Human Serum Proteins Research Articles

Bimodal Array-Based Fluorescence Sensor and Microfluidic Technology for Protein Fingerprinting and Clinical Diagnosis.

Proteins play a crucial role in determining disease states in humans, making them prime targets for the development of diagnostic sensors. The developed sensor array is used to investigate global proteomic changes by fingerprinting multifactorial disease states in model urine simulating phenylketonuria and in serum from preeclamptic pregnant women. Here, we report a fluorescence-based chemical sensing array that exploits the host-guest interaction between cucurbit[7]uril (CB[7]) and fluorescent triphenylamine derivatives (TPA) to detect a range of proteins. Using linear discriminant analysis, we identify fluorescence fingerprints of 14 proteins with over 98% accuracy in buffer and human serum. The array is optimized on an automated droplet microfluidic-based platform, for high-throughput sensing with controlled composition and lower sample volumes. This sensor enables the discrimination of proteins in physiological buffer and human serum, with promising applications in disease diagnosis.

Impact of pH-Dependent Dynamics of Human Serum Proteins on Dialysis Membranes: Cryptographic Structure Assessment, Synchrotron Imaging of Membrane-Protein Adsorption, and Molecular Docking Studies

Proteins are fundamental to biochemical processes and critical in hemodialysis. This study investigates the impact of pH on human serum albumin (HSA), fibrinogen (FB), and transferrin (TRF) interactions with polyarylethersulfone (PAES) hemodialysis membranes. A multi-method approach was utilized, including protein crystallography for structural insights, hydration layer analysis to explore solvation and interaction potentials, molecular docking using AutoDock 4.0 for binding affinity simulations, and in-situ X-ray synchrotron SR-μCT imaging to observe protein deposition dynamics.Molecular docking revealed that PAES demonstrated superior binding energies and interaction patterns with FB and TRF compared to cellulose triacetate (CTA), facilitated by specific hydrogen bonding within a water shell. CTA displayed weaker, hydration-sensitive interactions varying with pH. Imaging studies indicated that FB showed higher adsorption at pH 6 than at pH 7.2, predominantly in the middle membrane regions. Similarly, HSA and TRF exhibited increased adsorption at pH 6, suggesting a stronger affinity under acidic conditions. Mixed protein solutions also indicated higher adsorption at pH 6, emphasizing an increased risk of membrane fouling.These findings highlight the crucial role of pH in modulating protein-membrane interactions and enhancing the efficacy of hemodialysis. A deeper understanding of hydration environments and their effects on protein binding affinities provides valuable insights for optimizing membrane design and performance. Clinically, this research suggests that fine-tuning pH during hemodialysis could mitigate protein fouling on membranes, thereby improving procedural efficiency and potentially leading to better patient outcomes through enhanced dialysis effectiveness.

Pegylated gold nanoparticles interact with lipid bilayer and human serum albumin and transferrin

Gold nanoparticles (AuNPs) are potentially applicable in drug/nucleic acid delivery systems. Low toxicity, high stability, and bioavailability are crucial for the therapeutic use of AuNPs and they are mainly determined by their interactions with proteins and lipids on their route to the target cells. In this work, we investigated the interaction of two pegylated gold nanoparticles, AuNP14a and AuNP14b, with human serum proteins albumin (HSA) and transferrin (Tf) as well as dimyristoyl-phosphatidylcholine (DMPC) liposomes, which can be a representative of biomembranes. We showed that AuNP14a/b interacted with HSA and Tf changing their electrical, thermodynamic, and structural properties as evidenced by dynamic light scattering, zeta potential, transmission electron microscopy, circular dichroism, fluorescence quenching, and isothermal titration calorimetry. These nanoparticles penetrated the DMPC membrane suggesting their ability to reach a target inside the cell. In most of the effects, AuNP14b was more effective than AuNP14a, which might result from its more positive charge. Further studies are needed to evaluate whether the interaction of AuNP14a/b with HSA and Tf is safe for the cell/organism and whether they may safely penetrate natural membranes.

Proteomic Analysis of Human Serum Proteins Adsorbed onto Collagen Barrier Membranes.

Collagen barrier membranes are frequently used in guided tissue and bone regeneration. The aim of this study was to analyze the signature of human serum proteins adsorbed onto collagen membranes using a novel protein extraction method combined with mass spectrometry. Native porcine-derived collagen membranes (Geistlich Bio-Gide®, Wolhusen, Switzerland) were exposed to pooled human serum in vitro and, after thorough washing, subjected to protein extraction either in conjunction with protein enrichment or via a conventional surfactant-based method. The extracted proteins were analyzed via liquid chromatography with tandem mass spectrometry. Bioinformatic analysis of global profiling, gene ontology, and functional enrichment of the identified proteins was performed. Overall, a total of 326 adsorbed serum proteins were identified. The enrichment and conventional methods yielded similar numbers of total (315 vs. 309), exclusive (17 vs. 11), and major bone-related proteins (18 vs. 14). Most of the adsorbed proteins (n = 298) were common to both extraction groups and included several growth factors, extracellular matrix (ECM) proteins, cell adhesion molecules, and angiogenesis mediators involved in bone regeneration. Functional analyses revealed significant enrichment of ECM, exosomes, immune response, and cell growth components. Key proteins [transforming growth factor-beta 1 (TGFβ1), insulin-like growth factor binding proteins (IGFBP-5, -6, -7)] were exclusively detected with the enrichment-based method. In summary, native collagen membranes exhibited a high protein adsorption capacity in vitro. While both extraction methods were effective, the enrichment-based method showed distinct advantages in detecting specific bone-related proteins. Therefore, the use of multiple extraction methods is advisable in studies investigating protein adsorption on biomaterials.

Expedient measurement of total protein in human serum and plasma via the biuret method using fiber optic probe for patient samples and certified reference materials.

The biuret method is currently recognized as a reference measurement procedure for serum/plasma total protein by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). However, as the reaction involved in this method is highly time-dependent, to ensure identical measurement conditions for calibrator and samples for high accuracy, a fast and simple measurement procedure is critical to ensure the precision and trueness of this method. We measured serum/plasma total protein using a Cary 60 spectrophotometer coupled with a fiber optic probe, which was faster and simpler than the conventional cuvette method. The biuret method utilizing alkaline solutions of copper sulfate and potassium sodium tartrate was added to the sample and calibrator (NIST SRM 927e) incubated for 1h before measurement. A panel of samples consisting of pooled human serum, single donor serum, and certified reference materials (CRMs) from three sources were measured for method validation. Sixteen native patient samples were measured using the newly developed biuret method and compared against clinical analyzers. Additionally, the results of three cycles of a local External Quality Assessment (EQA) Programme submitted by participating clinical laboratories were compared against the biuret method. Our biuret method using fiber optic probe demonstrated good precision with within-day relative standard deviation (RSD) of 0.04 to 0.23% and between-day RSD of 0.58%. The deviations between the obtained values and the certified values for all three CRMs ranged from -0.38 to 1.60%, indicating good method trueness. The routine methods using clinical analyzers were also found to agree well with the developed biuret method using fiber optic probe for EQA samples and native patient samples. The biuret method using a fiber optic probe represented a convenient and reliable way of measuring serum total protein. It also demonstrated excellent precision and trueness using CRMs and patient samples, which made the method a simpler candidate reference method for serum protein measurement.

Simulation-based assessment of zwitterionic pendant group variations on the hemocompatibility of polyethersulfone membranes

In the realm of hemodialysis, Polyethersulfone (PES) membranes dominate due to their exceptional stability and mechanical properties, capturing 93% of the market. Despite their widespread usage, the hydrophobic nature of PES introduces complications in hemodialysis, potentially leading to severe adverse reactions in patients with end-stage renal disease (ESRD) through protein fouling. Addressing this issue, our study focused on enhancing hemocompatibility by modifying PES surfaces with zwitterionic materials, known for their hydrophilicity and biological membrane compatibility. We investigated the functionalization of PES membranes utilizing various zwitterions in different ratios. Utilizing molecular docking, we examined the interactions of three zwitterionic ligands—carboxybetaine methacrylate (CBMA), sulfobetaine methacrylate (SBMA), and (2-(methacryloyloxy)ethyl) phosphorylcholine (MPC)—with human serum proteins. Our analysis revealed that a 1:1 ratio of phosphobetaine and sulfobetaine exhibits the lowest affinity energy towards serum proteins, denoting an optimal hemocompatibility without the limitations associated with increased zwitterion ratios. This pivotal finding offers a new pathway for developing more efficient and safer hemodialysis membranes, promising improved care for ESRD patients.

Inflammation-Derived and Clinical Indicator-Based Predictive Model for Ischemic Stroke Recovery.

Neuroinflammatory responses are closely associated with poststroke prognosis severity. This study aimed to develop a predictive model, combining inflammation-derived markers and clinical indicators, for distinguishing functional outcomes in patients with subacute ischemic stroke. Based on activities of daily living assessments, ischemic stroke participants were categorized into groups with little effective (LE) recovery and obvious effective (OE) recovery. Initial biocandidates were identified by overlapping differentially expressed proteins from proteomics of clinical serum samples (5 LE, 5 OE, and 6 healthy controls) and differentially expressed genes from an RNA sequence of the ischemic cortex in middle cerebral artery occlusion mice (n=3). Multidimensional validations were conducted in ischemia-reperfusion models and a clinical cohort (15 LE, 11 OE, and 18 healthy controls). Models of robust biocandidates combined with clinical indicators were developed with machine learning in the training data set and prediction in another test data set (15 LE and 11 OE). We identified 194 differentially expressed proteins (LE versus healthy controls) and 174 differentially expressed proteins (OE versus healthy controls) in human serum, and 5121 differentially expressed genes (day 3) and 5906 differentially expressed genes (day 7) in middle cerebral artery occlusion mice cortex. Inflammation-derived biomarkers TIMP1 (tissue inhibitor metalloproteinase-1) and galactosidase-binding protein LGLAS3 (galectin-3) exhibited robust increases under ischemic injury in mice and humans. TIMP1 and LGALS3 coupled with clinical indicators (hemoglobin, low-density lipoprotein cholesterol, and uric acid) were developed into a combined model for differentiating functional outcome with high accuracy (area under the curve, 0.8). The combined model is a valuable tool for evaluating prognostic outcomes, and the predictive factors can facilitate development of better treatment strategies.

Ultrafast and highly sensitive detection of SARS-CoV-2 spike protein by field-effect transistor graphene-based biosensors

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), etiological agent for the coronavirus disease 2019 (COVID-19), has resulted in over 775 million global infections. Early diagnosis remains pivotal for effective epidemiological surveillance despite the availability of vaccines. Antigen-based assays are advantageous for early COVID-19 detection due to their simplicity, cost-effectiveness, and suitability for point-of-care testing (PoCT). This study introduces a graphene field-effect transistor-based biosensor designed for high sensitivity and rapid response to the SARS-CoV-2 spike protein. By functionalizing graphene with monoclonal antibodies and applying short-duration gate voltage pulses, we achieve selective detection of the viral spike protein in human serum within 100 µs and at concentrations as low as 1 fg ml−1, equivalent to 8 antigen molecules per µl of blood. Furthermore, the biosensor estimates spike protein concentrations in serum from COVID-19 patients. Our platform demonstrates potential for next-generation PoCT antigen assays, promising fast and sensitive diagnostics for COVID-19 and other infectious diseases.

Roughness affects the response of human fibroblasts and macrophages to sandblasted abutments

BackgroundA strong seal of soft-tissue around dental implants is essential to block pathogens from entering the peri-implant interface and prevent infections. Therefore, the integration of soft-tissue poses a challenge in implant-prosthetic procedures, prompting a focus on the interface between peri-implant soft-tissues and the transmucosal component. The aim of this study was to analyse the effects of sandblasted roughness levels on in vitro soft-tissue healing around dental implant abutments. In parallel, proteomic techniques were applied to study the interaction of these surfaces with human serum proteins to evaluate their potential to promote soft-tissue regeneration.ResultsGrade-5 machined titanium discs (MC) underwent sandblasting with alumina particles of two sizes (4 and 8 μm), resulting in two different surface types: MC04 and MC08. Surface morphology and roughness were characterised employing scanning electron microscopy and optical profilometry. Cell adhesion and collagen synthesis, as well as immune responses, were assessed using human gingival fibroblasts (hGF) and macrophages (THP-1), respectively. The profiles of protein adsorption to the surfaces were characterised using proteomics; samples were incubated with human serum, and the adsorbed proteins analysed employing nLC–MS/MS. hGFs exposed to MC04 showed decreased cell area compared to MC, while no differences were found for MC08. hGF collagen synthesis increased after 7 days for MC08. THP-1 macrophages cultured on MC04 and MC08 showed a reduced TNF-α and increased IL-4 secretion. Thus, the sandblasted topography led a reduction in the immune/inflammatory response. One hundred seventy-six distinct proteins adsorbed on the surfaces were identified. Differentially adsorbed proteins were associated with immune response, blood coagulation, angiogenesis, fibrinolysis and tissue regeneration.ConclusionsIncreased roughness through MC08 treatment resulted in increased collagen synthesis in hGF and resulted in a reduction in the surface immune response in human macrophages. These results correlate with the changes in protein adsorption on the surfaces observed through proteomics.

The mechanism of interaction between tri-para-cresyl phosphate and human serum protein: A multispectroscopic and in-silico study

Organophosphate flame retardants (OPFRs) pose the significant risks to the environment and human health and have become a serious public health issue. Tricresyl phosphates (TCPs), a group of aryl OPFRs, exhibit neurotoxicity and endocrine disrupting toxicity. However, the binding mechanisms between TCPs and human serum albumin (HSA) remain unknown. In this study, through fluorescence and ultraviolet–visible (UV–vis) absorption spectroscopy, molecular docking and molecular dynamics (MD), tri-para-cresyl phosphate (TpCP) was selected to explore potential interactions between HSA and TCPs. The results of the fluorescence spectroscopy demonstrated that a decrease in the fluorescence intensity of HSA and a blue shift were observed with the increasing concentrations of TpCP. The binding constant (Ka) was 2.575 × 104 L/mol, 4.701 × 104 L/mol, 5.684 × 104 L/mol and 9.482 × 104 L/mol at 293 K, 298 K, 303 K, and 310 K, respectively. The fluorescence process between HSA and TpCP involved a mix of static and dynamic quenching mechanism. The gibbs free energy (ΔG0) of HSA-TpCP system was −24.452 kJ/mol, −25.907 kJ/mol, -27.363 kJ/mol, and - 29.401 kJ/mol at 293 K, 298 K, 303 K, and 310 K, respectively, suggesting that the HSA-TpCP reaction was spontaneous. The enthalpy change (ΔH0) and thermodynamic entropy change (ΔS0) of the HSA-TpCP system were 60.83 kJ/mol and 291.08 J/(mol·>k), respectively, indicating that hydrophobic force was the major driving force in the HSA-TpCP complex. Furthermore, multispectral analysis also revealed that TpCP could alter the microenvironment of tryptophan residue and the secondary structure of HSA and bind with the active site I of HSA. Molecular docking and MD simulations confirmed that TpCP could spontaneously form a stable complex with HSA, which was consistent with the fluorescence experimental results. This study provides novel insights into the mechanisms of underlying the transportation and distribution of OPFRs in humans.

Necroptosis plays a role in TL1A-induced airway inflammation and barrier damage in asthma

BackgroundAirway epithelial cell (AEC) necroptosis contributes to airway allergic inflammation and asthma exacerbation. Targeting the tumor necrosis factor-like ligand 1 A (TL1A)/death receptor 3 (DR3) axis has a therapeutic effect on asthmatic airway inflammation. The role of TL1A in mediating necroptosis of AECs challenged with ovalbumin (OVA) and its contribution to airway inflammation remains unclear.MethodsWe evaluated the expression of the receptor-interacting serine/threonine-protein kinase 3(RIPK3) and the mixed lineage kinase domain-like protein (MLKL) in human serum and lung, and histologically verified the level of MLKL phosphorylation in lung tissue from asthmatics and OVA-induced mice. Next, using MLKL knockout mice and the RIPK3 inhibitor GSK872, we investigated the effects of TL1A on airway inflammation and airway barrier function through the activation of necroptosis in experimental asthma.ResultsHigh expression of necroptosis marker proteins was observed in the serum of asthmatics, and necroptosis was activated in the airway epithelium of both asthmatics and OVA-induced mice. Blocking necroptosis through MLKL knockout or RIPK3 inhibition effectively attenuated parabronchial inflammation, mucus hypersecretion, and airway collagen fiber accumulation, while also suppressing type 2 inflammatory factors secretion. In addition, TL1A/ DR3 was shown to act as a death trigger for necroptosis in the absence of caspases by silencing or overexpressing TL1A in HBE cells. Furthermore, the recombinant TL1A protein was found to induce necroptosis in vivo, and knockout of MLKL partially reversed the pathological changes induced by TL1A. The necroptosis induced by TL1A disrupted the airway barrier function by decreasing the expression of tight junction proteins zonula occludens-1 (ZO-1) and occludin, possibly through the activation of the NF-κB signaling pathway.ConclusionsTL1A-induced airway epithelial necroptosis plays a significant role in promoting airway inflammation and barrier dysfunction in asthma. Inhibition of the TL1A-induced necroptosis pathway could be a promising therapeutic strategy.

Induced Chirality in Sulfasalazine by Complexation With Albumins: Theoretical and Experimental Study.

Through molecular recognition, drugs can interact and complex with macromolecules circulating in the body. The serum albumin transport protein, found in several mammals, has several interaction sites where these molecules can be located. The drug sulfasalazine (SSZ) is known in the literature to complex at drug site 1 (DS1) in human serum (HSA) and bovine serum (BSA) proteins. This complexation can be studied using various spectroscopic techniques. With the techniques used in this work, absorption in the ultraviolet and visible regions (UV-Vis) and electronic circular dichroism (ECD), a significant difference was observed in the results involving HSA and BSA. The application of theoretical methodologies, such as TD-DFT and molecular docking, suggests that the conformation that SSZ assumes in DS1 of the two proteins is different, which exposes it to different amino acid residues and different hydrophobicities. This difference in conformation may be related to the location of DS1 where the drug interacts or to the possibility of SSZ moving in the BSA site, due to its larger size, and moving less freely in HSA.

Tailoring Fibroblast-Activation Protein Targeting for Theranostics: A Comparative Preclinical Evaluation of the 68Ga- and 177Lu-Labeled Monomeric and Dimeric Fibroblast-Activation Protein Inhibitors DOTA.SA.FAPi and DOTAGA.(SA.FAPi)2.

FAP radiopharmaceuticals show promise for cancer diagnosis; however, their limited tumor residency hinders treatment. This study compared two FAPi derivatives, DOTA.SA.FAPi and DOTAGA.(SA.FAPi)2, labeled with gallium-68 and lutetium-177, aiming to determine an optimum combination for creating theranostic pairs. The radiotracers were studied for lipophilicity, binding to human serum proteins, and binding to human cancer-associated fibroblasts (CAFs) in vitro, including saturation and internalization/externalization studies. PET/SPECT/CT and biodistribution studies were conducted in PC3 and U87MG xenografts for [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DOTAGA.(SA.FAPi)2. [177Lu]Lu-DOTA.SA.FAPi and [177Lu]Lu-DOTAGA.(SA.FAPi)2, were evaluated in PC3 xenografts. Biodistribution studies of [68Ga]Ga-DOTA.SA.FAPi were performed in healthy male and female mice. All radiotracers exhibited strong binding to FAP. Their internalization rate was fast while only [177Lu]Lu-DOTAGA.(SA.FAPi)2 was retained longer in CAFs. [68Ga]Ga-DOTAGA.(SA.FAPi)2 and [177Lu]Lu-DOTAGA.(SA.FAPi)2 displayed elevated lipophilicity and affinity for human serum proteins compared to [68Ga]Ga-DOTA.SA.FAPi and [177Lu]Lu-DOTA.SA.FAPi. In vivo studies revealed slower washout of [68Ga]Ga-DOTAGA.(SA.FAPi)2 within 3 h compared to [68Ga]Ga-DOTA.SA.FAPi. The tumor-to-tissue ratios of [68Ga]Ga-DOTAGA.(SA.FAPi)2 versus [68Ga]Ga-DOTA.SA.FAPi did not exhibit any significant differences. [177Lu]Lu-DOTAGA.(SA.FAPi)2 maintained a significant tumor uptake even after 96 h p.i. compared to [177Lu]Lu-DOTA.SA.FAPi. Dimeric compounds hold promise for therapy, while monomers are better suited for diagnostics. Finding the right combination is essential for effective disease management.

Soluble form of the MDA5 protein in human sera

Viral double-stranded RNA (dsRNA) is sensed by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including melanoma differentiation-associated gene 5 (MDA5). MDA5 recognizes the genome of dsRNA viruses and replication intermediates of single-stranded RNA viruses. MDA5 also plays an important role in the development of autoimmune diseases, such as Aicardi-Goutieres syndrome and type I diabetes. Patients with dermatomyositis with serum MDA5 autoantibodies (anti-CADM-140) are known to have a high risk of developing rapidly progressive interstitial lung disease and poor prognosis. However, there have been no reports on the soluble form of MDA5 in human serum. In the present study, we generated in-house monoclonal antibodies (mAbs) against human MDA5. We then performed immunohistochemical analysis and sensitive sandwich immunoassays to detect the MDA5 protein using two different mAbs (clones H27 and H46). As per the immunohistochemical analysis, the MDA5 protein was moderately expressed in the alveolar epithelia of normal lungs and was strongly expressed in the cytoplasm of lymphoid cells in the tonsils and acinar cells of the pancreas. Interestingly, soluble MDA5 protein was detectable in the serum, but not in the urine, of healthy donors. Soluble MDA5 protein was also detectable in the serum of patients with dermatomyositis. Immunoblot analysis showed that human cells expressed a 120 kDa MDA5 protein, while the 60 kDa MDA5 protein increased in the supernatant of peripheral mononuclear cells within 15 min after MDA5 agonist/double-strand RNA stimulation. Hydrogen deuterium exchange mass spectrometry revealed that an anti-MDA5 mAb (clone H46) bound to the epitope (415QILENSLLNL424) derived from the helicase domain of MDA5. These results indicate that a soluble MDA5 protein containing the helicase domain of MDA5 could be rapidly released from the cytoplasm of tissues after RNA stimulation.

The photophysical, photobiological, and DNA/HSA-binding properties of corroles containing carbazole and phenothiazine moieties

This study characterized four corrole derivatives, namely Cbz-Cor, MetCbz-Cor, PTz-Cor, and PTzEt-Cor, examining their photophysical, electrochemical, photobiological, and biomolecule-binding properties. Experimental photophysical data of absorption and emission elements correlated with a theoretical analysis obtained through time-dependent density functional theory (TD-DFT). As for the photophysical properties, we observed lower fluorescence quantum yields and discernible differences between the excited and ground states, as indicated by Stokes shift values. Natural Transition Orbit (NTO) plots presented high occupied molecular orbital - low unoccupied molecular orbital (HOMO-LUMO) densities around the tetrapyrrolic macrocycle in all examples. Our findings demonstrate that corroles maintain stability in solution and offer photostability (<20 %), predominantly in DMSO(5 %)/Tris-HCl (pH 7.4) buffer solution. Furthermore, the singlet oxygen (1O2) quantum yield and log POW values underscore their potential application in photoinactivation approaches, as these corroles serve as effective ROS generators with more lipophilic features. We also evaluated their biomolecular binding capacity towards salmon sperm DNA and human serum albumin using spectroscopic techniques and molecular docking analysis for sustenance. Concerning biomolecule interaction profiles, the corrole derivatives showed a propensity for interacting in the minor grooves of the double helix DNA due to secondary forces, which were more pronounced in site III of the human serum protein.

Precursor carboxy-silica for functionalization with interactive ligands. IV. Carbodiimide assisted preparation of immobilized antibody stationary phases for high performance immuno-affinity chromatography of human serum

In this Part IV of the article series dealing with the functionalization of the precursor carboxy silica with various chromatographic ligands, immuno affinity (IA) columns were prepared with immobilized anti-apolipoprotein B (AAP B) and anti-haptoglobin (AHP) antibodies for use in immuno affinity chromatography (IAC) in the aim of selectivily capturing their corresponding antigens from healthy and cancer human sera. Diseased human serum with adenocarcinoma cancer was selected as a typical diseased biological fluid. Besides preferentially capturing their corresponding antigens, the AAP B column captured from disease-free and cancer sera, 34 proteins and 33 proteins, respectively, while the AHP column enriched 38 and 47 proteins, respectively. This nonspecific binding can be attributed to the many proteins human serum have, which could mediate protein–protein interactions thus leading to the so-called “sponge effect”. This kind of behavior can be exploited positively in the determination of differentially expressed proteins (DEPs) for diseased serum with respect to healthy serum and in turn allow the identification of an array of potential biomarkers for cancer. In fact, For AHP column, 13 upregulated and 22 downregulated proteins were identified whereas for AAP B column the numbers were 23 and 10, respectively. The DEPs identified with both columns match those reported in the literature for other types of cancers. The different expression of proteins in each IAC column can be related to the variability of protein–protein interactions. In addition, an array of a few biomarkers is more indicative of a certain disease than a single biomarker.

Photosensitizer-thioglycosides enhance photodynamic therapy by augmenting cellular uptake

Photodynamic therapy (PDT) uses photosensitizing agents along with light to ablate tissue, including cancers. Such light-driven localized delivery of free-radical oxygen to kill target tissue depends on photosensitizer cell penetration efficacy. While the attachment of monosaccharides and disaccharides to photosensitizers has been shown to potentially provide improved photosensitizer delivery, the range of glycan entities tested thus far is limited. We sought to expand such knowledge by coupling N-acetylglucosamine (GlcNAc) to pyropheophorbides as thioglycosides, and then testing photosensitizer efficacy. To this end, GlcNAc was conjugated to both pyropheophorbide-a and methyl pyropheophorbide-a. Among the entities tested, the conjugation of N-acetylglucosamine to methyl pyropheophorbide-a (‘PSe’) as thioglycoside enhanced cell uptake both in the presence and absence of human serum proteins, relative to other compounds tested. The enhanced PSe penetrance into cells resulted in higher cell death upon illumination with 665 nm light. While acting as a potent photosensitizer, PSe did not affect cellular carbohydrate profiles. Overall, the study presents a new pyropheophorbide glycoconjugate with strong in vitro PDT efficacy.

Creating Anti‐Biofouling Surfaces by Degradable Main‐chain Polyphosphoester Polymer Brushes

AbstractThe implementation of polymer brush coatings has proven crucial in biomedical and biotechnological applications for controlling biological interactions. Despite the extensive research and use of synthetic polymer coatings, concerns regarding their long‐term non‐biodegradability have arisen. This issue is significant as non‐degradable polymers can lead to complications such as inflammatory responses and bioaccumulation, highlighting the need for polymers that degrade in a controlled manner. To address this, polyphosphonate brushes are synthesized as anti‐biofouling coatings on silicon surfaces via controlled surface‐initiated organocatalytic ring‐opening polymerization (SI‐ROP) of cyclic phospholanes. 2‐Ethyl‐2‐oxo‐1,3,2‐dioxaphospholane and 2‐hexyl‐2‐oxo‐1,3,2‐dioxaphospholane are chosen as the monomers to prepare hydrophilic and hydrophobic brushes with thicknesses up to 55 nm, with a proven degradability in aqueous media. In addition, protein adsorption, bacterial adhesion, and biofilm formation are investigated as a function of the polyphosphonate side chain. Compared to non‐coated surfaces and polyester brushes, hydrophilic poly(2‐ethyl‐2‐oxo‐1,3,2‐dioxaphospholane) brushes have an enhanced resistance toward fouling of albumin, fibrinogen, and diluted human serum proteins, as well as toward Escherichia coli and Staphylococcus aureus bacteria. The stability and anti‐biofouling performance of hydrophilic polyphosphonate brushes position them as an attractive option as degradable materials for anti‐biofouling matrices on medical devices and/or lubricious coatings for artificial implant technologies.

Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation

BackgroundThe use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties.ResultsIn this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv.ConclusionsThe antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.

Oliver Smithies. 23 June 1925—10 January 2017

Oliver Smithies was born in Copley, near Halifax in Yorkshire, UK. He received his doctorate from Oxford in 1951, then began working at the Connaught Laboratories in Toronto, where he developed starch gel electrophoresis. This technology allowed identification of genetic variants in human serum proteins and revolutionized protein analysis. After moving to the University of Wisconsin, he studied the genetics of antibody variability, then turned to nucleic acid methods, developing safe cloning vectors, driving production of software for genetic analysis, sequencing several human genes and finally creating genetically engineered animals, for which he later received the Nobel Prize. He then moved to the University of North Carolina, where he developed methods for altering gene dosage in mice, which he used to develop ways to attack complex physiological questions, including blood pressure regulation. Finally, he formulated a new hypothesis to explain kidney glomerular filtration, then devised methods that confirmed the hypothesis. Oliver collaborated with his wife, Nobuyo Maeda, during a long and happy marriage. While maintaining separate laboratories, they stimulated each other's scientific understanding and frequently published together. During a 70-year life in science, he mentored many students, postdoctoral fellows and collaborators, nearly all remaining his friends for life.