IL6-STAT3-C/EBPß-IL6 positive feedback loop in tumor-associated macrophages promotes the EMT and metastasis of lung adenocarcinoma.

BACKGROUND: Lung cancer is one of the most common tumors in the world, and metastasis is one of the major causes of tumor-related death in lung cancer patients. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME) and are frequently associated with tumor metastasis in human cancers. However, the regulatory mechanisms of TAMs in lung cancer metastasis remain unclear. METHODS: Single-cell sequencing analysis of lung cancer and normal tissues from public databases and from 14 patients who underwent surgery at Zhongshan Hospital was performed. In vitro co-culture experiments were performed to evaluate the effects of TAMs on lung cancer migration and invasion. Changes in the expression of IL-6, STAT3, C/EBPΒ, and EMT pathway were verified using RT-qPCR, western blotting, and immunofluorescence. Dual luciferase reporter assays and ChIP were used to reveal potential regulatory sites on the transcription factor sets. In addition, the effects of TAMs on lung cancer progression and metastasis were confirmed by in vivo models. RESULTS: TAM infiltration is associated with tumor progression and poor prognosis. IL-6 secreted by TAMs can activate the JAK2/STAT3 pathway through autocrine secretion, and STAT3 acts as a transcription factor to activate the expression of C/EBPß, which further promotes the transcription and expression of IL-6, forming positive feedback loops for IL6-STAT3-C/EBPß-IL6 in TAMs. IL-6 secreted by TAMs promotes lung cancer progression and metastasis in vivo and in vitro by activating the EMT pathway, which can be attenuated by the use of JAK2/STAT3 pathway inhibitors or IL-6 monoclonal antibodies. CONCLUSIONS: Our data suggest that TAMs promote IL-6 expression by forming an IL6-STAT3-C/EBPß-IL6 positive feedback loop. Released IL-6 can induce the EMT pathway in lung cancer to enhance migration, invasion, and metastasis. The use of IL-6-neutralizing antibody can partially counteract the promotion of LUAD by TAMs. A novel mechanism of macrophage-promoted tumor progression was revealed, and the IL6-STAT3-C/EBPß-IL6 signaling cascade may be a potential therapeutic target against lung cancer.

How the xerophytic moss Pogonatum inflexum tolerates desiccation.

KEY MESSAGE: Desiccation-tolerant process of xerophytic moss Pogonatum inflexum were identified through de novo transcriptome assembly , morphological structure and physiology analysis. Pogonatum inflexum (Lindb.) Lac. is a typical xerophytic moss and have been widely used in gardening and micro-landscape. However, the mechanisms underlying desiccation tolerance are still unclear. In this study, morphological,  physiological and trancriptomic analyses of P. inflexum to tolerate desiccation were carried out. Our results indicate that P. inflexum increase osmoregulation substances, shut down photosynthesis, and alter the content of membrane lipid fatty acids in response to desiccation, and the genes involved in these biological processes were changes in expression after desiccation. 12 h is the threshold for P. inflexum to tolerate desiccation and its photosynthesis has not been damaged within 12 h of desiccation and can still recover after rewater. We also proved that the gametocyte of P. inflexum has the ability to absorb and transport water, and contains lignin-synthesis genes in response to tolerant desiccation. Our findings not only explain the mechanisms of P. inflexum during desiccation, but also provide some attractive candidate genes for genetic breeding.

Dynamic effects of miR-20a-5p on hippocampal ripple energy after status epilepticus in rats.

To determine the dynamic effects of miR-20a-5p on hippocampal ripple energy in rats after status epilepticus (SE). A lithium pilocarpine (LiCl-PILO)-induced rat model of status epilepticus (SE) was established, and the rats were divided into the normal control (Control, CTL), epileptic control (PILO), valproic acid (VPA + PILO), miR-20a-5p overexpression lentivirus vector (miR + PILO), sponges blocking lentivirus vector (Sponges + PILO), and scramble sequence negative control (Scramble + PILO) groups (n = 6). Electroencephalograms (EEGs) were used to analyze changes in hippocampal ripple energy before and after SE. Quantitative polymerase chain reaction (q-PCR) analysis showed that miR-20a-5p levels gradually increased after miR-20a-5p overexpression lentivirus vector injection into the lateral ventricle, and the miR-20a-5p levels were significantly higher than that in CTL group on days 7 and 36 (P < 0.001). The miR-20a-5p levels decreased significantly on days 7 and 36 after blocking by sponges lentivirus vector injected into the lateral ventricle (P < 0.001). After injection of PILO, the average ripple energy expression in each group gradually increased, and reached the peak before chloral hydrate injection (compared with 1 day before SE, P < 0.05). The ripple energy in the VPA + PILO and Sponges + PILO groups was significantly lower than that in the PILO group at 60 min and 70 min after PILO injection and before chloral hydrate injection (P < 0.05), and maintained lower until 2 h after chloral hydrate injection in VPA + PILO (P < 0.05). Compared with the VPA + PILO group, the mean ripple energy of the Sponges + PILO group had no difference at all time points (P ≥ 0.05). After SE, ripple distribution of space and energy is closely related to the occurrence of epilepsy. Inhibition of miR20a-5p expression can downregulate ripple oscillation energy during seizure.

FSCN1 induced PTPRF-dependent tumor microenvironment inflammatory reprogramming promotes lung adenocarcinoma progression via regulating macrophagic glycolysis.

PURPOSE: Macrophages (MΦs) play a dual role in the promotion and suppression of lung adenocarcinoma (LUAD), the function of which is influenced by the metabolic status. The role of protein tyrosine phosphatase receptor type F (PTPRF) in cancer has not been elucidated, and its role in MΦs remains to be seen. METHODS: The Seahorse XFe 96 Cell Flow Analyzer detected glucose metabolism in tumor cells and macrophages. The expressions of FSCN1, M-CSF, IL4, PTPRF and IGF1 in macrophages were detected by Western blotting and qRT-PCR. Binding of FSCN1 and IGF1R was detected by co-immunoprecipitation. The tumor status in animals was observed using the IVIS Lumina III imaging system. RESULTS: We found that Fascin Actin-Bundling Protein 1 (FSCN1) activates the PI3K-AKT and JAK-STAT signaling pathways in LUAD cells via binding to IGF-1R, thereby promoting the secretion of cytokines such as IL4 and M-CSF. IL4 and M-CSF promote the expression of PTPRF in MΦs, leading to M2 polarization of MΦs by increasing glucose intake and lactate production. In return, M2-type MΦs act on LUAD cells by secreting cytokines such as IGF-1, CCL2, and IL10, which ultimately promote tumor progression. In vivo experiments proved that the knockdown of FSCN1 in A549 cells and PTPRF in MΦs greatly reduced LUAD proliferative and metastatic capacity, which was consistent with the in vitro findings. CONCLUSIONS: This study investigated the reprogramming effects of FSCN1 and PTPRF on inflammatory cytokines in the LUAD microenvironment, revealing potential mechanisms by which FSCN1 and PTPRF promote tumor progression and providing a new experimental basis for LUAD treatment.

De novo transcriptome assembly and analysis of genes involved in desiccation tolerance in Grimmia pilifera.

The anatomically simple plants that transition from the aquatic to the terrestrial have a certain mechanism to deal with the damage caused by the changing living environment. Grimmia pilifera is a type of resurrection plants that can survive a long period of desiccation. To understand the molecular mechanisms of desiccation tolerance, nine cDNA libraries were constructed in triplicate from mRNA obtained from G. pilifera for the 0 h, 6 h and 18 h desiccation treatment. RNA-Seq generated âˆ¼ 666 million reads which were assembled into 135,850 unigenes. The differentially expressed genes (DEGs) were identified between different period of time of desiccation. Gene ontology analysis showed that a significant number of genes involved in water deprivation, chloroplast organization, xyloglucan metabolic process, regulation of signaling pathway. In addition, genes involved in osmotic stress, oxidative stress, accumulation of soluble matter, such as gene encoding antioxidant enzymes, trehalose synthase and channel protein, were up-regulated in response to drought stress. These results will be helpful for further studying on the molecular mechanisms of desiccation responses in G. pilifera.

Anticonvulsant Effect of Carbenoxolone on Chronic Epileptic Rats and Its Mechanism Related to Connexin and High-Frequency Oscillations.

Objective: This study was designed to investigate the influence and mechanism of gap junction carbenoxolone (CBX) on dynamic changes in the spectral power of ripples and fast ripples (FRs) in the hippocampus of chronic epileptic rats. Methods: The lithium-pilocarpine (PILO) status epilepticus (SE) model (PILO group) and the CBX pretreatment model (CBX + PILO group) were established to analyze dynamic changes in the spectral power of ripples and FRs, and the dynamic expression of connexin (CX)26, CX32, CX36, and CX43 in the hippocampus of chronic epileptic rats. Results: Within 28 days after SE, the number of spontaneous recurrent seizures (SRSs) in the PILO group was significantly higher than that in the CBX + PILO group. The average spectral power of FRs in the PILO group was significantly higher than the baseline level at 1 and 7 days after SE. The average spectral power of FRs in the PILO group was significantly higher than that in the CBX + PILO group at 1, 7, and 14 days after SE. Seizures induced an increase in CX43 expression at 1 and 7 days after SE, but had no significant effect on CX26, CX36, or CX32. CBX pretreatment did not affect the expression of CXs in the hippocampus of normal rats, but it inhibited the expression of CX43 in epileptic rats. The number of SRSs at 2 and 4 weeks after SE had the highest correlation with the average spectral power of FRs; the average spectral power of FRs was moderately correlated with the expression of CX43. Conclusion: The results of this study indicate that the energy of FRs may be regulated by its interference with the expression of CX43, and thus, affect seizures. Blocking the expression of CX43 thereby reduces the formation of pathological high-frequency oscillations (HFOs), making it a promising strategy for the treatment of chronic epilepsy.

Pseudaestuariivita rosea sp. nov., isolated from Acmaea sp., a marine mollusk.

A Gram-stain-negative, pink-pigmented, aerobic, non-motile and rod-shaped bacterium, designated as strain H15T, was isolated from Acmaea sp., collected from Weihai, Shandong Province, China. The novel isolate was able to grow at 4-37 °C (optimum 33 °C), pH 5.5-9.0 (optimum 7.0) and with 0.0-7.0% NaCl (optimum 4%, w/v). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that the strain belonged to the family Rhodobacteraceae and was associated to the type strain of Pseudaestuariivita atlantica (96.7%). Genome analysis showed that the genome size was 3,893,398 bp and the DNA G + C content obtained from the draft genome sequence was 56.7%. The secondary metabolites predicated that the strain H15T contained one cluster of lasso peptide, one cluster of bacteriocin, two clusters of terpene production, two clusters of homoserine lactone and one cluster of beta lactone. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between genome sequences of strain H15T and all the related strains compared were lower than 63.1, 72.0 and 19.7%, respectively. Based on the analysis of chemical components, the predominant cellular fatty acids were summed featured 8 (C18:1ω7c/ω6c, 46.1%), C20:1 ω7c (17.1%), the major polar lipids contained phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid and the predominant menaquinone was Q10. Therefore, the combined chemotaxonomic, phenotypic and phylogenetic data indicated that the strain was considered to represent a novel species of the genus Pseudaestuariivita and the name Pseudaestuariivita rosea sp. nov. was proposed for strain H15T (MCCC 1K04420T = KCTC 82505T).

Differences in genetics and microenvironment of lung adenocarcinoma patients with or without TP53 mutation.

BACKGROUND: Differences in genetics and microenvironment of LUAD patients with or without TP53 mutation were analyzed to illustrate the role of TP53 mutation within the carcinogenesis of LUAD, which will provide new concepts for the treatment of LUAD. METHODS: In this study, we used genetics and clinical info from the TCGA database, including somatic mutations data, RNA-seq, miRNA-seq, and clinical data. More than one bioinformatics tools were used to analyze the unique genomic pattern of TP53-related LUAD. RESULTS: According to TP53 gene mutation status, we divided the LUAD patients into two groups, including 265 in the mutant group (MU) and 295 in the wild-type group (WT). 787 significant somatic mutations were detected between the groups, including mutations in titin (TTN), type 2 ryanodine receptor (RYR2) and CUB and Sushi multiple domains 3(CSMD3), which were up-regulated in the MU. However, no significant survival difference was observed. At the RNA level, we obtained 923 significantly differentially expressed genes; in the MU, α-defensin 5(DEFA5), pregnancy-specific glycoprotein 5(PSG5) and neuropeptide Y(NPY) were the most up-regulated genes, glucose-6-phosphatase (G6PC), alpha-fetoprotein (AFP) and carry gametocidal (GC) were the most down-regulated genes. GSVA analysis revealed 30 significant pathways. Compared with the WT, the expression of 12 pathways in the mutant group was up-regulated, most of which pointed to cell division. There were significant differences in tumor immune infiltrating cells, such as Macrophages M1, T cells CD4 memory activated, Mast cells resting, and Dendritic cells resting. In terms of immune genes, a total of 35 immune-related genes were screened, of which VGF (VGF nerve growth factor inducible) and PGC (peroxisome proliferator-activated receptor gamma coactivator) were the most significant up-regulated and down-regulated genes, respectively. Research on the expression pattern of immunomodulators found that 9 immune checkpoint molecules and 6 immune costimulatory molecules were considerably wholly different between the two groups. CONCLUSIONS: Taking the mutant group as a reference, LUAD patients in the mutant group had significant differences in somatic mutations, mRNA-seq, miRNA-seq, immune infiltration, and immunomodulators, indicating that TP53 mutation plays a crucial role in the occurrence and development of LUAD.

Effect of gap junction blockers on hippocampal ripple energy expression in rats with status epilepticus. / ç¼éš™è¿žæŽ¥é˜»æ–­å‰‚å¯¹å¤§é¼ ç™«ç—«åŽæµ·é©¬æ¶Ÿæ³¢æŒ¯è¡èƒ½é‡å˜åŒ–çš„å½±å“.

OBJECTIVES: To study the effect of gap junction blockers, quinine (QUIN) and carbenoxolone (CBX), on hippocampal ripple energy expression in rats with status epilepticus (SE). METHODS: A total of 24 rats were randomly divided into four groups: model, QUIN, valproic acid (VPA), and CBX (n=6 each). A rat model of SE induced by lithium-pilocarpine (PILO) was prepared. The QUIN, VPA, and CBX groups were given intraperitoneal injection of QUIN (50 mg/kg), VPA by gavage (200 mg/kg), and intraperitoneal injection of CBX (50 mg/kg) respectively, at 3 days before PILO injection. Electroencephalography was used to analyze the change in hippocampal ripple energy before and after modeling, as well as before and after chloral hydrate injection to control seizures. RESULTS: Ripple expression was observed in the hippocampal CA1, CA3, and dentate gyrus regions of normal rats. After 10 minutes of PILO injection, all groups had a gradual increase in mean ripple energy expression compared with 1 day before modeling, with the highest expression level before chloral hydrate injection in the model, VPA and CBX groups (P<0.05). The QUIN group had the highest expression level of mean ripple energy 60 minutes after PILO injection. The mean ripple energy returned to normal levels in the three intervention groups immediately after chloral hydrate injection, while in the model group, the mean ripple energy returned to normal levels 1 hour after chloral hydrate injection. The mean ripple energy remained normal till to day 3 after SE in the four groups. The changing trend of maximum ripple energy was similar to that of mean ripple energy. CONCLUSIONS: The change in ripple energy can be used as a quantitative indicator for early warning of seizures, while it cannot predict seizures in the interictal period. Gap junction blockers can reduce ripple energy during seizures.

Echinicola salinicaeni sp. nov., a novel bacterium isolated from saltern mud.

A novel gram-negative, aerobic, pink, motile, gliding, rod-shaped bacterium, designated P51T, was isolated from saline silt samples in Yantai, China. It was able to grow at 4-42 °C (optimum 33 °C), pH 4.0-9.0 (optimum 7.0), and in 0-11.0% NaCl (optimum 4.0%, w/v). It grew at 4 °C, which was lower than the minimum temperature for related strains. The genome consisted of 4111 genes with a total length of 5 139 782 bp. The 16S rRNA gene sequence analysis indicated that strain P51T was a member of the genus Echinicola and most closely related to 'Echinicola shivajiensis'. A genome analysis identified genes encoding proteins associated with carbon source utilisation, and the carotenoid biosynthesis and ß-lactam resistance pathways. Strain P51T shared an average nucleotide identity value below 84.7%, an average amino acid identity value between 70.8 and 89.3%, and a digital DNA-DNA hybridisation identity of between 17.9-28.2% with closely related type strains within the genus Echinicola. The sole menaquinone was MK-7, and the major fatty acids were iso-C15:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 4 (anteiso-C17:1 B and/or iso-C17:1 I), and summed feature 9 (iso-C17:1ω9c and/or 10-methyl C16:0). The polar lipids included one phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, three unidentified aminolipids, and one unknown lipid. The phenotypic, chemotaxonomic, and phylogenetic analyses suggest that strain P51T is a novel species of the genus Echinicola, for which the name Echinicola salinicaeni sp. nov. is proposed. The type strain was P51T (KCTC 82513T = MCCC 1K04413T).

Characterization of a New Biofunctional, Exolytic Alginate Lyase from Tamlana sp. s12 with High Catalytic Activity and Cold-Adapted Features.

Alginate, a major acidic polysaccharide in brown algae, has attracted great attention as a promising carbon source for biorefinery systems. Alginate lyases, especially exo-type alginate lyase, play a critical role in the biorefinery process. Although a large number of alginate lyases have been characterized, few can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G) at low temperatures by means of an exolytic mode. In this study, the gene of a new exo-alginate lyase-Alys1-with high activity (1350 U/mg) was cloned from a marine strain, Tamlana sp. s12. When sodium alginate was used as a substrate, the recombinant enzyme showed optimal activity at 35 °C and pH 7.0-8.0. Noticeably, recombinant Alys1 was unstable at temperatures above 30 °C and had a low melting temperature of 56.0 °C. SDS and EDTA significantly inhibit its activity. These data indicate that Alys1 is a cold-adapted enzyme. Moreover, the enzyme can depolymerize alginates polyM and polyG, and produce a monosaccharide as the minimal alginate oligosaccharide. Primary substrate preference tests and identification of the final oligosaccharide products demonstrated that Alys1 is a bifunctional alginate lyase and prefers M to G. These properties make Alys1 a valuable candidate in both basic research and industrial applications.

[Directional breeding of high oil content peanut variety Yuhua 9 by in vitro mutagenesis and screening].

Leaf water potential of peanut subjected to drought stress is positively related to the oil content of peanut kernels. The aim of this study was to directly screen the high oil mutants of peanut and create the new peanut varieties using hydroxyproline as water potential regulator. In vitro mutagenesis was carried out with the embryonic leaflets of peanut variety Huayu 20 as explants and pingyangmycin as a mutagen added into the somatic embryo formation medium. The formed somatic embryos were successively transferred to somatic embryo germination and selection medium containing 6 mmol/L hydroxyproline (at -2.079 MPa water potential ) to induce regeneration and directionally screen high oil content mutants. After that, these plantlets were grafted and transplanted to the experimental field and 132 high oil mutants with oil content over 55% were obtained from the offspring of regenerated plants. Finally, among them, the oil contents of 27 lines were higher than 58% and of 2 lines were higher than 60%. A new peanut variety Yuhua 9 with high yield and oil content was bred from the regenerated plant progenies combining the pedigree breeding method. The yield was 14.0% higher than that of the control cultivar in the testing new peanut varieties of Liaoning province, and also it has passed the national registration of non-major crop varieties. Yuhua 9 with an oil content of 61.05%, which was 11.55 percentage points higher than that of the parent Huayu 20, was the peanut cultivar with the highest oil content in the world. The result showed that it was an effective way for directional breeding of high oil peanut varieties by means of the three-step technique including in vitro mutagenesis, directional screening by reducing water potential in medium and pedigree selection of regenerated plant progenies.

Constitutive expression of GmF6'H1 from soybean improves salt tolerance in transgenic Arabidopsis.

Coumarin plays a pivotal role in plant response to biotic stress, as well as in the mediation of nutrient acquisition. However, its functions in response to abiotic stresses are largely unknown. In this work, a homologous gene, GmF6'H1, of AtF6'H1, which encodes the enzyme catalyzing the final rate-limiting step in the biosynthesis pathway of coumarin, was isolated from soybean. GmF6'H1 protein shares very high amino acid identity with AtF6'H1, and expression of GmF6'H1 in atf6'h1 can successfully restore the decreased coumarin production in the T-DNA insertion mutant. Further study revealed that the expression of GmF6'H1 in soybean was remarkably induced by salt stress. Constitutive expression of GmF6'H1 in Arabidopsis, driven by 35S promoter, significantly enhanced the resistance to salt of transgenic Arabidopsis. All these results suggest that GmF6'H1 can be used as a potential candidate gene for the engineering of plants with improved resistance to both biotic and abiotic stresses.

Flavihalobacter algicola gen. nov. sp. nov., a member of the family Flavobacteriaceae with alginate-degradation activity, isolated from marine alga Saccharina japonica.

A Gram-stain-negative, aerobic, yellow, non-motile, rod-shaped and alginate-degrading bacterium, designated Dm15T, was isolated from marine alga collected in Weihai, PR China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Dm15T represents a distinct line of the family Flavobacteriaceae. Strain Dm15T had the highest 16S rRNA gene sequence similarity to its closest phylogenetic neighbour Arcticiflavibacter luteus (96.7 %) and 93.7-96.4 % sequence similarity to other phylogenetic neighbours (Bizionia paragorgiae, Winogradskyella thalassocola, Ichthyenterobacterium magnum, Psychroserpens burtonensis and Arcticiflavibacter luteus) in the family Flavobacteriaceae. The novel isolate was able to grow at 10-40 °C (optimum, 30-33 °C), pH 7.0-9.0 (optimum, pH 7.0-7.5) and with 0.5-6.0 % NaCl (optimum 2.0-3.0 %, w/v). It could grow at 40 °C, and degrade alginate and cellulose, which were different from the neighbour genera. The draft genome consisted of 3395 genes with a total length of 3 798 431 bp and 34.1mol% G+C content. Especially, there were some specific genes coding for cellulase and alginate lyase, which provided a basis for the above phenotypic characteristics. The strain's genome sequence showed 71.1-80.2 % average amino acid identity values and 71.8-77.7 % average nucleotide identity values compared to the type strains of related genera within the family Flavobacteriaceae. It shared digital DNA-DNA hybridization identity of 19.8 and 20.9 % with I. magnum and A. luteus, respectively. The sole menaquinone was MK-6. The major fatty acids were iso-C15 : 0 and iso-C15 : 1 G. The polar lipids included six unidentified polar lipids, four unidentified aminolipids and phosphatidylethanolamine. Based on the results of phenotypic, chemotaxonomic and phylogenetic analyses, strain Dm15T represents a novel species of a new genus in the family Flavobacteriaceae, phylum Bacteroidetes, for which the name Flavihalobacter algicola gen. nov., sp. nov. is proposed. The type strain is Dm15T (KCTC 42256T=CICC 23815T).

A novel NOTCH3 mutation identified in patients with oral cancer by whole exome sequencing.

Oral cancer is a serious disease caused by environmental factors and/or susceptible genes. In the present study, in order to identify useful genetic biomarkers for cancer prediction and prevention, and for personalized treatment, we detected somatic mutations in 5 pairs of oral cancer tissues and blood samples using whole exome sequencing (WES). Finally, we confirmed a novel nonsense single-nucleotide polymorphism (SNP; chr19:15288426A>C) in the NOTCH3 gene with sanger sequencing, which resulted in a N1438T mutation in the protein sequence. Using multiple in silico analyses, this variant was found to mildly damaging effects on the NOTCH3 gene, which was supported by the results from analyses using PANTHER, SNAP and SNPs&GO. However, further analysis using Mutation Taster revealed that this SNP had a probability of 0.9997 to be 'disease causing'. In addition, we performed 3D structure simulation analysis and the results suggested that this variant had little effect on the solubility and hydrophobicity of the protein and thus on its function; however, it decreased the stability of the protein by increasing the total energy following minimization (-1,051.39 kcal/mol for the mutant and -1,229.84 kcal/mol for the native) and decreasing one stabilizing residue of the protein. Less stability of the N1438T mutant was also supported by analysis using I-Mutant with a DDG value of -1.67. Overall, the present study identified and confirmed a novel mutation in the NOTCH3 gene, which may decrease the stability of NOTCH3, and may thus prove to be helpful in cancer prognosis.

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

Basic Tilted Helix Bundle - a new protein fold in human FKBP25/FKBP3 and HectD1.

In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251-73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.

Responses to copper by the moss Plagiomnium cuspidatum: hydrogen peroxide accumulation and the antioxidant defense system.

Using both histochemical and cytochemical methods, we investigated the effects of copper (Cu) on the production of hydrogen peroxide (H2O2) and superoxide anion (O2(.-)) in the leaves of the moss Plagiomnium cuspidatum. Cu treatment significantly increased the contents of total thiobarbituric acid-reactive substances and H2O2, as well as the activity of guaiacol peroxidase and superoxide dismutase (SOD). Native PAGE detected all three forms of SOD (Mn-SOD, Fe-SOD and CuZn-SOD) in P. cuspidatum, and the increase in the total SOD activity appeared to be mainly caused by an increase in CuZn-SOD activity. According to cytochemical results, H2O2-dependent CeCl3 precipitates were primarily localized in the plasma membranes and cell walls, and O2(.-) was chiefly localized on the inner side of the plasma membrane and in the cytoplasm surrounding the chloroplasts. Experiments using imidazole as an inhibitor of NADPH oxidase, N-N-diethyldithiocarbamate as an inhibitor of CuZn-SOD, and 1,2-dihydroxybenzene-3,5-disulphonic acid as an O2(.-) scavenger indicated that a partial source of H2O2 in the cell walls may be NADPH oxidase. The results also showed that peroxidase (POD) is involved in the detoxification of H2O2. Increased POD activity induced by Cu may remove excess H2O2 caused by Cu.