3D organoid cultivation improves the maturation and functional differentiation of cholangiocytes from human pluripotent stem cells.

Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC-derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system-generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.

Inhibition of SARS-CoV-2 Nsp9 ssDNA-Binding Activity and Cytotoxic Effects on H838, H1975, and A549 Human Non-Small Cell Lung Cancer Cells: Exploring the Potential of Nepenthes miranda Leaf Extract for Pulmonary Disease Treatment.

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9's ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications.

Isolation and Characterization of Insecticidal Cyclotides from Viola communis.

Cyclotides are cysteine-rich plant-derived peptides composed of 28-37 amino acids with a head-to-tail cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Their beneficial biophysical properties make them promising molecules for pharmaceutical and agricultural applications. The Violaceae plant family is the major cyclotide-producing family, and to date, every examined plant from this family has been found to contain cyclotides. The presence of cyclotides in Viola communis was inferred by mass spectroscopy previously, but their sequences and properties had yet to be explored. In this study, the occurrence of cyclotides in this plant was investigated using proteomics and transcriptomics. Twenty cyclotides were identified at the peptide level, including two new members from the bracelet (Vcom1) and Möbius (Vcom2) subfamilies. Structural analysis of these newly identified peptides demonstrated a similar fold compared with cyclotides from the same respective subfamilies. Biological assays of Vcom1 and Vcom2 revealed them to be cytotoxic to Sf9 insect cell lines, with Vcom1 demonstrating higher potency than Vcom2. The results suggest that they could be further explored as insecticidal agents and confirm earlier general findings that bracelet cyclotides have more potent insecticidal activity than their Möbius relatives. Seven new cyclotide-like sequences were observed in the transcriptome of V. communis, highlighting the Violaceae as a rich source for new cyclotides with potential insecticidal activity. An analysis of sequences flanking the cyclotide domain in the various precursors from V. communis and other Violaceae plants revealed new insights into cyclotide processing and suggested the possibility of two alternative classes of N-terminal processing enzymes for cyclotide biosynthesis.

Repurposing a plant peptide cyclase for targeted lysine acylation.

Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine-leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.

Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins.

The carnivorous pitcher plants of the genus Nepenthes have long been known for their ethnobotanical applications. In this study, we prepared various extracts from the pitcher, stem, and leaf of Nepenthes miranda using 100% ethanol and assessed their inhibitory effects on key enzymes related to skin aging, including elastase, tyrosinase, and hyaluronidase. The cytotoxicity of the stem extract of N. miranda on H838 human lung carcinoma cells were also characterized by effects on cell survival, migration, proliferation, apoptosis induction, and DNA damage. The cytotoxic efficacy of the extract was enhanced when combined with the chemotherapeutic agent 5-fluorouracil (5-FU), indicating a synergistic effect. Flow cytometry analysis suggested that the stem extract might suppress H838 cell proliferation by inducing G2 cell cycle arrest, thereby inhibiting carcinoma cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 15 most abundant compounds in the stem extract of N. miranda. Notably, the extract showed a potent inhibition of the human RPA32 protein (huRPA32), critical for DNA replication, suggesting a novel mechanism for its anticancer action. Molecular docking studies further substantiated the interaction between the extract and huRPA32, highlighting bioactive compounds, especially the two most abundant constituents, stigmast-5-en-3-ol and plumbagin, as potential inhibitors of huRPA32's DNA-binding activity, offering promising avenues for cancer therapy. Overall, our findings position the stem extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and anti-skin-aging treatments, warranting further investigation into its molecular mechanisms and potential clinical applications.

Structure and Activity of Reconstructed Pseudo-Ancestral Cyclotides.

Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed 'pseudo-ancestral' sequences, named Ancy-m (for the ancestral cyclotide of the Möbius sub-family) and Ancy-b (for the bracelet sub-family), have well-defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re-engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl-ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy-b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.

Season of death, pathogen persistence and wildlife behaviour alter number of anthrax secondary infections from environmental reservoirs.

An important part of infectious disease management is predicting factors that influence disease outbreaks, such as R, the number of secondary infections arising from an infected individual. Estimating R is particularly challenging for environmentally transmitted pathogens given time lags between cases and subsequent infections. Here, we calculated R for Bacillus anthracis infections arising from anthrax carcass sites in Etosha National Park, Namibia. Combining host behavioural data, pathogen concentrations and simulation models, we show that R is spatially and temporally variable, driven by spore concentrations at death, host visitation rates and early preference for foraging at infectious sites. While spores were detected up to a decade after death, most secondary infections occurred within 2 years. Transmission simulations under scenarios combining site infectiousness and host exposure risk under different environmental conditions led to dramatically different outbreak dynamics, from pathogen extinction (R < 1) to explosive outbreaks (R > 10). These transmission heterogeneities may explain variation in anthrax outbreak dynamics observed globally, and more generally, the critical importance of environmental variation underlying host-pathogen interactions. Notably, our approach allowed us to estimate the lethal dose of a highly virulent pathogen non-invasively from observational studies and epidemiological data, useful when experiments on wildlife are undesirable or impractical.

Scanning mutagenesis identifies residues that improve the long-term stability and insecticidal activity of cyclotide kalata B1.

Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of ∼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.

Molecular dynamics simulations support a preference of cyclotide kalata B1 for phosphatidylethanolamine phospholipids.

Kalata B1 (kB1), a naturally occurring cyclotide has been shown experimentally to bind lipid membranes that contain phosphatidylethanolamine (PE) phospholipids. Here, molecular dynamics simulations were used to explore its interaction with two phospholipids, palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), and a heterogeneous membrane comprising POPC/POPE (90:10), to understand the basis for the selectivity of kB1 towards PE phospholipids. The simulations showed that in the presence of only 10 % POPE lipid, kB1 forms a stable binding complex with membrane bilayers. An ionic interaction between the E7 carboxylate group of kB1 and the ammonium group of PE headgroups consistently initiates binding of kB1 to the membrane. Additionally, stable noncovalent interactions such as hydrogen bonding (E7, T8, V10, G11, T13 and N15), cation-π (W23), and CH-π (W23) interactions between specific residues of kB1 and the lipid membrane play an important role in stabilizing the binding. These findings are consistent with a structure-activity relationship study on kB1 where lysine mutagenesis on the bioactive and hydrophobic faces of the peptide abolished membrane-dependent bioactivities. In summary, our simulations suggest the importance of residue E7 (in the bioactive face) in enabling kB1 to recognize and bind selectively to PE-containing phospholipids bilayers through ionic and hydrogen bonding interactions, and of W23 (in the hydrophobic face) for the association and insertion of kB1 into the lipid bilayer through cation-π and CH-π interactions. Overall, this work enhances our understanding of the molecular basis of the membrane binding and bioactivity of this prototypic cyclotide.

Delivery to, and Reactivation of, the p53 Pathway in Cancer Cells Using a Grafted Cyclotide Conjugated with a Cell-Penetrating Peptide.

Peptides are promising drug modalities that can modulate protein-protein interactions, but their application is hampered by their limited ability to reach intracellular targets. Here, we improved the cytosolic delivery of a peptide blocking p53:MDM2/X interactions using a cyclotide as a stabilizing scaffold. We applied several design strategies to improve intracellular delivery and found that the conjugation of the lead cyclotide to the cyclic cell-penetrating peptide cR10 was the most effective. Conjugation allowed cell internalization at micromolar concentration and led to elevated intracellular p53 levels in A549, MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells without causing membrane disruption. The lead peptide had >35-fold improvement in inhibitory activity and increased cellular uptake compared to a previously reported cyclotide p53 activator. In summary, we demonstrated the delivery of a large polar cyclic peptide in the cytosol and confirmed its ability to modulate intracellular protein-protein interactions involved in cancer.

Antiviral therapy reduces hepatocellular carcinoma through suppressing hepatitis B virus replication may improve ER stress, mitochondrial and metabolic dysfunctions and decrease p62 in hybridized mice with single HBV transgene and miR-122.

Hepatitis B virus (HBV) hijacks autophagy for its replication. Nucleos(t)ide analogs (NUCs) treatment suppressed HBV replication and reduced hepatocellular carcinoma (HCC) incidence. However, the use of NUCs in chronic hepatitis B (CHB) patients with normal or minimally elevated serum alanine aminotransferase (ALT) levels is still debated. Animal models are crucial for studying the unanswered issue and evaluating new therapies. MicroRNA-122 (miR-122), which regulates fatty acid and cholesterol metabolism, is downregulated during hepatitis and HCC progression. The reciprocal inhibition of miR-122 with HBV highlights its role in HCC development as a tumor suppressor. By crossbreeding HBV-transgenic mice with miR-122 knockout mice, we generated a hybrid mouse model with a high incidence of HCC up to 89% and normal ALT levels before HCC. The model exhibited early-onset hepatic steatosis, progressive liver fibrosis, and impaired late-phase autophagy. Metabolomics and microarray analysis identified metabolic signatures, including dysregulation of lipid metabolism, inflammation, genomic instability, the Warburg effect, reduced TCA cycle flux, energy deficiency, and impaired free radical scavenging. Antiviral treatment reduced HCC incidence in hybrid mice by approximately 30-35% compared to untreated mice. This effect was linked to the activation of ER stress-responsive transcription factor ATF4, clearance of autophagosome cargo p62, and suppression of the CHOP-mediated apoptosis pathway. In summary, this study suggests that despite minimal ALT elevation, HBV replication can lead to liver injury. Endoplasmic reticulum stress, reduced miR-122 levels, mitochondrial and metabolic dysfunctions, blocking protective autophagy resulting in p62 accumulation, apoptosis, fibrosis, and HCC. Antiviral may improve the above-mentioned pathogenesis through HBV suppression.