Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration.

Engineered human cardiac tissues have been utilized for various biomedical applications, including drug testing, disease modeling, and regenerative medicine. However, the applications of cardiac tissues derived from human pluripotent stem cells are often limited due to their immaturity and lack of functionality. Therefore, in this study, we establish a perfusable culture system based on in vivo-like heart microenvironments to improve human cardiac tissue fabrication. The integrated culture platform of a microfluidic chip and a three-dimensional heart extracellular matrix enhances human cardiac tissue development and their structural and functional maturation. These tissues are comprised of cardiovascular lineage cells, including cardiomyocytes and cardiac fibroblasts derived from human induced pluripotent stem cells, as well as vascular endothelial cells. The resultant macroscale human cardiac tissues exhibit improved efficacy in drug testing (small molecules with various levels of arrhythmia risk), disease modeling (Long QT Syndrome and cardiac fibrosis), and regenerative therapy (myocardial infarction treatment). Therefore, our culture system can serve as a highly effective tissue-engineering platform to provide human cardiac tissues for versatile biomedical applications.

Direct cardiac reprogramming has emerged as a promising therapeutic approach for cardiac regeneration. Full chemical reprogramming with small molecules to generate cardiomyocytes may be more amenable than genetic reprogramming for clinical applications as it avoids safety concerns associated with genetic manipulations. However, challenges remain regarding low conversion efficiency and incomplete cardiomyocyte maturation. Furthermore, the therapeutic potential of chemically induced cardiomyocytes (CiCMs) has not been investigated. Here, we report that a three-dimensional microenvironment reconstituted with decellularized heart extracellular matrix can enhance chemical reprogramming and cardiac maturation of fibroblasts to cardiomyocytes. The resultant CiCMs exhibit elevated cardiac marker expression, sarcomeric organization, and improved electrophysiological features and drug responses. We investigated the therapeutic potential of CiCMs reprogrammed in three-dimensional heart extracellular matrix in a rat model of myocardial infarction. Our platform can facilitate the use of CiCMs for regenerative medicine, disease modeling, and drug screening.

As demands for new antibiotics and strategies to control methicillin-resistant Staphylococcus aureus (MRSA) increase, there have been efforts to obtain more accurate and abundant information about the mechanism of the bacterial responses to antibiotics. However, most of the previous studies have investigated responses to antibiotics without considering the genetic differences between MRSA and methicillin-susceptible S. aureus (MSSA). Here, we initially applied a multi-omics approach into the clinical isolates (i.e., S. aureus WKZ-1 (MSSA) and S. aureus WKZ-2 (MRSA)) that are isogenic except for the mobile genetic element called staphylococcal cassette chromosome mec (SCCmec) type IV to explore the response to β-lactam antibiotics (oxacillin). First, the isogenic pair showed a similar metabolism without oxacillin treatment. The quantitative proteomics demonstrated that proteins involved in peptidoglycan biosynthesis (MurZ, PBP2, SgtB, PrsA), two-component systems (VrsSR, WalR, SaeSR, AgrA), oxidative stress (MsrA1, MsrB), and stringent response (RelQ) were differentially regulated after the oxacillin treatment of the isogenic isolates. In addition, targeted metabolic profiling showed that metabolites belonging to the building blocks (lysine, glutamine, acetyl-CoA, UTP) of peptidoglycan biosynthesis machinery were specifically decreased in the oxacillin-treated MRSA. These results indicate that the difference in metabolism of this isogenic pair with oxacillin treatment could be caused only by SCCmec type IV. Understanding and investigating the antibiotic response at the molecular level can, therefore, provide insight into drug resistance mechanisms and new opportunities for antibiotics development.

Methicillin-resistant Staphylococcus aureus (MRSA), one of the most well-known human pathogens, houses many virulence factors and regulatory proteins that confer resistance to diverse antibiotics. Although they have been investigated intensively, the correlations among virulence factors, regulatory proteins and antibiotic resistance are still elusive. We aimed to identify the most significant global MRSA regulator by concurrently analyzing protein-binding and several promoters under same conditions and at the same time point. DNA affinity capture assay (DACA) was performed with the promoters of mecA, sarA, and sarR, all of which significantly impact survival of MRSA. Here, we show that SarA protein binds to all three promoters. Consistent with the previous reports, ΔsarA mutant exhibited weakened antibiotic resistance to oxacillin and reduced biofilm formation. Additionally, production and activity of many virulence factors such as phenol-soluble modulins (PSM), α-hemolysin, motility, staphyloxanthin, and other related proteins were decreased. Comparing the sequence of SarA with that of clinical strains of various lineages showed that all sequences were highly conserved, in contrast to that observed for AgrA, another major regulator of virulence and resistance in MRSA. We have demonstrated that SarA regulates antibiotic resistance and the expression of various virulence factors. Our results warrant that SarA could be a leading target for developing therapeutic agents against MRSA infections.

Gut microbiota, a complex microbial community inhabiting human or animal intestines recently regarded as an endocrine organ, has a significant impact on human health. Probiotics can modulate gut microbiota and the gut environment by releasing a range of bioactive compounds. Escherichia coli (E. coli) strain Nissle 1917 (EcN), a Gram-negative bacterial strain, has been used to treat gastrointestinal (GI) disorders (i.e., inflammatory bowel disease, diarrhea, ulcerative colitis, and so on). However, endotoxicity of lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria in the gut, is known to have a strong influence on gut inflammation and maintenance of gut homeostasis. Therefore, characterizing the chemical structure of lipid A which determines the toxicity of LPS is needed to understand nonpathogenic colonization and commensalism properties of EcN in the gut more precisely. In the present study, MALDI multiple-stage mass spectrometry analysis of lipid A extracted from EcN demonstrates that hexaacylated lipid A (m/z 1919.19) contains a glucosamine disaccharide backbone, a myristate, a laurate, four 3-hydroxylmyristates, two phosphates, and phosphoethanolamine (PEA). PEA modification of lipid A is known to contribute to cationic antimicrobial peptide (CAMP) resistance of Gram-negative bacteria. To confirm the role of PEA in CAMP resistance of EcN, minimum inhibitory concentrations (MICs) of polymyxin B and colistin were determined using a wild-type strain and a mutant strain with deletion of eptA gene encoding PEA transferase. Our results confirmed that MICs of polymyxin B and colistin for the wild-type were twice as high as those for the mutant. These results indicate that EcN can more efficiently colonize the intestine through PEA-mediated tolerance despite the presence of CAMPs in human gut such as human defensins. Thus, EcN can be used to help treat and prevent many GI disorders.