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Introduction: Odontogenic keratocyst (OKC) and unicystic ameloblastoma (UA) are lesions of odontogenic origin. Both lesions are morphologically cysts. However, they are classified as developmental cysts and epithelial odontogenic tumours, respectively. Cyclin D1 (CCD1) dysregulation is associated with oncogenic activity and malignancies, while tumour protein p63 (p63) alterations are associated with tumourigenesis., Aim: To evaluate and compare the protein expression of CCD1 and p63 in sporadic OKC (OKC-sp), syndromic OKC (OKC-sy), and UA., Material and Methods: 45 cases from the Anatomical Pathology Department, Faculty of Dentistry, University of Chile were analysed and divided into groups: OKC-sp (n=15), OKC-sy (n=15) and UA (n=15), the latter categorised into intraluminal and/or luminal (n=7) and mural (n=8). Immunohistochemical staining for CCD1 and p63 proteins was performed from paraffin-embedded sections. Statistical analysis included the Shapiro-Wilk test, one-way ANOVA with Tukey's multiple comparisons, and Spearman's correlation coefficient (p<0.05)., Results: There was an involvement mainly in women in the mandibular area, and a high frequency of jaw expansion, especially in the mural UA. P63 protein expression was higher than CCD1 in all cystic lesions, particularly in mural UA (p<0.001). No correlation was found between CCD1 and p63 expression., Conclusion: P63 may serve as a valuable marker for evaluating cell proliferative activity in odontogenic cystic lesions, providing insights into the aggressive behaviour of mural UA., (Copyright © 2024 Sociedad Española de Anatomía Patológica. Publicado por Elsevier España, S.L.U. All rights reserved.)

Emerin and lamins not only influence nuclear morphology but are also involved in differentiation. We herein examined 82 resected cases of invasive lung adenocarcinoma using computer-assisted image analysis of nuclear morphology on Feulgen-stained and immunohistochemical sections of lamin A, B1, B2, and emerin (four proteins) to calculate the rank sum of the cell positivity rates for these four proteins. The rank sum of four proteins showed weak negative correlations with the nuclear area and perimeter and a weak positive correlation with the nuclear shape factor. Interestingly, the top three cases with the highest rank sum were papillary adenocarcinoma, and the bottom three cases were acinar adenocarcinomas containing cribriform patterns. We compared the rank sum for grading (differentiation: G1, G2, and G3) and predominant histological subtypes and found that the rank sum of G3 was lower than that of G1 and G2. Furthermore, the rank sum was lower for acinar adenocarcinoma with >20 % cribriform pattern (acinar+cribri) and solid adenocarcinoma than for lepidic and papillary adenocarcinoma. Individual examination of the four proteins revealed that emerin expression was lower in G3 than in G1, and lamin B2 expression was lower in G3 than in G1 and G2. Compared with lepidic adenocarcinoma, acinar+cribri showed significantly lower expression of all four proteins among histological subtypes. These data indicated that the expression of lamin A, B1, B2, and emerin was markedly decreased in poorly differentiated adenocarcinoma (i.e., G3), especially in acinar+cribri. Our data suggested that changes in these four proteins can not only affect nuclear morphology but also histological structure in lung adenocarcinoma., Competing Interests: Declaration of Competing Interest On behalf of all authors of our manuscript, I would like to declare that none of us has a conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Exosomes are of great significance in clinical diagnosis, due to their high homology with parental generation, which can reflect the pathophysiological status. However, the quantitative and classification detection of exosomes is still faced with the challenges of low sensitivity and complex operation. In this study, we develop an electrical and label-free method to directly detect exosomes with high sensitivity based on a Silicon nanowire field effect transistor biosensor (Si-NW Bio-FET). First, the impact of Debye length on Si-NW Bio-FET detection was investigated through simulation. The simulation results demonstrated that as the Debye length increased, the electrical response to Si-NW produced by charged particle at a certain distance from the surface of Si-NW was greater. A Si-NW Bio-FET modified with specific antibody CD81 on the nanowire was fabricated then used for detection of cell line-derived exosomes, which achieved a low limit of detection (LOD) of 1078 particles/mL in 0.01 × PBS. Furthermore, the Si-NW Bio-FETs modified with specific antibody CD9, CD81 and CD63 respectively, were employed to distinguish exosomes derived from human promyelocytic leukemia (HL-60) cell line in three different states (control group, lipopolysaccharide (LPS) inflammation group, and LPS + Romidepsin (FK228) drug treatment group), which was consistent with nano-flow cytometry. This study provides a highly sensitive method of directly quantifying exosomes without labeling, indicating its potential as a tool for disease surveillance and medication instruction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fibroblast activation protein-α (FAP) plays a crucial role in various physiological and pathological processes, making it a key target for cancer diagnostics and therapeutics. However, in vivo detection of FAP activity with fluorogenic probes remains challenging due to the rapid diffusion and clearance of fluorescent products from the target. Herein, we developed a self-immobilizing near-infrared (NIR) fluorogenic probe, Hcy-CF 2 H-PG, by introducing a difluoromethyl group to FAP substrate-caged NIR fluorophore. Upon selective activation by FAP, the fluorescence of Hcy-CF 2 H-PG was triggered, followed by the covalent labelling of FAP. Hcy-CF 2 H-PG demonstrated significantly improved sensitivity, selectivity, and long-lasting labelling capacity for FAP both in vitro and in vivo, compared to that of non-immobilized probes. This represents a noteworthy advancement in FAP detection and cancer diagnostics within complex physiological systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Cell-surface proteins, which are closely associated with various physiological and pathological processes, have drawn much attention in drug discovery and disease diagnosis. Thus, wash-free imaging of the target cell-surface protein under its native environment is critical and helpful for early detection and prognostic evaluation of diseases., Results: To minimize the interference from autofluorescence and fit the penetration depth towards tissue samples, we developed a fluorogenic antibody-based probe, Ab-Cy5.5, which will liberate > 5-fold turn-on near-infrared (NIR) emission in the presence of its target antigen within 10 min., Significance: By taking advantage of the fluorescence-quenched dimeric H-aggregation of Cy5.5, Ab-Cy5.5 with Cy5.5 attached at the N-terminus showed negligible background signal, allowing direct imaging of the target cell-surface protein in both living cells and tissue samples without washing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study demonstrates, for the first time, the proof-of-concept of a novel immunosensor, a touchpad-based immunochromatographic strip, that non-invasively extracts and detects skin surface proteins. The strip was composed of a nitrocellulose membrane at the center, where a spot of anti-human IgG capture antibody was physically adsorbed. The capture antibody spot was covered with a glass fiber membrane impregnated with phosphate-buffered saline (PBS) to extract skin surface proteins, avoiding direct contact of the human skin with the capture antibodies. Skin surface IgG was detected in two steps: (1) touching the capture antibody via a glass fiber membrane containing PBS, and (2) dipping the strip into the Au-nanoparticle-labeled secondary antibody to visualize the existence of the captured skin surface IgG on the strip. We qualitatively demonstrated that using a very small amount of PBS while maintaining contact with the skin, skin surface proteins can be concentrated and detected, even with a relatively low-sensitivity immunochromatographic chip. This sensor is expected to be a potential biosensor for the non-invasive diagnosis of the integrity of human skin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m / z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against Pf MATE, obtained from multiplexed ligand libraries.

Currently, no consensus exists regarding criteria required to designate a protein within a proteomic data set as a cell surface protein. Most published proteomic studies rely on varied ontology annotations or computational predictions instead of experimental evidence when attributing protein localization. Consequently, standardized approaches for analyzing and reporting cell surface proteome data sets would increase confidence in localization claims and promote data use by other researchers. Recently, we developed Veneer, a web-based bioinformatic tool that analyzes results from cell surface N -glycocapture workflows─the most popular cell surface proteomics method used to date that generates experimental evidence of subcellular location. Veneer assigns protein localization based on defined experimental and bioinformatic evidence. In this study, we updated the criteria and process for assigning protein localization and added new functionality to Veneer. Results of Veneer analysis of 587 cell surface N -glycocapture data sets from 32 published studies demonstrate the importance of applying defined criteria when analyzing cell surface proteomics data sets and exemplify how Veneer can be used to assess experimental quality and facilitate data extraction for informing future biological studies and annotating public repositories.

Oligomerization is an important feature of proteins, which gives a defined quaternary structure to complete the biological functions. Although frequently observed in membrane proteins, characterizing the oligomerization state remains complicated and time-consuming. In this study, 0.05% (w/v) sarkosyl-polyacrylamide gel electrophoresis (05SAR-PAGE) was used to identify the oligomer states of the membrane proteins CpxA, EnvZ, and Ma-Mscl with high sensitivity. Furthermore, two-dimensional electrophoresis (05SAR/sodium dodecyl sulfate-PAGE) combined with western blotting and liquid chromatography-tandem mass spectrometry was successfully applied to study the complex of CpxA/OmpA in cell lysate. The results indicated that 05SAR-PAGE is an efficient, economical, and practical gel method that can be widely used for the identification of membrane protein oligomerization and the analysis of weak protein interactions., (© 2024 Wiley‐VCH GmbH.)

Background and Objectives: Colorectal cancer (CRC) is the most frequently diagnosed malignant disease of the gastrointestinal system, and new diagnostic and prognostic markers are needed to elucidate the complete tumor profile. Materials and Methods : We used CRC tumor tissues (Dukes' A-D) and adjacent noncancerous tissues of 43 patients. Immunohistochemistry was used to examine the expression of phosphodiesterase 4B (PDE4B), phosphodiesterase 4D (PDE4D), and secreted frizzled related protein 5 (SFRP5) markers. We also analyzed the expression levels of PDE4B , PDE4D , and SFRP5 in CRC tissues compared to control tissues using RNA-sequencing data from the UCSC Xena browser. Results : In CRC stages, the distribution of PDE4B-positive cells varied, with differing percentages between epithelium and lamina propria. Statistically significant differences were found in the number of PDE4B-positive epithelial cells between healthy controls and all CRC stages, as well as between different CRC stages. Similarly, significant differences were observed in the number of PDE4B-positive cells in the lamina propria between healthy controls and all CRC stages, as well as between different CRC stages. CRC stage Dukes' C exhibited a significantly higher number of PDE4B-positive cells in the lamina propria compared to CRC stage Dukes' B. Significant differences were noted in the number of PDE4D-positive epithelial cells between healthy controls and CRC stages Dukes' A, B, and D, as well as between CRC stage Dukes' C and stages A, B, and D. CRC stage Dukes' A had significantly more PDE4D-positive cells in the lamina propria compared to stage D. Significant differences were also observed in the number of SFRP5-positive cells in the lamina propria between healthy controls and all CRC stages, as well as between CRC stages Dukes' A and D. While the expression of PDE4D varied across CRC stages, the expression of SFRP5 remained consistently strong in both epithelium and lamina propria, with significant differences noted mainly in the lamina propria. The expression levels of PDE4B , PDE4D , and SFRP5 reveal significant differences in the expression of these genes between CRC patients and healthy controls, with notable implications for patient prognosis. Namely, our results demonstrate that PDE4B, PDE4D, and SFRP5 are significantly under-expressed in CRC tissues compared to control tissues. The Kaplan-Meier survival analysis and the log-rank (Mantel-Cox) test revealed distinct prognostic implications where patients with lower expression levels of SFRP5 exhibited significantly longer overall survival. The data align with our immunohistochemical results and might suggest a potential tumor-suppressive role for these genes in CRC. Conclusions : Considering significantly lower gene expression, aligned with our immunohistochemical data in tumor tissue in comparison to the control tissue, as well as the significantly poorer survival rate in the case of its higher expression, we can hypothesize that SFRP5 is the most promising biomarker for CRC out of the observed proteins. These findings suggest alterations in PDE4B, PDE4D, and SFRP5 expression during CRC progression, as well as between different stages of CRC, with potential implications for understanding the molecular mechanisms involved in CRC development and progression.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has evolved as an essential technique in structural proteomics. The use of ion mobility separation (IMS) coupled to HDX-MS has increased the applicability of the technique to more complex systems and has been shown to improve data quality and robustness. The first step when running any HDX-MS workflow is to confirm the sequence and retention time of the peptides resulting from the proteolytic digestion of the nondeuterated protein. Here, we optimized the collision energy ramp of HDMS E experiments for membrane proteins using a Waters SELECT SERIES cIMS-QTOF system following an HDX workflow using Phosphorylase B, XylE transporter, and Smoothened receptor (SMO) as model systems. Although collision energy (CE) ramp 10-50 eV gave the highest amount of positive identified peptides when using Phosphorylase B, XylE, and SMO, results suggest optimal CE ramps are protein specific, and different ramps can produce a unique set of peptides. We recommend cIMS users use different CE ramps in their HDMS E experiments and pool the results to ensure maximum peptide identifications. The results show how selecting an appropriate CE ramp can change the sequence coverage of proteins ranging from 4 to 94%.

Purpose: Fibroblast activation protein (FAP) is a serine integral membrane protease, the expression of which has been confirmed in various cancer types. Solitary fibrous tumors of the pleura (SFTP) are rare mesenchymal fibroblastic neoplasms. We present a case of 18 F-labeled FAP inhibitor ([ 18 F]FAPI-74) PET imaging and its correlation with histological FAP expression and review an SFTP series at our institution in relation to the extent of FAP expression., Methods: This retrospective study included 13 patients who underwent surgery between March 2011 and December 2022 at our institute. One of the patients also underwent [ 18 F]FAPI-74 PET imaging. We semi-quantitatively evaluated FAP expression in SFTPs using immunohistochemical staining and H-scores., Results: Nine of the 13 patients were male, with a median age of 64 years (range, 28-79 years). The median tumor size was 6.6 cm (1.1, 16 cm). In the pathological findings, expression levels of Ki67 were 1-5% in 12 of 13 cases. Furthermore, FAP expression was observed in all patients, and the median H-score was 160 (range, 10-280). The H-score of FAP expression in two of the 13 patients was low (10 in both), and that in two of the 13 patients was high (240 and 280). The SUVmax value of [ 18 F]FAPI-74 PET was 3.57 in a patient in whom the H-score of FAP expression was 180., Conclusions: SFTPs expressed FAP to varying degrees in different patients and the [ 18 F]FAPI-74 PET results in one patient reflected FAP expression in the tumor tissue., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Toru Kimura reports financial support was provided by Japan Society for the Promotion of Science. Toru Kimura reports financial support was provided by The Japan Lung Cancer Society. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

The disruption of ribbon synapses in the cochlea impairs the transmission of auditory signals from the cochlear sensory receptor cells to the auditory cortex. Although cisplatin-induced loss of ribbon synapses is well-documented, and studies have reported nitration of cochlear proteins after cisplatin treatment, yet the underlying mechanism of cochlear synaptopathy is not fully understood. This study tests the hypothesis that cisplatin treatment alters the abundance of cochlear synaptosomal proteins, and selective targeting of nitrative stress prevents the associated synaptic dysfunction. Auditory brainstem responses of mice treated with cisplatin showed a reduction in amplitude and an increase in latency of wave I, indicating cisplatin-induced synaptic dysfunction. The mass spectrometry analysis of cochlear synaptosomal proteins identified 102 proteins that decreased in abundance and 249 that increased in abundance after cisplatin treatment. Pathway analysis suggested that the dysregulated proteins were involved in calcium binding, calcium ion regulation, synapses, and endocytosis pathways. Inhibition of nitrative stress by co-treatment with MnTBAP, a peroxynitrite scavenger, attenuated cisplatin-induced changes in the abundance of 27 proteins. Furthermore, MnTBAP co-treatment prevented the cisplatin-induced decrease in the amplitude and increase in the latency of wave I. Together, these findings suggest a potential role of oxidative/nitrative stress in cisplatin-induced cochlear synaptic dysfunction., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: This study aimed to investigate the potential of fibroblast activation protein (FAP) as a biomarker in the progression of oral leukoplakia (OLK) carcinogenesis. This was achieved by evaluating FAP expression at different levels of the organisation, namely oral normal mucosa (NM), OLK, and oral squamous cell carcinoma (OSCC)., Materials and Methods: Altogether, 88 paraffin-embedded tissue samples were examined, including 55 cases of OLK, 13 cases of OSCC, and 20 cases of NM (control group). An exhaustive investigation was performed to examine FAP expression in NM, OLK, and OSCC tissues via immunohistochemistry (IHC). The relationship between FAP expression and clinical pathologic characteristics was analysed. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB) also proved the expression of FAP in NM, OLK, and OSCC cells. Aberrant FAP expression in OLK and OSCC was explored using in vitro experiments., Results: Immunohistochemical results showed that high FAP expression was significantly correlated with histopathologic grade (P = .038) but not correlated with age, sex, or region (P = .953, .622, and .108, respectively). The expression level of FAP in NM tissues (0.15 ± 0.01) was minimal, whereas it was observed in OLK (0.28 ± 0.04) and OSCC (0.39 ± 0.02) tissues with a noticeable increase in expression levels (P < .001). The expression level of FAP in OLK with severe abnormal hyperplasia (S-OLK) tissues (0.33 ± 0.04) was significantly higher than in OLK with mild abnormal hyperplasia (MI-OLK, 0.26 ± 0.02) and OLK with moderate abnormal hyperplasia (MO-OLK, 0.28 ± 0.03) tissues (P < .001 and P = .039, respectively). The results of RT-PCR illustrated that the relative expression of FAP mRNA in OLK cells (2.63 ± 0.62) was higher than in NM cells (0.87 ± 0.14), but lower than in OSCC cells (5.63 ± 1.06; P = .027 and .012, respectively). FAP expression was minimal in NM cells (0.78 ± 0.06), modest in OLK cells (1.04 ± 0.06), and significantly elevated in OSCC cells (1.61 ± 0.09) based on the results of WB (P < .001)., Conclusions: Significant variations in FAP expression were observed in NM, OLK, and OSCC tissues and cells. These findings revealed that FAP may be a reliable biomarker for the early diagnosis and evaluation of OLK carcinogenesis., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Aberrant fucosylation observed in cancer cells contributes to an augmented release of fucosylated exosomes into the bloodstream, where miRNAs including miR-4732-3p hold promise as potential tumor biomarkers in our pilot study. However, the mechanisms underlying the sorting of miR-4732-3p into fucosylated exosomes during lung cancer progression remain poorly understood., Methods: A fucose-captured strategy based on lentil lectin-magnetic beads was utilized to isolate fucosylated exosomes and evaluate the efficiency for capturing tumor-derived exosomes using nanoparticle tracking analysis (NTA). Fluorescence in situ hybridization (FISH) and qRT-PCR were performed to determine the levels of miR-4732-3p in non-small cell lung cancer (NSCLC) tissue samples. A co-culture system was established to assess the release of miRNA via exosomes from NSCLC cells. RNA immunoprecipitation (RIP) and miRNA pull-down were applied to validate the interaction between miR-4732-3p and heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein. Cell functional assays, cell derived xenograft, dual-luciferase reporter experiments, and western blot were applied to examine the effects of miR-4732-3p on MFSD12 and its downstream signaling pathways, and the impact of hnRNPK in NSCLC., Results: We enriched exosomes derived from NSCLC cells using the fucose-captured strategy and detected a significant upregulation of miR-4732-3p in fucosylated exosomes present in the serum, while its expression declined in NSCLC tissues. miR-4732-3p functioned as a tumor suppressor in NSCLC by targeting 3'UTR of MFSD12, thereby inhibiting AKT/p21 signaling pathway to induce cell cycle arrest in G2/M phase. NSCLC cells preferentially released miR-4732-3p via exosomes instead of retaining them intracellularly, which was facilitated by the interaction of miR-4732-3p with hnRNPK protein for selective sorting into fucosylated exosomes. Moreover, knockdown of hnRNPK suppressed NSCLC cell proliferation, with the elevated levels of miR-4732-3p in NSCLC tissues but the decreased expression in serum fucosylated exosomes., Conclusions: NSCLC cells escape suppressive effects of miR-4732-3p through hnRNPK-mediated sorting of them into fucosylated exosomes, thus supporting cell malignant properties and promoting NSCLC progression. Our study provides a promising biomarker for NSCLC and opens a novel avenue for NSCLC therapy by targeting hnRNPK to prevent the "exosome escape" of tumor-suppressive miR-4732-3p from NSCLC cells., (© 2024. The Author(s).)

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

Mitochondria and peroxisomes are mutually dependent organelles that share several membrane proteins that carry out the same function in both organelles. To study the unique features of these dually localized proteins in each of the two organelles, it is essential to separate mitochondria from peroxisomes. Isolating organelles from cells of Baker's yeast, Saccharomyces cerevisiae, is crucial for our understanding of the biogenesis and functions of proteins. Traditionally, subcellular fractionation and isolation of individual organelles by differential centrifugation benefit from the specific and unique density of each organelle. However, when yeast cells are grown under normal conditions, certain organelles like mitochondria and peroxisomes share strikingly similar densities. This similarity challenges the separation of these organelles from one another. In this chapter, we describe an optimized procedure to address this task. We depict growth conditions that would favor stimulation of peroxisomes to increase their number and density, and portray organellar isolation followed by gradient centrifugation, enabling an improved separation of both organelles. Additionally, we illustrate the advantage of the procedure to study the dual localization of the membrane protein Fis1., (Copyright © 2024. Published by Elsevier Inc.)

Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins., (Copyright © 2024. Published by Elsevier Inc.)

Objective: To compare cervical stroma in advanced cervical cancer with the control group; to compare, in the pre-treatment period, hemogram parameters in patients with advanced cervical cancer with the same parameters as the control group; and to verify if there is an association of stromal markers with prognostic factors in cervical cancer., Materials and Methods: We prospectively evaluated 16 patients diagnosed with advanced invasive cervical cancer. A control group of 22 patients was used (uterine leiomyoma). Immunohistochemistry was performed to verify the stromal immunostaining of alpha-smooth muscle actin (SMA) and fibroblast activation protein alpha (FAP). Immunostainings and hemogram parameters were compared using Fisher's exact and Mann-Whitney Test, respectively., Results: Strong FAP immunostaining was more frequent in patients with cervical cancer when compared with patients with leiomyoma (P = 0.0002). Regarding SMA, strong immunostaining was also found more in the group of cancer patients compared to the control group (P &lt; 0.00001). The neutrophil-lymphocyte ratio (NLR) values were higher in the cancer patient group compared to the control group (P = 0.0019). There was no association of the parameters studied with prognostic factors., Conclusions: Strong FAP and SMA immunostaining was found more in patients with cervical cancer when compared to the control group. NLR values were also higher in cervical cancer.

Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest., (© 2023 Wiley-VCH GmbH.)

Milk fat globule membrane (MFGM) proteins are nutritional components with various biological functions. This study aimed to analyze and compare MFGM proteins in porcine colostrum (PC) and porcine mature milk (PM), via label-free quantitative proteomics. In total, 3917 and 3966 MFGM proteins were identified in PC and PM milk, respectively. A total of 3807 common MFGM proteins were found in both groups, including 303 significant differentially expressed MFGM proteins. Gene Ontology (GO) analysis revealed that the differentially expressed MFGM proteins were mainly related to the cellular process, cell, and binding. The dominant pathway of the differentially expressed MFGM proteins was related to the phagosome according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. These results reveal crucial insights into the functional diversity of MFGM proteins in porcine milk during lactation and provide theoretical guidance for the development of MFGM proteins in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Quantitative analysis of surface proteins on extracellular vesicles (EVs) has been considered to be a crucial approach for reflecting the status of diseases. Due to the diverse composition of surface proteins on EVs and the interference from nonvesicular proteins, accurately detecting the expression of surface proteins on EVs remains a challenging task. While membrane affinity molecules have been widely employed as EVs capture probes to address this issue, their inherent biochemical properties have not been effectively harnessed. In this paper, we found that the electrochemical redox activity of the DSPE-PEG molecule was diminished upon its insertion into the membrane of EVs. This observation establishes the DSPE-PEG molecule modified on the Au electrode surface as a capture and a redox probe for the electrochemical detection of EVs. By utilizing methylene blue-labeled aptamers, the targeted surface proteins of EVs can be detected by recording the ratio of the oxidation peak current of methylene blue and DSPE-PEG. Without complicated signal amplification, the detection limit for EVs is calculated to be 8.11 × 10 2 particles/mL. Using this platform, we directly analyzed the expression of CD63 and HER2 proteins on the surface of EVs in human clinical plasma samples, demonstrating its significant potential in distinguishing breast cancer patients from healthy individuals.

Extracellular vesicles (EVs), as a type of subcellular structure, have been extensively researched for their potential for developing advanced diagnostic technologies for various diseases. However, the biomolecular and biophysical heterogeneity of EVs has restricted their application in clinical settings. In this article, we developed a size-exclusion chromatography-based technique for simultaneous EV size subtyping and protein profiling. By eluting fluorescent aptamer-treated patient plasma through a size-exclusion column, the mixture can be classified into 50 nm aptamer-bound EVs, 100 nm aptamer-bound EVs and free-floating aptamers, which could further enable multiplex EV membrane protein profiling by analyzing the fluorescence intensities of EV-bound aptamers. Using this technique, we successfully identified EV size subtypes for differentiating gastrointestinal cancer prognosis states. Overall, we developed a rapid, user-friendly and low-cost EV size subtyping and protein profiling technique, which holds great potential for identifying crucial EV size subtypes for disease diagnosis in the clinic.

Fibrillary glomerulonephritis (FGN), a rare disease is pathologically characterized by glomerular fibril accumulation ranging from 12 to 24 nm in diameter with negative Congo red staining. Recently, the identification of DnaJ homolog subfamily B member 9 (DNAJB9) as a highly sensitive and specific marker for FGN has revolutionized diagnosis of this disease. However, few recent studies have reported DNAJB9-negative glomerulonephritis with fibrillar deposits. As such, it remains unclear whether DNAJB9-negative cases can be considered equivalent to FGN. Here, we report the case of a 70-year-old woman who developed renal impairment and nephrotic-range proteinuria. Renal biopsy and pathological examination revealed focal glomerulonephritis with fibrocellular crescents. Immunofluorescence microscopy showed IgA-dominant deposition of polytypic IgG in the glomerulus. Electron microscopy revealed hump-like subepithelial electron dense deposits with fibrils of 15-25 nm in diameter. These findings were consistent with FGN; thus, Congo red and direct fast scarlet (DFS) staining, and immunohistochemistry for DNAJB9 were performed. In addition to negative Congo red/DFS/DNAJB9 staining, laser microdissection (LMD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) resulted negative for DNAJB9, which is a highly sensitive and specific marker for FGN. The patient's renal function further declined, prompting administration of rituximab weekly for 2 weeks, similar to the treatment for FGN. This is a unique case of IgA-dominant glomerulonephritis with DNAJB9-negative fibrillar polytypic immunoglobulin deposits in the subepithelium, unlike previous DNAJB9-negative cases. Thus, DNAJB9-negative cases diagnosed based on accurate electron microscopic evaluation must be gathered, and LMD and LC-MS/MS must be used to analyze the organized fibrillar deposits to reveal the disease entity., (© 2022. The Author(s) under exclusive licence to The Japan Society of Nephrology.)

Cell surface proteins (CSPs) are often involved in various biological processes such as cell-cell interactions, immune responses, and molecular transport. The abnormal expression of CSP usually indicates the occurrence and development of human diseases. Most CSPs are glycosylated and have been explored as potential drug targets and disease biomarkers; however, efficient isolation of CSPs from intracellular proteins is difficult due to their low abundance and strong hydrophobicity. The comprehensive characterization of surface glycoproteins remains a great challenge and is often underrepresented in proteomics. In recent years, unprecedented progress has been made in the mass spectrometry analysis of surface proteins, and CSP capture methods and mass spectrometry have been greatly developed. In this article, we aim to give a comprehensive overview of innovative analytical methods that can enrich CSPs, including centrifugation-based separation, phase partitioning, adhesion-based capture of surface proteins, antibody or lectin affinity, and biotin-based chemical labeling. Surface glycoproteins are captured by chemical oxidation of glycans or click chemistry for carbohydrate metabolic labeling. These techniques offer a wide range of applications for studying the function of cell surface receptors and identifying markers for diagnostic and therapeutic development.

Detergents enable the investigation of membrane proteins by mass spectrometry. Detergent designers aim to improve underlying methodologies and are confronted with the challenge to design detergents with optimal solution and gas-phase properties. Herein, we review literature related to the optimization of detergent chemistry and handling and identify an emerging research direction: the optimization of mass spectrometry detergents for individual applications in mass spectrometry-based membrane proteomics. We provide an overview about qualitative design aspects including their relevance for the optimization of detergents in bottom-up proteomics, top-down proteomics, native mass spectrometry, and Nativeomics. In addition to established design aspects, such as charge, concentration, degradability, detergent removal, and detergent exchange, it becomes apparent that detergent heterogeneity is a promising key driver for innovation. We anticipate that rationalizing the role of detergent structures in membrane proteomics will serve as an enabling step for the analysis of challenging biological systems., (© 2023. The Author(s).)

Lactococcus lactis displaying recombinant proteins on its surface can be used as a potential drug delivery vector in prophylactic medication and therapeutic treatments for many diseases. These applications enable live-cell mucosal and oral administration, providing painless, needle-free solutions and triggering robust immune response at the site of pathogen entry. Immunization requires quantitative control of antigens and, ideally, a complete understanding of the bacterial processing mechanism applied to the target proteins. In this study, we propose a double-labeling method based on a conjugated dye specific for a recombinantly introduced polyhistidine tag (to visualize surface-exposed proteins) and a membrane-permeable dye specific for a tetra-cysteine tag (to visualize cytoplasmic proteins), combined with a method to block the labeling of surface-exposed tetra-cysteine tags, to clearly obtain location-specific signals of the two dyes. This allows simultaneous detection and quantification of targeted proteins on the cell surface and in the cytoplasm. Using this method, we were able to detect full-length peptide chains for the model proteins HtrA and BmpA in L. lactis , which are associated with the cell membrane by two different attachment modes, and thus confirm that membrane-associated proteins in L. lactis are secreted using the Sec-dependent post-translational pathway. We were able to quantitatively follow cytoplasmic protein production and accumulation and subsequent export and surface attachment, which provides a convenient tool for monitoring these processes for cell surface display applications.

A promising new class of biosensors leverages the sensing mechanisms of living cells by incorporating native transmembrane proteins into biomimetic membranes. Conducting polymers (CPs) can further improve the detection of electrochemical signals from these biological recognition elements through their low electrical impedance. Supported lipid bilayers (SLBs) on CPs mimic the structure and biology of the cell membrane to enable such sensing, but their extrapolation to new target analytes and healthcare applications has been difficult due to their poor stability and limited membrane properties. Blending native phospholipids with synthetic block copolymers to create a hybrid SLB (HSLB) may address these challenges by allowing for the tuning of chemical and physical properties during membrane design. We establish the first example of HSLBs on a CP device and show that polymer incorporation enhances bilayer resilience and thus offers important benefits toward bio-hybrid bioelectronics for sensing applications. Importantly, HSLBs outperform traditional phospholipid bilayers in stability by exhibiting strong electrical sealing after exposure to physiologically relevant enzymes that cause phospholipid hydrolysis and membrane degradation. We investigate the impact of HSLB composition on membranes and device performance and demonstrate the ability to finely adjust the lateral diffusivity of HSLBs with modest changes in block copolymer content through a large compositional range. The inclusion of the block copolymer into the bilayer does not disrupt electrical sealing on CP electrodes, an essential metric for electrochemical sensors, or the insertion of a representative transmembrane protein. This work interfacing tunable and stable HSLBs with CPs paves the way for future bioinspired sensors that combine the exciting developments from both bioelectronics and synthetic biology.

Pex30 is a peroxisomal protein whose role in peroxisome biogenesis via the endoplasmic reticulum has been established. It is a 58 KDa multi-domain protein that facilitates contact site formation between various organelles. The present study aimed to investigate the role of various domains of the protein in its sub-cellular localization and regulation of peroxisome number. For this, we created six truncations of the protein (1-87, 1-250, 1-352, 88-523, 251-523 and 353-523) and tagged GFP at the C-terminus. Biochemical methods and fluorescence microscopy were used to characterize the effect of truncation on expression and localization of the protein. Quantitative analysis was performed to determine the effect of truncation on peroxisome number in these cells. Expression of the truncated variants in cells lacking PEX30 did not cause any effect on cell growth. Interestingly, variable expression and localization of the truncated variants in both peroxisome-inducing and non-inducing medium was observed. Truncated variants depicted different distribution patterns such as punctate, reticulate and cytosolic fluorescence. Interestingly, lack of the complete dysferlin domain or C-Dysf resulted in increased peroxisome number similar to as reported for cells lacking Pex30. No contribution of this domain in the reticulate distribution of the proteins was also observed. Our results show an interesting role for the various domains of Pex30 in localization and regulation of peroxisome number., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To investigate the clinical implications of Golgi glycoprotein 73 (GP73) and granulocyte colony-stimulating factor (G-CSF) in children with bronchopneumonia (BP)., Methods: Seventy-two children with BP (observation group) and 81 healthy children (control group) consecutively brought to the present study's hospital between June 2019 and October 2020 were enrolled. GP73 and G-CSF levels were determined to analyze their diagnostic value for pediatric BP. High-sensitivity C-reactive protein (hs-CRP) was also measured. The clinical implications of GP73 and G-CSF in pediatric BP complicated with respiratory failure and their connections with the inflammatory response were discussed., Results: GP73 and G-CSF levels were remarkably higher in the observation group (p < 0.05). The sensitivity and specificity of combined detection (GP73+G-CSF) in predicting pediatric BP were 72.22% and 86.42%, respectively (p < 0.001). GP73 and G-CSF, which are closely related to X-ray classification and complications in the observation group, decreased after treatment and were positively correlated with hs-CRP (p < 0.05), especially in children complicated with respiratory failure. Regression analysis identified the independence of the course of the disease, hs-CRP, X-ray classification, GP73, and G-CSF as influencing factors of respiratory failure in children with BP (p < 0.05)., Conclusion: GP73 and G-CSF, with elevated levels in children with BP, are strongly linked to disease progression and are independent influencing factors of respiratory failure, which may be the key to diagnosing and treating pediatric BP in the future., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2022. Published by Elsevier Editora Ltda.)

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease. FGN is characterized by the deposition of randomly arranged, nonbranching microfibrils in the mesangium and glomerular basement membrane. The discovery of DNAJ homolog subfamily B member 9 (DNAJB9) in 2017 was a breakthrough, and DNAJB9 has been proven to be extremely useful for the definitive diagnosis of FGN. While FGN often occurs in middle-aged individuals, this case was diagnosed at a relatively young age of 17. We performed renal biopsy, and light microscopic study revealed mesangial proliferation with expansion and subepithelial deposits. Electron microscopic study showed glomerular deposition of randomly oriented nonbranching fibrils with a mean of 20 nm. However, direct first scarlet stain for amyloidosis was weakly positive. Therefore, we confirmed the diagnosis of FGN and eliminated the presence of amyloidosis with mass spectrometry. This is the first case in Japan in which the complication of amyloidosis was ruled out with mass spectrometry and FGN was diagnosed using immunostaining and mass spectrometry of DNAJB9. We began treatment with cyclosporine A. One and a half years after the start of the treatment, kidney function continues to be normal., (© 2022. The Author(s) under exclusive licence to The Japan Society of Nephrology.)

Membrane proteins constitute the filter that controls the cellular traffic of nutrients, ions and other essential molecules, as well as the transmission of signals across the membrane. These proteins interact with other proteins in the cytosol, cytoskeleton or the extracellular side of the membrane, giving rise to complex interactomes that are distributed throughout the various lipid microdomains of the membrane plane. In this manner, complex networks of protein-protein and protein-lipid interactions are formed which regulate the most diverse biological functions, and disturbance of these networks can lead to disease. Therefore, characterization of these interactomes is a priority for current biomedical sciences. Traditionally, such studies have largely depended on solubilization/dissociation of the essential components of multiprotein complexes with detergents of various strength. However, this technique may result in the loss of certain components of such complexes, especially those whose binding is weak or transient. Moreover, protein solubilization can lead to the formation of non-native spurious interactions. As an alternative, proximity labelling (PL) techniques have been developed in recent years that can identify interactors of the protein of interest in a native cellular environment, prior to solubilization. In this article, we review the recent advances in PL and explore the new possibilities they offer for the characterization of membrane interactomes. SIGNIFICANCE: Membranes establish a series of complex protein-protein and protein-lipid interactions that are essential for cell physiology. For decades, they have been one of the central objects of study in Cell and Molecular Biology. However, knowledge of the structure of membrane proteins and their respective interactomes lags far behind that of soluble proteins, mainly due to technical difficulties in their handling and characterization caused by their insolubility. Recent research has developed various techniques to study these proteins in their native cellular environment. In this review article we address the application to membrane proteins of the so-called 'proximity labeling methods', which allows neighborhood relationships to be established between proteins in intact cells. The scarcity of alternatives for study of the components of membrane complexes make these methods especially attractive for analyzing this type of membrane associated supramolecular structures., (Copyright © 2021. Published by Elsevier B.V.)

In addition to the angiotensin‑converting enzyme 2 (ACE2), a number of host cell entry mediators have been identified for severe acute respiratory syndrome coronavirus‑2 (SARS‑CoV‑2), including transmembrane protease serine 4 (TMPRSS4). The authors have recently demonstrated the upregulation of TMPRSS4 in 11 different cancers, as well as its specific expression within the central nervous system using in silico tools. The present study aimed to expand the initial observations and, using immunohistochemistry, TMPRSS4 protein expression in the gastrointestinal (GI) tract and lungs was further mapped. Immunohistochemistry was performed on tissue arrays and lung tissues of patients with non‑small cell lung cancer with concurrent coronavirus disease 2019 (COVID‑19) infection using TMPRSS4 antibody. The results revealed that TMPRSS4 was abundantly expressed in the oesophagus, stomach, small intestine, jejunum, ileum, colon, liver and pancreas. Moreover, the extensive TMPRSS4 protein expression in the lungs of a deceased patient with COVID‑19 with chronic obstructive pulmonary disease and bronchial carcinoma, as well in the adjacent normal tissue, was demonstrated for the first time, at least to the best of our knowledge. On the whole, the immunohistochemistry data of the present study suggest that TMPRSS4 may be implicated in the broader (pulmonary and extra‑pulmonary) COVID‑19 symptomatology; thus, it may be responsible for the tropism of this coronavirus both in the GI tract and lungs.

Objective: Clear cell renal cell carcinoma (ccRCC) carries significant morbidity and mortality globally and is often resistant to conventional radiotherapy and chemotherapy. Immune checkpoint blockade (ICB) has received attention in ccRCC patients as a promising anticancer treatment. Furthermore, competitive endogenous RNA (ceRNA) networks are crucial for the occurrence and progression of various tumors. This study was aimed at identifying reliable prognostic signatures and exploring potential mechanisms between ceRNA regulation and immune cell infiltration in ccRCC patients., Methods and Results: Gene expression profiling and clinical information of ccRCC samples were obtained from The Cancer Genome Atlas (TCGA) database. Through comprehensive bioinformatic analyses, differentially expressed mRNAs (DEmRNAs; n = 131), lncRNAs (DElncRNAs; n = 12), and miRNAs (DEmiRNAs; n = 25) were identified to establish ceRNA networks. The CIBERSORT algorithm was applied to calculate the proportion of 22 types of tumor-infiltrating immune cells (TIICs) in ccRCC tissues. Subsequently, univariate Cox, Lasso, and multivariate Cox regression analyses were employed to construct ceRNA-related and TIIC-related prognostic signatures. In addition, we explored the relationship between the crucial genes and TIICs via coexpression analysis, which revealed that the interactions between MALAT1, miR-1271-5p, KIAA1324, and follicular helper T cells might be closely correlated with the progression of ccRCC. Ultimately, we preliminarily validated that the potential MALAT1/miR-1271-5p/KIAA1324 axis was consistent with the ceRNA theory by qRT-PCR in the ccRCC cell lines., Conclusion: On the basis of the ceRNA networks and TIICs, we constructed two prognostic signatures with excellent predictive value and explored possible molecular regulatory mechanisms, which might contribute to the improvement of prognosis and individualized treatment for ccRCC patients., Competing Interests: The authors declare that no potential conflicts of interest exist., (Copyright © 2022 Lu Zhou et al.)

Background & Aims: Drug-induced liver injury (DILI) remains challenging to treat and is still a leading cause of acute liver failure. MG53 is a muscle-derived tissue-repair protein that circulates in the bloodstream and whose physiological role in protection against DILI has not been examined., Methods: Recombinant MG53 protein (rhMG53) was administered exogenously, using mice with deletion of Mg53 or Ripk3. Live-cell imaging, histological, biochemical, and molecular studies were used to investigate the mechanisms that underlie the extracellular and intracellular action of rhMG53 in hepatoprotection., Results: Systemic administration of rhMG53 protein, in mice, can prophylactically and therapeutically treat DILI induced through exposure to acetaminophen, tetracycline, concanavalin A, carbon tetrachloride, or thioacetamide. Circulating MG53 protects hepatocytes from injury through direct interaction with MLKL at the plasma membrane. Extracellular MG53 can enter hepatocytes and act as an E3-ligase to mitigate RIPK3-mediated MLKL phosphorylation and membrane translocation., Conclusions: Our data show that the membrane-delimited signaling and cytosolic dual action of MG53 effectively preserves hepatocyte integrity during DILI. rhMG53 may be a potential treatment option for patients with DILI., Lay Summary: Interventions to treat drug-induced liver injury and halt its progression into liver failure are of great value to society. The present study reveals that muscle-liver cross talk, with MG53 as a messenger, serves an important role in liver cell protection. Thus, MG53 is a potential treatment option for patients with drug-induced liver injury., Competing Interests: Conflict of interest JM and TT hold equity interest in TRIM-edicine, Inc. that develops MG53 for therapeutic applications. The Intellectual Properties related to MG53 are maintained by Rutgers University and The Ohio State University. All other authors declare that they have no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Cancer-associated fibroblasts (CAFs), a compartment of the tumor microenvironment, were previously thought to be a uniform cell population that promotes cancer progression. However, recent studies have shown that CAFs are heterogeneous and that there are at least two types of CAFs, that is, cancer-promoting and -restraining CAFs. We previously identified Meflin as a candidate marker of cancer-restraining CAFs (rCAFs) in pancreatic ductal adenocarcinoma (PDAC). The precise nature of rCAFs, however, has remained elusive owing to a lack of understanding of their comprehensive gene signatures. Here, we screened genes whose expression correlated with Meflin in single-cell transcriptomic analyses of human cancers. Among the identified genes, we identified matrix remodeling-associated protein 8 (MXRA8), which encodes a type I transmembrane protein with unknown molecular function. Analysis of MXRA8 expression in human PDAC samples showed that MXRA8 was differentially co-expressed with other CAF markers. Moreover, in patients with PDAC or syngeneic tumors developed in MXRA8-knockout mice, MXRA8 expression did not affect the roles of CAFs in cancer progression, and the biological importance of MXRA8 + CAFs is still unclear. Overall, we identified MXRA8 as a new CAF marker; further studies are needed to determine the relevance of this marker., (© 2022 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Background: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized., Objective: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease., Methods: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs., Results: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation., Conclusions: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With many food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families., (© 2022. The Author(s).)

An important aspect of understanding cancer biology is to connect the diverse repertoire of genotype-to-phenotype displays in individual specimens and ultimately resolve disease course outcome through informative datasets. A focus of cancer genomics has strived to provide predictive capabilities using genomic information to further inform therapeutic strategies. The advent of single-cell sequencing and analysis now provides a route to decipher high-resolution genomic diversity in individual samples and facilitate detailed understanding of clonal evolution in clinical research settings. In addition to generating high-throughput single-cell genomic SNV and CNV data, this protocol describes a new analytical ability that adds a second dimension which provides for interrogation of surface protein marker expression. The first immediate application of this technology is quite suitable to heme cancer cell studies. This multimodal approach allows for correlation of diverse genomic signatures to key phenotypic biomarkers such as immunophenotypes in leukemic diseases., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The dual marker algorithm Risk of Ovarian Malignancy Algorithm (ROMA) has been widely used in the clinic for the identification of equivocal pelvic masses in ovarian carcinoma. To obtain higher diagnostic efficiency, we created a new diagnostic index, Risk of Ovarian Malignancy Index (ROMI), by combing thymidine kinase 1 (TK1), HE4 and CA125., Methods: 335 patients with pelvic masses on imaging and 46 healthy controls were enrolled. Serum TK1 was analyzed before further study. ROMI and ROMA were evaluated for diagnostic efficiency., Results: The level of TK1 was elevated in malignant ovarian tumors compared to benign masses (p < 0.001) and healthy controls (p < 0.001). TK1 expression was positively correlated with stage, intrapelvic metastasis, lymphatic metastasis and distant metastasis (all p values < 0.001). The area under the receiver operating characteristic curve (AUC) of ROMI was higher than that of ROMA for both pre- and postmenopausal women. ROMI had better sensitivity, specificity, accuracy, and positive and negative predictive values than ROMA in diagnosis of all-stage or stage I + II ovarian carcinoma for both pre- and postmenopausal women., Conclusions: TK1 is a potential biomarker in detection of ovarian carcinoma. ROMI shows better diagnostic performance than ROMA in distinguishing malignant ovarian tumors from benign masses., (Copyright © 2021. Published by Elsevier B.V.)

Objective: Terminal 6q deletion is a rare genetic condition associated with a neurodevelopmental disorder characterized by intellectual disability and structural brain anomalies. Interestingly, a similar phenotype is observed in patients harboring pathogenic variants in the DLL1 gene. Our study aimed to further characterize the prenatal phenotype of this syndrome as well as to attempt to establish phenotype-genotype correlations., Method: We collected ultrasound findings from 22 fetuses diagnosed with a pure 6qter deletion. We reviewed the literature and compared our 22 cases with 14 fetuses previously reported as well as with patients with heterozygous DLL1 pathogenic variants., Results: Brain structural alterations were observed in all fetuses. The most common findings (>70%) were cerebellar hypoplasia, ventriculomegaly, and corpus callosum abnormalities. Gyration abnormalities were observed in 46% of cases. Occasional findings included cerebral heterotopia, aqueductal stenosis, vertebral malformations, dysmorphic features, and kidney abnormalities., Conclusion: This is the first series of fetuses diagnosed with pure terminal 6q deletion. Based on our findings, we emphasize the prenatal sonographic anomalies, which may suggest the syndrome. Furthermore, this study highlights the importance of chromosomal microarray analysis to search for submicroscopic deletions of the 6q27 region involving the DLL1 gene in fetuses with these malformations., (© 2021 John Wiley & Sons Ltd.)

Background: Local relapse of oral squamous cell carcinoma in non-involved mucosal surgical margins indicated possibility of field alteration in the margins, which could be predicted with certain biomarkers. The objectives were to evaluate the expression of Ki-67, Cornulin and ISG15 in non-involved mucosal surgical margins and the association of clinicopathological prognosticators with local relapse in oral squamous cell carcinoma., Methods: Surgical margins from the study (relapse) group (n = 23), control (non-relapse) group (n = 32) and normal oral mucosa (n = 5) were immunohistochemically stained using Ki-67, Cornulin and ISG15 antibodies. Association between expression of markers and clinicopathological prognosticators with local relapse in oral squamous cell carcinoma was analyzed statistically., Results: The study group surgical margins demonstrated significantly decreased Cornulin expression (p = 0.032). Low Cornulin expression was significantly associated with local relapse (p = 0.004) and non-tongue primary tumor (p = 0.013). Although not significantly associated with local relapse, expression of Ki-67 was significantly reduced in female patients (p = 0.041). Age above 57.5 years, Chinese & Indian ethnicity, alcohol consumption, epithelial dysplasia in surgical margins, and type III and IV patterns of invasion of tumor were also significantly related to local relapse. Regression analysis showed low expression of Cornulin (p = 0.018), and increased patient's age (p = 0.008) were predictors of local relapse in oral squamous cell carcinoma, with 34-fold risk and 18-fold risk, respectively. Expression of Ki-67 and ISG15 did not show significant association with local relapse in oral squamous cell carcinoma., Conclusion: Low expression of Cornulin is an independent predictor of relapse in oral squamous cell carcinoma., Competing Interests: The authors have declared that no competing interests exist.

Background: Development of an effective vaccine against blood-stage malaria requires the induction of long-term immune responses. Plasmodium vivax Reticulocyte Binding Protein 1a (PvRBP1a) is a blood-stage parasite antigen which is associated with invasion of red blood cells and induces antibody responses. Thus, PvRBP1a is considered as a target for design of a blood-stage vaccine against vivax malaria., Methods: Both cross-sectional and cohort studies were used to explore the development and persistence of long-lived antibody and memory B cell responses to PvRBP1a in individuals who lived in an area of low malaria endemicity. Antibody titers and frequency of memory B cells specific to PvRBP1a were measured during infection and following recovery for up to 12 months., Results: IgG antibody responses against PvRBP1a were prevalent during acute vivax malaria, predominantly IgG1 subclass responses. High responders to PvRBP1a had persistent antibody responses for at least 12-month post-infection. Further analysis of high responder found a direct relation between antibody titers and frequency of activated and atypical memory B cells. Furthermore, circulating antibody secreting cells and memory B cells specific to PvRBP1a were generated during infection. The PvRBP1a-specific memory B cells were maintained for up to 3-year post-infection, indicating the ability of PvRBP1a to induce long-term humoral immunity., Conclusion: The study revealed an ability of PvRBP1a protein to induce the generation and maintenance of antibody and memory B cell responses. Therefore, PvRBP1a could be considered as a vaccine candidate against the blood-stage of P. vivax., (© 2021. The Author(s).)

Objectives: In this study, clinical and biochemical methods were utilized to predict the final diagnosis of hereditary spherocytosis (HS), correlate the diagnosis with splenectomy, and examine the usefulness of this approach., Methods: We biochemically and cytochemically analysed erythrocyte membrane proteins before making a final HS diagnosis based on gene analysis to compare diagnostic approaches. The clinical features of six patients with various subtypes of HS and symptoms were observed by blood analysis using eosin-5'-maleimide staining, biochemical analysis using sodium dodecyl sulphate - polyacrylamide gel electrophoresis with western blotting, and mass spectrometry. Finally, diagnostic membrane gene analysis was performed., Results: Five of the six patients showed mild to moderate or severe anaemia, and the other patient was non-anaemic; all six patients showed faint eosin-5'-maleimide staining. In western blotting of erythrocyte membrane proteins, all six patients (three with β-spectrin, two with ankyrin, and one with SLC4A1 anomalies) showed low-molecular-weight peptide fragments, which were confirmed by mass spectrometry in the region corresponding to the band 3 protein. The two patients with an ankyrin gene anomaly exhibited severe anaemia, and two patients with simultaneous SLC4A1 , SPTB , and UGT1A1 anomalies exhibited mild anaemia and hyperbilirubinemia., Discussion: We determined the relationship among clinical features, cytochemical parameters, and gene anomalies in six patients with newly diagnosed HS while referring to previously published cases., Conclusion: These findings reveal a close relationship between clinical features and membrane characteristics in HS, which can facilitate diagnosis and inform treatment.

Background and Aims: NAFLD is the most prevalent chronic liver disease worldwide, but no effective pharmacological therapeutics are available for clinical use. NASH is the more severe stage of NAFLD. During this progress, dysregulation of endoplasmic reticulum (ER)-related pathways and proteins is one of the predominant hallmarks. We aimed to reveal the role of ring finger protein 5 (RNF5), an ER-localized E3 ubiquitin-protein ligase, in NASH and to explore its underlying mechanism., Approach and Results: We first inspected the expression level of RNF5 and found that it was markedly decreased in livers with NASH in multiple species including humans. We then introduced adenoviruses for Rnf5 overexpression or knockdown into primary mouse hepatocytes and found that palmitic acid/oleic acid (PAOA)-induced lipid accumulation and inflammation in hepatocytes were markedly attenuated by Rnf5 overexpression but exacerbated by Rnf5 gene silencing. Hepatocyte-specific Rnf5 knockout significantly exacerbated hepatic steatosis, inflammatory response, and fibrosis in mice challenged with diet-induced NASH. Mechanistically, we identified 3-hydroxy-3-methylglutaryl CoA reductase degradation protein 1 (HRD1) as a binding partner of RNF5 by systematic interactomics analysis. RNF5 directly bound to HRD1 and promoted its lysine 48 (K48)-linked and K33-linked ubiquitination and subsequent proteasomal degradation. Furthermore, Hrd1 overexpression significantly exacerbated PAOA-induced lipid accumulation and inflammation, and short hairpin RNA-mediated Hrd1 knockdown exerted the opposite effects. Notably, Hrd1 knockdown significantly diminished PAOA-induced lipid deposition, and up-regulation of related genes resulted from Rnf5 ablation in hepatocytes., Conclusions: These data indicate that RNF5 inhibits NASH progression by targeting HRD1 in the ubiquitin-mediated proteasomal pathway. Targeting the RNF5-HRD1 axis may provide insights into the pathogenesis of NASH and pave the way for developing strategies for NASH prevention and treatment., (© 2021 by the American Association for the Study of Liver Diseases.)

Transmembrane Channel-like (TMC) genes are critical in the carcinogenesis, proliferation, and cell cycle of human cancers. However, the multi-omics features of TMCs and their role in the prognosis and immunotherapeutic response of human cancer have not been explored. We discovered that TMCs 4-8 were commonly deregulated and correlated with patient survival in a variety of cancers. For example, TMC5 and TMC8 were correlated with the relapse and overall survival rates of breast cancer and skin melanoma, respectively. These results were validated by multiple independent cohorts. TMCs were regulated by DNA methylation and somatic alterations, such as TMC5 amplification in breast cancer (523/1062, 49.2%). Six algorithms concordantly uncovered the critical role of TMCs in the tumor microenvironment, potentially regulating immune cell toxicity and lymphocytes infiltration. Moreover, TMCs 4-8 were correlated with tumor mutation burden and expression of PD-1/PD-L1/CTLA4 in 33 cancers. Thus, we established an immunotherapy response prediction (IRP) score based on the signature of TMCs 4-8. Patients with higher IRP scores showed higher immunotherapeutic responses in five cohorts of skin melanoma (area under curve [AUC] = 0.90 in the training cohort, AUCs range from 0.70 to 0.83 in the validation cohorts). Together, our study highlights the great potential of TMCs as biomarkers for prognosis and immunotherapeutic response, which can pave the way for further investigation of the tumor-infiltrating mechanisms and therapeutic potentials of TMCs in cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Song, Tang, Luo, Shi, Song and Ran.)

Challenges in studying the structures and functions of cell membrane proteins lie in their lipophilicity, which makes them hard to be stabilized, crystallized, and expressed by E. coli . Herein, we propose an evanescent field excited surface-enhanced Raman scattering (EF-SERS) strategy for label-free analysis of membrane proteins in situ . Extracted cell membranes tightly wrapped the metal nanoparticles by an extruder, which ensures the SERS signals of the membrane proteins precisely benefit from the localized surface plasmons (LSPs). The leaky mode of a waveguide was employed to improve the plasmon excitation coupling. Thus, the LSPs and waveguide modes together enable the achievement of high-quality SERS profiles of membrane proteins. By spectral analysis, the structural changes of membrane proteins can be deeply understood at the molecular level. This method has broader applicability in establishing the Ramanomics of membrane proteins and unraveling the exact changes of membrane proteins during physiological processes.

There is no recognized serum biomarker to predict the recurrence of endometrial carcinoma (EC). We aimed to explore serum human epididymis protein 4 (HE4) and cancer antigen 125 (CA125) as the biomarkers to predict and monitor recurrence of type II EC. 191 patients diagnosed with type II EC were involved for this retrospective study. Comparing recurrent with non-recurrent patients, HE4 levels resulted a statistically significant difference at primary diagnosis and recurrence, respectively (P = 0.002 and P = < 0.001), while CA125 levels resulted statistically significant (P = < 0.001) at recurrence. According to receiver operating characteristic curve analysis, the areas under the curve were significant for HE4 levels at primary diagnosis and recurrence predicting recurrence. Furthermore, CA125 levels at recurrence were significant. And the combination of both markers showed the higher sensitivity and specificity than single one. Patients with higher HE4 levels were associated with worse disease-free survival and overall survival, the opposite was true for patients with lower HE4 levels. The preoperative HE4 levels could be used to evaluate the risk factors of type II EC. Which suggested that HE4 levels might associated with the prognosis of type II EC. And combination of HE4 and CA125 could be applied to monitor recurrence during follow-up., (© 2021. The Author(s).)