The SDS22:PP1:I3 complex: SDS22 binding to PP1 loosens the active site metal to prime metal exchange.

SDS22 and Inhibitor-3 (I3) are two ancient regulators of protein phosphatase 1 (PP1) that regulate multiple essential biological processes. Both SDS22 and I3 form stable dimeric complexes with PP1; however, and atypically for PP1 regulators, they also form a triple complex, where both proteins bind to PP1 simultaneously (SPI complex). Here we report the crystal structure of the SPI complex. While both regulators bind PP1 in conformations identical to those observed in their individual PP1 complexes, PP1 adopts the SDS22-bound conformation, which lacks its M1 metal. Unexpectedly, surface plasmon resonance (SPR) revealed that the affinity of I3 for the SDS22:PP1 complex is ∼10-fold lower than PP1 alone. We show that this change in binding affinity is solely due to the interaction of I3 with the PP1 active site, specifically PP1's M2 metal, demonstrating that SDS22 likely allows for PP1 M2 metal exchange and thus PP1 biogenesis.

Characterization of erythroferrone oligomerization and its impact on BMP antagonism.

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state.

The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.

Characterization of erythroferrone structural domains relevant to its iron-regulatory function.

Iron delivery to the plasma is closely coupled to erythropoiesis, the production of red blood cells, as this process consumes most of the circulating plasma iron. In response to hemorrhage and other erythropoietic stresses, increased erythropoietin stimulates the production of the hormone erythroferrone (ERFE) by erythrocyte precursors (erythroblasts) developing in erythropoietic tissues. ERFE acts on the liver to inhibit bone morphogenetic protein (BMP) signaling and thereby decrease hepcidin production. Decreased circulating hepcidin concentrations then allow the release of iron from stores and increase iron absorption from the diet. Guided by evolutionary analysis and Alphafold2 protein complex modeling, we used targeted ERFE mutations, deletions, and synthetic ERFE segments together with cell-based bioassays and surface plasmon resonance to probe the structural features required for bioactivity and BMP binding. We define the ERFE active domain and multiple structural features that act together to entrap BMP ligands. In particular, the hydrophobic helical segment 81 to 86 and specifically the highly conserved tryptophan W82 in the N-terminal region are essential for ERFE bioactivity and Alphafold2 modeling places W82 between two tryptophans in its ligands BMP2, BMP6, and the BMP2/6 heterodimer, an interaction similar to those that bind BMPs to their cognate receptors. Finally, we identify the cationic region 96-107 and the globular TNFα-like domain 186-354 as structural determinants of ERFE multimerization that increase the avidity of ERFE for BMP ligands. Collectively, our results provide further insight into the ERFE-mediated inhibition of BMP signaling in response to erythropoietic stress.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes.

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.

The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.

Disruption of the SP-A/SP-R210L (MYO18Aα) pathway prolongs gestation and reduces fetal survival during lipopolysaccharide-induced parturition in late gestation.

Prolonged labor can lead to infection, fetal distress, asphyxia, and life-threatening harm to both the mother and the baby. Surfactant protein A (SP-A) was shown to contribute to the maintenance of pregnancy and timing of term labor. SP-A modulates the stoichiometric expression of the SP-R210L and SP-R210S isoforms of the SP-R210 receptor on alveolar macrophages (AMs). Lack of SP-R210L dysregulates macrophage inflammatory responses. We asked whether SP-A alters normal and inflammation-induced parturition through SP-R210 using SP-A- and SP-R210L-deficient mice. Labor and delivery of time-pregnant mice were monitored in real time using a time-lapse infrared camera. Intrauterine injection with either vehicle or Escherichia coli lipopolysaccharide (LPS) on embryonic (E) day 18.5 post coitus was used to assess the effect of gene disruption in chorioamnionitis-induced labor. We report that either lack of SP-A or disruption of SP-R210L delays parturition by 0.40 and 0.55 days compared with controls, respectively. LPS induced labor at 0.60, 1.01, 0.40, 1.00, and 1.31 days earlier than PBS controls in wild type (WT), SP-A-deficient, littermate controls, heterozygous, and homozygous SP-R210L-deficient mice, respectively. Lack of SP-A reduced litter size in PBS-treated mice, whereas the total number of pups delivered was similar in all LPS-treated mice. The number of live pups, however, was significantly reduced by 50%-70% in SP-A and SP-R210L-deficient mice compared with controls. Differences in gestational length were not associated with intrauterine growth restriction. The present findings support the novel concept that the SP-A/SP-R210 pathway modulates timely labor and delivery and supports fetal lung barrier integrity during fetal-to-neonatal transition in term pregnancy.NEW & NOTEWORTHY To our knowledge, this study is the first to report that SP-A prevents delay of labor and inflammation-induced stillbirth through the receptor SP-R210L.

Pharmacokinetics and safety of EVER206, a novel polymyxin antimicrobial, in healthy Chinese subjects.

EVER206 (also known as SPR206) is a novel polymyxin analog that has shown in vitro potency and in vivo efficacy against multidrug-resistant (MDR) Gram-negative pathogens. This randomized, double-blinded, placebo-controlled, Phase I study evaluated the safety, tolerability, and pharmacokinetics of EVER206 in healthy Chinese subjects. After single administration of 50-300 mg EVER206, the Cmax ranged from 3.94 to 25.82 mg/L, and the AUC0-inf ranged from 12.42 to 101.67 h·mg/L. The plasma exposure displayed a linear relationship with the dose administered. After administration of 75 and 100 mg of EVER206 every 8 hours (q8 hour), a steady state was achieved on Day 2. The accumulation ratios of Cmax and AUC from Day 1 to Day 7 were in the range of 1.12 to 1.3. The elimination half-lives ranged from 2.86 to 4.32 hours in the single-ascending-dose (SAD) study and 4.71 to 6.18 hours in the multiple-ascending-dose (MAD) study. The urinary excretion of unchanged EVER206 increased with the dose, with the mean cumulative fraction ranging from 23.70% to 47.10%. EVER206 was safe and well-tolerated in Chinese healthy subjects. No severe treatment emerging adverse events (TEAEs), serious adverse events, or TEAEs leading to discontinuation were reported. The results of the present study demonstrated a similar safety profile of EVER206 with data reported in an earlier study on SPR206-101. The exposure of EVER206 in Chinese healthy subjects was higher than that in Australian healthy subjects. These results could enable further clinical development of EVER206 in Chinese patients with severe MDR Gram-negative pathogen infections.CLINICAL TRIALSThis study was registered at the Chinese Clinical Trial Registry under identifier ChiCTR2200056692.

Crystal structure combined with metabolomics and biochemical studies indicates that FAM3A participates in fatty acid beta-oxidation upon binding of acyl-L-carnitine.

As the first member of the family with sequence similarity 3 (FAM3), FAM3A promotes synthesis of ATP in mitochondria of hepatic cells and cells from other organs. Dysregulations of FAM3A are involved in the development of diabetes and nonalcoholic fatty liver disease (NAFLD). So far, the molecule mechanism under the physiological and pathological functions of FAM3A is largely unexplored. Here, we determined the crystal structure of FAM3A at high resolution of 1.38Å, complexed with an unknown-source compound which was characterized through metabolomics and confirmed as methacholine by thermal shift assay and surface plasmon resonance (SPR). Exploration for natural ligands of FAM3A was conducted through the same molecular interaction assays. The observed binding of acyl-L-carnitine molecules indicated FAM3A participating in fatty acid beta-oxidation. Knockdown and rescue assays coupled with fatty acid oxidation determination confirmed the role of FAM3A in beta-oxidation. This investigation reveals the molecular mechanism for the biological function of FAM3A and would provide basis for identifying drug target for treatment of diabetes and NAFLD.

Deciphering the drug delivery potential of Type1 lipid transfer protein from Citrus sinensis for enhancing the therapeutic efficacy of drugs.

Type1 Non-specific Lipid Transfer Protein (CsLTP1) from Citrus sinensis is a small cationic protein possessing a long tunnel-like hydrophobic cavity. CsLTP1 performing membrane trafficking of lipids is a promising candidate for developing a potent drug delivery system. The present work includes in-silico studies and the evaluation of drugs binding to CsLTP1 using biophysical techniques along with the investigation of CsLTP1's ability to enhance the efficacy of drugs employing cell-based bioassays. The in-silico investigations identified Panobinostat, Vorinostat, Cetylpyridinium Chloride, and Fulvestrant with higher affinities and stability of binding to the hydrophobic pocket of CsLTP1. SPR studies revealed strong binding affinities of anticancer drugs, Panobinostat (KD = 1.40 µM) and Vorinostat (KD = 2.17 µM) to CsLTP1 along with the binding and release kinetics. CD and fluorescent spectroscopy revealed drug-induced conformational changes in CsLTP1. CsLTP1-associated drug forms showed remarkably enhanced efficacy in MCF-7 cells, representing increased cell cytotoxicity, intracellular ROS, reduced mitochondrial membrane potential, and up-regulation of proapoptotic markers than the free drugs employing qRT-PCR and western blot analysis. The findings demonstrate that CsLTP1 binds strongly to hydrophobic drugs to facilitate their transport, hence improving their therapeutic efficacy revealed by the in-vitro investigations. This study establishes an excellent foundation for developing CsLTP1-based efficient drug delivery system.

Insights into Photopolymerization at the Nanoscale Using Surface Plasmon Resonance Imaging.

Near-field photopolymerization (NFPP) driven by surface plasmon resonance has attracted increasing attention in nanofabrication. This interest comes from the nanometer-scale control of polymer thickness, due to the confinement of the evanescent wave within a highly restricted volume at the surface. In this study, a novel approach using a multi-spectral surface plasmon resonance instrument is presented that gives access to real-time images of polymer growth during NFPP with nanometer sensitivity. Using the plasmonic evanescent wave for both polymerization and real-time sensing, the influence of irradiance, concentration of dye, and initiator are investigated on the threshold energy and kinetics of NFPP. How oxygen inhibition in the near field strongly affects photopolymerization is highlighted, more than in the far field.

Interface-Engineered 2D Heterojunction with Photoelectric Dual Gain: Mxene@MOF-Enhanced SPR Spectroscopy for Direct Sensing of Exosomes.

MXene is widely used in the construction of optoelectronic interfaces due to its excellent properties. However, the hydrophilicity and metastable surface of MXene lead to its oxidation behavior, resulting in the degradation of its various properties, which seriously limits its practical application. In this work, a 2D metal-organic framework (2D MOF) with matching 2D morphology, excellent stability performance, and outstanding optoelectronic performance is grown in situ on the MXene surface through heterojunction engineering to suppress the direct contact between reactive molecules and the inner layer material without affecting the original advantages of MXene. The photoelectric dual gain MXene@MOF heterojunction is confirmed. As a photoelectric material, its properties are highly suitable for the demand of interface sensitization layer materials of surface plasmon resonance (SPR). Therefore, using SPR as a platform for the application of this interface material, the performance of MXene@MOF and its potential mechanism to enhance SPR are analyzed in depth using experiments combined with simulation calculations (FDTD/DFT). Finally, the MXene@MOF/peptides-SPR sensor is constructed for rapid and sensitive detection of the cancer marker exosomes to explore its potential in practical applications. This work offers a forward-looking strategy for the design of interface materials with excellent photoelectric performance.

Restoration of sleep-wake behavior following short photoperiod exposure in ventral subicular lesioned male Wistar rats: A 24-h sleep-wake electroencephalographical study.

The ventral subiculum regulates emotion, stress responses, and spatial and social cognition. In our previous studies, we have demonstrated anxiety- and depression-like symptoms, deficits in spatial and social cognition in ventral subicular lesioned (VSL) rats, and restoration of affective and cognitive behaviors following photoperiod manipulation (short photoperiod regime, SPR; 6:18 LD cycle). In the present study, we have studied the impact of VSL on sleep-wake behavioral patterns and the effect of SPR on sleep-wakefulness behavior. Adult male Wistar rats subjected to VSL demonstrated decreased wake duration and enhanced total sleep time due to increased non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS). Power spectral analysis indicated increased delta activity during NREMS and decreased sigma band power during all vigilance states. Light is one of the strongest entrainers of the circadian rhythm, and its manipulation may have various physiological and functional consequences. We investigated the effect of 21-day exposure to SPR on sleep-wakefulness (S-W) behavior in VSL rats. We observed that SPR exposure restored S-W behavior in VSL rats, resulting in an increase in wake duration and a significant increase in theta power during wake and REMS. This study highlights the crucial role of the ventral subiculum in maintaining normal sleep-wakefulness patterns and highlights the effectiveness of photoperiod manipulation as a non-pharmacological treatment for reversing sleep disturbances reported in mood and neuropsychiatric disorders like Alzheimer's disease, bipolar disorder, and major depressive disorder, which also involve alterations in circadian rhythm.

Systematic Evaluation of Benchmark G4 Probes and G4 Clinical Drugs using three Biophysical Methods: A Guideline to Evaluate Rapidly G4-Binding Affinity.

G-quadruplex DNA structures (G4) are proven to interfere with most genetic and epigenetic processes. Small molecules binding these structures (G4 ligands) are invaluable tools to probe G4-biology and address G4-druggability in various diseases (cancer, viral infections). However, the large number of reported G4 ligands (>1000) could lead to confusion while selecting one for a given application. Herein we conducted a systematic affinity ranking of 11 popular G4 ligands vs 5 classical G4 sequences using FRET-melting, G4-FID assays and SPR. Interestingly SPR data globally align with the rankings obtained from the two semi-quantitative assays despite discrepancies due to limits and characteristics of each assay. In the whole, PhenDC3 emerges as the most potent binder irrespective of the G4 sequence. Immediately below PDS, PDC-360A, BRACO19, TMPyP4 and RHPS4 feature strong to medium binding again with poor G4 topology discrimination. More strikingly, the G4 drugs Quarfloxin, CX5461 and c-PDS exhibit weak affinity with all G4s studied. Finally, NMM and Cu-ttpy showed heterogeneous behaviors due, in part, to their physicochemical particularities poorly compatible with screening conditions. The remarkable properties of PhenDC3 led us to propose its use for benchmarking FRET-melting and G4-FID assays for rapid G4-affinity evaluation of newly developed ligands.

Utilizing fab fragment-conjugated surface plasmon resonance-based biosensor for detection of Salmonella Enteritidis.

Although antibodies, a key element of biorecognition, are frequently used as biosensor probes, the use of these large molecules can lead to adverse effects. Fab fragments can be reduced to allow proper antigen-binding orientation via thiol groups containing Fab sites that can directly penetrate Au sites chemically. In this study, the ability of the surface plasmon resonance (SPR) sensor to detect Salmonella was studied. Tris(2-carboxyethyl)phosphine was used as a reducing agent to obtain half antibody fragments. Sensor surface was immobilized with antibody, and bacteria suspensions were injected from low to high concentrations. Response units were changed by binding first reduced antibody fragments, then bacteria. The biosensor was able to determine the bacterial concentrations between 103 and 108 CFU/mL. Based on these results, the half antibody fragmentation method can be generalized for faster, label-free, sensitive, and selective detection of other bacteria species.

Peptidoglycan endopeptidase MepM of uropathogenic Escherichia coli contributes to competitive fitness during urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are common bacterial infections, primarily caused by uropathogenic Escherichia coli (UPEC), leading to significant health issues and economic burden. Although antibiotics have been effective in treating UPEC infections, the rise of antibiotic-resistant strains hinders their efficacy. Hence, identifying novel bacterial targets for new antimicrobial approaches is crucial. Bacterial factors required for maintaining the full virulence of UPEC are the potential target. MepM, an endopeptidase in E. coli, is involved in the biogenesis of peptidoglycan, a major structure of bacterial envelope. Given that the bacterial envelope confronts the hostile host environment during infections, MepM's function could be crucial for UPEC's virulence. This study aims to explore the role of MepM in UPEC pathogenesis. RESULTS: MepM deficiency significantly impacted UPEC's survival in urine and within macrophages. Moreover, the deficiency hindered the bacillary-to-filamentous shape switch which is known for aiding UPEC in evading phagocytosis during infections. Additionally, UPEC motility was downregulated due to MepM deficiency. As a result, the mepM mutant displayed notably reduced fitness in causing UTIs in the mouse model compared to wild-type UPEC. CONCLUSIONS: This study provides the first evidence of the vital role of peptidoglycan endopeptidase MepM in UPEC's full virulence for causing UTIs. MepM's contribution to UPEC pathogenesis may stem from its critical role in maintaining the ability to resist urine- and immune cell-mediated killing, facilitating the morphological switch, and sustaining motility. Thus, MepM is a promising candidate target for novel antimicrobial strategies.

Marine sulfated glycans inhibit the interaction of heparin with S-protein of SARS-CoV-2 Omicron XBB variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8 million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS­CoV­2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed Saccharina japonica, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5 S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents.

A portable surface plasmon resonance (SPR) sensor for the detection of immunoglobulin A in plasma.

BACKGROUND: A life-threatening anaphylactic shock can occur if a patient with undiagnosed immunoglobulin A (IgA) deficiency (i.e., IgA levels <500 ng/mL) receives IgA-containing blood, hence the need for a rapid, point-of-care (POC) method for IgA deficiency screening. Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect IgA, but this method requires trained specialists and ≥24 h to obtain a result. We developed a surface plasmon resonance (SPR)-based protocol to identify IgA-deficient patients or donors within 1 h. MATERIALS AND METHODS: The SPR sensor relies on the detection of IgAs captured by primary antibodies adsorbed on the SPR chip and quantified with secondary antibodies. The sensor was calibrated from 0 to 2000 ng/mL in buffer, IgA-depleted human serum, and plasma samples from IgA-deficient individuals. A critical concentration of 500 ng/mL was set for IgA deficiency. The optimized sensor was then tested on eight plasma samples with known IgA status (determined by ELISA), including five with IgA deficiency and three with normal IgA levels. RESULTS: The limit of detection was estimated at 30 ng/mL in buffer and 400 ng/mL in diluted plasma. The results obtained fully agreed with ELISA among the eight plasma samples tested. The protocol distinguished IgA-deficient from normal samples, even for samples with an IgA concentration closer to critical concentration. DISCUSSION: In conclusion, we developed a reliable POC assay for the quantification of IgA in plasma. This test may permit POC testing at blood drives and centralized centers to maintain reserves of IgA-deficient blood and in-hospital testing of blood recipients.

A low-temperature SPR-based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B.

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.

A rare olive compound oleacein functions as a TrkB agonist and mitigates neuroinflammation both in vitro and in vivo.

BACKGROUND: Neuroinflammation is widely acknowledged as a characteristic feature of almost all neurological disorders and specifically in depression- and anxiety-like disorders. In recent years, there has been significant attention on natural compounds with potent anti-inflammatory effects due to their potential in mitigating neuroinflammation and neuroplasticity. METHODS: In the present study, we aimed to evaluate the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. Our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response using human neuroblastoma cells (SH-SY5Y cells) and an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation. RESULTS: In SH-SY5Y cells, OC exhibited a significant dose-dependent increase in BDNF expression. This enhancement was absent when cells were co-treated with inhibitors of BDNF's receptor TrkB, as well as downstream molecules PI3K and MEK. Whole-transcriptomics analysis revealed that OC upregulated cell cycle-related genes under normal conditions, while downregulating inflammation-associated genes in LPS-induced conditions. Furthermore, surface plasmon resonance (SPR) assays demonstrated that OC exhibited a stronger and more stable binding affinity to TrkB compared to the positive control, 7,8-dihydroxyflavone. Importantly, bioluminescence imaging revealed that a single oral dose of OC significantly increased BDNF expression in the brains of Bdnf-IRES-AkaLuc mice. Furthermore, oral administration of OC at a dosage of 10 mg/kg body weight for 10 days significantly reduced immobility time in the tail suspension test compared to the LPS-treated group. RT-qPCR analysis revealed that OC significantly decreased the expression of pro-inflammatory cytokines Tnfα, Il6, and Il1ß, while simultaneously enhancing Bdnf expression, as well as both pro and mature BDNF protein levels in mice hippocampus. These changes were comparable to those induced by the positive control antidepressant drug fluoxetine. Additionally, microarray analysis of mouse brains confirmed that OC could counteract LPS-induced inflammatory biological events. CONCLUSION: Altogether, our study represents the first report on the potential antineuroinflammatory and antidepressant properties of OC via modulation of BDNF/TrkB neurotrophic activity. This finding underscores the potential of OC as a natural therapeutic agent for depression- and anxiety-related disorders.