CARD9S12N facilitates the production of IL-5 by alveolar macrophages for the induction of type 2 immune responses.

The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the TH1 and TH17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9S12N), is associated with several autoimmune diseases. However, the function of CARD9S12N has remained unknown. Here we generated CARD9S12N knock-in mice and found that CARD9S12N facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9S12N mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive TH2 cell-mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9S12N can turn alveolar macrophages into IL-5-producing cells and facilitates TH2 cell-mediated pathologic responses.

SALT OVERLY SENSITIVE2 stabilizes phytochrome-interacting factors PIF4 and PIF5 to promote Arabidopsis shade avoidance.

Sun-loving plants trigger the shade avoidance syndrome (SAS) to compete against their neighbors for sunlight. Phytochromes are plant red (R) and far-red (FR) light photoreceptors that play a major role in perceiving the shading signals and triggering SAS. Shade induces a reduction in the level of active phytochrome B (phyB), thus increasing the abundance of PHYTOCHROME-INTERACTING FACTORS (PIFs), a group of growth-promoting transcription factors. However, whether other factors are involved in modulating PIF activity in the shade remains largely obscure. Here, we show that SALT OVERLY SENSITIVE2 (SOS2), a protein kinase essential for salt tolerance, positively regulates SAS in Arabidopsis thaliana. SOS2 directly phosphorylates PIF4 and PIF5 at a serine residue close to their conserved motif for binding to active phyB. This phosphorylation thus decreases their interaction with phyB and posttranslationally promotes PIF4 and PIF5 protein accumulation. Notably, the role of SOS2 in regulating PIF4 and PIF5 protein abundance and SAS is more prominent under salt stress. Moreover, phyA and phyB physically interact with SOS2 and promote SOS2 kinase activity in the light. Collectively, our study uncovers an unexpected role of salt-activated SOS2 in promoting SAS by modulating the phyB-PIF module, providing insight into the coordinated response of plants to salt stress and shade.

TIME FOR COFFEE regulates phytochrome A-mediated hypocotyl growth through dawn-phased signaling.

To enhance plant fitness under natural conditions, the circadian clock is synchronized and entrained by light via photoreceptors. In turn, the circadian clock exquisitely regulates the abundance and activity of photoreceptors via largely uncharacterized mechanisms. Here we show that the clock regulator TIME FOR COFFEE (TIC) controls the activity of the far-red light photoreceptor phytochrome A (phyA) at multiple levels in Arabidopsis thaliana. Null mutants of TIC displayed dramatically increased sensitivity to light irradiation with respect to hypocotyl growth, especially to far-red light. RNA-sequencing demonstrated that TIC and phyA play largely opposing roles in controlling light-regulated gene expression at dawn. Additionally, TIC physically interacts with the transcriptional repressor TOPLESS (TPL), which was associated with the significantly increased PHYA transcript levels in the tic-2 and tpl-1 mutants. Moreover, TIC interacts with phyA in the nucleus, thereby affecting phyA protein turnover and the formation of phyA nuclear speckles following light irradiation. Genetically, phyA was found to act downstream of TIC in regulating far red light-inhibited growth. Taken together, these findings indicate that TIC acts as a major negative regulator of phyA by integrating transcriptional and post-translational mechanisms at multiple levels.

Mutual upregulation of HY5 and TZP in mediating phytochrome A signaling.

Phytochrome A (phyA) is the far-red (FR) light photoreceptor in plants that is essential for seedling de-etiolation under FR-rich environments, such as canopy shade. TANDEM ZINC-FINGER/PLUS3 (TZP) was recently identified as a key component of phyA signal transduction in Arabidopsis thaliana; however, how TZP is integrated into the phyA signaling networks remains largely obscure. Here, we demonstrate that ELONGATED HYPOCOTYL5 (HY5), a well-characterized transcription factor promoting photomorphogenesis, mediates FR light induction of TZP expression by directly binding to a G-box motif in the TZP promoter. Furthermore, TZP physically interacts with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), an E3 ubiquitin ligase targeting HY5 for 26S proteasome-mediated degradation, and this interaction inhibits COP1 interaction with HY5. Consistent with those results, TZP post-translationally promotes HY5 protein stability in FR light, and in turn, TZP protein itself is destabilized by COP1 in both dark and FR light conditions. Moreover, tzp hy5 double mutants display an additive phenotype relative to their respective single mutants under high FR light intensities, indicating that TZP and HY5 also function in largely independent pathways. Together, our data demonstrate that HY5 and TZP mutually upregulate each other in transmitting the FR light signal, thus providing insights into the complicated but delicate control of phyA signaling networks.

Evaporation-driven internal hydraulic redistribution alleviates root drought stress: Mechanisms and modeling.

Many tree species have developed extensive root systems that allow them to survive in arid environments by obtaining water from a large soil volume. These root systems can transport and redistribute soil water during drought by hydraulic redistribution (HR). A recent study revealed the phenomenon of evaporation-driven hydraulic redistribution (EDHR), which is driven by evaporative demand (transpiration). In this study, we confirmed the occurrence of EDHR in Chinese white poplar (Populus tomentosa) through root sap flow measurements. We utilized microcomputed tomography technology to reconstruct the xylem network of woody lateral roots and proposed conceptual models to verify EDHR from a physical perspective. Our results indicated that EDHR is driven by the internal water potential gradient within the plant xylem network, which requires 3 conditions: high evaporative demand, soil water potential gradient, and special xylem structure of the root junction. The simulations demonstrated that during periods of extreme drought, EDHR could replenish water to dry roots and improve root water potential up to 38.9% to 41.6%. This highlights the crucial eco-physiological importance of EDHR in drought tolerance. Our proposed models provide insights into the complex structure of root junctions and their impact on water movement, thus enhancing our understanding of the relationship between xylem structure and plant hydraulics.

Microbiological characteristics of the lower airway in adults with bronchiectasis: a prospective cohort study.

BACKGROUND: Microbial infection and colonization are frequently associated with disease progression and poor clinical outcomes in bronchiectasis. Identification of pathogen spectrum is crucial for precision treatment at exacerbation of bronchiectasis. METHODS: We conducted a prospective cohort study in patients with bronchiectasis exacerbation onset and stable state. Bronchoalveolar lavage fluid (BALF) was collected for conventional microbiological tests (CMTs) and metagenomic Next-Generation Sequencing (mNGS). Bronchiectasis patients were monitored for documenting the time to the next exacerbation during longitudinal follow-up. RESULTS: We recruited 168 eligible participants in the exacerbation cohorts, and 38 bronchiectasis patients at stable state at longitudinal follow-up. 141 bronchiectasis patients at exacerbation onset had definite or probable pathogens via combining CMTs with mNGS reports. We identified that Pseudomonas aeruginosa, non-tuberculous mycobacteria, Haemophilus influenzae, Nocardia spp, and Staphylococcus aureus were the top 5 pathogens with a higher detection rate in our cohorts via combination of CMTs and mNGS analysis. We also observed strong correlations of Pseudomonas aeruginosa, Haemophilus influenzae, non-tuberculous mycobacteria with disease severity, including the disease duration, Bronchiectasis Severity Index, and lung function. Moreover, the adjusted pathogenic index of potential pathogenic microorganism negatively correlated (r = -0.7280, p < 0.001) with the time to the next exacerbation in bronchiectasis. CONCLUSION: We have revealed the pathogenic microbial spectrum in lower airways and the negative correlation of PPM colonization with the time to the next exacerbation in bronchiectasis. These results suggested that pathogens contribute to the progression of bronchiectasis.

Spatial domain decomposition-based physics-informed neural networks for practical acoustic propagation estimation under ocean dynamics.

Practical acoustic propagation modeling is significantly affected by ocean dynamics, and then can be exploited in numerous oceanic applications, where "practical" refers to modeling acoustic propagation in simulations that mimic real-world ocean environments. Physics-based numerical models provide approximate solutions of wave equation and rely on accurate prior environmental knowledge while the environment of practical scenarios is largely unknown. In contrast, data-driven machine learning offers a promising avenue to estimate practical acoustic propagation by learning from dataset. However, collecting such a high-quality, noise-free, and dense dataset remains challenging. Under the practical hurdle posed by the above approaches, the emergence of physics-informed neural network (PINN) presents an alternative to integrate physics and data but with limited representation capacity. In this work, a framework, termed spatial domain decomposition-based physics-informed neural networks (SPINNs), is proposed to enhance the representation capacity in spatially dependent oceanic scenarios and effectively learn from incomplete and biased prior physics and noisy dataset. Experiments demonstrate SPINNs' advantages over PINN in practical acoustic propagation estimation. The learning capacity of SPINNs toward physics and dataset during training is further analyzed. This work holds promise for practical applications and future expansion.

Clinical and Genetic Study of Three Inherited Microdeletions of Chromosome 16p11.2.

Copy number variations (CNVs) in chromosome 16p11.2 are not rare. 16p11.2 microdeletion is among the most commonly known genetic etiologies of overweightness, autism spectrum disorder (ASD), and related neurodevelopmental disorders. We report the prenatal diagnosis and genetic counseling of three cases with inherited 16p11.2 microdeletions. In these families, mother/father and fetus have the same microdeletion. Following the use of molecular genetic techniques including array-based methods, the number of reported cases has rapidly increased. A combination of prenatal three-dimensional ultrasound, karyotype analysis, chromosomal microarray analysis (CMA), copy number variation sequencing (CNV-seq), whole-exome sequencing (WES), and genetic counseling is helpful for the prenatal diagnosis of chromosomal microdeletions/microduplications.

Identification of numerous novel disease-causing variants in patients with inherited retinal diseases, combining careful clinical-functional phenotyping with systematic, broad NGS panel-based genotyping.

Purpose: The widespread consensus is that genotyping is essential for patients with inherited retinal disease (IRD). Given the numerous ongoing gene therapy clinical trials for IRDs, identifying the pathogenic mutation in these patients has potential important therapeutic implications. In this study, we demonstrate how we identified with a high degree of confidence numerous novel disease-causing mutations, deletions, and duplications in a large consecutive IRD case series by using a judicious combination of careful, in-depth clinical-functional phenotyping to guide and integrate our genotyping approach. Methods: We conducted a retrospective analysis of data between November 2016 and March 2018 from the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases IRD patient database, which encompassed 378 IRD cases that had not yet been previously genotyped. With the exception of some patients who presented with classical clinical-functional phenotypes that allowed for targeted gene testing, all other subjects systematically underwent next-generation sequencing-based broad, IRD-focused panel testing. Most cases were also tested for parental allele phase. Results were reviewed vis-à-vis the clinical-functional phenotypes for reconciliation and potential addition of supplemental testing such as deletion/duplication microarrays or copy number variant (CNV) analysis. Supplemental testing was driven by an IRD specialist-laboratory consensus, and decisions were clinically or genetically driven or both. Results: By judiciously using this two-way approach and leveraging to its full potential the benefits of careful, in-depth clinical-functional phenotyping by an experienced IRD specialist, more than 80% of the cases in this series were successfully genotyped. We also identified with a high degree of confidence 52 novel disease-causing mutations, deletions, and duplications. Conclusions: The combination of meticulous, expert clinical-functional phenotyping studies with systematic next-generation sequencing panel-based genotyping and microarray deletion/duplication testing or CNV analysis as applicable in accordance with the above-mentioned consensus was extremely effective at the diagnostic end, reduced costs, and saved time. IRD specialist-laboratory two-way interactions and case discussions would augment the efficacy of this approach and improve the diagnostic yield in successfully solving and genotyping IRD cases.

Association of SGLT2 Inhibitors With Risk of Atrial Fibrillation and Stroke in Patients With and Without Type 2 Diabetes: A Systemic Review and Meta-Analysis of Randomized Controlled Trials.

ABSTRACT: Sodium-glucose cotransporter 2 (SGLT2) inhibitors have well-documented effects on reducing hospitalization for heart failure and cardiovascular mortality, although the effect on atrial fibrillation (AF) has not been comprehensively investigated. Therefore, we performed a meta-analysis to assess the association between SGLT2 inhibitors and AF risk by systematically searching PubMed, Embase, and ClinicalTrials.gov. Two investigators independently identified randomized controlled trials, which compared SGLT2 inhibitors with control in patients with type 2 diabetes, heart failure, or chronic kidney disease. Primary outcomes were incident AF and stroke. We included 20 randomized trials involving 63,604 patients. The SGLT2 inhibitors used were dapagliflozin (7 studies, 28,834 patients), canagliflozin (7 studies, 17,440 patients), empagliflozin (5 studies, 9082 patients), and ertugliflozin (1 study, 8246 patients). Follow-up ranged from 24 weeks to 202 weeks. SGLT2 inhibitors treatment was associated with a significant attenuation in the risk of incident AF (odds ratio = 0.82; 95% confidence interval, 0.72-0.93; P = 0.002) compared with control. No significant difference in stroke between SGLT2 inhibitors and control groups was found (odds ratio = 0.99; 95% confidence interval, 0.85-1.15; P = 0.908). This present meta-analysis indicates that SGLT2 inhibitors are associated with a lower risk of incident AF and do not significantly affect stroke risk for patients with and without type 2 diabetes.

A novel alphahypovirus that infects the fungal plant pathogen Sclerotinia sclerotiorum.

A novel positive single-stranded RNA virus, Sclerotinia sclerotiorum hypovirus 9 (SsHV9), was identified in the plant-pathogenic Sclerotinia sclerotiorum strain GB375, which was associated with a garden bean plant in the United States. The complete genome of SsHV9 is 14,067 nucleotides in length, excluding the poly(A) tail. It has a single large open reading frame encoding a putative polyprotein (4,196 amino acids), which is predicted to contain a papain-like protease, a protein of unknown function, an RNA-dependent RNA polymerase, and an RNA helicase. Phylogenetic analysis based on a multiple alignment of amino acid sequences of polyproteins that suggested SsHV9 belongs to the proposed genus "Alphahypovirus" in the family Hypoviridae.

Laparoscopic Resection of a High Grade Serous Ovarian Cancer that Recurred at the Vaginal Stump with Extensive Pelvic Adhesions after Complete Surgical Staging.

STUDY OBJECTIVE: To show laparoscopic resection of a high grade serous ovarian cancer that recurred at the vaginal stump with extensive pelvic adhesions after complete surgical staging. DESIGN: Stepwise demonstration of the procedure with narrated video footage. SETTING: University hospital. INTERVENTIONS: We reported a case of a 62-year-old woman with a history of complete surgical staging of high grade serous ovarian cancer staged IIB, which consisted of hysterectomy, bilateral salpingo-oophorectomy, pelvic and para-aortic lymph node dissection, and omentectomy, about 18 months before this admission. She received 6 courses of carboplatin/paclitaxel combination therapy after complete surgical staging and achieved complete remission. About 12 months after the last course of chemotherapy, she visited the local clinic because of irregular vaginal bleeding. Physical examination revealed a 3 × 3 × 2 cm3 mass at the vaginal vault. Biopsy of the mass was performed under colposcopy, and pathological reports showed recurrent high grade serous cancer. Her serum cancer antigen 125 level was in normal range. Positron emission tomographic/computed tomographic imaging (PET/CT) showed no evidence of disease dissemination. A diagnosis of recurrent high grade serous ovarian cancer was made. After the biopsy of the recurrent mass, there were no visible lesions, which made us believe that laparoscopy management would not contribute to intraperitoneal spread of the tumors. Therefore, laparoscopic resection of the vaginal stump was scheduled. The key steps of the procedure were summarized as follows. First, the bowels were released from the side abdominal wall to expose bilateral external iliac vessels. Second, bilateral ureters were identified and mobilized to avoid incidental ureter injuries. Third, we opened the rectovaginal space and detached the rectum from the posterior vaginal wall. Fourth, the posterior vesical wall was separated from the vaginal stump. After exposure of the key anatomic landmarks, laparoscopic resection of the vaginal stump was performed safely. Final pathologic report showed recurrent high grade serous ovarian cancer. The patient received 6 courses of carboplatin/paclitaxel combination therapy, and maintenance therapy with olaparib was suggested, but the patient refused to accept this suggestion. She is still in complete remission 8 months after surgery. CONCLUSION: Laparoscopic resection of a high grade serous ovarian cancer that recurred at the vaginal stump with extensive pelvic adhesions after complete surgical staging was achieved successfully in a logical way. The critical point of the procedure is to expose the key anatomic landmarks of the pelvis to avoid incidental injuries [1].

A Way to Reduce the Occurrence of Intraoperative Capsule Rupture in Presumed Clinically Early-stage Ovarian Cancer with Adhesions to the Abdominal Wall.

STUDY OBJECTIVE: To present a procedure to reduce the occurrence of intraoperative capsule rupture in presumed clinically early-stage ovarian cancer with adhesions to the abdominal wall. DESIGN: Stepwise presentation of the procedure with narrated video footage. SETTING: The occurrence of intraoperative capsule rupture exerts a negative effect on the prognosis of early-stage ovarian cancer [1,2]. Thus, it is important to reduce intraoperative capsule rupture to improve the oncologic outcome of such patients. In this video we describe a laparoscopic procedure to minimize the risk of intraoperative capsule rupture in presumed clinically early-stage ovarian cancer with adhesions to the abdominal wall. A 52-year-old woman was referred from a local clinic for a 6 × 6 × 4-cm left ovarian mass and a 7 × 6 × 6-cm right ovarian mass. Her serum cancer antigen 125 level was 214.4U/mL. Pelvic magnetic resonance imaging and positron emission tomographic/computed tomographic imaging showed no evidence of metastatic diseases or lymph node involvement. A diagnosis of ovarian malignancy was suspected. INTERVENTIONS: Laparoscopic evaluation suggested that the right adnexa was adhered to the right abdominal wall and there was no evidence of tumor seeding in the peritoneal cavity. We collected the peritoneal lavage fluid. Since pelvic adhesiolysis between the right adnexa and the abdominal wall may increase the occurrence of intraoperative capsule rupture of the ovarian tumor, leading to a worse clinical outcome, we decided to remove both the right adnexa as well as the adhered peritoneum. The key steps of the procedure are summarized as follows. First, the utero-ovarian ligament and tubal isthmus were coagulated and excised. Second, the pelvic peritoneum was excised, and the infundibulo-pelvic ligament and ureter were identified and mobilized. Third, the infundibulo-pelvic ligament was coagulated and excised. Fourth, the pelvic peritoneum which was adhered to the right adnexa was dissected off the ureter and excised. Then, the resected right adnexa as well as the adhered peritoneum were collected in a disposable pocket and removed to avoid further contamination. Adenocarcinoma was diagnosed by frozen section evaluation. So, surgical staging was performed laparoscopically, and consisted of hysterectomy, bilateral salpingo-oophorectomy, pelvic and para-aortic lymph node dissection, omentectomy, and random peritoneal biopsies from the pelvis, paracolic gutters, and undersurfaces of the diaphragm. Final pathologic reports showed ovarian clear cell carcinoma with involvement of both ovaries and the adhered peritoneum. CONCLUSION: Our method is effective for intact removal of the involved adnexa without rupture and the adhered pelvic peritoneum with potential for tumor seeding.

Folate-grafted glycyl-glycine-melphalan conjugate self-assembled amphilphilc nanomicelles augmented drug delivery, cytotoxicity and cellular uptake in human ovarian cancer cells.

OBJECTIVES: Folic acid was coupled to melphalan using glycyl-glycine (FA-Gly-Gly-Melphalan) to synthesise self-assembled nanomicelles for targeting ovarian cancer cells, SKOV3. METHODS AND RESULTS: FA-Gly-Gly-Melphalan self-assembled nanomicelles were prepared with critical micellar concentration (CMC) of 12-µg/ml. The mean particle size of FA-Gly-Gly-Melphalan self-assembled nanomicelles was measured to be 95.9 ± 3.4-nm significantly (p < 0.05) higher than 73.8 ± 6.3-nm of Gly-Gly-Melphalan self-assembled nanomicelles. Subsequently, zeta-potential of FA-Gly-Gly-Melphalan self-assembled nanomicelles was estimated to be -28.0 ± 1.5-mV significantly (p < 0.05) lower than -36.6 ± 2.7-mV of Gly-Gly-Melphalan self-assembled nanomicelles. The IC50 of FA-Gly-Gly-Melphalan self-assembled nanomicelles was estimated to be 4.1-µg/ml significantly (p < 0.001) lower than 14.2-µg/ml of Gly-Gly-Melphalan self-assembled nanomicelles and >18-µg/ml of melphalan. FA-Gly-Gly-Melphalan self-assembled nanomicelles preferentially accumulated in cytoplasm of SKOV3 cells nearby nucleus via receptor mediated endocytosis pathway after 24-h of incubation period, whilst Gly-Gly-Melphalan self-assembled nanomicelles were not incorporated sufficiently. CONCLUSION: FA-Gly-Gly-Melphalan self-assembled nanomicelles warrant in depth in vivo study for their safety, efficacy, and potency in clinical settings.

SICKLE represses photomorphogenic development of Arabidopsis seedlings via HY5- and PIF4-mediated signaling.

Arabidopsis CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and PHYTOCHROME INTERACTING FACTORs (PIFs) are negative regulators, and ELONGATED HYPOCOTYL5 (HY5) is a positive regulator of seedling photomorphogenic development. Here, we report that SICKLE (SIC), a proline rich protein, acts as a novel negative regulator of photomorphogenesis. HY5 directly binds the SIC promoter and activates SIC expression in response to light. In turn, SIC physically interacts with HY5 and interferes with its transcriptional regulation of downstream target genes. Moreover, SIC interacts with PIF4 and promotes PIF4-activated transcription of itself. Interestingly, SIC is targeted by COP1 for 26S proteasome-mediated degradation in the dark. Collectively, our data demonstrate that light-induced SIC functions as a brake to prevent exaggerated light response via mediating HY5 and PIF4 signaling, and its degradation by COP1 in the dark avoid too strong inhibition on photomorphogenesis at the beginning of light exposure.

Physiological and transcriptome analysis of Magnolia denudata leaf buds during long-term cold acclimation.

BACKGROUND: Magonlia denudata is an important perennial tree species of the Magnoliaceae family, known for its ornamental value, resistance to smoke pollution and wind, role in air purification, and robust cold tolerance. In this study, a high-throughput transcriptome analysis of leaf buds was performed, and gene expression following artificial acclimation 22 °C, 4 °C and 0 °C, was compared by RNA sequencing. RESULTS: Over 426 million clean reads were produced from three libraries (22 °C, 4 °C and 0 °C). A total of 74,503 non-redundant unigenes were generated, with an average length of 1173.7 bp (N50 = 1548). Based on transcriptional results, 357 and 235 unigenes were identified as being upregulated and downregulated under cold stress conditions, respectively. Differentially expressed genes were annotated using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses. The transcriptomic analysis focused on carbon metabolism and plant hormone signal transduction associated with cold acclimation. Transcription factors such as those in the basic helix-loop-helix and AP2/ERF families were found to play an important role in M. denudata cold acclimation. CONCLUSION: M. denudata exhibits responses to non-freezing cold temperature (4 °C) to increase its cold tolerance. Cold resistance was further strengthened with cold acclimation under freezing conditions (0 °C). Cold tolerance genes, and cold signaling transcriptional pathways, and potential functional key components for the regulation of the cold response were identified in M. denudata. These results provide a basis for further studies, and the verification of key genes involved in cold acclimation responses in M. denudata lays a foundation for developing breeding programs for Magnoliaceae species.

Hinge region of Arabidopsis phyA plays an important role in regulating phyA function.

Phytochrome A (phyA) is the only plant photoreceptor that perceives far-red light and then mediates various responses to this signal. Phosphorylation and dephosphorylation of oat phyA have been extensively studied, and it was shown that phosphorylation of a serine residue in the hinge region of oat phyA could regulate the interaction of phyA with its signal transducers. However, little is known about the role of the hinge region of Arabidopsis phyA. Here, we report that three sites in the hinge region of Arabidopsis phyA (i.e., S590, T593, and S602) are essential in regulating phyA function. Mutating all three of these sites to either alanines or aspartic acids impaired phyA function, changed the interactions of mutant phyA with FHY1 and FHL, and delayed the degradation of mutant phyA upon light exposure. Moreover, the in vivo formation of a phosphorylated phyA form was greatly affected by these mutations, while our data indicated that the abundance of this phosphorylated phyA form correlated well with the extent of phyA function, thus suggesting a pivotal role of the phosphorylated phyA in inducing the far-red light response. Taking these data together, our study reveals the important role of the hinge region of Arabidopsis phyA in regulating phyA phosphorylation and function, thus linking specific residues in the hinge region to the regulatory mechanisms of phyA phosphorylation.

Toward newborn screening of cerebrotendinous xanthomatosis: results of a biomarker research study using 32,000 newborn dried blood spots.

PURPOSE: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4), 5ß-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS). METHODS: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. CONCLUSION: The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.

The inhibitory effect and safety of GANT61 on HeLa cells in nude mice.

The Hedgehog (Hh) pathway effector Gli1 plays an important role in cervical cancer, and GANT61 is an Hh signaling inhibitor. In this study, we aimed to investigate the inhibitory effect of GANT61 on cervical cancer and to study its safety in nude mice. We used in vivo experiments to assess the effect of GANT61 on the growth of cervical cancer HeLa cells, and we measured the WBC, HGB, PLT, ALT, AST and Cre levels in nude mice. Next, we examined the organ and tumor morphology and distant metastasis by HE staining. We used immunohistochemistry to monitor the expression levels of Gli1, FoxM1, Ki-67, cyclinD1, E-cadherin, vimentin, survivin, caspase-3 and CD34+. Western blotting and RT-RCR were used to measure Gli1 expression. GANT61 inhibited the growth and metastasis of HeLa cervical cancer cells upon their transplantation into nude mice, and we preliminarily propose that GANT61 is safe for nude mice. These findings suggest that GANT61 could be used as a Hedgehog inhibitor to inhibit EMT and proliferation and to promote apoptosis via Gli1 downregulation.

Pharmacokinetics and metabolism of ulixertinib in rat by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

The purpose of this study was to develop and validate a simple and sensitive liquid chromatography tandem mass spectrometry method for the determination of ulixertinib in rat plasma. The plasma samples were precipitated with acetonitrile and then separated on a C18 column with water containing 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.3 mL/min. Analytes were monitored on a TSQ Vantage triple quadrupole tandem mass spectrometer operated in positive electrospray ionization mode. Selected reaction monitoring transitions were m/z 433.1→262.1 for ulixertinib and m/z 450.1→260.1 for internal standard. The assay achieved good linearity over the concentration range of 0.1-1000 ng/mL with correlation coefficient > 0.9991. The validated assay has been successfully applied to pharmacokinetic study of ulixertinib in rat after oral and intravenous administration. The results revealed that ulixertinib showed high exposure in rat plasma, low clearance, moderate oral bioavailability (45.13%), and dose-independent pharmacokinetic profiles over the oral dose range of 1-15 mg/kg. In addition, six metabolites from rat plasma and hepatocytes were detected and structurally identified by ultra-high performance liquid chromatography combined with high-resolution mass spectrometry. The metabolic pathways of ulixertinib referred to hydroxylation and dealkylation and glucuronidation.