Vaccination with structurally adapted fungal protein fibrils induces immunity to Parkinson's disease.

The pathological misfolding and aggregation of soluble α-synuclein into toxic oligomers and insoluble amyloid fibrils causes Parkinson's disease, a progressive age-related neurodegenerative disease for which there is no cure. HET-s is a soluble fungal protein that can form assembled amyloid fibrils in its prion state. We engineered HET-s(218-298) to form four different fibrillar vaccine candidates, each displaying a specific conformational epitope present on the surface of α-synuclein fibrils. Vaccination with these four vaccine candidates prolonged the survival of immunized TgM83+/- mice challenged with α-synuclein fibrils by 8% when injected into the brain to model brain-first Parkinson's disease or by 21% and 22% when injected into the peritoneum or gut wall, respectively, to model body-first Parkinson's disease. Antibodies from fully immunized mice recognized α-synuclein fibrils and brain homogenates from patients with Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Conformation-specific vaccines that mimic epitopes present only on the surface of pathological fibrils but not on soluble monomers, hold great promise for protection against Parkinson's disease, related synucleinopathies and other amyloidogenic protein misfolding disorders.

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.

Identification of a poly-cyclopropylglycine-containing peptide via bioinformatic mapping of radical S-adenosylmethionine enzymes.

Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.

Crystal Structure of DNA Replication Protein SsbA Complexed with the Anticancer Drug 5-Fluorouracil.

Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA metabolism by binding and stabilizing single-stranded DNA (ssDNA) intermediates. Through their multifaceted roles in DNA replication, recombination, repair, replication restart, and other cellular processes, SSB emerges as a central player in maintaining genomic integrity. These attributes collectively position SSBs as essential guardians of genomic integrity, establishing interactions with an array of distinct proteins. Unlike Escherichia coli, which contains only one type of SSB, some bacteria have two paralogous SSBs, referred to as SsbA and SsbB. In this study, we identified Staphylococcus aureus SsbA (SaSsbA) as a fresh addition to the roster of the anticancer drug 5-fluorouracil (5-FU) binding proteins, thereby expanding the ambit of the 5-FU interactome to encompass this DNA replication protein. To investigate the binding mode, we solved the complexed crystal structure with 5-FU at 2.3 Å (PDB ID 7YM1). The structure of glycerol-bound SaSsbA was also determined at 1.8 Å (PDB ID 8GW5). The interaction between 5-FU and SaSsbA was found to involve R18, P21, V52, F54, Q78, R80, E94, and V96. Based on the collective results from mutational and structural analyses, it became evident that SaSsbA's mode of binding with 5-FU diverges from that of SaSsbB. This complexed structure also holds the potential to furnish valuable comprehension regarding how 5-FU might bind to and impede analogous proteins in humans, particularly within cancer-related signaling pathways. Leveraging the information furnished by the glycerol and 5-FU binding sites, the complexed structures of SaSsbA bring to the forefront the potential viability of several interactive residues as potential targets for therapeutic interventions aimed at curtailing SaSsbA activity. Acknowledging the capacity of microbiota to influence the host's response to 5-FU, there emerges a pressing need for further research to revisit the roles that bacterial and human SSBs play in the realm of anticancer therapy.

Authentication of olive oil in commercial products using specific, sensitive, and rapid loop-mediated isothermal amplification.

Olive oil is an important and popularly used plant oil in the daily diet or chemical industry. Due to its biological benefits on human health and higher selling prices, adulteration of olive oil for commercial fraud by other plant oils is becoming a serious issue. In this study, a specific, sensitive and rapid loop-mediated isothermal amplification (LAMP) was first developed for the detection of Olea europaea DNA for olive oil authentication. The oleosin gene was used for the primer design of the LAMP assay. After primer validation, the results showed that the LAMP primers were specific and rapid to isothermally authenticate the oleosin gene of Olea europaea within 1 h at 62 °C and had no cross-reaction with other DNA of plant oils. The sensitivity of LAMP was 1 ng of genomic DNA in olive oil, and only 1% olive oil in the sample was requisite during DNA amplification. Additionally, positive detection by LAMP in all the collected commercial olive oil products was practically performed but not in PCR assays. In conclusion, herein, the established LAMP assay with specificity could not only be capable for rapid identification but also applicable for olive oil authentication for precluding adulteration in plant oil products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05726-y.

Specific, sensitive and rapid authentication of donkey-hide gelatine (Colla corii asini) in processed food using an isothermal nucleic acid amplification assay.

Donkey-hide gelatine (DHG) is a well-known, animal-derived traditional Chinese medicine material called Colla corii asini (known in Chinese as "E'jiao"). Because DHG is claimed to have properties that are beneficial to health, its consumption has increased, but its production has decreased. Thus, the incidence of DHG adulteration has become increasingly serious. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the authentication of DHG. Identification of donkey DNA from DHG was performed specifically and rapidly within one hour by LAMP primers. Moreover, the sensitivity of LAMP in authenticating DHG was 10-3 ng, which revealed a 105-fold higher sensitivity than that of conventional PCR. The relative detection limit was 0.1% DHG in the adulterants, including gelatines of horse, cow, pork, goat, sheep or chicken origins. When genomic DNAs extracted from heat-treated DHG samples, including boiling or autoclaving for 40 min, were used as templates, DHG detection by LAMP was unchanged and reproducible. In conclusion, the LAMP assay established herein could potentially be applied for the authentication of DHG and DHG-related products in herbal or food markets.

Identification of nuclear localization signal and nuclear export signal of VP1 from the chicken anemia virus and effects on VP2 shuttling in cells.

BACKGROUND: VP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1. METHODS: Two putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were co-expressed in cells using plasmid transfection. RESULTS: A functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm. CONCLUSION: Our data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm.

Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus.

BACKGROUND: Chicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules. RESULTS: In this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA. CONCLUSIONS: The results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.

The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice.

Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

Comorbidity of Atopic Disorders with Autism Spectrum Disorder and Attention Deficit/Hyperactivity Disorder.

OBJECTIVE: To assess the relationship between allergic manifestations in early life and the occurrence of newly diagnosed autism spectrum disorder (ASD) and attention deficit/hyperactivity disorder (ADHD) throughout childhood. STUDY DESIGN: We collected a population-based longitudinal cohort comprising children enrolled in Taiwan's National Health Insurance Program during 2000-2010. We first identified 387,262 children who had a diagnosis of atopic dermatitis (AD) before age 2 years, with 1:1 individualized matching to children without AD. Cox regression analyses were performed to estimate the early-onset and cumulative effects of allergic manifestations on ASD and ADHD. RESULTS: An estimated 0.5% of AD-exposed children received a diagnosis of ASD, and 3.7% were diagnosed with ADHD, significantly higher than the respective rates of 0.4% and 2.9% found in their nonexposed peers. Having AD before age 2 years was associated with an increased hazard ratio (HR) for ASD by 10% and that for ADHD by 16%; such increases were particularly prominent among those with earlier-onset or more severe AD. HRs were especially higher for children with persistent AD and emerging atopic respiratory diseases in childhood (eg, for ASD, adjusted HR, 1.75 and 2.13, respectively; P < .001). CONCLUSION: The observed increased risks of ASD and ADHD associated with AD in infancy suggest that a disordered immunologic response may exert effects on neurodevelopment and have implications for research into etiology and treatment strategies.

High molecular weight of polysaccharides from Hericium erinaceus against amyloid beta-induced neurotoxicity.

BACKGROUND: Hericium erinaceus (HE) is a well-known mushroom in traditional Chinese food and medicine. HE extracts from the fruiting body and mycelia not only exhibit immunomodulatory, antimutagenic and antitumor activity but also have neuroprotective properties. Here, we purified HE polysaccharides (HEPS), composed of two high molecular weight polysaccharides (1.7 × 10(5) Da and 1.1 × 10(5) Da), and evaluated their protective effects on amyloid beta (Aß)-induced neurotoxicity in rat pheochromocytoma PC12 cells. METHODS: HEPS were prepared and purified using a 95 % ethanol extraction method. The components of HEPS were analyzed and the molecular weights of the polysaccharides were determined using high-pressure liquid chromatography (HPLC). The neuroprotective effects of the polysaccharides were evaluated through a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and an MTT assay and by quantifying reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) of Aß-induced neurotoxicity in cells. RESULT: Our results showed that 250 µg/ml HEPS was harmless and promoted cell viability with 1.2 µM Aß treatment. We observed that the free radical scavenging rate exceeded 90 % when the concentration of HEPS was higher than 1 mg/mL in cells. The HEPS decreased the production of ROS from 80 to 58 % in a dose-dependent manner. Cell pretreatment with 250 µg/mL HEPS significantly reduced Aß-induced high MMPs from 74 to 51 % and 94 to 62 % at 24 and 48 h, respectively. Finally, 250 µg/mL of HEPS prevented Aß-induced cell shrinkage and nuclear degradation of PC12 cells. CONCLUSION: Our results demonstrate that HEPS exhibit antioxidant and neuroprotective effects on Aß-induced neurotoxicity in neurons.

Genome-wide annotation, expression profiling, and protein interaction studies of the core cell-cycle genes in Phalaenopsis aphrodite.

Orchidaceae is one of the most abundant and diverse families in the plant kingdom and its unique developmental patterns have drawn the attention of many evolutionary biologists. Particular areas of interest have included the co-evolution of pollinators and distinct floral structures, and symbiotic relationships with mycorrhizal flora. However, comprehensive studies to decipher the molecular basis of growth and development in orchids remain scarce. Cell proliferation governed by cell-cycle regulation is fundamental to growth and development of the plant body. We took advantage of recently released transcriptome information to systematically isolate and annotate the core cell-cycle regulators in the moth orchid Phalaenopsis aphrodite. Our data verified that Phalaenopsis cyclin-dependent kinase A (CDKA) is an evolutionarily conserved CDK. Expression profiling studies suggested that core cell-cycle genes functioning during the G1/S, S, and G2/M stages were preferentially enriched in the meristematic tissues that have high proliferation activity. In addition, subcellular localization and pairwise interaction analyses of various combinations of CDKs and cyclins, and of E2 promoter-binding factors and dimerization partners confirmed interactions of the functional units. Furthermore, our data showed that expression of the core cell-cycle genes was coordinately regulated during pollination-induced reproductive development. The data obtained establish a fundamental framework for study of the cell-cycle machinery in Phalaenopsis orchids.

High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression.

BACKGROUND: Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system. RESULTS: The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic activity when tested against PiCV-infected pigeon sera. CONCLUSIONS: These findings shows that the E. coli-expressed full-length PiCV Cap protein has great potential in terms of large-scaled production and this should allow in the future the development of a serodiagnostic kit that is able to clinically detect PiCV infection in pigeons.

Circulating hypervirulent Marek's disease viruses in vaccinated chicken flocks in Taiwan by genetic analysis of meq oncogene.

Marek's disease (MD) is an important neoplastic disease caused by serotype 1 Marek's disease virus (MDV-1), which results in severe economic losses worldwide. Despite vaccination practices that have controlled the MD epidemic, current increasing MD-suspected cases indicate the persistent viral infections circulating among vaccinated chicken farms in many countries. However, the lack of available information about phylogeny and molecular characterization of circulating MDV-1 field strains in Taiwan reveals a potential risk in MD outbreaks. This study investigated the genetic characteristics of 18 MDV-1 strains obtained from 17 vaccinated chicken flocks in Taiwan between 2018 and 2020. Based on the sequences of the meq oncogene, the phylogenetic analysis demonstrated that the circulating Taiwanese MDV-1 field strains were predominantly in a single cluster that showed high similarity with strains from countries of the East Asian region. Because the strains were obtained from CVI988/Rispens vaccinated chicken flocks and the molecular characteristics of the Meq oncoprotein showed features like vvMDV and vv+MDV strains, the circulating Taiwanese MDV-1 field strains may have higher virulence compared with vvMDV pathotype. In conclusion, the data presented demonstrates the circulation of hypervirulent MDV-1 strains in Taiwan and highlights the importance of routine surveillance and precaution strategies in response to the emergence of enhanced virulent MDV-1.

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.

UGT1A4 Polymorphism is not Associated with a Clinically Relevant Change in Giredestrant Exposure.

PURPOSE: Giredestrant is a potent, orally bioavailable, small-molecule selective estrogen receptor antagonist and degrader (SERD) that is being developed for the treatment of patients with estrogen receptor (ER)-positive breast cancer. In vitro, giredestrant was primarily metabolized by UGT1A4. The goal of this study was to investigate if UGT1A4 polymorphism had a clinically relevant impact on giredestrant exposure. METHODS: Genotyping and pharmacokinetic data were obtained from 118 and 61 patients in two clinical studies, GO39932 [NCT03332797] and acelERA Breast Cancer [NCT04576455], respectively. RESULTS: The overall allelic frequencies of UGT1A4*2 and UGT1A4*3 were 3.3% and 11%, respectively. Giredestrant exposure was consistent between patients with wild-type UGT1A4 and UGT1A4*2 and *3 polymorphisms, with no clinically relevant difference observed. In addition, haplotype analysis indicated that no other UGT1A4 variants were significantly associated with giredestrant exposure. CONCLUSION: Therefore, this study indicates that UGT1A4 polymorphism status is unlikely a clinically relevant factor to impact giredestrant exposure and giredestrant can be administered at the same dose level regardless of patients' UGT1A4 polymorphism status.

Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases.

The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.

Applications of Advanced Natural Language Processing for Clinical Pharmacology.

Natural language processing (NLP) is a branch of artificial intelligence, which combines computational linguistics, machine learning, and deep learning models to process human language. Although there is a surge in NLP usage across various industries in recent years, NLP has not been widely evaluated and utilized to support drug development. To demonstrate how advanced NLP can expedite the extraction and analyses of information to help address clinical pharmacology questions, inform clinical trial designs, and support drug development, three use cases are described in this article: (1) dose optimization strategy in oncology, (2) common covariates on pharmacokinetic (PK) parameters in oncology, and (3) physiologically-based PK (PBPK) analyses for regulatory review and product label. The NLP workflow includes (1) preparation of source files, (2) NLP model building, and (3) automation of data extraction. The Clinical Pharmacology and Biopharmaceutics Summary Basis of Approval (SBA) documents, US package inserts (USPI), and approval letters from the US Food and Drug Administration (FDA) were used as our source data. As demonstrated in the three example use cases, advanced NLP can expedite the extraction and analyses of large amounts of information from regulatory review documents to help address important clinical pharmacology questions. Although this has not been adopted widely, integrating advanced NLP into the clinical pharmacology workflow can increase efficiency in extracting impactful information to advance drug development.

Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications.

BACKGROUND: Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. RESULTS: Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other subunit proteins. Moreover, the VP2-396N and VP2-345 based ELISAs had better sensitivity (97.5%) and excellent specificity (100%) during serodiagnosis testing using a mean plus three standard deviations cut-off. The VP3-246M based ELISA showed a sensitivity of 85% and a specificity of 100% at the same cut-off value. CONCLUSIONS: This is the first report to systematically assess the antigenic characteristics of CAV viral proteins for sero-diagnosis purposes. Purified recombinant VP2-396N and VP2-345N subunit proteins, which span defined regions of VP2, were demonstrated to have good antigenicity and higher sensitivities than VP3-246M and were able to recognize CAV-positive chicken serum using an ELISA assay. The defined antigenicity potential of these chimeric subunit proteins produced by expression in E. coli seem to have potential and could be useful in the future for the development of the CAV diagnostic tests based on a subunit protein ELISA system.

The quest for balance between capturing data and model complexity: A quantitative clinical pharmacology approach applied to monoclonal antibodies.

The main objective of this tutorial is to provide the readers with a roadmap of how to establish increasingly complex target-mediated drug disposition (TMDD) models for monoclonal antibodies. To this end, we built mathematical models, each with a detailed visualization, starting from the basic TMDD model by Mager and Jusko to the well-established, physiologically based model by Li et al. in a step-wise fashion to highlight the relative importance of key physiological processes that impact mAb kinetics and system dynamics. As the models become more complex, the question of structural and parameter identifiability arises. To address this question, we work through a trastuzumab case example to guide the modeler's choice for model and parameter optimization in light of the context of use. We leave the readers of this tutorial with a brief summary of the advantages and limitations of each model expansion, as well as the model source codes for further self-guided exploration and hands-on analysis.