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Background and AimsDrug-induced liver injury (DILI), both intrinsic and idiosyncratic, causes frequent morbidity, mortality, clinical trial failures and post-approval withdrawal. This suggests an unmet need for improved in vitro models for DILI risk prediction that can account for diverse host genetics and other clinical factors. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system.MethodsHLOs were derived from 3 separate iPSC lines and benchmarked on two platforms for their ability to model in vitro liver function and identify hepatotoxic compounds using biochemical assays for albumin, ALT, and AST, microscopy-based morphological profiling, and single-cell transcriptomics: 1) HLOs dispersed in 384-well formatted plates and exposed to a library of compounds. 2) HLOs adapted to a liver-on-chip system.ResultsDispersed HLOs derived from the 3 iPSC lines had similar DILI predictive capacity to intact HLOs in a high-throughput screening format allowing for measurable IC50 values of compound cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of toxicity.On-chip HLOs significantly increased albumin production, CYP450 expression, and ALT/AST release when treated with known DILI drugs compared to dispersed HLOs and primary human hepatocytes. On-chip HLOs were able to predict the synergistic hepatotoxicity of tenofovir-inarigivir and showed steatosis and mitochondrial perturbation via phenotypic and transcriptomic analysis with exposure to FIAU and acetaminophen, respectively.ConclusionsThe high throughput and liver-on-chip system exhibit enhanced in vivo-like function and demonstrate the potential utility of these platforms for hepatotoxicity risk assessment. Tenofovir-inarigivr associated hepatotoxicity was observed and correlates with the clinical manifestation of DILI observed in patients.LAY SUMMARYIdiosyncratic (spontaneous, patient-specific) drug-induced liver injury (DILI) is difficult to study due to the lack of liver models that function as human liver tissue and are adaptable for large-scale drug screening. Human liver organoids grown from patient stem cells respond to known DILI-causing drugs in both a high-throughput and on a physiological “chip” culture system. These platforms show promise in their use as predictive model for novel drugs before entering clinical trials.

Dbait is a small double-stranded DNA molecule that has been utilized as a radiosensitizer to enhance the sensitivity of glioma to radiotherapy (RT). However, there is no effective drug delivery system to effectively overcome the blood—brain barrier (BBB). The aim of this study was to develop a gene delivery system by using the BBB and glioma dual-targeting and microenvironment-responsive micelles (ch-Kn(s-s)R8-An) to deliver Dbait into glioma for RT. Angiopep-2 can target the low-density lipoprotein receptor-related protein-1 (LRP1) that is overexpressed on brain capillary endothelial cells (BCECs) and glioma cells. In particular, due to upregulated matrix metalloproteinase 2 (MMP-2) in the tumor microenvironment, we utilized MMP-2-responsive peptides as the enzymatically degradable linkers to conjugate angiopep-2. The results showed that ch-Kn(s-s)R8-An micelles maintained a reasonable size (80–160 nm) with a moderate distribution and a decreased mean diameter from the cross-linking as well as exhibited low critical micelle concentration (CMC) with positive surface charge, ranging from 15 to 40 mV. The ch-K5(s-s)R8-An/pEGFP showed high gene transfection efficiency in vitro, improved uptake in glioma cells and good biocompatibility in vitro and in vivo. In addition, the combination of ch-K5(s-s)R8-An/Dbait with RT significantly inhibited the growth of U251 cells in vitro. Thus, ch-K5(s-s)R8-An/Dbait may prove to be a promising gene delivery system to target glioma and enhance the efficacy of RT on U251 cells., Graphical abstract Using Dbait as a radiosensitizer, dual-targeting and microenvironment-responsive micelle is developed as a gene delivery system to improve sensitivity of glioma to radiotherapy (RT). The micelle can effectively target glioma and further penetrate into the core of the tumor through exposing R8 to deliver Dbait into glioma cells.fx1

SummaryEuropean Americans (EA) are more susceptible to esophageal tissue damage and inflammation when exposed to gastric acid and bile acid reflux and have a higher incidence of esophageal adenocarcinoma when compared to African Americans (AA). Population studies have implicated specific genes for these differences; however, the underlying cause for these differences is not well understood. We describe a robust long-term culture system to grow primary human esophagus in vitro, use single cell RNA sequencing to compare primary human biopsies to their in vitro counterparts, identify known and new molecular markers of basal cell types, and demonstrate that in vivo cellular heterogeneity is maintained in vitro. We further developed an ancestrally diverse biobank and a high-content, image based, screening assay to interrogate bile-acid injury response. These results demonstrated that AA esophageal cells responded significantly differently than EA-derived cells, mirroring clinical findings, having important implications for addressing disparities in early drug development pipelines.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

Nonsynonymous mutations are well documented in TEM β-lactamases. The resulting amino acid changes often alter the conferred phenotype from broad spectrum (2b) conferred by TEM-1 to extended spectrum (2be), inhibitor resistant (2br), or both extended spectrum and inhibitor resistant (2ber). The encoding bla(TEM) genes also deviate in numerous synonymous mutations, which are not well understood. bla(TEM-3) (2be), bla(TEM-33) (2br), and bla(TEM-109) (2ber) were studied in comparison to bla(TEM-1). bla(TEM-33) was chosen for more detailed studies because it deviates from bla(TEM-1) by a single nonsynonymous mutation and three additional synonymous mutations. Genes encoding the enzymes with only nonsynonymous or all (including synonymous) mutations plus all permutations between bla(TEM-1) and bla(TEM-33) were expressed in Escherichia coli cells. In disc diffusion assays, genes encoding TEM-3, TEM-33, and TEM-109 with all synonymous mutations resulted in higher resistance levels than genes without synonymous mutations. Disc diffusion assays with the 16 genes carrying all possible nucleotide change combinations between bla(TEM-1) and bla(TEM-33) indicated different susceptibilities for different variants. Nucleotide BLAST searches did not identify genes without synonymous mutations but did identify some without nonsynonymous mutations. Energies of possible secondary mRNA structures calculated with mfold are generally higher with synonymous mutations, suggesting that their role could be to destabilize the mRNA and facilitate its unfolding for efficient translation. In summary, our data indicate that transition from bla(TEM-1) to other variant genes by simply acquiring the nonsynonymous mutations is not favored. Instead, synonymous mutations seem to support the transition to other variant genes with nonsynonymous mutations leading to different phenotypes.

The global rise of metallo-β-lactamases (MBLs) is problematic due to their ability to inactivate most β-lactam antibiotics. MBL inhibitors that could be co-administered with and restore the efficacy of β-lactams are highly sought after. In this study, we employ virtual screening of candidate MBL inhibitors without thiols or carboxylates to avoid off-target effects using the Avalanche software package, followed by experimental validation of the selected compounds. As target enzymes we chose the clinically relevant B1 MBLs NDM-1, IMP-1, and VIM-2. Among 32 compounds selected from a ~1.5 million compound library, 6 exhibited IC(50) values < 40 μM against NDM-1 and/or IMP-1. The most potent inhibitors of NDM-1, IMP-1, and VIM-2 had IC(50) values of 19 ± 2 μM, 14 ± 1 μM, and 50 ± 20 μM, respectively. While chemically diverse, the most potent inhibitors all contain combinations of hydroxyl, ketone, ester, amide, or sulfonyl groups. Docking studies suggest that these electron-dense moieties are involved in Zn(II) coordination and interaction with protein residues. These novel scaffolds could serve as the basis for further development of MBL inhibitors. A procedure for renaming NDM-1 residues to conform to the class B β-lactamase (BBL) numbering scheme is also included.

(1) Background: Metallo-&beta, lactamases (MBLs) have raised concerns due to their ability to inactivate carbapenems and newer generation cephalosporins and the absence of clinically available MBL inhibitors. Their genes are often transferred horizontally, and the number of MBL variants has grown exponentially, with many newer variants showing enhanced enzyme activity or stability. In this study, we investigated a closely related group of variants from the IMP family that all contain the combination of mutations S115T and S119G relative to IMP-1. (2) Methods: The effects of each individual mutation and their combination in the IMP-1 sequence background in comparison to IMP-1 were investigated. Their ability to confer resistance and their in-cell expression levels were determined. All enzymes were purified, and their secondary structure and thermal stability were determined with circular dichroism. Their Zn(II) content and kinetic constants with a panel of &beta, lactam antibiotics were determined. (3) Results: All four enzymes were viable and conferred resistance to all antibiotics tested except aztreonam. However, the single-mutant enzymes were slightly deficient, IMP-1S115T due to decreased enzyme activity and IMP-1-S119G due to decreased thermal stability and expression, while the double mutant did not show these defects. (4) Conclusions: These observations suggest that S119G was acquired due to its increased enzyme activity and S115T to suppress the thermal stability and expression defect introduced by S119G.

Significance Since its emergence in China in December 2019, SARS-CoV-2 has caused a global pandemic. Repurposing of FDA-approved drugs is a promising strategy for identifying rapidly deployable treatments for COVID-19. Herein, we developed a pipeline for quantitative, high-throughput, image-based screening of SARS-CoV-2 infection in human cells that led to the identification of several FDA-approved drugs and clinical candidates with in vitro antiviral activity., The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.