Intracellular Phosphatase Research Articles

3D-printing of dipyridamole/thermoplastic polyurethane materials for bone regeneration.

Tissue engineering combines biology and engineering to develop constructs for repairing or replacing damaged tissues. Over the last few years, this field has seen significant advancements, particularly in bone tissue engineering. 3D printing has revolutionised this field, allowing the fabrication of patient- or defect-specific scaffolds to enhance bone regeneration, thus providing a personalised approach that offers unique control over the shape, size, and structure of 3D-printed constructs. Accordingly, thermoplastic polyurethane (TPU)-based 3D-printed scaffolds loaded with dipyridamole (DIP) were manufactured to evaluate their in vitro osteogenic capacity. The fabricated DIP-loaded TPU-based scaffolds were fully characterised, and their physical and mechanical properties analysed. Moreover, the DIP release profile, the biocompatibility of scaffolds with murine calvaria-derived pre-osteoblastic cells, and the intracellular alkaline phosphatase (ALP) assay to verify osteogenic ability were evaluated. The results suggested that these materials offered an attractive option for preparing bone scaffolds due to their mechanical properties. Indeed, the addition of DIP in concentrations up to 10% did not influence the compression modulus. Moreover, DIP-loaded scaffolds containing the highest DIP cargo (10% w/w) were able to provide sustained drug release for up to 30 days. Furthermore, cell viability, proliferation, and osteogenesis of MC3T3-E1 cells were significantly increased with the highest DIP cargo (10% w/w) compared to the control samples. These promising results suggest that DIP-loaded TPU-based scaffolds may enhance bone regeneration. Combined with the flexibility of 3D printing, this approach has the potential to enable the creation of customized scaffolds tailored to patients' needs at the point of care in the future.

Orientation-dependent CD45 inhibition with viral and engineered ligands.

CD45 is a cell surface phosphatase that shapes the T cell receptor signaling threshold but does not have a known ligand. A family of adenovirus proteins, including E3/49K, exploits CD45 to evade immunity by binding to the extracellular domain of CD45, resulting in the suppression of T cell signaling. We determined the cryo-EM structure of this complex and found that the E3/49K protein is composed of three immunoglobulin domains assembled as "beads on a string" that compel CD45 into a closely abutted dimer by cross-linking the CD45 D3 domain, leading to steric inhibition of its intracellular phosphatase activity. Inspired by the E3/49K mechanism, we engineered CD45 surrogate ligands that can fine-tune T cell activation by dimerizing CD45 into different orientations and proximities. The adenovirus E3/49K protein has taught us that, despite a lack of a known ligand, CD45 activity can be modulated by extracellular dimerizing ligands that perturb its phosphatase activity and alter T cell responses.

IInduction of G2/M Phase Block and Apoptosis by Kaempferol in Human Sarcoma Cells

malignant cells has interested us to seek new strategies for the treatment of sarcoma. Flavonoids have been recently claimed to exert anti-cancer effects. We focused on the flavonoid kaempferol, to determine whether it induces apoptosis of sarcoma cells in vitro. Methods: We examined cell growth using a CCK-8 assay, morphology, cell cycle analysis, and the apoptosis or differentiation effects of kaempferol on cultured human osteosarcoma MG-63 cells. All experiments were performed at least three times in duplicates. The ANOVA and independent sample t-test were used for the comparison of the continuous variables Results: Kaempferol (50 μM), adversely affected the proliferation of MG-63 cells. Anti-proliferative action of kaempferol appeared to be linked to apoptotic cell death based on the morphological changes depicting nuclear fragmentation. Cell cycle analysis demonstrated the induction of G2/M phase arrest and an enhanced sub-G1 population. However, the intracellular alkaline phosphatase (ALP) activity was observed to have been stimulated. This apoptosis might be associated with cell differentiation of the cells in the early stage. Conclusions: This study demonstrates that kaempferol inhibited the growth of MG-63 cells in vitro. G2/M phase arrest and induction of intracellular ALP activities in the early phase are observed. These findings indicate that kaempferol may present as an additional pharmacological tool for the treatment of human sarcoma cells.

Antimicrobial effects and metabolomics analysis of the cell-free supernatant of Limosilactobacillus fermentum

In this study, Listeria monocytogenes and Escherichia coli were selected as reference bacteria, and the inhibitory effects and mechanisms of cell-free supernatants (CFS) of Limosilactobacillus fermentum 733 on its free-living bacteria and biofilm were investigated in depth. The findings demonstrated that L. fermentum 733 CFS displayed notable suppressive effects on L. monocytogenes and E. coli, which were caused by the loss of the cell membranes' structural integrity. This disturbance resulted in the release of intracellular adenosine triphosphate (ATP), alkaline phosphatase (ALP), and reactive oxygen species (ROS). Scanning electron microscopy and laser scanning confocal microscopy showed that L. fermentum 733 CFS disrupted L. monocytogenes and E. coli biofilm structure. L. fermentum 733 CFS inhibited partial population sensing and biofilm-associated gene expression of L. monocytogenes and E. coli, shown by fluorescence quantitative PCR (qPCR). Metabolomics analysis showed that L. fermentum 733 CFS strongly inhibited L. monocytogenes and E. coli by the disruption of both cellular structure and intracellular. These results offer novel perspectives and a theoretical foundation for the prevention of lactic acid bacteria (LAB) infections caused by L. monocytogenes and E. coli.

Helicobacter pylori East Asian type CagA hijacks more SHIP2 by its EPIYA-D motif to potentiate the oncogenicity

ABSTRACT CagA is a significant oncogenic factor injected into host cells by Helicobacter pylori, which is divided into two subtypes: East Asian type (CagAE), characterized by the EPIYA-D motif, and western type (CagAW), harboring the EPIYA-C motif. CagAE has been reported to have higher carcinogenicity than CagAW, although the underlying reason is not fully understood. SHIP2 is an intracellular phosphatase that can be recruited by CagA to perturb the homeostasis of intracellular signaling pathways. In this study, we found that SHIP2 contributes to the higher oncogenicity of CagAE. Co-Immunoprecipitation and Pull-down assays showed that CagAE bind more SHIP2 than CagAW. Immunofluorescence staining showed that a higher amount of SHIP2 recruited by CagAE to the plasma membrane catalyzes the conversion of PI(3,4,5)P3 into PI(3,4)P2. This alteration causes higher activation of Akt signaling, which results in enhanced IL-8 secretion, migration, and invasion of the infected cells. SPR analysis showed that this stronger interaction between CagAE and SHIP2 stems from the higher affinity between the EPIYA-D motif of CagAE and the SH2 domain of SHIP2. Structural analysis revealed the crucial role of the Phe residue at the Y + 5 position in EPIYA-D. After mutating Phe of CagAE into Asp (the corresponding residue in the EPIYA-C motif) or Ala, the activation of downstream Akt signaling was reduced and the malignant transformation of infected cells was alleviated. These findings revealed that CagAE hijacks SHIP2 through its EPIYA-D motif to enhance its carcinogenicity, which provides a better understanding of the higher oncogenic risk of H. pylori CagAE.

Isolation, purification, and antiosteoporosis activity of donkey bone collagen from discarded bone and its antioxidant peptides

Oxidative stress plays a crucial role in the development of osteoporosis. In this study, it was observed that donkey bone collagen (DC) at a concentration of 500 μg/mL scavenged 17.89 % of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals, indicating its antioxidant properties. Additionally, when an oxidative damage osteoblast model was created using H2O2, 100 μg/mL DC demonstrated the ability to enhance cell survival by 27.31 %. Furthermore, 50 μg/mL DC increased the intracellular differentiation marker alkaline phosphatase (ALP) level by 62.65 %. Additionally, the study revealed that DC significantly increased the expression of osteoporosis-related factors in serum and effectively restored the abnormal structure of spongy bone in mice osteoporosis model. Peptides (GGWFL, ANLGPA, and GWFK) isolated from DC through gastrointestinal digestion and subsequent enzymatic purification in vitro demonstrated the ability to safeguard osteoblasts from H2O2-induced damage by reducing intracellular reactive oxygen species (ROS). This protection resulted in enhanced cell survival and promoted osteoblast differentiation. This investigation underscores that DC can shield oxidative damage osteoblast model from oxidative stress, ameliorate osteoporosis, and enhance bone density in mice osteoporosis model. These findings suggest various DC applications in the food and medicine industries.

Antimicrobial Activity and Mechanisms of Walnut Green Husk Extract.

Walnut green husks (WGHs), by-products of walnut production, are believed to possess antimicrobial properties, making them a potential alternative to antibiotics. In this study, the antibacterial activities of three extracts, derived from WGH, against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were investigated, and the antibacterial mechanisms of an anhydrous ethanol extract of WGH (WGHa) were examined. The results showed that WGHa exhibited inhibitory effects on all tested bacteria. The ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis revealed that the major active compounds present in WGHa were terpenoids, phenols, and flavonoids. Treatment with WGHa resulted in the leakage of intracellular ions and alkaline phosphatase; a reduction in intracellular ATP content, ATPase activity, and nucleic acid content; as well as cellular metabolic viability. The transmission electron microscopy images showed varying degrees of cell deformation and membrane damage following WGHa treatment. The transcriptome sequencing and differentially expressed gene enrichment analyses revealed an up-regulation in pathways associated with RNA degradation, translation, protein export, and oxidative phosphorylation. Conversely, pathways involved in cell movement and localization, as well as cell wall organization and carbohydrate transport, were found to be down-regulated. These findings suggest that WGHa alters cell membrane permeability and causes damage to the cell wall. Additionally, WGHa interferes with cellular energy metabolism, compromises RNA integrity, and induces DNA replication stress, consequently inhibiting the normal growth and proliferation of bacteria. These findings unveiled the antimicrobial mechanisms of WGHa, highlighting its potential application as an antibiotic alternative.

Synergistic effect of FGF-2 and TGF-β1 on the mineralization of human umbilical cord perivascular cells

ObjectiveHuman umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-β1) on HUCPVC mineralization. DesignWe prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-β1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. ResultsFGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. ConclusionWe found that FGF-2 synergizes with TGF-β1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

Potential Use of Nucleic Acids as a Preceramic Polymer to Synthesize Nanodiamond-Embedded Phosphate Glass for Hard Tissue Engineering.

In recent years, nucleic acid has emerged as a versatile molecule that has been strategically used in material synthesis and biomedical applications. Keeping in mind the presence of the phosphate group, a glass former in the nucleic acids, we synthesized a transparent glass-like material by the thermal treatment of nucleic acids (DNA and RNA) at 900 °C at atmospheric pressure. Characterization of this material by transmission electron microscopy, X-ray photoelectron spectroscopy, and confocal fluorescence microscopy suggested the presence of in situ-formed nanodiamonds within the phosphate glass matrix. The molecular structure of glass investigated by X-ray photoelectron and infrared spectroscopy indicated a nearly equal proportion of metaphosphates and smaller phosphate units (pyro- and ortho-phosphate) that form the phosphate glass matrix. Thereafter, in vitro biological experiments showed that the nucleic acid-derived glass was non-toxic and cytocompatible, enhanced extracellular matrix secretion, and increased intracellular alkaline phosphatase activity, with potential application in hard tissue engineering. Our work offers insights into nanodiamond synthesis at atmospheric pressure and proves that nucleic acids could be used as a precursor to making an innovative glass-ceramic biomaterial.

Targeted inhibition of PTPN22 is a novel approach to alleviate osteogenic responses in aortic valve interstitial cells and aortic valve lesions in mice

BackgroundCalcific aortic valve disease (CAVD) is the most prevalent valvular disease and has high morbidity and mortality. CAVD is characterized by complex pathophysiological processes, including inflammation-induced osteoblastic differentiation in aortic valve interstitial cells (AVICs). Novel anti-CAVD agents are urgently needed. Protein tyrosine phosphatase nonreceptor type 22 (PTPN22), an intracellular nonreceptor-like protein tyrosine phosphatase, is involved in several chronic inflammatory diseases, including rheumatoid arthritis and diabetes. However, it is unclear whether PTPN22 is involved in the pathogenesis of CAVD.MethodsWe obtained the aortic valve tissue from human and cultured AVICs from aortic valve. We established CAVD mice model by wire injury. Transcriptome sequencing, western bolt, qPCR, and immunofluorescence were performed to elucidate the molecular mechanisms.ResultsHere, we determined that PTPN22 expression was upregulated in calcific aortic valve tissue, AVICs treated with osteogenic medium, and a mouse model of CAVD. In vitro, overexpression of PTPN22 induced osteogenic responses, whereas siRNA-mediated PTPN22 knockdown abolished osteogenic responses and mitochondrial stress in the presence of osteogenic medium. In vivo, PTPN22 ablation ameliorated aortic valve lesions in a wire injury-induced CAVD mouse model, validating the pathogenic role of PTPN22 in CAVD. Additionally, we discovered a novel compound, 13-hydroxypiericidin A 10-O-α-D-glucose (1 → 6)-β-D-glucoside (S18), in a marine-derived Streptomyces strain that bound to PTPN22 with high affinity and acted as a novel inhibitor. Incubation with S18 suppressed osteogenic responses and mitochondrial stress in human AVICs induced by osteogenic medium. In mice with aortic valve injury, S18 administration markedly alleviated aortic valve lesions.ConclusionPTPN22 plays an essential role in the progression of CAVD, and inhibition of PTPN22 with S18 is a novel option for the further development of potent anti-CAVD drugs.Graphical Therapeutic inhibition of PTPN22 retards aortic valve calcification through modulating mitochondrial dysfunction in AVICs.

The role of manganese in morphogenesis and pathogenesis of the opportunistic fungal pathogen Candida albicans.

Metals such as Fe, Cu, Zn, and Mn are essential trace nutrients for all kingdoms of life, including microbial pathogens and their hosts. During infection, the mammalian host attempts to starve invading microbes of these micronutrients through responses collectively known as nutritional immunity. Nutritional immunity for Zn, Fe and Cu has been well documented for fungal infections; however Mn handling at the host-fungal pathogen interface remains largely unexplored. This work establishes the foundation of fungal resistance against Mn associated nutritional immunity through the characterization of NRAMP divalent metal transporters in the opportunistic fungal pathogen, Candida albicans. Here, we identify C. albicans Smf12 and Smf13 as two NRAMP transporters required for cellular Mn accumulation. Single or combined smf12Δ/Δ and smf13Δ/Δ mutations result in a 10-80 fold reduction in cellular Mn with an additive effect of double mutations and no losses in cellular Cu, Fe or Zn. As a result of low cellular Mn, the mutants exhibit impaired activity of mitochondrial Mn-superoxide dismutase 2 (Sod2) and cytosolic Mn-Sod3 but no defects in cytosolic Cu/Zn-Sod1 activity. Mn is also required for activity of Golgi mannosyltransferases, and smf12Δ/Δ and smf13Δ/Δ mutants show a dramatic loss in cell surface phosphomannan and in glycosylation of proteins, including an intracellular acid phosphatase and a cell wall Cu-only Sod5 that is key for oxidative stress resistance. Importantly, smf12Δ/Δ and smf13Δ/Δ mutants are defective in formation of hyphal filaments, a deficiency rescuable by supplemental Mn. In a disseminated mouse model for candidiasis where kidney is the primary target tissue, we find a marked loss in total kidney Mn during fungal invasion, implying host restriction of Mn. In this model, smf12Δ/Δ and smf13Δ/Δ C. albicans mutants displayed a significant loss in virulence. These studies establish a role for Mn in Candida pathogenesis.

High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha.

The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we now provide the first experimental evidence for its site-specific glycosylation pattern. This includes the occurrence of O-glycan at the N-glycosylation sequon among the more than 30 O-glycosylation sites confidently identified beside the 7 N-glycosylation sites. The closely spaced N- and O-glycans appear to have mutually limited the extent of further galactosylation and sialylation. An immature smaller form of full-length PTPRA was found to be deficient in O-glycosylation, most likely due to failure to transit the Golgi. N-glycosylation, on the other hand, is dispensable for cell surface expression and contributes less than the extensive O-glycosylation to the overall solution structure of the ectodomain. The glycosylation information is combined with the overall structural features of the ectodomain derived from small-angle X-ray scattering and high-speed atomic force microscopy monitoring to establish a dynamic structural model of the densely glycosylated PTPRA ectodomain. The observed high structural flexibility, as manifested by continuous transitioning from fully to partially extended and fold-back conformations, suggests that the receptor-type phosphatase is anchored to the membrane and kept mostly at a monomeric state through an ectodomain shaped and fully shielded by glycosylation.

The Synergistic Antimicrobial Effect and Mechanism of Nisin and Oxacillin against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for skin and soft tissue infections with multi-resistance to many antibiotics. It is thus imperative to explore alternative antimicrobial treatments to ensure future treatment options. Nisin (NIS), an antibacterial peptide produced by Lactococcus lactis, was selected to combine with Oxacillin (OX), to evaluate the antimicrobial effect and potential mechanism against MRSA. The synergistic antimicrobial effect of OX and NIS was verified by Minimal Inhibitory Concentration (MIC) assays, checkerboard analysis, time-kill curve, biofilm producing ability, and mice skin infection model in vivo. For the potential synergistic antimicrobial mechanism, the microstructure and integrity change of MRSA cells were determined by Scanning and Transmission Electron Microscope (SEM and TEM), intracellular alkaline phosphatase activity and propidium iodide staining were assayed; And transcription of mecA, main gene of MRSA resistant to OX, were detected by qRT-PCR. The results showed NIS could restore the sensitivity of MRSA to OX and inhibit biofilm production; OX + NIS can make MRSA cell deform; NIS may recover OX sensitivity by inhibiting the transcription of mecA. In vivo, mice skin infection models indicate that OX + NIS can substantially alleviate MRSA infections. As a safe commercially available biological compound, NIS and the combination of antibiotics are worth developing as new anti-MRSA biomaterials.

THE FEATURES AND ROLE OF SHP2 PROTEIN IN POSTNATAL MUSCLE DEVELOPMENT

SHP-2 (encoded by PTPN11) is a ubiquitously expressed protein tyrosine phosphatase required for signal transduction by multiple different cell surface receptors. Humans with carry germline SHP-2 mutations develop Noonan syndrome or LEOPARD syndrome, which are characterized by cardiovascular, neurological and skeletal abnormalities.Shp2 is an important signaling agent for growth factors and cytokines. To investigate the Ptpn11 in postnatal myogenesis of mice  we analyzed using immunohistochemistry stem cell markers: Pax 7, Myo D, Myf 5 and Myo G in muscle cells isolated from wild type and Shp2 knockout mice. We used a conditional SHP-2 mouse mutant in which loss of expression of SHP-2 was induced in multiple tissues in response to drug administration. Our findings indicate that all these markers gradually decreased from birth to day 14 in mouse muscle cells. Here we show that Shp2, an intracellular tyrosine phosphatase with two SH2 domains, plays a critical role in mouse muscle development after birth. Our data demonstrate a molecular difference in the control and Sh2 knockout  postnatal myogenic stem cells, and assign to Ptpn11 signaling a key function in satellite cell activity. These findings illustrate an essential role for Shp-2 in muscle growth and remodeling in adults, and reveal some of the cellular and molecular mechanisms involved. The model is predicted to be of further use in understanding how Shp-2 regulates muscle morphogenesis, which could lead to the development of novel therapies for the treatment of malformations in human patients with SHP-2 mutations.

SHP-1 Protein Tyrosine Phosphatase Affects Early Postnatal Bone Development in Mice.

The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an intracellular tyrosine phosphatase that plays a negative regulatory role in immune cell signaling. Absent or diminished SHP-1 catalytic activity results in reduced bone mass with enhanced bone resorption. Here, we sought to investigate if Shp1 overexpression leads to increased bone mass and improved mechanical properties. Male and female wildtype (WT) and SHP1-transgenic (Tg) mice at 28, 56, and 84days of age were compared. We applied microcomputed tomography to assess femoral cortical bone geometry and trabecular architecture and 3-point mechanical bending to assess mid-diaphyseal structural and estimated material properties. Serum OPG, RANKL, P1NP, and CTX-1 concentrations were measured by enzyme-linked immunoassay. The majority of transgene effects were restricted to the 28-day-old mice. Trabecular bone volume per total volume, trabecular number, and connectivity density were greater in 28-day-old female SHP1-Tg mice when compared to WTs. SHP1-Tg female mice showed increased total and medullary areas, with no difference in cortical area and thickness. Cortical tissue mineral density was strongly genotype-dependent. Failure load, yield load, ultimate stress, and yield stress were all lower in 28-day-old SHP1-Tg females. In 28-day-old SHP1-Tg females, circulating levels of OPG and P1NP were higher and RANKL levels were lower than WT controls. Our study demonstrates a role for SHP-1 in early postnatal bone development; SHP-1 overexpression negatively impacted whole bone strength and material properties in females.

Barbaloin Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells: Involvement of Wnt/β-catenin Signaling Pathway.

Barbaloin, found in Aloe vera, exerts broad pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer. This study aims to investigate the effects of barbaloin on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Osteogenic induction of hBMSCs was performed in the presence or absence of barbaloin. Cell viability was determined by using the CCK-8 assay. The characteristic of hBMSCs was identified by using flow cytometry. Intracellular alkaline phosphatase (ALP) staining was performed to evaluate the ALP activity in hBMSCs. Alizarin Red S staining was performed to evaluate the matrix mineralization. The mRNA and protein levels of target genes were determined using qRT-PCR and western blotting, respectively. Treatment with barbaloin (10 and 20 μg/mL) significantly increased cell viability of hBMSCs after 72 hours. In addition, treatment with barbaloin increased the mRNA expression levels of ALP and its activities. Treatment with barbaloin also increased matrix mineralization and the mRNA and protein levels of late-differentiated osteoblast marker genes BMP2, RUNX2, and SP7 in hBMSCs. The underlying mechanisms revealed that barbaloin increased the protein expressions of Wnt/β-catenin pathway-related biomarkers. Barbaloin promotes osteogenic differentiation of hBMSCs by the regulation of the Wnt/β-catenin signaling pathway.

Epitope Editing in Hematopoietic Cells Enables CD45-Directed Immune Therapy

Background: Targeted immunotherapies such as CAR-T cells are limited to several well-known lineage markers for which the on-target off-tumor toxicities are clinically tolerated. CART cells are or have been developed individually for different hematologic malignancies based on the relevant lineage antigens (e.g. CD19 for B cell, BCMA for myeloma, CD7 for T cells, CD33 for AML). By targeting a pan-hematologic antigen, a single "drug" could be used for all hematologic indications, thereby accelerating clinical research. CD45 is expressed on most hematologic malignancies and is, therefore, an attractive target for pan-hematologic cancer immunotherapy. However, targeting CD45 with CART cells is limited by (i) expression of CD45 on hematopoietic stem cells (HSC) and their progeny (ii) T cell (including CART cell) fratricide, and (iii) the central function of CD45 in immune cell signaling. To render CD45-specific immunotherapy a potentially viable pan-hematologic CART option we built upon prior work in which we had deleted the pan-myeloid antigen CD33 in HSC. In the current work, however, we sought to modify the relevant CD45 epitope in both T-cells and HSCs in such a way that it is unrecognized by the anti-CD45 CAR-T cells while maintaining its surface expression and intracellular phosphatase function (Figure 1A). Methods: After mapping the amino acid residues on the extracellular domain of human CD45 that are required for binding by three different anti-CD45 scFv's, we devised a CRISPR base editing (BE) approach to install a single mutation at the identified epitope. This allowed us to generate a CD45 molecule that is "invisible" to CAR-T cells made from these anti-CD45 scFv's, thus rendering them resistant to fratricide or suicide. Controls were CD45WT (non-edited) or CD45KO (cells in which CD45 expression was deleted by Cas9-based indel formation). We tested the efficacy of the various CART against leukemia specimens in vitro and in vivo. We then installed the anti-CD45 resistant mutation in primary human CD34+ HSC and assessed the functional consequences of CD45edited hematopoiesis. Results: T-cells transduced with anti-CD45 CARs (CART-45) show substantially impaired ex vivo expansion compared to CD19-directed CAR-T cells due to fratricide, as expected. However, CART-45 cells in which the targeted epitope was altered through adenine base editing (CART-45edited) were resistant to fratricide, enabling the expansion of CD45-directed CAR-T cells comparable to that of CD45KO or CAR19 control T cells. Unlike CD45KO CAR-T cells, CD45editedcells retained CD45 phosphatase expression which is crucial for CAR-T cell function in xenograft tumor models. In mice engrafted with a CD19+ B-ALL cell line, CD45edited CART-19 function was equivalent to CD45WT CART-19, and superior to CD45KO CART-19 (Mock vs CD45KO, p = <0.001); Mock vs CD45edited, p=0.97. These results indicate that CD45 is needed for the proper function of CART cells. We then tested CART-45 in a patient-derived xenograft (PDX) model of human AML and found that CART-45 expanded in the peripheral blood and effectively eliminated tumor cells with no detectable blasts in peripheral blood or bone marrow 4 weeks post CART-45 injection. To protect the hematopoietic system from CD45-directed on-target/off-tumor toxicity, we base-edited the targeted CD45 epitope in human HSCs, achieving the desired Tyr->Cys conversion in 62% of human HSCs (median, 51%-91% range). CD45edited HSC maintained equivalent colony formation in vitro when compared to CD45WT HSC. Crucially, CD45edited HSC transplanted into immunodeficient mice led to equivalent hematopoietic engraftment and differentiation when compared with CD45WT cells, whereas CD45KO hematopoiesis was lost over time (Figure 1B). Conclusions: Here we developed a novel platform that combines pan-hematologic anti-CD45 CAR-T cells with an engineered hematopoietic stem cell transplant endowed with selective resistance to CD45-specific immunotherapy while maintaining CD45 expression and function. The CD45 phosphatase domain is necessary and sufficient to maintain hematopoiesis and T cell function in humanized mice. This platform creates a cancer-specific antigen in all residual host hematopoietic cells, paving the way to profound myeloablation. This platform could be used to treat most hematopoietic malignancies and perhaps other diseases requiring profound hematopoietic ablation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antibacterial Effect and Possible Mechanism of Salicylic Acid Microcapsules against Escherichia coli and Staphylococcus aureus.

Microcapsules serve as a feasible formulation to load phenolic substances such as salicylic acid, a natural and safe antimicrobial agent. However, the antibacterial efficacy of salicylic acid microcapsules (SAMs) remains to be elucidated. Here, salicylic acid/β-cyclodextrin inclusion microcapsules were subjected to systematic antibacterial assays and preliminary antibacterial mechanism tests using Escherichia coli and Staphylococcus aureus as target organisms. It was found that the core-shell rhomboid-shaped SAMs had a smooth surface. SAMs exhibited a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 4 mg/mL against both bacteria. In the growth inhibition assay, 1/4 × MIC, 1/2 × MIC, and 1 × MIC of SAMs effectively retarded bacterial growth, and this effect was more prominent with the rise in the level of SAMs. Practically, SAMs possessed a rapid bactericidal effect at the 1 × MIC level with a reduction of more than 99.9% bacterial population within 10 min. A pronounced sterilization activity against E. coli and S. aureus was also observed when SAMs were embedded into hand sanitizers as antimicrobial agents. Moreover, exposure of both bacteria to SAMs resulted in the leakage of intracellular alkaline phosphatases and macromolecular substances (nucleic acids and proteins), which indicated the disruption of bacterial cell walls and cell membranes. In conclusion, SAMs were able to inactivate E. coli and S. aureus both in vitro and in situ, highlighting the promising utilization of this formulation for antimicrobial purposes in the area of food safety and public health.

Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

BackgroundGemcitabine (GEM) is one of the first-line chemotherapies for bladder cancer (BC), but the GEMs cannot recognize cancer cells and have a low long-term response rate and high recurrence rate with side effects during the treatment of BC. Targeted transport of GEMs to mediate cytotoxicity to tumor and avoid the systemic side effects remains a challenge in the treatment of BC.MethodsBased on a firstly confirmed biomarker in BC-protein tyrosine kinase 7 (PTK7), which is overexpressed on the cell membrane surface in BC cells, a novel targeting system protein tyrosine kinase 7 aptamer-Gemcitabine conjugate (PTK7-GEMs) was designed and synthesized using a specific PTK7 aptamer and GEM through auto-synthesis method to deliver GEM against BC. In addition, the antitumor effects and safety evaluation of PTK7-GEMs was assessed with a series of in vitro and in vivo assays.ResultsPTK7-GEMs can specifically bind and enter to BC cells dependent on the expression levels of PTK7 and via the macropinocytosis pathway, which induced cytotoxicity after GEM cleavage from PTK7-GEMs respond to the intracellular phosphatase. Moreover, PTK7-GEMs showed stronger anti-tumor efficacy and excellent biosafety in three types of tumor xenograft mice models.ConclusionThese results demonstrated that PTK7-GEMs is a successful targeted aptamer-drug conjugates strategy (APDCs) to treat BC, which will provide new directions for the precision treatment of BC in the field of biomarker-oriented tumor targeted therapy.

Maternal high-fat diet promotes calcified atherosclerotic plaque formation in adult offspring by enhancing transformation of VSMCs to osteochondrocytic-like phenotype

AimMaternal high-fat diet (HFD) is associated with the development of cardiovascular disease (CVD) in adult offspring. Atherosclerotic vascular calcification is well documented in patients with CVD. We examined the effect of maternal HFD on calcified plaque formation. Methods and resultsSeven-week-old female apo-E−/− mice (C57BL6/J) were nourished either an HFD or a normal diet (ND) a week before mating, and during gestation and lactation. Offspring of both the groups were fed a high-cholesterol diet (HCD) from 8 weeks of age. Osteogenic activity of the thoracic aorta, assessed using an ex vivo imaging system, was significantly increased after 3 months of HCD in male offspring of HFD-fed dams (O-HFD) as compared with those of ND-fed dams (O-ND). Alizarin-red-positive area in the aortic root was significantly increased after 6 months of HCD in male O-HFD as compared to that of O-ND. Plaque size and Oil Red O-positive staining were comparable between the two groups. Primary cultured vascular smooth muscle cells (VSMCs) of the thoracic aorta were treated with phosphate and interleukinL-1β (IL-1β) to transform them into an osteochondrocytic-like phenotype. Intracellular calcium content and alkaline phosphatase activity were markedly higher in the VSMCs of O-HFD than in O-ND. IL-1β concentration in the supernatant of bone marrow-derived macrophages was markedly higher in O-HFD than in O-ND. ConclusionOur findings indicate that maternal HFD accelerates the expansion of atherogenic calcification independent of plaque progression. In vitro phosphate- and IL-1β-induced osteochondrocytic transformation of VSMCs was augmented in O-HFD. Inhibition of VSMCs, skewing toward osteochondrocytic-like cells, might be a potential therapeutic strategy for preventing maternal HFD-associated CVD development.