IXPE detection of polarized X-rays from magnetars and photon mode conversion at QED vacuum resonance.

The recent observations of the anomalous X-ray pulsars 4U 0142+61 and 1RXS J170849.0-400910 by the Imaging X-ray Polarimetry Explorer (IXPE) opened up a new avenue to study magnetars, neutron stars endowed with superstrong magnetic fields (B ≳ 1014 G). The detected polarized X-rays from 4U 0142+61 exhibit a 90° linear polarization swing from low photon energies (E ≲ 4 keV) to high energies (E ≳ 5.5 keV). We show that this swing can be explained by photon polarization mode conversion at the vacuum resonance in the magnetar atmosphere; the resonance arises from the combined effects of plasma-induced birefringence and Quantum electrodynamics (QED)-induced vacuum birefringence in strong magnetic fields. This explanation suggests that the atmosphere of 4U 0142 is composed of partially ionized heavy elements and that the surface magnetic field is comparable to or less than 1014 G, consistent with the dipole field inferred from the measured spindown. It also implies that the spin axis of 4U 0142+61 is aligned with its velocity direction. The polarized X-rays from 1RXS J170849.0-400910 do not show such 90° swing, consistent with magnetar atmospheric emission with B ≳ 5 × 1014 G.

Comprehensive Proteomics Analysis Identifies CD38-Mediated NAD+ Decline Orchestrating Renal Fibrosis in Pediatric Patients With Obstructive Nephropathy.

Obstructive nephropathy is one of the leading causes of kidney injury and renal fibrosis in pediatric patients. Although considerable advances have been made in understanding the pathophysiology of obstructive nephropathy, most of them were based on animal experiments and a comprehensive understanding of obstructive nephropathy in pediatric patients at the molecular level remains limited. Here, we performed a comparative proteomics analysis of obstructed kidneys from pediatric patients with ureteropelvic junction obstruction and healthy kidney tissues. Intriguingly, the proteomics revealed extensive metabolic reprogramming in kidneys from individuals with ureteropelvic junction obstruction. Moreover, we uncovered the dysregulation of NAD+ metabolism and NAD+-related metabolic pathways, including mitochondrial dysfunction, the Krebs cycle, and tryptophan metabolism, which led to decreased NAD+ levels in obstructed kidneys. Importantly, the major NADase CD38 was strongly induced in human and experimental obstructive nephropathy. Genetic deletion or pharmacological inhibition of CD38 as well as NAD+ supplementation significantly recovered NAD+ levels in obstructed kidneys and reduced obstruction-induced renal fibrosis, partially through the mechanisms of blunting the recruitment of immune cells and NF-κB signaling. Thus, our work not only provides an enriched resource for future investigations of obstructive nephropathy but also establishes CD38-mediated NAD+ decline as a potential therapeutic target for obstruction-induced renal fibrosis.

MicroRNA hsa-let-7e-5p in hUC-MSC-EVs alleviates oral mucositis by targeting TAB2.

Oral mucositis (OM) is a severe side effect of anti-cancer therapy, with limited available treatments. Mesenchymal stem cells (MSCs) and their secreted extracellular vesicles (EVs) have demonstrated effective protection against OM. However, the underlying mechanism remains elusive. In the current study, we purified EVs secreted by human umbilical cord MSCs (hUC-MSC-EVs) and investigated their role in lipopolysaccharide (LPS)-induced human oral keratinocytes (HOKs). We observed that treatment with hUC-MSC-EVs significantly reduced the inflammatory response of HOKs to LPS induction. Through small RNA-seq using miRNAs extracted from hUC-MSC-EVs, we identified hsa-let-7e-5p as one of the most highly expressed miRNAs. Bioinformatic analysis data indicated that hsa-let-7e-5p may inhibit the NF-κB signalling pathway by targeting TAB2. Overexpression of the hsa-let-7e-5p inhibitor significantly attenuated the anti-inflammatory effect of hUC-MSC-EVs in LPS-induced HOKs, which could be reversed by the knockdown of TAB2. In addition, we administered hUC-MSC-EVs in a hamster model for OM and observed that these EVs alleviated OM phenotypes. Taken together, our observations suggest that hsa-let-7e-5p in hUC-MSC-EVs could protect the oral mucosa from OM by repressing TAB2 expression.

Extreme Resonant Eccentricity Excitation of Stars around Merging Black-Hole Binary.

We study the dynamics of a star orbiting a merging black-hole binary (BHB) in a coplanar triple configuration. During the BHB's orbital decay, the system can be driven across the apsidal precession resonance, where the apsidal precession rate of the stellar orbit matches that of the inner BHB. As a result, the system gets captured into a state of resonance advection until the merger of the BHB, leading to extreme eccentricity growth of the stellar orbit. This resonance advection occurs when the inner binary has a nonzero eccentricity and unequal masses. The resonant driving of the stellar eccentricity can significantly alter the hardening rate of the inner BHB and produce observational signatures to uncover the presence of nearby merging or merged BHBs.

Construction and application of tuberculosis medical and nursing integration cooperation model.

OBJECTIVE: To explore the clinical effect of the tuberculosis (TB) doctor-nurse integration management model METHODS: This study is a retrospective historical cohort study. The clinical data of 180 patients with TB in our hospital from 2019 to 2020 were analyzed retrospectively. In a control group, 90 cases were treated with the traditional medical care model. An observation group of 90 cases received clinical diagnoses, treatments, and nursing under a doctor-nurse integration management model. Comparative analyses between the two groups were conducted on various aspects, including the awareness level of TB prevention and control, medication compliance and patient satisfaction. Comparisons between the two groups were evaluated using independent-sample t-tests or Chi-squared tests RESULTS: Compared with the control group, the knowledge awareness levels of TB prevention and medication compliance in the observation group were significantly higher (p < .05). The appointment waiting times and hospitalization times in the observation group were significantly lower than in the control group (p < .05). The total average satisfaction score of the patients in the observation group was significantly higher than in the control group (p < .05). Compared with the control group, the patients in the observation group were significantly more satisfied with their nursing methods, operating techniques, psychological techniques, service attitudes, and ward management (p < .05). In addition, in the observation group, medical-nursing relationships and doctor-patient communication were better than in the control group; additionally, the satisfaction of doctors with nursing work was also higher than in the control group, which was a statistically significant difference (p < .05) CONCLUSION: The implementation of an integrated medical-nursing cooperation model for TB will help increase the awareness of health knowledge in patients with TB, improve patient medication compliance and enhance patient satisfaction.

GTSE1 promotes tumor growth and metastasis by attenuating of KLF4 expression in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignant tumors and is characterized by a poor prognosis. Although G2- and S -phase expressed-1 (GTSE1) is known to be involved in the progression and metastasis of various cancers, its significance and mechanism in ccRCC remain unknown. In the present study, we found that GTSE1 was overexpressed in ccRCC tissues, especially in metastatic samples. Moreover, high GTSE1 expression was positively correlated with higher pT stage, tumor size, clinical stage, and WHO/ISUP grade and worse prognosis. And GTSE1 expression served as an independent prognostic factor for overall survival (OS). In addition, GTSE1 knockdown inhibited ccRCC cell proliferation, migration, and invasion, and enhanced cell apoptosis in vitro and in vivo. GTSE1 was crucial for epithelial-mesenchymal transition (EMT) in ccRCC. Mechanistically, GTSE1 depletion could upregulate the expression of Krüppel-like factor 4 (KLF4), which acts as a tumor suppressor in ccRCC. Downregulation of KLF4 effectively rescued the inhibitory effect induced by GTSE1 knockdown and reversed the EMT process. Overall, our results revealed that GTSE1 served as an oncogene regulating EMT through KLF4 in ccRCC, and that GTSE1 could also serve as a novel prognostic biomarker and may represent a promising therapeutic target for ccRCC.

Next-Generation Sequencing-Based Analysis of Urine Cell-Free mtDNA Reveals Aberrant Fragmentation and Mutation Profile in Cancer Patients.

BACKGROUND: Many studies have demonstrated the high efficacy of cell-free nuclear DNA in cancer diagnostics. Compared to nuclear DNA, mitochondrial DNA (mtDNA) exhibits distinct characteristics, including multiple copies per cell and higher mutation frequency. However, the potential applicability of cell-free mtDNA (cf-mtDNA) in plasma and urine remains poorly investigated. METHODS: Here, we comprehensively analyzed the fragmentomic and mutational characteristics of cf-mtDNA in urine and plasma samples from controls and cancer patients using next-generation sequencing. RESULTS: Compared to plasma cf-mtDNA, urine cf-mtDNA exhibited increased copy numbers and wider spread in fragment size distributions. Based on 2 independent animal models, urine cf-mtDNA originated predominantly from local shedding and transrenal excretion. Further analysis indicated an enhanced fragmentation of urine cf-mtDNA in renal cell carcinoma (RCC) and colorectal cancer (CRC) patients. Using the mtDNA sequence of peripheral blood mononuclear cells for reference, the mutant fragments were shorter than wild-type fragments in urine cf-mtDNA. Size selection of short urine cf-mtDNA fragments (<150 bp) significantly enhanced the somatic mutation detection. Our data revealed remarkably different base proportions of fragment ends between urine and plasma cf-mtDNA that also were associated with fragment size. Moreover, both RCC and CRC patients exhibited significantly higher T-end and lower A-end proportions in urine cf-mtDNA than controls. By integrating the fragmentomic and mutational features of urine cf-mtDNA, our nomogram model exhibited a robust efficacy for cancer diagnosis. CONCLUSIONS: Our proof-of-concept findings revealed aberrant fragmentation and mutation profiles of urine cf-mtDNA in cancer patients that have diagnostic potential.

Hyperendogenous Heparinization Suggests a Guideline for the Management of Massive Wasp Stings in Two Victims.

Bees and wasps (order Hymenoptera) are commonly encountered worldwide and often deliver defensive stings when in contact with humans. Massive envenomation resulting from >50 stings causes a toxic reaction and life-threatening complications that typically result in rhabdomyolysis and disseminated intravascular coagulation. Two male patients who were stung over 80 times by wasps experienced severe coagulation abnormality. Consecutive examination by thromboelastography (TEG) guided by heparinase treatment during their hospitalization evidenced heparin-like coagulation dysfunction despite no clinical use of heparin-like substances. Numerous tests were also conducted to confirm whether the coagulation abnormalities could be attributed to hyperendogenous heparinization and allergic reaction, rhabdomyolysis, and vascular endothelial cell injury without apparent disseminated intravascular coagulation, which might all be affected by the production of endogenous heparin. The reduced coagulation potential caused by hyperendogenous heparinization was associated with the binding of antithrombin and the activation of fibrinolysis. In addition, TEG-identified coagulopathy was moderated using protamine for heparin neutralization. The massively envenomed patients survived and were discharged after completion of medical care. We also review clinical manifestations from other published case reports, including topical treatment. Our study provides clinical evidence and guidance for diagnosis via TEG and appropriate intervention with protamine for patients with massive wasp envenomation.

Development and validation of a prognostic nomogram for patients with intravesical recurrence after radical nephroureterectomy for non-metastatic upper tract urothelial carcinoma.

PURPOSE: To develop and validate a prognostic nomogram for patients with intravesical recurrence (IVR) after radical nephroureterectomy (RNU) for non-metastatic upper tract urothelial carcinoma (UTUC). METHODS: The clinical data of 468 patients registered in the surveillance, epidemiology and end results database between 2010 and 2015 were retrospectively analyzed. Multivariate analysis using the Cox proportional hazard model was used to determine independent prognostic factors for the development of a nomogram to predict the 1-, 3-, and 5-year probability of individual cancer-specific survival (CSS). Moreover, the nomogram was internally validated using receiver operating characteristic curves and calibration plots. RESULTS: Age at IVR > 80 years, UTUC stage ≥ T3, bladder cancer (BC) stage T1, and muscle-invasive BC (stage ≥ T2) were identified as independent risk factors for CSS in patients with IVR after RNU, whereas a time interval of > 24 months between UTUC and BC was an independent protective factor. The 1-, 3-, and 5-year predictive accuracies of our nomogram were 0.74, 0.70, and 0.71, respectively. Additionally, 1-, 3-, and 5-year calibration curves demonstrated perfect agreement between the nomogram-predicted and the actual CSS. CONCLUSIONS: This study developed and internally validated the first nomogram to date to predict individual prognosis in patients with IVR after RUN for UTUC. This nomogram can be used for patient counseling and for designing clinical trials.

Effect of Oyster Meat Preload on Postmeal Glycemic Control in Healthy Young Adults.

Objective: Evidence suggests that food preload improves postmeal glycemic profiles, but the effects of marine food are poorly understood. Our study aims to verify the regulating effects of premeal oyster meat (OM) on postprandial blood glucose.Method: Edible parts of the flesh of oyster were prepared for a randomized crossover experiment. After overnight fasting, 20 healthy young men consumed 300 mL of preload drinks with 0 g/kg body weight (BW) (control), 0.1 g/kg BW, and 0.2 g/kg BW. Peripheral blood concentrations of glucose and gastrointestinal hormones were measured before preloading at baseline (0 minutes) and at intervals after the preload and after a preset rice meal. The nutrient composition of OM was analyzed.Results: Compared with other doses, 0.2 g/kg BW OM preload induced higher plasma premeal insulin (p < 0.05), C-peptide (p < 0.05), and glucagon-like peptide-1 (GLP-1; p < 0.05) without altering the glucose concentrations during premeal times. By contrast, 0.2 g/kg BW OM induced less secretion of glucose (p < 0.05) and gastric inhibitory peptide (GIP; p < 0.05), but higher secretion of GLP-1 (p < 0.05) than 0.1 g/kg BW of OM after a meal. During the entire experiment (0-170 minutes), OM reduced the blood glucose (p < 0.05) and GIP (p < 0.05), but increased GLP-1 (p < 0.05). OM was rich in protein (78.4%) and low in fat (6%). Glutamic acid, aspartic acids, glycine, and taurine are the amino acids with high content found in OM.Conclusions: OM preload reduces postmeal glycemia in healthy young people with associated changes in gastrointestinal hormone responses. This effect may be attributed to the rich contents of protein and amino acids of OM.

Biological characterization of human amniotic epithelial cells in a serum-free system and their safety evaluation.

Human amniotic epithelial cells (hAECs), derived from the innermost layer of the term placenta closest to the fetus, have been shown to be potential seed cells for allogeneic cell therapy. Previous studies have shown a certain therapeutic effect of hAECs. However, no appropriate isolation and culture system for hAECs has been developed for clinical applications. In the present study, we established a serum-free protocol for hAEC isolation and cultivation, in which better cell growth was observed compared with that in a traditional culture system with serum. In addition to specific expression of cell surface markers (CD29, CD166 and CD90), characterization of the biological features of hAECs revealed expression of the pluripotent markers SSEA4, OCT4 and NANOG, which was greater than that in human mesenchymal stem cells, whereas very low levels of HLA-DR and HLA-DQ were detected, suggesting the weak immunogenicity of hAECs. Intriguingly, CD90+ hAECs were identified as a unique population with a powerful immunoregulatory capacity. In a systemic safety evaluation, intravenous administration of hAEC did not result in hemolytic, allergy, toxicity issues or, more importantly, tumorigenicity. Finally, the therapeutic effect of hAECs was demonstrated in mice with radiation-induced damage. The results revealed a novel function of hAECs in systemic injury recovery. Therefore, the current study provides an applicable and safe strategy for hAEC cell therapy administration in the clinical setting.

Survival of planets around shrinking stellar binaries.

The discovery of transiting circumbinary planets by the Kepler mission suggests that planets can form efficiently around binary stars. None of the stellar binaries currently known to host planets has a period shorter than 7 d, despite the large number of eclipsing binaries found in the Kepler target list with periods shorter than a few days. These compact binaries are believed to have evolved from wider orbits into their current configurations via the so-called Lidov-Kozai migration mechanism, in which gravitational perturbations from a distant tertiary companion induce large-amplitude eccentricity oscillations in the binary, followed by orbital decay and circularization due to tidal dissipation in the stars. Here we explore the orbital evolution of planets around binaries undergoing orbital decay by this mechanism. We show that planets may survive and become misaligned from their host binary, or may develop erratic behavior in eccentricity, resulting in their consumption by the stars or ejection from the system as the binary decays. Our results suggest that circumbinary planets around compact binaries could still exist, and we offer predictions as to what their orbital configurations should be like.

Size distribution, directional source contributions and pollution status of PM from Chengdu, China during a long-term sampling campaign.

Long-term and synchronous monitoring of PM10 and PM2.5 was conducted in Chengdu in China from 2007 to 2013. The levels, variations, compositions and size distributions were investigated. The sources were quantified by two-way and three-way receptor models (PMF2, ME2-2way and ME2-3way). Consistent results were found: the primary source categories contributed 63.4% (PMF2), 64.8% (ME2-2way) and 66.8% (ME2-3way) to PM10, and contributed 60.9% (PMF2), 65.5% (ME2-2way) and 61.0% (ME2-3way) to PM2.5. Secondary sources contributed 31.8% (PMF2), 32.9% (ME2-2way) and 31.7% (ME2-3way) to PM10, and 35.0% (PMF2), 33.8% (ME2-2way) and 36.0% (ME2-3way) to PM2.5. The size distribution of source categories was estimated better by the ME2-3way method. The three-way model can simultaneously consider chemical species, temporal variability and PM sizes, while a two-way model independently computes datasets of different sizes. A method called source directional apportionment (SDA) was employed to quantify the contributions from various directions for each source category. Crustal dust from east-north-east (ENE) contributed the highest to both PM10 (12.7%) and PM2.5 (9.7%) in Chengdu, followed by the crustal dust from south-east (SE) for PM10 (9.8%) and secondary nitrate & secondary organic carbon from ENE for PM2.5 (9.6%). Source contributions from different directions are associated with meteorological conditions, source locations and emission patterns during the sampling period. These findings and methods provide useful tools to better understand PM pollution status and to develop effective pollution control strategies.

Boosted photocatalytic efficiency through plasmonic field confinement with bowtie and diabolo nanostructures under LED irradiation.

Photoresist and electron beam lithography techniques were used to fabricate embedded Ag bowtie and diabolo nanostructures with various apex angles on the surface of a TiO2 film. The reinforced localized surface plasmon resonance (LSPR) and electric field generated at both the Ag/TiO2 and air/TiO2 interfaces enabled high light absorbance in the TiO2 nanostructure. Results for both the bowtie and diabolo nanostructures showed that a reduction in the apex angle enhances both LSPR and Raman intensity. The maximum electric current density observed at the apex indicates that the strongest SPR confines at the tip gap of the bowtie and corners of the diabolo. In a long-wavelength region, as the apex angle increases, the resonant peak wavelength of the standing wave matches the increased length of the prism edges of the bowtie and diabolo to create a redshift. In a short-wavelength region, as the apex angle increases, the blueshift of the resonant peak wavelength is presumably attributable to the increase in the effective index of the local surface plasmon polariton standing wave mainly residing along both the bowtie and diabolo axes. The redshift and blueshift trend in the simulation results for the resonant peak wavelength agrees well with the experimental results. The fastest photocatalytic rate was obtained by placing the Ag/TiO2 bowtie at an apex angle of 30° in the methylene blue solution, revealing that the plasmonic photocatalysis causes the highest degradation efficiency. This is because the Schottky junction and LSPR can stimulate many valid radicals for the environmental improvement.

Glutamate protects against Ca(2+) paradox-induced injury and inhibits calpain activity in isolated rat hearts.

This study determined the effects of glutamate on the Ca(2+) paradoxical heart, which is a model for Ca(2+) overload-induced injury during myocardial ischaemia and reperfusion, and evaluated its effect on a known mediator of injury, calpain. An isolated rat heart was retrogradely perfused in a Langendorff apparatus. Ca(2+) paradox was elicited via perfusion with a Ca(2+) -free Krebs-Henseleit (KH) solution for 3 minutes followed by Ca(2+) -containing normal KH solution for 30 minutes. The Ca(2+) paradoxical heart exhibited almost no viable tissue on triphenyltetrazolium chloride staining and markedly increased LDH release, caspase-3 activity, cytosolic cytochrome c content, and apoptotic index. These hearts also displayed significantly increased LVEDP and a disappearance of LVDP. Glutamate (5 and 20 mmol/L) significantly alleviated Ca(2+) paradox-induced injury. In contrast, 20 mmol/L mannitol had no effect on Ca(2+) paradox. Ca(2+) paradox significantly increased the extent of the translocation of µ-calpain to the sarcolemmal membrane and the proteolysis of α-fodrin, which suggests calpain activation. Glutamate also blocked these effects. A non-selective inhibitor of glutamate transporters, dl-TBOA (10 µmol/L), had no effect on control hearts, but it reversed glutamate-induced cardioprotection and reduction in calpain activity. Glutamate treatment significantly increased intracellular glutamate content in the Ca(2+) paradoxical heart, which was also blocked by dl-TBOA. We conclude that glutamate protects the heart against Ca(2+) overload-induced injury via glutamate transporters, and the inhibition of calpain activity is involved in this process.

Subcellular localization of proline-rich tyrosine kinase 2 during oocyte fertilization and early-embryo development in mice.

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase, is a member of the focal adhesion kinase family and is highly expressed in oocytes. Using a combination of confocal microscopy and RNAi, we localized and studied the function of both Pyk2 and tyrosine-phosphorylated Pyk2 (p-Pyk2) during mouse oocyte fertilization and early embryo development. At the onset of fertilization, Pyk2 and p-Pyk2 were detected predominantly in sperm heads and the oocyte cytoplasm. Upon formation of male and female pronuclei, Pyk2 and its activated form leave the cytoplasm and accumulate in the two pronuclei. We detected Pyk2 in blastomere nuclei and found both Pyk2 and p-Pyk2 in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as throughout early embryo development.

Unique MicroRNA signatures associated with early coronary atherosclerotic plaques.

The pathophysiology of coronary atherosclerotic plaques is a complex process. Early detection of coronary atherosclerotic plaques is critical in the prevention, prognostic and therapeutic intervention of cardiovascular disease. MicroRNAs (miRNAs), endogenous short non-coding RNAs, have been reported to play an important role in cardiovascular diseases and are also used as disease markers. However, the miRNA expression profile in early coronary atherosclerotic plaques has yet been reported. We hypothesize that miRNAs can be used as effective disease markers for detection of early coronary atherosclerotic plaques. In this analysis, coronary artery samples from three patients with early coronary atherosclerosis were harvested and miRNA expression profile determined using microarray analysis. Compared with healthy controls, a total of 44 miRNAs were upregulated and 57 miRNAs were downregulated. Among the dysregulated miRNAs, eight were significantly upregulated while five miRNAs were significantly downregulated, as determined by t-test (P < 0.05). Four of the significantly dysregulated miRNAs, including miR-221, miR-155, miR-100 and hsa-miR-1273, were selected and verified by real-time PCR. The real-time PCR results were consistent with the microarray data that miR-221, miR-155 and miR-100 were significantly downregulated in plaques, whereas miR-1273 was significantly upregulated. These results indicate that miRNAs expression level can be used as potential markers for early coronary atherosclerotic plaque formation.

Computed tomography and magnetic resonance imaging findings of malignant solid pseudopapillary tumors of the pancreas with macroscopic venous tumor thrombosis: a report of 4 cases.

PURPOSE: The purpose of this study was to report the imaging findings of malignant pancreatic solid pseudopapillary tumors (SPTs) with macroscopic venous tumor thrombi. METHODS: The clinical features and imaging findings of 4 cases of malignant pancreatic SPT with venous tumor thrombi were retrospectively reviewed. RESULTS: The tumor thrombi were located in the splenic vein (n = 3) or the main portal vein and the proximal splenic vein (n = 1). Venous thrombi were connected with the main pancreatic tumors and showed venous filling defects on computed tomography and magnetic resonance imaging. Tumor thrombi primarily consisting of necrotic component and/or hemorrhage displayed no enhancement after contrast injection (n = 3). The enhancement pattern of the tumor thrombi that consisted mainly of tumor nests was consistent with pancreatic SPT (n = 1), that is, a slight enhancement in the arterial phase and a progressive enhancement in the portal venous phase and the equilibrium phase. Venous tumor thrombi associated with hemorrhage were hyperintense on both T1-weighted and T2-weighted images. CONCLUSIONS: It is uncommon for pancreatic SPTs to spread by invading the venous system and forming macroscopic venous tumor thrombi.

Mutation of Disulfide Bond Sites Reduces the Immunoreactivity of Cra a 4 by Changing the Structural Characteristics.

Sarcoplasmic calcium-binding protein (Cra a 4) from Crassostrea angulata belongs to the EF-hand superfamily, and understanding of its structure-allergenicity relationship is still insufficient. In this study, chemical denaturants were used to destroy the structure of Cra a 4, showing that disruption of the structure reduced its IgG-/IgE-binding activity. To explore which critical amino acid site affects the allergenicity of Cra a 4, the mutants were obtained by site-directed mutations in the disulfide bonds site (C97), conformational epitopes (I105, D114), or Ca2+-binding region (D106, D110) and their IgG-/IgE-binding activity was reduced significantly using serological tests. Notably, C97A had the lowest immunoreactivity. In addition, two conformational epitopes of Cra 4 were verified. Meanwhile, the increase of the α-helical content, surface hydrophobicity, and surface electrostatic potential of C97A affected its allergenicity. Overall, the understanding of the structure-allergenicity relationship of Cra a 4 allowed the development of a hypoallergenic mutant.

Development of Hypoallergenic Derivatives of Cra a 1 with B Cell Epitope Deletion and T Cell Epitope Retention.

Tropomyosin was reported as an important allergen in Crassostrea angulata and designated as Cra a 1. The localization of the T cell epitopes and the reduction of the immunoreactivity of Cra a 1 are still lacking. In this study, four T cell epitopes were identified by using wild-type Cra a 1 (wtCra a 1)-immunized mouse splenocytes cultured with synthetic peptides. The immunoreactivity was maintained after chemical denaturation treatment, indicating that the linear epitope is an immunodominant epitope of wtCra a 1. Furthermore, the hypoallergenic derivative (mCra a 1) was developed by the deletion of linear B cell epitopes and retention of T cell epitopes. mCra a 1 could stimulate CD4+T cell proliferation and upregulate interleukin-10 secretion. Overall, basophil activation by mCra a 1 was low, but its ability to induce T cell proliferation was retained, suggesting that mCra a 1 may serve as a viable candidate for treating oyster allergy.