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Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

There are no targeted medical therapies for Acute Lung Injury (ALI) or its most severe form acute respiratory distress syndrome (ARDS). Infections are the most common cause of ALI/ARDS and these disorders present clinically with alveolar inflammation and barrier dysfunction due to the influx of neutrophils and inflammatory mediator secretion. We designed the C6 peptide to inhibit voltage-gated proton channels (Hv1) and demonstrated that it suppressed the release of reactive oxygen species (ROS) and proteases from neutrophils in vitro. We now show that intravenous C6 counteracts bacterial lipopolysaccharide (LPS)-induced ALI in mice, and suppresses the accumulation of neutrophils, ROS, and proinflammatory cytokines in bronchoalveolar lavage fluid. Confirming the salutary effects of C6 are via Hv1, genetic deletion of the channel similarly protects mice from LPS-induced ALI. This report reveals that Hv1 is a key regulator of ALI, that Hv1 is a druggable target, and that C6 is a viable agent to treat ALI/ARDS.

We recently established a role for the stretch-activated two-pore-domain K+ (K2P) channel TREK-1 (K2P2.1) in inflammatory cytokine secretion using models of hyperoxia-, mechanical stretch-, and TNF-α-induced acute lung injury. We have now discovered the expression of large conductance, Ca2+-activated K+ (BK) channels in human pulmonary microvascular endothelial cells and primary human alveolar epithelial cells using semiquantitative real-time PCR, IP and Western blot, and investigated their role in inflammatory cytokine secretion using an LPS-induced acute lung injury model. As expected, LPS induced IL-6 and CCL-2 secretion from pulmonary endothelial and epithelial cells. BK activation with NS1619 decreased LPS-induced CCL-2 but not IL-6 secretion from endothelial cells and had no effect on epithelial cells, although fluorometric assays revealed that BK activation hyperpolarized the plasma membrane potential (Em) of both cell types. Interestingly, BK inhibition (Paxilline) did not alter cytokine secretion or the Em in either cell type. Furthermore, LPS treatment by itself did not affect the Em or intracellular Ca2+ concentrations. Therefore, we propose BK channel activation as a novel targeted approach to counteract LPS-induced CCL-2 secretion from endothelial cells. This protective effect appears to occur via Em hyperpolarization but independent of intracellular Ca2+ concentrations.

No targeted therapies exist to counteract Hyperoxia (HO)-induced Acute Lung Injury (HALI). We previously found that HO downregulates alveolar K2P2.1 (TREK-1) K+ channels, which results in worsening lung injury. This decrease in TREK-1 levels leaves a subset of channels amendable to pharmacological intervention. Therefore, we hypothesized that TREK-1 activation protects against HALI. We treated HO-exposed mice and primary alveolar epithelial cells (AECs) with the novel TREK-1 activators ML335 and BL1249, and quantified physiological, histological, and biochemical lung injury markers. We determined the effects of these drugs on epithelial TREK-1 currents, plasma membrane potential (Em), and intracellular Ca2+ (iCa) concentrations using fluorometric assays, and blocked voltage-gated Ca2+ channels (CaV) as a downstream mechanism of cytokine secretion. Once-daily, intra-tracheal injections of HO-exposed mice with ML335 or BL1249 improved lung compliance, histological lung injury scores, broncho-alveolar lavage protein levels and cell counts, and IL-6 and IP-10 concentrations. TREK-1 activation also decreased IL-6, IP-10, and CCL-2 secretion from primary AECs. Mechanistically, ML335 and BL1249 induced TREK-1 currents in AECs, counteracted HO-induced cell depolarization, and lowered iCa2+ concentrations. In addition, CCL-2 secretion was decreased after L-type CaV inhibition. Therefore, Em stabilization with TREK-1 activators may represent a novel approach to counteract HALI.

Background: Following yttrium‐90 radioembolization (90Y‐RE), 90Y‐PET/CT and 90Y‐SPECT/CT imaging provide the means to calculate the voxelized absorbed dose distribution. Given the widespread use of the two imaging modalities and lack of well‐established standardized dosimetry protocols for 90Y‐RE, there is a clinical need to systematically investigate and evaluate differences in the performance of voxel‐based dosimetry between 90Y‐PET/CT and 90Y‐SPECT/CT. Purpose: To quantitatively analyze and compare 90Y‐PET/CT and 90Y‐SPECT/CT‐based dosimetry following 90Y‐RE. Methods: 90Y‐PET/CT and 90Y‐SPECT/CT imaging was acquired for 35 patients following 90Y‐RE with TheraSphere for the treatment of unresectable hepatocellular carcinoma. Dosimetry was performed using the local deposition method with known activity and the mean dose (Dmean) was calculated for perfused liver volumes (PV), tumors (T), and perfused normal livers (NL). Additionally, the absorbed dose to x% of the volume (Dx, x ∈$ \in $ [5%, 10%, ..., 90%, 95%]) and the volume receiving y Gy (Vy, y ∈$ \in $ [10 Gy, 20 Gy, ..., 190 Gy, 200 Gy]) were calculated for T and NL, respectively. Dose metrics were compared using linear regression, Bland‐Altman analysis, and statistical testing. Results: Both 90Y‐SPECT/CT and 90Y‐PET/CT‐based tumor Dmean were strongly correlated (R2 ≥ 0.90) with Dx, excluding metrics on the extrema. Intra‐modality comparisons of various Dx and Vy metrics yielded statistically significant differences (ANOVA, p < 0.001) for both90Y‐PET/CT and 90Y‐SPECT/CT. Based on statistical testing, only Dx metrics separated by greater than 20%–30% coverage, and only Vy metrics separated by greater than 40–70 Gy, reported significant differences. For PV, there was a strong correlation (R2 ≥ 0.99) between Dmean derived separately from 90Y‐PET/CT and 90Y‐SPECT/CT imaging. The strength of the correlation was slightly reduced for T and NL with R2 = 0.91 and R2 = 0.95, respectively. For PV, the mean bias ± standard error (SE) and 95% limits of agreement (LOA) between Dmean from the two modalities was effectively zero with ‐0.8 ± 0.4% (± 2.5%). For T and NL, the mean bias ± SE (± LOA) was ‐14.5 ± 3.7% (± 24%) and 9.4 ± 4.7% (± 27%), respectively. Conclusion: The strong correlation between Dmean and Dx suggests information from multiple dose metrics (e.g., D70 and Dmean) is largely redundant when establishing dose‐response relationships in 90Y‐RE. Dmean is highly correlated between 90Y‐PET/CT and 90Y‐SPECT/CT‐based dosimetry, for all liver VOIs. Relative to 90Y‐SPECT/CT, 90Y‐PET/CT, on average, yielded higher Dmean to tumors (14%) and lower Dmean to perfused normal livers (9%). Absorbed dose differences for perfused liver volumes between 90Y‐SPECT/CT and 90Y‐PET/CT were negligible. [ABSTRACT FROM AUTHOR]

Wheat is used for making many food products due to its diverse quality profile among different wheat classes. Since laboratory analysis of these end-use quality traits is costly and time-consuming, genetic dissection of the traits is preferential. This study used a genome-wide association study (GWAS) of ten end-use quality traits, including kernel protein, flour protein, flour yield, softness equivalence, solvent's retention capacity, cookie diameter, and top-grain, in soft red winter wheat (SRWW) adapted to US southeast. The GWAS included 266 SRWW genotypes that were evaluated in two locations over two years (2020–2022). A total of 27,466 single nucleotide markers were used, and a total of 80 significant marker-trait associations were identified. There were 13 major-effect quantitative trait loci (QTLs) explaining >10% phenotypic variance, out of which, 12 were considered to be novel. Five of the major-effect QTLs were found to be stably expressed across multiple datasets, and four showed associations with multiple traits. Candidate genes were identified for eight of the major-effect QTLs, including genes associated with starch biosynthesis and nutritional homeostasis in plants. These findings increase genetic comprehension of these end-use quality traits and could potentially be used for improving the quality of SRWW. [ABSTRACT FROM AUTHOR]

A new 90Y SIR-Spheres delivery kit (SIROS D-vial and shield) has been introduced with a different physical form from the legacy V-Vial kit. Here, we establish the dose calibrator settings and exposure-rate–to–activity conversion factor to assay 90Y SIR-Spheres activity in the new SIROS kit. Methods: Eight D-vials with initial 90Y activities from 1.2 to 6.6 GBq within acrylic shields were assayed with dose calibrators and exposure-rate meters until activities decayed to approximately 0.1 GBq. The dose calibrator settings resulting in the lowest median activity errors and the best-fit slope of exposure rate versus activity were identified. Results: SIROS D-vial 90Y activity can be accurately and reliably estimated directly using setting 51 × 10 on both the CRC-15R and the CRC-55tR dose calibrators (errors within ±0.5%) and indirectly with an exposure-rate reading at 30 cm using conversion factor 0.664 ± 0.003 GBq/(mR/h) (R2 = 0.985). Conclusion: Dose calibrator settings and exposure-rate–to–activity conversion factor for 90Y activity assays with new SIROS kit should be updated from legacy V-Vial parameters to avoid an approximately 10% underestimation. [ABSTRACT FROM AUTHOR]

Context.--Distinguishing the different types of amyloid is clinically important because treatments and outcomes are different. Mass spectrometry is the new gold standard for amyloid typing, but it is costly and not widely available. Therefore, immunolabeling remains the first step in identifying the most common types of amyloidosis. In amyloid subtyping, direct immunofluorescence works well when applied to frozen sections, but immunohistochemistry on formalin-fixed, paraffin-embedded material often yields poor results, particularly for light chain amyloidosis. Recently, paraffin immunofluorescence has been described as a valuable salvage technique in renal pathology when frozen sections are not available but it has not been evaluated for extra-renal diseases. Objectives.--To evaluate the use of paraffin immunofluorescence for light-chain detection in extra-renal amyloidosis and other light-chain-associated diseases. Design.--First, we compared the staining intensity of both light chains between paraffin immunofluorescence and immunohistochemistry on a retrospective cohort of 28 cases of amyloidosis that have been previously typed. Then, we studied the role of paraffin immunofluorescence as an addition to our classical immunohistochemistry panel for amyloidosis typing. Results.--In the retrospective cohort, we found that paraffin immunofluorescence outperformed immunohistochemistry for light-chain detection. Then, in the prospective part of the study, we showed that the proportion of correctly classified cases increased from 50% to 71.9% with the adjunction of second-intention paraffin immunofluorescence to the immunohistochemistry procedure. Conclusions.--We therefore view paraffin immunofluorescence as a significant addition to the routine workflow for detection of light-chain-related diseases. (Arch Pathol Lab Med. 2021;145:343-351; doi: 10.5858/arpa.2020-0124-OA), Amyloidosis is a group or disorders associated with the presence of extracellular amyloid deposits constituted by fibrillar aggregates of misfolded protein. More than 35 protein types have been identified as [...]

BACKGROUND: Dengue is a major public health problem worldwide. Assessment of adaptive immunity is important to understanding immunopathology and to define correlates of protection against dengue virus (DENV). To enable global assessment of CD4+ T cell responses, we mapped HLA-DRB1-restricted DENV-specific CD4+ T cell epitopes in individuals previously exposed to DENV in the general population of the dengue-endemic region of Managua, Nicaragua. METHODS: HLA class II epitopes in the population of Managua were identified by an in vitro IFNγ ELISPOT assay. CD4+ T cells purified by magnetic bead negative selection were stimulated with HLA-matched epitope pools in the presence of autologous antigen-presenting cells, followed by pool deconvolution to identify specific epitopes. The epitopes identified in this study were combined with those previously identified in the DENV endemic region of Sri Lanka, to generate a megapool (MP) consisting of 180 peptides specifically designed to achieve balanced HLA and DENV serotype coverage. The DENV CD4MP180 was validated by intracellular cytokine staining assays. RESULTS: We detected responses directed against a total of 431 epitopes, representing all 4 DENV serotypes, restricted by 15 different HLA-DRB1 alleles. The responses were associated with a similar pattern of protein immunodominance, overall higher magnitude of responses, as compared to what was observed previously in the Sri Lanka region. Based on these epitope mapping studies, we designed a DENV CD4 MP180 with higher and more consistent coverage, which allowed the detection of CD4+ T cell DENV responses ex vivo in various cohorts of DENV exposed donors worldwide, including donors from Nicaragua, Brazil, Singapore, Sri Lanka, and U.S. domestic flavivirus-naïve subjects immunized with Tetravalent Dengue Live-Attenuated Vaccine (TV005). This broad reactivity reflects that the 21 HLA-DRB1 alleles analyzed in this and previous studies account for more than 80% of alleles present with a phenotypic frequency ≥5% worldwide, corresponding to 92% phenotypic coverage of the general population (i.e., 92% of individuals express at least one of these alleles). CONCLUSION: The DENV CD4 MP180 can be utilized to measure ex vivo responses to DENV irrespective of geographical location.

This qualitative study investigated the lived experiences of six community college dance students. The theoretical framework of social cognitive career theory was used but was modified to include only the tenets most frequently cited in the literature that directly influenced the career identity of dancers particularly in relation to their motivation to academically persist and graduate. The research provides insight into the individual experiences of the participants in their dance journey. The goal was to obtain current data on the student experience so that dance programs can further enhance the student experience. The phenomenological analysis produced an early thematic matrix of 18 codes that were reduced to six major themes. Of those six themes, this study will focus on academic commitment, dance identification, and challenges.

Agro‐morphological traits play a significant role in the adaptation of wheat to diverse agroecosystems. Genetic understanding of these traits is crucial to develop cultivars adapted to specific environments and maximize their productivity. This is a comprehensive genome‐wide association study (GWAS) of 230 diverse lines of soft red winter wheat for identifying quantitative trait loci (QTLs) related to eight key agro‐morphological traits. The diversity panel was evaluated in two locations for three consecutive years (2020–2023). A total of 150 significant marker–trait associations were detected, including 65 for three flag leaf traits, 35 for peduncle length, 33 for plant height, 16 for heading date, and one for plant vigor using 27,466 single nucleotide polymorphism (SNP) markers. Eleven high‐confidence major‐effect QTLs explaining greater than 10% phenotypic variance were detected, of which seven were stable, and one showed an association with plant height and peduncle length. QTLs possibly allelic for important dwarfing (Rht23) and vernalization (Vrn‐B1) genes were identified. Six QTLs, QFlw.uga‐1A, QPdl.uga‐1A, QFlw.uga‐2B.2, QPdl.uga‐5A, QPdl.uga‐7A, and QPht.uga‐7B, are presumed to be novel, and nearby candidate gene(s) were identified for all except QPdl.uga‐1A. The pyramiding of favorable alleles from major‐effect QTLs was found to have significant improvement in peduncle length (shortened by 5 cm), flag leaf width (increased by 0.18 cm), and plant height (shortened by 11 cm). This study has improved our genetic understanding of important agro‐morphological traits. These results, upon further validation, can be used in breeding for desirable plant architecture to improve wheat yield potential. Core Ideas: Genome‐wide association study (GWAS) was conducted using 230 wheat lines evaluated for eight agro‐morphological traits.One hundred fifty significant marker–trait associations were found using 27,466 single nucleotide polymorphism (SNP) markers.Eleven major‐effect quantitative trait loci (QTLs) including seven stable QTLs were identified.Six major‐effect QTLs were presumed to be novel, and candidate genes were identified for five of them.Pyramiding favorable alleles from these QTLs led to significant improvement in traits. [ABSTRACT FROM AUTHOR]

Abstract Background Alveolar septation marks the beginning of the transition from the saccular to alveolar stage of lung development. Inflammation can disrupt this process and permanently impair alveolar formation resulting in alveolar hypoplasia as seen in bronchopulmonary dysplasia in preterm newborns. NF-κB is a transcription factor central to multiple inflammatory and developmental pathways including dorsal-ventral patterning in fruit flies; limb, mammary and submandibular gland development in mice; and branching morphogenesis in chick lungs. We have previously shown that epithelial overexpression of NF-κB accelerates lung maturity using transgenic mice. The purpose of this study was to test our hypothesis that targeted deletion of NF-κB signaling in lung epithelium would impair alveolar formation. Methods We generated double transgenic mice with lung epithelium-specific deletion of IKKβ, a known activating kinase upstream of NF-κB, using a cre-loxP transgenic recombination strategy. Lungs of resulting progeny were analyzed at embryonic and early postnatal stages to determine specific effects on lung histology, and mRNA and protein expression of relevant lung morphoreulatory genes. Lastly, results measuring expression of the angiogenic factor, VEGF, were confirmed in vitro using a siRNA-knockdown strategy in cultured mouse lung epithelial cells. Results Our results showed that IKKβ deletion in the lung epithelium transiently decreased alveolar type I and type II cells and myofibroblasts and delayed alveolar formation. These effects were mediated through increased alveolar type II cell apoptosis and decreased epithelial VEGF expression. Conclusions These results suggest that epithelial NF-κB plays a critical role in early alveolar development possibly through regulation of VEGF.

Background: The calculation of the net administered activity (Aadmin) in patients undergoing 90Y‐radioembolization is essential for dosimetry and radiation safety, yet current methods for measuring residual 90Y activity are often associated with high uncertainty. Therefore, an accurate, robust, and clinically viable method for the determination of Aadmin across approved 90Y microsphere devices is desirable. Purpose: We report on a novel method to determine Aadmin by leveraging the quantitative capabilities of SPECT/CT to measure 90Y‐emission in vivo from patients following 90Y‐radioembolization with glass or resin microspheres. Methods: 90Y‐SPECT/CT attenuation‐corrected count data from 147 sequential 90Y‐radioembolization patients was used for this analysis. Aadmin was calculated as part of routine clinical practice via the exposure rate differences between the initial 90Y‐vial and the 90Y‐residual jar. This served as our gold standard measure of Aadmin. Patient data for each microsphere device were separated into training and testing cohorts to first develop regression models and then to independently assess model performance. The training cohorts were divided into four groups: first, based on the microsphere device (glass or resin), and second, based on the SPECT volume used to calculate counts (the full SPECT field of view (FOV) or liver only (VOIliver)). Univariate linear regression models were generated for each group to predict Aadmin based on 90Y‐SPECT data from the training cohorts. Leave‐one‐out cross validation was implemented to estimate variability in model parameters. To assess performance, linear models derived from the training cohort were applied to 90Y‐SPECT data from the testing cohort. A comparison of the models between microspheres devices was also performed. Results: Linear models derived from the glass and resin training cohorts demonstrated a strong, positive correlation between 90Y‐SPECT image counts and Aadmin for VOIliver and FOV with R2 > 0.98 in all cases. In the glass training cohort, model accuracy (100%—absolute mean prediction error) and precision (95% prediction intervals of mean prediction error) were 99.0% and 15.4% for the VOIliver and 99.7% and 17.5% for the FOV models, respectively. In the resin training cohort, the corresponding values were 98.6% and 16.7% for VOIliver and > 99.9% and 11.4% for the FOV models, respectively. The application of these linear models to 90Y‐SPECT data from the testing cohort showed Aadmin prediction errors to have high accuracy and precision for both microsphere devices. For the glass testing cohort, accuracy (precision) was 96.9% (19.6%) and 98.8% (21.1%) for the VOIliver and FOV models, respectively. The corresponding values for the resin training cohort were 97.3% (26.2%) and 98.5% (25.7%) for the VOIliver and FOV models, respectively. The slope of the linear models between the two microsphere devices was observed to be significantly different with resin microspheres generating 48%–49% more SPECT counts for equivalent 90Y activity based on each device manufacturer's activity calibration process. Conclusion: 90Y‐SPECT image counts can reliably predict (accuracy > 95% and precision < 18%) Aadmin after 90Y‐radioembolization, with performance characteristics essentially equivalent for both glass and resin microspheres. There is a clear indication that activity calibrations are fundamentally different between the two microsphere devices. [ABSTRACT FROM AUTHOR]