The switch from client holding to folding in the Hsp70/Hsp90 chaperone machineries is regulated by a direct interplay between co-chaperones.

Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here, we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfold a stringent client, the glucocorticoid receptor ligand-binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. Current concepts assume that to proceed to client folding, Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 functionally interacts with Hop, relieves the stalling Hsp90-Hop interaction, and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle.

RETICULON-LIKE PROTEIN B2 is a proviral factor co-opted for the biogenesis of viral replication organelles in plants.

Endomembrane remodeling to form a viral replication complex (VRC) is crucial for a virus to establish infection in a host. Although the composition and function of VRCs have been intensively studied, host factors involved in the assembly of VRCs for plant RNA viruses have not been fully explored. TurboID-based proximity labeling (PL) has emerged as a robust tool for probing molecular interactions in planta. However, few studies have employed the TurboID-based PL technique for investigating plant virus replication. Here, we used Beet black scorch virus (BBSV), an endoplasmic reticulum (ER)-replicating virus, as a model and systematically investigated the composition of BBSV VRCs in Nicotiana benthamiana by fusing the TurboID enzyme to viral replication protein p23. Among the 185 identified p23-proximal proteins, the reticulon family of proteins showed high reproducibility in the mass spectrometry data sets. We focused on RETICULON-LIKE PROTEIN B2 (RTNLB2) and demonstrated its proviral functions in BBSV replication. We showed that RTNLB2 binds to p23, induces ER membrane curvature, and constricts ER tubules to facilitate the assembly of BBSV VRCs. Our comprehensive proximal interactome analysis of BBSV VRCs provides a resource for understanding plant viral replication and offers additional insights into the formation of membrane scaffolds for viral RNA synthesis.

Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer.

In our previous research, we proved that ailanthone (AIL) inhibits the growth of gastric cancer (GC) cells and causes apoptosis by inhibiting P23. However, we still find some GC organoids are insensitive to AIL. We have done some sequencing analysis and found that the insensitive strains are highly expressed in PARP1. In this study, we investigated whether AIL can enhance the anti-tumour effect of PARPi in GC. CCK8 and spheroid colony formation assay were used to measure anti-tumour effects. SynergyFinder software was used to calculate the synergy score of the drug combination and flow cytometry was used to detect apoptosis. Western blot, IHC, IF tests were used to measure protein expression. Finally, nude mouse xenograft models were used to verify the in vitro mechanisms. High expression of PARP1 was found to be the cause of drug insensitivity. When AIL is paired with a PARP1 inhibitor, olaparib (OLP), drug sensitivity improves. We discovered that this combination functions by blocking off HSP90-BRCA1 interaction and inhibiting the activity of PARP1, thus in turn inhibiting the homologous recombination deficiency and base excision repair pathway to finally achieve synthetic lethality through increased sensitivity. Moreover, P23 can regulate BRCA1 in GC in vitro. This study proves that the inhibitory effect of AIL on BRCA1 allowed even cancer cells with normal BRCA1 function to be sensitive to PARP inhibitors when it is simultaneously administered with OLP. The results greatly expanded the scope of the application of PARPi.

Mite allergen sensitization patterns in Turkish children: Age-related changes and molecular correlations.

BACKGROUND: Mites are ubiquitous aeroallergens found worldwide. Elucidating individual mite allergen sensitization patterns provides critical insights for managing allergic diseases. This study aimed to investigate molecular allergen (MA) sensitization patterns across different age groups and explore cluster relationships among mite-sensitized children. METHODS: We analyzed 76 children who exhibited sensitization to at least one of the 17 distinct mite MAs through microarray testing. RESULTS: Dermatophagoides farinae exhibited a slightly higher prevalence of sensitization compared with Dermatophagoides pteronyssinus. Der p 1/2 and Der f 1/2 demonstrated an almost 40% sensitization rate, while Der p 10/Blo t 10, Der p 20, Der p 23, and Gly d 2/Lep d 2 displayed an approximately 20% sensitization rate. Sensitization levels and ratios increased significantly with age for Der p 23 but showed numerical rises for other MAs, except for Der p 10/Blo t 10. The presence of various types of atopic diseases had only a minimal impact on sensitization profiles. Strong correlations emerged between Der f 2 and Der p 2, Der p 10 and Blo t 10, Der p 21 and Blo t 5, as well as Gly d 2 and Lep d 2. Hierarchical cluster analysis substantiated these relationships. Der p 10 and its homolog Blo t 10-sensitive patients (15/76) were mostly seen as mono sensitization(12/15). Ten patients exhibited monosensitization to Der p 20, suggesting a possible association with scabies infection. CONCLUSION: In children, mite sensitization diversity and levels increased with age. The presence of significant correlations/cluster relationships among these sensitizations underscores homologies among specific MAs.

Characteristics of Allergen Components of Dermatophagoides pteronyssinus in Shrimp and Mite Co-Sensitized Patients in Southern China.

INTRODUCTION: Dermatophagoides pteronyssinus (Dp) and shrimp are common air allergens and allergenic food sources, respectively, in southern China. This study aimed to analyze the specific immunoglobulin E (sIgE) characteristics and relationships of Dp components among co-sensitized patients with Dp and shrimp. MATERIALS AND METHODS: Serum samples were collected from 112 patients with Dp sensitization (61 with shrimp sensitization and 51 without) from southern China. The sIgE concentrations of Dp and shrimp crude extracts were determined by ImmunoCAP, and the sIgE of Dp allergen components (Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Der p 21, and Der p 23) was detected by protein chip. RESULTS: Overall, in the Dp-allergic patients, Der p 1 had the highest positive rate (72.3%), followed by Der p 2 (65.2%), Der p 23 (46.4%), Der p 7 (32.14%), Der p 21 (29.46%), Der p 5 (22.32%), and Der p 10 (17.86%). Compared with that in the shrimp nonsensitized group, the positive rate of sIgE for Der p 10 (27.87% vs. 5.88%, p = 0.002) in the shrimp sensitization group was significantly higher; however, the positive rate of sIgE for Der p 7 (22.95% vs. 43.14%, p = 0.023) was significantly lower. Moreover, the concentration of sIgE for Der p 10 increased statistically in the shrimp-sensitized group. The correlation analysis also showed that shrimp sensitization was significantly correlated with Der p 10. CONCLUSION: Among patients with Dp sensitization, Der p 1 had the highest positive rate, followed by Der p 2 and Der p 23. Meanwhile, Der p 10 may play an important role in patients with shrimp sensitization, while Der p 7 may be the meaningful allergen component in patients with Dp sensitization alone. In general, component-resolved diagnosis technology in clinical practice can effectively guide patients with polysensitization to avoid allergic substances.

The relationship of D. pteronyssinus allergic component sIgE and sIgG4 in house dust mite allergic rhinitis or/and allergic asthma patients.

Objective: House-dust mite sensitization is an important cause of allergic asthma and/or rhinitis in southern China. This study aimed to analyze the immune effect and relationship between the Dermatophagoides pteronyssinus components specific immunoglobulin E (sIgE) and sIgG4. Methods: The serum levels of sIgE and sIgG4 to D. pteronyssinus allergen components Der p 1, 2, 3, 5, 7, 10, and 23 were detected in 112 patients with allergic rhinitis (AR) and/or allergic asthma (AA). Results: Overall, Der p 1 had the highest positive rate of sIgE (72.3%), followed by Der p 2 (65.2%) and Der p 23 (46.4%). Meanwhile, the highest positive rates of sIgG4 were for Der p 2 (47.3%), Der p 1 (33.0%), and Der p 23 (25.0%). The patients with AR and AA had a higher positive rate (43.4%) of sIgG4 than that in the patients with AR (42.4%) and the patients with AA (20.4%; p = 0.043). In patients with AR, the positive rate of sIgE in Der p 1 (84.8%) was higher than that in sIgG4 (42.4%; p = 0.037), but the positive rate of sIgG4 in Der p 10 (21.2%) was higher than that in sIgE (18.2%; p < 0.001). Most of the patients were positive for sIgE and sIgG4 of Der p 2 and Der p 10 at the same time. However, positive results for sIgE alone were just found in Der p 7 and Der p 21. Optimal scale analysis showed that Der p 2, Der p 7, and Der p 21 sIgG4 were closely related to AR and AA (Cronbach α = 0.917). Conclusion: Herein, the D. pteronyssinus allergen components showed different characteristics among the patients with AR, patients with AA, and patients with AR and AA in southern China. Thus, sIgG4 may be play an important role in allergic reactions.

Molecular reactivity profiling upon immunotherapy with a 300 IR sublingual house dust mite tablet reveals marked humoral changes towards major allergens.

BACKGROUND: Molecular antibody reactivity profiles have not yet been studied in depth in patients treated by sublingual house dust mite (HDM) tablet immunotherapy. Humoral immune responses to a large panel of HDM mite allergens were studied using allergen microarray technology in a subset of clinically defined high and low responder patients from a double-blind placebo-controlled allergen-specific immunotherapy (AIT) trial using sublingual 300 IR HDM tablets. METHODS: Serum levels of IgE, IgG and IgG4 to 13 Dermatophagoides pteronyssinus molecules were measured at baseline and after 1-year AIT, using allergen microarrays in 100 subjects exhibiting high or low clinical benefit. RESULTS: Der p 1, Der p 2 and Der p 23 were the most frequently recognized allergens in the study population. Patients with HDM-related asthma had significantly higher allergen-specific IgE levels to Der p 1 and Der p 23. No significant difference in the distribution of allergen sensitization pattern was observed between high and low responders. An increase in serum allergen-specific IgG and IgG4 occurred upon AIT, in particular to allergens Der p 1, Der p 2 and Der p 23 (p < 0.0001). CONCLUSIONS: We confirm for our study population that Der p 1- and Der p 23-specific IgE levels are associated with asthma. IgE reactivity profiles were not predicitive of sublingual AIT outcomes, with 300 IR tablets as efficacious in pauci- and multi-sensitized subjects. Our study is the first to demonstrate the induction of IgG and IgG4  specific for the HDM allergens Der p 1, Der p 2 and Der p 23 by sublingual AIT.

Serodominance Profile in a Dust Mite Complex Region.

BACKGROUND/OBJECTIVE: The objective of this study was to describe the molecular sensitization profile of mite allergy in an area with a high environmental exposure of house dust mites (HDM) and storage mites. METHODS: Skin prick tests were performed with standardized extracts (DIATER, Madrid, Spain). A specific commercial molecular panel (MADx) for Dermatophagoides pteronyssinus (Dpt), Dermatophagoides farinae (Dfar), Lepidoglyphus destructor (Ldt), Tyrophagus putrescentiae (Tput), and Blomia tropicalis (Blot) was correlated with clinical parameters in Galician (northwestern of Spain) HDM allergic patients. RESULTS: Fifty patients (60% female) met the inclusion and exclusion criteria. All of the patient's present rhinitis (50), 28% (14) rhinitis and asthma, and 18% (9) atopic dermatitis (AD). Hundred patients had a positive prick test for Dpt, followed by Dfar (92%), Ldt and Tput (74%), and Blot (68%). More than 50% recognized specific IgE for Der p 1, Der p 2, reaching 86% in the case of Der p 23. No statistically significant differences in IgE levels were found between patients with/without asthma and those with mild or moderate-severe rhinitis. Der p 7 was higher among rhinitis patients (p value 0.05). AD relative risk (RR) was increased in patients sensitized to Der f 2, Der p 2, and Der p 23. Der p 10 decreases the risk to have AD (RR 0.80). CONCLUSION: The evaluation of IgE results in a comprehensive panel of allergens allows differentiation of serological reactivity profiles with their clinical expression, to perform an optimal management. Improvements in component resolved diagnosis and more research on the clinical relevance of mite allergens are needed to achieve a genuine diagnosis leading to specific immunotherapy.

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail.

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species.

P23-A137†Cross-border cooperation for corneal donation between lions eye bank Saar-Lor-Lux, Trier/Westpfalz in Homburg/Saar and Luxembourg.

PURPOSE: With the increasing demand for corneas, eye banks must optimize and extend their sources of tissue donation. On the other hand, corneal transplantation is a specialized procedure performed in hospitals with high quality standards and ideally an integrated eye bank. In this report we would like to focus on an international win-win-win agreement between the Department of Ophthalmology at Saarland University Medical Center (Homburg/Saar, Germany), the LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz and the four major non-university hospitals without corneal transplantation competence in Luxembourg. METHODS: In 2012, at the initiative of the Luxembourgish Ministry of Health and Department of Ophthalmology (Homburg/Saar, Germany), an international agreement was established with the Centre Hospitalier du Luxembourg (Luxembourg). Administrative and legislative rules were developed. Luxembourgish nursing personnel attended a practical training program for corneal excision at the Department of Ophthalmology in Homburg/Saar allowing them to harvest the two first corneal donors on site by themselves during the first year. In the following years two more hospitals, the Centre Hospitalier Emile Mayrisch (Esch-sur-Alzette, Luxembourg) and the Hôpitaux Robert Schuman (Kirchberg, Luxembourg), joined the cooperation. RESULTS: From 2012 until 2021, three hospitals in Luxembourg donated 779 corneas to the LIONS Eye Bank of the Saarland University Medical Center in Homburg/Saar (Germany). In return, 308 Luxembourgish patients have received a corneal transplantation at the Department of Ophthalmology in Homburg/Saar. In 2022, the extension continued and an agreement with a fourth hospital in Luxembourg at the Centre Hospitalier du Nord (Ettelbruck, Luxembourg) was signed providing even more donations. CONCLUSION: The cross-border collaboration for corneal donation and patient treatment has proven to be successful with both numbers of harvested donors and transplanted patients rising. However, international legislation for tissue donation needs to be accurately respected and a quality management system established to provide continuous quality of the donor tissue.

[Characteristics of allergen component in dust mite-induced allergic rhinitis patients].

Objective:To investigate the characteristics of allergen component in dust mite（DM） -induced allergic rhinitis（AR） patients, and provide reference for the diagnosis and treatment of AR. Methods:DM-induced AR patients with or without allergic asthma（AA） who visited the Allergy Department of Tongji Hospital, Huazhong University of Science and Technology between 2021 and 2022 were enrolled. Patients'age, gender, and visual analog scale（VAS） for symptoms were recorded. sIgE and sIgG4 levels of allergen components such as Der f1, Der f2, Der p1, Der p2, Der p7, Der p10, Der p21, and Der p23 were detected using a protein chip method. The sensitization characteristics of the allergen components in the patients were observed, and the correlation between sIgE, sIgG of each component and VAS as well as the component differences between AR and AR with AA（AR&AA） were evaluated. Results:A total of 87 DM-induced AR patients were enrolled, with 42.5% of them were AR&AA, their VAS scores were significantly higher than those of AR patients（6.38±1.95 vs 5.25±1.85, P=0.009 8）. The order of sensitization rates for DM components was as follows: Der p2（82.8%）, Der f2（81.6%）, Der p1（74.7%）, Der f1（70.1%）, and Der p23（35.6%）. The order of positive rates for sIgG4 was: Der p2（21.8%）, Der f2（13.8%）, Der p21（8.0%）, and Der p7（6.9%）. There were no correlation between the sIgE, sIgG4 levels or positive numbers of components and VAS scores, but there were positive correlations between sIgE, sIgG4 concentrations of components. Compared with AR patients, AR&AA patients had higher levels of sIgE for Der p（60.5[7.2-91.1]vs 14.0[4.8-45.1], P=0.02）, Der f（49.8[15.7-81.6]vs 21.3[7.0-50.2], P=0.04）, Der p1（27.2[0.7-51.5]vs 2.6[0.2-24.9], P=0.02）, Der p2（20.0[1.4-60.6]vs 5.5[0.6-19.1], P=0.004）, and Der f2（58.9[16.0-89.2]vs 23.4[0.9-56.8], P=0.009）, and a higher proportion of AR with AA patients had sIgE levels of Der p1（70.3% vs 48.0%, P=0.038） and Der p23（27.0% vs 14.0%, P=0.039） that were ≥3 grades. Conclusion:Der p1/f1, Der p2/f3, and Der p23 are the major components of DM sensitized AR patients. Multiple component sensitization and sIgE, sIgG4 levels of each component are not correlated with the severity of AR. The sIgE levels of the Der p1/f1, Der p2/f3, and Der p23 components in AR&AA patients are higher than AR.

Molecular cytogenetic characterization of de novo concomitant distal 8p deletion of 8p23.3p23.1 and Xp and Xq deletion of Xp22.13q28 due to an unbalanced X;8 translocation detected by amniocentesis.

OBJECTIVE: We present molecular cytogenetic characterization of de novo concomitant distal 8p deletion of 8p23.3p23.1 and Xp and Xq deletion of Xp22.13q28 due to an unbalanced X;8 translocation detected by amniocentesis. CASE REPORT: A 33-year-old primigravid woman underwent amniocentesis at 18 weeks of gestation because of a Down syndrome risk of 1/52 at the first-trimester maternal serum screening calculated from 0.29 multiples of the median (MoM) of pregnancy associated plasma protein-A (PAPP-A), 1.14 MoM of free ß-hCG and 0.46 MoM of placental growth factor (PlGF). Amniocentesis revealed a karyotype of 45,X,add(8)(p23.1). The parental karyotypes were normal. Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from cultured amniocytes revealed a 137-Mb deletion of Xp22.13q28 and a 10.53-Mb deletion of 8p23.3p23.1. The karyotype thus was 45,X,der(8)t(X;8)(p22.13;p23.1). Prenatal ultrasound revealed pericardial effusion and skin edema. The pregnancy was subsequently terminated, and a 568-g malformed fetus was delivered with hypertelorism and low-set ears. The cord blood had a karyotype of 45,X,der(8)t(X;8)(p22.13;p23.1). aCGH analysis of the cord blood revealed the result of arr [GRCH37 (hg19)] 8p23.3p23.1 (191,530-10,724,642) × 1.0, arr Xp22.13q28 (18,194,098-155,232,907) × 1.0. CONCLUSION: aCGH analysis is useful elucidating the genetic nature of an aberrant chromosome with an additional maternal of unknown origin attached to a chromosome terminal region.

Biodegradation of PET by the membrane-anchored PET esterase from the marine bacterium Rhodococcus pyridinivorans P23.

Evidence for microbial biodegradation of polyethylene terephthalate (PET) has been reported, but little is known about the PET biodegradation process and molecular mechanism by marine microorganisms. Here, we show the biodegradation of PET by the membrane-anchored PET esterase from the marine bacterium Rhodococcus pyridinivorans P23, elucidate the properties of this enzyme, and propose the PET biodegradation by this strain in biofilm. We identify the PET-degrading enzyme dubbed PET esterase through activity tracking. In addition to depolymerizing PET, it hydrolyzes MHET into TPA under acid conditions. We prove that it is a low and constitutively transcribed, membrane-anchored protein displayed on the cell surface. Furthermore, we also investigate the microbial groups possessing PET esterase coupled with the TPA degradation pathway, mainly in the phyla Proteobacteria and Actinobacteriota. Clarification of the microbial PET biodegradation in the marine environment will contribute to the understanding of bioremediation of marine PET pollution.

Serological analysis of allergic components of house dust mite provides more insight in epidemiological characteristics and clinical symptom development in North China.

Background: House dust mite (HDM) is the most common airborne source causing complex allergy symptoms. There are geographic differences in the allergen molecule sensitization profiles. Serological testing with allergen components may provide more clues for diagnosis and clinical management. Objective: This study aims to investigate the sensitization profile of eight HDM allergen components in a large number of patients enrolled in the clinic and to analyze the relation of gender, age, and clinical symptoms in North China. Methods: The 548 serum samples of HDM-allergic patients (ImmunoCAP® d1 or d2 IgE ≥0.35) were collected in Beijing City and divided in four different age groups and three allergic symptoms. The specific IgE of HDM allergenic components, Der p 1/Der f 1, Der p 2/Der f 2, Der p 7, Der p 10, Der p 21, and Der p 23, was measured using the micro-arrayed allergen test kit developed by Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd. The new system was validated by comparing to single-component Der p 1, Der p 2, and Der p 23 tests by ImmunoCAP in 39 sera. The epidemiological study of these IgE profiles and the relation to age and clinical phenotypes were analyzed. Results: A greater proportion of male patients was in the younger age groups, while more female patients were in the adult groups. Both the sIgE levels and the positive rates (approximately 60%) against Der p 1/Der f 1 and Der p 2/Der f 2 were higher than for the Der p 7, Der p 10, and Der p 21 components (below 25%). The Der f 1 and Der p 2 positive rates were higher in 2-12-year-old children. The Der p 2 and Der f 2 IgE levels and positive rates were higher in the allergic rhinitis group. The positive rates of Der p 10 increased significantly with age. Der p 21 is relevant in allergic dermatitis symptom, while Der p 23 contributes to asthma development. Conclusion: HDM groups 1 and 2 were the major sensitizing allergens, with group 2 being the most important component relevant to respiratory symptoms in North China. The Der p 10 sensitization tends to increase with age. Der p 21 and Der p 23 might be associated with the development of allergic skin disease and asthma, respectively. Multiple allergen sensitizations increased the risk of allergic asthma.

The p23 co-chaperone is a succinate-activated COX-2 transcription factor in lung adenocarcinoma tumorigenesis.

P23, historically known as a heat shock protein 90 (HSP90) co-chaperone, exerts some of its critical functions in an HSP90-independent manner, particularly when it translocates into the nucleus. The molecular nature underlying how this HSP90-independent p23 function is achieved remains as a biological mystery. Here, we found that p23 is a previously unidentified transcription factor of COX-2, and its nuclear localization predicts the poor clinical outcomes. Intratumor succinate promotes p23 succinylation at K7, K33, and K79, which drives its nuclear translocation for COX-2 transcription and consequently fascinates tumor growth. We then identified M16 as a potent p23 succinylation inhibitor from 1.6 million compounds through a combined virtual and biological screening. M16 inhibited p23 succinylation and nuclear translocation, attenuated COX-2 transcription in a p23-dependent manner, and markedly suppressed tumor growth. Therefore, our study defines p23 as a succinate-activated transcription factor in tumor progression and provides a rationale for inhibiting p23 succinylation as an anticancer chemotherapy.

Dust mite component Analysis: Identifying key allergens components for effective immunotherapy in allergic rhinitis.

BACKGROUND: The aim of this study was to examine the frequency of sensitization to house dust mite (HDM) components among allergic rhinitis patients receiving subcutaneous immunotherapy (SCIT), and to assess the correlation between SCIT efficacy and specific IgE (sIgE) levels for allergenic HDM components. METHODS: Serum samples and clinical data were collected from 38 allergic rhinitis patients receiving HDM-SCIT at baseline and after 1 year of treatment. Effective treatment was defined as a therapeutic index (TI) of at least 50% after 1 year. Cytokine levels were analyzed using commercial ELISA kits, while serum total and specific IgE levels were determined by the fluoroenzymeimmunoassay technique. The ALLEOS 2000 magnetic particle chemiluminescence system was used to measure sIgE levels for Der f, Der p 1, Der p 2, Der p 10, and Der p 23. RESULTS: Allergic rhinitis patients undergoing HDM-SCIT had a high rate of allergic sensitization to the HDM major allergens Der p (100%), Der f (100%), Der p 1 (94.74%), Der p 2 (94.74%), and Der p 23 (36.84%). Patients who responded to SCIT had higher levels of IgE for HDM components at baseline, while those with ineffective treatment showed an opposite performance, particularly for Der p 1 (P＜0.05). After 1 year of treatment, effective and ineffective patients showed opposite trends in sIgE for dust mite components (decreased in effective patients, increased in ineffective patients). HDM-SCIT led to a significant reduction in IL-2, IL-4, IL-6, and EOS% (P＜0.05). IgE for Der p, Der f, Der p 1, Der p 2, and HDM sIgE were significantly positively correlated (P < 0.001). The correlation heatmap analysis based on changes in values reveals a negative correlation between CSMS score changes and sIgE for Der f and Der p 1, and a positive correlation with IL-2, IL-10, and TNF (P < 0.05). CONCLUSIONS: The molecular sensitization profiles during HDM-SCIT are variable and relate to treatment efficacy. Molecular diagnosis can assist allergists in identifying patients eligible for HDM-SCIT, thereby enhancing the treatment's clinical efficacy. Serum cytokine levels of IL-2, IL-4, IL-6，and EOS% may serve as useful biomarkers for monitoring HDM-SCIT efficacy.

Antibody reactivity to Mycobacterium tuberculosis-specific regions of differences 1 and regions of differences 9 proteins and peptides in rabbits, mice, and humans.

Background: The major antigens encoded by Mycobacterium tuberculosis-specific genomic regions of differences (RDs) could be useful in the development of new vaccines and/or diagnostic reagents using T-cell and/or antibody assays. In particular, RD1 proteins PE35, PPE68, ESXA, ESXB, and RD9 protein ESXV and their peptides have been identified as major T-cell antigens. However, little is known about their antibody reactivities in different mammalian species. This study aims to determine the antigen-specific antibody reactivities to the above antigens and their peptides in three different mammalian species, i.e., rabbits, mice, and humans. Methods: Sera were obtained from (i) rabbits immunized with purified recombinant proteins PE35, PPE68, ESXA, ESXB, and ESXV; (ii) mice immunized with recombinant DNA vaccine constructs of pUMVC6 and pUMVC7 containing RD1 and RD9 genes; and (iii) tuberculosis (TB) patients and healthy humans. Enzyme-linked immunosorbent assays (ELISAs) were performed with the sera to determine the antibody reactivity to purified recombinant proteins, peptide pools, and individual peptides of RD1 and RD9 proteins. Results: The ELISA results with sera from rabbits immunized with pure recombinant proteins showed positive antibody reactivity with all of the immunizing proteins and their synthetic peptide pools. Testing of the sera with individual peptides showed positive antibody reactivity with PE35 peptides P1 (aa 1-25), P2 (aa 16-40), P5 (aa 61-85), and P6 (aa 76-99); PPE68 peptides P9 (aa 121-145), P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371); all peptides (P1 to P6) of ESXA and ESXB; and ESXV peptides P1 (aa 1-25), P2 (aa 16-40), P3 (aa 31-55), P5 (aa 61-85), and P6 (aa 76-94). The sera from mice immunized with DNA vaccine constructs showed antibody reactivity to all proteins and the peptide P6 (aa 76-99) of PE35 and peptides P19 (aa 271-295) and P24 (aa 346-371) of PPE68. In humans, the peptides P11 (aa 151-175), P14 (aa 196-220), P22 (aa 316-340), P23 (aa 331-355), and P24 (aa 346-371) of PPE68 and the peptides P4 (aa 46-70), P5 (aa 61-85), and P6 (aa 76-94) of ESXV showed positive reactivity with sera from TB patients and healthy controls. Conclusion: The results demonstrate the presence of several antibody epitopes in each protein, but variations in the epitopes recognized were observed among mice, rabbits, and humans, which could be due to mammalian species differences and/or mode of antigen delivery.

Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

BACKGROUND: The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. METHODS: nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. RESULTS: In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. CONCLUSIONS: Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

A novel strategy for active compound efficacy status identification in multi-tropism Chinese herbal medicine (Scutellaria baicalensis Georgi) based on multi-indexes spectrum-effect gray correlation analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bioactive substance identification is always the focal point and the main challenge in Chinese herbal medicine (CHM). Most CHM present multiple efficacies and multiple tropisms, which has improved the application accuracy of CHM, and is worthy of further study. In this article, the concept of "multi-tropism efficacy of CHM" has been proposed for the first time. In addition, it is hypothesized that the different components in CHM can be classified based on their efficacy status. AIM OF THE STUDY: The spectrum-effect relationship between the fingerprint and efficacy was established to identify the efficacy status of components. This provided a practical, efficient and accurate way to identify the bioactive substances from a complex CHM system. MATERIALS AND METHODS: The network pharmacology approach was applied to preliminarily analyze the potential antibacterial compounds and mechanisms of HQ. Furthermore, its chemical fingerprint was established and the characteristic peaks were identified by LC-MS/MS. The antibacterial and anti-inflammatory bioactivities of HQ were determined to evaluate its pharmacological effect of heat-clearing and detoxification, and its anticoagulation activity was determined to evaluate its heat-clearing and tocolysis effects. The spectrum-effect relationships were assessed by gray correlation analysis to discriminate the status of active components in HQ with different efficacies. RESULTS: Network pharmacology analysis revealed apigenin, wogonin, baicalein, acacetin, ß-sitosterol, baicalin, eugenol, moslosooflavone, palmitic acid, oroxylin-A 7-O-glucuronide, and scutevulin as the potential active compounds responsible for the efficacy of HQ against both E. coli and S. aureus. The spectrum-effect relationship was utilized to reveal the orientation activities, with the results as follows: 1) The main basic-efficacy components in HQ with antibacterial, anti-inflammatory, and anticoagulant effects were P5, P8, P9, P15, P18, P19, P20; while the general basic-efficacy components were P2, P3, P6, P7, P11, P14, P21, P22, P28. 2) The main efficacy-oriented components in HQ with antibacterial effects on E. coli were P1, P12, P17, while the general efficacy-oriented compound was P10, P24, P25, P26, P27; the main efficacy-oriented in HQ with antibacterial effects on S. aureus were P14 and the general efficacy-oriented components were P1, P12, P26, P29, P30, respectively. 3) The main efficacy-oriented components with anti-inflammatory activity were P14, P24, P25, P27, and P30, while the general efficacy-oriented components were P13, P23, P26. 4) The main efficacy-oriented compounds in HQ with effects on anticoagulation were P6 and P22; these acted by prolonging APTT through the intrinsic coagulation pathway and PT through the extrinsic coagulation pathway, respectively. 5) The pharmacodynamic status classification of Scutellaria baicalensis ingredients were confirmed by nine reference compounds exemplarily. CONCLUSION: This work established a novel strategy for active compound efficacy status identification in multi-tropism Chinese herbal medicine (Scutellaria baicalensis Georgi) based on multi-indexes spectrum-effect gray correlation analysis, the method is scientific feasible and can be applied to the effective substances identification and quality control of other CHM.

Long-read Oxford nanopore sequencing reveals a de novo case of complex chromosomal rearrangement involving chromosomes 2, 7, and 13.

BACKGROUND: Complex chromosomal rearrangements (CCRs) are associated with high reproductive risk, infertility, abnormalities in offspring, and recurrent miscarriage in women. It is essential to accurately characterize apparently balanced chromosome rearrangements in unaffected individuals. METHODS: A CCR young couple who suffered two spontaneous abortions and underwent labor induction due to fetal chromosomal abnormalities was studied using long-read sequencing(LRS), single-nucleotide polymorphism (SNP) array, G-banding karyotype analysis (550-band resolution), and Sanger sequencing. RESULTS: SNP analysis of the amniotic fluid cells during the third pregnancy revealed a 9.9-Mb duplication at 7q21.11q21.2 and a 24.8-Mb heterozygous deletion at 13q21.1q31.1. The unaffected female partner was a carrier of a three-way CCR [46,XX,? ins(7;13)(q21.1;q21.1q22)t(2;13)(p23;q22)]. Subsequent LRS analysis revealed the exact breakpoint locations on the derivative chromosomes and the specific method of chromosome rearrangement, indicating that the CCR carrier was a more complex structural rearrangement comprising five breakpoints. Furthermore, LRS detected an inserted fragment of chromosome 13 in chromosome 7. CONCLUSIONS: LRS is effective for analyzing the complex structural variations of the human genome and may be used to clarify the specific CCRs for effective genetic counseling and appropriate intervention.