Mesoscale DNA feature in antibody-coding sequence facilitates somatic hypermutation.

Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.

The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women.

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non-small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

A multimodal atlas of hepatocellular carcinoma reveals convergent evolutionary paths and 'bad apple' effect on clinical trajectory.

BACKGROUND & AIMS: Hepatocellular Carcinoma (HCC) is a highly fatal cancer characterized by high intra-tumor heterogeneity (ITH). A panoramic understanding of its tumor evolution, in relation to its clinical trajectory, may provide novel prognostic and treatment strategies. METHODS: Through the Asia-Pacific Hepatocellular Carcinoma (AHCC) trials group (NCT03267641), we recruited one of the largest prospective cohorts of HCC with over 600 whole genome and transcriptome samples from 123 treatment-naïve patients. RESULTS: Using a multi-region sampling approach, we revealed seven convergent genetic evolutionary paths governed by the early driver mutations, late copy number variations and viral integrations, which stratify patient clinical trajectories after surgical resection. Furthermore, such evolutionary paths shaped the molecular profiles, leading to distinct transcriptomic subtypes. Most significantly, although we found the coexistence of multiple transcriptomic subtypes within certain tumors, patient prognosis was best predicted by the most aggressive cell fraction of the tumor, rather than by overall degree of transcriptomic ITH level - a phenomenon we termed the 'bad apple' effect. Finally, we found that characteristics throughout early and late tumor evolution provide significant and complementary prognostic power in predicting patient survival. CONCLUSIONS: Taken together, our study generated a comprehensive landscape of evolutionary history for HCC and provided a rich multi-omics resource for understanding tumor heterogeneity and clinical trajectories. CLINICAL TRIAL NUMBER: NCT03267641 (Observational cohort) IMPACT AND IMPLICATIONS: This prospective study, utilizing comprehensive multi-sector, multi-omics sequencing and clinical data from surgically resected HCC, reveals critical insights into the role of tumor evolution and intra-tumor heterogeneity (ITH) in determining the prognosis of Hepatocellular Carcinoma (HCC). These findings are invaluable for oncology researchers and clinicians, as they underscore the influence of distinct evolutionary paths and the 'bad apple' effect, where the most aggressive tumor fraction dictates disease progression. These insights not only enhance prognostic accuracy post-surgical resection but also pave the way for developing personalized therapies tailored to specific tumor evolutionary and transcriptomic profiles. The co-existence of multiple sub-types within the same tumor prompts a re-appraisal of the utilities of depending on single samples to represent the entire tumor and suggests the need for clinical molecular imaging. This research thus marks a significant step forward in the clinical understanding and management of HCC, underscoring the importance of integrating tumor evolutionary dynamics and multi-omics biomarkers into therapeutic decision-making.

Highly Stable Lithium Metal Batteries Enabled by Tuning the Molecular Polarity of Diluents in Localized High-Concentration Electrolytes.

Electrolyte plays a crucial role in ensuring stable operation of lithium metal batteries (LMBs). Localized high-concentration electrolytes (LHCEs) have the potential to form a robust solid-electrolyte interphase (SEI) and mitigate Li dendrite growth, making them a highly promising electrolyte option. However, the principles governing the selection of diluents, a crucial component in LHCE, have not been clearly determined, hampering the advancement of such a type of electrolyte systems. Herein, the diluents from the perspective of molecular polarity are rationally designed and developed. A moderately fluorinated solvent, 1-(1,1,2,2-tetrafluoroethoxy)propane (TNE), is employed as a diluent to create a novel LHCE. The unique molecular structure of TNE enhances the intrinsic dipole moment, thereby altering solvent interactions and the coordination environment of Li-ions in LHCE. The achieved solvation structure not only enhances the bulk properties of LHCE, but also facilitates the formation of more stable anion-derived SEIs featured with a higher proportion of inorganic species. Consequently, the corresponding full cells of both Li||LiFePO4 and Li||LiNi0.8 Co0.1 Mn0.1 O2 cells utilizing Li thin-film anodes exhibit extended long-term stability with significantly improved average Coulombic efficiency. This work offers new insights into the functions of diluents in LHCEs and provides direction for further optimizing the LHCEs for LMBs.

Umpolung Flow Chemistry for the Synthesis of a 3-Oxo-3H-spiro[benzofuran-2,4'-piperidine] Building Block.

An efficient and scalable route to tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate, a central prochiral intermediate in the synthesis of SHP2 inhibitor GDC-1971 (migoprotafib), was achieved. Preparation of the title compound from readily available 2-fluorobenzaldehyde included formation of a modified Katritzky benzotriazole hemiaminal, which, upon deprotonation by n-butyllithium, participated in umpolung reactivity via 1,2-addition to tert-butyl 4-oxopiperidine-1-carboxylate (N-Boc-4-piperidone). Most notably, this reaction was developed as a robust plug-flow process that could be executed on multiple kilograms without the need for pilot-scale reaction vessels operating at low cryogenic temperatures. Treatment of the resulting tetrahedral intermediate with oxalic acid resulted in collapse to the corresponding 4-(2-fluorobenzoyl)-4-hydroxypiperidine, which was isolated as a solid via crystallization. The synthesis concluded with an optimized intramolecular SNAr reaction and final crystallization to generate tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate as a stable, high-quality intermediate suitable for further functionalization toward GDC-1971.

Hypoxia-driven immunosuppression by Treg and type-2 conventional dendritic cells in HCC.

BACKGROUND AND AIMS: Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS: We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS: We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.

MAE-Based Self-Supervised Pretraining Algorithm for Heart Rate Estimation of Radar Signals.

Noncontact heart rate monitoring techniques based on millimeter-wave radar have advantages in unique medical scenarios. However, the accuracy of the existing noncontact heart rate estimation methods is still limited by interference, such as DC offsets, respiratory harmonics, and environmental noise. Additionally, these methods still require longer observation times. Most deep learning methods related to heart rate estimation still need to collect more heart rate marker data for training. To address the above problems, this paper introduces a radar signal-based heart rate estimation network named the "masked phase autoencoders with a vision transformer network" (MVN). This network is grounded on masked autoencoders (MAEs) for self-supervised pretraining and a vision transformer (ViT) for transfer learning. During the phase preprocessing stage, phase differencing and interpolation smoothing are performed on the input phase signal. In the self-supervised pretraining step, masked self-supervised training is performed on the phase signal using the MAE network. In the transfer learning stage, the encoder segment of the MAE network is integrated with the ViT network to enable transfer learning using labeled heart rate data. The innovative MVN offers a dual advantage-it not only reduces the cost associated with heart rate data acquisition but also adeptly addresses the issue of respiratory harmonic interference, which is an improvement over conventional signal processing methods. The experimental results show that the process in this paper improves the accuracy of heart rate estimation while reducing the requisite observation time.

Conditional Knockout of Hypoxia-Inducible Factor 1-Alpha in Tumor-Infiltrating Neutrophils Protects against Pancreatic Ductal Adenocarcinoma.

Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1α) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1α (HIF-1αΔNφ) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1α in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer.

The underlying molecular mechanisms and biomarkers between periodontitis and COVID-19.

OBJECTIVE: Emerging evidence shows the clinical consequences of patient with COVID-19 and periodontitis are not promising, and periodontitis is a risk factor. Periodontitis and COVID-19 probably have a relationship. Hence, this study aimed to identify the common molecular mechanism that may help to devise potential therapeutic strategies in the future. MATERIAL AND METHODS: We analyzed two RNA-seq datasets for differential expressed genes, enrichment of biological processes, transcription factors (TFs) and deconvolution-based immune cell types in periodontitis, COVID-19 and healthy controls. Relationships between TFs and mRNA were established by Pearson correlation analysis, and the common TFs-mRNA regulatory network and nine co-upregulated TFs of the two diseases was obtained. The RT-PCR detected the TFs. RESULTS: A total of 1616 and 10201 differentially expressed gene (DEGs) from periodontitis and COVID-19 are found. Moreover, nine shared TFs and common biological processes associated with lymphocyte activation involved in immune response were identified across periodontitis and COVID-19. The cell type enrichment revealed elevated plasma cells among two diseases. The RT-PCR further confirmed the nine TFs up-regulation in periodontitis. CONCLUSION: The pathogenesis of periodontitis and COVID-19 is closely related to the expression of TFs and lymphocyte activation, which can provide potential targets for treatment.

Highly efficient removal of ozone by amorphous manganese oxides synthesized with a simple hydrothermal method.

Amorphous manganese oxides (MnOx) were synthesized by facile hydrothermal reactions between potassium permanganate and manganese acetate. Synthesis parameters, including hydrothermal time and temperature and molar ratio of precursors, significantly affected the ozone removal performance and structure property of MnOx. Amorphous MnOx-1.5, which was prepared at the Mn2+/Mn7+ molar ratio of 1.5 under hydrothermal conditions of 120°C and 2 hr, showed the highest ozone removal rate of 93% after 480 min at the room temperature, RH (relative humidity) = 80% and WHSV (weight hourly space velocity) = 600 L/(g·hr). The morphology, composition and structure of catalysts were investigated with X-ray diffractometer (XRD), Raman spectra, N2 physisorption, field emission scanning electron microscope (FESEM), X-ray photoelectron spectroscopy (XPS), H2 temperature-programmed reduction (H2-TPR), O2 temperature-programmed desorption (O2-TPD) and in situ diffuse reflectance infrared Fourier transform spectroscopy (in situ DRIFTS). It was confirmed that high catalytic activity of amorphous MnOx for ozone removal was mainly ascribed to its abundant oxygen vacancies, high oxygen mobility and large specific surface area.

Cerium-modified amorphous manganese oxides for efficient catalytic removal of ozone.

Manganese-based catalysts were widely developed for catalytic removal of ozone, and the low stability and water inactivation are major challenges. To improve removal performance of ozone, three methods were applied to modify amorphous manganese oxides, including acidification, calcination and Ce modification. The physiochemical properties of prepared samples were characterized, and the catalytic activity for ozone removal was evaluated. All modification methods can promote the removal of ozone by amorphous manganese oxides, and Ce modification showed the most significant enhancement. It was confirmed that the introduction of Ce markedly changed the amount and property of oxygen vacancies in amorphous manganese oxides. Superior catalytic activity of Ce-MnOx can be ascribed to its more content and enhanced formation ability of oxygen vacancies, larger specific surface area and higher oxygen mobility. Furthermore, the durability tests under high relative humidity (80%) determined that Ce-MnOx showed excellent stability and water resistance. These demonstrate the promising potential of amorphously Ce-modified manganese oxides for catalytic removal of ozone.

Atroposelective Negishi Coupling Optimization Guided by Multivariate Linear Regression Analysis: Asymmetric Synthesis of KRAS G12C Covalent Inhibitor GDC-6036.

An efficient asymmetric synthesis of a potent KRAS G12C covalent inhibitor, GDC-6036 (1), is reported. The synthesis features a highly atroposelective Negishi coupling to construct the key C-C bond between two highly functionalized pyridine and quinazoline moieties by employing a Pd/Walphos catalytic system. Statistical modeling by comparing computational descriptors of a range of Walphos chiral bisphosphine ligands to a training set of experimental results was used to inform the selection of the best ligand, W057-2, which afforded the desired Negishi coupling product (Ra)-3 in excellent selectivity. A subsequent telescoped reaction sequence of alkoxylation, global deprotection, and acrylamide formation, followed by a final adipate salt formation, furnished GDC-6036 (1) in 40% overall yield from starting materials pyridine 5 and quinazoline 6.

Efficient RNA Virus Targeting via CRISPR/CasRx in Fish.

The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. IMPORTANCE RNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.

Analysis and Validation of Human Targets and Treatments Using a Hepatocellular Carcinoma-Immune Humanized Mouse Model.

BACKGROUND AND AIMS: Recent development of multiple treatments for human hepatocellular carcinoma (HCC) has allowed for the selection of combination therapy to enhance the effectiveness of monotherapy. Optimal selection of therapies is based on both HCC and its microenvironment. Therefore, it is critical to develop and validate preclinical animal models for testing clinical therapeutic solutions. APPROACH AND RESULTS: We established cell line-based or patient-derived xenograft-based humanized-immune-system mouse models with subcutaneous and orthotopic HCC. Mice were injected with human-specific antibodies (Abs) to deplete human immune cells. We analyzed the transcription profiles of HCC cells and human immune cells by using real-time PCR and RNA sequencing. The protein level of HCC tumor cells/tissues or human immune cells was determined by using flow cytometry, western blotting, and immunohistochemistry. The HCC tumor size was measured after single, dual-combination, and triple-combination treatment using N-(1',2-Dihydroxy-1,2'-binaphthalen-4'-yl)-4-methoxybenzenesulfonamide (C188-9), bevacizumab, and pembrolizumab. In this study, human immune cells in the tumor microenvironment were strongly selected and modulated by HCC, which promoted the activation of the IL-6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in tumor cells and led to augmented HCC proliferation and angiogenesis by releasing angiogenic cytokines in humanized-immune-system mice with HCC. In particular, intratumor human cluster of differentiation-positive (hCD14+ ) cells could produce IL-33 through damage-associated molecular pattern/Toll-like receptor 4/activator protein 1, which up-regulated IL-6 in other intratumor immune cells and activated the JAK2/STAT3 pathway in HCC. Specific knockdown of the CD14 gene in human monocytes could impair IL-33 production induced by cell lysates. Subsequently, we evaluated the in vivo anti-HCC effect of C188-9, bevacizumab, and pembrolizumab. The results showed that the anti-HCC effect of triple-combination therapy was superior to that of single or dual treatments. CONCLUSIONS: Humanized-immune-system HCC mouse models are suitable for identifying targets from cancer and immune components and for testing combinational therapies.

Alterations in mitochondrial biogenesis and respiratory activity, inflammation of the senescence-associated secretory phenotype, and lipolysis in the perirenal fat and liver of rats following lifelong exercise and detraining.

The primary aims of this study were to determine the effects of lifelong exercise and detraining on age-related alterations in mitochondrial function, inflammation associated with senescence-associated secretory phenotype (SASP), and lipolysis in the perirenal fat and liver of rats. Female Sprague-Dawley rats were randomly assigned to four groups: young control (n = 12), old control (n = 12), detraining (n = 12), and lifelong exercise (n = 12). We then investigated mitochondrial function, SASP-associated inflammation, and lipolysis in the perirenal fat and liver using qRT-PCR and western blotting to assess the expression of AKT, hypoxia-inducible factor 1α (HIF-1α), nuclear factor-kappa B (NF-κB), c-jun kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK). In the tissues of both the perirenal fat and liver, lifelong exercise significantly improved mitochondrial function, SASP-associated inflammation, and lipolysis. Meanwhile, pathways associated with inflammatory regulation were inhibited, predominantly via the activation of phosphorylated-AKT (p-AKT) and suppression of HIF-1α in both tissues, and via JNK in the perirenal fat and p38MAPK in the liver. Furthermore, detraining activated NF-κB expression in both tissues and induced the upregulation of serum high-sensitivity C-reactive protein (hsCRP) levels. Collectively, lifelong exercise was found to exert beneficial effects by ameliorating age-related alterations in mitochondrial function, SASP-associated inflammation, and lipolysis in perirenal fat and liver tissues, potentially inhibiting inflammation via the JNK and p38 MAPK pathways, respectively, as well as the HIF-1α and AKT pathways in both tissues. In contrast, detraining induced high levels of circulating hsCRP by activating the NF-κB signaling pathway in both tissues.

Monocytic Myeloid-Derived Suppressor Cells Underpin Resistance to Adoptive T Cell Therapy in Nasopharyngeal Carcinoma.

Advanced, late-stage Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) is incurable, and its treatment remains a clinical and therapeutic challenge. Results from a phase II clinical trial in advanced NPC patients employing a combined chemotherapy and EBV-specific T cell (EBVST) immunotherapy regimen showed a response rate of 71.4%. Longitudinal analysis of patient samples showed that an increase in EBV DNA plasma concentrations and the peripheral monocyte-to-lymphocyte ratio negatively correlated with overall survival. These parameters were combined into a multivariate analysis to stratify patients according to risk of death. Immunophenotyping at serial time points showed that low-risk individuals displayed significantly decreased amounts of monocytic myeloid-derived suppressor cells postchemotherapy, which subsequently influenced successful cytotoxic T-lymphocyte (CTL) immunotherapy. Examination of the low-risk group, 2 weeks post-EBVST infusion, showed that individuals with a greater overall survival possessed an increased frequency of CD8 central and effector memory T cells, together with higher levels of plasma interferon (IFN)-γ, and cytotoxic lymphocyte-associated transcripts. These results highlight the importance of the rational selection of chemotherapeutic agents and consideration of their impact on both systemic immune responses and downstream cellular immunotherapy outcomes.

Elucidating the structure and cytochrome P450-mediated mechanism for novel metabolites of GDC-0575 in rats.

1. GDC-0575 is an ATP-competitive small-molecule inhibitor of ChK1 that is being developed by Genentech for the treatment of various human malignancies.2. In a radiolabeled mass balance study of GDC-0575 in rats, two novel metabolites, named M12 (-71 Da,) and M17 (+288 Da), were detected as abundant circulating metabolites.3. Subsequent mass spectrometry and nuclear magnetic resonance analysis showed that M12 was a cyclized metabolite of GDC-0575, whereas M17 was its heterodimer to the parent. We further determined that M12 was mainly generated by cytochrome P450 (Cyp) 2d2.4. We proposed the potential mechanism was initiated by the oxidation on the pyrrole ring and subsequent cyclisation of the free primary amine onto C-3 of the pyrrole ring. This was followed by expulsion of cyclopropylcarboxamide and a loss of water to form intermediate I, which can be further oxidised to form M12, or dimerise with another molecule of GDC-0575 as nucleophile to form M17.5. To verify this hypothesis, we attempted to trap the intermediate I with glutathione (GSH) trapping assay and the GSH conjugate on the pyrrole ring was identified. This suggests the oxidation on the pyrrole led to reactive metabolite formation and supported this proposed mechanism.

Overexpression of miR-146a promotes cell proliferation and migration in a model of diabetic foot ulcers by regulating the AKAP12 axis.

In the current study, we aimed to study the effect of miR-146a on proliferation and migration in an in vitro diabetic foot ulcer (DFU) model by targeting A-kinase-anchoring protein 12 (AKAP12). An in vitro DFU model was initially established using HaCaT cells derived from human keratinocytes and induced by advanced glycation end products (AGEs). The effects of overexpression of miR-146a on proliferation and migration ability were analysed. The expression levels of miR-146a and AKAP12 were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and AKAP12, hypoxia-inducible factor-1α (HIF-1α), Wnt3a and ß-catenin protein levels were measured by western blotting. The cell proliferation ability was measured by MTT, and the migration ability was analysed by a cell scratch assay. The binding between miR-146a and AKAP12 was identified using a luciferase reporter assay. The results demonstrated that AGEs significantly suppressed cell proliferation and migration, while the expression of miR-146a decreased and the expression of AKAP12 increased. A luciferase reporter assay revealed that miR-146a could directly target AKAP12. Overexpression of miR-146a promoted cell proliferation and migration in an in vitro DFU model and also promoted the expression of HIF-1α, Wnt3a and ß-catenin but suppressed the expression of AKAP12. Co-overexpression of miR-146a and AKAP12 reversed the effect of miR-146a on cell proliferation and migration. Our findings revealed that miR-146a directly targeted AKAP12 and promoted cell proliferation and migration in an in vitro DFU model. This study provides a new perspective for the study of miR-146a in the treatment of DFU.

Transcriptome analysis of the spleen provides insight into the immunoregulation of Scortum barcoo under Streptococcus agalactiae infection.

Jade perch (Scortum barcoo) is a freshwater fish with substantial economic value, which has been widely cultivated all over the world. However, with the intensification and expansion of farming, several bacterial and viral diseases have occurred in jade perch. To understand the immune response of jade perch against Streptococcus agalactiae (Group B Streptococcus, GBS), we performed a histopathological examination and transcriptome sequencing of jade perch spleen after artificial bacterial infection. GBS infection can cause structural changes and even necrosis of the jade perch spleen, which may affect the survival of infected individuals. A total of 144,458 unigenes were obtained through de novo assembly of spleen transcriptome. Among them, 1821 unigenes were identified as DEGs, including 1415 up-regulated and 406 down-regulated unigenes in the infection group. Moreover, the analysis of GO and KEGG revealed that many GO terms and pathways were involved in the host immune response, such as Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, and TNF signaling pathway. In addition, according to transcriptome data and qRT-PCR analysis, the expression levels of many cytokines that participate in the inflammatory response changed a lot after GBS infection. Overall, this transcriptomic analysis provided valuable information for studying the immune response of jade perch against bacterial infection.

Stereoconvergent and -divergent Synthesis of Tetrasubstituted Alkenes by Nickel-Catalyzed Cross-Couplings.

We report the development of a method to diastereoselectively access tetrasubstituted alkenes via nickel-catalyzed Suzuki-Miyaura cross-couplings of enol tosylates and boronic acid esters. Either diastereomeric product was selectively accessed from a mixture of enol tosylate starting material diastereomers in a convergent reaction by judicious choice of the ligand and reaction conditions. A similar protocol also enabled a divergent synthesis of each product isomer from diastereomerically pure enol tosylates. Notably, high-throughput optimization of the monophosphine ligands was guided by chemical space analysis of the kraken library to ensure a diverse selection of ligands was examined. Stereoelectronic analysis of the results provided insight into the requirements for reactive and selective ligands in this transformation. The synthetic utility of the optimized catalytic system was then probed in the stereoselective synthesis of various tetrasubstituted alkenes, with yields up to 94% and diastereomeric ratios up to 99:1 Z/E and 93:7 E/Z observed. Moreover, a detailed computational analysis and experimental mechanistic studies provided key insights into the nature of the underlying isomerization process impacting selectivity in the cross-coupling.