Defining the T cell transcriptional landscape in pediatric liver transplant rejection at single cell resolution.

Acute cellular rejection (ACR) affects >80% of pediatric liver transplant recipients within 5 years, and late ACR is associated with graft failure. Traditional anti-rejection therapy for late ACR is ineffective and has remained unchanged for six decades. Although CD8+ T cells promote late ACR, little has been done to define their specificity and gene expression. Here, we used single-cell sequencing and immune repertoire profiling (10X Genomics) on 30 cryopreserved 16G liver biopsies from 14 patients (5 pre-transplant or with no ACR, 9 with ACR). We identified expanded intragraft CD8+ T cell clonotypes (CD8EXP) and their gene expression profiles in response to anti-rejection treatment. Notably, we found that expanded CD8+ clonotypes (CD8EXP) bore markers of effector and CD56hiCD161- 'NK-like' T cells, retaining their clonotype identity and phenotype in subsequent biopsies from the same patients despite histologic ACR resolution. CD8EXP clonotypes localized to portal infiltrates during active ACR, and persisted in the lobule after histologic ACR resolution. CellPhoneDB analysis revealed differential crosstalk between KC and CD8EXP during late ACR, with activation of the LTB-LTBR pathway and downregulation of TGFß signaling. Therefore, persistently-detected intragraft CD8EXP clones remain active despite ACR treatment and may contribute to long-term allograft fibrosis and failure of operational tolerance.

T-cell infiltrate intensity is associated with delayed response to treatment in late acute cellular rejection in pediatric liver transplant recipients.

BACKGROUND: Late acute cellular rejection (ACR) is associated with donor-specific antibodies (DSA) development, chronic rejection, and allograft loss. However, accurate predictors of late ACR treatment response are lacking. ACR is primarily T-cell mediated, yet B cells and plasma cells (PC) also infiltrate the portal areas during late ACR. To test the hypothesis that the inflammatory milieu is associated with delayed response (DR) to rejection therapy, we performed a single-center retrospective case-control study of pediatric late liver ACR using multiparameter immunofluorescence for CD4, CD8, CD68, CD20, and CD138 to identify immune cell subpopulations. METHODS: Pediatric liver transplant recipients transplanted at <17 years of age and treated for biopsy-proven late ACR between January 2014 and 2019 were stratified into rapid response (RR) and DR based on alanine aminotransferase (ALT) normalization within 30 days of diagnosis. All patients received IV methylprednisolone as an initial rejection treatment. Immunofluorescence was performed on archived formalin-fixed paraffin embedded (FFPE) liver biopsy tissue. RESULTS: Liver biopsies from 60 episodes of late ACR in 54 patients were included in the analysis, of which 33 were DR (55%). Anti-thymocyte globulin was only required in the DR group. The frequency of liver-infiltrating CD20+ and CD8+ lymphocytes and the prevalence of autoantibodies were higher in the DR group. In univariate logistic regression analysis, serum gamma-glutamyl transpeptidase (GGT) level at diagnosis, but not ALT, Banff score or presence of DSA, predicted DR. CONCLUSIONS: Higher serum GGT level, presence of autoantibodies, and increased CD8+ T-cell infiltration portends DR in late ACR treatment in children.

Spatial Confinement of Enzyme and Nanozyme in Silica-Based Hollow Microreactors.

Designing a strategy for encasing enzymes and nanozymes in microreactors with spatial confinement in a way to improve the selectivity and activity of nanozymes is an exciting goal. In the present work, we report a facile route to encapsulate glucose oxidase (GOx) and poly(ethylenimine) (PEI)-conjugated magnetite nanoparticles (Fe3O4-PEI) in the hollow interior of hybrid microreactors. The microreactors are prepared by polyallylamine hydrochloride (PAH)-mediated silica (SiO2) nanoparticle assembly on calcium carbonate (CaCO3) particles as a removable core. By tuning both shape and phase (vaterite/calcite and pure calcite) of CaCO3, it allows generation of GOx and Fe3O4-PEI-encapsulated silica hollow microspheres (GOx-Fe3O4@SHS) and microcubes (GOx-Fe3O4@SHC). As observed, in a biomimetic cascade catalysis, the confined GOx in the microreactors is able to catalyze oxidation of glucose to gluconic acid and hydrogen peroxide (H2O2), followed by the activation of H2O2 by Fe3O4-PEI for the oxidation of the chromogenic substrate o-phenylenediamine (oPD) to 2,3-diaminophenazine. Comparison of the peroxidase-like activity of the encapsulated Fe3O4-PEI shows that the hollow microspheres (GOx-Fe3O4@SHS) result in activity 14 times higher than that of the hollow microcubes (GOx-Fe3O4@SHC), which in turn is corroborated to the differential loading capacity of GOx in microspheres and microcubes. The evaluation of kinetic parameters indicates a fivefold increase in the catalytic constant (kcat) of Fe3O4-PEI confined in hollow microspheres (GOx-Fe3O4@SHS) compared to the mixture comprising free GOx and Fe3O4-PEI in solution. It suggests that the confined space in the microreactors allows the tandem reactions of GOx and Fe3O4-PEI to take place in close proximity, leading to an improved overall activity. This indeed is seen in the kcat obtained for Fe3O4@SHS (GOx added externally during the assay), which is 14 times lower than that of GOx-Fe3O4@SHS.

Tunable Surface Wrinkling by a Bio-Inspired Polyamine Anion Coacervation Process that Mediates the Assembly of Polyoxometalate Nanoclusters.

A bio-inspired method is used to render controlled wrinkling surface patterns on supramolecular architectures assembled from polyoxometalate (POM) clusters. It involves a polyamine-multivalent anion interaction generating positively charged coacervates, which while dictating the assembly of POM into spherical structures further facilitate an interesting surface morphogenesis with wrinkling patterns. This spontaneous surface wrinkling depends on the type of multivalent anion and the pH. As the polyamine-anion interaction becomes stronger, the wrinkles turn denser with lesser depth, which eventually undergoes post-buckling to engender a complex surface pattern. Interestingly, the order of influence exerted by different anions on the morphology follows the Hofmeister series. Moreover, the mild synthesis conditions keep the functional POM units dispersed in the sphere with a structural transformability to their lacunary form.

Designing Microreactors Resembling Cellular Microenvironment via Polyamine-Mediated Nanoparticle-Assembly for Tuning Glucose Oxidase Kinetics.

Spatial confinement of glucose oxidase (GOx) in the hollow interior of a bioinspired matrix via polyamine mediated silica nanoparticle assembly under environmentally benign conditions is demonstrated herein. In a similarity to the biosilicification processes in diatoms, we use poly(allylamine hydrochloride) (PAH) to direct the assembly of silica nanoparticles on CaCO3 spheres as the removable core. When this assembly process is performed on the CaCO3 spheres, which are preloaded with GOx in a postsynthesis method, microspheres encapsulating GOx are formed. Interestingly, the encapsulated GOx in these microreactors exhibits activity with a Michaelis-Menten constant ( KM) that is 2- to 3-fold less compared with the free enzyme in the solution. While the microenvironment of the organic (PAH)-inorganic (silica) hybrid system can be advantageous for the substrate to interact with enzyme, the effective pH in the vicinity of the entrapped enzyme may also be accountable for the improved activity, resulting in the lower apparent KM and enhanced specificity constant ( kcat/ KM). A 2-fold higher thermal stability of the encapsulated GOx compared with free GOx in solution further demonstrates the efficacy of the integrated architecture. Additionally, the PAH by virtue of its buffering capability allows the microspheres in imparting pH stability to the encapsulated GOx. Therefore, the method is not only a greener process for performing enzyme immobilization but also anticipated to aid in designing microreactors for enhanced enzyme activity.

Potential of the Bioinspired CaCO3 Microspheres Loaded with Tetracycline in Inducing Differential Cytotoxic Effects toward Noncancerous and Cancer Cells: A Cytogenetic Toxicity Assessment Using CHO Cells in Vitro.

Calcium carbonate (CaCO3)-based materials as feasible pH-sensitive drug carriers, which can actively dissolve in an acidic microenvironment of cancer cells, are finding increasing importance. This has drawn our interest in the development of a bioinspired polypeptide- mediated method to design calcium carbonate microspheres loaded with tetracycline (CaCO3-TC) with an aim to explore its safe application in cancer therapeutics. Its therapeutic application in cancer patients essentially demands its safety information on the normal cells. Herein our study presents the in vitro genetic toxicological information on CaCO3-TC using noncancerous mammalian CHO cells in comparison to bare TC at three different concentrations (100, 200, and 300 µM) selected based on the cytotoxicity data (MTT). Assessment of various end points like chromosome aberrations, micronucleus, mitotic index and effects on cell cycle distribution after 24 h post-treatment demonstrates a significant reduction in clastogenic ( P < 0.001), aneugenic potential ( P < 0.05), and nonmitotoxic nature of CaCO3-TC than that of bare TC. Noticeably, as inferred from the FACS analysis on cancer cells, G2/M phase accumulation in breast cancer cells (MDA-MB-231), and at G1 phase in cervical cancer cells (HeLa) reveal its potential anticancer property. On the other hand, the genotoxicity studies illustrate protective effects of CaCO3-TC on noncancerous cells. While the pH-dependent dissolution property of the CaCO3 matrix encasing tetracycline results in higher toxicity on cancer cells, the near neutral pH in the case of normal cells prevents complete dissolution of CaCO3 thereby not allowing the encapsulated TC to adequately interact with the cells. Therefore, thus assembled CaCO3 spheres not only provide a way for facile encapsulation of tetracycline under mild conditions but also result in an effective matrix for differential toxicity toward normal and cancer cells justifying its clinical development as a novel target-specific drug in therapeutic applications for metastatic cancers.

A bio-inspired strategy for the interfacial assembly of graphene oxide with in situ generated Ag/AgCl: designing sustainable hybrid photocatalysts.

Herein, we report a polyamine-mediated assembly to integrate graphene oxide (GO) sheets with Ag/AgCl to fabricate a hybrid nanocomposite (GO-Ag/AgCl) at nearly neutral pH and ambient temperature. Inspired by the role of polyamines in the excellent integration of components to generate hierarchical nanostructures in biominerals such as diatoms, we showed that our strategy enabled the fabrication of GO-semiconductor composites with a well-integrated structure. The polyamines not only facilitated the in situ generation of Ag/AgCl, but also simultaneously allowed their interaction with GO suitable for visible light active photocatalysis, as revealed by the detailed characterization of the synthesized materials. Consequently, the GO-Ag/AgCl exhibited nearly 5 times higher photocatalytic activity and better photostability than Ag/AgCl under visible light irradiation. The nanocomposite reached its highest activity at the graphene content of 4.16 wt%. Thus, the assembly process represented an effective way to design hybrid composites. Moreover, as a sustainable photocatalyst, it facilitates effective separation of the photogenerated charge carriers at the interface, thereby improving activity and stability.

In Situ Strategy to Encapsulate Antibiotics in a Bioinspired CaCO3 Structure Enabling pH-Sensitive Drug Release Apt for Therapeutic and Imaging Applications.

Herein we demonstrate a bioinspired method involving macromolecular assembly of anionic polypeptide with cationic peptide-oligomer that allows for in situ encapsulation of antibiotics like tetracycline in CaCO3 microstructure. In a single step one-pot process, the encapsulation of the drug occurs under desirable environmentally benign conditions resulting in drug loaded CaCO3 microspheres. While this tetracycline-loaded sample exhibits pH dependent in vitro drug-release profile and excellent antibacterial activity, the encapsulated drug or the dye-conjugated peptide emits fluorescence suitable for optical imaging and detection, thereby making it a multitasking material. The efficacy of tetracycline loaded calcium carbonate microspheres as pH dependent drug delivery vehicles is further substantiated by performing cell viability experiments using normal and cancer cell lines (in vitro). Interestingly, the pH-dependent drug release enables selective cytotoxicity toward cancer cell lines as compared to the normal cells, thus having the potential for further development of therapeutic applications.

Compartmentalisation of enzymes for cascade reactions through biomimetic layer-by-layer mineralization.

Cellular metabolic pathways are paradigms for the rapid and waste-free conversion of molecules into useful products through multiple enzyme-catalyzed steps (cascade reactions). Attempts to establish efficient cascade reactions for technological applications have focused on mimicking nature's high degree of organization by controlling the positioning of enzymes through immobilization in tailor-made compartments. The present work utilized peptide-mediated layer-by-layer mineralization as a facile and generic method for the compartmentalisation of multi-enzyme systems in nanoscale silica layers. It is demonstrated that, in a multilayer system, the overall rate of the reaction cascade was primarily affected by the placement of the enzyme catalyzing the first step, with the placement of the enzyme possessing the lowest catalytic efficiency also being an important factor. As the rate-limiting enzymes were positioned closer to the external silica surface, the overall rate of cascade reactions increased. Furthermore, distributing the enzymes into different adjacent silica compartments yielded higher overall cascade reaction rates compared to placement of the enzymes into the same silica layer. The synthetic methods and kinetic analyses presented here provide guidance for improving the performance of immobilized multi-enzyme systems for a wide range of technological applications.

Enabling antibacterial coating via bioinspired mineralization of nanostructured ZnO on fabrics under mild conditions.

Herein, we present an environmentally benign method capable of mineralization and deposition of nanomaterials to introduce antibacterial functionalities into cotton fabrics under mild conditions. Similar to the way in which many naturally occurring biominerals evolve around the living organism under ambient conditions, this technique enables flexible substrates like the cotton fabric to be coated with inorganic-based functional materials. Specifically, our strategy involves the use of long-chain polyamines known to be responsible in certain biomineralization processes, to nucleate, organize, and deposit nanostructured ZnO on cotton bandage in an aqueous solution under mild conditions of room temperature and neutral pH. The ZnO-coated cotton bandages as characterized by SEM, confocal micro-Raman spectroscopy, XRD, UV-DRS, and fluorescence microscopy demonstrate the importance of polyamine in generating a stable and uniform coating of spindle-shaped ZnO particles on individual threads of the fabric. As the coating process requires only mild conditions, it avoids any adverse effect on the thermal and mechanical properties of the substrate. Furthermore, the ZnO particles on cotton fabric show efficient antibacterial activity against both gram-positive and gram-negetive bacteria. Therefore, the developed polyamine mediated bioinspired coating method provides not only a facile and "green" synthesis for coating on flexible substrate but also the fabrication of antibacterial enabled materials for healthcare applications.

Bio-inspired motifs via tandem assembly of polypeptides for mineralization of stable CaCO3 structures.

A macromolecular-assembly of polypeptides constructs a network of anionic and cationic charges vital for recognizing and coassembling Ca(2+) and CO(3)(2-) ions to mineralize and stabilize different mineral forms of CaCO(3) with core-shell or solid morphologies in an aqueous solution.

Controlled orientation in a bio-inspired assembly of Ag/AgCl/ZnO nanostructures enables enhancement in visible-light-induced photocatalytic performance.

In a bio-inspired approach, polyamine-mediated mineralization of ZnO was explored to develop an environmentally benign methodology for synthesizing Ag/AgCl/ZnO nanostructures. The assembling properties displayed by the polyamines to create composite structures was utilized to have the nanocomponents effectively interact with each other in a way that is desirable for the application envisaged. The polyamines, which act as a mineralizing agent for ZnO nanoparticles, also facilitate the formation of Ag/AgCl within ZnO under ambient conditions. Thus synthesized Ag/AgCl/ZnO nanostructures represent a multi-heterojunction system in which the nanocomponents lead in a synergistic way to enhancement in the photocatalytic activity under visible-light irradiation.

Morphology-controlled assembly of ZnO nanostructures: a bioinspired method and visible luminescence.

In a bioinspired methodology, positively charged polypeptides and polyamines directly catalyse ZnO mineralization under "green" conditions of room temperature and neutral pH. The polyamines not only act as mineralizing agents for the formation of ZnO nanoparticles, but also self-assemble the nanoparticles to form spindle-like morphologies at these very ambient conditions. Both the directional growth of ZnO and its luminescent property have a pH dependency. At higher pH, the ZnO shape changes to a rodlike morphology that exhibits green photoluminescence with different intensity than that for ZnO spindles.

Kinetics of dissociation of tris-(2,2'-bipyridyl) iron(II) in water solubilized by Triton X-100 reverse micelles.

The dissociation of tris-(2,2'-bipyridyl) iron(II) ([Fe(bipy)3]2+) has been studied in the Triton X-100/hexanol/cyclohexane reverse micellar medium. The reaction obeys simple first-order kinetics with no evidence of autoinhibition. The first-order rate constant (k1) has been determined at different values of W ([H2O]/[Triton X-100]). The rate (k1) decreases with increasing value of W. k1 also increases with increase in Triton X-100 concentration at constant values of W, showing that the reaction takes place at greater speed at the micellar interface. The kinetic results can be interpreted by the monomolecular pseudo-phase model. The effect of W on rate (k1) is more pronounced in the range of W from 1.55 to 4.2 but less pronounced at higher W. The reaction is further accelerated by Cl- and SCN- ions and the kinetic results provide evidence for the formation of ion pairs between the cation [Fe(bipy)3]2+ and each of these anions. The formation of such ion pairs has not been observed in aqueous medium but has been reported earlier in aqueous-alcohol mixtures. This result therefore provides evidence for the lower micropolarity of solubilized water compared to ordinary water.