Loss-free tensile ductility of dual-structure titanium composites via an interdiffusion and self-organization strategy.

The deformation-coordination ability between ductile metal and brittle dispersive ceramic particles is poor, which means that an improvement in strength will inevitably sacrifice ductility in dispersion-strengthened metallic materials. Here, we present an inspired strategy for developing dual-structure-based titanium matrix composites (TMCs) that achieve 12.0% elongation comparable to the matrix Ti6Al4V alloys and enhanced strength compared to homostructure composites. The proposed dual-structure comprises a primary structure, namely, a TiB whisker-rich region engendered fine grain Ti6Al4V matrix with a three-dimensional micropellet architecture (3D-MPA), and an overall structure consisting of evenly distributed 3D-MPA "reinforcements" and a TiBw-lean titanium matrix. The dual structure presents a spatially heterogeneous grain distribution with 5.8 µm fine grains and 42.3 µm coarse grains, which exhibits excellent hetero-deformation-induced (HDI) hardening and achieves a 5.8% ductility. Interestingly, the 3D-MPA "reinforcements" show 11.1% isotropic deformability and 66% dislocation storage, which endows the TMCs with good strength and loss-free ductility. Our enlightening method uses an interdiffusion and self-organization strategy based on powder metallurgy to enable metal matrix composites with the heterostructure of the matrix and the configuration of reinforcement to address the strength-ductility trade-off dilemma.

Prevalence, predictors, and outcomes of acute respiratory distress syndrome in severe stroke.

OBJECTIVES: Patients with severe stroke are at high risk of developing acute respiratory distress syndrome (ARDS), but this severe complication was often under-diagnosed and rarely explored in stroke patients. We aimed to investigate the prevalence, early predictors, and outcomes of ARDS in severe stroke. METHODS: This prospective study included consecutive patients admitted to neurological intensive care unit (neuro-ICU) with severe stroke, including acute ischemic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage. The incidence of ARDS was examined, and baseline characteristics and severity scores on admission were investigated as potential early predictors for ARDS. The in-hospital mortality, length of neuro-ICU stay, the total cost in neuro-ICU, and neurological functions at 90 days were explored. RESULTS: Of 140 patients included, 35 (25.0%) developed ARDS. Over 90% of ARDS cases occurred within 1 week of admission. Procalcitonin (OR 1.310 95% CI 1.005-1.707, P = 0.046) and PaO2/FiO2 on admission (OR 0.986, 95% CI 0.979-0.993, P < 0.001) were independently associated with ARDS, and high brain natriuretic peptide (OR 0.994, 95% CI 0.989-0.998, P = 0.003) was a red flag biomarker warning that the respiratory symptoms may be caused by cardiac failure rather than ARDS. ARDS patients had longer stays and higher expenses in neuro-ICU. Among patients with ARDS, 25 (62.5%) were moderate or severe ARDS. All the patients with moderate to severe ARDS had an unfavorable outcome at 90 days. CONCLUSIONS: ARDS is common in patients with severe stroke, with most cases occurring in the first week of admission. Procalcitonin and PaO2/FiO2 on admission are early predictors of ARDS. ARDS worsens both short-term and long-term outcomes. The conflict in respiratory support strategies between ARDS and severe stroke needs to be further studied.

Highly Robust and Strain-Resilient Thin Film Conductors Featuring Brittle Materials.

Stretchable conductors with stable electrical conductivity under various deformations are essential for wearable electronics, soft robots, and biointegrated devices. However, brittle film-based conductors on elastomeric substrates often suffer from unexpected electrical disconnection due to the obvious mechanical incompatibility between stiff films and soft substrates. We proposed a novel out-of-plane crack control strategy to achieve the strain-insensitive electrical performance of thin-film-based conductors, featuring conductive brittle materials, including nanocrystalline metals (Cu, Ag, Mo) and transparent oxides (ITO). Our metal film-based conductors exhibit an ultrahigh initial conductivity (1.3 × 105 S cm-1) and negligible resistance change (R/R0 = 1.5) over wide strain range from 0 to 130%, enabled by film-induced substrate cracking and liquid metal-induced electrical self-repairing. They could function well under multimodal deformations (stretching, bending, and twisting) and severe mechanical damage (cutting and puncturing). We demonstrated the strain-resilient electrical functionality of metal film-based conductors in a flexible light-emitting diode display that shows high mechanical compliance.

Tal2 is required for generation of GABAergic neurons in the zebrafish midbrain.

BACKGROUND: In the zebrafish midbrain, GABAergic neurons develop from precursors located in the nucleus of the medial longitudinal fasciculus (nMLF). However, the precise mechanisms that underline generation of the nMLF GABAergic neuron are poorly understood. RESULTS: GABAergic neurons in the nMLF co-express transcription factors tal2, gata2a, gata3, and nkx1.2lb. The Nodal-related gene and shh signaling are required for differentiation of nMLF GABAergic neuron precursors. Tal2 is important for nMLF GABAergic neurogenesis. Disruption of Tal2, embryos completely lack the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) expressing cells in the nMLF, and the whole nkx1.2lb expressing cells in the midbrain. Although almost all tal2-expressing cells in the diencephalon and/or nMLF are gata2a- and gata3-positive, simultaneous knockdown of gata2a and gata3 does not affect either tal2 or gad67 expression. CONCLUSIONS: In the zebrafish midbrain, expression of tal2, gata2a, and/or gata3 is independent of each other. The function of gata2a and gata3 is dispensable for generation of GABAergic neuron in the nMLF. This suggests that the functional connections of the regulatory genes leading to generation of nMLF GABAergic neurons have diverged between mouse and zebrafish.

Blood-brain barrier damage and new onset refractory status epilepticus: An exploratory study using dynamic contrast-enhanced magnetic resonance imaging.

OBJECTIVE: This study was undertaken to characterize the blood-brain barrier (BBB) dysfunction in patients with new onset refractory status epilepticus (NORSE) using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). METHODS: This study included three groups of adult participants: patients with NORSE, encephalitis patients without status epilepticus (SE), and healthy subjects. These participants were retrospectively included from a prospective DCE-MRI database of neurocritically ill patients and healthy subjects. The BBB permeability (Ktrans) in the hippocampus, basal ganglia, thalamus, claustrum, periventricular white matter, and cerebellum were measured and compared between these three groups. RESULTS: A total of seven patients with NORSE, 14 encephalitis patients without SE, and nine healthy subjects were included in this study. Among seven patients with NORSE, only one had a definite etiology (autoimmune encephalitis), and the rest were cryptogenic. Etiology of encephalitis patients without SE included viral (n = 2), bacterial (n = 8), tuberculous (n = 1), cryptococcal (n = 1), and cryptic (n = 2) encephalitis. Of these 14 encephalitis patients without SE, three patients had seizures. Compared to healthy controls, NORSE patients had significantly increased Ktrans values in the hippocampus (.73 vs. .02 × 10-3 /min, p = .001) and basal ganglia (.61 vs. .003 × 10-3 /min, p = .007) and a trend in the thalamus (.24 vs. .08 × 10-3 /min, p = .017). Compared to encephalitis patients without SE, NORSE patients had significantly increased Ktrans values in the thalamus (.24 vs. .01 × 10-3 /min, p = .002) and basal ganglia (.61 vs. .004 × 10-3 /min, p = .013). SIGNIFICANCE: This exploratory study demonstrates that BBBs of NORSE patients were impaired diffusely, and BBB dysfunction in the basal ganglia and thalamus plays an important role in the pathophysiology of NORSE.

Identification of pivotal genes with prognostic evaluation value in lung adenocarcinoma by bioinformatics analysis.

Lung cancer remains the leading cause of cancer morbidity and mortality worldwide, and over-diagnosis causes various unnecessary losses in patients' lives and health. How to more effectively screen lung cancer patients and their potential prognostic risk become the focus of our current study. By analyzing the LUAD expression profile in The Cancer Genome Atlas (TCGA), we constructed a weighted gene co-expression network using differentially expressed genes (DEGs) to find the key modules and pivotal genes. A COX proportional risk regression model based on the least absolute shrinkage and selection operator (LASSO) was used to assess the predictive value of the model for the prognosis of LUAD patients. A total of 4107 up-regulated DEGs and 2022 down-regulated DEGs were identified in this study, and enrichment analysis showed that these analyzes were associated with the extracellular matrix of cells and adhesion. Ten gene markers consisting of LDHA, TOP2A, UBE2C, TYMS, TRIP13, EXO1, TTK, TPX2, ZWINT, and UHRF1 were established by extracting the central genes in the key modules, and the upregulation of these genes was accompanied by an increased prognostic risk of patients. Among them, high expression of LDHA, TRIP13, and TTK in LUAD was associated with shorter overall survival and could be used as independent prognostic factors to participate in metabolic processes such as tumor NAD. The present study provides a powerful molecular target for the study of LUAD prognosis and provides a theoretical basis for the diagnosis and treatment of LUAD and the development of targeted inhibitors.

Characterization and Comparative Analysis of Chloroplast Genomes of Medicinal Herb Scrophularia ningpoensis and Its Common Adulterants (Scrophulariaceae).

Scrophularia ningpoensis, a perennial medicinal plant from the Scrophulariaceae family, is the original species of Scrophulariae Radix (SR) in the Chinese Pharmacopoeia. This medicine is usually deliberately substituted or accidentally contaminated with other closely related species including S. kakudensis, S. buergeriana, and S. yoshimurae. Given the ambiguous identification of germplasm and complex evolutionary relationships within the genus, the complete chloroplast genomes of the four mentioned Scrophularia species were sequenced and characterized. Comparative genomic studies revealed a high degree of conservation in genomic structure, gene arrangement, and content within the species, with the entire chloroplast genome spanning 153,016-153,631 bp in full length, encoding 132 genes, including 80 protein-coding genes, 4 rRNA genes, 30 tRNA genes, and 18 duplicated genes. We identified 8 highly variable plastid regions and 39-44 SSRs as potential molecular markers for further species identification in the genus. The consistent and robust phylogenetic relationships of S. ningpoensis and its common adulterants were firstly established using a total of 28 plastid genomes from the Scrophulariaceae family. In the monophyletic group, S. kakudensis was determined to be the earliest diverging species, succeeded by S. ningpoensis. Meanwhile, S. yoshimurae and S. buergeriana were clustered together as sister clades. Our research manifestly illustrates the efficacy of plastid genomes in identifying S. ningpoensis and its counterfeits and will also contribute to a deeper understanding of the evolutionary processes within Scrophularia.

A Review on the Morphology, Cultivation, Identification, Phytochemistry, and Pharmacology of Kitagawia praeruptora (Dunn) Pimenov.

Kitagawia praeruptora (Dunn) Pimenov, commonly known as Qianhu in China, is a widely used folk Chinese herbal medicine. This article reviews its botanical traits, ethnopharmacology, cultivation techniques, identification, phytochemical compositions, and pharmacological effects. Over 70 coumarin compounds, including simple coumarins, pyranocoumarins, and furanocoumarins, have been isolated within this plant. Additionally, K. praeruptora contains other components such as flavonoids, fatty acids, benzoic acids, and sterols. This information highlights the importance of utilizing active ingredients and excavating pharmacological effects. With its remarkable versatility, K. praeruptora exhibits a wide range of pharmacological effects. It has been found to possess expectorant and bronchodilator properties, cardiovascular protection, antimicrobial and antioxidant activities, anti-tumor effects, and even antidiabetic properties. It is recommended to focus on the development of new drugs that leverage the active ingredients of K. praeruptora and explore its potential for new clinical applications and holistic utilization.

Anti-inflammation of isoliquiritigenin via the inhibition of NF-κB and MAPK in LPS-stimulated MAC-T cells.

BACKGROUND: The application of plant extracts has received great interest for the treatment of bovine mastitis. Isoliquiritigenin (ISL) is a rich dietary flavonoid that has significant antioxidative, anti-inflammatory and anticancer activities. This study was conducted to explore the protective efficacy and related mechanism of ISL against lipopolysaccharide (LPS)-stimulated oxidation and inflammation in bovine mammary epithelial cells (MAC-T) by in vitro experiments. RESULTS: Real-time PCR and ELISA assays indicated that ISL treatment at 2.5, 5 and 10 µg/mL significantly reduced the mRNA and protein expression of the oxidative indicators cyclooxygenase-2 and inducible nitric oxide synthase (P < 0.01), and of the inflammatory cytokines interleukin-6 (P < 0.05), interleukin-1ß (P < 0.01) and tumor necrosis factor-α (P < 0.01) in LPS-stimulated MAC-T cells. Moreover, Western blotting and immunofluorescence tests indicated that the phosphorylation levels of nuclear factor kappa (NF-κB) p65 and the inhibitor of NF-κB were significantly decreased by ISL treatment, thus blocking the nuclear transfer of NF-κB p65. In addition, ISL attenuated the phosphorylation levels of p38, extracellular signal-regulated kinase and c-jun NH2 terminal kinase. CONCLUSIONS: Our data demonstrated that ISL downregulated the LPS-induced inflammatory response in MAC-T cells. The anti-inflammatory and antioxidative activity of ISL involves the NF-κB and MAPK cascades.

Mechanochemical Solvent-Free Suzuki-Miyaura Cross-Coupling of Amides via Highly Chemoselective N-C Cleavage.

Although cross-coupling reactions of amides by selective N-C cleavage are one of the most powerful and burgeoning areas in organic synthesis due to the ubiquity of amide bonds, the development of mechanochemical, solid-state methods remains a major challenge. Herein, we report the first mechanochemical strategy for highly chemoselective, solvent-free palladium-catalyzed cross-coupling of amides by N-C bond activation. The method is conducted in the absence of external heating, for short reaction time and shows excellent chemoselectivity for σ N-C bond activation. The reaction shows excellent functional group tolerance and can be applied to late-stage functionalization of complex APIs and sequential orthogonal cross-couplings exploiting double solventless solid-state methods. The results extend mechanochemical reaction environments to advance the chemical repertoire of N-C bond interconversions to solid-state environmentally friendly mechanochemical methods.

Anti-inflammation of epicatechin mediated by TMEM35A and TMPO in bovine mammary epithelial cell line cells and mouse mammary gland.

Epicatechin (EC) has significant antiinflammation, antioxidation, and anticancer activities. It also provides a new alternative treatment for mastitis, which can result in great economic losses in the dairy industry if left untreated. The purpose of this study was to investigate the anti-inflammatory effects of EC on mastitis and the underlying mechanism using in vivo and in vitro systems. The use of ELISA and immunohistochemistry assays showed that EC treatment at 1.5, 7.5, 15, and 30 mg/mL decreased protein expression of inflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase; inflammatory cytokines, which were composed of IL-1ß, TNF-α, and IL-6 in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cell line (MAC-T); and mouse mammary gland, together with reduced filtration of T lymphocytes in the mouse mammary gland. Furthermore, EC treatment reduced LPS-induced phosphorylation levels of p65 and inhibitor of NF-κB, and blocked nuclear translocation of p65 as revealed by western blot and immunofluorescence test in MAC-T cells and the mouse mammary gland. Epicatechin also attenuated LPS-induced phosphorylation levels of mitogen-activated protein kinase members (i.e., p38, c-Jun N-terminal kinase 1/2 and extracellular regulated protein kinases 1/2). Using RNA-seq and tandem mass tag analyses, upregulation of TMEM35A and TMPO proteins was disclosed in MAC-T cells cotreated with LPS and EC. Although clustered regularly interspaced short palindromic repeats/Cas9-based knockdown of TMEM35A and TMPO attenuated abundance of phosphorylated (p)-p65, p-p38, TNF-α, and iNOS, overexpression of TMEM35A reversed EC-mediated effects in TMPO knockdown cells. Moreover, interaction between TMEM35A and TMPO was detected using the co-immunoprecipitation method. In conclusion, our data demonstrated that EC inhibited LPS-induced inflammatory response in MAC-T cells and the mouse mammary gland. Importantly, TMEM35A mediated the transmembrane transport of EC, and the interaction between TMEM35A and TMPO inhibited MAPK and NF-κB pathways.

Development of 19 novel microsatellite markers of lily-of-the-valley (Convallaria, Asparagaceae) from transcriptome sequencing.

The lily-of-the-valley Convallaria (Asparagaceae) consists of three herbaceous perennial species. The plants are commonly found in northern hemisphere, and are best-known for their ornamental and pharmaceutical value. In order to assess the genetic structure, diversity and demographic history of Convallaria species, 19 novel microsatellite markers were developed based on transcriptome data of C. keiskei. Polymorphism and cross-amplification of the markers were tested in three populations of C. keiskei and one population each of C. majalis and C. montana. The transferability rate in two species was both 89.5%. The average number of alleles detected per locus was 7.7, 3.3 and 2.7 in C. keiskei, C. majalis and C. montana, respectively, and the polymorphism information content correspondingly varied from 0.067 to 0.730, from 0.071 to 0.637 and from 0.195 to 0.680 at the population level. The observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.833, respectively. Seven of the 19 loci showed significant deviation from Hardy-Weinberg equilibrium. The availability of these markers will provide a useful molecular tool for further population genetics, phylogeographic and breeding studies of Convallaria species.

The effectiveness of aquatic physical therapy intervention on disease activity and function of ankylosing spondylitis patients: a meta-analysis.

This study aimed (i) to complement existing research by focusingon aquatic physical therapy was potentially beneficial to patients with AS; (ii)tosystematically analyze all evidence available in the literature about effectiveness of the aquatic physical therapy intervention on pain and disease activity in AS patients. A systematic search was performed in major electronic databasesto identify studies reporting aquatic physical therapy intervention on pain and disease activity of AS patients. Three independent investigators screened the identified articles, extracted the data, and assessed the methodological quality of the included studies. Qualitative descriptions were conducted, and quantitative analysis was performed with RevMan software (version 5.3).The results were expressed in terms of mean difference(MD) and the corresponding 95% confidence interval.A total of five studies comprising 1,393 participants were included in the study. Meta-analyses showed that aquatic physical therapy interventions significantly reduced the pain scores(SMD=-0.44, 95 % CI:-0.84,-0.04, p=0.03) and BASDAI scores (MD=-0.40, 95% CI:-0.73,-0.06, p=0.02) because of follow up time among these studies; therefore, a subgroup analysis should be conducted for comparison. Aquatic physical therapy can statistically significantly reduce pain and disease activity in patients with AS compared with controls.

Combining transcriptomics and metabolomics to reveal the underlying molecular mechanism of ergosterol biosynthesis during the fruiting process of Flammulina velutipes.

BACKGROUND: Flammulina velutipes has been recognized as a useful basidiomycete with nutritional and medicinal values. Ergosterol, one of the main sterols of F. velutipes is an important precursor of novel anticancer and anti-HIV drugs. Therefore, many studies have focused on the biosynthesis of ergosterol and have attempted to upregulate its content in multiple organisms. Great progress has been made in understanding the regulation of ergosterol biosynthesis in Saccharomyces cerevisiae. However, this molecular mechanism in F. velutipes remains largely uncharacterized. RESULTS: In this study, nine cDNA libraries, prepared from mycelia, young fruiting bodies and mature fruiting bodies of F. velutipes (three replicate sets for each stage), were sequenced using the Illumina HiSeq™ 4000 platform, resulting in at least 6.63 Gb of clean reads from each library. We studied the changes in genes and metabolites in the ergosterol biosynthesis pathway of F. velutipes during the development of fruiting bodies. A total of 13 genes (6 upregulated and 7 downregulated) were differentially expressed during the development from mycelia to young fruiting bodies (T1), while only 1 gene (1 downregulated) was differentially expressed during the development from young fruiting bodies to mature fruiting bodies (T2). A total of 7 metabolites (3 increased and 4 reduced) were found to have changed in content during T1, and 4 metabolites (4 increased) were found to be different during T2. A conjoint analysis of the genome-wide connection network revealed that the metabolites that were more likely to be regulated were primarily in the post-squalene pathway. CONCLUSIONS: This study provides useful information for understanding the regulation of ergosterol biosynthesis and the regulatory relationship between metabolites and genes in the ergosterol biosynthesis pathway during the development of fruiting bodies in F. velutipes.

Physiological and transcriptome analysis reveal molecular mechanism in Salvia miltiorrhiza leaves of near-isogenic male fertile lines and male sterile lines.

BACKGROUND: Our previous study finds that male sterility in Salvia miltiorrhiza could result in stunted growth and reduced biomass, but their molecular mechanisms have not yet been revealed. In this article, we investigate the underlying mechanism of male sterility and its impact on plant growth and metabolic yield by using physiological analysis and mRNA sequencing (RNA-Seq). RESULTS: In this study, transcriptomic and physiological analysis were performed to identify the mechanism of male sterility in mutants and its impact on plant growth and metabolic yield. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, it is found that the pathways are mainly enriched in processes including organ development, primary metabolic process and secondary metabolic process. Physiological analysis show that the chloroplast structure of male sterile mutants of S. miltiorrhiza is abnormally developed, which could result in decrease in leaf gas exchange (A, E and gs), chlorophyll fluorescence (Fv, Fm and Fv/Fm), and the chlorophyll content. Expression level of 7 differentially expressed genes involved in photosynthesis-related pathways is downregulated in male sterile lines of S. miltiorrhiza, which could explain the corresponding phenotypic changes in chlorophyll fluorescence, chlorophyll content and leaf gas exchange. Transcriptomic analysis establishes the role of disproportionating enzyme 1 (DPE1) as catalyzing the degradation of starch, and the role of sucrose synthase 3 (SUS3) and cytosolic invertase 2 (CINV2) as catalyzing the degradation of sucrose in the S. miltiorrhiza mutants. The results also confirm that phenylalanine ammonialyase (PAL) is involved in the biosynthesis of rosmarinic acid and salvianolic acid B, and flavone synthase (FLS) is an important enzyme catalyzing steps of flavonoid biosynthesis. CONCLUSIONS: Our results from the physiological and transcriptome analysis reveal underlying mechanism of plant growth and metabolic yield in male sterile mutants, and provide insight into the crop yield of S. miltiorrhiza.

Various Phyllosphere and Soil Bacterial Communities of Natural Grasses and the Impact Factors in a Copper Tailings Dam.

Copper mining caused severe damage to the ecological environment of mining areas. The combination of microbe and plant remediation has an application potential in improving the absorption and transformation efficiency of heavy metals. The phyllosphere is the largest biointerface on the planet, and bacteria are the dominant microbial inhabitants of the phyllosphere, believed to be critical to plant growth and health. This study investigated the phyllospheric and soil bacteria communities using high-throughput sequencing, and endophyte infection statuses of four natural grasses by toluidine blue heparin assay. Results showed variation in phyllospheric bacterial community structure. Gammaproteobacteria were the most abundant bacterial population. Bacilli were found in the phyllosphere of Bothriochloa ischaemum and Imperata cylindrica, while Clostridia were only found in Calamagrostis epigejos. Alphaproteobacteria were the dominant bacteria in soil. In addition, bacterial communities were influenced by endophytic infection statuses. Oxalobacteraceae was associated with soil carbon and sulfur. Enterobacteriaceae had negative correlation with the ratio of soil carbon and nitrogen, and had positive correlation with Cd content. These results offer useful insights into phyllospheric bacterial community variance in four different natural grasses in a copper tailings dam.

A widely compatible expression system for the production of highly O-GlcNAcylated recombinant protein in Escherichia coli.

O-GlcNAcylation is a ubiquitous and dynamic post-translational modification on serine/threonine residues of nucleocytoplasmic proteins in metazoa, which plays a critical role in numerous physiological and pathological processes. But the O-GlcNAcylation on most proteins is often substoichiometric, which hinders the functional study of the O-GlcNAcylation. This study aimed to improve the production of highly O-GlcNAcylated recombinant proteins in Escherichia coli (E. coli). To achieve this goal, we constructed a bacterial artificial chromosome-based chloramphenicol-resistant expression vector co-expressing O-GlcNAc transferase (OGT) and key enzymes (phosphoglucose mutase, GlmM and N-acetylglucosamine-1-phosphate uridyltransferase, GlmU) of the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis pathway in E. coli, which can effectively increase the O-GlcNAcylation of the OGT target protein expressed by another vector. The results revealed that the expression of GlmM and GlmU increases the cellular concentration of UDP-GlcNAc in E. coli, which markedly enhanced the activity of the co-expressed OGT to its target proteins, such as H2B, p53 and TAB1. Altogether, we established a widely compatible E. coli expression system for producing highly O-GlcNAcylated protein, which could be used for modifying OGT target proteins expressed by almost any commercial expression vectors in E. coli. This new expression system provides possibility for investigating the roles of O-GlcNAcylation in the enzymatic activity, protein-protein interaction and structure of OGT target proteins.

A selective requirement for 53BP1 in the biological response to genomic instability induced by Brca1 deficiency.

The molecular pathways leading from genomic instability to cellular senescence and/or cell death remain incompletely characterized. Using mouse embryonic fibroblasts with constitutively increased DNA damage due to the absence of the full-length form of the tumor suppressor Brca1 (Brca1(Delta 11/Delta 11)), we show that deletion of p53 binding protein 1 (53BP1) selectivity abrogates senescence and cell death stimulated by reduced Brca1 activity. Furthermore, the embryonic lethality induced by Brca1 mutation can be alleviated by 53BP1 deletion. Adult Brca1(Delta 11/Delta 11)53BP1(-/-) manifest constitutively high levels of genomic instability, yet age relatively normally, with a surprisingly low incidence of overall tumor formation. Together, these in vitro and in vivo data suggest that 53BP1 is specifically required for the development of premature senescence and apoptosis induced by Brca1 deficiency. These observations may have important implications for Brca1-mediated tumor formation as well as for the molecular pathway leading from genomic instability to organismal aging.

New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro.

O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3',5,5'-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.

Interplay among BRCA1, SIRT1, and Survivin during BRCA1-associated tumorigenesis.

Germline mutations of BRCA1 predispose women to breast and ovarian cancers. However, the downstream mediators of BRCA1 function in tumor suppression remain elusive. We found that human BRCA1-associated breast cancers have lower levels of SIRT1 than their normal controls. We further demonstrated that mammary tumors from Brca1 mutant mice have low levels of Sirt1 and high levels of Survivin, which is reversed by induced expression of Brca1. BRCA1 binds to the SIRT1 promoter and increases SIRT1 expression, which in turn inhibits Survivin by changing the epigenetic modification of histone H3. Absence of SIRT1 blocks the regulation of Survivin by BRCA1. Furthermore, we demonstrated that activation of Sirt1 and inhibition of Survivin expression by resveratrol elicit a more profound inhibitory effect on Brca1 mutant cancer cells than on Brca1-wild-type cancer cells both in vitro and in vivo. These findings suggest that resveratrol treatment serves as an excellent strategy for targeted therapy for BRCA1-associated breast cancer.