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Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV‐associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field‐flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high‐purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV‐associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell‐line‐derived EV markers are incompatible with the identification of plasma‐EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma‐EV markers. Benefiting from the high‐purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma‐ and serum‐derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications. [ABSTRACT FROM AUTHOR]

Nanostructured surfaces are effective at physically killing bacterial cells, highlighting their prospective application as biomaterials. The benefits of application of mechano‐bactericidal nanostructures as an alternative to chemical functionalisation are well documented, however, the effects of protein adsorption are not well understood. In this work, theoretical and experimental analyses are conducted by studying the adsorption of human serum proteins (HSP) to nanosheet titanium (Ti) and its subsequent effect on the mechano‐bactericidal efficacy toward Staphylococcus aureus and Pseudomonas aeruginosa cells. The nanosheet pattern exhibits enhanced antibiofouling behaviour mantaining high bactericidal efficiency toward both Gram‐negative and Gram‐positive cells in the presence of adsorbed HSP. To ascertain the immunomodulatory response, S. aureus cells are introduced to protein‐conditioned Ti nanosheet surfaces prior to introducing RAW 264.7 macrophages. On the pre‐infected nanostructured surfaces, macrophages exhibit wound healing behaviour with superior activation of M2‐like macrophage polarization and secretion of anti‐inflammatory cytokines. By contrast, macrophages attached to infected smooth surfaces activated the M1‐like polarized phenotype via the high expression of pro‐inflammatory cytokines, indicating persistent inflammation. The outcomes of this work demonstrate the suitability of Ti nanosheets as a potential biomaterial surface whereby the mechano‐bactericidal activity is not compromised by HSP adsorption and, furthermore, positively influenced an anti‐inflammatory immune response. [ABSTRACT FROM AUTHOR]

Background: To date, most existing models for predicting neuromyelitis optica spectrum disorder (NMOSD) are based primarily on clinical characteristics. Blood‐based NMOSD severity and prognostic predictive immune‐ and inflammation‐related biomarkers are needed. We aimed to investigate the associations between plasma inflammatory biomarkers and relapse and attack severity in NMOSD. Methods: This two‐step, single‐center prospective cohort study included discovery and validation cohorts. We quantified 92 plasma inflammatory proteins by using Olink's proximity extension assay and identified differentially expressed proteins in the relapse group (relapse within 1 year of follow‐up) and severe attack group. To define a new molecular prognostic model, we calculated the risk score of each patient based on the key protein signatures and validated the results in the validation cohort. Results: The relapse prediction model, including FGF‐23, DNER, GDNF, and SLAMF1, predicted the 1‐year relapse risk. The severe attack prediction model, including PD‐L1 and MCP‐2, predicted the severe clinical attack risk. Both the relapse and severe attack prediction models demonstrated good discriminative ability and high accuracy in the validation cohort. Conclusions: Our discovered biomarker signature and prediction models may complement current clinical risk stratification approaches. These inflammatory biomarkers could contribute to the discovery of therapeutic interventions and prevent NMOSD progression. [ABSTRACT FROM AUTHOR]

Superficial, systemic microcirculations, distinct from the pulmonary circulation, supply the mucosae of human nasal and conducting airways. Non‐injurious, inflammatory challenges of the airway mucosa cause extravasation without overt mucosal oedema. Instead, likely reflecting minimal increases in basolateral hydrostatic pressure, circulating proteins/peptides of all sizes are transmitted paracellularly across the juxtaposed epithelial barrier. Thus, small volumes of extravasated, unfiltered bulk plasma appear on the mucosal surface at nasal and bronchial sites of challenge. Importantly, the plasma‐exuding mucosa maintains barrier integrity against penetrability of inhaled molecules. Thus, one‐way epithelial penetrability, strict localization, and well‐controlled magnitude and duration are basic characteristics of the plasma exudation response in human intact airways. In vivo experiments in human‐like airways demonstrate that local plasma exudation is also induced by non‐sanguineous removal of epithelium over an intact basement membrane. This humoral response results in a protective, repair‐promoting barrier kept together by a fibrin‐fibronectin net. Plasma exudation stops once the provisional barrier is substituted by a new cellular cover consisting of speedily migrating repair cells, which may emanate from all types of epithelial cells bordering the denuded patch. Exuded plasma on the surface of human airways reflects physiological microvascular‐epithelial cooperation in first line mucosal defense at sites of intact and regenerating epithelium. [ABSTRACT FROM AUTHOR]

One of the key pharmacokinetic properties of most small molecule drugs is their ability to bind to serum proteins. Unbound or free drug is responsible for pharmacological activity while the balance between free and bound drug can impact drug distribution, elimination, and other safety parameters. In the hepatic impairment (HI) and renal impairment (RI) clinical studies, unbound drug concentration is often assessed; however, the relevance and impact of the protein binding (PB) results is largely limited. We analyzed published clinical safety and pharmacokinetic studies in subjects with HI or RI with PB assessment up to October 2022 and summarized the contribution of PB results on their label dose recommendations. Among drugs with HI publication, 32% (17/53) associated product labels include PB results in HI section. Of these, the majority (9/17, 53%) recommend dose adjustments consistent with observed PB change. Among drugs with RI publication, 27% (12/44) of associated product labels include PB results in RI section with the majority (7/12, 58%) recommending no dose adjustment, consistent with the reported absence of PB change. PB results were found to be consistent with a tailored dose recommendation in 53% and 58% of the approved labels for HI and RI section, respectively. We further discussed the interpretation challenges of PB results, explored treatment decision factors including total drug concentration, exposure–response relationships, and safety considerations in these case examples. Collectively, comprehending the alterations in free drug levels in HI and RI informs treatment decision through a risk‐based approach. [ABSTRACT FROM AUTHOR]

Introduction: Recent evidence recommends the use of biologics in patients with severe uncontrolled type 2 chronic rhinosinusitis with nasal polyp (CRSwNP) owing to its propensity for recurrence after functional endoscopic sinus surgery (FESS). Among the type 2 biologics used for the treatment of nasal polyps, dupilumab (Dupi, anti‐IL‐4) exhibited superior efficacy and safety in indirect comparison studies. Objective: This study aimed to evaluate the objective and subjective outcomes of patients with CRSwNP treated with and without adjuvant Dupi therapy after FESS. Methods: Adult patients with type 2 CRSwNP who underwent FESS with adjuvant Dupi after surgery were enrolled. A matched control group without adjuvant Dupi therapy were recruited during the same period. All patients underwent nasal endoscopy and completed the sinonasal outcome test‐22 questionnaire evaluations at baseline and 3 months after surgery. Results: A total of 10 patients who received postoperative adjuvant therapy with Dupi and 20 patients who underwent surgery only were included. Patients with add‐on Dupi therapy had significantly higher eosinophil cationic protein levels in the serum, eosinophil counts in peripheral blood, prevalence of asthma, and nasal polyp score at baseline. Both treatments were effective in reducing the patient's symptoms by SNOT‐22 at 3 months postoperatively. However, patients with adjuvant Dupi therapy exhibited significantly better endoscopic scores than those with surgery only (p =.022). Conclusion: Surgery plays an important role in treating patients with CRSwNP, and adjuvant Dupi use may facilitate objective mucosal recovery postoperatively. Level of Evidence: 4. [ABSTRACT FROM AUTHOR]

Summary: The plant hormone auxin and its directional intercellular transport play a major role in diverse aspects of plant growth and development. The establishment of auxin gradients requires the asymmetric distribution of members of the auxin efflux carrier PIN‐FORMED (PIN) protein family to the plasma membrane. An endocytic pathway regulates the recycling of PIN proteins between the plasma membrane and endosomes, providing a mechanism for dynamic localisation.N‐Ethylmaleimide‐sensitive factor adaptor protein receptors (SNAP receptors, SNAREs) mediate fusion between vesicles and target membranes and are classed as Q‐ or R‐SNAREs based on their sequence. We analysed gain‐ and loss‐of‐function mutants, dominant‐negative transgenics and localisation of the Arabidopsis R‐SNARE VAMP714 protein to understand its function.We demonstrate that VAMP714 is essential for the insertion of PINs into the plasma membrane, for polar auxin transport, root gravitropism and morphogenesis. VAMP714 gene expression is upregulated by auxin, and the VAMP714 protein co‐localises with endoplasmic reticulum and Golgi vesicles and with PIN proteins at the plasma membrane.It is proposed that VAMP714 mediates the delivery of PIN‐carrying vesicles to the plasma membrane, and that this forms part of a positive regulatory loop in which auxin activates a VAMP714‐dependent PIN/auxin transport system to control development. [ABSTRACT FROM AUTHOR]

The primary structure of the 2265 residues of bovine plasma fibronectin has been completed. The new sequences reported in this paper are residues 600-868 (269 residues), 1138-1217 (80 residues), 1518 -1599 (82 residues) and 1868-2061 (194 residues). These sequences constitute six type II homology units and two non-monologous connecting strands. Thus, there are fifteen type III homology units in plasma fibronectin. Evidence for two of the three splice variant found in rat liver cels [Schwarzbauer et al. (1983) Cell 35, 421-431] was obtained. No indications of the 'extra' type III domain present in some human fibroblast fibronectins [Kornblihtt et al. (1984) EMBO J. 3, 221-226] was found. Three carbohydrate groups (two glucosamine-based and one galactosamine-based) were identified, giving a total of eight carbohydrate groups in the lowest splice variant of bovine plasma fibronectin. A second free sulfhydryl group (cysteine) was identified. This cysteine, like the first, is in a type III homology unit. HPLC patterns of peptides obtained from the C-terminal 6-kDa fragment suggested that the interchain bridge pattern of fibronectin is antiparallel. The bovine plasma fibronectin sequence is highly homologous to the human cellular fibronectin sequence deduced from the cDNA sequence [Kornblihtt et al. (1985) EMBO J. 4, 1755-1759]. [ABSTRACT FROM AUTHOR]

The expression of human transferrin and lactoferrin binding activity in Haemophilus influenzae, detected by a binding assay using human transferrin or lactoferrin conjugated to peroxidase, was regulated by the level of available iron in the medium. Transferrin binding activity was present in all H. influenzae isolates tested but not detected in other Haemophilus species or in species of Pasteurella or Actinobacillus. Lactoferrin binding activity was only detected in 1⁄15 H. influenzae isolates tested. The transferrin and lactoferrin receptors were shown to be specific for the respective human proteins by means of a competition binding assay. Competition binding assays also showed that iron-loaded transferrin was more effective at blocking the transferrin receptor than apotransferrin, but no differences in receptor blocking were observed between iron-loaded lactoferrin and apolactoferrin. [ABSTRACT FROM AUTHOR]

Introduction: Intervertebral disc degeneration and Modic change are the main spinal structural changes associated with chronic low back pain (LBP). Both conditions are thought to manifest local inflammation and if inflammatory proteins translocate to the blood circulation could be detected systemically. The work here assesses whether the presence of disc degeneration is associated with detectable blood level changes of five inflammatory markers and whether chronic LBP is associated with these changes. Materials and Methods: Two hundred and forty TwinsUK cohort participants with both MRI disc degeneration grade and Modic change extent, and IL‐6, IL‐8, IL‐8 TNF, and CX3CL1 protein blood concentration measurements were included in this work. Linear mixed effects models were used to test the association of blood cytokine concentration with disc degeneration score and Modic change volumetric score. Association of chronic LBP status from questionnaires with disc degeneration, Modic change, and cytokine blood concentration was also tested. Results: No statistically significant association between disc degeneration or Modic change with cytokine blood concentration was found. Instead, regression analysis pointed strong association between cytokine blood concentration with body mass index for IL‐6 and with age for IL‐6 and TNF. Mild association was found between IL‐8 blood concentration and body mass index. Additionally, LBP status was associated with Modic change volumetric score but not associated with any cytokine concentration. Conclusions: We found no evidence that Modic change and disc degeneration are able to produce changes in tested blood cytokine concentration. However, age and body mass index have strong influence on cytokine concentration and both are associated with the conditions studied which may confound associations found in the literature. It is then unlikely that cytokines produced in the disc or vertebral bone marrow induce chronic LBP. [ABSTRACT FROM AUTHOR]

Protein glycosylation responds sensitively to disease states. It is implicated in every hallmark of cancer and has recently started to be considered as a hallmark itself. Changes in N‐glycosylation microheterogeneity are more dramatic than those of protein expression due to the non‐template nature of protein glycosylation. This enables their potential use in serum‐based diagnostics. Here, we perform glycopeptidomics on serum from patients with oropharyngeal squamous cell carcinoma (OPSCC), compared to controls and comparing between cancers based on etiology (human papilloma virus‐ positive or negative). Using MS2, we then targeted glycoforms, significantly different between the groups, to identify their glycopeptide compositions. Simultaneously we investigate the same serum proteins, comparing whether N‐glycosylation changes reflect protein‐level changes. Significant glycoforms were identified from proteins such as alpha‐1‐antitrypsin (SERPINA1), haptoglobin, and different immunoglobulins. SERPINA1 had glycovariance at 2 N‐glycosylation sites, that were up to 35 times more abundant in even early‐stage OPSCCs, despite minimal differences between SERPINA1 protein levels between groups. Some identified glycoforms' fold changes (FCs) were in line with serum protein level FCs, others were less abundant in early‐stage cancers but with great variance in higher‐stage cancers, such as on immunoglobulin heavy constant gamma 2, despite no change in protein levels. Such findings indicate that glycovariant analysis might be more beneficial than proteomic analysis, which is yet to be fruitful in the search for biomarkers. Highly sensitive glycopeptide changes could potentially be used in the future for cancer screening. Additionally, characterizing the glycopeptide changes in OPSCC is valuable in the search for potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Glycidol fatty acid esters that are present in foods are degraded in vivo to the animal carcinogen glycidol, which binds to the N‐terminal valine of hemoglobin (Hb) to form N‐(2,3‐dihydroxypropyl)valine (diHOPrVal) adducts. The existence of other chemicals that are converted to glycidol is unknown. To determine the effect of different exposure conditions on the formation of diHOPrVal adducts, several glycidol‐related chemicals (3‐monochloropropane‐1,2‐diol; 3‐MCPD, epichlorohydrin, glyceraldehyde, acrylic acid, and 1,2‐propanediol) were evaluated using in vitro and in vivo (single/repeated dose) methods. In vitro, the reaction of 3‐MCPD or epichlorohydrin with human Hb produced 17% and 0.7% of diHOPrVal, as compared to equimolar glycidol, respectively. Following a single administration of glycidol‐related compounds to ICR mice, diHOPrVal formation was observed only in the epichlorohydrin‐treated group after day 5 of exposure. After 14 days of repeated dosing, the amounts of diHOPrVal produced by epichlorohydrin and 3‐MCPD in vivo were <1% of diHOPrVal produced by an equal molar concentration of glycidol. Furthermore, glyceraldehyde group produced 0.2% of diHOPrVal at the same molar concentration of glycidol equivalents, in which diHOPrVal formation could not be confirmed by the in vitro assay. The results indicate the usefulness of diHOPrVal as an exposure marker for glycidol; however, the contribution of its formation in vivo by exposure to various chemicals will be necessary to validate and interpret the results. [ABSTRACT FROM AUTHOR]

Introduction: Severe COVID‐19 management is still challenging. Having a laboratory factor to predict the severity of a patient's condition can be very useful in how to approach each patient. There have been studies concentrating on the correlation between serum C‐reactive protein (CRP) level and COVID‐19 severity but we aim to reach a threshold for CRP in disease severity determination. Methods: We conducted a thorough search on PubMed, Web of Science and Google Scholar databases from early 2019 to October 2021, and 323 studies were assessed for eligibility in three phases. We used the Newcastle‐Ottawa Scale to examine the validity of the studies. The t‐test was applied for the CRP level cutoffs. Results: Eventually, 11 articles and 1615 patients were included in this systematic review. Our analysis evaluated combined mean, median, and standard deviation of severe patients' CRP to be respectively 73.37, 53.80, and 47.936. Based on the combined mean, 75 mg/dL was suggested as an initial threshold for baseline CRP in hospitalized patients for developing severe conditions. Conclusion: This study recommends that COVID‐19 patients with on‐admission serum CRP levels of 75 mg/dL and more are likely associated with severe conditions. Thus, anti‐inflammatory agents and further following may be helpful in these patients. [ABSTRACT FROM AUTHOR]

Aims: Patients with heart failure (HF) exhibit poor prognosis, which is further deteriorated by pulmonary hypertension (PH), with negative impact on morbidity and mortality. As PH due to left HF (LHF‐PH) is among the most common causes of PH, there is an urge according to the 2021 European Society of Cardiology HF guidelines to find new biomarkers that aid in prognostication of this patient cohort. Given the role of tumour necrosis factor‐alpha (TNF‐α) in HF progression, we aimed to investigate the prognostic value of plasma proteins related to TNF‐α in patients with LHF‐PH, in relation to haemodynamic changes following heart transplantation (HT). Methods and results: Twenty TNF‐α‐related plasma proteins were analysed using proximity extension assay in healthy controls (n = 20) and patients with LHF‐PH (n = 67), before and 1 year after HT (n = 19). Plasma levels were compared between the groups, and the prognostic values were determined using Kaplan–Meier and Cox regression analyses. Plasma levels of lymphotoxin‐beta receptor (LTBR), TNF receptor superfamily member 6B (TNFRSF6B), and TNF‐related apoptosis‐inducing ligand receptors 1 and 2 (TRAIL‐R1 and TRAIL‐R2, respectively) were higher in LHF‐PH pre‐HT vs. controls (P < 0.0001), as well as higher in pre‐HT vs. post‐HT (P < 0.001). The elevated pre‐HT levels of LTBR, TNFRSF6B, TRAIL‐R1, and TRAIL‐R2 decreased towards the levels of healthy controls after HT. Higher preoperative levels of LTBR, TNFRSF6B, TRAIL‐R1, and TRAIL‐R2 in LHF‐PH were associated with worse survival rates (P < 0.002). In multivariate Cox regression models, each adjusted for age and sex, LTBR, TNFRSF6B, TRAIL‐R1, and TRAIL‐R2 predicted mortality (P < 0.002) [hazard ratio (95% confidence interval): 1.12 (1.04–1.19), 1.01 (1.004–1.02), 1.28 (1.14–1.42), and 1.03 (1.02–1.04), respectively]. Conclusions: Elevated pre‐HT plasma levels of the TNF‐α‐related proteins LTBR, TNFRSF6B, TRAIL‐R1, and TRAIL‐R2 in LHF‐PH decreased 1 year after HT, displaying a normalization pattern towards the levels of the healthy controls. These proteins were also prognostic, where higher levels were associated with worse survival rates in LHF‐PH, providing new insight in their potential role as prognostic biomarkers. Larger studies are warranted to validate our findings and to investigate their possible pathobiological mechanisms in LHF‐PH. [ABSTRACT FROM AUTHOR]

Proteomic approaches have unique advantages in the identification of biological pathways that influence physical frailty, a multifactorial geriatric syndrome predictive of adverse health outcomes in older adults. To date, proteomic studies of frailty are scarce, and few evaluated prefrailty as a separate state or examined predictors of incident frailty. Using plasma proteins measured by 4955 SOMAmers in the Atherosclerosis Risk in Community study, we identified 134 and 179 proteins cross‐sectionally associated with prefrailty and frailty, respectively, after Bonferroni correction (p < 1 × 10−5) among 3838 older adults aged ≥65 years, adjusting for demographic and physiologic factors and chronic diseases. Among them, 23 (17%) and 82 (46%) were replicated in the Cardiovascular Health Study using the same models (FDR p < 0.05). Notably, higher odds of prefrailty and frailty were observed with higher levels of growth differentiation factor 15 (GDF15; pprefrailty = 1 × 10−15, pfrailty = 2 × 10−19), transgelin (TAGLN; pprefrailty = 2 × 10−12, pfrailty = 6 × 10−22), and insulin‐like growth factor‐binding protein 2 (IGFBP2; pprefrailty = 5 × 10−15, pfrailty = 1 × 10−15) and with a lower level of growth hormone receptor (GHR, pprefrailty = 3 × 10−16, pfrailty = 2 × 10−18). Longitudinally, we identified 4 proteins associated with incident frailty (p < 1 × 10−5). Higher levels of triggering receptor expressed on myeloid cells 1 (TREM1), TAGLN, and heart and adipocyte fatty‐acid binding proteins predicted incident frailty. Differentially regulated proteins were enriched in pathways and upstream regulators related to lipid metabolism, angiogenesis, inflammation, and cell senescence. Our findings provide a set of plasma proteins and biological mechanisms that were dysregulated in both the prodromal and the clinical stage of frailty, offering new insights into frailty etiology and targets for intervention. [ABSTRACT FROM AUTHOR]

β2‐microglobulin (β2‐m) is a plasma protein derived from physiological shedding of the class I major histocompatibility complex (MHCI), causing human systemic amyloidosis either due to persistently high concentrations of the wild‐type (WT) protein in hemodialyzed patients, or in presence of mutations, such as D76N β2‐m, which favor protein deposition in the adulthood, despite normal plasma levels. Here we describe a new transgenic Caenorhabditis elegans (C. elegans) strain expressing human WT β2‐m at high concentrations, mimicking the condition that underlies dialysis‐related amyloidosis (DRA) and we compare it to a previously established strain expressing the highly amyloidogenic D76N β2‐m at lower concentrations. Both strains exhibit behavioral defects, the severity of which correlates with β2‐m levels rather than with the presence of mutations, being more pronounced in WT β2‐m worms. β2‐m expression also has a deep impact on the nematodes' proteomic and metabolic profiles. Most significantly affected processes include protein degradation and stress response, amino acids metabolism, and bioenergetics. Molecular alterations are more pronounced in worms expressing WT β2‐m at high concentration compared to D76N β2‐m worms. Altogether, these data show that β2‐m is a proteotoxic protein in vivo also in its wild‐type form, and that concentration plays a key role in modulating pathogenicity. Our transgenic nematodes recapitulate the distinctive features subtending DRA compared to hereditary β2‐m amyloidosis (high levels of non‐mutated β2‐m vs. normal levels of variant β2‐m) and provide important clues on the molecular bases of these human diseases. [ABSTRACT FROM AUTHOR]

Diet can affect the inflammatory state of the body. Accordingly, the dietary inflammatory index (DII) has been developed to quantify the inflammatory properties of food items. This study sought to investigate the association between dietary inflammation index (DII) and the odds ratio of elevated CRP (E‐CRP) through a systematic review and meta‐analysis study. The International electronic databases of PubMed, Web of Science (ISI), and Scopus were searched until May 2023 to find related articles. From 719 studies found in the initial search, 14 studies, with a total sample size of 59,941 individuals, were included in the meta‐analysis. The calculated pooled odds ratio (OR) of E‐CRP in the highest DII category was 1.39 (95% CI: 1.06, 1.14, test for heterogeneity: p =.63, and I2 =.0%) in comparison with the lowest DII category. Also, the results of this study showed that each unit increase in DII as a continuous variable generally elicited a 10% increase in the odds of E‐CRP (OR 1.10, 95% CI 1.06, 1.14, test for heterogeneity: p =.63, and I2 =.0%). Subgroup meta‐analyses showed that there is a higher E‐CRP odds ratio for the articles that reported energy‐adjusted DII (E‐DII) instead of DII, the studies that measured CRP instead of hs‐CRP, and the studies that used 24‐h recall instead of FFQ as the instrument of dietary intake data collection. Individuals with a higher DII were estimated to have higher chances of developing elevated serum CRP. This value was influenced by factors such as the participants' nationality, instruments of data collection, methods used to measure inflammatory biomarkers, study design, and data adjustments. However, future well‐designed studies can help provide a more comprehensive understanding of the inflammatory properties of diet and inflammatory serum biomarkers. [ABSTRACT FROM AUTHOR]

Antibodies from malaria-exposed individuals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)-anchored merozoite surface protein 1 (MSP1) and particularly its C-terminal fragment (MSP119) comprised of two epidermal growth factor (EGF)-like modules. In this paper, we revisit the role of agglutination and immunity using transgenic fluorescent marker proteins. We describe expression of heterologous MSP119‘miniproteins’ on the surface of Plasmodium falciparum merozoites. To correctly express these proteins, we determined that GPI-anchoring and the presence of a signal sequence do not allow default export of proteins from the endoplasmic reticulum to merozoite surface and that extra sequence elements are required. The EGFs are insufficient for correct trafficking unless they are fused to additional residues that normally reside upstream of this fragment. Antibodies specifically targeting the surface-expressed miniprotein can inhibit erythrocyte invasion in vitro despite the presence of endogenous MSP1. Using a line expressing a green fluorescent protein–MSP1 fusion protein, we demonstrate that one mode of inhibition by antibodies targeting the MSP119 domain is the rapid agglutinating of merozoites prior to erythrocyte attachment. [ABSTRACT FROM AUTHOR]

The platelet-derived growth factor (PDGF) family was for more than 25 years assumed to consist of only PDGF-A and -B. The discovery of the novel family members PDGF-C and PDGF-D triggered a search for novel activities and complementary fine tuning between the members of this family of growth factors. Since the expansion of the PDGF family, more than 60 publications on the novel PDGF-C and PDGF-D have been presented, highlighting similarities and differences to the classical PDGFs. In this paper we review the published data on the PDGF family covering structural (gene and protein) similarities and differences among all four family members, with special focus on PDGF-C and PDGF-D expression and functions. Little information on the protein structures of PDGF-C and -D is currently available, but the PDGF-C protein may be structurally more similar to VEGF-A than to PDGF-B. PDGF-C contributes to normal development of the heart, ear, central nervous system (CNS), and kidney, while PDGF-D is active in the development of the kidney, eye and brain. In adults, PDGF-C is active in the kidney and the central nervous system. PDGF-D also plays a role in the lung and in periodontal mineralization. PDGF-C is expressed in Ewing family sarcoma and PDGF-D is linked to lung, prostate and ovarian cancers. Both PDGF-C and -D play a role in progressive renal disease, glioblastoma/medulloblastoma and fibrosis in several organs. [ABSTRACT FROM AUTHOR]

Background: Attainment of adequate transfer of passive immunity (TPI) is critical to health of calves; however, studies comparing available tools for measurement of TPI in individual beef animals are limited. Objectives: To report agreement between 4 tests evaluating individual TPI status in beef calves. Animals: One hundred ninety‐six beef calves born to cows and heifers presenting for calving management or dystocia. Methods: Retrospective study to assess serum immunoglobulin (IgG) concentrations via turbidimetric immunoassay (TI), gamma‐glutamyl transferase (GGT), serum total protein (TP), and single radial immunodiffusion (RID; reference standard). Test agreement was evaluated using Passing‐Bablok regression, Bland‐Altman analysis, Cohen's kappa, and receiver operating characteristic (ROC) curves with and without covariate adjustment to determine optimal thresholds. Results: Correlation between RID and test results varied: TI, ρ = 0.757; TP, ρ = 0.715; GGT: ρ = 0.413. For the TI compared to RID, regression analysis identified a constant (intercept = −0.51 [CI: −2.63, 3.05]) and proportional (slope = 1.87 [CI: 1.69, 2.08]) bias. Based on ROC, TI concentrations of ≤9.89 and ≤13.76 g/L, and TP concentrations of ≤5.5 and ≤6.0 g/dL, indicated IgG concentrations <18.0 and <25.0 g/L, respectively. Conclusions and Clinical Importance: Within this cohort of calves, TI demonstrated the best correlation with RID; however, significant bias was identified which led to frequent underestimation of IgG concentration. Serum total protein demonstrated less correlation with RID but had less misclassification than TI. Both TI and TP demonstrated less correlation for calves that received colostrum replacement prompting clinical awareness of colostrum type when evaluating individual TPI in beef calves. [ABSTRACT FROM AUTHOR]

During skeletal development, bone growth and mineralization require transport of substantial amounts of calcium, while maintaining very low concentration. How an organism overcomes this major logistical challenge remains mostly unexplained. To shed some light on the dynamics of this process, cryogenic focused ion beam‐scanning electron microscopy (cryo‐FIB/SEM) is used to image forming bone tissue at day 13 of a chick embryo femur. Both cells and matrix in 3D are visualized and observed as calcium‐rich intracellular vesicular structures. Counting the number of these vesicles per unit volume and measuring their calcium content based on the electron back‐scattering signal, the intracellular velocity at which these vesicles need to travel to transport all the calcium required for the mineral deposited in one day within the collagenous tissue can be estimated. This velocity at 0.27 µm s−1 is estimated, which is too large for a diffusion process and rather suggests active transport through the cellular network. It is concluded that calcium logistics is hierarchical and based on several transport mechanisms: first through the vasculature using calcium‐binding proteins and the blood flow, then active transport over tens of micrometers through the network of osteoblasts and osteocytes, and finally diffusive transport over the last one or two microns. [ABSTRACT FROM AUTHOR]

Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nmin diameter) in the bloodstreamcarry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, and fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1,DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs obtained from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. [ABSTRACT FROM AUTHOR]

Objective: To retrospectively analyze the efficacy and safety of dupilumab in the treatment of bullous pemphigoid. Methods: From October 2020 to October 2022, the medical records of patients with bullous pemphigoid who were treated with dupilumab in our department were collected retrospectively to analyze the therapeutic effect and changes in laboratory indexes. Results: The records of a total of 11 patients with bullous pemphigoid who were treated with dupilumab was reviewed. Within 2 weeks of the treatment, 10 (90.9%) of the 11 patients had complete or substantial control of the disease. The BPDAI scores of the patients decreased from baseline 113 (62, 181) to 37 (6, 130) at 2 weeks (p =.001) and 4 (0, 37) at 12 weeks after treatment (p <.001). In the 11 patients treated with dupilumab, the relief time of pruritus was 0–3 days (0.5, 7) days, and the pruritus was significantly alleviated after 2 weeks (t = 15.925, p <.001). The DLQI score decreased from (25.5 ± 2.5) before treatment, to (11.8 ± 4.4) at 2 weeks (t = 10.764, p <.001) and (2.1 ± 1.9) at 12 weeks (t = 30.038, p <.001). The patients had high eosinophil counts, high serum IgE levels, low serum total protein levels, and abnormal blood coagulation function. The aforementioned indicators gradually returned to normal after treatment. No adverse reactions occurred during the treatment. Conclusion: Dupilumab can effectively control the condition of bullous pemphigoid, efficiently relieve pruritus symptoms, and is relatively safe. [ABSTRACT FROM AUTHOR]

We have tested the hypothesis that dense cell formation in sickle cell disease is associated with increased binding of calpromotin to the membrane, an event that occurs during the activation of calcium-dependent potassium transport. By SDS polyacrylamide gel electrophoresis, we found that sickle cell membranes contained more calpromotin than did normal membranes when stained with Coomassie brilliant blue or when transferred to nitrocellulose paper and immunostained with horseradish peroxidase. Also, the membranes from dense sickle cells contained significantly (P = 0.00055) higher levels of calpromotin, 2.62+/-1.59 microg/mg membrane protein, compared to light sickle cells, 1.40+/-0.70 microg/mg membrane protein, when measured by an enzyme-linked immunosorbent assay. The ratio of calpromotin associated with dense cell membranes to light cell membranes was significantly greater than 1.0 (P < 0.00005). Transmission electron micrographs of immunogold-labelled membranes supported the increase in calpromotin binding in dense sickle cell membranes. In addition, the immunogold probe demonstrated clustering, which was not observed in light sickle cell membranes nor in normal membranes. Finally, we incubated HbSS cells in vitro using a repetitive deoxygenation/ reoxygenation procedure to produce dense cells and then measured the levels of calpromotin associated with their membranes. As expected, the levels of calpromotin bound to the membrane doubled during the procedure relative to the basal levels at the beginning of the incubation. The correlation coefficient, calculated between the increase in dense cell formation and the increase in calpromotin associated with the membrane, was statistically significant (P = 0.038). The results demonstrate that an increase in calpromotin binding to the membrane is associated with dense cell formation presumably through the activation of the calcium-dependent potassium channel.

The reaction of human hemoglobin with carbon monoxide has been investigated near the equilibrium isosbestic wavelength (i.e. 426 nm). As previously reported by others [Gray, R. D. & Gibson, Q. H. (1971) J. Biol. Chem. 246, 5176-5178], in the presence of 0.1 M phosphate pH 7.0 a rise-and-fall kinetic pattern can be observed at this wavelength, which indicates the presence of at least one spectroscopically detectable intermediate species. In this paper we demonstrate that (a) the intermediate species is thermodynamically stable; (b) both phases refer to bimolecular processes; (c) only the initial fast phase is observed when deoxyhemoglobin is reacted with substoichiometric amounts of CO (i. e. final [CO]/[heme] ≤ 0.5); (d) only the second slow phase is observed when hemoglobin that is partially saturated with CO (Y ≤ 0.5) is reacted with saturating CO concentrations; (e) the CO dissociation rate constant measured on the intermediate formed after a partial CO saturation at a final Y ≈ 0.4 has a value similar to that observed starting from the fully liganded form. These results can be accounted for by a two-state allosteric model [Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118] under the assumption that either (a) 426 nm is an isosbestic wavelength for the T0-R spectral changes but not for the T0-T liganded reaction; or (b) a functional heterogeneity of the two types of subunits is present in the T state and at this wavelength this feature is spectroscopically detectable. [ABSTRACT FROM AUTHOR]

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/ 50 ± 10 mol NF-L and an apparent dissociation constant Kd = 4.3 × 10-7 M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 × 10-6 M with I mol fodrin bound/80 ± 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 × 10-7 M and the maximum binding was I mol fodrin/55 ± 10 mol GFP. [ABSTRACT FROM AUTHOR]

Adult bovine aortic endothelial (ABAE) cells, exposed to serum-free medium, specifically bind 125I-labeled human high-density lipoprotein (125I-HDL). Addition of human lipoprotein-deficient serum (LPDS) reduces the specific binding of 125I-HDL in a concentration-dependent manner, such that LPDS at a concentration of 6 mg protein/ml almost completely inhibits the specific binding of 125I-HDL. ABAE cultures exposed to 125I-labeled LPDS (125I-LPDS) specifically bind two peptides, which appear as minor iodinated components in 125I-LPDS. The binding of these two components is abolished in the presence of excess amounts of unlabeled LPDS or HDL. Preincubation of ABAE cells with 25-hydroxycholesterol (25-HC) results in an increase in the binding of the two 125I-LPDS components, similar to the increase observed in 125I-HDL binding in the presence of 25-HC. These two LPDS components comigrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with apolipoproteins A-I and A-IV of molecular masses 28 kDa and 43 kDa respectively. Furthermore, these two proteins were transfered from the SDS gel to nitrocellulose paper and interacted specifically with anti-(A-I) and anti-(A-IV) sera respectively. When ABAE cultures, pretreated with 25-HC in the presence of LPDS, are subjected to cell-surface iodination, the A-IV appears as one of the major proteins on the cell surface accessible to iodination. The interaction of A-IV with the cell surface of 25-HC-treated cells is not specific to ABAE cells and appears also in human skin fibroblasts. Analysis of the relative amounts of various apolipoproteins in the 125I-HDL bound to ABAE cells demonstrates a decrease in the relative amount of iodinated A-II concomitant with increase in the relative amounts of the other iodinated apolipoproteins, when compared to the composition of the native 125I-HDL. These changes are similar whether the binding is done in the presence or absence of LPDS. It indicates that the decrease in 125I-HDL binding in the presence of LPDS is not due to displacement of the iodinated apolipoproteins A-I and A-IV in the 125I-HDL by unlabeled A-I and A-IV present in LPDS. The results indicate that free apolipoproteins A-I and A-IV, present in LPDS, can displace HDL on the cell surface of ABAE cells. Thus, free A-I and A-IV, present in plasma, control the binding of HDL to endothelial cells and may regulate the process of cholesterol removal from the cells performed by HDL. [ABSTRACT FROM AUTHOR]

The serum glycoprotein α2-macroglobulin can be converted into a potent macrophage-activating factor that promotes the Fcγ receptor-mediated phagocytosis of macrophages, through modification of the sugar moiety with liposome-treated B-cell glycosidase(s). This paper discusses the activation mechanism of B-cell membranous glycosidase by liposomes using mouse splenic B cells. B-cell membranous β-galactosidase and βN-acetylglucosaminidase were significantly activated by liposome treatment, and this process can be regulated by trypsin-sensitive protein. To clarify the contribution of trypsin-sensitive protein to enzyme activities, the B-cell surface antigen receptor was studied. With the addition of a Fab′ fragment of anti-mouse IgM but not IgD antibody, the activation of both glycosidases induced by liposomes was significantly inhibited and was essentially the same as that of saline-treated glycosidase activities. Consequently, interactions of liposomes with cell-surface IgM may cause B-cell membranous glycosidase activation. A significant decrease in membrane fluidity, particularly near the membrane surface rather than deep within the membrane, was observed in liposome-treated B cells using electron spin resonance. Liposomes would thus appear to interact with B cells via cell-surface IgM, with a consequent decrease in membrane fluidity, as well as the activation of B-cell membranous glycosidases, causing α2-macroglobulin to be converted into a macrophage activating factor. [ABSTRACT FROM AUTHOR]

The intravenous injection into mice of small amounts of heat-aggregated bovine serum albumin (BSA) results in deposition of BSA within the glomerulus. Injection of rabbit anti-BSA antibody at a time when all circulating BSA has been cleared results in in situ formation of BSA anti-BSA immune complexes with fixation of mouse C3. Subsequent injection of passively-prepared immune complexes gives much enhanced deposition of immune material when com- pared with mice given immune complexes alone. This paper shows that this is an antigen-specific phenomenon and not a result of non-specific trapping by damaged glomeruli. The implications of these observations on the pathogenesis of immune complex glomerulonephritis are discussed. [ABSTRACT FROM AUTHOR]

As demonstrated by a simple procedure based on indirect immunoprecipitation, proteins retained on heparin- Sepharose 4B from postrnitochondrial supernatants of rat liver and Zajdela hepatoma catalyse the translation of rabbit serum albumin rnRNA in the presence of ribosomal subunits from rather, Zajdela hepatoma or rabbit ceticulocytes. The albumin synthesis shows an optimum at 1.5 mM MgCI2 and 25 mM KCl and requires ATP and GTP. It is significantly stimulated by tRNA and proceeds for more than 2 h. suggesting a high rate of reini- tiation. At the optimum ribosomes : rnRNA ratio of 13: 1, the immunoprecipitable radioactivity exceeds 15 - 20-times the blank values. Fluorography of polyacrylamide slabs after electrophoresis of immunoprecipitates revealed the presence of only complete full size serum albumin without any smaller peptides resulting from premature terminations of polypeptide chains, demonstrating faithful translation. In stained gels only, both heavy and light chains of immunoglobulin G were found, indicating that the assay procedure is highly specific and reliable. The fractionated heterologous protein-synthesizing system described in this paper may be generally useful for studies on the synthesis of specific proteins and factors affecting their rates since, unlike comparable translation assays. a precise calculation of the balance of newly synthesized proteins is possible. [ABSTRACT FROM AUTHOR]

Peptide sequences obtained from hen ovotransferrin are compared with the complete amino acid sequence of the protein deduced from a cDNA sequence (Jeltsch and Chambon, preceding paper). Of the 705 positions of the whole protein 605 can be matched by the peptide sequences. Some possible discrepancies between the two methods are pointed out. The two halves of the chain show marked similarities in their sequences with 37 % identical residues. The positions of the 15 disulphide bridges are shown; there are 6 homologous bridges in each half of the molecule and 3 extra bridges which occur only in the C-terminal half. The terminal residues of the half- molecule fragments obtained by limited proteolysis are identified. The two domains are joined by a 9-residue connecting peptide. Sequence variability has been found at 9 positions. The sequence of hen ovotransfernn is compared with the partial sequence available for human transferrin. From this some tentative conclusions about the identities of the metal-binding residues and about the evolution of transferrin are reached. [ABSTRACT FROM AUTHOR]

The present paper reports data for the effect of pH and D-glycerate 2,3-bisphosphate (D-glycerate-2,3-P2) on the oxygen equilibrium of normal and SH(β93)-modified human hemoglobin. At sufficiently high D-glycerate- 2,3-P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at all pH values between 6 and 9. Furthermore the difference in the affinity for D-glycerate-2,3-P2 between deoxy and oxy hemoglobin remains constant with pH, implying that the pK values of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of D-glycerate-2,3-P2 on the hemoglobin molecule. In the hemoglobins modified in position β93, the difference in affinity between deoxy and oxy hemoglobin for D-glycerate-2.3-P2 decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for D-glycerate-2,3-P2. The effect of the chemical modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especially pronounced in the presence of phosphates. [ABSTRACT FROM AUTHOR]

Sheep-serum transferrin shows marked polymorphism and more than 20 alleles have been identified although only 4 or 5 of these have a frequency higher than 1%. Each of the alleles has two bands in starch-gel electrophoresis, corresponding to a major and a minor fraction. This paper describes the isolation and partial characterisation of the two fractions from the transferrin of sheep homozygous for Tf B. The purification consisted of: (a) precipitation by ammonium sulphate, (b) chromatography on CM-cellulose and, finally (c) chromatography on DEAE-Sephadex. The purification procedure had no effect on the electrophoretic mobility of the two fractions and they appeared homogeneous by starch-gel and polyamide-gel electrophoresis and by immuno-electrophoresis. The amino-acid compositions of both fractions were very similar and the sequences of the first eight amino-acid residues: Ser-Pro-Glu-Lys-Thr-Val-Arg-Trp-were identical for both bands. These results, and the fact that the two fractions are found in all genetic variants and always have the same relative mobility strongly suggest that the differences do not lie in the polypeptide chain. From the results of hydrolysis by neuraminidase and assay of sialic acid, the major and minor fractions most probably contain two and three sialic acid residues (exclusively N-acetylneuraminic acid) respectively, thus explaining the different electrophoretic mobilities. The sialic-acid content has been calculated on the basis of a molecular weight of 77500 as determined both by low-speed equilibrium ultracentrifugation and by Archibald's method. The following physico-chemical constants have been established: absorption coefficient at 280nm, A1 mg/cm³1 cm = 1.25 ± 0.01 cm² · mg-1; partial specific volume, &vmacr; = 0.734 cm³ · g-1; sedimentation coefficient s20.wo = 5.15 S; refractive-index increment, dn/dc = 0.173 cm³ · g-1. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,χ) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vχ chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of CNBr fragment H4 of the murine immunoglobulin MOPC 173 (IgG2a,χ) has been determined, thus completing the sequence determination of the entire heavy chain. The H4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CH1 domain, the hinge region and the beginning of CH2. Sequence comparisons suggest that the CH1 domain is highly conserved in evolution, and allows the definition of two additional isotypic-specific regions. [ABSTRACT FROM AUTHOR]

This paper reports some physico-chemical and functional properties of two of the isolated hemoglobin components from trout (Salmo irideus) blood (Hb trout I and Hb trout IV). Both hemoglobins in the oxygenated state are tetrameric at pH between 7 and 7.8 and protein concentration above &assymp; 0.1 mg/ml. The tetramer is extremely stable towards dissociation into subunits and the stability constants, estimated by differential gel filtration, are between 10 and 50 times larger than that of human hemoglobin in phosphate buffer. A very small degree of dissociation is also suggested by the very small extent of hybrid formation even in concentrated KI or triethanolamine. The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. Hb trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity (θ); furthermore upon deoxygenation the value of θ at 222 nm becomes more negative by about 50%. On the ether hand Hb trout I shows a different circular dichroism spectrum and in particular the ellipticity at 222 nm is lower than that of Hb trout IV. Moreover the spectrum is insensitive to the presence of heme ligands. Hb trout IV binds human haptoglobin (Hp) as shown by quenching of the fluorescence of the tryptophanyl residues of Hp upon addition of hemoglobin. The apparent stoichiometry of complex formation corresponds to two Hb tetramers per Hp molecule (while for human hemoglobin the stoichiometry corresponds to &assymp; one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O2 and especially CO combination as studied by flash photolysis. The effect of several third components on the O2 equilibrium of both Hb trout I and IV has been investigated. In general the effect of adding ions or other molecules to a solution of lib trout I is very minor. On the other hand Hb trout IV is more susceptible to changes in solvent composition. Of particular importance is the action of ATP, the physiological organic phosphate, which is without effect on Hb trout I, while it has a clear effect on Hb trout IV. The addition of ATP shifts the Root effect, the characteristic property of Hb trout IV, to higher pH values, thereby acting as one of the allosteric effectors. [ABSTRACT FROM AUTHOR]

Rat liver microsomes were separated into three subfractions by means of ultracentrifugation through sucrose media of different densities and in the presence of different cations. Detergent extracts of these preparations were analysed by double diffusion in agar gel and immunoelectrophoresis with rabbit antisera against each of the subfractions. One subfraction was derived from the ‘rough’ and another from the ‘smooth’ part of the endoplasmic reticulum of the liver parenchymal cells. Their extracts contained at least thirteen soluble antigens in common. However, fraction-specific antigens were also present. Thus, the extracts of the rough (R) membranes contained at least one typical antigen, not found in any of the other fractions. An antiserum against this component also precipitated a non-migrating antigen present only in the extracts of smooth (Sa) membranes. These two antigens may represent different molecular forms of a substance involved in binding of the ribosomes or in the assembly of membrane subunits. At least eleven of the antigens common for the two fractions exhibited enzymatic activities when assayed after precipitation with antibody in the immunoelectro- phoretic plates. Six immunologically and electrophoretically distinct antigens had esterase activity with α-naphthyl propionate as substrate. Two of these esterases also split indoxyl acetate. Three other antigens with acid phosphatase activity split both α-naphthyl acid phosphate and β-glycerophosphate. Three antigens had nucleoside diphosphatase activity when tested with uridine- or inosine-diphosphate. Preliminary experiments also suggested that two additional antigens possessed NADH-diaphorase activity and thus could belong to the microsomal electron trans- port systems. The third subfraction consisted of electron-microscopically smooth membranes (Sb), enzymatically different from those of the endoplasmic reticulum. The antigens typical for the latter were either absent or present only in minor and variable concentrations. Its extracts contained at least two typical antigens. One of these was identified as contaminating ferritin. The nature and origin of the other antigen has not yet been established. All antigens described in this paper were immunologically different from the common rat serum proteins. [ABSTRACT FROM AUTHOR]

Phylogenic research on the haptoglobin of different animal species led us to study the structure of rabbit haptoglobin. Rabbit haptoglobin, whose homogeneity was checked according to several criteria, was submitted to hydrolysis by pronase (72 h, 39°, pH 8.2); the hydrolysate was then filtered through Sephadex G-25, and the resulting glycopeptide fraction was separated on Dowex 50-X2 (H+) into two parts, one (A) not retained by the resin, the other one (B) collected after elution with 0.5 M NaCl. Amino-acid and carbohydrate composition, nature of end groups, and timing of sialic acid release, were determined on fraction A; from the amount of aspartic acid, the molecular weight was estimated to be no less than 2860. Fraction B, which amounted to one third of fraction A, could not be analysed on account of the insufficient quantity available. Quantitative paper electrophoresis at pH 3.5 and 6.2 yielded 8 glycopeptides from fraction A and 2 from fraction B. These various glycopeptides showed notable differences in their amino-acid composition, mainly with respect to glutamic acid, proline, glycine and histidine. On the contrary, no significant differences were found in the carbohydrate composition, with the exception of the oses N-acetylneuraminic acid ratio, which varies from 2 to 10. These results suggest 6 oligosaccharide units to be present in rabbit haptoglobin, as compared to 7 in human haptoglobin, bound to peptidic fractions varying in their amino-acid composition in the neighbourhood of the carbohydrate-peptide linkage. Although the precise nature of this bond has not been ascertained, it does not appear to be an O-glycosidic one. [ABSTRACT FROM AUTHOR]

This paper describes a method for simultaneous measurement of the accumulation of plasma protein and erythrocytes in skin reactions of hypersensitivity to bovine γ-globulin (BGG) and tuberculin PPD in the guinea-pig. The procedure consists in giving 125I-labelled plasma albumin and 51Cr-labelled autologous erythrocytes together by intravenous injection into guinea-pigs bearing skin lesions of allergic inflammation at different times and for different periods during the development of the skin reactions. Isotope accumulation in excised skin reactions is measured by scintillation counting at different stages during the evolution of hypersensitivity responses. Skin reactions of combined anaphylactic and Arthus hypersensitivity to BGG were characterized by pronounced increased vascular permeability principally in the first hour. In established 24-hour hypersensitivity reactions to both antigens (BGG and PPD) there was continuing accumulation of both plasma albumin and erythrocytes. During the development of the tuberculin reaction, an intermediate phase of isotope accumulation occurred between 6 and 9 hours after skin testing; serum transfer studies showed that this intermediate peak was not attributable to circulating antibody alone. These isotope tracer techniques were also applied to study vascular permeability in systemically transferred reactions of delayed hypersensitivity and in the vascular response to the intradermal injection of an inflammatory factor generated by antigen-activation of sensitized lymphocytes. It was concluded that isotope tracing provided objective and sensitive methods for analyzing microvascular responses in allergic inflammation. [ABSTRACT FROM AUTHOR]

This paper describes a new method for the morphological study of individual antibody-forming cells (AFC) on cell smears of the quality of normal haematological preparations. The early AFC response to polymerized flagellin of S. adelaide was studied in vivo using C57BL mice, which have very low background levels of AFC and in vitro using dispersed spleen cell cultures from CBA mice. AFC, arising as a result of in vivo or in vitro stimulation were found to comprise a heterogeneous population, including basophilic mononuclear cells, lymphocytes of most sizes, immature blast cells and occasional plasma cells. The earliest AFC detected comprised a high percentage (28 per cent in vivo, 31 per cent in vitro) of small lymphocyte-like cells. Studies of the incorporation of [³H]thymidine showed that most AFC arose by proliferation but that a proportion of AFC, the small lymphocyte-like cells, arose by differentiation of precursor cells not involving cell division. The effects of antigen concentration on the kinetics of AFC were investigated in vitro. Subtolerogenic antigen doses caused a delayed and decreased AFC response. [ABSTRACT FROM AUTHOR]

Summary: Pattern recognition receptors (PRRs) are plasma membrane‐localised proteins that sense molecular patterns to initiate pattern‐triggered immunity (PTI). Receptor‐like cytoplasmic kinases (RLCKs) function downstream of PRRs to propagate signal transduction via the phosphorylation of substrate proteins. The identification and characterisation of RLCK‐regulated substrate proteins are critical for our understanding of plant immunity.We showed that SHOU4 and SHOU4L are rapidly phosphorylated upon various patterns elicitation and are indispensable for plant resistance to bacterial and fungal pathogens. Protein–protein interaction and phosphoproteomic analysis revealed that BOTRYTIS‐INDUCED KINASE 1, a prominent protein kinase of RLCK subfamily VII (RLCK‐VII), interacted with SHOU4/4L and phosphorylated multiple serine residues on SHOU4L N‐terminus upon pattern flg22 treatment. Neither phospho‐dead nor phospho‐mimic SHOU4L variants complemented pathogen resistance and plant development defect of the loss‐of‐function mutant, suggesting that reversible phosphorylation of SHOU4L is critical to plant immunity and plant development.Co‐immunoprecipitation data revealed that flg22 induced SHOU4L dissociation from cellulose synthase 1 (CESA1) and that a phospho‐mimic SHOU4L variant inhibited the interaction between SHOU4L and CESA1, indicating the link between SHOU4L‐mediated cellulose synthesis and plant immunity.This study thus identified SHOU4/4L as new components of PTI and preliminarily revealed the mechanism governing SHOU4L regulation by RLCKs. [ABSTRACT FROM AUTHOR]

Objective: The level of cartilage acidic protein 1 (CRTAC1) in plasma was recently discovered to be associated with osteoarthritis (OA) risk and progression to joint replacement in Iceland. This study was undertaken to validate these findings in an independent population. Methods: In this study, 1,462 plasma proteins were measured in 54,265 participants from the UK Biobank on the Olink Explore platform. We analyzed the association of plasma proteins with prevalent OA, incident OA, and progression to joint replacement. We assessed the specificity of OA association through comparison of associations with inflammatory joint diseases and with previous joint replacement. Results: The CRTAC1 protein showed the strongest association with prevalent knee OA (odds ratio [OR] 1.34 [95% confidence interval (95% CI) 1.27, 1.41]) and was associated with hip OA (OR 1.19 [95% CI 1.11, 1.28]). It predicted incident diagnosis of OA in the knee (hazard ratio [HR] 1.40 [95% CI 1.35, 1.46]) and hip (HR 1.25 [95% CI 1.19, 1.31]), as well as progression to joint replacement (HR 1.20 [95% CI 1.08, 1.33] for the knee and HR 1.22 [95% CI 1.08, 1.38] for the hip), while no association was found with inflammatory joint diseases. Individuals in the highest quintile of risk based on CRTAC1 level, age, sex, and body mass index had a 10‐fold risk of knee or hip OA within 5 years compared to those in the lowest quintile. Adding aggrecan core protein (ACAN) and neurocan core protein (NCAN) to the model improved the prediction of OA but not joint replacement. Furthermore, we replicated the association of CUB domain–containing protein 1 with prior joint replacement. Conclusion: Plasma CRTAC1 is a specific biomarker for OA and a predictor of OA risk and progression to joint replacement. Adding ACAN and NCAN protein levels to the CRTAC1 model improved the prediction of OA. [ABSTRACT FROM AUTHOR]

Background: Obese asthma is a complex phenotype and further characterization of the pathophysiology is needed. This study aimed to explore inflammation‐related plasma biomarkers in lean and overweight/obese asthmatics. Methods: We elucidated levels of inflammation‐related plasma proteins in obese asthma phenotypes in the population‐based cohort BAMSE (Swedish: Children, Allergy, Milieu, Stockholm, Epidemiology) using data from 2069 24‐26‐year‐olds. Subjects were divided into lean asthma (n = 166), lean controls (n = 1440), overweight/obese asthma (n = 73) and overweight/obese controls (n = 390). Protein levels (n = 92) were analysed using the Olink Proseek Multiplex Inflammation panel. Results: Of the 92 included proteins, 41 were associated with lean and/or overweight/obese asthma. The majority of proteins associated with overweight/obese asthma also associated with overweight/obesity among non‐asthmatics. Beta‐nerve growth factor (BetaNGF), interleukin 10 (IL‐10), and matrix metalloproteinase 10 (MMP10) were associated only with lean asthma while C‐C motif chemokine 20 (CCL20), fibroblast growth factor 19 (FGF19), interleukin 5 (IL‐5), leukemia inhibitory factor (LIF), tumor necrosis factor ligand superfamily member 9 (TNFRSF9), and urokinase‐type plasminogen activator (uPA) were associated only with overweight/obese asthma. Overweight/obesity modified the association between asthma and 3 of the proteins: fibroblast growth factor 21 (FGF21), interleukin 4 (IL‐4), and urokinase‐type plasminogen activator (uPA). In the overweight/obese group, interleukin‐6 (IL‐6) was associated with non‐allergic asthma but not allergic asthma. Conclusion: These data indicate distinct plasma protein phenotypes in lean and overweight/obese asthmatics which, in turn, can impact upon therapeutic approaches. [ABSTRACT FROM AUTHOR]

Some studies proposed the anti‐inflammatory effect of soy protein and soy isoflavones by changing the serum adiponectin and resistin levels. The purpose of this research was to determine the impact of soy isoflavones and soy protein on blood adiponectin and resistin levels in adults. Scopus, PubMed, Cochrane Library, ISI Web of Science, and ClinicalTrials.gov databases were searched until April 2022. The effect size was computed by the mean changes from the beginning for intervention and comparison groups and their standard deviation. In the case of significant heterogeneity, DerSimonian and Laird random‐effects model was used. Six and five clinical trials were selected for the systematic review and meta‐analysis, respectively. The overall estimate indicated that soy isoflavones in combination with soy protein did not significantly change serum adiponectin level (weighted mean differences (WMD) = 0.36 μg/ml; 95% confidence interval (CI): −0.26, 0.99; p =.25), but significantly increased serum resistin level (WMD = 0.64 ng/ml, 95% CI: 0.25, 1.04; p =.001). In combination with soy protein, soy isoflavones nonsignificantly increased serum adiponectin levels, but significantly increased resistin levels. New intervention studies with a high dose of soy isoflavones and soy protein in different parts of the world and an updated meta‐analysis are needed to confirm the results of our study. [ABSTRACT FROM AUTHOR]

Background and Purpose: The constitutive androstane receptor (CAR), a known xenobiotic sensor, plays an important role in drug metabolism by regulating numerous genes. The polycyclic aromatic hydrocarbon pyrene, an environmental pollutant, is a CAR activator and induces mouse hepatotoxicity via CAR. Here, we investigate the molecular mechanisms of the inflammatory response in pyrene-caused mice liver injury.Experimental Approach: Effects of pyrene on the liver were investigated in wild-type and CAR knockout (KO) mice. Levels of pyrene and its urinary metabolite were analysed by high performance liquid chromatography (HPLC). Inflammatory responses were measured by qRT-PCR, western blotting, and ELISA for cytokines.Key Results: Serum amyloid A proteins (SAAs) were markedly increased in the liver and serum of pyrene-exposed wild-type mice. IL-17-producing helper T cells (Th17 cells) and IL-17 levels were increased in the liver of pyrene-exposed wild-type mice. Hepatic mRNA levels of inflammatory cytokines including IL-1β, IL-6 and TNFα, and serum IL-6 levels were significantly elevated in pyrene-treated wild-type mice. However, these changes were not observed in CAR KO mice.Conclusion and Implications: CAR plays a crucial role in pyrene-caused mice liver inflammatory response with increased SAAs and Th17 cells. Our results suggest that serum SAAs may be a convenient biomarker for early diagnosis of liver inflammatory response caused by polycyclic aromatic hydrocarbons, including pyrene. CAR and Th17 cells may be potential targets for novel therapeutic strategies for xenobiotic-induced liver inflammation. [ABSTRACT FROM AUTHOR]

SUMMARY: Drought is a major environmental factor that limits the production of alfalfa (Medicago sativa). In the present study, M. sativa NUCLEAR TRANSPORT FACTOR 2‐LIKE (MsNTF2L) was identified as a nucleus‐, cytoplasm‐, and plasma membrane‐localized protein. Its transcriptional expression was highly induced by ABA and drought stress. Overexpression of MsNTF2L in Arabidopsis resulted in hypersensitivity to ABA during both the seed germination and seedling growth stages. However, transgenic Arabidopsis plants exhibited enhanced tolerance to drought stress by reducing the levels of reactive oxygen species (ROS) and increasing the expression of stress/ABA‐inducible genes. Consistently, analysis of MsNTF2L overexpression (OE) and RNA interference (RNAi) alfalfa plants revealed that MsNTF2L confers drought tolerance through promoting ROS scavenging, a decrease in stomatal density, ABA‐induced stomatal closure, and epicuticular wax crystal accumulation. MsNTF2L highly affected epicuticular wax deposition, as a large group of wax biosynthesis and transport genes were influenced in the alfalfa OE and RNAi lines. Furthermore, transcript profiling of drought‐treated alfalfa WT, OE, and RNAi plants showed a differential drought response for genes related to stress/ABA signaling, antioxidant defense, and photosynthesis. Taken together, these results reveal that MsNTF2L confers drought tolerance in alfalfa via modulation of leaf water loss (by regulating both stomata and wax deposition), antioxidant defense, and photosynthesis. Significance Statement: By studying the drought‐inducible genes of alfalfa (Medicago sativa), we discovered that the nucleus‐, cytoplasm‐, and plasma membrane‐localized protein NUCLEAR TRANSPORT FACTOR 2‐LIKE improves drought tolerance by modulating leaf water loss (by regulating both stomata and wax deposition), antioxidant defense, and photosynthesis. This gene might be targeted in future breeding programs to improve drought tolerance in alfalfa. [ABSTRACT FROM AUTHOR]

Patient‐derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin‐associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin‐ER interactions increased levels of PKR‐like ER kinase effectors and ER‐plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension‐mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis. Synopsis: How three‐dimensional spheroid cultures of mammary epithelial cells retain superior long‐term viability and stress resilience remains unclear. This work uncovers coupling between extracellular matrix (ECM) topology, cortical actin tension and endoplasmic reticulum (ER) function, which serves to safeguard protein secretion and cellular stress response. High cortical actin tension in 2D mammary epithelial cell cultures facilitates filamin‐PERK interaction and compromises ER function.In 3D spheroid cultures, mammary epithelial cell ‐ ECM interactions reduce cortical actin tension and filamin binding to PERK, maintaining ER homeostasis.Reduced cortical actin tension increases plasma membrane protrusion size and duration, favoring recruitment of negative curvature‐binding proteins that facilitate protein secretion. [ABSTRACT FROM AUTHOR]

Background and Purpose: Acute intermittent porphyria (AIP) is a rare disease caused by a genetic mutation in the hepatic activity of the porphobilinogen‐deaminase. We aimed to develop a mechanistic model of the enzymatic restoration effects of a novel therapy based on the administration of different formulations of recombinant human‐PBGD (rhPBGD) linked to the ApoAI lipoprotein. This fusion protein circulates in blood, incorporating into HDL and penetrating hepatocytes. Experimental Approach: Single i.v. dose of different formulations of rhPBGD linked to ApoAI were administered to AIP mice in which a porphyric attack was triggered by i.p. phenobarbital. Data consist on 24 h urine excreted amounts of heme precursors, 5‐aminolevulinic acid (ALA), PBG and total porphyrins that were analysed using non‐linear mixed‐effects analysis. Key Results: The mechanistic model successfully characterized over time the amounts excreted in urine of the three heme precursors for different formulations of rhPBGD and unravelled several mechanisms in the heme pathway, such as the regulation in ALA synthesis by heme. Treatment with rhPBGD formulations restored PBGD activity, increasing up to 51 times the value of the rate of tPOR formation estimated from baseline. Model‐based simulations showed that several formulation prototypes provided efficient protective effects when administered up to 1 week prior to the occurrence of the AIP attack. Conclusion and Implications: The model developed had excellent performance over a range of doses and formulation type. This mechanistic model warrants use beyond ApoAI‐conjugates and represents a useful tool towards more efficient drug treatments of other enzymopenias as well as for acute intermittent porphyria. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitor (ICI)‐based immunotherapy brought new hope for gastric cancer (GC) treatment. However, due to the lack of proper biomarkers, patient selection and outcome prediction for GC's immunotherapy remain unsatisfying. In this study, through applying an extracellular vesicle (EV) protein expression array, we assessed the correlation of plasma EV‐derived protein spectrum with outcomes of ICI‐related therapeutic combinations. Plasma from 112 GC patients received ICI‐related therapies were investigated retrospectively/prospectively as three cohorts. We identified four plasma EV‐derived proteins (ARG1/CD3/PD‐L1/PD‐L2) from 42 crucial candidate proteins and combined them as an EV‐score that robustly predicting immunotherapeutic outcomes at baseline and dynamically monitoring disease progression along with treatment. High EV‐score reflected microenvironmental features of stronger antitumour immunity, characterized by more activated CD8+ T/NK cells, higher TH1/TH2 ratio and higher expressions of IFN‐γ/perforin/granzymes in paired peripheral blood, which were verified by dataset analysis and in vivo experiments. EV‐score≥1 GC received more therapeutic benefits from ICIs, while EV‐score < 1 GC potentially benefited more from ICIs combining HER2‐targeted therapies. Collectively, through proposing a plasma EV‐score on protein level that powerfully predicting and monitoring GC's immunotherapeutic outcomes, our work facilitated clinical patient selection and decision‐makings, and provided mechanistical insights for immunotherapy‐related microenvironmental changes and improvements for current ICI‐regimens. [ABSTRACT FROM AUTHOR]

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell‐derived extracellular vesicles (EVs). Here we demonstrate that nano‐sized EVs from therapy‐grade human placental‐expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell‐secreted soluble factors via tangential flow‐filtration (TFF) and subtractive tandem mass‐tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein‐based flow cytometry, super‐resolution and electron microscopy verified EV identity. PLX‐EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose‐dependently inhibited T cell proliferation in vitro. Corona removal by size‐exclusion or ultracentrifugation abrogated angiogenesis. Re‐establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super‐resolution microscopy, electron microscopy and zeta‐potential shift, and served as a proof‐of‐concept. Understanding EV corona formation will improve rational EV‐inspired nano‐therapy design. [ABSTRACT FROM AUTHOR]