Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis.

The liver has multiple regeneration modes, including hepatocellular hypertrophy and self-renewal of hepatocytes. When hepatocyte proliferation is impaired, hepatic progenitor cells may proliferate through ductular reaction (DR), differentiate into hepatocytes, and contribute to fibrosis. However, the three-dimensional spatial relationship between DR and regenerating hepatocytes and dynamic changes in DR associated with fibrosis remain poorly understood. Here, we performed three-dimensional (3D) imaging of cleared 42 liver explants with chronic and acute liver diseases and 4 normal livers to visualize DR. In chronic hepatic liver diseases, such as viral hepatitis, steatohepatitis, autoimmune hepatitis, and cryptogenic cirrhosis, the total length and number of branches of DR showed a significant positive correlation. We studied the spatial relationship between DR and GS-expressing cells using glutamine synthetase (GS) and cytokeratin 19 (CK19) as markers of liver regeneration and DR, respectively. The percentage of CK19-positive cells that co-expressed GS was less than 10% in chronic liver diseases. In contrast, nearly one-third of CK19-positive cells co-expressed GS in acute liver diseases, and chronic cholestatic liver diseases, including primary biliary cholangitis and primary sclerosing cholangitis, showed no co-expression. We also found that DR was longer and had more branching in livers with progressive fibrosis compared to those with regressive fibrosis. Our results suggest that DR displays varying degrees of spatial complexity and contribution to liver regeneration. DR may serve as hepatobiliary junctions that maintain continuity between hepatocytes and bile ducts rather than hepatocyte regeneration in chronic liver diseases.

Metal-Like Stretchable Nanocomposite Using Locally-Bundled Nanowires for Skin-Mountable Devices.

Stretchable conductive nanocomposites have been intensively studied for wearable bioelectronics. However, development of nanocomposites that simultaneously feature metal-like conductivity(> 100 000 S cm-1 ) and high stretchability (> 100%) for high-performance skin-mountable devices is still extremely challenging. Here a material strategy for such a nanocomposite is presented by using local bundling of silver nanowires stabilized with dual ligands (i.e., 1-propanethiols and 1-decanethiols). When the nanocomposite is solidified via solvent evaporation under a highly humid condition, the nanowires in the organic solution are bundled and stabilized. The resulting locally-bundled nanowires lower contact resistance while maintain their percolation network, leading to high conductivity. Dual ligands of 1-propanethiol and 1-decanethiol further boost up the conductivity. As a result, a nanocomposite with both high conductivity of ≈122,120 S cm-1 and high stretchability of ≈200% is obtained. Such superb electrical and mechanical properties are critical for various applications in skin-like electronics, and herein, a wearable thermo-stimulation device is demonstrated.

Stretchable Low-Impedance Conductor with Ag-Au-Pt Core-Shell-Shell Nanowires and in Situ Formed Pt Nanoparticles for Wearable and Implantable Device.

Mechanically soft metallic nanocomposites have gained much attention as a key material for intrinsically stretchable biointegrated devices. However, it has been challenging to develop a stretchable conductive nanocomposite with all the desired material characteristics including high conductivity, high stretchability, low cytotoxicity, and low impedance. Here, we present a material strategy for the stretchable conductive nanocomposite, particularly emphasizing low impedance, by combining silver-gold-platinum core-shell-shell nanowires and homogeneously dispersed in situ synthesized platinum nanoparticles (Pt NPs). The highly embossed structure of the outermost Pt shell, together with the intrinsic electrical property of Pt, contributes to minimizing the impedance. The gold-platinum double-layer sheath prevents leaching of cytotoxic Ag ions, thus improving biocompatibility. Homogeneously dispersed Pt NPs, synthesized in situ during fabrication of the nanocomposite, simultaneously enhance conductivity, reduce impedance, and improve stretchability by supporting the percolation network formation. This intrinsically stretchable nanocomposite conductor can be applied to wearable and implantable bioelectronics for recording biosignals and delivering electrical stimulations in vivo.

Ventricular tachyarrhythmia treatment and prevention by subthreshold stimulation with stretchable epicardial multichannel electrode array.

The implantable cardioverter-defibrillator (ICD) is an effective method to prevent sudden cardiac death in high-risk patients. However, the transvenous lead is incompatible with large-area electrophysiological mapping and cannot accommodate selective multichannel precision stimulations. Moreover, it involves high-energy shocks, resulting in pain, myocardial damage, and recurrences of ventricular tachyarrhythmia (VTA). We present a method for VTA treatment based on subthreshold electrical stimulations using a stretchable epicardial multichannel electrode array, which does not disturb the normal contraction or electrical propagation of the ventricle. In rabbit models with myocardial infarction, the infarction was detected by mapping intracardiac electrograms with the stretchable epicardial multichannel electrode array. Then, VTAs could be terminated by sequential electrical stimuli from the epicardial multichannel electrode array beginning with low-energy subthreshold stimulations. Last, we used these subthreshold stimulations to prevent the occurrence of additional VTAs. The proposed protocol using the stretchable epicardial multichannel electrode array provides opportunities toward the development of innovative methods for painless ICD therapy.

Frequent Intraluminal Growth of Large Muscular Veins in Surgically Resected Colorectal Cancer Tissues: A 3-Dimensional Pathologic Reconstruction Study.

Although venous invasion (VI) is common in colorectal cancers (CRCs) and is associated with distant metastasis, the 3-dimensional (3D) microscopic features and associated mechanisms of VI are not well elucidated. To characterize the patterns of VI, 103 tissue slabs were harvested from surgically resected CRCs with ≥pT2. They were cleared using the modified immunolabeling-enabled 3D imaging of solvent-cleared organs method, labeled with multicolor fluorescent antibodies, including antibodies against cytokeratin 19, desmin, CD31, and E-cadherin, and visualized by confocal laser scanning microscopy. VI was classified as intravasation, intraluminal growth, and/or extravasation, and 2-dimensional and 3D microscopic features were compared. VI was detected more frequently in 3D (56/103 [54.4%]) than in conventional 2-dimensional hematoxylin and eosin-stained slides (33/103 [32%]; P < .001). When VI was present, it was most commonly in the form of intraluminal growth (51/56), followed by extravasation (13/56) and intravasation (5/56). The mean length of intraluminal growth was 334.0 ± 212.4 µm. Neoplastic cell projections extended from cancer cell clusters in the connective tissue surrounding veins, penetrated the smooth muscle layer, and then grew into and filled the venous lumen. E-cadherin expression changed at each invasion phase; intact E-cadherin expression was observed in the cancer cells in the venous walls, but its expression was lost in small clusters of intraluminal neoplastic cells. In addition, reexpression of E-cadherin was observed when cancer cells formed well-oriented tubular structures and accumulated and grew along the luminal side of the venous wall. In contrast, singly scattered cancer cells and cancer cells with poorly defined tubular structures showed loss of E-cadherin expression. E-cadherin expression was intact in the large cohesive clusters of extravasated cancer cells. However, singly scattered cells and smaller projections of neoplastic cells in the stroma outward of venous wall showed a loss of E-cadherin expression. In conclusion, VI was observed in more than half of the CRCs analyzed by 3D histopathologic image reconstruction. Once inside a vein, neoplastic cells can grow intraluminally. The epithelial-mesenchymal transition is not maintained during VI of CRCs.

Adaptive Self-Organization of Nanomaterials Enables Strain-Insensitive Resistance of Stretchable Metallic Nanocomposites.

Highly conductive and stretchable nanocomposites are promising material candidates for skin electronics. However, the resistance of stretchable metallic nanocomposites highly depends on external strains, often deteriorating the performance of fabricated electronic devices. Here, a material strategy for the highly conductive and stretchable nanocomposites comprising metal nanomaterials of various dimensions and a viscoelastic block-copolymer matrix is presented. The resistance of the nanocomposites can be well retained under skin deformations (<50% strain). It is demonstrated that silver nanomaterials can self-organize inside the viscoelastic media in response to external strain when their surface is conjugated with 1-decanethiol. Distinct self-organization behaviors associated with nanomaterial dimensions and strain conditions are found. Adopting the optimum composition of 0D/1D/2D silver nanomaterials can render the resistance of the nanocomposites insensitive to uniaxial or biaxial strains. As a result, the resistance can be maintained with a variance of < 1% during 1000 stretching cycles under uniaxial and biaxial strains of <50% while a high conductivity of ≈31 000 S cm-1 is achieved.

Multifunctional Injectable Hydrogel for In Vivo Diagnostic and Therapeutic Applications.

Injectable hydrogels show high potential for in vivo biomedical applications owing to their distinctive mode of administration into the human body. In this study, we propose a material design strategy for developing a multifunctional injectable hydrogel with good adhesiveness, stretchability, and bioresorbability. Its multifunctionality, whereupon multiple reactions occur simultaneously during its injection into the body without requiring energy stimuli and/or additives, was realized through meticulous engineering of bioresorbable precursors based on hydrogel chemistry. The multifunctional injectable hydrogel can be administered through a minimally invasive procedure, form a conformal adhesive interface with the target tissue, dynamically stretch along with the organ motions with minimal mechanical constraints, and be resorbed in vivo after a specific period. Further, the incorporation of functional nanomaterials into the hydrogel allows for various in vivo diagnostic and therapeutic applications, without compromising the original multifunctionality of the hydrogel. These features are verified through theranostic case studies on representative organs, including the skin, liver, heart, and bladder.

Highly conductive and elastic nanomembrane for skin electronics.

Skin electronics require stretchable conductors that satisfy metallike conductivity, high stretchability, ultrathin thickness, and facile patternability, but achieving these characteristics simultaneously is challenging. We present a float assembly method to fabricate a nanomembrane that meets all these requirements. The method enables a compact assembly of nanomaterials at the water-oil interface and their partial embedment in an ultrathin elastomer membrane, which can distribute the applied strain in the elastomer membrane and thus lead to a high elasticity even with the high loading of the nanomaterials. Furthermore, the structure allows cold welding and bilayer stacking, resulting in high conductivity. These properties are preserved even after high-resolution patterning by using photolithography. A multifunctional epidermal sensor array can be fabricated with the patterned nanomembranes.

Three-dimensional analysis of extrahepatic cholangiocarcinoma and tumor budding.

Advances in tissue clearing and microscopy make it possible to study human diseases in three dimensions (3D). High-grade tumor budding is known to be associated with poor prognosis in various cancers; however, little is known about the 3D architecture of tumor budding. Using tissue clearing, we analyzed the 3D structure of tumor budding and E-cadherin expression in 31 extrahepatic cholangiocarcinomas. A total of 31 thick slabs (up to 5 mm) were harvested from surgically resected tumor tissue, including 27 hilar and 4 distal cholangiocarcinomas. Twenty-eight cases were adenocarcinoma, and three were undifferentiated carcinoma. After clearing, the tissues were immunolabeled with antibodies to cytokeratin 19 and to E-cadherin, and then visualized using light-sheet and confocal laser scanning microscopy. Tumor budding was evaluated in hematoxylin and eosin-stained sections (2D) using standard pathological criteria. Of the 31 cancers, 13 showed low-grade tumor budding and 18 showed high-grade tumor budding. First, 3D analysis revealed that the neoplastic cells in tumor buds of adenocarcinoma were typically not individual islands of cells, but rather tips of attenuated protrusions connected to the main tumor. Second, adenocarcinomas with low-grade tumor budding were composed predominantly of tubules that only focally form cords at the periphery. By contrast, adenocarcinomas with high-grade tumor budding predominantly formed cords in both centers and peripheries of the tumors. Third, adenocarcinoma with low-grade tumor budding was characterized by a few short protrusions with few branches, whereas adenocarcinoma with high-grade tumor budding was characterized by longer protrusions with more branching. Finally, immunolabeling of E-cadherin was stronger in the center of the adenocarcinoma but decreased at the tips of protrusions. E-cadherin loss was more extensive in the protrusions of high-grade tumor budding than in the protrusions of low-grade tumor budding. Our findings suggest that tumor buds as seen in 2D are, in fact, cross-sections of attenuated but contiguous protrusions extending from the main tumor. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Material Design and Fabrication Strategies for Stretchable Metallic Nanocomposites.

Stretchable conductive nanocomposites fabricated by integrating metallic nanomaterials with elastomers have become a vital component of human-friendly electronics, such as wearable and implantable devices, due to their unconventional electrical and mechanical characteristics. Understanding the detailed material design and fabrication strategies to improve the conductivity and stretchability of the nanocomposites is therefore important. This Review discusses the recent technological advances toward high performance stretchable metallic nanocomposites. First, the effect of the filler material design on the conductivity is briefly discussed, followed by various nanocomposite fabrication techniques to achieve high conductivity. Methods for maintaining the initial conductivity over a long period of time are also summarized. Then, strategies on controlled percolation of nanomaterials are highlighted, followed by a discussion regarding the effects of the morphology of the nanocomposite and postfabricated 3D structures on achieving high stretchability. Finally, representative examples of applications of such nanocomposites in biointegrated electronics are provided. A brief outlook concludes this Review.

Three-dimensional visualization of cleared human pancreas cancer reveals that sustained epithelial-to-mesenchymal transition is not required for venous invasion.

Venous invasion is three times more common in pancreatic cancer than it is in other major cancers of the gastrointestinal tract, and venous invasion may explain why pancreatic cancer is so deadly. To characterize the patterns of venous invasion in pancreatic cancer, 52 thick slabs (up to 5 mm) of tissue were harvested from 52 surgically resected human ductal adenocarcinomas, cleared with a modified iDISCO method, and labeled with fluorescent-conjugated antibodies to cytokeratin 19, desmin, CD31, p53 and/or e-cadherin. Labeled three-dimensional (3D) pancreas cancer tissues were visualized with confocal laser scanning or light sheet microscopy. Multiple foci of venous and even arterial invasion were visualized. Venous invasion was detected more often in 3D (88%, 30/34 cases) than in conventional 2D slide evaluation (75%, 25/34 cases, P < 0.001). 3D visualization revealed pancreatic cancer cells crossing the walls of veins at multiple points, often at points where preexisting capillary structures bridge the blood vessels. The neoplastic cells often retained a ductal morphology (cohesive cells forming tubes) as they progressed from a stromal to intravenous location. Although immunolabeling with antibodies to e-cadherin revealed focal loss of expression at the leading edges of the cancers, the neoplastic cells within veins expressed e-cadherin and formed well-oriented glands. We conclude that venous invasion is almost universal in pancreatic cancer, suggesting that even surgically resectable PDAC has access to the venous spaces and thus the ability to disseminate widely. Furthermore, we observe that sustained epithelial-mesenchymal transition is not required for venous invasion in pancreatic cancer.

Highly conductive, stretchable and biocompatible Ag-Au core-sheath nanowire composite for wearable and implantable bioelectronics.

Wearable and implantable devices require conductive, stretchable and biocompatible materials. However, obtaining composites that simultaneously fulfil these requirements is challenging due to a trade-off between conductivity and stretchability. Here, we report on Ag-Au nanocomposites composed of ultralong gold-coated silver nanowires in an elastomeric block-copolymer matrix. Owing to the high aspect ratio and percolation network of the Ag-Au nanowires, the nanocomposites exhibit an optimized conductivity of 41,850 S cm-1 (maximum of 72,600 S cm-1). Phase separation in the Ag-Au nanocomposite during the solvent-drying process generates a microstructure that yields an optimized stretchability of 266% (maximum of 840%). The thick gold sheath deposited on the silver nanowire surface prevents oxidation and silver ion leaching, making the composite biocompatible and highly conductive. Using the nanocomposite, we successfully fabricate wearable and implantable soft bioelectronic devices that can be conformally integrated with human skin and swine heart for continuous electrophysiological recording, and electrical and thermal stimulation.

Glycoepitopes of staphylococcal wall teichoic acid govern complement-mediated opsonophagocytosis via human serum antibody and mannose-binding lectin.

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or ß-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, ß-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that ß-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact ß-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-ß-GlcNAc-specific WTA IgGs, anti-ß-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.

Intradermal immunization with wall teichoic acid (WTA) elicits and augments an anti-WTA IgG response that protects mice from methicillin-resistant Staphylococcus aureus infection independent of mannose-binding lectin status.

The objectives of this study were to investigate the immune response to intradermal immunization with wall teichoic acid (WTA) and the effect of MBL deficiency in a murine model of infection with methicillin-resistant Staphylococcus aureus (MRSA). WTA is a bacterial cell wall component that is implicated in invasive infection. We tested susceptibility to MRSA infection in wild type (WT) and MBL deficient mice using two strains of MRSA: MW2, a community-associated MRSA (CA-MRSA); and COL, a healthcare-associated MRSA (HA-MRSA). We also performed in vitro assays to investigate the effects of anti-WTA IgG containing murine serum on complement activation and bacterial growth in whole blood. We found that MBL knockout (KO) mice are relatively resistant to a specific MRSA strain, MW2 CA-MRSA, compared to WT mice, while both strains of mice had similar susceptibility to a different strain, COL HA-MRSA. Intradermal immunization with WTA elicited and augmented an anti-WTA IgG response in both WT and MBL KO mice. WTA immunization significantly reduced susceptibility to both MW2 CA-MRSA and COL HA-MRSA, independent of the presence of MBL. The protective mechanisms of anti-WTA IgG are mediated at least in part by complement activation and clearance of bacteria from blood. The significance of these findings is that 1) Intradermal immunization with WTA induces production of anti-WTA IgG; and 2) This anti-WTA IgG response protects from infection with both MW2 CA-MRSA and COL HA-MRSA even in the absence of MBL, the deficiency of which is common in humans.

Human SAP is a novel peptidoglycan recognition protein that induces complement-independent phagocytosis of Staphylococcus aureus.

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer--a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an FcγR-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain-like receptors.

Specific serum Ig recognizing staphylococcal wall teichoic acid induces complement-mediated opsonophagocytosis against Staphylococcus aureus.

Wall teichoic acid (WTA) of Staphylococcus aureus is a major cell envelope-associated glycopolymer that is a key molecule in promoting colonization during S. aureus infection. The complement system plays a key role in the opsonization and clearance of pathogens. We recently reported that S. aureus WTA functions as a ligand of human serum mannose-binding lectin (MBL), a recognition molecule of the lectin complement pathway. Intriguingly, serum MBL in adults does not bind to WTA because of an inhibitory effect of serum anti-WTA-IgG. In this study, serum anti-WTA-IgG was purified to homogeneity using a purified S. aureus WTA-coupled affinity column to examine the biological function of human anti-WTA-IgG. The purified anti-WTA-IgG contained the IgG2 subclass as a major component and specifically induced C4 and C3 deposition on the S. aureus surface in the anti-WTA-IgG-depleted serum, but not in C1q-deficient serum. Furthermore, the anti-WTA-IgG-dependent C3 deposition induced phagocytosis of S. aureus cells by human polymorphonuclear leukocytes. These results demonstrate that serum anti-WTA-IgG is a real trigger for the induction of classical complement-dependent opsonophagocytosis against S. aureus. Our results also support the fact that a lack of the lectin complement pathway in MBL-deficient adults is compensated by Ag-specific, Ab-mediated adaptive immunity.

Human serum mannose-binding lectin senses wall teichoic acid Glycopolymer of Staphylococcus aureus, which is restricted in infancy.

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.