[Mechanism of total glucosides of White Paeony Capsules in ameliorating acute lung injury based on NLRP3 inflammasome].

This study deciphered the ameliorating effect and molecular mechanism of the total glucosides of White Paeony Capsules(TGP) in the treatment of mice model with acute lung injury(ALI) via NOD-like receptor thermal protein domain associated protein 3(NLRP3) signaling pathway of the inflammasome. The study established an inflammasome activation model of primed bone marrow-derived macrophages(BMDMs), and its molecular mechanism was investigated by Western blot(WB), immunofluorescence staining, enzyme-linked immunosorbent assay(ELISA), and flow cytometry. C57BL/6J mice were randomly divided into a blank control group, a TGP group, a model group(LPS group), LPS+low-and high-dose TGP groups, LPS+MCC950 group, and LPS+MCC950+TGP group, with eight mice per group. The ALI model was induced in mice. Finally, bronchoalveolar lavage fluid(BALF) and lung tissue were collected. Lung index and lung weight wet-to-dry ratio were determined for each group of mice. The pathological changes in lung tissue were observed through hematoxylin-eosin(HE) staining. The number of neutrophils in the BALF of each group was detected using flow cytometry. The levels of interleukin(IL)-1ß, IL-6, and tumor necrosis factor(TNF)-α in the BALF were determined by ELISA. The expressions of IL-1ß, IL-18, IL-6, and TNF-α in the lung tissue were determined by real-time quantitative PCR(RT-qPCR). This study demonstrated that TGP dramatically blocked the activation of the NLRP3 inflammasome by inhibiting the production of upstream mitochondrial reactive oxygen species(mtROS) and the subsequent oligomerization of apoptosis-associated specks(ASC). Additionally, in the ALI mice model, compared with the blank control group, the model group showed alveolar structure rupture, thic-kening of alveolar septa, and dramatically increased lung index, lung weight wet-to-dry ratio in lung tissue, neutrophil count, and inflammatory factor levels. Compared with the model group, the pathological morphology of lung tissue was significantly ameliorated in the TGP and MCC950 groups, and the lung index and lung weight wet-to-dry ratio were significantly reduced. Neutrophil counts were reduced, and levels of inflammatory factors were significantly downregulated. Notably, compared with the MCC950 group, there was no significant difference in effect in the MCC950+TGP group. Collectively, the study reveals that TGP may ameliorate ALI in mice by inhibiting the activation of NLRP3 inflammasome, providing a safe and effective drug candidate for the prevention or treatment of ALI/ARDS.

Novel mutations and molecular pathways identified in patients with brain iron accumulation disorders.

Brain iron accumulation disorders (BIADs) are a group of diseases characterized by iron overload in deep gray matter nuclei, which is a common feature of neurodegenerative diseases. Although genetic factors have been reported to be one of the etiologies, much more details about the genetic background and molecular mechanism of BIADs remain unclear. This study aimed to illustrate the genetic characteristics of BIADs and clarify their molecular mechanisms. A total of 84 patients with BIADs were recruited from April 2018 to October 2022 at Xuanwu Hospital. Clinical characteristics including family history, consanguineous marriage history, and age at onset (AAO) were collected and assessed by two senior neurologists. Neuroimaging data were conducted for all the patients, including cranial magnetic resonance imaging (MRI) and susceptibility-weighted imaging (SWI). Whole-exome sequencing (WES) and capillary electrophoresis for detecting sequence mutation and trinucleotide repeat expansion, respectively, were conducted on all patients and part of their parents (whose samples were available). Variant pathogenicity was assessed according to the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP). The NBIA and NBIA-like genes with mutations were included for bioinformatic analysis, using Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genome (KEGG). GO annotation and KEGG pathway analysis were performed on Metascape platform. In the 84 patients, 30 (35.7%) were found to carry mutations, among which 20 carried non-dynamic mutations (missense, stop-gained, frameshift, inframe, and exonic deletion) and 10 carried repeat expansion mutations. Compared with sporadic cases, familial cases had more genetic variants (non-dynamic mutation: P=0.025, dynamic mutation: P=0.003). AAO was 27.85±10.42 years in cases with non-dynamic mutations, which was significantly younger than those without mutations (43.13±17.17, t=3.724, P<0.001) and those with repeated expansions (45.40±8.90, t=4.550, P<0.001). Bioinformatic analysis suggested that genes in lipid metabolism, autophagy, mitochondria regulation, and ferroptosis pathways are more likely to be involved in the pathogenesis of BIADs. This study broadens the genetic spectrum of BIADs and has important implications in genetic counselling and clinical diagnosis. Patients diagnosed as BIADs with early AAO and family history are more likely to carry mutations. Bioinformatic analysis provides new insights into the molecular pathogenesis of BIADs, which may shed lights on the therapeutic strategy for neurodegenerative diseases.

Peripheral Blood Monocytosis Is Associated With Long-Term Disease Severity in Pediatric-Onset Inflammatory Bowel Disease.

OBJECTIVES: Peripheral blood monocytosis (PBM) is a marker of increased disease severity in adults with inflammatory bowel diseases (IBDs). We sought to determine whether PBM serves as a prognostic biomarker in patients with pediatric-onset IBD for a more aggressive long-term disease course when followed into adulthood. METHODS: Patients with pediatric-onset inflammatory bowel disease were identified within an adult tertiary care center, within a consented, prospectively collected natural history disease registry, to compare clinical outcomes between patients with and without PBM from the years 2009 to 2019. Patients demonstrating elevation in PBM at any time defined membership and long-term clinical trajectories were compared with pediatric-onset patients without PBM. RESULTS: A total of 581 patients with IBD, diagnosed by 18 years of age, were identified for inclusion, of which 440 patients were diagnosed with Crohn disease and 141 with ulcerative colitis. Monocytosis was detected by complete blood cell counts in 40.1% of patients. PBM was associated with steroid and biologic exposure, number of IBD-related surgeries, and increased health care utilization. Multivariate logistic regression analyses, accounting for elevation of inflammatory markers and other values associated with acute disease activity as well as steroid use, showed persistently increased odds of biologic exposure, emergency department visits, and hospitalizations, but not surgeries, after detection of monocytosis. CONCLUSIONS: Within patients with pediatric-onset IBD, the sub-cohort with PBM had associated worse clinical outcomes and other markers of increased disease severity.

Pancreatic Enzyme Replacement Therapy in Patients with Non-pancreatic Digestive Conditions: A Nationwide Claims Analysis.

BACKGROUND AND AIMS: Pancreatic enzyme replacement therapy (PERT) is most commonly used to treat exocrine insufficiency related to pancreatic diseases, but can be used for non-pancreatic digestive conditions (NPDC). We aimed to determine the prevalence of PERT use and describe prescription patterns in individuals with NPDC. METHODS: A nationally representative claims database of 48.6 million enrollees was used to identify individuals who received PERT prescription(s) in the absence of any pancreas-related diagnosis. Data on demographics, enrolment, comorbidities, exocrine function testing, treatment and potential indications for PERT were retrieved, and compared with individuals who received PERT for primary diagnosis of chronic pancreatitis (CP). RESULTS: A total of 29,234 individuals (64.1% female, mean age 52.4 ± 16.5 years) received PERT for NPDC. The overall estimated US population prevalence rate for PERT use for NDPC was 60.2/100,000 persons. Rates increased significantly with age and were higher in women in all age groups except 1-20 years old. When compared with CP, individuals with NPDC receiving PERT were more likely to be older (52.4 vs. 50.1 years), female (64.1% vs. 51.0%), have lower prevalence of alcoholism (3.6% vs. 25.0%), tobacco abuse (8.4% vs. 30.1%), and received PERT for shorter mean duration (5.3 vs. 8.2 months) (all p < 0.001). Median dose of PERT in individuals with NPDC was 2880 lipase units/day. CONCLUSIONS: Although proportionally low, a sizable population receives PERT for NPDC. PERT for NPDC is usually prescribed at a low dose and for shorter duration, suggesting it is used mostly as a trial for or until resolution of symptoms.

Hartnup disease presenting as hereditary spastic paraplegia and severe peripheral neuropathy.

Hartnup disease cases were rare, and the genotype-phenotype correlation was not fully understood. Here we reported two unrelated young men diagnosed as Hartnup disease, who carried novel compound heterozygote mutations in the SLC6A19 gene and presented with new phenotypes. Other than intermittent encephalopathy and photosensitive rashes, they displayed symptoms and signs of spastic paraplegia and severe peripheral nerve damages. Magnetic resonance imaging showed mild bilateral cerebellar atrophy and thinning of the thoracic spinal cord. Electromyogram detected mixed sensorimotor polyneuropathy in lower limbs. Sural nerve biopsy and pathological study indicated the moderately reduced neural fibers in the periphery nerves. Urinary amino acid analysis showed increased levels of multiple neutral amino acids. Moreover, muscle strengths in the lower limbs and the walking ability have been improved in both cases (MRC 3/5 to 4/5 in Patient 1; walking distance elongated from 50 to 100 m in Patient 2) after the treatment with oral nicotinic acid and intravenous injection of multiple amino acids. Exome sequencing revealed and confirmed the existence of the novel compound heterozygous SLC6A19 mutations: c.533G>A (p.Arg178Gln) and c.1379-1G>C mutations in patient1, and c.1433delG (p.Gly478AlafsTer44) and c.811G>A (p.Ala271Thr) in patient 2. Taken together, these findings expanded the clinical, neuroimaging, pathology, and genetic spectrum of Hartnup disease. However, the co-existence of HSP and peripheral neuropathy was only inferred based on clinical observations, and pathological and molecular studies are needed to further dissect the underlying mechanisms.

Characteristics of Interferon-Associated Diabetes Mellitus in Past 30 Years: A Review.

Interferon (IFN) is a broad-spectrum antiviral agent that activates cell surface receptors and causes cells to produce antiviral proteins, inhibiting viral replication. Interferon use has long been associated with diabetes. The PubMed database was searched for articles related to diabetes and interferon from March 30, 2020. Patients were divided into type 1 diabetes group and type 2 diabetes group. We reviewed the relevant literature to compare interferon-associated T1D and interferon-associated T2D differences. Interferon treatment shortened the incubation period of T2D and changed the original T2D to T1D. The onset of interferon-associated T1D required longer periods of IFN treatment than interferon-associated T2D, and the interferon-associated T1D group had higher GADA positive rates, lower BMI, lower fasting blood glucose, and greater insulin dependence (p<0.05). More patients in the T1D group were positive for HLA-DRB1*04, DRB1*03, DRB1*09, DRB1*14, HLA-DQB1*04, HLA-DQB1*02, HLA-DQB1*03, and HLA-DQB1*05. The combined detection of GAD antibodies and HLA alleles may be an effective method to predict the incidence of T1D after IFN treatment.

CD155/TIGIT signaling regulates the effector function of tumor-infiltrating CD8+ T cell by NF-κB pathway in colorectal cancer.

BACKGROUND AND AIM: CD155/T-cell immunoglobulin and ITIM domain (TIGIT) suppressed anti-cancer immunity in several cancers, but its roles in colorectal cancer (CRC) were not clear. Here, we investigated its roles in CRC. METHODS: The percentages of CD8+ T cells expressing TIGIT and secreting cytokines (IL-2, TNF-α, and IFNγ) were evaluated by flow cytometry. The expression level of CD155 was determined by western blot and immunohistochemistry. The levels of cytokines were determined by enzyme-linked immunosorbent assay. The activation of the nuclear factor-kappa B (NF-κB) pathway was examined by western blot and immunofluorescent assay. RESULTS: T-cell immunoglobulin and ITIM domain was overexpressed on CD8+ T cells of CRC patients and mice. CD155 was overexpressed in mice CRC tissues and cells. The addition of CD155 recombinant protein could decrease the percentages of CD8+ T cells secreting cytokines. Blocking TIGIT could increase the percentages of cytokine-secreting CD8+ T cells. Coculturing with CD155-knockdown CRC cells could upregulate the percentages of CD8+ T cells secreting cytokines. Blocking TIGIT partially counteracted the effect of the knockdown of CD155. Besides, coculturing with CD155-knockdown CRC cells could promote the secretion of cytokines, activate the NF-κB pathway, and enhance the nuclear translocation of p65. And these effects were counteracted by the application of an NF-κB inhibitor. Finally, blocking TIGIT played anti-cancer roles such as suppression of tumor growth, increasing the percentages of cytokine-secreting CD8+ T cells and activation of the NF-κB signaling pathway. CONCLUSION: Suppressing CD155/TIGIT exerted anti-cancer effects against CRC, and our findings provided a potential therapeutic approach to treat CRC.

Factors Associated with the Development of Gastrointestinal Symptoms in Patients Hospitalized with Covid-19.

BACKGROUND AND AIMS: The most common symptoms of Covid-19 are respiratory; however, gastrointestinal symptoms are present in up to 50% of patients. We aimed to determine characteristics associated with the development of gastrointestinal symptoms in patients with Covid-19. METHODS: A case-control study of adults hospitalized for Covid-19 was conducted across a geographically diverse alliance of 36 US and Canadian medical centers. Data were manually abstracted from electronic health records and analyzed using regression analyses to determine characteristics associated with any gastrointestinal symptoms and diarrhea specifically. RESULTS: Of 1406 patients, 540 (38%) reported at least one gastrointestinal symptom and 346 (25%) reported diarrhea. Older patients (≥ 80 years) had significantly lower rates of any gastrointestinal symptoms and diarrhea (vs. patients 18-79 years, OR 0.41, p < 0.01 and OR 0.43 p = 0.01, respectively), while those with IBS (OR 7.70, p = 0.02 and OR 6.72, p < 0.01, respectively) and on immunosuppressive therapy (OR = 1.56, p = 0.02) had higher rates of any gastrointestinal symptom and diarrhea. Patients with constitutional symptoms exhibited significantly higher rates (OR 1.91, p < 0.01), while those with pulmonary disease alone had lower rates of gastrointestinal symptoms (OR 0.23, p = 0.01). A significant interaction between constitutional symptoms and pre-existing pulmonary conditions was observed. CONCLUSIONS: Several patient- and disease-specific characteristics associate with gastrointestinal symptoms in patients with Covid-19. Knowledge of these may provide insights into associated pathophysiologic mechanisms, and help health care professionals provide targeted attention to reduce morbidity related to Covid-19.

Arabidopsis ADF1 is Regulated by MYB73 and is Involved in Response to Salt Stress Affecting Actin Filament Organization.

Actin cytoskeleton and transcription factors play key roles in plant response to salt stress; however, little is known about the link between the two regulators in response to salt stress. Actin-depolymerizing factors (ADFs) are conserved actin-binding proteins in eukaryotes. Here, we revealed that the expression level of ADF1 was induced by salt stress. The adf1 mutants showed significantly reduced survival rate, increased percentage of actin cable and reduced density of actin filaments, while ADF1 overexpression seedlings displayed the opposite results when compared with WT under the same condition. Furthermore, biochemical assays revealed that MYB73, a R2R3 MYB transcription factor, binds to the promoter of ADF1 and represses its expression via the MYB-binding site core motif ACCTAC. Taken together, our results indicate that ADF1 participates in salt stress by regulating actin organization and may also serve as a potential downstream target of MYB73, which is a negative regulator of salt stress.

GLABRA2 Regulates Actin Bundling Protein VILLIN1 in Root Hair Growth in Response to Osmotic Stress.

Actin binding proteins and transcription factors are essential in regulating plant root hair growth in response to various environmental stresses; however, the interaction between these two factors in regulating root hair growth remains poorly understood. Apical and subapical thick actin bundles are necessary for terminating rapid elongation of root hair cells. Here, we show that Arabidopsis (Arabidopsis thaliana) actin-bundling protein Villin1 (VLN1) decorates filaments in shank, subapical, and apical hairs. vln1 mutants displayed significantly longer hairs with longer hair growing time and defects in the thick actin bundles and bundling activities in the subapical and apical regions, whereas seedlings overexpressing VLN1 showed different results. Genetic analysis showed that the transcription factor GLABRA2 (Gl2) played a regulatory role similar to that of VLN1 in hair growth and actin dynamics. Moreover, further analyses demonstrated that VLN1 overexpression suppresses the gl2 mutant phenotypes regarding hair growth and actin dynamics; GL2 directly recognizes the promoter of VLN1 and positively regulates VLN1 expression in root hairs; and the GL2-mediated VLN1 pathway is involved in the root hair growth response to osmotic stress. Our results demonstrate that the GL2-mediated VLN1 pathway plays an important role in the root hair growth response to osmotic stress, and they describe a transcriptional mechanism that regulates actin dynamics and thereby modulates cell tip growth in response to environmental signals.

Serum biomarkers for chronic pancreatitis pain patterns.

OBJECTIVES: Chronic pancreatitis (CP) is associated with debilitating refractory pain. Distinct subtypes of CP pain have been previously characterized based on severity (none, mild-moderate, severe) and temporal (none, intermittent, constant) nature of pain, but no mechanism-based tools are available to guide pain management. This exploratory study was designed to determine if potential pain biomarkers could be detected in patient serum and whether they associate with specific pain patterns. METHODS: Cytokines, chemokines, and peptides associated with nociception and pain were measured in legacy serum samples from CP patients (N = 99) enrolled in the North American Pancreatitis Studies. The unsupervised hierarchical cluster analysis was applied to cluster CP patients based on their biomarker profile. Classification and regression tree was used to assess whether these biomarkers can predict pain outcomes. RESULTS: The hierarchical cluster analysis revealed a subset of patients with predominantly constant, mild-moderate pain exhibited elevated interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-2 (IL-2), tumor necrosis factor alpha (TNFα), and monocyte chemoattractant protein-1 (MCP1) whereas patients with higher interleukin-4 (IL-4), interleukin-8 (IL-8) and calcitonin gene related peptide (CGRP) were more likely to have severe pain. Interestingly, analyses of each individual biomarker revealed that patients with constant pain had reduced circulating TNFα and fractalkine. Patients with severe pain exhibited a significant reduction in TNFα as well as trends towards lower levels of IL-6 and substance P. DISCUSSION: The observations from this study indicate that unique pain experiences within the chronic pancreatitis population can be associated with distinct biochemical signatures. These data indicate that further hypothesis-driven analyses combining biochemical measurements and detailed pain phenotyping could be used to develop precision approaches for pain management in patients with chronic pancreatitis.

Meg3-DMR, not the Meg3 gene, regulates imprinting of the Dlk1-Dio3 locus.

The imprinted delta like 1 homolog (DLK1) - thyroxine deiodinase type III (DIO3) locus regulates development and growth. Its imprinting regulation involves two differentially methylated regions (DMRs), intergenic-DMR (IG-DMR) and maternally expressed gene 3-DMR (Meg3-DMR). In mice, a maternal deletion of the IG-DMR leads to LOI in the locus, proving that the IG-DMR is a cis-acting imprinting control region of the locus. However, the Meg3-DMR overlaps with the promoter, exon 1 and intron 1 of the Meg3 gene. Because deletion of the Meg3-DMR inactivates the Meg3 gene, their roles in imprinting regulation of Meg3-DMR mice is unknown. Therefore, we generated two mouse models: Meg3Δ(1-4) and Meg3Δ(2-4), respectively targeting exons 1-4 and exons 2-4 of the Meg3 gene. A maternal deletion of Meg3Δ(1-4) caused embryonic death and LOI in both embryos and placentas, but did not affect methylation status of the IG-DMR. In contrast, mice carrying a maternal deletion of Meg3Δ(2-4) were born normally and did not have LOI. These data indicate that it is the Meg3-DMR, not the Meg3 gene, which regulates imprinting of the Dlk1-Dio3 locus.

Patterns of seizure prophylaxis after oncologic neurosurgery.

BACKGROUND: Evidence supporting routine postoperative antiepileptic drug (AED) prophylaxis following oncologic neurosurgery is limited, and actual practice patterns are largely unknown beyond survey data. OBJECTIVE: To describe patterns and predictors of postoperative AED prophylaxis following intracranial tumor surgery. METHODS: The MarketScan Database was used to analyze pharmacy claims data and clinical characteristics in a national sample over a 5-year period. RESULTS: Among 5895 patients in the cohort, levetiracetam was the most widely used AED for prophylaxis (78.5%) followed by phenytoin (20.5%). Prophylaxis was common but highly variable for patients who underwent open resection of supratentorial intraparenchymal tumors (62.5%, reference) or meningiomas (61.9%). In multivariate analysis, biopsies were less likely to receive prophylaxis (44.8%, OR 0.47, 95% CI 0.33-0.67), and there was near consensus against prophylaxis for infratentorial (9.7%, OR 0.07, CI 0.05-0.09) and transsphenoidal procedures (0.4%, OR 0.003, CI 0.001-0.010). Primary malignancies (52.1%, reference) and secondary metastases (42.2%) were more likely to receive prophylaxis than benign tumors (23.0%, OR 0.63, CI 0.48-0.83), as were patients discharged with home services and patients in the Northeast. There was a large spike in duration of AED use at approximately 30 days. CONCLUSIONS: Use of seizure prophylaxis following intracranial biopsies and supratentorial resections is highly variable, consistent with a lack of guidelines or consensus. Current practice patterns do not support a clear standard of care and may be driven in part by geographic variation, availability of post-discharge services, and electronic prescribing defaults rather than evidence. Given uncertainty regarding effectiveness, indications, and appropriate duration of AED prophylaxis, well-powered trials are needed.

The effect of Funneliformis mosseae on the plant growth, Cd translocation and accumulation in the new Cd-hyperaccumulator Sphagneticola calendulacea.

The screening and identification of hyperaccumulators is the key to the phytoremediation of soils contaminated by heavy metal (HM). Arbuscular mycorrhizal fungus (AMF) can improve plant growth and tolerance to HM; therefore, AMF-assisted phytoextraction has been regarded as a potential technique for the remediation of HM-polluted soils. A greenhouse pot experiment was conducted to determine whether Sphagneticola calendulacea is a Cd-hyperaccumulator and to investigate the effect of the AMF-Funneliformis mosseae (FM) on plant growth and on the accumulation, subcellular distribution and chemical form of Cd in S. calendulacea grown in soils supplemented with different Cd levels. At 25, 50 and 100 mg Cd kg-1 level, S. calendulacea showed high Cd tolerance, the translocation factor and the bioconcentration factor exceeded 1, and accumulation of more than 100 mg Cd kg-1 was observed in the aboveground parts of the plant, meeting the requirements for a Cd-hyperaccumulator. Moreover, FM colonization significantly increased both biomasses and Cd concentration in S. calendulacea. After FM inoculation, the Cd concentrations and proportions increased in the cell walls, but exhibited no significant change in the organelles of the shoots. Meanwhile, FM symbiosis contributed to the conversion of Cd from highly toxic chemical forms (extracted by 80% ethanol and deionized water) to less toxic chemical forms (extracted by 1 M NaCl, 2% acetic acid, 0.6 M HCl) of Cd in the shoots. Overall, S. calendulacea is a typical Cd-hyperaccumulator, and FM symbiosis relieved the phytotoxicity of Cd and promoted plant growth and Cd accumulation, and thus greatly increasing the efficiency of phytoextraction for Cd-polluted soil. Our study provides a theoretical basis and application guidance for the remediation of Cd-contaminated soil by the symbiont of S. calendulacea with FM.

QRank: a novel quantile regression tool for eQTL discovery.

MOTIVATION: Over the past decade, there has been a remarkable improvement in our understanding of the role of genetic variation in complex human diseases, especially via genome-wide association studies. However, the underlying molecular mechanisms are still poorly characterized, impending the development of therapeutic interventions. Identifying genetic variants that influence the expression level of a gene, i.e. expression quantitative trait loci (eQTLs), can help us understand how genetic variants influence traits at the molecular level. While most eQTL studies focus on identifying mean effects on gene expression using linear regression, evidence suggests that genetic variation can impact the entire distribution of the expression level. Motivated by the potential higher order associations, several studies investigated variance eQTLs. RESULTS: In this paper, we develop a Quantile Rank-score based test (QRank), which provides an easy way to identify eQTLs that are associated with the conditional quantile functions of gene expression. We have applied the proposed QRank to the Genotype-Tissue Expression project, an international tissue bank for studying the relationship between genetic variation and gene expression in human tissues, and found that the proposed QRank complements the existing methods, and identifies new eQTLs with heterogeneous effects across different quantile levels. Notably, we show that the eQTLs identified by QRank but missed by linear regression are associated with greater enrichment in genome-wide significant SNPs from the GWAS catalog, and are also more likely to be tissue specific than eQTLs identified by linear regression. AVAILABILITY AND IMPLEMENTATION: An R package is available on R CRAN at https://cran.r-project.org/web/packages/QRank . CONTACT: xs2148@cumc.columbia.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

NTL8 Regulates Trichome Formation in Arabidopsis by Directly Activating R3 MYB Genes TRY and TCL1.

The NAM, ATAF1/2, and CUC (NAC) are plant-specific transcription factors that regulate multiple aspects of plant growth and development and plant response to environmental stimuli. We report here the identification of NTM1-LIKE8 (NTL8), a membrane-associated NAC transcription factor, as a novel regulator of trichome formation in Arabidopsis (Arabidopsis thaliana). From an activation-tagged Arabidopsis population, we identified a dominant, gain-of-function mutant with glabrous inflorescence stem. By using plasmid rescue and RT-PCR analyses, we found that NTL8 was tagged; thus, the mutant was named ntl8-1 Dominant (ntl8-1D). Recapitulation experiment further confirmed that the phenotype observed in the ntl8-1D mutant was caused by elevated expression of NTL8 Quantitative RT-PCR results showed that the expression level of the single-repeat R3 MYB genes TRIPTYCHON (TRY) and TRICHOMELESS1 (TCL1) was elevated in the ntl8-1D mutant. Genetic analyses demonstrated that NTL8 acts upstream of TRY and TCL1 in the regulation of trichome formation. When recruited to the promoter region of the reporter gene Gal4:GUS by a fused GAL4 DNA-binding domain, NTL8 activated the expression of the reporter gene. Chromatin immunoprecipitation results indicated that TRY and TCL1 are direct targets of NTL8. However, NTL8 did not interact with SQUAMOSA PROMOTER BINDING PROTEIN LIKE9, another transcription factor that regulates the expression of TRY and TCL1, in yeast and plant cells. Taken together, our results suggest that NTL8 negatively regulates trichome formation in Arabidopsis by directly activating the expression of TRY and TCL1.

Whole-genome sequencing revealed armadillo repeat containing 5 (ARMC5) mutation in a Chinese family with ACTH-independent macronodular adrenal hyperplasia.

Primary macronodular adrenal hyperplasia (PMAH), also known in the past as bilateral macronodular adrenalhyperplasia or adrenocorticotropin (ACTH)-independent macronodular adrenal hyperplasia, is a rare type of Cushing's syndrome (CS) and is associated with bilateralenlargement of the adrenal glands. It accounts for <1% of all endogenous cases of CS. In order toidentify the pathogenic mutations in the causative gene of (AIMAH pedigrees, Whole-genome sequencing of three patients in family I was used to retrieve candidate causative genes. Meanwhile, the causative gene was identified by Sanger sequencing from the two pedigrees. Sequencing of ARMC5 exons of three patients was carried out to identify somatic mutations. Moreover, haploid clone of one tumor DNA sample was conducted. ARMC5 was the causative gene of two pedigrees confirmed by whole-genome sequencing (WGA) and Sanger sequencing. The variant sites of the two families were c.C943T (p.R315W) and c.C1960T (p.R654X), respectively. Autosomal dominant inheritance of AIMAH was confirmed by genotypes of one family member. Several somatic mutations were discovered in tumor DNA samples. In addition, haploid clone of tumor DNA was confirmed by germline mutation and somaticmutation, which suggested the pathogenic mechanism of "two-hit-model." ARMC5 was the causative gene of AIMAH pedigrees. This AIMAH in this study presented autosomal dominant inheritance, fitting to Mendelian inheritance law. However, the pathogenic mode of this disease showed as compound heterozygote.

Characterization of an activation-tagged mutant uncovers a role of GLABRA2 in anthocyanin biosynthesis in Arabidopsis.

In Arabidopsis, anthocyanin biosynthesis is controlled by a MYB-bHLH-WD40 (MBW) transcriptional activator complex. The MBW complex activates the transcription of late biosynthesis genes in the flavonoid pathway, leading to the production of anthocyanins. A similar MBW complex regulates epidermal cell fate by activating the transcription of GLABRA2 (GL2), a homeodomain transcription factor required for trichome formation in shoots and non-hair cell formation in roots. Here we provide experimental evidence to show that GL2 also plays a role in regulating anthocyanin biosynthesis in Arabidopsis. From an activation-tagged mutagenized population of Arabidopsis plants, we isolated a dominant, gain-of-function mutant with reduced anthocyanins. Molecular cloning revealed that this phenotype is caused by an elevated expression of GL2, thus the mutant was named gl2-1D. Consistent with the view that GL2 acts as a negative regulator of anthocyanin biosynthesis, gl2-1D seedlings accumulated less whereas gl2-3 seedlings accumulated more anthocyanins in response to sucrose. Gene expression analysis indicated that expression of late, but not early, biosynthesis genes in the flavonoid pathway was dramatically reduced in gl2-1D but elevated in gl2-3 mutants. Further analysis showed that expression of some MBW component genes involved in the regulation of late biosynthesis genes was reduced in gl2-1D but elevated in gl2-3 mutants, and chromatin immunoprecipitation results indicated that some MBW component genes are targets of GL2. We also showed that GL2 functions as a transcriptional repressor. Taken together, these results indicate that GL2 negatively regulates anthocyanin biosynthesis in Arabidopsis by directly repressing the expression of some MBW component genes.

Malignant insulinoma: Report of 6 patients and literature review.

OBJECTIVE: Patients with malignant insulinoma always present with symptoms of severe hypoglycemia and have poor life expectancy. In addition, inoperable metastatic malignant insulinomas are very difficult to manage. The aim of this report is to present our successful experiences in diagnosis and treatment of this disease in 6 patients. PATIENTS/METHODS: Six patients (male 2, female 4) with malignant insulinomas were admitted into our hospital. Their clinical histories, including clinical presentations, endocrine evaluations, radiological images, pathological examination and treatments, were reviewed. RESULTS: The diagnosis of malignant insulinoma combined with liver metastases was confirmed in all patients by endocrine evaluation and radiological images. Patients 1-3 underwent surgical management. The primary and metastasized tumors were completely resected. After successful surgery, no hypoglycemia recurred. Patients 4-6 did not undergo surgery because of systemic disease and poor health. Instead, they were administrated with diazoxide 50 mg Three Times a Day (TID), with final doses up to 200-300 mg TID. These 3 patients had good responses to diazoxide administration. After treatment, the frequency and severity of hypoglycemia were improved significantly. All 6 patients had better life quality than previously expected. CONCLUSION: Combination of surgical and medical approaches can improve life quality and prolong survival of patients with malignant insulinomas.

Arabidopsis microtubule destabilizing protein40 is involved in brassinosteroid regulation of hypocotyl elongation.

The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana microtubule destabilizing protein40 (MDP40) as a positive regulator of hypocotyl cell elongation. Brassinazole-resistant1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation.