Transmembrane Transport Research Articles

Molecular engineering of fluorescent dyes for long-term specific visualization of the plasma membrane based on alkyl-chain-regulated cell permeability

Long-term visualization of changes in plasma membrane dynamics during important physiological processes can provide intuitive and reliable information in a 4D mode. However, molecular tools that can visualize plasma membranes over extended periods are lacking due to the absence of effective design rules that can specifically track plasma membrane fluorescent dye molecules over time. Using plant plasma membranes as a model, we systematically investigated the effects of different alkyl chain lengths of FMR dye molecules on their performance in imaging plasma membranes. Our findings indicate that alkyl chain length can effectively regulate the permeability of dye molecules across plasma membranes. The study confirms that introducing medium-length alkyl chains improves the ability of dye molecules to target and anchor to plasma membranes, allowing for long-term imaging of plasma membranes. This provides useful design rules for creating dye molecules that enable long-term visualization of plasma membranes. Using the amphiphilic amino-styryl-pyridine fluorescent skeleton, we discovered that the inclusion of short alkyl chains facilitated rapid crossing of the plasma membrane by the dye molecules, resulting in staining of the cell nucleus and indicating improved cell permeability. Conversely, the inclusion of long alkyl chains hindered the crossing of the cell wall by the dye molecules, preventing staining of the cell membrane and demonstrating membrane impermeability to plant cells. The FMR dyes with medium-length alkyl chains rapidly crossed the cell wall, uniformly stained the cell membrane, and anchored to it for a long period without being transmembrane. This allowed for visualization and tracking of the morphological dynamics of the cell plasma membrane during water loss in a 4D mode. This suggests that the introduction of medium-length alkyl chains into amphiphilic fluorescent dyes can transform them from membrane-permeable fluorescent dyes to membrane-staining fluorescent dyes suitable for long-term imaging of the plasma membrane. In addition, we have successfully converted a membrane-impermeable fluorescent dye molecule into a membrane-staining fluorescent dye by introducing medium-length alkyl chains into the molecule. This molecular engineering of dye molecules with alkyl chains to regulate cell permeability provides a simple and effective design rule for long-term visualization of the plasma membrane, and a convenient and feasible means of chemical modification for efficient transmembrane transport of small molecule drugs.

Influence of genetic polymorphisms on pharmacokinetics and treatment response of mycophenolic acid: a scoping review.

This scoping review explores the impact of genetic polymorphisms on the pharmacokinetics and treatment responses of mycophenolic acid (MPA), an immunosuppressant. The study includes 83 articles from 1226 original studies, focusing on transplantation (n=80) and autoimmune disorders (n=3). Genetic variants in uridine 5'-diphospho-glucuronosyltransferase (UGT1A9, UGT1A8 and UGT2B7) and transmembrane transporters (ABCC2, SLCO1B1, SLCO1B3 and ABCB1) significantly affected MPA's pharmacokinetics and susceptibility to its adverse effect. Whereas variants in several genes including UGT1A9, UGT2B7, IMPDH1 and IMPDH2 have been associated with a higher risk of transplant rejection. However, there is a lack of studies on MPA's impact on autoimmune disorders and limited research on the Asian population. The findings underscore the need for further research on MPA's impact across different populations and diseases, particularly among other Asian ethnic groups, to advance personalized medicine in MPA therapy.

Reversing Anticancer Drug Resistance by Synergistic Combination of Chemotherapeutics and Membranolytic Antitumor β-Peptide Polymer.

Multidrug resistance is the main obstacle to cancer chemotherapy. Overexpression of drug efflux pumps causes excessive drug efflux from cancer cells, ultimately leading to drug resistance. Hereby, we raise an effective strategy to overcome multidrug resistance using a synergistic combination of membranolytic antitumor β-peptide polymer and chemotherapy drugs. This membrane-active β-peptide polymer promotes the transmembrane transport of chemotherapeutic drugs by increasing membrane permeability and enhances the activity of chemotherapy drugs against multidrug-resistant cancer cells. As a proof-of-concept demonstration, the synergistic combination of β-peptide polymer and doxorubicin (DOX) is substantially more effective than DOX alone against drug-resistant cancer both in vitro and in vivo. Notably, the synergistic combination maintains a potent anticancer activity after continuous use. Collectively, this combination therapy using membrane lytic β-peptide polymer appears to be an effective strategy to reverse anticancer drug resistance.

Responsive Anionophores with AND Logic Multi-Stimuli Activation.

Artificial ion transport systems have emerged as an important class of compounds that promise applications in chemotherapeutics as anticancer agents or to treat channelopathies. Stimulus-responsive systems that offer spatiotemporally controlled activity for targeted applications remain rare. Here we utilize dynamic hydrogen bonding interactions of a 4,6-dihydroxy-isophthalamide core to generate a modular platform enabling access to stimuli-responsive ion transporters that can be activated in response to a wide variety of external stimuli, including light, redox, and enzymes, with excellent OFF-ON activation profiles. Alkylation of the two free hydroxyl groups with stimulus-responsive moieties locks the amide bonds through intramolecular hydrogen bonding and hence makes them unavailable for anion binding and transport. Triggering using a particular stimulus to cleave both cages reverses the hydrogen bonding arrangement, to generate a highly preorganized anion binding cavity for efficient transmembrane transport. Integration of two cages that are responsive to orthogonal stimuli enables multi-stimuli activation, where both stimuli are required to trigger transport in an AND logic process. Importantly, the strategy provides a facile method to post-functionalize the highly active transporter core with a variety of stimulus-responsive moieties for targeted activation with multiple triggers.

Responsive Anionophores with AND Logic Multi‐Stimuli Activation

AbstractArtificial ion transport systems have emerged as an important class of compounds that promise applications in chemotherapeutics as anticancer agents or to treat channelopathies. Stimulus‐responsive systems that offer spatiotemporally controlled activity for targeted applications remain rare. Here we utilize dynamic hydrogen bonding interactions of a 4,6‐dihydroxy‐isophthalamide core to generate a modular platform enabling access to stimuli‐responsive ion transporters that can be activated in response to a wide variety of external stimuli, including light, redox, and enzymes, with excellent OFF‐ON activation profiles. Alkylation of the two free hydroxyl groups with stimulus‐responsive moieties locks the amide bonds through intramolecular hydrogen bonding and hence makes them unavailable for anion binding and transport. Triggering using a particular stimulus to cleave both cages reverses the hydrogen bonding arrangement, to generate a highly preorganized anion binding cavity for efficient transmembrane transport. Integration of two cages that are responsive to orthogonal stimuli enables multi‐stimuli activation, where both stimuli are required to trigger transport in an AND logic process. Importantly, the strategy provides a facile method to post‐functionalize the highly active transporter core with a variety of stimulus‐responsive moieties for targeted activation with multiple triggers.

Proteomics-Derived Biomarker Panel Facilitates Distinguishing Primary Lung Adenocarcinomas With Intestinal or Mucinous Differentiation From Lung Metastatic Colorectal Cancer

The diagnosis of primary lung adenocarcinomas with intestinal or mucinous differentiation (PAIM) remains challenging due to the overlapping histomorphological, immunohistochemical (IHC), and genetic characteristics with lung metastatic colorectal cancer (lmCRC). This study aimed to explore the protein biomarkers that could distinguish between PAIM and lmCRC. To uncover differences between the two diseases, we used tandem mass tagging–based shotgun proteomics to characterize proteomes of formalin-fixed, paraffin-embedded tumor samples of PAIM (n = 22) and lmCRC (n = 17).Then three machine learning algorithms, namely support vector machine (SVM), random forest, and the Least Absolute Shrinkage and Selection Operator, were utilized to select protein features with diagnostic significance. These candidate proteins were further validated in an independent cohort (PAIM, n = 11; lmCRC, n = 19) by IHC to confirm their diagnostic performance. In total, 105 proteins out of 7871 proteins were significantly dysregulated between PAIM and lmCRC samples and well-separated two groups by Uniform Manifold Approximation and Projection. The upregulated proteins in PAIM were involved in actin cytoskeleton organization, platelet degranulation, and regulation of leukocyte chemotaxis, while downregulated ones were involved in mitochondrial transmembrane transport, vasculature development, and stem cell proliferation. A set of ten candidate proteins (high-level expression in lmCRC: CDH17, ATP1B3, GLB1, OXNAD1, LYST, FABP1; high-level expression in PAIM: CK7 (an established marker), NARR, MLPH, S100A14) was ultimately selected to distinguish PAIM from lmCRC by machine learning algorithms. We further confirmed using IHC that the five protein biomarkers including CDH17, CK7, MLPH, FABP1 and NARR were effective biomarkers for distinguishing PAIM from lmCRC. Our study depicts PAIM-specific proteomic characteristics and demonstrates the potential utility of new protein biomarkers for the differential diagnosis of PAIM and lmCRC. These findings may contribute to improving the diagnostic accuracy and guide appropriate treatments for these patients.

Boosting Osmotic Energy Harvesting from Organic Solutions by Ultrathin Covalent Organic Framework Membranes.

Extracting osmotic energy from waste organic solutions via reverse electrodialysis represents a promising approach to reuse such industrial wastes and helps to mitigate the ever-growing energy needs. Herein, a molecularly thin membrane of covalent organic frameworks is engineered via interfacial polymerization to investigate its ion transport behavior in organic solutions. Interestingly, a significant deviation from linearity between ion conductance and reciprocal viscosity is observed, attributed to the nanoscale confinement effect on intermolecular interactions. This finding suggests a potential strategy to modulate the influence of apprarent viscosity on transmembrane transport. The osmotic energy harvesting of the ultrathin membrane in organic systems was studied, achieving an unprecedented output power density of over 84.5 W m-2 at a 1000-fold salinity gradient with a benign conversion efficiency and excellent stability. These findings provide a meaningful stepping stone for future studies seeking to fully leverage the potentials of organic systems in energy harvesting applications.

Glycosylated Antibiotics: New Promising Bacterial Efflux Pumps Inhibitors

Antimicrobial resistance is considered a major concern problem; bacteria have evolved mechanisms to overcome antibiotics’ action through evolutionary process. One main resistance mechanism that bacteria developed is the pumping of the antibiotics out of bacterial cells by transmembrane transporter proteins known as efflux pumps. To overcome bacterial resistance guided by efflux pumps, efflux pumps inhibitors (EPIs) are small molecules that obstruct efflux pumps binding sites and its structural assembly leading to disability in the efflux pumps normal function, new EPIs which under the current study are created by modifying the chemical structure of most common antibiotics including Ampicillin, Penicillin, Chloramphenicol, Ciprofloxacin and Tetracycline, such antibiotics are modified by adding N-acetyl glucose amine moiety to acceptor OH group of the respective antibiotic, the newly modified antibiotics are glycosylated EPIs. To test the effectiveness of the new EPIs in inhibiting AcrB-TolC and MexA-OprM efflux pumps functions, ADME properties for all of glycosylated antibiotics have been measured through applying Lipinski’s role of 5, docking and simulation studies have been included as well. Docked glycosylated tetracycline has given the highest binding energy in the active sites of both pumps, with -9.4 against AcrB and -8.8 against MexA. The simulation study has confirmed the binding of the glycosylated tetracycline in the active sites of both pumps, as well as its stability during the biological dynamicity of both pumps (opening and closing channels). The results validation requires a long simulation time about 50 ns or more which was un applicable due to cost limitation, however, the newly glycosylated antibiotics have promising results that might make it eligible as drug candidates to overcome bacterial resistance.

Intensive ammonium fertilizer addition activates iron and carbon conversion coupled cadmium redistribution in a paddy soil under gradient redox conditions

While over-fertilization and nitrogen deposition can lead to the enrichment of nitrogen in soil, its effects on heavy metal fractions under gradient moisture conditions remains unclear. Here, the effect of intensive ammonium (NH4+) addition on the conversion and interaction of cadmium (Cd), iron (Fe) and carbon (C) was studied. At relatively low (30–80 %) water hold capacity (WHC) NH4+ application increased the carbonate bound Cd fraction (F2Cd), while at relatively high (80–100 %) WHC NH4+ application increased the organic matter bound Cd fraction (F4Cd). Iron‑manganese oxide bound Cd fractions (F3Cd) and oxalate-Fe decreased, but DCB-Fe increased in NH4+ treatments, indicating that amorphous Fe was the main carrier of F3Cd. The variations in F1Cd and F4Cd observed under the 100–30-100 % WHC treatment were similar to those observed under low moisture conditions (30–60 % WHC). The C=O/C-H ratio of organic matter in soil decreased under the 30–60 % WHC treatment, but increased under the 80–100 % WHC treatment, which was the dominant factor influencing F4Cd changes. The conversion of NH4+ declined with increasing soil moisture content, and the impact on oxalate-Fe was greater at 30–60 % WHC than at 80–100 % WHC. Correspondingly, genetic analysis showed the effect of NH4+ on Fe and C metabolism at 30–60 % WHC was greater than at 80–100 % WHC. Specifically, NH4+ treatment enhanced the expression of genes encoding extracellular Fe complexation (siderophore) at 30–80 % WHC, while inhibiting genes encoding Fe transmembrane transport at 30–60 % WHC, indicating that siderophores simultaneously facilitated Cd detoxification and Fe complexation. Furthermore, biosynthesis of sesquiterpenoid, steroid, butirosin and neomycin was significantly correlated with F4Cd, while glycosaminoglycan degradation metabolism and assimilatory nitrate reduction was significantly correlated with F2Cd. Overall, this study gives a more comprehensive insight into the effect of NH4+ on activated Fe and C conversion on soil Cd redistribution under gradient moisture conditions.

Symplasmic and transmembrane zinc transport is modulated by cadmium in the Cd/Zn hyperaccumulator Sedum alfredii

This study investigated the influence of Cd (25 µM) on Zn accumulation in a hyperaccumulating (HE) and a non-hyperaccumulating (NHE) ecotype of Sedum alfredii Hance at short-term supply of replete (Zn5, 5 µM) and excess (Zn400, 400 µM) Zn. Cd inhibited Zn accumulation in both ecotypes, especially under Zn400, in organs with active metal sequestration, i.e. roots of NHE and shoots of HE. Direct biochemical Cd/Zn competition at the metal-protein interaction and changes in transporter gene expression contributed to the observed accumulation patterns in the roots. Specifically, in HE, Cd stimulated SaZIP4 and SaPCR2 under Zn5, but downregulated SaIRT1 and SaZIP4 under Zn400. However, Cd downregulated related transporter genes, except for SaNRAMP1, in NHE, irrespective of Zn. Cadmium stimulated casparian strip (CSs) development in NHE, as part of the defense response, while it had a subtle effect on the (CS) in HE. Moreover, Cd delayed the initiation of the suberin lamellae (SL) in HE, but stimulated SL deposition in NHE under both Zn5 or Zn400. Changes in suberization were mainly ascribed to suberin-biosynthesis-related genes and hormonal signaling. Altogether, Cd regulated Zn accumulation mainly via symplasmic and transmembrane transport in HE, while Cd inhibited both symplasmic and apoplasmic Zn transport in NHE.

Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

Functional connectivity subtypes of MDD and their associations with gene expression profile, neurotransmitter, and cognition

IntroductionThere’s large heterogeneity present in major depressive disorder (MDD) and controversial evidence on alterations of brain functional connectivity (FC), making it hard to elucidate the neurobiological basis of MDD. Subtyping is one promising solution to characterize this heterogeneity.ObjectivesTo identify neurophysiological subtypes of MDD based on FC derived from resting-state functional magnetic resonance imaging using large multisite data and investigate the differences in genetic mechanisms and neurotransmitter basis of FC alterations, and the differences of FC-related cognition between each subtype.MethodsConsensus clustering of FC patterns was applied to a population of 829 MDD patients from REST-Meta-MDD database after data cleaning and image quality control. Gene transcriptomic data derived from Allen Human Brain Atlas and neurotransmitter receptor/transporter density data acquired by using neuromap toolbox were used to characterize the molecular mechanism underlying each FC-based subtype by identifying the gene set and neurotransmitters/transporters showing high spatial similarity with the profiles of FC alterations between each subtype and 770 healthy controls. The FC-related cognition in each subtype was also selected by lasso regression.ResultsTwo stable neurophysiological MDD subtypes were found and labeled as hypoconnectivity (n=527) and hyperconnectivity (n=299) characterized by the FC differences in each subtype relative to controls, respectively. The two subtypes did not differ in age, sex, and scores of Hamilton Depression/Anxiety Scale.The genes related to FC alterations were enriched in ion transmembrane transport, synaptic transmission/organization, axon development, and regulation of neurotransmitter level for both subtypes, but specifically enriched in glial cell differentiation for hypoconnectivity subtype, while enriched in regulation of presynaptic membrane and regulation of neuron differentiation for hyperconnectivity subtype.FC alterations were associated with the density of 5-HT2a receptor in both subtypes. For hyperconnectivity subtype, FC alterations were also correlated with the density of norepinephrine transporter, glutamate receptor, GABA receptor, 5-HT1b receptor, and cannabinoid receptor.Both subtypes showed correlations between FC and categorization, motor inhibition, and localization. The FC in hypoconnectivity subtype correlated with response inhibition, selective attention, face recognition, sleep, empathy, expertise, uncertainty, and anticipation, while that was related to inference, speech perception, and reward anticipation in hyperconnectivity subtype.ConclusionsOur findings suggested the presence of two neuroimaging subtypes of MDD characterized by hypo or hyper-connectivity. The two subtypes had both shared and distinct genetic mechanisms, neurotransmitter receptor/transporter profiles, and cognition types.Disclosure of InterestNone Declared

Comprehensive pan-cancer analysis: essential role of ABCB family genes in cancer.

The adenosine triphosphate-binding-cassette (ABC) transporter orchestrates the transmembrane transport of diverse substrates with the aid of ATP as an energy source. ABC transporter constitutes a widespread superfamily of transporters prominently present on the cellular membrane of organisms. Advancements in understanding have unveiled additional roles beyond mere intracellular or extracellular transport functions for the ABC protein family, encompassing involvement in DNA repair, protein translation, and gene expression regulation. Yet its role in tumors is still unknown. This study drew support from multiple databases, including Gene Expression Omnibus (GEO), European Genome-phenome Archive (EGA), The Cancer Genome Atlas (TCGA), and employed multidimensional bioinformatics analyses, incorporating online databases and the R-project. Through a comprehensive analysis, we seek to discern transcriptional-level disparities among genes and their consequential impacts on prognosis, tumor microenvironment (TME), stemness score, immune subtypes, clinical characteristics, and drug sensitivity across human cancers. ABC transporter subfamily B (ABCB) family genes exhibited heightened expression across diverse tumors, demonstrating a significant correlation with overall prognosis in pan-cancer contexts. Notably, gene expression levels manifested substantial associations with TME, stemness score, immune subtypes, clinical characteristics, and drug sensitivity in specific cancers, including kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), and pancreatic adenocarcinoma (PAAD). Within this subset, transporter associated with antigen processing 1 (TAP1), TAP2, and ABCB6 emerged as noteworthy oncogenes. The outcomes of this study contribute to a comprehensive understanding of the implications of ABCB family genes in tumor progression, offering insights into potential therapeutic targets for cancer. Notably, the identification of ABCB6 as a significant oncogene suggests promising avenues for targeted therapies in KIRP, LIHC, and PAAD.

Multi-functional D-alpha-tocopheryl polyethylene glycol succinate surface modified nanocrystals improve the stability and oral bioavailability of pueraria flavonoids

Pueraria flavonoids (PF), the main component of “Getong Tongluo capsule”, were applied in the clinical treatment of cardiovascular diseases. However, the complex nature of multiple-components, along with poor stability and low oral bioavailability of PF, hinders its widespread clinical use. This study aimed to develop D-alpha-tocopheryl polyethylene glycol succinate (TPGS) surface-modified PF-loaded nanocrystals (PF–NCs)，and investigate their physico-chemical properties and oral absorption mechanism. The prepared PF-NCs showed as rod shape crystal with an average particle size and polydispersity index of 259.0 ± 7.80 nm and 0.195 ± 0.046. Compared with PF, the stability of PF-NCs was significantly improved. Furthermore, the transport of PF-NCs in Madin-Darby canine kidney (MDCK) monolayers was time-, concentration- and energy-dependent. The endocytosis of PF-NCs was mediated mainly through macropinocytosis, caveolin, clathrin and lipid rafts/caveolae. The involvement of multidrug resistance-associated protein 2 (MRP2) transporter and P-gp in the transmembrane transport of PF-NCs was also significant. Pharmacokinetic studies demonstrated that PF-NCs exhibited a substantial enhancement of PF's oral bioavailability. Overall, the prepared PF-NCs demonstrated enhanced stability and bioavailability, offering valuable insights for the development of multi-component nanocrystals and investigation into their mechanisms of oral absorption.

Effects of grape seed procyanidins on the lipid metabolism of growing-finishing pigs based on transcriptomics and metabolomics analyses

This study investigated how lipid metabolism in the longissimus thoracis is influenced by the diet supplemented with grape seed procyanidins (GSPs) in growing-finishing pigs. Forty-eight crossbred pigs were randomly assigned to four groups, each receiving a basal diet, or basal diet added with 150, 200, and 250 mg/kg GSPs. Transcriptomics and metabolomics were employed to explore differential gene and metabolite regulation. The expression of key lipid metabolism-related genes was tested via qRT-PCR, and the lipid and fatty acid composition of the longissimus thoracis were determined. Dietary GSPs at different concentrations upregulated lipoprotein lipase (LPL), which is involved in lipolysis, and significantly increased the mRNA expression levels of carnitine palmitoyltransferase-1B (CPT1B) and cluster of differentiation 36 (CD36), implicated in transmembrane transport of fatty acids. Dietary supplementation of GSPs at 200 or 250 mg/kg markedly reduced total cholesterol and triglyceride content in longissimus thoracis. Dietary GSPs significantly decreased the contents of low-density lipoprotein cholesterol and saturated fatty acids, while increasing unsaturated fatty acids. In conclusion, GSPs may regulate lipid metabolism, reducing cholesterol level, and improving fatty acid composition in the longissimus thoracis of growing-finishing pigs. Our findings provide evidence for the beneficial effects of GSPs as pig feed additives for improving lipid composition.

The decreased interface tension increased the transmembrane transport of soy hull polysaccharide-derived SCFAs in the Caco-2 cells

Polysaccharides impact intestinal fermentation and regulate interfacial properties which affect absorption and transportation. Short-chain fatty acids (SCFAs), the main metabolites of soy hull polysaccharide lysate, are readily absorbed by the body and perform various physiological functions. We analysed the interfacial properties and transport of soy hull polysaccharide-derived SCFAs in the Caco-2 cell model to clarify the transmembrane transport mechanism. The results showed that the interfacial properties of the co-culture system were influenced by both transit time and concentration of SCFAs, the uptake and transit rates of SCFAs at 1–3 h increased significantly with time (P < 0.05). With increasing transit time and concentration, the transit rates of SCFAs on the apical side (AP) → basolateral side (BL) and BL → AP sides increased and then stabilised, the transit rate of the AP → BL side was higher than that of the BL → AP side. Proteomic analysis showed that soy hull polysaccharide-derived SCFAs resulted in the differential expression of 285 upregulated and 501 downregulated after translocation across Caco-2 cells. The differentially expressed proteins were mainly enriched in ribosomes, oxidative phosphorylation, nuclear transport, and SNARE vesicular transport. This study lays the theoretical foundation for understanding the structure-activity relationship of soy hull polysaccharides in the intestine.

Analysis of the Mycotoxin Levels and Expression Pattern of SWN Genes at Different Time Points in the Fungus Slafractonia leguminicola.

The fungal plant pathogen Slafractonia leguminicola produces two mycotoxins that affect animals: slaframine, which causes slobbers, and swainsonine, which causes locoism. Slafractonia leguminicola contains the swainsonine-associated orthologous gene clusters, "SWN", which include a multifunctional swnK gene (NRPS-PKS hybrid), swnH1 and swnH2 (nonheme iron dioxygenase genes), swnN and swnR (reductase genes), and swnT (transmembrane transporter). In addition to these genes, two paralogs of swnK, swnK1 (paralog1) and swnk2 (paralog2), are found in S. leguminicola. cDNAs from total mRNA were isolated from the S. leguminicola mycelia grown in the culture plates as well as from leaves inoculated with the fungal mycelia at different time points, and expression pattern of the SWN genes were analyzed using RT-qPCR. The concentrations of swainsonine and slaframine production from this fungus at different time points were also examined using liquid chromatography-mass spectrometry. The timing of gene expression was similar in cultured fungus and inoculated leaves and agreed with our proposed biosynthetic pathway. Substantially more swainsonine was produced than slaframine during time course studies.

Myricetin Oligomer Triggers Multi-Receptor Mediated Penetration and Autophagic Restoration of Blood-Brain Barrier for Ischemic Stroke Treatment.

Restoration of blood-brain barrier (BBB) dysfunction, which drives worse outcomes of ischemic stroke, is a potential target for therapeutic opportunities, whereas a sealed BBB blocks the therapeutics entrance into the brain, making the BBB protection strategy paradoxical. Post ischemic stroke, hypoxia/hypoglycemia provokes the up-regulation of transmembrane glucose transporters and iron transporters due to multiple metabolic disorders, especially in brain endothelial cells. Herein, we develop a myricetin oligomer-derived nanostructure doped with Ce to bypass the BBB which is cointermediated by glucose transporters and iron transporters such as glucose transporters 1 (GLUT1), sodium/glucose cotransporters 1 (SGLT1), and transferrin(Tf) reporter (TfR). Moreover, it exhibits BBB restoration capacity by regulating the expression of tight junctions (TJs) through the activation of protective autophagy. The myricetin oligomers scaffold not only acts as targeting moiety but is the prominent active entity that inherits all diverse pharmacological activities of myricetin. The suppression of oxidative damage, M1 microglia activation, and inflammatory factors makes it a multitasking nanoagent with a single component as the scaffold, targeting domain and curative components.

Optimizing Paclitaxel Oral Absorption and Bioavailability: TPGS Co-Coating via Supercritical Anti-Solvent Fluidized Bed Technology.

Supercritical anti-solvent fluidized bed (SAS-FB) coating technology has the advantages of reducing particle size, preventing high surface energy particle aggregation, improving the dissolution performance and bioavailability of insoluble drugs. The poor solubility of Biopharmaceutics Classification System (BCS) class IV drugs poses challenges in achieving optimal bioavailability. Numerous anti-cancer drugs including paclitaxel (PTX) belong to the BCS class IV, hindering their therapeutic efficacy. To address this concern, our study explored SAS-FB technology to coat PTX with D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) onto lactose. Under our optimized conditions, we achieved a PTX coating efficiency of 96.8%. Further characterization confirmed the crystalline state of PTX in the lactose surface coating by scanning electron microscopy and X-ray powder diffraction. Dissolution studies indicated that SAS-FB processed samples release over 95% of the drug within 1 min. Moreover, cell transmembrane transport assays demonstrated that SAS-FB processed PTX samples co-coated with TPGS had an enhanced PTX internalization into cells and a higher permeability coefficient compared to those without TPGS. Finally, compared to unprocessed PTX, SAS-FB (TPGS) and SAS-FB processed samples showed a 2.66- and 1.49-fold increase in oral bioavailability in vivo, respectively. Our study highlights the efficacy of SAS-FB co-coating for PTX and TPGS as a promising strategy to overcome bioavailability challenges inherent in BCS class IV drugs. Our approach holds broader implications for enhancing the performance of similarly classified medications.

Proteomics reveals the significance of vacuole Pi transporter in the adaptability of Brassica napus to Pi deprivation.

Vacuolar Pi transporters (VPTs) have recently been identified as important regulators of cellular Pi status in Arabidopsis thaliana and Oryza sativa. In the oil crop Brassica napus, BnA09PHT5;1a and BnC09PHT5;1a are two homologs of AtPHT5;1, the vacuolar Pi influx transporter in Arabidopsis. Here, we show that Pi deficiency induces the transcription of both homologs of PHT5;1a genes in B. napus leaves. Brassica PHT5;1a double mutants (DM) had smaller shoots and higher cellular Pi concentrations than wild-type (WT, Westar 10), suggesting the potential role of BnPHT5;1a in modulating cellular Pi status in B. napus. A proteomic analysis was performed to estimate the role of BnPHT5;1a in Pi fluctuation. Results show that Pi deprivation disturbs the abundance of proteins in the physiological processes involved in carbohydrate metabolism, response to stimulus and stress in B. napus, while disruption of BnPHT5;1a genes may exacerbate these processes. Besides, the processes of cell redox homeostasis, lipid metabolic and proton transmembrane transport are supposed to be unbalanced in BnPHT5;1a DM under the -Pi condition. Noteworthy, disruption of BnPHT5;1a genes severely alters the abundance of proteins related to ATP biosynthesis, and proton/inorganic cation transmembrane under normal Pi condition, which might contribute to B. napus growth limitations. Additionally, seven new protein markers of Pi homeostasis are identified in B. napus. Taken together, this study characterizes the important regulatory role of BnPHT5;1a genes as vacuolar Pi influx transporters in Pi homeostasis in B. napus.