Uptake Of Small Molecules Research Articles

Differential Effect of Simulated Microgravity on the Cellular Uptake of Small Molecules.

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

Trivalent Disulfide Unit-Masked System Efficiently Delivers Large Oligonucleotide.

Oligonucleotide drugs are shining in clinical therapeutics, but efficient and safe delivery systems severely limit their widespread use. A disulfide unit technology platform based on dynamic thiol exchange chemistry at the cell membrane has the potential for drug delivery. However, the alteration of the disulfide unit CSSC dihedral angle induced by different substituents directly affects the effectiveness of this technology and its stability. Previously, we constructed a trivalent low dihedral angle disulfide unit that can effectively promote the cellular uptake of small molecules. Here, we constructed a novel disulfide unit-masked oligonucleotide hybrid based on a low dihedral angle disulfide unit, motivated by prodrug design. Cellular imaging results showed that such a system exhibited superior cellular delivery efficiency than the commercial Lipo2000 without cytotoxicity. The thiol reagents significantly reduced its cellular uptake (57-74%), which proved to be endocytosis-independent. In addition, in vivo distribution experiments in mice showed that such systems can be rapidly distributed in liver tissues with a duration of action of more than 24 h, representing a potential means of silencing genes involved in the pathogenesis of liver-like diseases. In conclusion, this trivalent disulfide unit-masked system we constructed can effectively deliver large oligonucleotide drugs.

β-barrel proteins dictate the effect of core oligosaccharide composition on outer membrane mechanics.

The outer membrane is the defining cellular structure of Gram-negative bacteria, a group that contains many important pathogens like Escherichia coli , Vibrio cholerae , and Pseudomonas aeruginosa . One role of the outer membrane is to block the uptake of small molecules like antibiotics. However, it is becoming increasingly clear that it also functions as a structural exoskeleton that is critical for the cell's ability to cope with internal and external mechanical forces. Here, we carefully dissect the molecular basis for the load-bearing capacity of the outer membrane by screening a set of mutants with a new cell biophysics assay.

Engineering the Spatial Distribution of Amphiphilic Molecule within Complex Coacervate Microdroplet via Modulating Charge Strength of Polyelectrolytes.

The investigation of the interplay between complex coacervate microdroplets and amphiphilic molecules offers valuable insights into the processes of prebiotic compartmentalization on the early Earth and presents a promising avenue for future advancements in biotechnology. Herein, the interaction between complex coacervate microdroplets and amphiphilic molecule (decanoic acid) is systematically investigated by varying charge strengths of negatively charged polyelectrolytes (DNA and PAA) and positively charged polyelectrolytes (PDDA and DEAE-Dextran). It is found that the interaction between amphiphilic molecule and complex coacervate microdroplets depended on the delicate balance between the interaction between decanoic acid and polyelectrolyte and the interaction between two polyelectrolytes. The different spatial distribution of amphiphilic molecule can result in differences in the internal microenvironment, which can further alter the uptake or exclusion of small molecules and biomolecules with different charges and polarities and functional biological process.

Abstract 6033: Preclinical evaluation of an actinium-225 labeled PSMA-targeting small molecule (225Ac-PSMA-Trillium (BAY 3563254)) for the treatment of metastatic castration resistant prostate cancer (mCRPC)

Abstract PSMA is a clinically validated target for the treatment of mCRPC. Since the linear energy transfer and degree of induced DNA damage of alpha particles far exceeds that of beta particles, several investigational PSMA-targeted small molecules labeled with the alpha-emitter actinium-225 (225Ac) have shown encouraging clinical signs of efficacy. However, xerostomia is often found as a dose-limiting side effect, presumably due to low levels of PSMA expression and efficient uptake of small molecules in salivary glands. To reduce these unwanted side effects a PSMA-targeted radioligand comprising a triad of important features was investigated. It consists of a high-affinity PSMA binder for specific tumor-targeting; a customized albumin-binding moiety designed to prolong plasma residence time and increase the therapeutic index by improving tumor uptake while reducing salivary gland uptake; and a chelator for efficient complexation of 225Ac. From a series of compounds containing the same high-affinity PSMA-targeting moiety, but different chain lengths and albumin-binding domains, compound RPS-072 containing a PEG-3 and PEG-8 chain and an iodobenzyl moiety as albumin-binder was selected for further evaluation (J Nucl Med 2019; 60:656-663). This compound was the basis for the PSMA-Trillium with a DOTA chelator for imaging applications in humans (NCT05773703) or a macropa chelator for 225Ac labeling resulting in 225Ac-PSMA-Trillium. In vitro preclinical characterization of 225Ac-PSMA-Trillium was performed to test affinities for PSMA and albumin. In a SPR assay, Kd was measured as 4.88x1011 M, Kon as 1.4x107 1/Ms and Koff as 6.6x10-4 1/Ms. 225Ac-PSMA-Trillium demonstrated good stability up to 48 h and induced potent in vitro cytotoxicity in the LNCaP subclone C4-2 (0.114 kBq/ml). In vivo biodistribution of 225Ac-PSMA-Trillium showed consistent with the albumin binding property a relatively slow pharmacokinetics profile with 5 % ID/g still in the blood after 24 h and tumor accumulation peaking at ~ 20 % ID/g after 5-7 days. Tumor penetration was investigated using autoradiography which demonstrated fast, homogeneous, and even distribution across the tumor within 15 mins after injection. This property was also reflected by strong therapeutic efficacy in several PrCa models. In the LNCaP model a tumor growth inhibition was observed at a single dose dependent efficacy at 150 kBq/kg and 300 kBq/kg and increased the time to reach 400 mm3 by 35 days for the 300 kBq/kg group. In the androgen independent PDX KuCaP-1 model, a single dose of 250 kBq/kg 225Ac-PSMA-Trillium induced strong tumor growth inhibition over the course of 35 days. A first-in-human trial with 225Ac-PSMA-Trillium is anticipated to start in the first half of 2024. Citation Format: Sabine Zitzmann-Kolbe, Alex Papple, Thorsten Poethko, Ingrid Moen, Christoph Schatz, Shawn Hillier, John W. Babich, Alan Cuthbertson, Urs B. Hagemann. Preclinical evaluation of an actinium-225 labeled PSMA-targeting small molecule (225Ac-PSMA-Trillium (BAY 3563254)) for the treatment of metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6033.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

Proteomic Analysis of the Mycobacterium tuberculosis Outer Membrane for Potential Implications in Uptake of Small Molecules.

Increased resistance to current antimycobacterial agents and a potential bias toward relatively hydrophobic chemical entities highlight an urgent need to understand how current anti-TB drugs enter the tubercle bacilli. While inner membrane proteins are well-studied, how small molecules cross the impenetrable outer membrane remains unknown. Here, we employed mass spectrometry-based proteomics to show that octyl-β-d-glucopyranoside selectively extracts the outer membrane proteins of Mycobacterium tuberculosis. Differentially expressed proteins between nutrient-replete and nutrient-depleted conditions were enriched to identify proteins involved in nutrient uptake. We demonstrate cell surface localization of seven new proteins using immunofluorescence and show that overexpression of the proteins LpqY and ProX leads to hypersensitivity toward streptomycin, while overexpression of SubI, SpmT, and Rv2041 exhibited higher membrane permeability, assessed through an EtBr accumulation assay. Further, proton NMR metabolomics suggests the role of six outer membrane proteins in glycerol uptake. This study identifies several outer membrane proteins that are involved in the permeation of small hydrophilic molecules and are potential targets for enhancing the uptake and efficacy of anti-TB drugs.

Biogenesis of EVs in Trypanosomatids.

Trypanosomes are protozoan parasites responsible for human diseases such as Chagas disease, African trypanosomiasis, and leishmaniasis. These organisms' growth in various environments and exhibit multiple morphological stages, while adapting their surface components. They acquire and release materials extensively to get nutrients and manage interactions with the extracellular environment. They acquire and utilize proteins, lipids, and carbohydrates for growth via using membrane transport and endocytosis. Endocytosis takes place through distinct membrane areas known as the flagellar pocket and cytostome, depending on the parasite species and its developmental stage. Some forms establish a complex endocytic system to either store or break down the absorbed materials. In contrast, membrane transport facilitates the uptake of small molecules like amino acids, carbohydrates, and iron via particular receptors on the plasma membrane. Concurrently, these parasites secrete various molecules such as proteins, enzymes, nucleic acids, and glycoconjugates either in soluble form or enclosed in extracellular vesicles, which significantly contribute to their parasitic behavior. These activities require exocytosis through a secretory pathway in certain membrane domains such as the flagellum, flagellar pocket, and plasma membrane, which are controlled at various developmental stages. The main features of the endocytic and exocytic mechanisms, as well as the organelles involved, are discussed in this chapter along with their connection to the formation of exosomes and extracellular vesicles in the Tritryp species.

Analysis of reproducibility and robustness of a renal proximal tubule microphysiological system OrganoPlate 3-lane 40 for in vitro studies of drug transport and toxicity.

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.

A169 THE DIRECT COSTS OF INFLAMMATORY BOWEL DISEASE IN CANADA: A POPULATION-BASED ANALYSIS OF HISTORICAL AND CURRENT COSTS

A169 THE DIRECT COSTS OF INFLAMMATORY BOWEL DISEASE IN CANADA: A POPULATION-BASED ANALYSIS OF HISTORICAL AND CURRENT COSTS

Abstract 3936: Orally bioavailable 'lymphatropic' kinase inhibitors

Abstract Cancer signaling pathways can readily adapt to evade therapy through intrinsic resistance or compensatory mechanisms driving a resistant state. The prominence of oncogenic kinase signaling in a multitude of cancer types has inspired development of molecularly targeted drugs, facilitating combination treatment strategies designed to overcome adaptations such as signaling crosstalk and activation of downstream effectors. Treatments using inhibitor combinations, however, remain a challenge as clinical trials have been fraught with dose-limiting toxicities. Avoidance of hepatic first-pass metabolism by transport and sequestration into mesenteric gastrointestinal lymphatic vessels may improve drug exposure and reduce dose-limiting toxicities often observed in combination therapy studies. Despite potential advantages, lymphatically directed kinase inhibitors have remained unexplored due in part to limited understanding of the physicochemical properties required for lymphatic uptake of small molecules. We demonstrate a first-in-class series of orally bioavailable small molecule kinase inhibitors which are intrinsically ‘lymphatropic’, undergoing lymphatic absorption to achieve sustained circulatory drug levels and therapeutic benefit, with no observable toxicity. Single agent multifunctional inhibitors of high-value targets (MEK/PI3K, MEK/PI3K/mTOR, &amp; MEK/mTOR) were designed and synthesized. Compounds had unique physiochemical properties, demonstrating high lymphatic uptake in a mesenteric lymphatic vessel cannulation model and in isolated lymph nodes extracts. Pharmacokinetic studies in mice following oral administration revealed that sustained plasma blood levels were achieved within the therapeutic window for up to 24 hours. Oral bioavailability in mice was found to be 80-85% of drug administered and was constant over a single-dose range study (100 to 1,000 mg/kg). On-target in vitro and in vivo inhibition of activated MEK and PI3K was also demonstrated, revealing high potency of both the parent molecule and therapeutically active metabolites. Together, these studies revealed that lymphatic sequestration of orally dosed compounds serves to provide a reservoir that circumvents pharmacotoxicity through controlled physiological drug release into the systemic circulation and avoidance of first-pass hepatic metabolism. This platform provides a template for further development of additional lymphatropic small molecule inhibitors presenting opportunities in which multi-targeted cancer treatment options can now be evaluated. Exciting new therapeutic prospects offered by lymphatropic agent development is anticipated to transform treatment options for a wide variety of cancer types such as metastasis and lymphoma along with a myriad of other diseases including autoimmune disorders. Citation Format: Brian D. Ross, Youngsoon Jang, Amanda Welton, Christopher A. Bonham, Kevin Heist, Lucas McDonald, Gary D. Luker, Thomas L. Chenevert, Marcian Van Dort. Orally bioavailable 'lymphatropic' kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3936.

Insights into the role of N‐linked glycosylation in directing plasma membrane localization of GPCRs expressed in Saccharomyces cerevisiae

Glycosylation is the biochemical process of the attachment of sugar moieties or carbohydrates to proteins during co‐translational or post‐translational modification (PTM). It is one of many types of protein modifications that naturally occurs during the process of protein expression in organisms like Saccharomyces cerevisiae (Baker’s yeast). For natively expressed proteins, glycosylation can be critical for proper protein processing and can also be important for the production of proteins in a stable and biologically active state. For proteins that are non‐natively, or heterologously, expressed in these organisms, glycosylation can lead to improper processing and ultimately produce proteins with little to no biological relevance. This proves to be an undesirable effect for the elucidation of protein structure. However, large quantities of protein are required for structural analysis, presenting a significant obstacle for structural biologists and those who use heterologous expression as a means to acquire large quantities of protein. This problem becomes significantly more challenging for membrane proteins.Membrane proteins are an elusive class of biomolecules that reside or closely associate with the lipid bilayers of cells and encompass transporters, receptors and channel pores, to name a few. They play an intrinsic role in maintaining proper cell function including signaling and selective uptake of extracellular ions and small molecules. G‐protein coupled receptors (GPCRs) constitute one of the largest groups of membrane proteins across the plant and animal kingdoms. Despite being of scientific and medical interest, GPCRs and many integral membrane proteins are notoriously difficult to isolate and work with because of their hydrophobicity, structural flexibility and instability in the absence of the cell membrane. Optimized experimental conditions often vary from protein to protein and necessitate a systematic approach to achieve. This typically requires labor and resource intensive efforts. The goal of this work is to uncover new insights into the role of post‐translational processing and protein localization in the cell. Using a combination of fluorescence microscopy and bioinformatics we formulated an approach to investigate the effects of N‐linked glycosylation on plasma membrane localization of GPCRs expressed in S. cerevisiae. We postulate a critical link between plasma membrane localization and expression of “ligand active” or structurally relevant GPCRs. Previous studies of the human adenosine A2A receptor (hA2AR) show it readily localizes to the plasma membrane during expression in yeast, while the human dopamine D2 receptor (hD2R) does not. A sequence comparison of the two reveals three putative N‐linked glycosylation sites at the N‐terminal extracellular region of hD2R which is lacking in hA2AR. Using a chimera of these two receptors prepared with a C‐terminal GFP tag, we show the chimera does not traffic to the plasma membrane similarly to full‐length hA2AR, suggesting N‐linked glycosylation strongly influences functional high‐level expression of GPCRs and possibly other membrane proteins in yeast.

Boron clusters as broadband membrane carriers

The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes1–4. To remedy this, charged amphiphilic molecules have been classically used as carriers3,5. However, such amphiphilic carriers may cause aggregation and non-specific membrane lysis6,7. Here we show that globular dodecaborate clusters, and prominently B12Br122−, can function as anionic inorganic membrane carriers for a broad range of hydrophilic cargo molecules (with molecular mass of 146–4,500 Da). We show that cationic and neutral peptides, amino acids, neurotransmitters, vitamins, antibiotics and drugs can be carried across liposomal membranes. Mechanistic transport studies reveal that the carrier activity is related to the superchaotropic nature of these cluster anions8–12. We demonstrate that B12Br122− affects cytosolic uptake of different small bioactive molecules, including the antineoplastic monomethyl auristatin F, the proteolysis targeting chimera dBET1 and the phalloidin toxin, which has been successfully delivered in living cells for cytoskeleton labelling. We anticipate the broad and distinct delivery spectrum of our superchaotropic carriers to be the starting point of conceptually distinct cell-biological, neurobiological, physiological and pharmaceutical studies.

Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models.

Multicellular tumor spheroids have emerged as well-structured, three-dimensional culture models that resemble and mimic the complexity of the dense and hypoxic cancer microenvironment. However, in brain tumor studies, a variety of glioblastoma multiforme (GBM) cell lines only self-assemble into loose cellular aggregates, lacking the properties of actual glioma tumors in humans. In this study, we used type-I collagen as an extracellular matrix component to promote the compaction of GBM aggregates forming tight spheroids to understand how collagen influences the properties of tumors, such as their growth, proliferation, and invasion, and collagenase to promote collagen degradation. The GBM cell lines U87MG, T98G, and A172, as well as the medulloblastoma cell line UW473, were used as standard cell lines that do not spontaneously self-assemble into spheroids, and GBM U251 was used as a self-assembling cell line. According to the findings, all cell lines formed tight spheroids at collagen concentrations higher than 15.0 μg mL-1. Collagen was distributed along the spheroid, similarly to that observed in invasive GBM tumors, and decreased cell migration with no effect on the cellular uptake of small active molecules, as demonstrated by uptake studies using the photosensitizer verteporfin. The enzymatic cleavage of collagen affected spheroid morphology and increased cell migration while maintaining cell viability. Such behaviors are relevant to the physiological models of GBM tumors and are useful for better understanding cell migration and the in vivo infiltration path, drug screening, and kinetics of progression of GBM tumors.

Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria.

Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the “Trojan horse strategy”. In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E. coli. However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565.

Genome-wide identification of candidate aquaporins involved in water accumulation of pomegranate outer seed coat.

Aquaporins (AQPs) are a class of highly conserved integral membrane proteins that facilitate the uptake and transport of water and other small molecules across cell membranes. However, little is known about AQP genes in pomegranate (Punica granatum L.) and their potential role in water accumulation of the outer seed coat. We identified 38 PgrAQP genes in the pomegranate genome and divided them into five subfamilies based on a comparative analysis. Purifying selection played a role in the evolution of PgrAQP genes and a whole-genome duplication event in Myrtales may have contributed to the expansion of PgrTIP, PgrSIP, and PgrXIP genes. Transcriptome data analysis revealed that the PgrAQP genes exhibited different tissue-specific expression patterns. Among them, the transcript abundance of PgrPIPs were significantly higher than that of other subfamilies. The mRNA transcription levels of PgrPIP1.3, PgrPIP2.8, and PgrSIP1.2 showed a significant linear relationship with water accumulation in seed coats, indicating that PgrPIP1.3/PgrPIP2.8 located in the plasma membrane and PgrSIP1.2 proteins located on the tonoplast may be involved in water accumulation and contribute to the cell expansion of the outer seed coat, which then develops into juicy edible flesh. Overall, our results provided not only information on the characteristics and evolution of PgrAQPs, but also insights on the genetic improvement of outer seed coats.

Using membrane perturbing small molecules to target chronic persistent infections.

After antibiotic treatment, a subpopulation of bacteria often remains and can lead to recalcitrant infections. This subpopulation, referred to as persisters, evades antibiotic treatment through numerous mechanisms such as decreased uptake of small molecules and slowed growth. Membrane perturbing small molecules have been shown to eradicate persisters as well as render these populations susceptible to antibiotic treatment. Chemotype similarities have emerged suggesting amphiphilic heteroaromatic compounds possess ideal properties to increase membrane fluidity and such molecules warrant further investigation as effective agents or potentiators against persister cells.

Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.

SecY-mediated quality control prevents the translocation of non-gated porins

OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.

Le rôle des porines dans la résistance aux antibiotiques

The transport of small molecules across membranes is essential for the import of nutrients and other energy sources into the cell and for the export of waste and other potentially harmful byproducts out of the cell. While hydrophobic molecules are permeable to membranes, ions and other small polar molecules require transport via specialized membrane transport proteins. The two major classes of membrane transport proteins are transporters and channels. They are clearly involved in antibiotic resistance resulting in decreased intracellular penetration and a multidrug resistance phenotype.Recent structural biology reports and functional assays have substantially contributed to our understanding of the mechanism that mediates uptake of small molecules, including antibiotics.