Searching for Suitable Kojic Acid Coformers: From Cocrystals and Salt to Eutectics.

The possibility of obtaining cocrystals of kojic acid with organic coformers has been investigated by both computational and experimental approaches. Cocrystallization attempts have been carried out with about 50 coformers, in different stoichiometric ratios, by solution, slurry, and mechanochemical methods. Cocrystals were obtained with 3-hydroxybenzoic acid, imidazole, 4-pyridone, DABCO, and urotropine, while piperazine yielded a salt with the kojiate anion; cocrystallization with theophylline and 4-aminopyridine resulted in stoichiometric crystalline complexes that could not be described with certainty as cocrystals or salts. In the cases of panthenol, nicotinamide, urea, and salicylic acid the eutectic systems with kojic acid were investigated via differential scanning calorimetry. In all other preparations the resulting materials were constituted of a mixture of the reactants. All compounds were investigated by powder X-ray diffraction; the five cocrystals and the salt were fully characterized via single crystal X-ray diffraction. The stability of the cocrystals and the intermolecular interactions in all characterized compounds have been investigated by computational methods based on the electronic structure and pairwise energy calculations, respectively.

Exploring the Co-Crystallization of Kojic Acid with Silver(I), Copper(II), Zinc(II), and Gallium(III) for Potential Antibacterial Applications.

Co-crystallization of kojic acid (HKA) with silver(I), copper(II), zinc(II), or gallium(III) salts yielded three 1D coordination polymers and one 0D complex in which kojic acid was present as a neutral or anionic terminal or bridging ligand. All reactions were conducted mechanochemically via ball milling and manual grinding, or via slurry. All solids were fully characterized via single-crystal and/or powder X-ray diffraction. As kojic acid is a mild antimicrobial compound that is widely used in cosmetics, and the metal cations possess antibacterial properties, their combinations were tested for potential antibacterial applications. The minimal inhibition concentrations (MICs) and minimal biocidal concentrations (MBCs) for all compounds were measured against standard strains of the bacteria P. aeruginosa, S. aureus, and E. coli. All compounds exerted appreciable antimicrobial activity in the order of silver, zinc, copper, and gallium complexes.

Humanized mice for investigating SARS-CoV-2 lung infection and associated human immune responses.

There is an urgent need for animal models of coronavirus disease 2019 to study immunopathogenesis and test therapeutic intervenes. In this study, we showed that NOD/SCID IL2rg-/- (NSG) mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-Sequencing identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, IFN stimulation and pulmonary fibrosis, between HL grafts from infected and control NSG-L mice. Furthermore, when infected with SARS-CoV-2, humanized mice with both human immune system (HIS) and autologous HL grafts (HISL mice) had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.

Proteomic and Metabolomic Characterization of SARS-CoV-2-Infected Cynomolgus Macaque at Early Stage.

Although tremendous effort has been exerted to elucidate the pathogenesis of severe COVID-19 cases, the detailed mechanism of moderate cases, which accounts for 90% of all patients, remains unclear yet, partly limited by lacking the biopsy tissues. Here, we established the COVID-19 infection model in cynomolgus macaques (CMs), monitored the clinical and pathological features, and analyzed underlying pathogenic mechanisms at early infection stage by performing proteomic and metabolomic profiling of lung tissues and sera samples from COVID-19 CMs models. Our data demonstrated that innate immune response, neutrophile and platelet activation were mainly dysregulated in COVID-19 CMs. The symptom of neutrophilia, lymphopenia and massive "cytokines storm", main features of severe COVID-19 patients, were greatly weakened in most of the challenged CMs, which are more semblable as moderate patients. Thus, COVID-19 model in CMs is rational to understand the pathogenesis of moderate COVID-19 and may be a candidate model to assess the safety and efficacy of therapeutics and vaccines against SARS-CoV-2 infection.

Steps towards a nature inspired inorganic crystal engineering.

This Perspective outlines the results obtained at the University of Bologna by applying crystal engineering strategies to develop nature inspired organic-inorganic materials to tackle challenges in the health and environment sectors. It is shown by means of a number of examples that co-crystallization of inorganic salts, such as alkali and transition metal halides, with organic compounds, such as amino acids, urea, thiourea and quaternary ammonium salts, can be successfully used for (i) chiral resolution and conglomerate formation from racemic compounds, (ii) inhibition of soil enzyme activity in order to reduce urea decomposition and environmental pollution, and (iii) preparation of novel agents to tackle antimicrobial resistance. All materials described in this Perspective have been obtained by mechanochemical solvent-free or slurry methods and characterized by solid state techniques. The fundamental idea is that a crystal engineering approach based on the choice of intermolecular interactions (coordination and hydrogen bonds) between organic and inorganic compounds allows obtaining materials with collective properties that are different, and often very much superior to those of the separate components. It is also demonstrated that the success of this strategy depends crucially on cross-disciplinary synergistic exchange with expert scientists in the areas of bioinorganics, microbiology, and chirality application-oriented developments of these novel materials.

Generation of immunodeficient pig with hereditary tyrosinemia type 1 and their preliminary application for humanized liver.

BACKGROUND: Mice with humanized livers are important models to study drug toxicology testing, development of hepatitis virus treatments, and hepatocyte transplantation therapy. However, the huge difference between mouse and human in size and anatomy limited the application of humanized mice in investigating human diseases. Therefore, it is urgent to construct humanized livers in pigs to precisely investigate hepatocyte regeneration and human hepatocyte therapy. CRISPR/Cas9 system and somatic cell cloning technology were used to generate two pig models with FAH deficiency and exhibiting severe immunodeficiency (FAH/RAG1 and FAH/RAG1/IL2RG deficiency). Human primary hepatocytes were then successfully transplanted into the FG pig model and constructed two pigs with human liver. RESULTS: The constructed FAH/RAG1/IL2RG triple-knockout pig models were characterized by chronic liver injury and severe immunodeficiency. Importantly, the FG pigs transplanted with primary human hepatocytes produced human albumin in a time dependent manner as early as 1 week after transplantation. Furthermore, the colonization of human hepatocytes was confirmed by immunochemistry staining. CONCLUSIONS: We successfully generated pig models with severe immunodeficiency that could construct human liver tissues.

Incompatibility between recipient CD47 and donor SIRPα is not a key risk factor for thrombocytopenia or anemia following rat liver xenotransplantation in mice.

Liver xenotransplantation (LXT) is greatly impeded by severe thrombocytopenia, anemia, and coagulopathy. Hepatic phagocytic cells are thought to play an important role in LXT-induced thrombocytopenia and anemia. In this study, we investigated whether the lack of recipient CD47-donor SIRPα interaction, which is known to induce xenograft rejection by macrophages, exacerbates platelet and RBC depletion following LXT. We first addressed this question in the absence of anti-donor immune responses using a syngeneic mouse liver transplantation (LT) model. Neither wild-type (WT) nor CD47KO B6 mice developed thrombocytopenia following LT from WT B6 donors. Although a moderate decline in RBCs was detected following LT, there was no significant difference in RBC counts between WT and CD47KO recipients. Because mouse CD47 is cross-reactive with rat SIRPα, we then compared thrombocytopenia and anemia between WT and CD47KO mice following rat LXT. Unlike syngeneic mouse LT, significant thrombocytopenia and anemia were detected following rat LXT. However, the severities of both platelet and RBC depletions were comparable between WT and CD47KO recipients. Furthermore, WT and CD47KO recipients showed a similar extent of early platelet activation. Our results indicate that CD47-SIRPα signaling does not significantly affect the loss of platelets or RBCs following LXT, suggesting that the limited cross-reactivity between recipient CD47 and donor SIRPα is not a significant risk factor for LXT-induced thrombocytopenia and anemia.

IL2RG-deficient minipigs generated via CRISPR/Cas9 technology support the growth of human melanoma-derived tumours.

OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development. MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice. CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.

Different effects of lysophosphatidic acid on L-type calcium current in neonatal rat ventricular myocytes with and without H2O2 treatment.

L-type calcium current (I(Ca-L)) alterations are implicated in various cardiac diseases, and the lysophosphatidic acid (LPA) level increases in several ischemic heart diseases. We investigated the effects of LPA on I(Ca-L) in normal and H2O2-treated neonatal rat ventricular myocytes. LPA treatment (24h) increased the action potential duration (APD) and I(Ca-L) in normal ventricular myocytes, but it decreased these parameters in H2O2-treated myocytes. LPA increased the single-channel open probability of L-type calcium channels in both normal and H2O2-treated myocytes. LPA activated calcineurin (CaN) and induced the cytoplasm-to-nucleus translocation of nuclear factor of activated T-cells (NFAT) in H2O2-treated cardiomyocytes. In H2O2-treated cardiomyocytes, LPA decreased Ca(v)1.2 mRNA and protein expression levels at 4 and 8h, respectively. A CaN inhibitor (FK-506) prevented LPA-induced APD, I(Ca-L), and Ca(v)1.2 mRNA and protein down-regulation. The LPA-induced I(Ca-L) increase in normal cardiomyocytes was CaN-NFAT signaling-independent, and LPA did not affect Ca(v)1.2 mRNA or protein expression. In conclusion, LPA increases the I(Ca-L) in normal ventricular myocytes by increasing the single-channel open probability of L-type calcium channels, and LPA decreases I(Ca-L) in H2O2-treated cardiomyocytes via the CaN-NFAT pathway.