Agreement of lymphocyte subsets detection permits reference intervals transference between flow cytometry systems: direct validation using established reference intervals.

OBJECTIVES: With the increasing demand and application of lymphocyte subsets detection in clinical laboratories, different single-platform flow cytometer (FCM) systems have been developed. There is an urgent need to establish the reference intervals (RIs) for different single-platform FCMs and transferring them from one FCM system to another provides a much more feasible and convenient approach. This study aimed to explore the transferability of RIs for lymphocyte subsets across different flow cytometry platforms. METHODS: We first conducted the pairwise method comparison across four FCM platforms, including NovoCyte, BriCyteE6, DxFLEX, and FACSCantoII systems. Next, the transferability of RIs of lymphocyte subsets was evaluated. Furthermore, we conducted the RIs transference based on the FACSCantoII system, BriCyteE6 system and DxFLEX system, except for NK cells. The transferred RIs were further verified by calculating the bias (CV) between the established ones. RESULTS: The results of lymphocyte subsets detection based on the NovoCyte, BriCyteE6, DxFLEX, and FACSCantoII systems were comparable and it was feasible to transfer the RIs of lymphocyte subsets detected by the four FCM systems. The RIs of lymphocyte subsets detection using FACSCantoII, DxFLEX, and BriCyteE6 systems were established. Upon transferring the RIs of lymphocyte subsets from the FACSCantoII system to the BriCyteE6 system, and DxFLEX system except for NK cells, the CV between the transferred RIs and the established ones was below 20 % for all parameters. CONCLUSIONS: The present study illustrated that the RIs of lymphocyte subsets could be transferred across different flow cytometry systems except for NK cells with different definition strategies.

How students' writing motivation, teachers' personal and professional attributes, and writing instruction impact student writing achievement: a two-level hierarchical linear modeling study.

Student motivation to write is a pivotal factor influencing their writing achievement. However, individual motivation to write is not independent of the learning environment. It also is crucial for teachers to develop their own efficacy, knowledge, and ability in writing and writing instruction to help them utilize effective instructional methods that stimulate students' motivation to write and further promote their writing achievement. Given these considerations, we utilized a two-level hierarchical linear model to examine the relationships among student motivation, teacher personal and professional traits, teacher writing instruction, and writing achievement at student and teacher levels. Our analysis of the dataset, which included 346 fourth and fifth graders nested within 41 classrooms, found that motivation had a positive predictive effect on writing ability at both student and teacher levels. Moreover, female students, fifth graders, and typically achieving students demonstrated higher writing achievement than their counterparts. While there were no significant effects of teacher efficacy, knowledge, ability, or professional development on student writing achievement, we observed that higher frequency of classroom management practices during writing instruction had a significant negative effect on student writing achievement. Our full model revealed that the relationship between student motivation and achievement was negatively moderated by teachers' increased use of instructional practices related to process features and using writing instruction materials, but positively moderated by increased use of varied teaching tactics. Overall, our findings emphasize the importance of contextual factors in understanding the complexity of student writing achievement and draw attention to the need for effective instructional practices to support students' writing development.

DPPC-coated lipid nanoparticles as an inhalable carrier for accumulation of resveratrol in the pulmonary vasculature, a new strategy for pulmonary arterial hypertension treatment.

In this study, we investigated the feasibility of dipalmitoylphosphatidylcholine-coated lipid nanoparticles (DPPC-LNs) as a carrier for preferential accumulation into lungs of Resveratrol (Res), a potentially promising drug for the treatment of pulmonary arterial hypertension (PAH). Res-loaded DPPC-LNs were prepared following a thin film hydration-ultrasonic dispersion technique using glyceryl monostearate as lipid core. DPPC can reduce the interactions between nanoparticles and pulmonary surfactant. The optimal formulation was prepared and characterized for physicochemical properties, storage stability and in vitro release profiles. The optimal formulation was evaluated for uptake by pulmonary arterial smooth muscle cells (PASMCs) using fluorescence microscopy. The efficacy of Res-loaded DPPC-LNs in reducing hyperplasia was tested in 5-HT induced proliferated PASMCs. The drug absorption profiles upon intratracheal administration were monitored in healthy rats. Optimized spherical DPPC-LNs - with mean size of 123.7 nm, zeta potential of -19.4 mV and entrapment efficiency of 94.40% - exhibited an 80% cumulative drug release over 48 h. Fluorescence microscopic study revealed an time-dependent enhancement of cellular uptake of Rh123-labeled DPPC-LNs by PASMCs. PASMC proliferation induced by 5-HT was significantly inhibited by Res-loaded DPPC-LNs. Optimized DPPC-LNs appeared to be safe when incubated with PASMCs. Besides, plasma and lung tissue data analysis indicated higher value of accumulation after intratracheal administration of Res-loaded DPPC-LNs in comparison with the intravenously dosed Res solution, indicating longer retention of Res in the lungs and their slower entry to the systemic blood circulation. DPPC-LNs could be a viable delivery system for site-specific treatment of PAH.

Screening of Nanobody Specific for Peanut Major Allergen Ara h 3 by Phage Display.

Peanut allergy is a major health problem worldwide. Detection of food allergens is a critical aspect of food safety. The VHH domain of single chain antibody from camelids, also known as nanobody (Nb), showed its advantages in the development of biosensors because of its high stability, small molecular size, and ease of production. However, no nanobody specific to peanut allergens has been developed. In this study, we constructed a library with random triplets (NNK) in its CDR regions of a camel nanobody backbone. We screened the library with peanut allergy Ara h 3 and obtained several candidate nanobodies. One of the promising nanobodies, Nb16 was further biochemical characterization by gel filtration, isothermal titration calorimetry (ITC), cocrystallization, and Western blot in terms of its interaction with Ara h 3. Nb16 specifically binds to peanut major allergen Ara h 3 with a dissociation constant of 400 nM. Furthermore, we obtained the Ara h 3-Nb16 complex crystals. Structure analysis shows the packing mode is completely different between the Ara h 3-Nb16 complex crystal and the native Ara h 3 crystal. Structural determination of Ara h 3-Nb16 will provide the necessary information to understand the allergenicity of this important peanut allergen. The nanobody Nb16 may have application in the development of biosensors for peanut allergen detection.

Purification, Characterization, and Crystal Structure of Parvalbumins, the Major Allergens in Mustelus griseus.

Fish play important roles in human nutrition and health, but also trigger allergic reactions in some population. Parvalbumin (PV) represents the major allergen of fish. While IgE cross-reactivity to PV in various bony fish species has been well characterized, little information is available about allergens in cartilaginous fish. In this study, two shark PV isoforms (named as SPV-I and SPV-II) from Mustelus griseus were purified. Their identities were further confirmed by mass spectroscopic analysis. IgE immunoblot analysis showed that sera from fish-allergic patients reacted to both SPV-I and SPV-II, but the majority of sera reacted more intensely to SPV-I than SPV-II. Thermal denaturation monitored by CD spectrum showed that both of the SPV allergens are highly thermostable. SPV-I maintained its IgE-binding capability after heat denaturation, while the IgE-binding capability of SPV-II was reduced. The results of crystal structure showed that SPV-I and SPV-II were similar in their overall tertiary structure, but their amino acid sequences shared lower similarities, indicating that the differences in the IgE-binding capabilities of SPV-I and SPV-II might be due to differential antigen epitopes in these two isoforms.