SERS-based microdevices for use as in vitro diagnostic biosensors.

Advances in surface-enhanced Raman scattering (SERS) detection have helped to overcome the limitations of traditional in vitro diagnostic methods, such as fluorescence and chemiluminescence, owing to its high sensitivity and multiplex detection capability. However, for the implementation of SERS detection technology in disease diagnosis, a SERS-based assay platform capable of analyzing clinical samples is essential. Moreover, infectious diseases like COVID-19 require the development of point-of-care (POC) diagnostic technologies that can rapidly and accurately determine infection status. As an effective assay platform, SERS-based bioassays utilize SERS nanotags labeled with protein or DNA receptors on Au or Ag nanoparticles, serving as highly sensitive optical probes. Additionally, a microdevice is necessary as an interface between the target biomolecules and SERS nanotags. This review aims to introduce various microdevices developed for SERS detection, available for POC diagnostics, including LFA strips, microfluidic chips, and microarray chips. Furthermore, the article presents research findings reported in the last 20 years for the SERS-based bioassay of various diseases, such as cancer, cardiovascular diseases, and infectious diseases. Finally, the prospects of SERS bioassays are discussed concerning the integration of SERS-based microdevices and portable Raman readers into POC systems, along with the utilization of artificial intelligence technology.

VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

Dynamic metabolism of endothelial triglycerides protects against atherosclerosis in mice.

Blood vessels are continually exposed to circulating lipids, and elevation of ApoB-containing lipoproteins causes atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FAs) into triglyceride-rich (TG-rich) lipid droplets (LDs) is not known. In this study, we showed that deletion of the enzyme adipose TG lipase (ATGL) in the endothelium led to neutral lipid accumulation in vessels and impaired endothelial-dependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD affects endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state.

Recent advances in point-of-care testing of COVID-19.

Advances in microfluidic device miniaturization and system integration contribute to the development of portable, handheld, and smartphone-compatible devices. These advancements in diagnostics have the potential to revolutionize the approach to detect and respond to future pandemics. Accordingly, herein, recent advances in point-of-care testing (POCT) of coronavirus disease 2019 (COVID-19) using various microdevices, including lateral flow assay strips, vertical flow assay strips, microfluidic channels, and paper-based microfluidic devices, are reviewed. However, visual determination of the diagnostic results using only microdevices leads to many false-negative results due to the limited detection sensitivities of these devices. Several POCT systems comprising microdevices integrated with portable optical readers have been developed to address this issue. Since the outbreak of COVID-19, effective POCT strategies for COVID-19 based on optical detection methods have been established. They can be categorized into fluorescence, surface-enhanced Raman scattering, surface plasmon resonance spectroscopy, and wearable sensing. We introduced next-generation pandemic sensing methods incorporating artificial intelligence that can be used to meet global health needs in the future. Additionally, we have discussed appropriate responses of various testing devices to emerging infectious diseases and prospective preventive measures for the post-pandemic era. We believe that this review will be helpful for preparing for future infectious disease outbreaks.

Loss of cis-PTase function in the liver promotes a highly penetrant form of fatty liver disease that rapidly transitions to hepatocellular carcinoma.

Obesity-linked fatty liver is a significant risk factor for hepatocellular carcinoma (HCC)1,2; however, the molecular mechanisms underlying the transition from non-alcoholic fatty liver disease (NAFLD) to HCC remains unclear. The present study explores the role of the endoplasmic reticulum (ER)-associated protein NgBR, an essential component of the cis-prenyltransferases (cis-PTase) enzyme3, in chronic liver disease. Here we show that genetic depletion of NgBR in hepatocytes of mice (N-LKO) intensifies triacylglycerol (TAG) accumulation, inflammatory responses, ER/oxidative stress, and liver fibrosis, ultimately resulting in HCC development with 100% penetrance after four months on a high-fat diet. Comprehensive genomic and single cell transcriptomic atlas from affected livers provides a detailed molecular analysis of the transition from liver pathophysiology to HCC development. Importantly, pharmacological inhibition of diacylglycerol acyltransferase-2 (DGAT2), a key enzyme in hepatic TAG synthesis, abrogates diet-induced liver damage and HCC burden in N-LKO mice. Overall, our findings establish NgBR/cis-PTase as a critical suppressor of NAFLD-HCC conversion and suggests that DGAT2 inhibition may serve as a promising therapeutic approach to delay HCC formation in patients with advanced non-alcoholic steatohepatitis (NASH).

Effect of Nanoparticle Size on Plasmon-Driven Reaction Efficiency.

Hot electron chemistry is of paramount significance because of its applicability to photocatalytic reactions, solar energy conversion, and waste decomposition. The nonradiative decay of excited plasmons in gold nanoparticles (AuNPs) generates highly energetic nonthermal electrons and holes that can induce chemical reactions when transferred to nearby molecules. In this study, we explore the relationship between AuNP size (26-133 nm) and the plasmon-induced reaction yield. To isolate the size from other structural parameters, we prepare perfectly round gold nanospheres (AuNSs) with narrow size distributions. The use of a nanoparticle-on-mirror configuration, in which the reactant molecules (4-mercaptobenzoic acid) are positioned in nanogaps between the AuNSs and a Au film, promotes the generation of hot carriers and allows the highly sensitive detection of the reaction products (benzenethiol) using surface-enhanced Raman spectroscopy. We show that the reaction yield increases as the AuNS size increases up to 94 nm and then decreases for larger AuNSs. This peculiar Λ-shaped size-dependent reactivity can be explained by considering both the plasmonic absorption efficiency of AuNSs and the decay rate of plasmons via electron-surface scattering. The product of the calculated absorption cross section and the inverse of the AuNS size reproduces our experimental results remarkably well. These findings will contribute to the design of highly efficient plasmonic photocatalysts and photovoltaic devices.

Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. METHODS: Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. RESULTS: ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. CONCLUSIONS: Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.

Eruptive xanthoma model reveals endothelial cells internalize and metabolize chylomicrons, leading to extravascular triglyceride accumulation.

Although tissue uptake of fatty acids from chylomicrons is primarily via lipoprotein lipase (LpL) hydrolysis of triglycerides (TGs), studies of patients with genetic LpL deficiency suggest additional pathways deliver dietary lipids to tissues. Despite an intact endothelial cell (EC) barrier, hyperchylomicronemic patients accumulate chylomicron-derived lipids within skin macrophages, leading to the clinical finding eruptive xanthomas. We explored whether an LpL-independent pathway exists for transfer of circulating lipids across the EC barrier. We found that LpL-deficient mice had a marked increase in aortic EC lipid droplets before and after a fat gavage. Cultured ECs internalized chylomicrons, which were hydrolyzed within lysosomes. The products of this hydrolysis fueled lipid droplet biogenesis in ECs and triggered lipid accumulation in cocultured macrophages. EC chylomicron uptake was inhibited by competition with HDL and knockdown of the scavenger receptor-BI (SR-BI). In vivo, SR-BI knockdown reduced TG accumulation in aortic ECs and skin macrophages of LpL-deficient mice. Thus, ECs internalize chylomicrons, metabolize them in lysosomes, and either store or release their lipids. This latter process may allow accumulation of TGs within skin macrophages and illustrates a pathway that might be responsible for creation of eruptive xanthomas.

Plasmon-driven protodeboronation reactions in nanogaps.

Boronic acids are the key compounds in Suzuki coupling reactions and in the detection of monosaccharides. The C-B bond cleavage deboronation is an important side reaction that lowers the Suzuki coupling reaction yield and even disables saccharide detection. Here, we report that protodeboronation occurs for 4-mercaptophenylboronic acid (MPBA) within narrow nanogaps between gold nanoparticles (AuNPs) and planar gold substrates. The irradiation of such nanoparticle-on-mirror (NPoM) systems at 785 nm drives the protodeboronation reaction to form benzenethiol (BT). Wavelength-dependence experiments, combined with dark-field single-particle scattering spectroscopy, reveal that excitation of the bonding dipole plasmon mode of the NPoM leads to the best efficiency. Among the excited plasmon decay pathways, the generation of hot charge carriers induces the protodeboronation of MPBA. The possibility of plasmonic thermal reactions is ruled out because external heating of the substrates does not cause the reaction to take place. A comparison of the reaction yield under ambient, Ar, and oxygen gas conditions reveals that hot charge carriers directly transfer to MPBA, which subsequently produces BT, but the presence of oxygen promotes the reaction by opening another hot-electron transfer channel. The protodeboronation reaction of MPBA is an important addition to the catalog of plasmon-driven chemical reactions, not only because the reaction is relevant to organic and analytical chemistry but also because it deepens our understanding of the hot carrier dynamics at the interface between plasmonic nanoparticles and molecules.

BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9-mediated internalization of ALK-1, BMP-9-dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9-induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9-mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation.

BACKGROUND: Ischemic stroke is a main cause of mortality. Blood-brain barrier (BBB) breakdown appears to play a critical role in inflammation in patients with ischemic stroke and acceleration of brain injury. The BBB has a protective function and is composed of endothelial cells, pericytes, and astrocytes. In ischemic stroke treatments, regulation of vascular endothelial growth factor (VEGF)-A and vascular endothelial growth factor receptor (VEGFR)-2 is a crucial target despite adverse effects. Our previous study found that loss of C-type lectin family 14 member A (CLEC14A) activated VEGF-A/VEGFR-2 signaling in developmental and tumoral angiogenesis. Here, we evaluate the effects of BBB impairment caused by CLEC14A deficiency in ischemia-reperfusion injury. METHODS: In vitro fluorescein isothiocyanate (FITC)-dextran permeability, transendothelial electrical resistance (TEER) assay, and immunostaining were used to evaluate endothelial integrity. BBB permeability was assessed using Evans blue dye and FITC-dextran injection in Clec14a-/- (CLEC14A-KO) mice and wild-type mice. Middle cerebral artery occlusion surgery and behavioral assessments were performed to evaluate the neurologic damage. The change of tight junctional proteins, adhesion molecules, pro-inflammatory cytokines, and microglial were confirmed by immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction of brain samples. RESULTS: In endothelial cells, knockdown of CLEC14A increased FITC-dextran permeability and decreased transendothelial electrical resistance; the severity of this effect increased with VEGF treatment. Immunofluorescence staining revealed that tight junctional proteins were attenuated in the CLEC14A knockdown endothelial cells. Consistent with the in vitro results, CLEC14A-KO mice that were injected with Evans blue dye had cerebral vascular leakage at postnatal day 8; wild-type mice had no leakage. We used a middle cerebral artery occlusion model and found that CLEC14A-KO mice had severe infarcted brain and neurological deficits with upregulated VEGFR-2 expression. FITC-dextran leakage was present in CLEC14A-KO mice after ischemia-reperfusion, and the numbers of tight junctional molecules were significantly decreased. Loss of CLEC14A increased the pro-inflammatory response through adhesion molecule expression, and glial cells were activated. CONCLUSIONS: These results suggest that activation of VEGFR-2 in CLEC14A-KO mice aggravates ischemic stroke by exacerbating cerebral vascular leakage and increasing neuronal inflammation after ischemia-reperfusion injury.

Effect of Nanogap Morphology on Plasmon Coupling.

Plasmon coupling is the fundamental principle by which the optical resonances in nanoparticle assemblies are tuned. Interactions of plasmons among nanoparticles in close proximity create plasmon coupling modes whose energies are sensitive to the nanogap parameters. Whereas many studies have focused on the gap distances, we herein probe the effect of gap morphology on plasmon coupling. Dimers that are prepared by adsorbing perfectly round ultrauniform Au nanospheres (AuNSs) onto the faces, edges, and vertices of Au nanocubes (AuNCs) present distinctly different nanogap morphologies. Dark-field single-particle scattering spectroscopy reveals that the longitudinal plasmon coupling mode shifts to lower energies as the AuNS forms a nanogap with parts of the AuNC with higher curvature. Simulation spectra are also consistent with this observation. Our calculations indicate that the much larger charge density at the vertex or edge of a AuNC lowers the plasmon coupling energy through the contribution of the Coulomb interaction when the AuNC combines with the AuNS. In comparison, the plasmon energies or anisotropic polarizability along the face, edge, and vertex directions of a AuNC differ only slightly and thus do not cause a shift in the plasmon coupling mode.

Colour and SERS patterning using core-satellite nanoassemblies.

We explore spatial control of the formation of core-satellite nanoassemblies on glass substrates. UV irradiation leads to the photooxidative desorption of thiol linkers from gold nanoparticles deposited on the substrates, thereby prohibiting further assembly in the irradiated region. The distribution of assemblies and monomers yields a pattern with stark contrasts in colour and Raman enhancement. Our findings can be utilised in the fabrication of microfluidic SERS sensors, colour displays, photonic devices, and metamaterials.

Carbohydrate-binding protein CLEC14A regulates VEGFR-2- and VEGFR-3-dependent signals during angiogenesis and lymphangiogenesis.

Controlled angiogenesis and lymphangiogenesis are essential for tissue development, function, and repair. However, aberrant neovascularization is an essential pathogenic mechanism in many human diseases, including diseases involving tumor growth and survival. Here, we have demonstrated that mice deficient in C-type lectin family 14 member A (CLEC14A) display enhanced angiogenic sprouting and hemorrhage as well as enlarged jugular lymph sacs and lymphatic vessels. CLEC14A formed a complex with VEGFR-3 in endothelial cells (ECs), and CLEC14A KO resulted in a marked reduction in VEGFR-3 that was concomitant with increases in VEGFR-2 expression and downstream signaling. Implanted tumor growth was profoundly reduced in CLEC14A-KO mice compared with that seen in WT littermates, but tumor-bearing CLEC14A-KO mice died sooner. Tumors in CLEC14A-KO mice had increased numbers of nonfunctional blood vessels and severe hemorrhaging. Blockade of VEGFR-2 signaling suppressed these vascular abnormalities and enhanced the survival of tumor-bearing CLEC14A-KO mice. We conclude that CLEC14A acts in vascular homeostasis by fine-tuning VEGFR-2 and VEGFR-3 signaling in ECs, suggesting its relevance in the pathogenesis of angiogenesis-related human disorders.

AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation.

The phosphoinositide 3-kinase-Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide-dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465-474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465-474 residues abrogated the AMIGO2-PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1-Akt pathway in ECs and suggest that interference of the PDK1-AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor.

Anti-angiogenic activity of thienopyridine derivative LCB03-0110 by targeting VEGFR-2 and JAK/STAT3 Signalling.

Vascular endothelial growth factor receptor-2 (VEGFR-2) and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signalling are important for tumor angiogenesis and metastasis. In this study, we identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB03-0110) as a potent angiogenesis inhibitor. LCB03-0110 inhibited VEGFR-2 and JAK/STAT3 signalling in primary cultured human endothelial cells and cancer cells. An in vitro kinase assay and molecular modelling revealed that LCB03-0110 inhibited VEGFR-2, c-SRC and TIE-2 kinase activity via preferential binding at the ATP-binding site of their kinases. LCB03-0110 successfully occupied the hydrophobic pocket of VEGFR-2, c-SRC and TIE-2. LCB03-0110 also inhibited hypoxia-induced HIF/STAT3 and EGF- or angiopoietin-induced signalling cascades. In addition, LCB03-0110 inhibited VEGF-induced proliferation, viability, migration and capillary-like tube formation. LCB03-0110 also suppressed the sprouting of endothelial cells in the rat aorta and the formation of new blood vessels in the mouse Matrigel plug assay, but also suppressed pulmonary metastasis and tumor xenograft in mice. Our results suggest that LCB03-0110 is a potential candidate small molecule for blocking angiogenesis mediated by aberrant activation of VEGFR-2 and JAK/STAT3 signalling.

Combined effect of vascular-leakage-blocker Sac-1004 and antiangiogenic drug sunitinib on tumor angiogenesis.

Tumor blood vessels are often leaky because of poor covering by mural cells and loose cell-to-cell contacts. Leaky vessels result in hemorrhage and limited vascular perfusion, which lead to hypoxic tumor microenvironment. Antiangiogenic agents have been shown to normalize the tumor blood vessels, albeit temporarily. Continued administration has been found to be associated with increased tumor hypoxia, a major driving force behind chemoresistance and metastasis. Sac-1004 was recently demonstrated to prevent vascular leakage, normalize tumor vessels and prevent metastasis in sustained manner. Here, we sought that combining antiangiogenic agent, sunitinib with Sac-1004 could have better inhibitory effect upon tumor growth. We found that B16F10 tumor growth was significantly reduced and tumor-bearing mice survival was increased upon combining sunitinib therapy with Sac-1004. In concordance with this observation, tumor vascular perfusion was substantially improved in tumors receiving combination therapy. In addition, tumor vascular leakage was reduced to higher extent in combination treatment group as compared to either therapy alone, an effect attributed to improved vascular junction. Interestingly, hypoxia in tumor environment was significantly reduced, when sunitinib was combined with Sac-1004. Taken together, our data demonstrates that combining antiangiogenic therapy with vascular-leakage inhibiting agent might be a beneficial strategy to combat cancer.