Search

The spatiotemporal pattern of the spread of pathologically modified tau through brain regions in Alzheimer’s disease (AD) can be explained by prion-like cell-to-cell seeding and propagation of misfolded tau aggregates. Hence, to develop targeted therapeutic antibodies, it is important to identify the seeding- and propagation-competent tau species. The hexapeptide 275VQIINK280 of tau is a critical region for tau aggregation, and K280 is acetylated in various tauopathies, including AD. However, the mechanism that links tau acetylated on lysine 280 (tau-acK280) to subsequent progression to neurodegenerative disease remains unclear. Here, we demonstrate that tau-acK280 is critical for tau propagation processes including secretion, aggregation, and seeding. We developed an antibody, Y01, that specifically targets tau-acK280 and solved the crystal structure of Y01 in complex with an acK280 peptide. The structure confirmed that Y01 directly recognizes acK280 and the surrounding residues. Strikingly, upon interaction with acetylated tau aggregates, Y01 prevented tauopathy progression and increased neuronal viability in neuron cultures and in tau-Tg mice through antibody-mediated neutralization and phagocytosis, respectively. Based on our observations that tau-acK280 is a core species involved in seeding and propagation activities, the Y01 antibody that specifically recognizes acK280 represents a promising therapeutic candidate for AD and other neurodegenerative diseases associated with tauopathy.

Uncontrolled activation of transforming growth factor (TGF)-β results in a wide range of pathologic conditions. Therapeutic interventions to regulate TGF-β signaling during fibrosis have been developed but the effectiveness is still limited. Here, we show that developmental endothelial locus-1 (Del-1) ameliorates fibrosis in mice by inhibiting αv integrin-mediated activation of TGF-β. Del-1 bound to αvβ6 integrin, an important activator of TGF-β, and inhibited the binding of αvβ6 integrin to the latency-associated peptide (LAP), thereby suppressing αv integrin-mediated activation of TGF-β. Lack of Del-1 increased colocalization of αv integrin and LAP in the lungs, which was reversed by Del-1 supplementation. The crucial role of Del-1 in regulating TGF-β activity was recapitulated in a mouse model of fibrosis using an adenovirus expressing inactive TGF-β1. Del-1 supplementation improved the pathological characteristics of the mice and reduced mortality. Thus, we propose that Del-1 is a negative regulator of TGF-β activation and a potential anti-fibrotic factor.

Abstract Human granulocyte colony-stimulating factor (GCSF) is a well-known cytokine for neutropenia treatment. However, daily injections are required due to the short circulating half-life of the protein. To overcome this bottleneck, we fused GCSF with the Fc domain of IgG1 at the C terminus (GCSF-Fc) and with the maltose binding protein (MBP) tag at the N-terminus and expressed it as a soluble protein in the cytoplasm of E. coli. We also conjugated PEG aldehyde to GCSF to make PEG-GCSF. The bioactivities of GCSF-Fc and PEG-GCSF were similar to native GCSF using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50 dose-response curves for GCSF, GCSF-Fc and PEG-GCSF were 37 ± 12 pM, 75 ± 13.5 pM and 46 ± 5.5 pM, respectively. When the proteins were injected into neutropenic rats, the group injected with PEG-GCSF showed the highest and fastest recovery of neutrophils, followed by GCSF-Fc and GCSF. ELISA assay revealed the PEG-GCSF had the longest plasma circulation (>72 h), followed by GCSF-Fc (>48 h) and GCSF (~24 h), which is consistent with the in vivo activities of the proteins. In summary, the GCSF-Fc purified from E. coli was not as efficient as PEG-GCSF in treating neutropenic rats.

ObjectivePrognosis of myocardial infarction tends to be worse when serum C-reactive protein (CRP) level is high. miRNAs are also known to be involved in different pathogeneses of heart diseases such as myocardial infarction. However, how CRP is involved in myocardial infarction has not been fully elucidated. We hypothesized that serum CRP changes the miRNA profile during ischemia-reperfusion injury (IRI) of the myocardium. To confirm this hypothesis, we performed global miRNA expression profiling of myocardium using IRI and CRP infusion rat model.MethodsAfter ligation of the coronary artery of rat hearts, human serum CRP was intravenously injected, and reperfusion was performed (I/R+CRP group, n = 6). Control group consisted of the sham group (n = 3), IV CRP infusion group (CRP only, n = 3), and the I/R-only group (I/R only, n = 5). We evaluated 423 miRNA expression in non-ischemic areas and areas at risk (AAR) of each group using NanoString nCounter miRNA expression assay.ResultsMiR-124 was downregulated in non-ischemic myocardium in CRP-only group. In AAR, 7 miRNAs were commonly upregulated in both I/R-only and I/R+CRP groups. And additional 6 miRNAs were upregulated in the I/R+CRP group (miR-33, miR-409-3p, miR-384-3p, miR-3562, miR-101a, and miR-340-5p). Similarly, in the non-ischemic areas, 6 miRNAs were commonly upregulated in both I/R-only and I/R+CRP groups, and additional 5 miRNAs changed in the I/R+CRP group (upregulation of miR-3559-5p, miR-499, and miR-21 and downregulation of miR-500 and miR-532-3p).ConclusionWe showed that when serum CRP level is high, IRI results in multiple miRNA profile changes not only in ischemic areas but also in non-ischemic myocardium. Our results may provide a strong basis for studying the role of CRP and miRNAs in ischemic heart disease.

[This corrects the article DOI: 10.1371/journal.pone.0156296.].

Aim: This study determined the characteristics of patients with early-stage melanoma (IA-IIA) who later had stage IV recurrence.Patients & methods: We retrospectively examined 880 melanoma patients and identified those who progressed to stage IV disease from an initial early-stage (n = 50).Results: We observed a median latent period of 4 years between early-stage diagnosis and metastatic disease. More patients (54%) developed metastatic disease 4 years or later from the initial diagnosis. 34% had regular dermatology appointments, and 30% had regular oncology follow-up. Lung and brain were the most common metastatic sites.Conclusion: Long term monitoring beyond 4 years and a low threshold for performing symptom-guided imaging, particularly if pulmonary or neurologic symptoms occur, may be prudent after early-stage melanoma diagnosis.

Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

The primary objective of this study was to demonstrate the effectiveness of machine learning (ML) in predicting the mechanical behavior of sandwich structures. To achieve this, the study focused on assessing the bending response of environmentally friendly sandwich beams consisting of auxetic cores made from polylactic acid (PLA) and flax/epoxy composite face sheets reinforced with halloysite nanotubes (HNTs). Two ML techniques, specifically shallow neural networks (SNNs) and deep neural networks (DNNs), were employed to predict the specific energy absorption (SEA) and load-deflection curves of these sandwich beams, respectively. The key design parameters under consideration included the HNT content in the face sheets and three geometric characteristics of the auxetic cells. Subsequently, 16 distinct specimens were meticulously designed for manufacturing, following Taguchi's experimental design principles. The cores of the structures were produced using 3D printing techniques, while the face sheets were meticulously fabricated using a hand layup process. These prepared specimens were then subjected to a three-point bending test, and the collected data were employed to train the aforementioned neural networks. The outcomes of this study revealed that an SNN with a single hidden layer comprising seven neurons effectively predicted the SEA of the structures across various design parameter values. Additionally, the remarkable performance of a DNN, consisting of five hidden layers with 128, 64, 32, 16, and 8 units, respectively, was demonstrated by comparing its predicted results with the experimental results for a randomly designed sandwich beam. Highlights • ML techniques were used for bending behavior of sandwich beams. • The beams were made of flax/epoxy/HNT face sheets and 3D-printed auxetic core. • SNN was successfully employed to predict the SEA of the structures. • DNN effectively predicted the load-deflection curves. • HNT content and auxetic cells significantly impacted the bending properties. [ABSTRACT FROM AUTHOR]