Correlation between the dynamic changes of γδT cells, Th17 cells, CD4+CD25+ regulatory T cells in peripheral blood and pharmacological interventions against bleomycin-induced pulmonary fibrosis progression in mice.

The involvement of γδT cells, Th17 cells, and CD4+CD25+ regulatory T cells (Tregs) is crucial in the progression of pulmonary fibrosis (PF), particularly in maintaining immune tolerance and homeostasis. However, the dynamics of these cells in relation to PF progression, especially under pharmacological interventions, remains poorly understood. This study aims to unravel the interplay between the dynamic changes of these cells and the effect of pharmacological agents in a mouse model of PF induced by intratracheal instillation of bleomycin. We analyzed changes in lung histology, lung index, hydroxyproline levels, and the proportions of γδT cells, Th17 cells, and Tregs on the 3rd, 14th, and 28th days following treatment with Neferine, Isoliensinine, Pirfenidone, and Prednisolone. Our results demonstrate that these drugs can partially or dynamically reverse weight loss, decrease lung index and hydroxyproline levels, and ameliorate lung histopathological damage. Additionally, they significantly modulated the abnormal changes in γδT, Th17, and Treg cell proportions. Notably, on day 3, the proportion of γδT cells increased in the Neferine and Prednisolone groups but decreased in the Isoliensinine and Pirfenidone groups, while the proportion of Th17 cells decreased across all treated groups. On day 14, the Neferine group showed an increase in all three cell types, whereas the Pirfenidone group exhibited a decrease. In the Isoliensinine group, γδT and Th17 cells increased, and in the Prednisolone group, only Tregs increased. By day 28, an increase in Th17 cell proportion was observed in all treatment groups, with a decrease in γδT cells noted in the Neferine group. These shifts in cell proportions are consistent with the pathogenesis changes induced by these anti-PF drugs, suggesting a correlation between cellular dynamics and pharmacological interventions in PF progression. Our findings imply potential strategies for assessing the efficacy and timing of anti-PF treatments based on these cellular changes.

Inhibiting sphingosine 1-phosphate lyase: From efficacy to mechanism.

Sphingosine-1 phosphate (S1P) is a lipid metabolite regulating diverse biological processes, including proliferation, differentiation, migration, and apoptosis, highlighting its physiological and therapeutic significance. Current S1P-based therapeutic approaches primarily focus on modulating the downstream signalling via targeting S1P receptors, however, this is challenged by incomplete receptor internalisation. Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that "gatekeeps" the final step of S1P degradation. Cognisant of the complex ligand and receptor interaction and dynamic metabolic networks, the selective modulation of SPL activity presents a new opportunity to regulate S1P biosynthesis and reveal its role in various systems. Over the past decade, an evolving effort has been made to identify new molecules that could block SPL activity in vitro or in vivo. This review focuses on summarising the current understanding of the reported SPL inhibitors identified through various screening approaches, discussing their efficacy in diverse model systems and the possible mechanism of action. Whilst effective modulation of S1P levels via inhibiting SPL is feasible, the specificity of those inhibitors remains inconclusive, presenting a clear challenge for future implications. Yet, none of the currently available SPL inhibitors is proven effective in elevating S1P levels within the central nervous system. This review article embraces future research focusing on investigating selective SPL inhibitors with high potency and possibly blood-brain-barrier permeability, which would aid the development of new S1P-based therapeutics for neurological disorders.

Low cycle number multiplex PCR: A novel strategy for the construction of amplicon libraries for next-generation sequencing.

Multiplex PCR is a critical step when preparing amplicon library for next-generation sequencing. However, there are several challenges related to multiplex PCR including poor uniformity, nonspecific amplification, and primer-dimers. To address these issues, we propose a novel solution strategy that involves using a low cycle number (<10 cycles) in multiplex PCR and then employing carrier DNAs and magnetic beads for the selection of targeted products. This technique improves the amplicon uniformity while also reducing primer-dimers and PCR artifacts. To evaluate our technique, we initially utilized 120 DNA fragments from mouse genome containing single nucleotide polymorphism (SNP) sites. Sequencing results demonstrated that with only 7 cycles of multiplex PCR, 95.8% of the targeted SNP sites were mapped, with a coverage of at least 1×. The average sequencing depth of all amplicons was 1705.79 ± 1205.30×; 87% of them reached a coverage depth that exceeded 0.2-fold of the average sequencing depth. Our method had a greater uniformity (87%) when compared to Hi-Plex PCR (53.3%). Furthermore, we validated our strategy by randomly selecting 90 primer pairs twice from the initial set of 120 primer-pairs. Next, we used the same protocol to prepare amplicon libraries. The two groups had an average sequencing depth of 1013.30 ± 585.57× and 219.10 ± 158.27×, respectively; over 84% of the amplicons had a sequencing depth that exceeded 0.2-fold of average depth. These results suggest that the use of a low cycle number in multiplex PCR is a cost-effective and efficient approach for the preparation of amplicon libraries.

Multiplex Detection of RNA Viruses Based on Ligation Reaction and Universal PCR Amplification.

To detect several RNA viruses simultaneously, a method based on multiplex ligation reaction combined with multiplex qPCR or multiplex PCR+capillary electrophoresis was established to detect four RNA viruses: human immunodeficiency virus (HIV), hepatitis C (HCV), influenza A virus (IAV) H1N1 and H5N1. The experimental conditions including ligation probe concentration, annealing procedure, ligation temperature and ligase dosage were optimized intensively. We found that the specificity of the ligation reaction was affected by the probe concentration predominantly, high-probe concentration (100 nM) resulted in splint-independent ligation with efficiency comparable to that with RNA splint. The sensitivity of the ligation reaction was affected by the annealing mode apparently as the sensitivity of the step-down annealing mode was 100 times higher than that of the isothermal annealing at 37 °C. Under the optimized condition, this assay could detect virus RNA as low as 16 viral copies per reaction in doubleplex and triplex real-time quantitative PCR detection with satisfactory specificity and precision. By multiplex PCR+capillary electrophoresis, four RNA viruses could be detected in one tube with the sensitivity of 10 copies per reaction.

Junior and senior students possess differential preferences towards multimodal digital anatomy resources.

Digital technologies are changing how anatomy is taught tremendously. However, little is known about the effective integration of multimodal digital resources when concurrently provided in an anatomy course. To address this question, an array of digital anatomy resources including Augmented Reality (AR) and Virtual Reality (VR) anatomy resources were concurrently trialed by a total of 326 undergraduate and postgraduate students across three undergraduate (systemic anatomy, neuroanatomy, and regional anatomy) and one postgraduate anatomy (applied musculoskeletal anatomy) curricula in 2022. A five-point Likert scale learning and teaching survey was conducted to evaluate students' experiences, preferences, and perceptions. Most undergraduate (81% systemic anatomy, 76% neuroanatomy, and 87% regional anatomy) and postgraduate (97%) participants across the four cohorts felt confident in studying anatomy using digital resources and the majority (>80% undergraduate and >90% postgraduate) found the multimodal digital anatomy resources interactive and stimulating. The response showed that undergraduate (77% systemic anatomy, 81% neuroanatomy, and 97% regional anatomy) and postgraduate students (92%) consistently enjoyed their experience of using multimodal digital anatomy resources and thought that these resources enhanced their interest in studying anatomy. However, there are significant differences in ratings of specific digital resources among the junior (first-year undergraduates) and senior (third-year undergraduates and postgraduates) students. The virtual dissection table was uniformly preferred by the four cohorts of students across the board. Interestingly, however, VR anatomy and radiographic-based digital anatomy resources received diverse ratings. VR anatomy was valued most by junior undergraduate students (84%) who studied systemic anatomy compared to their senior counterparts (73%) who studied regional anatomy, whereas radiographic-based digital anatomy resources were more valued by the postgraduate students (93%) compared to undergraduates (65% systemic anatomy, 73% neuroanatomy, and 48% regional anatomy). This study identifies that while students uniformly appreciate the value of multimodal digital anatomy teaching, there is a clear difference in their perceptions towards individual resources, likely in a course-specific manner. We conclude that the selection and adoption of digital anatomy tools must be tailored as part of course design and that digital anatomy tools should be used in combination to provide an effective learning experience for students.

QuanDB: a quantum chemical property database towards enhancing 3D molecular representation learning.

Previous studies have shown that the three-dimensional (3D) geometric and electronic structure of molecules play a crucial role in determining their key properties and intermolecular interactions. Therefore, it is necessary to establish a quantum chemical (QC) property database containing the most stable 3D geometric conformations and electronic structures of molecules. In this study, a high-quality QC property database, called QuanDB, was developed, which included structurally diverse molecular entities and featured a user-friendly interface. Currently, QuanDB contains 154,610 compounds sourced from public databases and scientific literature, with 10,125 scaffolds. The elemental composition comprises nine elements: H, C, O, N, P, S, F, Cl, and Br. For each molecule, QuanDB provides 53 global and 5 local QC properties and the most stable 3D conformation. These properties are divided into three categories: geometric structure, electronic structure, and thermodynamics. Geometric structure optimization and single point energy calculation at the theoretical level of B3LYP-D3(BJ)/6-311G(d)/SMD/water and B3LYP-D3(BJ)/def2-TZVP/SMD/water, respectively, were applied to ensure highly accurate calculations of QC properties, with the computational cost exceeding 107 core-hours. QuanDB provides high-value geometric and electronic structure information for use in molecular representation models, which are critical for machine-learning-based molecular design, thereby contributing to a comprehensive description of the chemical compound space. As a new high-quality dataset for QC properties, QuanDB is expected to become a benchmark tool for the training and optimization of machine learning models, thus further advancing the development of novel drugs and materials. QuanDB is freely available, without registration, at https://quandb.cmdrg.com/ .

CAMDOL-enabled diastereoselective synthesis of α-substituted phosphonates.

Enantiopure α-substituted phosphonic acids are widely utilized as drugs, pesticides, and ligands. Despite numerous synthetic approaches having been investigated, precise construction of P-adjacent chiral tertiary carbon centres by the employment of recoverable chiral auxiliaries is traditional and still one of the most reliable and practical synthetic methodologies so far. Herein, we present a highly diastereoselective synthesis of α-substituted phosphonates via the unique CAMDOL-derived P-substrates by an efficient sequential deprotonation with LiHMDS and alkylation/arylation with RI. A wide range of 30 structurally diverse α-substituted phosphonate products, including the well-known P-analogues of naproxen and ibuprofen, were thus afforded conveniently in up to 92% yields and 99 : 1 diastereomeric ratios. The related chiral phosphonic acid could be easily obtained by simple acidic hydrolysis with fully recovered auxiliary. This CAMDOL-enabled asymmetric synthetic protocol exhibits comparative advantages over known chiral-induction methods with easy accessibility and compatibility of furnishing a variety of C-stereogenic centres in the proximity of the phosphorus atom, including some rare examples.

AlphaFold-guided redesign of a plant pectin methylesterase inhibitor for broad-spectrum disease resistance.

Plant cell walls are a critical site where plants and pathogens continuously struggle for physiological dominance. Here we show that dynamic remodeling of pectin methylesterification of plant cell walls is a component of the physiological and co-evolutionary struggles between hosts and pathogens. A Phytophthora sojae secreted pectin methylesterase (PsPME1) decreases the degree of pectin methylesterification, thus synergizing with an endo-polygalacturonase (PsPG1) to weaken plant cell walls. To counter PsPME1-mediated susceptibility, a plant-derived pectin methylesterase inhibitor protein, GmPMI1, protects pectin to maintain a high methylesterification status. GmPMI1 protects plant cell walls from enzymatic degradation by inhibiting both soybean and P. sojae pectin methylesterases during infection. However, constitutive expression of GmPMI1 disrupted the tradeoff between host growth and defense responses. So, we used AlphaFold structure tools to design a modified form of GmPMI1 (GmPMI1R) which specifically targets and inhibits pectin methylesterases secreted from pathogens but not from the plants. Transient expression of GmPMI1R enhanced plant resistance to oomycetes and fungal pathogens. In summary, our work highlights biochemical modification of the cell wall as an important focal point in the physiological and co-evolutionary conflict between the hosts and microbes and serves as an important proof-of-concept for how rapid advancements in AI-driven structure-based tools can accelerate the prediction of new strategies for plant protection.

Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction

OBJECTIVES: Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1 percent) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100 percent) at codon 180 and codon 204. Concordant results were observed for 99.4 percent of the 158 samples at codon 181 and 98.7 percent at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1 percent mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.

A modified simple RFLP-PCR method for single nucleotide polymorphism (SNP) typing

We describe a modified single nucleotide polymorphism (SNP) typing method based on the restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). This is a simple, economical method without the need for special equipment. For most SNP loci, a common restriction endonuclease (Hind III, EcoR I or BamH I) recognizing site (RER) can be introduced into one allelic form, but not the other by two rounds of mismatched PCR. The flanking regions can be changed by as many as five bases after PCR amplification with specially designed mismatching primers so the genotypes can be distinguished after digestion of the PCR products with corresponding endonucleases.