Roboticized AI-assisted microfluidic photocatalytic synthesis and screening up to 10,000 reactions per day.

The current throughput of conventional organic chemical synthesis is usually a few experiments for each operator per day. We develop a robotic system for ultra-high-throughput chemical synthesis, online characterization, and large-scale condition screening of photocatalytic reactions, based on the liquid-core waveguide, microfluidic liquid-handling, and artificial intelligence techniques. The system is capable of performing automated reactant mixture preparation, changing, introduction, ultra-fast photocatalytic reactions in seconds, online spectroscopic detection of the reaction product, and screening of different reaction conditions. We apply the system in large-scale screening of 12,000 reaction conditions of a photocatalytic [2 + 2] cycloaddition reaction including multiple continuous and discrete variables, reaching an ultra-high throughput up to 10,000 reaction conditions per day. Based on the data, AI-assisted cross-substrate/photocatalyst prediction is conducted.

Evolution and functional divergence of glycosyltransferase genes shaped the quality and cold tolerance of tea plants.

Plant glycosyltransferases (UGTs) play a key role in plant growth and metabolism. Here, we examined the evolutionary landscape among UGTs in 28 fully sequenced species from early algae to angiosperms. Our findings revealed a distinctive expansion and contraction of UGTs in the G and H groups in tea (Camellia sinensis), respectively. Whole-genome duplication and tandem duplication events jointly drove the massive expansion of UGTs, and the interplay of natural and artificial selection has resulted in marked functional divergence within the G group of the sinensis-type tea population. In Cluster II of group G, differences in substrate selection (e.g., Abscisic Acid) of the enzymes encoded by UGT genes led to their functional diversification, and these genes influence tolerance to abiotic stresses such as low temperature and drought via different modes of positive and negative regulation, respectively. UGTs in Cluster III of the G group have diverse aroma substrate preferences, which contributes a diverse aroma spectrum of the sinensis-type tea population. All Cluster III genes respond to low-temperature stress, whereas UGTs within Cluster III-1, shaped by artificial selection, are unresponsive to drought. This suggests that artificial selection of tea plants focused on improving quality and cold tolerance as primary targets.

The mitochondrial genome of Lavandula angustifolia Mill. (Lamiaceae) sheds light on its genome structure and gene transfer between organelles.

BACKGROUND: Lavandula angustifolia holds importance as an aromatic plant with extensive applications spanning the fragrance, perfume, cosmetics, aromatherapy, and spa sectors. Beyond its aesthetic and sensory applications, this plant offers medicinal benefits as a natural herbal remedy and finds use in household cleaning products. While extensive genomic data, inclusive of plastid and nuclear genomes, are available for this species, researchers have yet to characterize its mitochondrial genome. This gap in knowledge hampers deeper understanding of the genome organization and its evolutionary significance. RESULTS: Through the course of this study, we successfully assembled and annotated the mitochondrial genome of L. angustifolia, marking a first in this domain. This assembled genome encompasses 61 genes, which comprise 34 protein-coding genes, 24 transfer RNA genes, and three ribosomal RNA genes. We identified a chloroplast sequence insertion into the mitogenome, which spans a length of 10,645 bp, accounting for 2.94% of the mitogenome size. Within these inserted sequences, there are seven intact tRNA genes (trnH-GUG, trnW-CCA, trnD-GUC, trnS-GGA, trnN-GUU, trnT-GGU, trnP-UGG) and four complete protein-coding genes (psbA, rps15, petL, petG) of chloroplast derivation. Additional discoveries include 88 microsatellites, 15 tandem repeats, 74 palindromic repeats, and 87 forward long repeats. An RNA editing analysis highlighted an elevated count of editing sites in the cytochrome c oxidase genes, notably ccmB with 34 editing sites, ccmFN with 32, and ccmC with 29. All protein-coding genes showed evidence of cytidine-to-uracil conversion. A phylogenetic analysis, utilizing common protein-coding genes from 23 Lamiales species, yielded a tree with consistent topology, supported by high confidence values. CONCLUSIONS: Analysis of the current mitogenome resource revealed its typical circular genome structure. Notably, sequences originally from the chloroplast genome were found within the mitogenome, pointing to the occurrence of horizontal gene transfer between organelles. This assembled mitogenome stands as a valuable resource for subsequent studies on mitogenome structures, their evolution, and molecular biology.

Zinc pyrithione ameliorates colitis in mice by interacting on intestinal epithelial TRPA1 and TRPV4 channels.

AIMS: Although zinc pyrithione (ZPT) has been studied as topical antimicrobial and cosmetic consumer products, little is known about its pharmacological actions in gastrointestinal (GI) health and inflammation. Our aims were to investigate the effects of ZPT on transient receptor potential (TRP) channels and Ca2+ signaling in intestinal epithelial cells (IECs) and its therapeutic potential for colitis. MAIN METHODS: Digital Ca2+ imaging and patch-clamp electrophysiology were performed on human colonic epithelial cells (HCoEpiC) and rat small intestinal epithelial cells (IEC-6). The transcription levels of proinflammatory cytokines such as IL-1ß were detected by RTq-PCR. Dextran sulfate sodium (DSS) was used to induce colitis in mice. KEY FINDINGS: ZPT dose-dependently induced Ca2+ signaling and membrane currents in IECs, which were attenuated by selective blockers of transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 4 (TRPV4) channels, respectively. Interestingly, Ca2+ entry via TRPA1 channels inhibited the activity of TRPV4 channels in HCoEpiC, but not vice versa. ZPT significantly promoted migration of IECs by activating TRPA1 and TRPV4 channels. ZPT reversed lipopolysaccharides (LPS)-induced changes in mRNA expression of TRPA1 and TRPV4. Moreover, ZPT decreased mRNA levels of pro-inflammatory factors promoted by LPS in HCoEpiC, which were restored by selective TRPA1 blocker. In whole animal studies in vivo, ZPT significantly ameliorated DSS-induced body weight loss, colon shortening and increases in stool score, serum calprotectin and lactic acid (LD) in mouse model of colitis. SIGNIFICANCE: ZPT exerts anti-colitic action likely by anti-inflammation and pro-mucosal healing through TRP channels in IECs. The present study not only expands pharmacology spectrum of ZPT in GI tract, but also repurposes it to a potential drug for colitis therapy.

Biosensor development for diabetes diagnosis: Determining relevant miRNA using a newly developed N-annulated perylene fluorescent dye.

MicroRNAs (miRNAs) have emerged as essential biomarkers for disease diagnosis, and several techniques are available to determine type 2 diabetes (T2D) relevant miRNAs. However, detecting circulating miRNAs can be challenging due to their small size, low abundance, and high sequence similarity, often requiring sensitive detection approaches combined with additional amplification processes. Laser-induced fluorescence (LIF) is a classic analytical method suitable for sensitively detecting trace amounts of nucleotide acid. Duplex-specific nuclease (DSN)-mediated amplification recently gained attention due to its catalytic activity based on target recycling, demonstrating a promising approach for miRNA amplification. This work developed a novel N-annulated perylene fluorescent dye to create a biosensor to analyze the miRNA (miR-223) relevant to T2D. The amine-reactive fluorescent dye assists the amidation reaction for nucleotide labeling, giving the oligonucleotide probe a high fluorescence quantum yield and sufficient water solubility. By combining the locked nucleic acid (LNA) modified oligonucleotide fluorescent probe to enhance the stability of LNA/RNA hybrids, thereby improving the DSN-mediated target miR-223 recycling for signal amplification, the proposed biosensor can highly selectively determine miR-223 with a limit of detection (LOD, S/N = 3) of 9.5 pM. When applied to real-world samples, the biosensor demonstrated its potential to distinguish between T2D patients and healthy controls.

Impact of a Potent Strain of Plant Growth-Promoting Bacteria (PGPB), Bacillus subtilis S1 on Bacterial Community Composition, Enzymatic Activity, and Nitrogen Content in Cucumber Rhizosphere Soils.

Antagonistic bacterial strains from Bacillus spp. have been widely studied and utilized in the biocontrol of phytopathogens and the promotion of plant growth, but their impacts on the rhizosphere microecology when applied to crop plants are unclear. Herein, the effects of applying the antagonistic bacterium Bacillus subtilis S1 as a biofertilizer on the rhizosphere microecology of cucumbers were investigated. In a pot experiment on cucumber seedlings inoculated with S1, 3124 bacterial operational taxonomic units (OTUs) were obtained from the rhizosphere soils using high-throughput sequencing of 16S rRNA gene amplicons, and the most abundant phylum was Proteobacteria that accounted for 49.48% in the bacterial community. S1 treatment significantly reduced the abundances of soil bacterial taxa during a period of approximately 30 days but did not affect bacterial diversity in the rhizosphere soils of cucumbers. The enzymatic activities of soil nitrite reductase (S-Nir) and dehydrogenase (S-DHA) were significantly increased after S1 fertilization. However, the activities of soil urease (S-UE), cellulase (S-CL), and sucrase (S-SC) were significantly reduced compared to the control group. Additionally, the ammonium- and nitrate-nitrogen contents of S1-treated soil samples were significantly lower than those of the control group. S1 fertilization reshaped the rhizosphere soil bacterial community of cucumber plants. The S-CL activity and nitrate-nitrogen content in rhizosphere soil affected by S1 inoculation play important roles in altering the abundance of rhizosphere soil microbiota.

High-density lipoprotein cholesterol trajectory and new-onset metabolic dysfunction-associated fatty liver disease incidence: a longitudinal study.

BACKGROUND: Although high-density lipoprotein cholesterol (HDL-C) exerts a significant influence on the development of metabolic dysfunction-associated fatty liver disease (MAFLD), the association of dynamic changes in HDL-C levels with the risk of MAFLD remains unclear. Thus, the aim of the current study was to explore the association between the changing trajectories of HDL-C and new-onset MAFLD. The findings of this study may provide a theoretical basis for future personalized intervention and prevention targeting MAFLD. METHODS: A total of 1507 participants who met the inclusion criteria were recruited from a community-based physical examination population in Nanjing, China from 2017 to 2021. Group-based trajectory models were constructed to determine the heterogeneous HDL-C trajectories. The incidence of MAFLD in each group in 2022 was followed up, and the Cox proportional hazards regression model was applied to investigate the associations between different HDL-C trajectories and the risk of new-onset MAFLD. RESULTS: The incidences of MAFLD in the low-stable, moderate-stable, moderate-high-stable, and high-stable groups of HDL-C trajectory were 26.5%, 13.8%, 7.2% and 2.6%, respectively. The incidence rate of MAFLD in the order of the above trajectory groups exhibited a decreasing trend (χ2 = 72.55, Ptrend<0.001). After adjusting for confounders, the risk of MAFLD onset in HDL-C low-stable group was still 5.421 times (95%CI: 1.303-22.554, P = 0.020) higher than that in the high-stable group. Subgroup analyses of the combined (moderate high-stable and high-stable groups combined), moderate-stable and low-stable groups showed that sex, age, and overweight/obesity did not affect the association between HDL-C trajectory and MAFLD risk. CONCLUSIONS: Persistently low HDL-C level is a risk factor for the onset of MAFLD. Long-term monitoring of HDL-C levels and timely intervention for those experiencing persistent declines are crucial for early prevention of MAFLD.

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) gene polymorphisms and five related serum molecular levels in 2587 patients: Associated differentially with adverse pregnancy.

BACKGROUND: The aim of the study is to investigate the relationship between Methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) polymorphisms, 5 serum related molecular levels and the risk of adverse pregnancies in different genders. METHODS: Patients aged from 22 to 38 with a history of adverse pregnancy treated in our genetic eugenics clinic of Henan Provincial People's Hospital are selected. The controls aged from 20 to 34 undergoing eugenics examinations in our genetic eugenics clinic that had no history of adverse pregnancy and at least one healthy child are selected. Sanger sequencing and Chemiluminescence Microparticle Immuno Assay (CMIA) are used for detecting the mutations of MTHFR and MTRR and the 5 serum molecular serum levels. RESULTS: In the female group, MTHFR 677 C > T is associated with Recurrent spontaneous abortion (RSA) (P = 0.0017), Chromosomal abnormality (CA) (P = 0.0053), Cleft lip and palate (CLP) (P = 0.0326) and Brain dysplasia (BD) (P = 0.0072); MTHFR 1298 A > C is associated with Infertility (P = 0.0026) and BD (P = 0.0382); MTRR 66 A > G is associated with CLP (P = 0.0131). In the male group, MTHFR 677 C > T is associated with RSA (P = 0.0003), Infertility (P = 0.0013), CA (P = 0.0027) and BD (P = 0.0293). In the female group, the genotype of MTHFR 677 C > T is associated with RSA (P = 0.0017), CA (P = 0.0014) and BD (P = 0.0021); MTHFR 1298 A > C is associated with Infertility (P = 0.0081) and MTRR 66 A > G is associated with Infertility (P = 0.0309). In the male group, the genotype of MTHFR 677 C > T is associated with RSA (P = 0.0008), Infertility (P = 0.0096) and CA (P = 0.0165) and MTRR 66 A > G is associated with Infertility (P = 0.0158) and congenital heart disease (CHD) (P = 0.0218). In the male group, there is statistically significant difference of the serum Homocysteine (Hcy) levels (P < 0.0001) between adverse pregnancy group and controls. In the female group, there is statistically significant difference of the serum vitamin D levels (P = 0.0015) between adverse pregnancy group and controls. CONCLUSIONS: Polymorphic variants in MTHFR and MTRR, serum Folic acid (FA), Hcy and B12 levels in the male group and vitamin D levels in the female group are associated differentially with adverse pregnancy.

A novel de novo synonymous variant in GREB1L impacts the mRNA splicing associated with aplasia of the urogenital system.

GREB1-like retinoic acid receptor coactivator (GREB1L) gene is associated with autosomal dominant renal hypodysplasia/aplasia 3 (RHDA3) and deafness, autosomal dominant 80 (DFNA80). Among the GREB1L variants reported, most of them are missense or frameshift, while no pathogenic synonymous variants have been recorded. Classical theory paid little attention to synonymous variants and classified it as nonpathogenic; however, recent studies suggest that the variants might be equally important. Here, we report a 7-year-old girl with new symptoms of clitoromegaly, uterovaginal, and ovarian agenesis as well as right kidney missing. A novel de novo GREB1L synonymous variant (NM_001142966: c.4731C>T, p.G1577=) was identified via whole exome sequencing. The variant was predicted to be disease-causing through in silico analysis and was classified as likely pathogenic. Minigene splicing assays confirmed a 6 bp deletion in mutant cDNA comparing with the wild type, leading to two amino acids lost in GREB1L protein. Secondary and tertiary structure modeling showed alterations in protein structure. Our finding reveals a novel GREB1L variant with a new phenotype of urogenital system and is the first to report a pathogenic synonymous variant in GREB1L which affects mRNA splicing, suggesting synonymous variants cannot be ignored in prenatal diagnosis and genetic counseling.

B7-H3 promotes the migration and invasion of colorectal cancer cells via regulating the actin cytoskeleton and RhoA/ROCK1/LIMK1 signaling pathway.

BACKGROUND AND AIMS: Aberrant expression of B7 homolog 3 protein (B7-H3) has been detected in various cancers including colorectal cancer (CRC) and implicated in modulating multiple biological functions of CRC cells. However, its role in CRC metastasis has not yet been determined. This study aims to explore and unravel the underlying mechanisms through which B7-H3 contributes to migration, invasion and actin cytoskeleton in CRC. METHODS: The expression of B7-H3 and LIMK1 in CRC tumor samples was determined by IHC staining. Transwell and F-actin immunofluorescence staining assays were performed to explore the role of B7-H3 in migration, invasion and actin filament accumulating of CRC cells. RNA-seq and Western blot assays were used to investigate the molecular mechanisms. RESULTS: B7-H3 was highly expressed in CRC tissues and positively associated with poor prognosis of CRC patients by immunohistochemistry. Migration and invasion assays showed that B7-H3 knockdown significantly inhibited the migration and invasion of CRC cells. B7-H3 overexpression had the opposite effect. Moreover, we determined that B7-H3 could regulate actin cytoskeleton and the RhoA/ROCK1/LIMK1 pathway by F-actin immunofluorescence staining and Western blot. Importantly, the BDP5290, an inhibitor of the RhoA/ROCK1/(LIM domain kinase 1) LIMK1 axis, reversed the effects of B7-H3 overexpression on actin filament accumulating, migration, and invasion of CRC cells. CONCLUSIONS: Our study concluded that B7-H3 facilitated CRC cell actin filament accumulating, migration, and invasion through the RhoA/ROCK1/LIMK1 axis.

Mesoporous Graphene Oxide Nanocomposite Effective for Combined Chemo/Photo Therapy Against Non-Small Cell Lung Cancer.

Introduction: Lung cancer is the most common cancer worldwide, among which non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers. Chemotherapy, a mainstay modality for NSCLC, has demonstrated restricted effectiveness due to the emergence of chemo-resistance and systemic side effects. Studies have indicated that combining chemotherapy with phototherapy, such as photodynamic therapy (PDT) and photothermal therapy (PTT), can enhance efficacy of therapy. In this work, an aminated mesoporous graphene oxide (rPGO)-protoporphyrin IX (PPIX)-hyaluronic acid (HA)@Osimertinib (AZD) nanodrug delivery system (rPPH@AZD) was successfully developed for combined chemotherapy/phototherapy for NSCLC. Methods: A pH/hyaluronidase-responsive nanodrug delivery system (rPPH@AZD) was prepared using mesoporous graphene oxide. Its morphology, elemental composition, surface functional groups, optical properties, in vitro drug release ability, photothermal properties, reactive oxygen species production, cellular uptake and cell viability were evaluated. In addition, the in vivo therapeutic effect, biocompatibility, and imaging capabilities of rPPH@AZD were verified by a tumor-bearing mouse model. Results: Aminated mesoporous graphene oxide (rPGO) plays a role as a drug delivery vehicle owing to its large specific surface area and ease of surface functionalization. rPGO exhibits excellent photothermal conversion properties under laser irradiation, while PPIX acts as a photosensitizer to generate singlet oxygen. AZD acts as a small molecule targeted drug in chemotherapy. In essence, rPPH@AZD shows excellent photothermal and fluorescence imaging effects in tumor-bearing mice. More importantly, in vitro and in vivo results indicate that rPPH@AZD can achieve hyaluronidase/pH dual response as well as combined chemotherapy/PTT/PDT anti-NSCLC treatment. Conclusion: The newly prepared rPPH@AZD can serve as a promising pH/hyaluronidase-responsive nanodrug delivery system that integrates photothermal/fluorescence imaging and chemo/photo combined therapy for efficient therapy against NSCLC.

Kinetically Controlled Self-Assembly of Ag Nanoclusters with Enhanced Luminescence.

Constructing self-assembly with definite assembly structure-property correlation is of great significance for expanding the property richness and functional diversity of metal nanoclusters (NCs). Herein, a well-designed liquid reaction strategy was developed through which a highly ordered nanofiber superstructure with enhanced green photoluminescence (PL) was obtained via self-assembly of the individual silver nanoclusters (Ag NCs). By visual monitoring of the kinetic reaction process using time-dependent and in situ spectroscopy measurements, the assembling structure growth and the structure-determined luminescence mechanisms were revealed. The as-prepared nanofibers featured a series of advantages involving a high emission efficiency, large Stokes shift, homogeneous chromophore, excellent photostability, high temperature, and pH sensibility. By virtue of these merits, they were successfully employed in various fields of luminescent inks, encryption and anticounterfeiting platforms, and optoelectronic light-emitting diode (LED) devices.

Identification of Matrine as a Kirsten rats Arcomaviral oncogene homolog inhibitor alleviating chemotherapy-induced neuropathic pain.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) represents a prevailing and severe clinical concern, characterized by limited availability of clinically effective treatment strategies. Current evidence endorses matrine's potential as a neuroprotective and analgesic agent for CIPN. Nevertheless, the precise targets and mechanisms of action of matrine remain insufficiently explored, impeding comprehensive pharmacological investigation and clinical application. OBJECTIVE: This study endeavors to elucidate the analgesic and neuroprotective effects of matrine in mice with vincristine-induced neuropathic pain. A focal point is the identification of matrine's specific target and the underlying molecular mechanisms governing its analgesic and neuroprotective actions. METHODS: To discern matrine's analgesic effects in CIPN mice, we conducted behavioral experiments encompassing the Von Frey filament test and Hargreaves Test. Furthermore, we conducted electrophysiological and histopathological assessments involving HE staining, Nissl staining, and Fluoro-Jade B staining to evaluate matrine's effects on neuroprotection within dorsal root ganglia and the spinal cord of CIPN mice. Sequentially, thermal shift assay, GTP hydrolysis assay, and nucleotide exchange assay were executed to validate matrine's inhibitory effects on KRAS. Molecular docking and site-directed mutagenesis experiments were implemented to identify the precise binding pocket of matrine on KRAS. Lastly, matrine's inhibitory effects on downstream signaling pathways of KRAS were confirmed through experiments conducted at animal model. RESULTS: Matrine exhibited a notable increase in mechanical withdrawal threshold and thermal withdrawal latency in vincristine-treated mice. This compound substantially ameliorated the neurofunctional blockade associated with sensory and motor functions induced by vincristine. Moreover, matrine mitigated pathological damage within DRG and the L4-L5 spinal cord regions. The study's MST experiments indicated matrine's substantial elevation of KRAS's melting temperature. The GTP hydrolysis and nucleotide exchange assays revealed concentration-dependent inhibition of KRAS activity by matrine. Molecular docking provided insight into the binding mode of matrine with KRAS, while site-directed mutagenesis verified the specific binding site of matrine on KRAS. Lastly, matrine's inhibition of downstream Raf/Erk1/2 and PI3K/Akt/mTOR signaling pathways of KRAS was confirmed in VCR mice. CONCLUSION: Compared to previous studies, our research has identified matrine as a natural inhibitor of the elusive protein KRAS, often considered "undruggable." Furthermore, this study has revealed that matrine exerts its therapeutic effects on chemotherapy-induced peripheral neuropathy (CIPN) by inhibiting KRAS activation, subsequently suppressing downstream signaling pathways such as Raf/Erk1/2 and PI3K/Akt/mTOR. This investigation signifies the discovery of a novel target for matrine, thus expanding the potential scope of its involvement in KRAS-related biological functions and diseases. These findings hold the promise of providing a crucial experimental foundation for forthcoming drug development initiatives centered around matrine, thereby advancing the field of pharmaceutical research.

Superoxide signal orchestrates tetrathiomolybdate-induced longevity via ARGK-1 in Caenorhabditis elegans.

PURPOSE: While reactive oxygen species (ROS) have been identified as key redox signaling agents contributing to aging process, which and how specific oxidants trigger healthy longevity remain unclear. This paper aimed to explore the precise role and signaling mechanism of superoxide (O2•-) in health and longevity. METHODS: A tool for precise regulation of O2•- levels in vivo was developed based on the inhibition of superoxide dismutase 1 (SOD1) by tetrathiomolybdate (TM) in Caenorhabditis elegans (C. elegans). Then, we examined the effects of TM on lifespan, reproduction, lipofuscin accumulation, mobility, and stress resistance. Finally, the signaling mechanism for longevity induced by TM-O2•- was screened by transcriptome analysis and tested in sod-1 and argk-1 RNAi strains, sod-2, sod-3, and daf-16 mutants. RESULTS: TM promoted longevity in C. elegans with a concomitant extension of healthy lifespan as indicated by increasing fertility and mobility and reducing lipofuscin accumulation, as well as enhanced resistance to different abiotic stresses. Mechanically, TM could precisely regulate O2•- levels in nematodes via modulating SOD1 activity. An O2•- scavenger Mn(III)TBAP abolished TM-induced lifespan extension, while an O2•- generator paraquat at low concentration mimicked the life prolongation effects. The longevity in TM-treated worms was abolished by sod-1 RNAi but was not affected in sod-2 or sod-3 mutants. Further transcriptome analysis revealed arginine kinase ARGK-1 and its downstream insulin/insulin-like growth factor 1 signaling (IIS) as potential effectors for TM-O2•‾-induced longevity, and argk-1 RNAi or daf-16 mutant nullified the longevity. CONCLUSIONS: These findings indicate that it is feasible to precisely control specific oxidant in vivo and O2•- orchestrates TM-induced health and longevity in C. elegans via ARGK-1-IIS axis.

A new chemical derivatization reagent sulfonyl piperazinyl for the quantification of fatty acids using LC-MS/MS.

In our previous study, a chemical derivatization reagent named 5-(dimethylamino) naphthalene-1-sulfonyl piperazine (Dns-PP) was developed to enhance the chromatographic retention and the mass spectrometric response of free fatty acids (FFAs) in reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (RPLC-ESI-MS). However, Dns-PP exhibited strong preferences for long-chain FFAs, with limited improvement for short- or medium-chain FFAs. In this study, a new series of labeling reagents targeting FFAs were designed, synthesized, and evaluated. Among these reagents, Tmt-PP (N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine) exhibited the best MS response and was selected for further evaluations. We compared Tmt-PP with Dns-PP and four commonly used carboxyl labeling reagents from existing studies, demonstrating the advantages of Tmt-PP. Further comparisons between Tmt-PP and Dns-PP in measuring FFAs from biological samples revealed that Tmt-PP labeling enhanced the MS response for about 80 % (30/38) of the measured FFAs, particularly for short- and medium-chain FFAs. Moreover, Tmt-PP labeling significantly improved the chromatographic retention of short-chain FFAs. To ensure accurate quantification, we developed a stable isotope-labeled Tmt-PP (i.e., d12-Tmt-PP) to react with chemical standards and serve as one-to-one internal standards (IS). The method was validated for accuracy, precision, sensitivity, linearity, stability, extraction efficiency, as well as matrix effect. Overall, this study introduced a new chemical derivatization reagent Tmt-PP (d12-Tmt-PP), providing a sensitive and accurate option for quantifying FFAs in biological samples.

Development of a Quorum Sensing-Mediated Bacterial Autolytic System in Escherichia coli for Automatic Release of Intracellular Products.

Escherichia coli, one of the most efficient expression hosts for recombinant proteins, is widely used in chemical, medical, food, and other industries. De novo engineering of gene regulation circuits and cell density-controlled E. coli cell lysis are promising directions for the release of intracellular bioproducts. Here, we developed an E. coli autolytic system, named the quorum sensing-mediated bacterial autolytic (QS-BA) system, by incorporating an acyl-homoserine lactone (AHL)-based YasI/YasR-type quorum sensing circuit from Pseudoalteromonas into E. coli cells. The results showed that the E. coli QS-BA system can release the intracellular bioproducts into the cell culture medium in terms of E. coli cell density, which offers an environmentally-friendly, economical, efficient, and flexible E. coli lysis platform for production of recombinant proteins. The QS-BA system has the potential to serve as an integrated system for the large-scale production of target products in E. coli for medical and industrial applications.

A real-world disproportionality analysis of semaglutide: Post-marketing pharmacovigilance data.

AIM/INTRODUCTION: The recent adverse reactions associated with semaglutide have led the Food and Drug Administration (FDA) to issue a "black box warning", and it is necessary to analyze all reports of adverse reactions to improve the safety of its clinical use. MATERIALS AND METHODS: Statistical analyses and signal mining were performed by obtaining the adverse event reports related to semaglutide in the FAERS database from the first quarter of 2018 to the fourth quarter of 2023. We used disproportionality and Bayesian analysis to examine clinical and demographic attributes, trends reported quarterly, and contrasts between two distinct indications (obesity and type 2 diabetes). RESULTS: We found 10 unexpected adverse signals related to "pancreatic cancer", "intestinal obstruction", "cholecystitis", and "polycystic ovary" and both the two different indications had the same serious adverse reaction events occurring. CONCLUSIONS: This study identified many unexpected signals of serious adverse reactions, suggesting the importance of continuous post-marketing surveillance of semaglutide to understand its potential risks.

PRRSV GP5 inhibits the antivirus effects of chaperone-mediated autophagy by targeting LAMP2A.

Autophagy is an important biological process in host defense against viral infection. However, many viruses have evolved various strategies to disrupt the host antiviral system. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus with a large economic impact on the swine industry. At present, studies on the escape mechanism of PRRSV in the autophagy process, especially through chaperone-mediated autophagy (CMA), are limited. This study confirmed that PRRSV glycoprotein 5 (GP5) could disrupt the formation of the GFAP-LAMP2A complex by inhibiting the MTORC2/PHLPP1/GFAP pathway, promoting the dissociation of the pGFAP-EF1α complex, and blocking the K63-linked polyubiquitination of LAMP2A to inhibit the activity of CMA. Further research demonstrated that CMA plays an anti-PRRSV role by antagonizing nonstructural protein 11 (NSP11)-mediated inhibition of type I interferon (IFN-I) signaling. Taken together, these results indicate that PRRSV GP5 inhibits the antiviral effect of CMA by targeting LAMP2A. This research provides new insight into the escape mechanism of immunosuppressive viruses in CMA. IMPORTANCE: Viruses have evolved sophisticated mechanisms to manipulate autophagy to evade degradation and immune responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus that causes enormous economic losses in the swine industry. However, the mechanism by which PRRSV manipulates autophagy to defend against host antiviral effects remains unclear. In this study, we found that PRRSV GP5 interacts with LAMP2A and disrupts the formation of the GFAP-LAMP2A complex, thus inhibiting the activity of CMA and subsequently enhancing the inhibitory effect of the NSP11-mediated IFN-I signaling pathway, ultimately facilitating PRRSV replication. Our study revealed a novel mechanism by which PRRSV escapes host antiviral effects through CMA, providing a potential host target, LAMP2A, for developing antiviral drugs and contributing to understanding the escape mechanism of immunosuppressive viruses.

Programmable Tetrahedral DNA-RNA Nanocages Woven with Stimuli-Responsive siRNA for Enhancing Therapeutic Efficacy of Multidrug-Resistant Tumors.

Multidrug resistance (MDR) is a major obstacle limiting the effectiveness of chemotherapy against cancer. The combination strategy of chemotherapeutic agents and siRNA targeting drug efflux has emerged as an effective cancer treatment to overcome MDR. Herein, stimuli-responsive programmable tetrahedral DNA-RNA nanocages (TDRN) have been rationally designed and developed for dynamic co-delivery of the chemotherapeutic drug doxorubicin and P-glycoprotein (P-gp) siRNA. Specifically, the sense and antisense strand sequences of the P-gp siRNA, which are programmable bricks with terminal disulfide bond conjugation, are precisely embedded in one edge of the DNA tetrahedron. TDRN provides a stimuli-responsive release element for dynamic control of functional cargo P-gp siRNA that is significantly more stable than the "tail-like" TDN nanostructures. The stable and highly rigid 3D nanostructure of the siRNA-organized TDRN nanocages demonstrated a notable improvement in the stability of RNase A and mouse serum, as well as long-term storage stability for up to 4 weeks, as evidenced by this study. These biocompatible and multifunctional TDRN nanocarriers with gold nanocluster-assisted delivery (TDRN@Dox@AuNCp) are successfully used to achieve synergistic RNAi/Chemo-therapy in vitro and in vivo. This programmable TDRN drug delivery system, which integrates RNAi therapy and chemotherapy, offers a promising approach for treating multidrug-resistant tumors.

Hybrid mRMR and multi-objective particle swarm feature selection methods and application to metabolomics of traditional Chinese medicine.

Metabolomics data has high-dimensional features and a small sample size, which is typical of high-dimensional small sample (HDSS) data. Too high a dimensionality leads to the curse of dimensionality, and too small a sample size tends to trigger overfitting, which poses a challenge to deeper mining in metabolomics. Feature selection is a valuable technique for effectively handling the challenges HDSS data poses. For the feature selection problem of HDSS data in metabolomics, a hybrid Max-Relevance and Min-Redundancy (mRMR) and multi-objective particle swarm feature selection method (MCMOPSO) is proposed. Experimental results using metabolomics data and various University of California, Irvine (UCI) public datasets demonstrate the effectiveness of MCMOPSO in selecting feature subsets with a limited number of high-quality features. MCMOPSO achieves this by efficiently eliminating irrelevant and redundant features, showcasing its efficacy. Therefore, MCMOPSO is a powerful approach for selecting features from high-dimensional metabolomics data with limited sample sizes.