Search

Programmed cell death‐ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single‐chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof‐of‐concept data for the effect of scFv using PDL1‐expressing cells was lacking. In this study, we conducted two kinds of cell‐based immunoassays, western blotting and enzyme‐linked immunosorbent assay, using anti‐PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell‐based immunoassays to detect PDL1. [ABSTRACT FROM AUTHOR]

Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis. [ABSTRACT FROM AUTHOR]

Meibomian gland dysfunction (MGD), a chronic abnormality of meibomian glands, causes various dry eye symptoms. Principal treatments for MGD are warm compression and mechanical squeezing of the eyelids. In this study, the immediate impact of this treatment on tear film lipid layer thickness (TFLLT) and the meibomian gland (MG) structure in MGD and normal groups was investigated to establish its efficacy and potential side effects. Nineteen MGD patients and seven normal subjects were enrolled. TFLLT and blinking parameters were evaluated before and after warm compression. Morphological changes of MG structures after mechanical squeezing were analyzed using Image J and Fiji. Differential analysis of the MGD and the normal groups of TFLLT changes after warm compression showed a significant increase in the normal group. In normal eyes, the average, maximum, and minimum TFLLT were significantly increased, and in the MGD group, only the minimum TFLLT was improved. Blinking parameters showed no significant change in either group. Morphometric analysis showed no damages of the MG after MG squeezing. A significant increase in MG length was observed in normal eyes. Warm compression immediately increased TFLLT more significantly in the normal group than in the MGD patients. Mechanical expression is a safe therapeutic option without remarkable structural MG damages. [ABSTRACT FROM AUTHOR]

Serum is one of the most commonly used samples in many studies to identify protein biomarkers to diagnose cancer. Although conventional enzyme-linked immunosorbent assay (ELISA) or liquid chromatography-mass spectrometry (LC-MS)-based methods have been applied as clinical tools for diagnosing cancer, there have been troublesome problems, such as inferior multiplexing capabilities, high development costs and long turnaround times, which are inappropriate for high-throughput analytical platforms. Here, we developed a simple and robust cancer diagnostic method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based total serum protein fingerprinting. First, serum samples were simply diluted with distilled water and subsequently spotted onto a MALDI plate without prior chromatographic purification or separation. The sample preparation method was enough to collect reproducible total serum protein fingerprints and would be highly advantageous for high-throughput assay. Each of the integrated main spectrum profiles (MSPs), which are representative of liver cancer patients (n = 40) or healthy controls (n = 80), was automatically generated by the MALDI Biotyper 3 software. The reliability of the integrated MSPs was successfully evaluated in comparison with a blind test set (n = 31), which consisted of 13 liver cancer patients and 18 healthy controls. Additionally, our partial least squares discriminant analysis (PLS-DA) demonstrated a statistically significant difference in MALDI-TOF MS-based total serum protein fingerprints between liver cancer patients and healthy controls. Taken together, this work suggests that this method may be an effective high-throughput platform technology for various cancer diagnoses and disease evaluations. [ABSTRACT FROM AUTHOR]

Background/aims: This study aimed to establish a wide-field optical coherence tomography (OCT) deviation map obtained from swept-source OCT (SS-OCT) scans. Moreover, it also aimed to compare the diagnostic ability of this wide-field deviation map with that of the peripapillary and macular deviation maps currently being used for the detection of early glaucoma (EG)., Methods: Four hundred eyes, including 200 healthy eyes and 200 eyes with EG were enrolled in this retrospective observational study. Patients underwent a comprehensive ocular examination, including wide-field SS-OCT (DRI-OCT Triton; Topcon, Tokyo, Japan). The individual wide-field scan was converted into a uniform template using the fovea and optic disc centres as fixed landmarks. Subsequently, the wide-field deviation map was obtained via the comparison between individual wide-field data and a normative wide-field database that had been created by combining images of healthy eyes into a uniform template in a previous study. The ability of the new wide-field deviation map to distinguish between EG and healthy eyes was assessed by comparing it with conventional deviation maps based on the area under the receiver operating characteristic curve (AUC)., Results: The wide-field deviation map obtained using the normative wide-field database showed the highest diagnostic ability for the diagnosis of EG (AUC=0.980 and 961 for colour-coded pixels presenting <5% and <1%, respectively) among various deviation maps. Its AUC was significantly superior to that of most conventional deviation maps (p<0.05). The wide-field deviation map demonstrated early structural glaucomatous damage well over a wider area., Conclusion: The wide-field SS-OCT deviation map exhibited good performance for distinguishing between eyes with EG and healthy eyes. The visualisation of the wider damaged area on the wide-field deviation map could be useful for the diagnosis of EG in clinical settings., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values ( R 2 = 0.6-0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C ( R = 0.5) and glycated ApoA-I with higher fasting glucose ( R = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single N -acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference ( R ∼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes.

Purpose: One purpose of this study was to collect wide-field swept-source optical coherence tomography (SS-OCT) data from healthy eyes and build a wide-filed normative database. Another purpose was to compare the glaucoma diagnostic ability of new parameters based on this normative database to the parameters that are currently in use, such as the peripapillary retinal nerve fiber layer (RNFL), macular ganglion cell-inner plexiform layer, and ganglion cell complex (GCC) thickness., Methods: This study had 220 healthy eyes and 292 eyes with early-stage glaucoma (EG) and moderate-stage glaucoma (MG) enrolled. Using the wide-field SS-OCT images (12 × 9 mm) of healthy eyes, a wide-field normative database was constructed by transforming and combining the individual images into a uniform template using the fovea and optic disc centers as fixed landmarks. Adjustment for the disc size was conducted. With this normative database, new parameters based on the ratio of the fovea-disc distance (FDD) consisting of the fovea-disc relationship were evaluated. The glaucoma diagnostic ability was assessed based on the area under the receiver operating characteristic curve (AUC)., Results: Among the new peripapillary parameters, the RNFL of the circumference of the circle with diameter 0.8 FDD showed the highest AUC value for EG and MG, but the value was not significantly superior to that of the initial RNFL (AUC = 0.940 vs. 0.937, P = 0.631). Among the macular parameters, the GCC of the area of the circle of 1.5 FDD showed the highest AUC value for EG and MG, and the value was significantly superior to that of initial GCC (AUC = 0.929 vs. 0.919, P = 0.033). However, there was no significant difference between the initial and adjusted GCC thickness in patients included in the EG or MG groups separately., Conclusions: A wide-field normative database was built to consider the relationship between the fovea and the optic disc. Considering this aspect, we found that the GCC analysis using a broader area presented a significantly greater glaucoma diagnostic performance for EG and MG in the macula than the initial parameter for the GCC., Translational Relevance: Based on this wide-field normative database, the clinical use of a wide-field deviation map may help diagnose the patients with EG and MG in the future.

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.

Permethylation is one of the most practical modifications for mass spectrometric analysis of carbohydrates. In this study, we showed a facile platform for the on-line permethylation and analysis of acidic N-glycans using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry to elucidate the N-glycan structures derived from glycoprotein samples. Two capillaries, individually packed with NaOH and C18 powder, were coupled to electrospray ion trap mass spectrometer. Prior to mass spectrometry, oligosaccharides were permethylated in NaOH-packed capillary by methyl iodide, and the permethylated glycans were consequently extracted from residual reagents, such as NaOH and methyl iodide, in C18-capillary, instead of using conventional chloroform extraction. After permethylation and purification, the permethylated glycans were released from C18 capillary by acetonitrile, and sprayed into mass spectrometer. Using this on-line strategy, a picomolar range of sialylated N-glycans from bovine fetuin were analyzed by tandem mass spectrometry.

Decellularization of tissues or organs can provide an efficient strategy for preparing functional scaffolds for tissue engineering. Microstructures of native extracellular matrices and their biochemical compositions can be retained in the decellularized matrices, providing tissue-specific microenvironments for efficient tissue regeneration. Here, we report the versatility of liver extracellular matrix (LEM) that can be used for two-dimensional (2D) coating and three-dimensional (3D) hydrogel platforms for culture and transplantation of primary hepatocytes. Collagen type I (Col I) has typically been used for hepatocyte culture and transplantation. In this study, LEM was compared with Col I in terms of biophysical and mechanical characteristics and biological performance for enhancing cell viability, differentiation, and hepatic functions. Surface properties of LEM coating and mechanical properties and gelation kinetics of LEM hydrogel could be manipulated by adjusting the LEM concentration. In addition, LEM hydrogel exhibited improved elastic properties, rapid gelation, and volume maintenance compared to Col I hydrogel. LEM coating significantly improved hepatocyte functions such as albumin secretion and urea synthesis. More interestingly, LEM coating upregulated hepatic gene expression of human adipose-derived stem cells, indicating enhanced hepatic differentiation of these stem cells. The viability and hepatic functions of primary hepatocytes were also significantly improved in LEM hydrogel compared to Col I hydrogel both in vitro and in vivo. Albumin and hepatocyte transcription factor expression was upregulated in hepatocytes transplanted in LEM hydrogels. In conclusion, LEM can provide functional biomaterial platforms for diverse applications in liver tissue engineering by promoting survival and maturation of hepatocytes and hepatic commitment of stem cells. This study demonstrates the feasibility of decellularized matrix for both 2D coating and 3D hydrogel in liver tissue engineering.