In pursuit of degenerative brain disease diagnosis: Dementia biomarkers detected by DNA aptamer-attached portable graphene biosensor.

Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aß), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aß, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aß, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.

Development of neonatal connectome dynamics and its prediction for cognitive and language outcomes at age 2.

The functional brain connectome is highly dynamic over time. However, how brain connectome dynamics evolves during the third trimester of pregnancy and is associated with later cognitive growth remains unknown. Here, we use resting-state functional Magnetic Resonance Imaging (MRI) data from 39 newborns aged 32 to 42 postmenstrual weeks to investigate the maturation process of connectome dynamics and its role in predicting neurocognitive outcomes at 2 years of age. Neonatal brain dynamics is assessed using a multilayer network model. Network dynamics decreases globally but increases in both modularity and diversity with development. Regionally, module switching decreases with development primarily in the lateral precentral gyrus, medial temporal lobe, and subcortical areas, with a higher growth rate in primary regions than in association regions. Support vector regression reveals that neonatal connectome dynamics is predictive of individual cognitive and language abilities at 2  years of age. Our findings highlight network-level neural substrates underlying early cognitive development.

Ultrasensitive and Highly Selective Detection of Staphylococcus aureus at the Single-Cell Level Using Bacteria-Imprinted Polymer and Vancomycin-Conjugated MnO2 Nanozyme.

Pathogenic bacterial infections, even at extremely low concentrations, pose significant threats to human health. However, the challenge persists in achieving high-sensitivity bacterial detection, particularly in complex samples. Herein, we present a novel sandwich-type electrochemical sensor utilizing bacteria-imprinted polymer (BIP) coupled with vancomycin-conjugated MnO2 nanozyme (Van@BSA-MnO2) for the ultrasensitive detection of pathogenic bacteria, exemplified by Staphylococcus aureus (S. aureus). The BIP, in situ prepared on the electrode surface, acts as a highly specific capture probe by replicating the surface features of S. aureus. Vancomycin (Van), known for its affinity to bacterial cell walls, is conjugated with a Bovine serum albumin (BSA)-templated MnO2 nanozyme through EDC/NHS chemistry. The resulting Van@BSA-MnO2 complex, serving as a detection probe, provides an efficient catalytic platform for signal amplification. Upon binding with the captured S. aureus, the Van@BSA-MnO2 complex catalyzes a substrate reaction, generating a current signal proportional to the target bacterial concentration. The sensor displays remarkable sensitivity, capable of detecting a single bacterial cell in a phosphate buffer solution. Even in complex milk matrices, it maintains outstanding performance, identifying S. aureus at concentrations as low as 10 CFU mL-1 without requiring intricate sample pretreatment. Moreover, the sensor demonstrates excellent selectivity, particularly in distinguishing target S. aureus from interfering bacteria of the same genus at concentrations 100-fold higher. This innovative method, employing entirely synthetic materials, provides a versatile and low-cost detection platform for Gram-positive bacteria. In comparison to existing nanozyme-based bacterial sensors with biological recognition materials, our assay offers distinct advantages, including enhanced sensitivity, ease of preparation, and cost-effectiveness, thereby holding significant promise for applications in food safety and environmental monitoring.

NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Chromosome-scale genome assembly-assisted identification of Mi-9 gene in Solanum arcanum accession LA2157, conferring heat-stable resistance to Meloidogyne incognita.

Root-knot nematodes (RKNs) are infamous plant pathogens in tomato production, causing considerable losses in agriculture worldwide. Mi-1 is the only commercially available RKN-resistance gene; however, the resistance is inactivated when the soil temperature is over 28 °C. Mi-9 in wild tomato (Solanum arcanum LA2157) has stable resistance to RKNs under high temperature but has not been cloned and applied. In this study, a chromosome-scale genome assembly of S. arcanum LA2157 was constructed through Nanopore and Hi-C sequencing. Based on molecular markers of Mi-9 and comparative genomic analysis, the localization region and candidate Mi-9 genes cluster consisting of seven nucleotide-binding sites and leucine-rich repeat (NBS-LRR) genes were located. Transcriptional expression profiles confirmed that five of the seven candidate genes were expressed in root tissue. Moreover, virus-induced gene silencing of the Sarc_034200 gene resulted in increased susceptibility of S. arcanum LA2157 to Meloidogyne incognita, and genetic transformation of the Sarc_034200 gene in susceptible Solanum pimpinellifolium conferred significant resistance to M. incognita at 25 °C and 30 °C and showed hypersensitive responses at nematode infection sites. This suggested that Sarc_034200 is the Mi-9 gene. In summary, we cloned, confirmed and applied the heat-stable RKN-resistance gene Mi-9, which is of great significance to tomato breeding for nematode resistance.

Meloidogyne enterolobii MeMSP1 effector targets the glutathione-S-transferase phi GSTF family in Arabidopsis to manipulate host metabolism and promote nematode parasitism.

Meloidogyne enterolobii is an emerging root-knot nematode species that overcomes most of the nematode resistance genes in crops. Nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we show the MeMSP1 effector is secreted into giant cells and promotes M. enterolobii parasitism. Using co-immunoprecipitation and bimolecular fluorescent complementation assays, we identified glutathione-S-transferase phi GSTFs as host targets of the MeMSP1 effector. This protein family plays important roles in plant responses to abiotic and biotic stresses. We demonstrate that MeMSP1 interacts with all Arabidopsis GSTF. Moreover, we confirmed that the N-terminal region of AtGSTF9 is critical for its interaction, and atgstf9 mutant lines are more susceptible to root-knot nematode infection. Combined transcriptome and metabolome analyses showed that MeMSP1 affects the metabolic pathways of Arabidopsis thaliana, resulting in the accumulation of amino acids, nucleic acids, and their metabolites, and organic acids and the downregulation of flavonoids. Our study has shed light on a novel effector mechanism that targets plant metabolism, reducing the production of plant defence-related compounds while favouring the accumulation of metabolites beneficial to the nematode, and thereby promoting parasitism.

An adaptive three-dimensional hydrodynamic focusing microfluidic impedance flow cytometer.

Microfluidic impedance cytometry is emerging as a label-free, low-cost and portable solution for cell analysis. Impedance-based cell or particle characterization is provided by microfluidic and electronic devices. We report the design and characterization of a miniaturized flow cytometer based on a 3-dimensional (3D) hydrodynamic focusing mechanism. A sheath adaptively concentrated the sample laterally and vertically at the bottom of the microchannel, reducing the variance of particle translocation height and increasing the signal-to-noise ratio of the particle impedance pulse. Through simulation and confocal microscopy experiments, it has been verified that an increase in the ratio of sheath to sample decreased the cross-sectional area of the concentrated stream, which can be reduced to 26.50% of the pre-focusing value. The appropriate sheath flow settings increased the impedance pulse amplitude for different particles, and the coefficient of variation reduced by at least 35.85%, contributing to a more accurate representation of the particle impedance characteristic distribution. The system displayed the difference of HepG2 cell impedance before and after drug treatment, which is consistent with the results of flow cytometry, providing a convenient and inexpensive solution for monitoring cell status.

An integrated microfluidic chip for nucleic acid extraction and continued cdPCR detection of pathogens.

This paper introduces an enclosed microfluidic chip that integrates sample preparation and the chamber-based digital polymerase chain reaction (cdPCR). The sample preparation of the chip includes nucleic acid extraction and purification based on magnetic beads, which adsorb nucleic acids by moving around the reaction chambers to complete the reactions including lysis, washing, and elution. The cdPCR area of the chip consists of tens of thousands of regularly arranged microchambers. After the sample preparation processes are completed, the purified nucleic acid can be directly introduced into the microchambers for amplification and detection on the chip. The nucleic acid extraction performance and digital quantification performance of the system were examined using synthetic SARS-CoV-2 plasmid templates at concentrations ranging from 101-105 copies per µL. Further on, a simulated clinical sample was used to test the system, and the integrated chip was able to accurately detect SARS-CoV-2 virus particle samples doped with interference (saliva) with a detection limit of 10 copies per µL. This integrated system could provide a promising tool for point-of-care testing of pathogenic infections.

A microfluidic immunosensor for automatic detection of carcinoembryonic antigen based on immunomagnetic separation and droplet arrays.

Diagnosis of cancer by biomarkers plays an important role in human health and life. However, current laboratory techniques for detecting cancer biomarkers still require laborious and time-consuming operation by skilled operators and associated laboratory instruments. This work presents a colorimetric biosensor for the rapid and sensitive detection of carcinoembryonic antigen (CEA) based on an automated immunomagnetic separation platform and a droplet array microfluidic chip with the aid of an image analysis system. Immunomagnetic nanoparticles (MNPs) were used to capture CEA in the samples. CEA-detecting antibodies and horseradish peroxidase (HRP) were modified on polystyrene microspheres (PS), catalysing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) as signal outputs. Color reaction data were analyzed to establish a CEA concentration standard curve. The movement of MNPs between droplets in the microfluidic chip is achieved using an automatically programmable magnetic control system. This colorimetric biosensor has been used for the simultaneous detection of six CEA samples ranging from 100 pg mL-1 to 100 ng mL-1 with a detection limit of 14.347 pg mL-1 in 10 min, following the linear equation: y = -4.773 ln(x) + 156.26 with a correlation of R2 = 0.9924, and the entire workflow can be completed within 80 minutes. The microfluidic immunosensor designed in this paper has the advantages of low cost, automation, low sample consumption, high throughput, and promising applications in biochemistry.

Methylorubrum rhodesianum M520 as a biocontrol agent against Meloidogyne incognita (Tylenchida: Heteroderidae) J2s infecting cucumber roots.

AIMS: Root-knot nematodes (RKNs) are plant pathogens that cause huge economic losses worldwide. The biological management of RKNs may be a sustainable alternative to chemical control methods. Here, the biocontrol potential of Methylorubrum rhodesianum M520 against the RKN Meloidogyne incognita was investigated to theoretically support its application as a biocontrol agent in field production. METHODS AND RESULTS: In-vitro assays showed 91.9% mortality of M. incognita second-stage juveniles in the presence of strain M520 and that the hatching rate of M. incognita eggs was 21.7% lower than that of eggs treated with sterile water. In pot experiments, the M520 treatment caused 70.8% reduction in root-knots and increased plant shoot length and stem and root fresh weights, compared to control plant values. In split-root experiments, cucumber roots treated with M520 showed 25.6% decrease in root gall number, compared to that in control roots. CONCLUSION: M520 has multiple mechanisms against RKNs and might be used as a biocontrol agent against M. incognita in cucumber, laying a foundation for further studying M520 biocontrol against RKNs.

The Genome of Fusarium oxysporum f. sp. phaseoli Provides Insight into the Evolution of Genomes and Effectors of Fusarium oxysporum Species.

Fusarium oxysporum f. sp. phaseoli, the causal agent of cowpea fusarium wilt, is a serious threat to cowpea production in China. In this study, a sample of cowpea fusarium wilt was identified as Fusarium oxysporum f. sp. phaseoli using the methods of morphological characters and molecular detection. We further reported the first genome assembly for Fusarium oxysporum f. sp. phaseoli, with 53.7 Mb genome sequence comprising 14,694 genes. Comparative genomic analysis among five Fusarium oxysporum genomes showed that four accessory chromosomes in the five Fusarium oxysporum display similar characteristics, with low sequence similarity (55.35%, vs. overall average of 81.76%), low gene density (2.18 genes/10 kb vs. 3.02 genes/Mb) and highly transposable element density (TEs) (15.01/100 kb vs. 4.89/100 kb), indicating that variable accessory chromosomes are the main source of Fusarium oxysporum evolution. We identified a total of 100 Fusarium oxysporum f. sp. phaseoli-specific effectors in the genome and found 13 specific effector genes located in large insertion or deletion regions, suggesting that insertion or deletion events can cause the emergence of species-specific effectors in Fusarium oxysporum. Our genome assembly of Fusarium oxysporum f. sp. phaseoli provides a valuable resource for the study of cowpea fusarium wilt, and the comparative genomic study of Fusarium oxysporum could contribute to the knowledge of genome and effector-associated pathogenicity evolution in Fusarium oxysporum study.

High-quality chromosome-level genomes of Cucumis metuliferus and Cucumis melo provide insight into Cucumis genome evolution.

Cucumis metuliferus (African horned cucumber), a wild relative of Cucumis sativus (cucumber) and Cucumis melo (melon), displays high-level resistance to several important plant pathogens (e.g., root-knot nematodes and several viruses). Here, we report a chromosome-level genome assembly for C. metuliferus, with a 316 Mb genome sequence comprising 29 039 genes. Phylogenetic analysis of related species in family Cucurbitaceae indicated that the divergence time between C. metuliferus and melon was 17.8 million years ago. Comparisons between the C. metuliferus and melon genomes revealed large structural variations (inversions and translocations >1 Mb) in eight chromosomes of these two species. Gene family comparison showed that C. metuliferus has the largest number of resistance-related nucleotide-binding site leucine-rich repeat (NBS-LRR) genes in Cucurbitaceae. The loss of NBS-LRR loci caused by large insertions or deletions (indels) and pseudogenization caused by small indels explained the loss of NBS-LRR genes in Cucurbitaceae. Population structure analysis suggested that C. metuliferus originated in Zimbabwe, then spread to other southern African regions where it likely underwent similar domestic selection as melon. This C. metuliferus reference sequence will accelerate the understanding of the molecular evolution of resistance-related genes and enhance cucurbit crop improvement efforts.

Detection of EGFR gene with a droplet digital PCR chip integrating a double-layer glass reservoir.

The lack of reliable and practical method for detecting rare hot mutation of epidermal growth factor receptor (EGFR) in circulating tumor DNA (ctDNA) for lung cancer has remained a challenge for general clinical application due to excess wild type DNA in clinical samples. In this study, we developed a droplet digital PCR (ddPCR) platform, integrating a PDMS chip and double-layer glass reservoir. The duplex T-junction droplet generators in PDMS chip can produce about one million uniform droplets of 4.187 pL within ∼10 min, which were then stored in the glass reservoir. The double-layer glass reservoir can protect droplets from evaporation and breaking, solving the problem of instability during thermal-cycling. The quantitative capabilities of the ddPCR chip were evaluated by testing EGFR exon gene 21, with a good linear correlation in the wide range of 101 to 106 copies/µL (R2 = 0.9998). We then demonstrated that the proposed ddPCR device can recognize rare EGFR L858R mutation under a background of 106 copies/µL wild-type DNA at a sensitivity of 0.0001%. Finally, we demonstrated this ddPCR platform could identify low amount of EGFR L858R mutation in ctDNA and CTCs of patients with lung cancer.

Identifying the grade of bladder cancer cells using microfluidic chips based on impedance.

Quantification of tumor cell heterogeneity is critical for clinical diagnostic and therapeutic applications, including evaluation of the cancerous stage of tumors. In this work, we presented a novel method to effectively distinguish the grade of bladder cancer at a single-cell level in both cell line and clinical cell samples. This was achieved by taking advantage of microdroplets and microelectrodes, which can encapsulate and then trap single cells for measuring their impedance in a label-free and non-invasive manner. These findings suggested that this impedance analysis device based on droplet microfluidics is promising in the fields of clinical and point-of-care diagnostics.

A Meloidogyne incognita C-type lectin effector targets plant catalases to promote parasitism.

Root-knot nematodes, Meloidogyne spp., secrete effectors to modulate plant immune responses and establish a parasitic relationship with host plants. However, the functions and plant targets of C-type lectin (CTL)-like effectors of Meloidogyne incognita remain unknown. Here, we characterized a CTL-like effector of M. incognita, MiCTL1a, and identified its target and role in nematode parasitism. In situ hybridization demonstrated the expression of MiCTL1 in the subventral glands; and in planta, immunolocalization showed its secretion during M. incognita parasitism. Virus-induced gene silencing of the MiCTL1 reduced the infection ability of M. incognita in Nicotiana benthamiana. The ectopic expression in Arabidopsis not only increased susceptibility to M. incognita but also promoted root growth. Yeast two-hybrid and co-immunoprecipitation assays revealed that MiCTL1a interacts with Arabidopsis catalases, which play essential roles in hydrogen peroxide homeostasis. Knockout or overexpression of catalases showed either increased or reduced susceptibility to M. incognita, respectively. Moreover, MiCTL1a not only reduced catalase activity in vitro and in planta but also modulated stress-related gene expressions in Arabidopsis. Our data suggest that MiCTL1a interacts with plant catalases and interferes with catalase activity, allowing M. incognita to establish a parasitic relationship with its host by fine-tuning responses mediated by reactive oxygen species.

Microfluidic integrated capacitive biosensor for C-reactive protein label-free and real-time detection.

A microfluidic chip has been integrated with a capacitive biosensor based on mass-producible three-dimensional (3D) interdigital electrode arrays. To achieve the monitoring of biosensor preparation and cardiac- and periodontitis-related biomarkers, all the processes were detected in a continuously on-site way. Fabrication steps for the microfluidic chip-bonded 3D interdigital capacitor biosensor include gold thiol modification, the activation of EDC/sulfo-NHS, and the bioconjugation of antibodies. Fluorescent characterization and X-ray photoelectron spectroscopy analysis were applied to assess the successful immobilization of the C-reactive protein (CRP) antibody. The experimental results indicate the good specificity and high sensitivity of the microfluidic integrated 3D capacitive biosensor. The limit of detection of the 3D capacitive biosensor for CRP label-free detection was about 1 pg mL-1. This 3D capacitive biosensor with integrated microfluidics is mass-producible and has achieved the on-site continuous detection of cardiac- and periodontitis-related biomarkers with high performance.

Biocontrol Efficacy of Bacillus cereus Strain Bc-cm103 Against Meloidogyne incognita.

Root-knot nematodes (Meloidogyne spp.) are soilborne pathogens that infect vegetable crops and cause major economic losses worldwide annually. Therefore, there is an urgent need for novel nematicides or biological control agents to reduce the damage caused by root-knot nematodes. In this study, we tested efficacy of the Bacillus cereus strain Bc-cm103, isolated from the rhizoplane of Cucumis metuliferus, against Meloidogyne incognita. Strain Bc-cm103 fermentation broth caused 100% mortality of the nematode second-stage juveniles within 12 h and decreased the egg hatching rate by 40.06% within 72 h compared with sterile water. Confocal laser-scanning microscopy revealed that strain Bc-cm103 formed a biofilm on cucumber (C. sativus) roots, which protected the roots from the infection of M. incognita. Additionally, strain Bc-cm103 activated the defense-responsive genes PR1, PR2, LOX1, and CTR1 in cucumber. Furthermore, strain Bc-cm103 significantly reduced the appearance of root galls in pot, split-root, and field tests. These results indicated that B. cereus strain Bc-cm103 had a strong suppressive effect on M. incognita and therefore could be used as a potential biocontrol agent against this pathogen.

Volatile Organic Compounds of Bacillus cereus Strain Bc-cm103 Exhibit Fumigation Activity against Meloidogyne incognita.

Bacillus cereus strain Bc-cm103 shows nematicidal activity and, therefore, has been used as a biological control agent to control the root-knot nematode Meloidogyne incognita. However, it remains unknown whether volatile organic compounds (VOCs) produced by B. cereus strain Bc-cm103 are effective in biocontrol against M. incognita. Therefore, in this study, we investigated the activity of Bc-cm103 VOCs against M. incognita. The B. cereus strain Bc-cm103 significantly repelled the second-stage juveniles (J2s) of M. incognita. In vitro evaluation of VOCs produced by the fermentation of Bc-cm103 in a three-compartment Petri dish revealed the mortality rates of M. incognita J2s as 90.8% at 24 h and 97.2% at 48 h. Additionally, evaluation of the ability of Bc-cm103 VOCs to suppress M. incognita infection in a double-layered pot test showed that root galls on cucumber roots decreased by 46.1%. Furthermore, 21 VOCs were identified from strain Bc-cm103 by solid-phase microextraction gas chromatography-mass spectrometry, including alkanes, alkenes, esters, and sulfides. Among them, dimethyl disulfide (30.63%) and S-methyl ester butanethioic acid (30.29%) were reported to have strong nematicidal activity. Together, these results suggest that B. cereus strain Bc-cm103 exhibits fumigation activity against M. incognita.

Recent development of antibiotic detection in food and environment: the combination of sensors and nanomaterials.

In recent years, the abuse of antibiotics has led to the pollution of soil and water environment, not only poultry husbandry and food manufacturing will be influenced to different degree, but also the human body will produce antibody. The detection of antibiotic content in production and life is imperative. In this review, we provide comprehensive information about chemical sensors and biosensors for antibiotic detection. We classify the currently reported antibiotic detection technologies into chromatography, mass spectrometry, capillary electrophoresis, optical detection, and electrochemistry, introduce some representative examples for each technology, and conclude the advantages and limitations. In particular, the optical and electrochemical methods based on nanomaterials are discussed and evaluated in detail. In addition, the latest research in the detection of antibiotics by photosensitive materials is discussed. Finally, we summarize the pros and cons of various antibiotic detection methods and present a discussion and outlook on the expansion of cross-scientific areas. The synthesis and application of optoelectronic nanomaterials and aptamer screening are discussed and prospected, and the future trends and potential impact of biosensors in antibiotic detection are outlined.Graphical abstract.

[A review on cell-based models of human liver disease in vitro].

Unhealthy diet, habits and drug abuse cause a variety of liver diseases, including steatohepatitis, liver fibrosis, liver cirrhosis and liver cancer, which seriously affect human health. The fabrication of highly simulated cell models in vitro is important in the treatment of liver diseases and drug development. This article summarized the common strategies for the construction of liver pathology models in vitro. It introduced four typical cell models in vitro related to liver disease and provided a reference for the study of liver disease models.