Serum Protein Binding Research Articles

Robust Reversible Cross-Linking Strategy for Intracellular Protein Delivery with Excellent Serum Tolerance.

Intracellular protein delivery has attracted increasing attentions in biomedical applications. However, current delivery systems usually have poor serum stability due to the competitive binding of serum proteins to the polymers during delivery. Here, we report a reversible cross-linking strategy to improve the serum stability of polymers for robust intracellular protein delivery. In the proposed delivery system, nanoparticles are assembled by cargo proteins and cationic polymers and further stabilized by a glutathione-cleavable and traceless cross-linker. The cross-linked nanoparticles show high stability and efficient cell internalization in serum containing medium and can release the cargo proteins in response to intracellular glutathione and acidic pH in a traceless manner. The generality and versatility of the proposed strategy were demonstrated on different types of cationic polymers, cargo proteins, as well as cell lines. The study provides a facile and efficient method for improving the serum tolerance of cationic polymers in intracellular protein delivery.

Binding of serum-derived amyloid-associated proteins to amyloid fibrils

Background Amyloid signature proteins such as serum amyloid P component, apolipoprotein E (ApoE), and ApoA-IV generally co-localise with amyloid, regardless of the types of amyloid precursor protein or the organs. Most of these proteins derive from serum and have reportedly been involved in amyloid fibril formation and stabilisation, as well as in excretion and degradation of amyloid precursor proteins. However, the processes and mechanisms by which these specific proteins deposit together with amyloid fibrils have not been clarified. Methods We analysed the binding of serum proteins to amyloid fibrils derived from amyloid β and insulin in vitro by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Specific serum proteins including ApoA-I, ApoE, ApoA-IV, ApoC-III and vitronectin adhered to amyloid fibrils at high concentrations in vitro. In addition, the profile of these proteins commonly occurred in both amyloid β and insulin amyloid fibrils and was mostly consistent with the composition of amyloid signature proteins. We also showed that high concentrations of serum proteins can adhere to amyloid fibrils in a short time. Conclusions Our in vitro results suggest that amyloid signature proteins coexist with amyloid primarily dependent on the binding of each serum protein, in the extracellular fluid, to amyloid fibrils.

Impact of Vitamin D Elements and Osteoporosis Factors in Postmenopausal Iraqi Women with T2DM

Osteoporosis is a systematic bone disorder characterized by bone mass loss and bone tissue architectural degeneration. The incidence of osteoporosis in women increases with age, reflecting the considerable increase in bone loss rate in postmenopausal women once estrogen loses its protective influence. The purpose of this study was to determine the levels of Osteocalcin, Vitamin D, and other parameters that influence bone quality and increased bone fragility in postmenopausal Iraqi women with type 2 diabetes mellitus (T2DM). The levels of vitamin D receptor and vitamin D binding protein in serum are studied for the first time in our study. The present study included 89 postmenopausal women aged 50-70 years old, 62 T2DM patients, and 27 controls. Ten of the T2DM patients were considered osteoporotic, 28 were considered osteopenia, and 22 were normal; this classification is according to the WHO criterion. After matching for body mass index (BMI) and age for patients and controls, results show a significant difference in serum Osteocalcin, vitamin D, and vitamin D binding protein levels in patients compared to controls. In our study, diabetic patients appear with high levels of Osteocalcin, vit.D, and vitamin D binding protein compared with non-diabetic control. The processes underlying diabetes mellitus-induced skeletal problems are unknown. Anti-diabetic medications might have an adverse or favorable effect on bone metabolism. The study concludes that managing skeletal health in postmenopausal women entails screening fracture risk factors, lowering modifiable risk variables through dietary and lifestyle modifications, and using a pharmacologic treatment for individuals at high risk of osteoporosis or fracture. Women with osteoporosis must be managed for the rest of their lives.

Heparin Binding Protein in Early Differential Diagnosis of Bacterial Meningitis.

Heparin-binding protein is a serine protease that is mobilized rapidly from emigrating polymorphonuclear leukocytes that acts as a chemoattractant activator of monocyte and macrophages. We investigated the potential role and efficacy of serum and cerebrospinal fluid heparin binding protein in differentiating bacterial meningitis from tuberculosis and viral meningitis. A case diagnosed with acute bacterial meningitis (n:37), viral meningitis (n:30) and tuberculous meningitis (n:30) was included in this study. The diagnosis was based on history, clinical criteria, cerebrospinal fluid examination, latex agglutination and culture, and response to therapy. Heparin-binding protein was measured using enzyme-linked immunosorbent technique in both cerebrospinal fluid and serum. Cerebrospinal fluid heparin-binding protein levels were 7.81 ± 0.23ng/mL in bacterial meningitis, 6.11 ± 0.3ng/mL in tuberculosis meningitis and 5.75 ± 0.1ng/mL in viral meningitis. The mean serum level was 14.98 ± 1.1ng/mL in bacterial meningitis, 6.89 ± 0.4ng/mL in tuberculosis meningitis, and 6.02 ± 0.4ng/mL in viral meningitis. Both heparin-binding protein levels were significantly higher in patients with bacterial meningitis. We found that serum and cerebrospinal fluid heparin binding protein is a useful marker for differentiating bacterial meningitis from non-bacterial meningitis.

Purple red rice anthocyanins alleviate intestinal damage in cyclophosphamide-induced mice associated with modulation of intestinal barrier function and gut microbiota

The regulatory effects of purple red rice bran anthocyanins (PRBA) on intestinal barrier function and gut microbiota in mice were investigated. Results showed that PRBA had an ameliorative effect on intestinal barrier damage, including restoration of villus length, improvement in the number of cupped cells and promotion of sIgA secretion. PRBA stimulated the production of cytokines, reduced the levels of endotoxin (ET) and lipopolysaccharide binding protein (LBP) in serum, as well as upregulated the expression of tight junction proteins (TJs) and NF-κB pathway proteins. Furthermore, PRBA not only promoted the production of short-chain fatty acids (SCFAs), but also regulated the intestinal microbiota by increasing beneficial bacteria (Lachnospiraceae, Bacteroidaceae, Ruminococcaceae) and reducing pathogenic bacteria (Shigella) to maintained intestinal homeostasis. Above results indicated that PRBA could ameliorate cyclophosphamide-induced impairment of intestinal barrier function and dysregulation of the gut microbiota, which provides a new idea for broadening the exploitation of PRBA.

Mesona chinensis Benth Polysaccharides Alleviate DSS-Induced Ulcerative Colitis via Inhibiting of TLR4/MAPK/NF-κB Signaling Pathways and Modulating Intestinal Microbiota.

Ulcerative colitis (UC) is a severe disease of the intestinal tract. To investigate the role of TLR4/Mitogen-activated protein kinase (MAPK)/Nuclear factor kappa-B(NF-κB) pathways and intestinal flora in UC, and the protective mechanisms of Mesona chinensis Benth polysaccharides (MBP), potential therapeutic agents due to their diabetes-relieving, cancer-suppressing, and immunomodulatory properties. A dextran sulfate sodium (DSS)-induced mouse colitis model is used for experiments; the histopathology, immunohistochemistry, and Western blotting's results suggest that MBP can alleviate the colitis symptoms, inhibits the overproduction of TNF-α, IL-1β, promote IL-10, reduces myeloperoxidase activity, and alleviates the inflammatory response probably by inhibiting the activation of TLR4/MAPK/NF-κB pathways. Furthermore, MBP improvs the ratio of Bcl-2/BAX, maintains the intestinal integrity by promoting the levels of zonulin occludin-1 (ZO-1), occluding and mucin mucin-2 (MUC-2), reduces the levels of endotoxin (ET), lipopolysaccharide binding protein (LBP) in serum, and oxidative stress in liver. Moreover, using 16S rRNA Gene Sequencing analysis, MBP regulates gut microbiota by decreasing the abundances of Helicobacter and Prevotella and increasing the abundances of Lactobacillus and Coprococcus, reverses microbiota dysbiosis caused by DSS. These findings confirm the anti-inflammatory effects of MBP, restoration of the intestinal barrier and intestinal flora, and have therapeutic potential to attenuate the development of UC.

Serum protein level as a predictor of therapeutic response and adverse effects associated with afatinib use.

BackgroundAfatinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor with significant serum protein binding. Total protein level was found to be associated with plasma afatinib level. The trough serum concentration was shown to be associated with development of adverse effects. This study aims to explore the association between serum protein levels and clinical responses and adverse effects in advanced stage non-small cell lung cancer (NSCLC) treated with afatinib.MethodsThis was a retrospective cohort study of Chinese patients with metastatic NSCLC harboring EGFR mutations who received first or second line afatinib treatment. The association of serum protein and album levels, as well as their ratio, and the development of adverse effects was investigated.ResultsAmong 217 patients included, 91 were on afatinib as first line treatment and 126 as second line treatment. Higher serum protein levels, albumin levels and albumin to globulin ratio, were found to be associated with clinical response to afatinib among patients on first or second line setting. Among patients on first line afatinib with Eastern Cooperative Oncology Group Performance State (ECOG PS) at 1 or above, those with lower serum protein levels at baseline had higher risks of developing grade 2 or above gastrointestinal adverse effects. Among patients on second line afatinib and with ECOG PS at 1 or above, patients with lower serum protein levels at baseline had higher risks of developing grade 3 or above cutaneous adverse effects. Lower serum albumin to globulin ratio at baseline was associated with increased risks of grade 3 or above gastrointestinal adverse effects among patients with ECOG PS at 1 or above and no prior systemic chemotherapy.ConclusionsSerum protein, albumin level and serum albumin to globulin ratio may predict the response to afatinib and occurrence of adverse effects with afatinib treatment.

Pharmacokinetics of isavuconazole in healthy cats after oral and intravenous administration.

BackgroundIsavuconazole is a triazole antifungal drug that has shown good efficacy in human patients. Absorption and pharmacokinetics have not been evaluated in cats.ObjectivesTo determine the pharmacokinetics of isavuconazole in cats given a single IV or PO dose.AnimalsEight healthy, adult research cats.MethodsFour cats received 100 mg capsules of isavuconazole PO. Four cats received 5 mg/kg isavuconazole solution IV. Serum was collected at predetermined intervals for analysis using ultra‐high performance liquid chromatography‐tandem mass spectrometry. Data were analyzed using a 2‐compartment uniform weighting pharmacokinetic analysis with lag time for PO administration and a 2 compartment, 1/y2 weighting for IV administration. Predicted 24 and 48‐hour dosing intervals of 100 mg isavuconazole administered PO were modeled and in vitro plasma protein binding was assessed.ResultsBoth PO and IV drug administration resulted in high serum concentrations. Intravenous and PO formulations of isavuconazole appear to be able to be used interchangeably. Peak serum isavuconazole concentrations occurred 5 ± 3.8 hours after PO administration with an elimination rate half‐life of 66.2 ± 55.3 hours. Intersubject variability was apparent in both the PO and IV groups. Two cats vomited 6 to 8 hours after PO administration. No adverse effects were observed in the IV group. Oral bioavailability was estimated to be approximately 88%. Serum protein binding was calculated to be approximately 99.0% ± 0.03%.Conclusions and Clinical ImportanceIsavuconazole might prove to be useful in cats with fungal disease given its favorable pharmacokinetics. Additional studies on safety, efficacy, and tolerability of long‐term isavuconazole use are needed.

Galactosylated Prodrugs: A Strategy to Improve the Profile of Nonsteroidal Anti-Inflammatory Drugs.

Carbohydrates are one of the most abundant and important classes of biomolecules. The variety in their structures makes them valuable carriers that can improve the pharmaceutical phase, pharmacokinetics and pharmacodynamics of well-known drugs. D-galactose is a simple, naturally occurring monosaccharide sugar that has been extensively studied for use as a carrier and has proven to be valuable in this role. With the aim of validating the galactose-prodrug approach, we have investigated the galactosylated prodrugs ibuprofen, ketoprofen, flurbiprofen and indomethacin, which we have named IbuGAL, OkyGAL, FluGAL and IndoGAL, respectively. Their physicochemical profiles in terms of lipophilicity, solubility and chemical stability have been evaluated at different physiological pH values, as have human serum stability and serum protein binding. Ex vivo intestinal permeation experiments were performed to provide preliminary insights into the oral bioavailability of the galactosylated prodrugs. Finally, their anti-inflammatory, analgesic and ulcerogenic activities were investigated in vivo in mice after oral treatment. The present results, taken together with those of previous studies, undoubtedly validate the galactosylated prodrug strategy as a problem-solving technique that can overcome the disadvantages of NSAIDs.

A Comparative Assessment of Human Serum Proteins Interactions with Hemodialysis Clinical Membranes using Molecular Dynamics Simulation

AbstractEnd stage renal disease (ESRD) affects ≈10% of the world's population. Hemodialysis (HD) is a life‐sustaining extracorporeal blood purifying treatment for ESRD patients. Despite the advances in technology, the hemoincompatibility of the dialyzers leads to a high morbidity and mortality rate. Decreasing interactions between polymers and blood constituents will result in controlled binding of human serum proteins to the surface and consequently enhanced biocompatibility. This study aims to assess the interaction energy between common hemodialysis polymer structures and human serum proteins using molecular dynamics simulation to offer a framework for understanding the dominant interactions as a reference for material development. Molecular dynamics and molecular docking simulations are conducted for calculating polymerprotein binding energies. Common serum proteins are selected for protein models. Poly aryl ether sulfone (PAES) with and without polyvinyl pyrrolidone (PVP), polyvinylidene fluoride (PVDF), cellulose triacetate (CTA), polyacrylonitrile (PAN), and polymethyl methacrylate (PMMA) membrane structures are chosen as the different classes of dialyzers. The van der Waals interactions between polymers and proteins dominate the binding energies sequence. Molecular docking results of the affinity between protein receptors‐ligands are aligned with the MD binding interaction.

Study of Renal Accumulation of Targeted Polycations in Acute Kidney Injury.

Acute kidney injury (AKI) is a global healthcare burden characterized by rapid loss of renal function and high morbidity and mortality. Chemokine receptor CXCR4 participates in the renal infiltration of immune cells following injury and in local inflammatory enhancement. Injured renal tubule cells overexpress CXCR4, which could be used as a target for improved drug delivery in AKI. Plerixafor is a small-molecule CXCR4 antagonist that has shown beneficial effects against AKI and has been previously developed into a polymeric analog (polymeric plerixafor, PP). With the goal of gaining a better understanding of how overall charge and hydrophilicity affect renal accumulation of PP, we have synthesized PP copolymers containing hydroxyl, carboxyl, primary amine, and alkyl moieties using Michael-type addition copolymerization. All synthesized copolymers showed excellent CXCR4-binding and inhibiting ability in vitro and improved cellular uptake in hypoxia-reoxygenation stimulated mouse tubule cells. Analysis of serum protein binding revealed that polymers with hydroxyl group modification showed the least amount of protein binding. Biodistribution of the polymers was tested in a unilateral ischemia reperfusion-induced AKI mouse model. The results showed significant differences in accumulation in the injured kidneys depending on the net charge and hydrophilicity of the polymers. The findings of this study will guide the development of polymeric drug carriers for targeted delivery to injured kidneys for better AKI therapy.

Solution speciation and human serum protein binding of indium(III) complexes of 8-hydroxyquinoline, deferiprone and maltol

Solution speciation and serum protein binding of selected In(III) complexes bearing O,O and O,N donor sets were studied to provide comparative data for In(III) and analogous Ga(III) complexes. Aqueous stability of the In(III) complexes of maltol, deferiprone, 8-hydroxyquinoline (HQ) and 8-hydroxyquinoline-5-sulfonate (HQS) was characterized by a combined pH-potentiometric and UV–visible spectrophotometric approach. Formation of mono, bis and tris-ligand complexes was observed. The tris-ligand complexes of HQ (InQ3) and deferiprone (InD3) are present in solution in ca. 90% at 10 µM concentration at pH = 7.4, while the tris-maltolato complex (InM3) displays insufficient stability under these conditions. Binding towards human serum albumin (HSA) and (apo)transferrin ((apo)Tf) of InQ3, InD3 and InM3 complexes and Ga(III) analogue of InQ3 (GaQ3) together with InCl3 was investigated by a panel of methods: steady-state and time-resolved spectrofluorometry, UV–visible spectrophotometry and membrane ultrafiltration. Moderate binding of InQ3 to HSA was found (log K′ = 5.0–5.1). InD3 binds to HSA to a much lower extent in comparison to InQ3. ApoTf is able to displace HQ, deferiprone and maltol effectively from their In(III) complexes. Protein binding of non-dissociated InQ3 was also observed at high complex-to-apoTf ratios. Studies conducted with the InQ3/GaQ3 – HSA – Tf ternary systems revealed the more pronounced Tf binding of In(III) via ligand release, while the original GaQ3 scaffold is preferably retained upon protein interactions and significant albumin binding occurs. Significant dissociation of InQ3 was detected in human blood serum as well.Graphical abstract

Magnetic solid phase extraction followed by in-situ derivatization with core–shell structured magnetic graphene oxide nanocomposite for the accurate quantification of free testosterone and free androstenedione in human serum

Circulating free androgens are important indicators for a variety of diseases, so accurate determination of these hormones in serum is of great clinical significance. However, there are still many challenges for the accurate quantification of free androgens using mass methods because of their very low levels and complex interferences in serum, as well as the high serum protein binding rate and high nonspecific binding (NSB) rate. Here, an HPLC–MS/MS method coupled with magnetic solid phase extraction (MSPE) and in-situ derivatization was developed to quantify two free androgens—free testosterone (FT) and free androstenedione (FA4)—in human serum simultaneously and accurately. Ultrafiltration was used to obtain free androgens in serum. To minimize the NSB rate and obtain accurate results, the ultrafiltration membrane was doubly modified with surfactant followed by a silane-coupling agent. Multiple pre-saturation with the tested samples was also used. With these strategies, the ultrafiltration recoveries were up to 95.4% for FT and 94.0% for FA4, so the NSB was negligible. After that, the extremely low levels of free androgens in ultrafiltrates were extracted and enriched using MSPE with core–shell structured ferroferric oxide coated with graphene oxide. Hydroxylamine hydrochloride was used to derivatize the analytes and the reaction took place on the surface of the adsorbent. All the extraction and derivatization conditions were optimized. Under such conditions, the assays were linear for FT within the range of 2–100 pg mL−1 and for FA4 within the range of 5–500 pg mL−1. The intra- and inter-run CV was less than 12.3% and 10.9% for FT and less than 7.2% and 8.3% for FA4, respectively. For the intra- and inter-run accuracies, the relative error of the mean was 9.9% and 8.4% for FT, and 11.5% and 7.3% for FA4, respectively. The total extraction recoveries with MSPE in-situ derivatization were 93.2% for FT, 93.8% for FA4 and 95.7% for the internal standard. The method was validated and was used to quantify the trace level of these two free androgens in serum samples from female patients suspected of having polycystic ovary syndrome (PCOS) accurately. It is expected to improve the diagnosis accuracy of PCOS when combined with other clinical indicators.

P-NCS-Bn-NODAGA as a bifunctional chelator for radiolabeling with the 186Re/99mTc-tricarbonyl core: Radiochemistry with model complexes and a GRPR-targeting peptide

IntroductionWith the goal of developing theranostic agents for application in radiopharmaceutical chemistry, in this work, we studied p-NCS-Bn-NODAGA (1) as a bifunctional chelator for the fac-[M(CO)3]+ core (M = natRe, 186Re, 99mTc). Specifically, we studied complexes of the formula [M(CO)3(L)]+, where L denotes either Bn-NODAGA-Pyr (2) or Bn-NODAGA-Ser-Ser-RM2 (3). MethodsThe model bioconjugate molecule 2 was synthesized by conjugating pyrrolidine with 1, while 3 was derived from the conjugation of the gastrin-releasing peptide receptor (GRPR)-targeting peptide Ser-Ser-RM2 with 1. Labeling of 2 and 3 was performed with [M(CO)3(OH2)3]+ (where M = natRe, 186Re, or 99mTc). The stability of the radioactive complexes was studied against l-histidine and l-cysteine (1 mM in PBS; pH 7.4, 37 °C). GRPR affinity of both peptide 3 and its metallated counterpart, Re-3, were determined with in vitro competitive binding assays in GRPR-expressing PC-3 cells using [125I]I-Tyr4-BBN as the competitor. ResultsAfter a thorough radiolabeling optimization process, the [M(CO)3(2)]+ model complexes (M = 186Re and 99mTc) were synthesized with 94 ± 2% radiochemical yield (RCY; estimated by radio-HPLC). In stability studies, [186Re]Re-2 remained intact through 7 d in l-cysteine and l-histidine. Similarly, stability studies in rat serum at 37 °C showed 99 ± 1% intact [186Re]Re-2 through 4 h. Non-specific rat serum protein binding of [186Re]Re-2 was found to be 33 ± 4% at 4 h. The [99mTc]Tc-2 complex was found to be stable in l-histidine and l-cysteine at 37 °C through 24 h. [99mTc]Tc-2 was also stable in rat serum, with 38 ± 3% non-specific protein binding, at 4 h. The [M(CO)3(3)]+ peptide radiometal complex (M = 186Re and 99mTc) syntheses were also optimized, resulting in RCYs of 35% for [186Re]Re-3 and 47% for [99mTc]Tc-3 (estimated by radio-HPLC). [186Re]Re-3 showed 98 ± 2% and 84 ± 5% stability in l-histidine and l-cysteine, respectively, through 48 h. Similarly, stability studies in rat serum at 37 °C showed 85 ± 3% intact [186Re]Re-3 through 4 h, with 29 ± 7% non-specific protein binding in rat serum. [99mTc]Tc-3 was found to be 84 ± 3% and 82 ± 4% stable in l-histidine and l-cysteine at 24 h, respectively. [99mTc]Tc-3 in rat serum at 37 °C showed 88 ± 2% stability through 4 h, with 25 ± 2% non-specific protein binding. Both 3 and Re-3 demonstrated high GRPR affinity, with IC50 values of 3.1 nM and 3.9 nM, respectively. ConclusionsThe low nanomolar IC50 values obtained for 3 and Re-3 demonstrate high affinity of this novel [M(CO)3]-labeled bioconjugate for GRPR. The encouraging stability studies and receptor affinity results demonstrate promise for further development of these metal complexes as a theranostic matched pair for targeting GRPR.

Probing multifunctional azure B conjugated gold nanoparticles with serum protein binding properties for trimodal photothermal, photodynamic, and chemo therapy: Biophysical and photophysical investigations

Multimodal or combination therapy has been considered as a powerful approach for treatment of complex diseases like cancer. The fascinating physicochemical and optoelectronic properties of gold nanoparticles make them potential candidate for cancer therapeutic and diagnostic applications. Herein, we design a multifunctional nanosystem by conjugating a photosensitizer, Azure B (AB) with citrate reduced gold nanoparticles (CI-Au NPs) through non-covalent interactions. The conjugation of AB with CI-Au NPs was confirmed through UV–Visible absorption spectroscopy and Fourier Transform Infra-red (FT-IR) spectroscopy. The morphology, size, and charge of the prepared nano-conjugates (AB@CI-Au NPs) were determined by transmission electron microscopy (TEM), Dynamic light scattering (DLS), and zeta potential measurements. The proficiency of AB@CI-Au NPs for cancer photo-therapies was demonstrated by evaluating their potential for photothermal heating and singlet oxygen generation in solution upon Visible laser (635 nm, 500 mW/cm2) irradiation. The AB@CI-Au NPs display superior heating efficiency than CI-Au NPs alone or free AB, and offer better photostability as well as singlet oxygen generation rate compared to free photosensitizer. The interaction of AB@CI-Au NPs with Calf thymus DNA (Ct-DNA) and transport protein Bovine Serum Albumin (BSA) were studied using various biophysical techniques including Circular dichroism, UV–Visible and fluorescence spectroscopic studies. AB@CI-Au NPs show intercalative binding with Ct-DNA by inducing local perturbations in double helical structure and hence can exert chemotherapeutic action by targeting DNA. AB@CI-Au NPs also display moderate binding with BSA without any adverse effect on BSA structure, which is desirable for significant biodistribution and pharmacokinetics of AB@CI-Au NPs. Also, in vitro cytotoxicity of the AB@CI-Au NPs with and without laser irradiation (635 nm, 500 mW/cm2) was demonstrated using the hepatocellular carcinoma (HepG2) cell lines. Our findings through photophysical and biophysical studies strongly recommend the exploitation of AB@CI-Au NPs nanoconjugates as a multifunctional probe for trimodal anticancer therapy.

Fluorescent sensors of siderophores produced by bacterial pathogens

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Remifentanil Promotes PDIA3 Expression by Activating p38MAPK to Inhibit Intestinal Ischemia/Reperfusion-Induced Oxidative and Endoplasmic Reticulum Stress.

Background: Remifentanil protects against intestinal ischemia/reperfusion (I/R) injury; however, its exact mechanism remains to be elucidated. The objective of this study was to investigate the underlying molecular mechanism of remifentanil in intestinal I/R injury in mice. Methods: We evaluated the intestine-protective effect of remifentanil in adult male mice with 45 min superior mesenteric artery occlusion followed by 4 h reperfusion by determining the following: intestinal Chiu’s scores, diamine oxidase, and intestinal fatty acid binding protein in serum; the apoptotic index, lipid peroxidation product malondialdehyde (MDA), and superoxide dismutase (SOD) activity in the intestinal mucosa; and the intestinal mRNA and protein expressions of Bip, CHOP, caspase-12, and cleaved caspase-3, reflecting endoplasmic reticulum (ER) stress. Furthermore, conditional knockout mice, in which the protein disulfide isomerase A3 (PDIA3) gene was deleted from the intestinal epithelium, and SB203580 (a selective p38MAPK inhibitor) were used to determine the role of PDIA3 and p38MAPK in I/R progression and intestinal protection by remifentanil. Results: Our data showed that intestinal I/R induced obvious oxidative stress and endoplasmic reticulum stress–related cell apoptosis, as evidenced by an increase in the intestinal mucosal malondialdehyde, a decrease in the intestinal mucosal SOD, and an increase in the apoptotic index and the mRNA and protein expression of Bip, CHOP, caspase-12, and cleaved caspase-3. Remifentanil significantly improved these changes. Moreover, the deletion of intestinal epithelium PDIA3 blocked the protective effects of remifentanil. SB203580 also abolished the intestinal protection of remifentanil and downregulated the mRNA and protein expression of PDIA3. Conclusion: Remifentanil appears to act via p38MAPK to protect the small intestine from intestinal I/R injury by its PDIA3-mediated antioxidant and anti-ER stress properties.

Surface-regulated injection dose response of ultrasmall luminescent gold nanoparticles.

With the rapid growth of the use of renal-clearable nanomedicines in disease targeting and therapy, a fundamental understanding of their injection dose responses is of great importance for future translation to clinical settings. Using glutathione-coated gold nanoparticles (GS-AuNPs) as a renal-clearable nanomedicine model for the construction of ultrasmall AuNPs with different serum protein binding abilities, we discover that the concentration-dependent serum protein binding capabilities endow GS-AuNPs with a more sensitive response to injection dose than NPs resistant to serum protein binding, resulting in greatly improved tumor-targeting efficiencies during both single and repeated low-dose injections; the performance is also distinct from nonrenal-clearable AuNPs coated with serum protein, which show decreased tumor-targeting efficiency with a decrease in the injection dose.

Combined Hemoperfusion and Continuous Veno-Venous Hemofiltration for Carbamazepine Intoxication

Introduction: Carbamazepine (CBZ) is a widely used anticonvulsant with a low molecular weight that allows for extracorporeal removal of free drug by both dialytic and hemoperfusion techniques, particularly in a massive overdose where serum protein binding is saturated. This report presents a case of CBZ intoxication where we were able to compare the mass removal of CBZ using hemoperfusion, with the mass removal of CBZ achieved with continuous renal replacement therapy (CRRT) during combined treatment. Methods: The Jafron HA230 resin hemoperfusion cartridge was applied in series with the continuous veno-venous hemofiltration (CVVH) circuit. Baseline and ongoing serum drug levels along with further samples from pre- and post-hemoperfusion cartridges and from CVVH effluent were collected. Results: Combined CVVH and resin hemoperfusion therapy in series was associated with a 50% reduction in the CBZ level from 16 mg/L to 8 mg/L over 3 h, far more rapid than that observed with CVVH alone or in the absence of extracorporeal drug clearance in the preceding hours. The combination therapy removed close to 35 mg/h of CBZ. Conclusion: The combination of CRRT and hemoperfusion can be easily deployed, appears safe, and is able to combine the CBZ mass removal achieved with each technique, thus to maximize CBZ extraction.

Quaternary Ammonium Compounds: Bioaccumulation Potentials in Humans and Levels in Blood before and during the Covid-19 Pandemic.

Quaternary ammonium compounds (QACs) are commonly used in a variety of consumer, pharmaceutical, and medical products. In this study, bioaccumulation potentials of 18 QACs with alkyl chain lengths of C8–C18 were determined in the in vitro–in vivo extrapolation (IVIVE) model using the results of human hepatic metabolism and serum protein binding experiments. The slowest in vivo clearance rates were estimated for C12-QACs, suggesting that these compounds may preferentially build up in blood. The bioaccumulation of QACs was further confirmed by the analysis of human blood (sera) samples (n = 222). Fifteen out of the 18 targeted QACs were detected in blood with the ΣQAC concentrations reaching up to 68.6 ng/mL. The blood samples were collected during two distinct time periods: before the outbreak of the COVID-19 pandemic (2019; n = 111) and during the pandemic (2020, n = 111). The ΣQAC concentrations were significantly higher in samples collected during the pandemic (median 6.04 ng/mL) than in those collected before (median 3.41 ng/mL). This is the first comprehensive study on the bioaccumulation and biomonitoring of the three major QAC groups and our results provide valuable information for future epidemiological, toxicological, and risk assessment studies targeting these chemicals.