Scalable Functionalization of Polyaniline-Grafted rGO Field-Effect Transistors for a Highly Sensitive Enzymatic Acetylcholine Biosensor

For decades, acetylcholine (Ach) has been considered a critical biomarker for several degenerative brain diseases, including Alzheimer’s, Parkinson’s disease, Huntington’s disease, and schizophrenia. Here, we propose a wafer-scale fabrication of polyaniline (PAni)-grafted graphene-based field-effect transistors (PGFET) and their biosensing applications for highly sensitive and reliable real-time monitoring of Ach in flow configuration. The grafted PAni provides suitable electrostatic binding sites for enzyme immobilization and enhances the pH sensitivity (2.68%/pH), compared to that of bare graphene-FET (1.81%/pH) for a pH range of 3–9 without any pH-hysteresis. We further evaluated the PGFET’s sensing performance for Ach detection with a limit of detection at the nanomolar level and significantly improved sensitivity (~103%) in the concentration range of 108 nM to 2 mM. Moreover, the PGFET exhibits excellent selectivity against various interferences, including glucose, ascorbic acid, and neurotransmitters dopamine and serotonin. Finally, we investigated the effects of an inhibitor (rivastigmine) on the AchE activity of the PGFET. From the results, we demonstrated that the PGFET has great potential as a real-time drug-screening platform by monitoring the inhibitory effects on enzymatic activity

EphA2 Interacts with Tim-4 through Association between Its FN3 Domain and the IgV Domain of Tim-4

Tim-4 promotes the engulfment of apoptotic cells or exogenous particles by securing them on phagocytes. It is unable to transduce signals by itself but helps other engulfment receptors sense and internalize them. However, the identity of the engulfment receptors collaborating with Tim-4 is still incompletely understood. In this study, we searched for a candidate transmembrane protein with a FN3 domain, important for interaction with Tim-4, in silico and investigated whether it indeed interacts with Tim-4 and is involved in Tim-4-mediated phagocytosis. We found that EphA2 containing a FN3 domain in the extracellular region interacted with Tim-4, which was mediated by the IgV domain of Tim-4 and the FN3 domain of EphA2. Nevertheless, we found that EphA2 expression failed to alter Tim-4-mediated phagocytosis of apoptotic cells or polystyrene beads. Taken together, our findings suggest that EphA2, a new Tim-4 interacting protein, may intervene in a Tim-4-mediated cellular event even if it is not phagocytosis of endogenous or exogenous particles and vice versa

Effects of mechanical properties of gelatin methacryloyl hydrogels on encapsulated stem cell spheroids for 3D tissue engineering

Cell spheroids are three-dimensional cell aggregates that have been widely employed in tissue engineering. Spheroid encapsulation has been explored as a method to enhance cell-cell interactions. However, the effect of hydrogel mechanical properties on spheroids, specifically soft hydrogels (<1 kPa), has not yet been studied. In this study, we determined the effect of encapsulation of stem cell spheroids by hydrogels crosslinked with different concentrations of gelatin methacryloyl (GelMA) on the functions of the stem cells. To this end, human adipose-derived stem cell (ADSC) spheroids with a defined size were prepared, and spheroid-laden hydrogels with various concentrations (5, 10, 15%) were fabricated. The apoptotic index of cells from spheroids encapsulated in the 15% hydrogel was high. The migration distance was five-fold higher in cells encapsulated in the 5% hydrogel than the 10% hydrogel. After 14 days of culture, cells from spheroids in the 5% hydrogel were observed to have spread and proliferated. Osteogenic factor and pro-angiogenic factor production in the 15% hydrogel was high. Collectively, our results indicate that the functionality of spheroids can be regulated by the mechanical properties of hydrogel, even under 1 kPa. These results indicate that spheroid-laden hydrogels are suitable for use in 3D tissue construction

Bioengineered amyloid peptide for rapid screening of inhibitors against main protease of SARS-CoV-2

Abstract The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has evoked a worldwide pandemic. As the emergence of variants has hampered the neutralization capacity of currently available vaccines, developing effective antiviral therapeutics against SARS-CoV-2 and its variants becomes a significant challenge. The main protease (Mpro) of SARS-CoV-2 has received increased attention as an attractive pharmaceutical target because of its pivotal role in viral replication and proliferation. Here, we generated a de novo Mpro-inhibitor screening platform to evaluate the efficacies of Mpro inhibitors based on Mpro cleavage site-embedded amyloid peptide (MCAP)-coated gold nanoparticles (MCAP-AuNPs). We fabricated MCAPs comprising an amyloid-forming sequence and Mpro-cleavage sequence, mimicking in vivo viral replication process mediated by Mpro. By measuring the proteolytic activity of Mpro and the inhibitory efficacies of various drugs, we confirmed that the MCAP-AuNP-based platform was suitable for rapid screening potential of Mpro inhibitors. These results demonstrated that our MCAP-AuNP-based platform has great potential for discovering Mpro inhibitors and may accelerate the development of therapeutics against COVID-19

Cytokine-Like 1 Regulates Cardiac Fibrosis via Modulation of TGF-β Signaling

<div><p>Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in <i>cytl1</i> knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of <i>cytl1</i> alone led to the development of CF <i>in vivo</i>. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of <i>cytl1</i> was sufficient to induce these two critical CF-related processes <i>in vitro</i>, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-β receptor 1. Cytl1 induced the expression of TGF-β2 both <i>in vivo</i> and <i>in vitro</i>. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 <i>in vitro</i>. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-β-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies.</p></div

Cytl1 is upregulated under pathological conditions.

<p>Three groups of WT mice were subjected to TAC for 6 wks (TAC), ligation of coronary artery for 4 wks (MI), or ligation of coronary artery for 30 min followed by reperfusion for 24 hrs (I/R). Hearts were harvested and qRT-PCR was performed to determine the transcript levels of Cytl1. n = 3 for sham, n = 3 for TAC, n = 4 for MI, n = 4 for I/R. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

AAV-mediated overexpression of Cytl1 induces CF.

<p>Control virus or AAV-Cytl1 (5 × 10¹⁰ viral genome) was injected into the tail vein of WT mice, and the phenotype of the heart was examined after 8 wks. (A) Picrosirius staining of heart cross-sections from WT mice injected with control virus or AAV-Cytl. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

CF is attenuated in <i>cytl1</i> KO mice.

<p>WT and <i>cytl1</i> KO mice were subjected to TAC for 6 wks and the extent of fibrosis in the heart was analyzed. (A) Picrosirius staining of heart cross-sections from WT and <i>cytl1</i> KO mice subjected to TAC. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Cytl1 functions independently of CCR2.

<p>Adult primary cardiac FBs were treated with CCL2 (20 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with the CCR2 antagonist CAS445679-97-0 (6 nM). (A) The cells were immunostained with an antibody against α-SMA. (B) Quantification of the mRNA levels of TGF-β2, Cytl1, collagen 1 and α-SMA by qRT-PCR. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p