unclear. In the current study, we found that BALB/c and Kunming mice are susceptible to PDCoV. Our results showed that there were obvious lesions in intestinal and lung tissues from the infected mice. PDCoV RNAs were detected in the lung, kidney, and intestinal tissues from the infected mice of both strains, and there existed wider tissue tropism in the PDCoV-infected BALB/c mice. The RNA and protein levels of aminopeptidase N from mice were relatively high in the kidney and intestinal tissues and obviously increased after PDCoV infection. The viral-specific IgG and neutralizing antibodies against PDCoV were detected in the serum of infected mice. An interesting finding was that two key amino acid mutations, D138H and Q641K, in the S protein were identified in the PDCoV-infected mice. The essential roles of these two mutations for PDCoV-adaptive evolution were confirmed by cryo-electron microscope structure model analysis. The evolutionary characteristics of PDCoV among Deltacoronaviruses (δ-CoVs) were further analyzed. δ-CoVs from multiple mammals are closely related based on the phylogenetic analysis. The codon usage analysis demonstrated that similar codon usage patterns were used by most of the mammalian δ-CoVs at the global codon, synonymous codon, and amino acid usage levels. These results may provide more insights into the evolution, host ranges, and cross-species potential of PDCoV.

Lymphatic metastasis is a crucial mechanism by which the cancer cells break away from the primary (original) tumor and travel to the closest regional lymph node(s) and ultimately to other organs or parts of the body, which is closely associated with tumor recurrence and reduced survival. Thus, tracking tumor lymphatic metastasis and realizing imaging-guided lymphoma resection surgery is of great significance. In this study, an activatable nanoprobe is developed for precisely tracking lymphatic metastasis of tumors and imaging-guided resection of the primary tumor and metastatic lymphoma. The molecular probe contains tricyanofuran as the electron-accepting unit (electron acceptor), xanthene as the electron-donating unit (electron donor), and alanine as the responsive unit (recognition moiety) for aminopeptidase N, and the probe molecules form the nanoprobe with bovine serum albumin as the matrix. The nanoprobe can respond specifically to aminopeptidase N overproduced in the tumor, thereby transmuting the alanine into an amino group, and correspondingly the nanoprobe is activated. Strong optoacoustic and NIR-II fluorescence signals emitted by the activated nanoprobe can be utilized for visualizing the lymphatic metastasis of tumors. Moreover, the nanoprobe with the aid of three-dimensional multispectral optoacoustic tomography (3D MSOT) imaging can accurately locate the tumor site of lymphatic metastasis, and ultimately, both the primary tumor and the metastatic lymphoma can be excised with resection surgery under the guidance of NIR-II fluorescence imaging.

Background: The aminopeptidase N (APN) over-expressed in tumor cells plays a critical role in angiogenesis which makes the development of APN inhibitors an attractive strategy for cancer research. Aims and Objectives: It is clinically significant to develop potential APN inhibitors for cancer treatment. The design, synthesis, biological evaluation and molecular simulation of mandelic acid peptidomimetic derivatives as APN inhibitors are reported. Materials and Methods: Analysis of the binding mode of bestatin to APN led to the design and synthesis of mandelic acid peptidomimetic derivatives. APN inhibitory activities in vitro were evaluated by the spectrophotometric method. The binding mode of the target compounds with the APN binding site was studied relying on docking studies, molecular dynamics simulation experiments and binding energies calculation. Results: The structures of target compounds were confirmed by IR, 1H-NMR and MS. All compounds exhibited a different range of inhibitory ability with IC50 values lying in the micromolar level. The compound 9m was found to be most potent as compared to other target derivatives. The molecular simulation revealed that ligand coordinating with the catalytic zinc ion is very important for inhibitory activities. Conclusion: The compound 9m might represent a promising scaffold for the further development of novel anti-cancer drugs.

Non-small cell lung cancer (NSCLC) is the most common type in lung cancer in the world, and it severely threatens the life of patients. Resveratrol has been reported to inhibit cancer. However, mechanisms of resveratrol inhibiting NSCLC were unclear. The aim of this study was to identify differentially expressed genes (DEGs) of NSCLC treated with resveratrol and reveal the potential targets of resveratrol in NSCLC. We obtained mRNA expression profiles of two datasets from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI-GEO) and 271 DEGs were selected for further analysis. Data from STRING shown that 177 nodes and 342 edges were in the protein-protein interaction (PPI) network, and 10 hub genes (ANPEP, CD69, ITGAL, PECAM1, PTPRC, CD34, ITGA1, CCL2, SOX2, and EGFR) were identified by Cytoscape plus-in cytoHubba. Survival analysis revealed that NSCLC patients showing low expression of PECAM1, ANPEP, CD69, ITGAL, and PTPRC were associated with worse overall survival (OS) (P < 0.05), and high expression of SOX2 and EGFR was associated with worse OS for NSCLC patients (P < 0.05). Overall, we identified ANPEP, CD69, ITGAL, and PTPRC as potential candidate genes which were main effects of resveratrol on the treatment of NSCLC. ANPEP, ITGAL, CD69, and PTPRC are all clusters of differentiation (CD) antigens, might be the targets of resveratrol. The bioinformatic results suggested that the inhibitory effect of resveratrol on lung cancer may be related to the immune signaling pathway. Further studies are needed to validate these findings and to explore their functional mechanisms.

Spin hyperpolarization enables real-time metabolic imaging of carbon-13-labeled substrates. While hyperpolarized l-(1

In this study, pyrralylisoleucine (Pyrr-Ile) and pyrralylalanine (Pyrr-Ala), two dipeptide-bound pyrralines with different C-termini were synthesized as the representatives of dietary advanced glycation end products (dAGEs). The structures of Pyrr-Ile and Pyrr-Ala were characterized by high-resolution mass spectrometry, nuclear magnetic resonance, and Fourier transform infrared spectroscopy. Then, the transport of Pyrr-Ile and Pyrr-Ala across intestinal epithelial cells was investigated using Caco-2 cell monolayers, and their interaction with aminopeptidase N (APN) was analyzed. The results showed that the apparent permeability coefficient (Papp) of Pyrr-Ala was (14.1 ± 2.26) × 10-7 cm·s-1 calculated by free pyrraline, while the Papp values of Pyrr-Ile were (32.4 ± 5.35) × 10-7 and (19.1 ± 1.46) × 10-7 cm·s-1 when they were, respectively, calculated according to their dipeptide-bound or free form. Both Pyrr-Ala and Pyrr-Ile were potential substrates of APN, and their hydrolysis by APN may make the intact transmembrane transport of Pyrr-Ala and Pyrr-Ile more difficult, especially for Pyrr-Ala. Besides, the occurrence of product inhibition in hydrolysis of Pyrr-Ile was possible. Pyrr-Ile and Pyrr-Ala were different in Papp values and transport forms, which suggested that the C-terminus may play an important role in their transport across the Caco-2 cell monolayers. In addition, the results highlight the intact transmembrane transport of dipeptide-bound pyrraline.

Polarized epithelial cells can organize into complex structures with a characteristic central lumen. Lumen formation requires that cells coordinately orient their polarity axis so that the basolateral domain is on the outside and apical domain inside epithelial structures. Here we show that the transmembrane aminopeptidase, CD13, is a key determinant of epithelial polarity orientation. CD13 localizes to the apical membrane and associates with an apical complex with Par6. CD13-deficient cells display inverted polarity in which apical proteins are retained on the outer cell periphery and fail to accumulate at an intercellular apical initiation site. Here we show that CD13 is required to couple apical protein cargo to Rab11-endosomes and for capture of endosomes at the apical initiation site. This role in polarity utilizes the short intracellular domain but is independent of CD13 peptidase activity., Epithelial cells that organise into structures that contain a lumen are polarised. Here, the authors show that the short intracellular domain of transmembrane protein CD13 is required to capture endosomes at the apical site and is required for the polarisation of cells.

Many fluorophores/probes suffer from the interference of albumin in biosystems. Herein, we propose an effective strategy to overcome this interference by virtue of both an albumin-insensitive fluorophore and its changeable π-conjugation, and demonstrate the strategy by designing an oxazine-based fluorogenic probe for aminopeptidase N (APN). The modification on the N atom in the oxazine fluorophore with alanine through a cleavable linker locks the resulting probe in a non-conjugated, colorless and non-fluorescent state, so the non-specific interaction of albumin produces no spectroscopic response. APN can selectively cleave the alanine moiety, restoring the large π-conjugation and strong fluorescence. The capability of the probe to eliminate the albumin influence has been demonstrated by imaging APN in different cell lines, and by quantitatively determining APN in human serum and mouse urine. The present strategy may be useful for developing more specific fluorogenic probes for other enzymes.

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

Sepsis-induced acute lung injury (ALI) is a serious sepsis complication and the prevailing cause of death. Circulating plasma exosomes might exert a key role in regulating intercellular communication between immunological and structural cells, as well as contributing to sepsis-related organ damage. However, the molecular mechanisms by which exosome-mediated intercellular signaling exacerbate ALI in septic infection remains undefined. Therefore, we investigated the effect of macrophage-derived exosomal APN/CD13 on the induction of epithelial cell necrosis. Exosomal APN/CD13 levels in the plasma of septic mice and patients with septic ALI were found to be higher. Furthermore, increased plasma exosomal APN/CD13 levels were associated with the severity of ALI and fatality in sepsis patients. We found remarkably high expression of APN/CD13 in exosomes secreted by LPS-stimulated macrophages. Moreover, c-Myc directly induced APN/CD13 expression and was packed into exosomes. Finally, exosomal APN/CD13 from macrophages regulated necroptosis of lung epithelial cells by binding to the cell surface receptor TLR4 to induce ROS generation, mitochondrial dysfunction and NF-κB activation. These results demonstrate that macrophage-secreted exosomal APN/CD13 can trigger epithelial cell necroptosis in an APN/CD13-dependent manner, which provides insight into the mechanism of epithelial cell functional disorder in sepsis-induced ALI.

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN-specific substrate group to the ortho-acrylated phenoxy-dioxetane scaffold. In this way, two CL probes (APN-CL and BAPN-CL) were designed with noncapped leucine and butoxy-carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN-CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN-CL was still restrained even with the high-level APN. The result was further elucidated by molecular docking simulations. Probe APN-CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN-CL imaged abnormal levels of APN in tumour cells and tumour-bearing mice. Moreover, this probe APN-CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost-effective probe, APN-CL will be a promising alternative in the early diagnosis of pathologies and for cost-effective screening of inhibitors.

Background: Sacubitril/valsartan (S/V) treatment is beneficial in patients with heart failure with reduced ejection fraction (HFrEF), but its mode of action remains elusive, although it involves the increase in ANP (atrial natriuretic peptide). Methods: Combining mass spectrometry and enzymatic assay in the plasma of 73 HFrEF patients treated with S/V and controls, we deciphered proANP processing that converts proANP into 4 vasoactive peptides. Results: We found that proANP processing is sequential and involved meprin B, ECE (endothelin-converting enzyme) 1, and ANPEP (aminopeptidase N). This processing is limited in HFrEF patients via the downregulation of proANP production, corin, and meprin B activities by miR-425 and miR1-3p. S/V restored or compensated proANP processing by downregulating miR-425 and miR1-3p, hence increasing levels of proANP-derived bioactive peptides. In contrast, S/V directly and indirectly partially inhibited ECE1 and ANPEP. ECE1 partial inhibition resulted in a lower-than-expected increase in ET1 (endothelin 1), tilting the vasoactive balance toward vasodilation, and possibly hypotension. Furthermore, proANP glycosylation interferes with the midregional proANP assay –a clinical surrogate for proANP production, preventing any pathophysiological interpretation of the results. The analysis of S/V dose escalation with respect to baseline treatments suggests S/V-specific effects. Conclusions: These findings offer mechanistic evidence to the natriuretic peptide -defective state in HFrEF, which is improved by S/V. These data also strongly suggests that S/V increases plasma ANP by multiple mechanisms that involve 2 microRNAs, besides its protection from NEP (neprilysin) cleavage. Altogether, these data provide new insights on HFrEF pathophysiology and the mode of action of S/V.

Astrocytes are major supportive glia and immune modulators in the brain; they are highly secretory in nature and interact with other cell types via their secreted proteomes. To understand how astrocytes communicate during neuroinflammation, we profiled the secretome of human astrocytes following stimulation with proinflammatory factors. A total of 149 proteins were significantly upregulated in stimulated astrocytes, and a bioinformatics analysis of the astrocyte secretome revealed that the brain renin-angiotensin system (RAS) is an important mechanism of astrocyte communication. We observed that the levels of soluble form of aminopeptidase N (sANPEP), an RAS component that converts angiotensin (Ang) III to Ang IV in a neuroinflammatory milieu, significantly increased in the astrocyte secretome. To elucidate the role of sANPEP and Ang IV in neuroinflammation, we first evaluated the expression of Ang IV receptors in human glial cells because Ang IV mediates biological effects through its receptors. The expression of angiotensin type 1 receptor was considerably upregulated in activated human microglial cells but not in human astrocytes. Moreover, interleukin-1β release from human microglial cells was synergistically increased by cotreatment with sANPEP and its substrate, Ang III, suggesting the proinflammatory action of Ang IV generated by sANPEP. In a mouse neuroinflammation model, brain microglial activation and proinflammatory cytokine expression levels were increased by intracerebroventricular injection of sANPEP and attenuated by an enzymatic inhibitor and neutralizing antibody against sANPEP. Collectively, our results indicate that astrocytic sANPEP-induced increase in Ang IV exacerbates neuroinflammation by interacting with microglial proinflammatory receptor angiotensin type 1 receptor, highlighting an important role of indirect crosstalk between astrocytes and microglia through the brain RAS in neuroinflammation.

Although feline coronavirus (FCoV) infection is extremely common in cats, there are currently few effective treatments. A peptide derived from the heptad repeat 2 (HR2) domain of the coronavirus (CoV) spike protein has shown effective for inhibition of various human and animal CoVs

Tumour revascularization and the consequent radioresistance activated by the up-regulated angiogenic pathway after radiation exposure remain a major bottleneck for improving the tumouricidal effect of radiotherapy (RT) in hepatocellular carcinoma (HCC). Herein, we show that fabricated aminopeptidase N (ANP/CD13)-targeting Gd-hybridized gold nanomolecules (tGd-GNMs) can efficaciously suppress tumour revascularization and the consequent radioresistance, and then synergize in augmenting the RT response. Both in vitro and in vivo experiments demonstrate that the targeted delivery of vascular endothelial growth factor (VEGF) siRNA into the tumour site and the generation of an abundance of intratumourally cytotoxic reactive oxygen species (ROS) under X-ray radiation by the tGd-GNMssiRNA complex has the capability to down-regulate VEGF gene expression and strengthen the radiation response. Furthermore, the tGd-GNMssiRNA complex contributes to excellent active tumour targeting ability, remarkably enhancing tumour contrast in the fluorescence, computed tomography (CT) and magnetic resonance (MR) imaging modalities in real-time with a long imaging time window. Overall, the synthesized tGd-GNMssiRNA complex with excellent potentiation of the antitumour ability and real-time multimodal imaging ability represents a promising visualized theranostic nanoplatform for the treatment of HCC.

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design., The spike protein of coronaviruses (S-protein) is an envelope-anchored trimeric type I transmembrane glycoprotein that mediates receptor binding and the fusion of the viral and host cell membranes. Here the authors report the conformational states of HCoV-229E S trimer and observe the conformational changes in S1 subunit from closed state to activated state for receptor binding.

Cancer is one of the most serious diseases that are harmful to human health. Systemic chemotherapy is an optimal therapeutic strategy for the treatment of cancer, but great difficulty has been encountered in its administration in the form of multidrug resistance (MDR). As an enzyme on the outer cell surface, CD13 is documented to be involved in the MDR development of tumor cells. In this review, we will focus on the role of CD13 in MDR generation based on the current evidence.

Objectives Patients with clonal cytopenia of undetermined significance (CCUS) are at increased risk of developing myeloid neoplasia (MN). We evaluated whether a simple flow cytometry immunophenotyping (FCIP) assay could differentiate the risk of development of MN in patients with CCUS. Methods Bone marrow aspirates were assessed by FCIP panel in a cohort of 80 patients identified as having CCUS based on next-generation sequencing or cytogenetics from March 2015 to May 2020, with available samples. Flow cytometric assay included CD13/HLA-DR expression pattern on CD34-positive myeloblasts; CD13/CD16 pattern on maturing granulocytic precursors; and aberrant expression of CD2, CD7, or CD56 on CD34-positive myeloblasts. Relevant demographic, comorbidity, and clinical and laboratory data, including the type and extent of genetic abnormalities, were extracted from the electronic health record. Results In total, 17 (21%) patients with CCUS developed MN over the follow-up period (median survival follow-up, 28 months [95% confidence interval, 19-31]). Flow cytometry immunophenotyping abnormalities, including the aberrant pattern of CD13/HLA-DR expression, as detected at the time of the diagnosis of CCUS, were significantly associated with risk of developing MN (hazard ratio, 2.97; P = .006). Additional FCIP parameters associated with the development of MN included abnormal expression of CD7 on myeloblasts and the presence vs absence of any FCIP abnormality. Conclusions A simple FCIP approach that includes assessment of CD13/HLA-DR pattern on CD34-positive myeloblasts can be useful in identifying patients with CCUS at higher risk of developing MN.

To investigate the expressions of CD33 and CD13 in newly diagnosed multiple myeloma (MM) patients and its relationship with prognosis.It was retrospectively observed that the expression of CD33 and CD13 in 121 MM patients who were newly diagnosed from January 2014 to January 2020, and the relationship between the expressions of CD33 and CD13 and patients prognosis was analyzed.Among the 121 newly diagnosed MM patients, there were 30 patients (24.8%) in the CD33CD33 and CD13 are prognostic risk factors in patients with MM.CD33和CD13表达与多发性骨髓瘤患者预后的关系.探讨初诊多发性骨髓瘤（MM）患者中CD33和CD13的表达情况及其与患者预后的关系.回顾性分析2014年1月至2020年1月初诊的121例MM患者中CD33和CD13的表达情况及CD33和CD13表达与患者预后的关系.121例初诊MM患者中，CD33CD33和CD13均是MM患者的预后不良危险因素.

Background Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial. Results We have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a > 34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains. Conclusion Our results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of action of Bt toxins, which can compensate for the absence of individual receptors, and are consistent with an interplay among diverse midgut receptors in the toxins’ mechanism of action in a super pest.

Background: Truncated tissue factor (tTF) retargeted by NGR-peptides to aminopeptidase N (CD13) in tumor vasculature is effective in experimental tumor therapy. tTF-NGR induces tumor growth inhibition in a variety of human tumor xenografts of different histology. To improve on the therapeutic efficacy we have combined tTF-NGR with radiotherapy. Methods: Serum-stimulated human umbilical vein endothelial cells (HUVEC) and human HT1080 sarcoma cells were irradiated in vitro, and upregulated early-apoptotic phosphatidylserine (PS) on the cell surface was measured by standard flow cytometry. Increase of cellular procoagulant function in relation to irradiation and PS cell surface concentration was measured in a tTF-NGR-dependent Factor X activation assay. In vivo experiments with CD-1 athymic mice bearing human HT1080 sarcoma xenotransplants were performed to test the systemic therapeutic effects of tTF-NGR on tumor growth alone or in combination with regional tumor ionizing radiotherapy. Results: As shown by flow cytometry with HUVEC and HT1080 sarcoma cells in vitro, irradiation with 4 and 6 Gy in the process of apoptosis induced upregulation of PS presence on the outer surface of both cell types. Proapoptotic HUVEC and HT1080 cells both showed significantly higher procoagulant efficacy on the basis of equimolar concentrations of tTF-NGR as measured by FX activation. This effect can be reverted by masking of PS with Annexin V. HT1080 human sarcoma xenografted tumors showed shrinkage induced by combined regional radiotherapy and systemic tTF-NGR as compared to growth inhibition achieved by either of the treatment modalities alone. Conclusions: Irradiation renders tumor and tumor vascular cells procoagulant by PS upregulation on their outer surface and radiotherapy can significantly improve the therapeutic antitumor efficacy of tTF-NGR in the xenograft model used. This synergistic effect will influence design of future clinical combination studies.

STUDY QUESTION Is there a relationship between human sperm aminopeptidase N (APN) and embryo development in humans? SUMMARY ANSWER Human sperm APN could possibly become a new molecular biomarker for identifying the potential for high-quality and usable embryos. WHAT IS KNOWN ALREADY The diagnosis of male fertility is one of the major concerns of reproductive medicine. Approximately 30–40% of men with otherwise normal fertility parameters are still unable to achieve pregnancy. The predictive clinical value of semen analysis to identify fertile or infertile males is limited; therefore, new diagnostic methodologies for sperm are urgently required. Sperm APN may be a relevant molecular marker due to its high concentration in sperm cells and its important roles in sperm physiology, such as its functions in motility, acrosome reaction and embryo development. STUDY DESIGN, SIZE, DURATION This study included 81 couples who underwent oocyte-donation cycles at Clínica IVI Bilbao (Spain), yielding 611 embryos, between September 2014 and July 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was conducted in an assisted reproduction unit and an academic research laboratory. All the semen samples were examined and classified following World Health Organization guidelines. Spermatozoa were isolated from semen using the discontinuous colloidal silica gradient (45–90%) technique. Embryo quality and development were determined according to the Spanish Association of Reproduction Biology Studies (ASEBIR) criteria. Human sperm APN levels were analyzed by quantitative and semiquantitative flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE The most well-developed and usable blastocysts were associated with low sperm APN levels. Semen samples that had lower APN levels generated more expanded, hatched and usable blastocysts and fewer early, arrested and non-usable blastocysts. The cumulative probability of having well-developed blastocysts increased by 1.38-fold at Day 5 and 1.90-fold at Day 6 of embryo development, and the likelihood of having usable embryos increased by 1.48-fold, when semen samples with low APN levels were used during the ICSI technique. LIMITATIONS, REASONS FOR CAUTION The data were obtained from a single fertility clinic. A multicentre study will be required to confirm the results. WIDER IMPLICATIONS OF THE FINDINGS Human sperm APN has the potential to become a new molecular biomarker to help identify the potential for high-quality embryos and diagnose male infertility, especially when seminal parameters are close to the threshold values. It could be a crucial tool for couples for whom the number of usable blastocysts is critical for ART success. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Basque Government (GIC15/165) and the University of the Basque Country (UPV/EHU) (EHUA14/17). The authors declare that they have no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

We enriched a specific subpopulation of liver cancer stem cells using multiple surface markers and then cultured the cells with modified chemically defined medium to maintain the cancer stem cell properties for 2 weeks in vitro. This study may provide an effective in vitro model for the study of the biological properties of liver cancer stem cells and their use in targeted drug screening., Liver cancer stem cells (L‐CSCs) are considered to be an important therapeutic target for hepatocellular carcinoma (HCC). This study provides a new in vitro long‐term culture model for a specific subpopulation of L‐CSCs enriched by cell surface markers. We combined CD13, CD133 and EpCAM to selectively enrich L‐CSCs, which we then cultured in modified chemically defined medium. The enriched L‐CSCs exhibited enhanced proliferation, self‐renewal and long‐term clonal maintenance ability as compared with non‐CSCs. Compared with wild‐type hepatocellular carcinoma, the expression of stemness surface markers, oncogenes, drug resistance and tumorigenicity in enriched L‐CSCs was significantly increased. In summary, the subpopulation of L‐CSCs still maintains cancer stem cell‐related phenotypes after 14 days of culture.

The therapeutic index of cytokines in cancer therapy can be increased by targeting strategies based on protein engineering with peptides containing the CNGRC (NGR) motif, a ligand that recognizes CD13-positive tumor vessels. We show here that the targeting domain of recombinant CNGRC-cytokine fusion proteins, such as NGR-TNF (a CNGRC-tumor necrosis factor-α (TNF) conjugate used in clinical studies) and NGR-EMAP-II, undergoes various post-translational modification and degradation reactions that lead to the formation of markedly heterogeneous products. These modifications include N-terminal cysteine acetylation or the formation of various asparagine degradation products, the latter owing to intramolecular interactions of the cysteine α-amino group with asparagine and/or its succinimide derivative. Blocking the cysteine α-amino group with a serine (SCNGRC) reduced both post-translational and degradation reactions. Furthermore, the serine residue reduced the asparagine deamidation rate to isoaspartate (another degradation product) and improved the affinity of NGR for CD13. Accordingly, genetic engineering of NGR-TNF with the N-terminal serine produced a more stable and homogeneous drug (called S-NGR-TNF) with improved antitumor activity in tumor-bearing mice, either when used alone or in combination with chemotherapy. In conclusion, the targeting domain of NGR-cytokine conjugates can undergo various untoward modification and degradation reactions, which can be markedly reduced by fusing a serine to the N-terminus. The SCNGRC peptide may represent a ligand for cytokine delivery to tumors more robust than conventional CNGRC. The S-NGR-TNF conjugate (more stable, homogeneous, and active than NGR-TNF) could be rapidly developed for clinical trials.

Objective. To investigate the effects of Apatinib on the “stemness” of lung cancer cells in vivo and to explore its related mechanisms. Methods. A xenograft model of lung cancer cells A549 was established in nude mice and randomized into a control group (n = 4) and an Apatinib group (n = 4). Tumor tissues were harvested after 2 weeks, and mRNA was extracted to detect changes in stemness-related genes (CD133, EPCAM, CD13, CD90, ALDH1, CD44, CD45, SOX2, NANOG, and OCT4) and Wnt/β-catenin, Hedgehog, and Hippo signal pathways. Results. Compared with the control group, the volume and weight of nude mice treated with Apatinib were different and had statistical significance. Apatinib inhibited the expressions of ABCG2, CD24, ICAM-1, OCT4, and SOX2 and upregulated the expressions of CD44, CD13, and FOXD3. Apatinib treatment also inhibited the Wnt/β-catenin, Hedgehog, and Hippo signaling pathways. Conclusion. Apatinib suppressed the growth of non-small-cell lung cancer cells by repressing the stemness of lung cancer through the inhibition of the Hedgehog, Hippo, and Wnt signaling pathways.

Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

The chemoresistance of hepatocellular carcinoma (HCC) is a serious problem that directly hinders the effect of chemotherapeutic agents. We previously reported that Aminopeptidase N (CD13) inhibition can enhance the cytotoxic efficacy of chemotherapy agents. In the present study, we use liver cancer cells to explore the molecular mechanism accounting for the relationship between CD13 and chemoresistance. We demonstrate that CD13 overexpression activates the P38/heat shock protein 27/cAMP response element-binding protein (CREB) signaling pathway to limit the efficacy of cytotoxic agents. Moreover, blockade of P38 or CREB sensitizes HCC cells to 5-fluorouracil. Then we reveal that CREB binds to the autophagy related 7 (ATG7) promoter to induce autophagy and promote HCC cell chemoresistance. CD13 inhibition also downregulates the expression of ATG7, autophagy, and tumor cell growth in vivo. Overall, the combination a CD13 inhibitor and chemotherapeutic agents may be a potential strategy for overcoming drug resistance in HCC. SIGNIFICANCE STATEMENT: Our study demonstrates that Aminopeptidase N (CD13) promotes hepatocellular carcinoma (HCC) cell chemoresistance via the P38/heat shock protein 27/cAMP response element-binding protein (CREB) pathway. CREB regulates autophagy related 7 transcription and expression to induce autophagy. Our results collectively suggest that CD13 may serve as a potential target for overcoming HCC resistance.

The arms race between entomopathogenic bacteria and their insect hosts is an excellent model for decoding the intricate coevolutionary processes of host-pathogen interaction. Here, we demonstrate that the MAPK signaling pathway is a general switch to trans-regulate differential expression of aminopeptidase N and other midgut genes in an insect host, diamondback moth (Plutella xylostella), thereby countering the virulence effect of Bacillus thuringiensis (Bt) toxins. Moreover, the MAPK cascade is activated and fine-tuned by the crosstalk between two major insect hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH) to elicit an important physiological response (i.e. Bt resistance) without incurring the significant fitness costs often associated with pathogen resistance. Hormones are well known to orchestrate physiological trade-offs in a wide variety of organisms, and our work decodes a hitherto undescribed function of these classic hormones and suggests that hormonal signaling plasticity is a general cross-kingdom strategy to fend off pathogens., Bacillus thuringiensis (Bt) is an important bioinsecticide, but high-level resistance has been rapidly evolving in agricultural pests. Here, Guo et al. show that the MAPK cascade can be activated by enhanced upstream insect hormone signals to counter Bt virulence in the diamondback moth.

Objectives To screen and identify ideal leading compounds from a drug library (ZINC15 database) with potential inhibition of aminopeptidase N(CD13) to contribute to medication design and development. Results Two novel natural compounds, ZINC000000895551 and ZINC000014820583, from the ZINC15 database were found to have a higher binding affinity and more favorable interaction energy binding with CD13 with less rodent carcinogenicity, Ames mutagenicity, and non-inhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis suggested that the 2 complexes, ZINC000000895551-CD13 and ZINC000014820583-CD13, have favorable potential energy, and exist stably in the natural circumstances. Conclusion This study discovered that ZINC000000895551 and ZINC000014820583 were ideal leading compounds to be inhibitions targeting to CD13. These compounds were selected as safe drug candidates as CD13 target medication design and improvement. Materials and method Potential inhibitors of CD13 were identified using a series of computer-aided structural and chemical virtual screening techniques. Structure-based virtual screening was carried out to calculate LibDock scores, followed by analyzing their absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to reveal binding affinity between the selected compounds and CD13. Molecular dynamics simulation was applied to evaluate stability of the ligand-CD13 complex under natural environment.

Hepatocellular carcinoma (HCC) is one of the most prevalent human malignancies worldwide and has high morbidity and mortality. Elucidating the molecular mechanisms underlying HCC recurrence and metastasis is critical to identify new therapeutic targets. This study aimed to determine the roles of aminopeptidase N (APN, also known as CD13) in HCC proliferation and metastasis and its underlying mechanisms. We detected APN expression in clinical samples and HCC cell lines using immunohistochemistry, flow cytometry, real-time PCR, and enzyme activity assays. The effects of APN on HCC metastasis and proliferation were verified in both in vitro and in vivo models. RNA-seq, phosphoproteomic, western blot, point mutation, co-immunoprecipitation, and proximity ligation assays were performed to reveal the potential mechanisms. We found that APN was frequently upregulated in HCC tumor tissues and high-metastatic cell lines. Knockout of APN inhibited HCC cell metastasis and proliferation in vitro and in vivo. Functional studies suggested that a loss of APN impedes the ERK signaling pathway in HCC cells. Mechanistically, we found that APN might mediate the phosphorylation at serine 31 of BCKDK (BCKDKS31), promote BCKDK interacting with ERK1/2 and phosphorylating it, thereby activating the ERK signaling pathway in HCC cells. Collectively, our findings indicate that APN mediates the phosphorylation of BCKDKS31 and activates its downstream pathway to promote HCC proliferation and metastasis. Therefore, the APN/BCKDK/ERK axis may serve as a new therapeutic target for HCC therapy, and these findings may be helpful to identify new biomarkers in HCC progression.

PurposeCancer stem cells (CSCs) are naturally resistant to chemotherapy, explaining why tumor relapse frequently occurs after initial regression upon administration of chemotherapeutic agents in most cases. A CSC population characterized by CD13 expression has been identified in hepatocellular carcinoma (HCC). In the current study, we aimed to clarify the molecular mechanism by which it escapes conventional therapies. Materials and MethodsHere, we used flow cytometry to examine the percentage of CD13+ CSCs in HepG2 and HuH7 cells after chemotherapy. Using in vitro isotope labeling technique, we compared metabolic pathways between CD13+ and CD13– subpopulations. Using co-immunoprecipitation and western blotting, we determined the target expressions in protein levels under different conditions. We also performed immunohistochemistry to detect the target proteins under different conditions. Animal models were constructed to verify the potential role of tyrosine metabolism in post-chemotherapeutic relapse in vivo.ResultsWe observed that quiescent CD13+ CSCs are enriched after chemotherapy in HCCs, and serve as a reservoir for recurrence. Mechanistically, CD13+ CSCs were dependent on aerobic metabolism of tyrosine rather than glucose as energy source. Tyrosine metabolism also generated nuclear acetyl-CoA to acetylate and stabilize Foxd3, thereby allowing CD13+ CSCs cells to sustain quiescence and resistance to chemotherapeutic agents.ConclusionThese findings encourage further exploration of eliminating CD13+ cells by targeting specific metabolic pathways to prevent recurrence in HCCs.

Using a minimalist approach, an 11-residue peptide (Peptide 1) tagged with rhodamine fluorophore was designed and synthesized for selective detection of cancer cells. Peptide 1 contains RGD and NGR motifs to bind, respectively, integrins and aminopeptidase CD13, which are over expressed in cancer cells. Surface tension measurements revealed that peptide 1 possess surface-active property owing to the overall hydrophobicity and cationic nature of the peptide. Peptide 1 displays cancer cell-selective binding at ≤5.0 µM concentrations, while peptide 2 (randomized sequence of 1) shows non-selective binding to normal and cancer cells. Fluorescence microscopy and FACS analysis demonstrated the intracellular localization of peptide 1 in three different cancer cell lines, confirming the role of RGD and NGR motifs. Cytotoxicity assay exhibited the viability of normal and cancer cells up to 100 µM concentrations of peptide 1. Steady-state fluorescence measurements disclosed the preferential interactions of the peptide 1 with anionic POPC/POPG bilayers rather than with zwitterionic POPC lipid bilayers. Circular dichroism studies showed minimal changes in the secondary structure of peptide 1 upon binding with the anionic lipid bilayers. Peptide 1 is largely unordered, non-toxic, and useful for identification of cancer cells. Peptide 1 provides a template for designing drug-loaded peptides for targeted delivery into cancer cells.

Squamous cell/adenosquamous carcinoma (SC/ASC) is a rarely identified form of gallbladder cancer with poorly understood clinical features. As such, there is an urgent need to identify novel prognostic biomarkers for such gallbladder SC/ASC cases, and for gallbladder adenocarcinomas (ACs). The levels of ACO2 and ANPEP proteins were assessed via an EnVision-based immunohistochemical approach using 46 SC/ASC and 80 AC patient samples. There was a marked reduction in levels of ACO2 and ANPEP in gallbladder AC relative to normal adjacent tissue or benign gallbladder lesions. The was a significant correlation between lack of ACO2 and ANPEP and larger tumors, higher tumor-node-metastasis (TNM) staging, invasion, metastasis to regional lymph nodes, and ineligibility for surgical resection in both SC/ASC and AC tumor samples. Kaplan–Meier survival analyses further confirmed a relationship between ACO2 and ANPEP negativity and decreased overall survival in patients with these diseases (p

Nonspecific adsorption is of great concern for electrochemical biosensors performing in complex biological media, and various antifouling materials have been introduced into the sensing interfaces to improve the antifouling capability of different biosensors. However, for most of the biosensors with antifouling materials and sensing probes coexisting in the sensing interfaces, either the antifouling materials will impair the sensing performances or the sensing probes will affect the antifouling ability. Herein, a facile and efficient antifouling biosensor was developed based on a newly designed three-in-one peptide with anchoring, antifouling, and recognizing capabilities. One end of the designed peptide is a unique anchoring part that is rich in amine groups, and this part can be anchored to the poly(3,4-ethylenedioxythiophene) (PEDOT)-citrate film electrodeposited on a glassy carbon electrode. The other end of the peptide is a recognizing part that can specifically bind to the aminopeptidase N (APN) and human hepatocellular carcinoma cells (HepG2 cells). Meanwhile, the middle part of the peptide, together with the anchoring part, was designed to be antifouling. With this designed multifunctional peptide, highly sensitive and low-fouling biosensors capable of assaying target APN and HepG2 cells in complex biological media can be easily prepared, with detection limits of 0.4 ng·mL

Background: Emerging evidence suggests that secretome of mesenchymal stem cells has many anti-inflammatory and regenerative properties, which makes it a suitable candidate for the treatment of autoimmune and degenerative diseases. Dipeptidyl Peptidase-IV (DPP-IV)/CD26 and Aminopeptidase N (APN)/CD13 are ubiquitous ecto-enzymes which can digest various substrates including some chemokines and neuropeptides that are involved in inflammatory conditions. Objective: To evaluate the enzymatic activity of DPP-IV/CD26 and APN/CD13 in MSC conditioned media (MSC-CM). Methods: The MSCs were isolated from the mouse’s abdominal adipose tissues and were cultured without or with preconditioning by adding 2 µg/mL phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). The levels of interleukin-10 (IL-10), nitric oxide (NO), as well as the enzymatic activities of DPP-IV/CD26 and APN/CD13 were measured in MSC-CM. Results: The level of IL-10 and the enzyme activity of APN/CD13 did not show any changes in the MSC-CM of stimulated and non-stimulated cells. However, NO production was increased after treatment by LPS or PMA; nevertheless, the DPP-IV/CD26 activity was decreased in MSC-CM merely following the stimulation of cells with LPS. Conclusion: Our results indicated that MSC‐secretome had DPP-IV/CD26 and APN/CD13 activity. The DPP-IV/CD26 activity was decreased following stimulation of MSCs by toll-like receptor 4 agonist. Further studies are needed to reveal the possible contribution of DPP-IV/CD26 and APN/CD13 in the anti-inflammatory functions of MSC-CM.