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To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFβ pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies. Autosomal recessive polycystic kidney disease (ARPKD) is a genetic disorder which is associated with kidney and liver pathology, including liver fibrosis. Here the authors develop and characterize human liver organoids with a ARPKD mutation, and find that they show aspects of the pathology, including fibrosis.

Background Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae . Results We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets. Conclusions Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.

Individual variation in pain sensation makes pain clinical trials quite challenging to interpret. Now, Michael Salter and colleagues report that genetic variation in the P2RX7 gene affects pore formation of the protein and pain sensation in humans. Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary 1 . Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da 2 , 3 . Using genome-wide linkage analyses, we discovered an association between nerve-injury–induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7 . Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.

Background ‘ Long read ’ sequencing methods have been used to identify previously uncharacterized structural variants that cause human genetic diseases. Therefore, we investigated whether long read sequencing could facilitate genetic analysis of murine models for human diseases. Results The genomes of six inbred strains (BTBR T + Itpr3tf/J, 129Sv1/J, C57BL/6/J, Balb/c/J, A/J, SJL/J) were analyzed using long read sequencing. Our results revealed that (i) Structural variants are very abundant within the genome of inbred strains (4.8 per gene) and (ii) that we cannot accurately infer whether structural variants are present using conventional short read genomic sequence data, even when nearby SNP alleles are known. The advantage of having a more complete map was demonstrated by analyzing the genomic sequence of BTBR mice. Based upon this analysis, knockin mice were generated and used to characterize a BTBR-unique 8-bp deletion within Draxin that contributes to the BTBR neuroanatomic abnormalities, which resemble human autism spectrum disorder. Conclusion A more complete map of the pattern of genetic variation among inbred strains, which is produced by long read genomic sequencing of the genomes of additional inbred strains, could facilitate genetic discovery when murine models of human diseases are analyzed.

Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to these response differences, here we analyze in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis indicates that the p53 family protein, p73, affects Th1 differentiation. In cells differentiated under Th1 conditions in vitro, p73 negatively regulates IFNγ production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes such as Ifng and Il12rb2 . Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice have increased IFNγ production and less disease severity, whereas in an adoptive transfer model of inflammatory bowel disease, transfer of p73-deficient naïve CD4 + T cells increases Th1 responses and augments disease severity. Our results thus identify p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease. Heterogeneous helper T (Th) cell responses contribute to differential susceptibility to immunological disorders. Here the authors perform haplotype-based computational screens of 16 inbred mouse strains to identify a transcription factor, p73, as an important negative regulator of Th1 differentiation, with p73 deficient mice manifesting alterations in two inflammatory disease models.

Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants. Please see later in the article for the Editors' Summary.

Ondansetron is an anti‐emetic 5‐HT3 receptor antagonist being investigated for treating neonatal opioid withdrawal syndrome (NOWS). Sparse PK data were analyzed from a multicenter, double‐blind clinical trial with 98 mother/neonate dyads. Pregnant women with opioid use disorder were randomized to receive either placebo or ondansetron 8 mg intravenously within 4 h of delivery. Neonates born to mothers who were randomized to ondansetron received 0.07 mg/kg orally once every 24 h for up to five doses. Using current PK data, model parameters from a two‐compartmental structural model from the literature (i.e., a priori model) were updated with the Metropolis‐Hastings Markov‐chain Monte Carlo estimation algorithm in NONMEM. The updated Bayesian model indicated a slower absorption rate (KA) but no differences in model parameters (CL, V, V2, Q) after including body weight and postmenstrual age. Sensitivity analyses on CL prior revealed statistical improvement favoring larger body weights, but not changes in postmenstrual age. However, further model development using larger body weights did not illustrate superior performance through visual inspection of diagnostic plots. Overall, a cumulative AUC of at least 1000 ng*h/mL appears to be the threshold for reductions in symptom severity. Exposure‐response analyses suggest the total number of doses to be the primary driver for efficacy with respect to AUC, which reasonably aligns with the literature. Overall, it is suggested that at least three doses of the current oral ondansetron regimen are required to reduce symptom severity in neonates.

Administration of a widely used 5‐hydroxytryptamine receptor (5HT3AR) antagonist (ondansetron) potently inhibited the development of experimentally induced opioid dependence and withdrawal responses in mice and humans. However, in several studies examining withdrawal symptoms in subjects with chronic opioid use disorders (OUDs), ondansetron exhibited reduced or absent efficacy. Because attenuation of opioid withdrawal symptomatology is mediated within the brain, this study examined single‐dose ondansetron pharmacokinetics in the blood and brain of mice. We demonstrate that ondansetron concentrations in the brain (Cbrain ng/mg) are 1000‐fold lower than the blood concentrations (Cblood ng/ml) and decrease rapidly after ondansetron administration; and that a large percentage of brain ondansetron remains in the ventricular fluid. These results indicate that the ondansetron dose, and the time window between ondansetron and opioid administration, and when withdrawal is assessed are critical considerations for clinical studies involving subjects with chronic OUD. The pharmacokinetic results and the dosing considerations discussed here can be used to improve the design of subsequent clinical trials, which will test whether a more prolonged period of ondansetron administration can provide a desperately needed therapy that can prevent the development of neonatal opioid withdrawal syndrome in babies born to mothers with chronic OUD.

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin‐like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue‐derived, epithelial‐only intestinal organoids. HELP enables the encapsulation of dissociated patient‐derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal‐derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin‐ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid–matrix interactions and potential patient‐specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G’ ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10−3–1 × 10−3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal‐derived products or synthetic polyethylene glycol for potential clinical translation. A tunable, designer matrix, termed hyaluronan elastin‐like protein (HELP) that enables the formation, differentiation, and passaging of adult primary tissue‐derived organoids is reported. HELP matrices allow stiffness, stress relaxation rate, and integrin‐ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid–matrix interactions and potential patient‐specific optimization.

Nonalcoholic fatty liver disease (NAFLD) is characterized by increased lipid accumulation and excessive deposition of extracellular matrix (ECM) that results in tissue stiffening. The potential interplay between matrix stiffness and hepatocyte lipid accumulation during NAFLD has not been established. Here, an in vitro NAFLD model is developed using chemically defined, engineered hydrogels and human induced pluripotent stem cell‐derived hepatic organoids (HOs). Specifically, dynamic covalent chemistry crosslinking, along with transient small molecule competitors, are used to create dynamic stiffening hydrogels that enable the reproducible culture of HOs. Within matrices that mimic the stiffness of healthy to diseased tissue (≈1–6 kPa), lipid droplet accumulation in HOs is triggered by exposure to an NAFLD‐associated free fatty acid. These NAFLD model suggests that higher stiffness microenvironments result in increased hepatic lipid droplet accumulation, increased expression of fibrosis markers, and increased metabolic dysregulation. By targeting the ROCK mechanosignaling pathway, the synergy between matrix stiffness and lipid droplet accumulation is disrupted. The in vitro model of NAFLD has the potential to understand the role of mechanosignaling in disease progression and identify new pathways for therapeutic intervention. Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation and extracellular matrix stiffening. An in vitro NAFLD model is developed using engineered hydrogels (G′ ≈ 1–6 kPa) and iPSC‐derived hepatic organoids. It is demonstrated that higher stiffness can increase lipid droplet accumulation, fibrosis, and metabolic dysregulation, which can be reversible with ROCK inhibitor Y27632. This model highlights mechanosignaling's role in NAFLD progression and potential therapeutic targets.