Genetic breakthroughs in the Brassica napus-Sclerotinia sclerotiorum interactions.

Sclerotinia sclerotiorum (Lib.) de Bary is a highly destructive fungal pathogen that seriously damages the yield and quality of Brassica napus worldwide. The complex interaction between the B. napus and S. sclerotiorum system has presented significant challenges in researching rapeseed defense strategies. Here, we focus on the infection process of S. sclerotiorum, the defense mechanisms of rapeseed, and recent research progress in this system. The response of rapeseed to S. sclerotiorum is multifaceted; this review aims to provide a theoretical basis for rapeseed defense strategies.

Extracellular enzyme stoichiometry reveals carbon and nitrogen limitations closely linked to bacterial communities in China's largest saline lake.

Saline lakes possess substantial carbon storage and play essential roles in global carbon cycling. Benthic microorganisms mine and decompose sediment organic matter via extracellular enzymes to acquire limiting nutrients and thus meet their element budgets, which ultimately causes variations in sediment carbon storage. However, current knowledge about microbial nutrient limitation and the associated organic carbon changes especially in saline lake remains elusive. Therefore, we took Qinghai Lake, the largest saline lake of China, as an example to identify the patterns and drivers of microbial metabolic limitations quantified by the vector analyses of extracellular enzyme stoichiometry. Benthic microorganisms were dominantly colimited by carbon (C) and nitrogen (N). Such microbial C limitation was aggravated upon the increases in water salinity and sediment total phosphorus, which suggests that sediment C loss would be elevated when the lake water is concentrated (increasing salinity) and phosphorus becomes enriched under climate change and nutrient pollution, respectively. Microbial N limitation was predominantly intensified by water total nitrogen and inhibited by C limitation. Among the microbial drivers of extracellular enzyme investments, bacterial community structure consistently exerted significant effects on the C, N, and P cycles and microbial C and N limitations, while fungi only altered the P cycle through species richness. These findings advance our knowledge of microbial metabolic limitation in saline lakes, which will provide insights towards a better understanding of global sediment C storage dynamics under climate warming and intensified human activity.

[Vertical Distribution Characteristics and Influencing Factors of Phytoplankton Community Structure in Qiandao Lake].

Clear vertical variations in phytoplankton community structure are usually observed in deep-water lakes and reservoirs, which is one of the key components of water quality and ecosystem functioning. However, the vertical patterns and ecological drivers of phytoplankton communities in deep-water lakes and reservoirs are still understudied. In this study, we took Qiandao Lake, a deep-water reservoir, as an example to reveal the vertical distribution characteristics of phytoplankton communities and its influencing factors by investigating phytoplankton community structure and the associated water quality index at 12 sites across the whole lake in two seasons (spring and autumn). The results showed that the phytoplankton abundance and chlorophyll a were highest in the surface layer in autumn and then decreased toward deep water, whereas in spring, the maximum value occurred in the subsurface layer (2-5 m), and the dominant phytoplankton species showed obvious vertical stratification characteristics. Specifically, in spring, Cryptomonas and Pseudanabaena dominated the surface and subsurface layers, Cryptomonas dominated in the middle layer, and the abundance of Cyclotella at the bottom layer was significantly higher than that of the other algae genera. The dominant genera in autumn were Pseudanabaena and Aphanizomenon. In the subsurface and middle layers, Leptolyngbya and Pseudanabaena occupied the dominant position, and Leptolyngbya became the only dominant genus. In the bottom layer, Leptolyngbya was the only dominant genus. The key environmental indicators of the water also had obvious vertical changes. The contents of N and P nutrients had a negative correlation with the water depth in spring, whereas the reverse trend was observed in autumn. The correlation analysis showed that the vertical variation in phytoplankton abundance in spring was significantly positively correlated with phosphate concentration, whereas the vertical distribution of phytoplankton abundance in autumn was significantly positively correlated with intensity of light, and the water temperature, NH4+-N, and total nitrogen were the main factors driving the vertical changes in the dominant genera of phytoplankton community in the two seasons. To summarize, environmental conditions such as water temperature, light, and nutrients had strong effects on the vertical distribution of phytoplankton. In the ecological investigation and quality assessment of deep-water lakes and reservoirs, the vertical distribution characteristics of the phytoplankton community structure and the influence of environmental conditions should be fully considered.

Gene expression in mouse brain following chronic hypoxia: role of sarcospan in glial cell death.

Hypoxia is a hallmark of respiratory, neurological, or hematological diseases as well as life at high altitude. For example, chronic constant hypoxia (CCH) occurs in chronic lung diseases or at high altitude, whereas chronic intermittent hypoxia (CIH) occurs in diseases such as sleep apnea or sickle cell disease. Despite the fact that such conditions are frequent, the cellular and molecular mechanisms underlying the effect of hypoxia, whether constant or intermittent, are not well understood. In this study, we first determined the effect of CCH and CIH on global gene expression in different regions of mouse brain using microarrays and then investigated the biological role of genes of interest. We found that: 1) in the cortical region, the expression level of 80 genes was significantly altered by CIH (16 up- and 64 downregulated), and this number increased to 137 genes following CCH (34 up- and 103 downregulated); 2) a similar number of gene alterations was identified in the hippocampal area, and the majority of the changes in this region were upregulations; 3) two genes (Sspn and Ttc27) were downregulated in both brain regions and following both treatments; and 4) RNA interference-mediated knockdown of Sspn increased cell death in hypoxia in a cell culture system. We conclude that CIH or CCH induced significant and distinguishable alterations in gene expression in cortex and hippocampus and that Sspn seems to play a critical role in inducing cell death under hypoxic conditions.

Prognostic value of humbug gene overexpression in stage II colon cancer.

Overexpression of aspartyl (asparaginyl) beta-hydroxylase (AAH) has been demonstrated in hepatocellular carcinoma, cholangiocarcinoma, and pancreatic carcinoma. AAH has an important role in regulating cell motility and invasiveness. Humbug is a truncated homolog of AAH, with a role in calcium regulation. The present study examines the prognostic use of AAH and humbug gene expression in stage II colon cancer. One hundred thirty cases of TNM stage II colon carcinoma were retrieved from the Rhode Island Hospital pathology archives. Tissue microarrays were immunostained with the FB50 and 15C7 monoclonal antibodies generated to recombinant AAH. However, FB50 also recognizes humbug. In addition, AAH and humbug expression was analyzed in samples of colon cancer and adjacent normal mucosa by real-time quantitative reverse transcriptase-polymerase chain reaction. Humbug (FB50) expression was localized to the tumor cytoplasm, whereas normal colonic epithelium did not exhibit significant immunoreactivity. Humbug staining was detected in 85% of the neoplasms, 23% of which stained strongly. Strong humbug immunoreactivity positively correlated with nuclear grade (P = .006) and inversely with survival (P = .027). In contrast to humbug, AAH (15C7) immunoreactivity was seen in normal and neoplastic epithelium. There was no correlation between AAH immunoreactivity and tumor grade, or survival. Correspondingly, reverse transcriptase-polymerase chain reaction studies demonstrated up-regulation of humbug but not AAH in 95% of colon carcinomas relative to adjacent colon cancer-free mucosa (P < .0001). This study demonstrates that high levels of humbug immunoreactivity in colon carcinomas correlate with histologic grade and tumor behavior, suggesting that humbug can serve as a prognostic biomarker of TNM stage II colon cancers. In addition, molecular studies demonstrated that the increased levels of FB50 detected were due to humbug, as opposed to AAH overexpression.

[Effect of insulin on the expression of platelet membrane glycoprotein II b/III a and its mechanisms].

OBJECTIVE: To investigate the effects of insulin on the expression of platelet membrane glycoprotein (GP) IIb/III a and its mechanism in normal subjects. METHODS: Flow cytometry was used to detect the expression of the whole-blood platelet membrane GP b/ a. The respective effects of insulin, L-nitro-arginine methyl ester (L-NAME) and methylene blue (MB) and their combination on the expression of GP b/ a were also studied. RESULTS: Insulin had no effect on the expression of GP II b/III a in healthy subjects, but significantly inhibited the up-regulated expression of GP II b/III a induced respectively by thrombin and collagen. The inhibitory effects of insulin on thrombin- or collagen-induced expression of platelet membrane GP II b/III a were dose- and time-dependent, and could be completely blocked by L-NAME or methylene blue. CONCLUSION: Insulin may inhibit the over-expression of platelet membrane GP IIb/III a by NOS-->NO-->GC-->GMP pathway.

[Effect of insulin on expression of whole-blood platelet membrane P-selectin].

OBJECTIVE: To investigate the effects of insulin on the expression of whole-blood platelet membrane P-selectin. METHODS: Flow cytometry (FCM) was used to detect the expression of whole-blood platelet membrane P-selectin, and the effect of insulin on the expression on quiescent and activated plasma platelet observed in normal young subjects. RESULTS: Insulin had no effect on the P-selectin expression on quiescent platelet membrane, but significantly inhibited the up-regulation of P-selectin expression induced by thrombin and collagen respectively, exhibiting dose- and time-dependent effects. CONCLUSION: Insulin can inhibit P-selectin expression on activated platelet membrane.

Prognostic value of aspartyl (asparaginyl)-beta-hydroxylase/humbug expression in non-small cell lung carcinoma.

Despite improvements in the detection and use of biomarkers, including epidermal growth factor receptor, ERCC1, and p16, the 5-year survival rate with non-small cell lung cancer remains at 15%. This suggests that additional biomarkers are needed to better prognosticate clinical course and guide therapeutic approaches. Previous studies showed that increased levels of aspartyl (asparaginyl)-beta-hydroxylase and a highly related molecule, humbug, correlate with clinical course and survival with hepatic, biliary, pancreatic, and colon carcinomas. We now characterize the prognostic use of aspartyl (asparaginyl)-beta-hydroxylase/humbug immunoreactivity in different subtypes of non-small cell lung cancer. Tissue microarrays including 375 paraffin-embedded non-small cell lung cancers (195 adenocarcinomas; 18 bronchioloalveolar carcinomas; 113 squamous cell carcinomas; and 49 large cell carcinomas) were immunostained with FB50 monoclonal antibody, which recognizes human aspartyl (asparaginyl)-beta-hydroxylase/humbug. Immunoreactivity (intensity and distribution) in neoplastic cells were scored under code, and data were subjected to univariate and Cox multivariate analyses, adjusting for age, stage, and treatment. High levels of FB50 immunoreactivity were more often detected in adenocarcinomas (28% for adenocarcinoma, 17% for bronchioloalveolar carcinoma), compared with squamous cell carcinomas (10%) and large cell carcinomas (10%). Univariate analysis demonstrated inverse relationships between intensity of FB50 immunoreactivity and survival with squamous cell carcinoma (P = .004), and a strong trend with respect to large cell carcinoma (P = .057). Cox multivariate test showed that FB50 immunoreactivity (P = .025), clinical stage (P = .029), and tumor size (P = .0001) were all independent predictors of survival with squamous cell carcinoma. High levels of FB50 immunohistochemical staining correlate with poor prognosis in non-small cell lung cancer, particularly squamous cell carcinoma subtype. Therefore, FB50 immunoreactivity may be useful in defining patient subsets that are likely to benefit from adjuvant therapy.

Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane.

Drs2p, a P-type adenosine triphosphatase required for a phosphatidylserine (PS) flippase activity in the yeast trans Golgi network (TGN), was first implicated in protein trafficking by a screen for mutations synthetically lethal with arf1 (swa). Here, we show that SWA4 is allelic to CDC50, encoding a membrane protein previously shown to chaperone Drs2p from the endoplasmic reticulum to the Golgi complex. We find that cdc50Delta exhibits the same clathrin-deficient phenotypes as drs2Delta, including delayed transport of carboxypeptidase Y to the vacuole, mislocalization of resident TGN enzymes and the accumulation of aberrant membrane structures. These trafficking defects precede appearance of cell polarity defects in cdc50Delta, suggesting that the latter are a secondary consequence of disrupting Golgi function. Involvement of Drs2p-Cdc50p in PS translocation suggests a role in restricting PS to the cytosolic leaflet of the Golgi and plasma membrane. Annexin V binding and papuamide B hypersensitivity indicate that drs2Delta or cdc50Delta causes a loss of plasma membrane PS asymmetry. However, clathrin and other endocytosis null mutants also exhibit a comparable loss of PS asymmetry, and studies with drs2-ts and clathrin (chc1-ts) conditional mutants suggest that loss of plasma membrane asymmetry is a secondary consequence of disrupting protein trafficking.

Genomic signatures of human versus avian influenza A viruses.

Position-specific entropy profiles created from scanning 306 human and 95 avian influenza A viral genomes showed that 228 of 4591 amino acid residues yielded significant differences between these 2 viruses. We subsequently used 15,785 protein sequences from the National Center for Biotechnology Information (NCBI) to assess the robustness of these signatures and obtained 52 "species-associated" positions. Specific mutations on those points may enable an avian influenza virus to become a human virus. Many of these signatures are found in NP, PA, and PB2 genes (viral ribonucleoproteins [RNPs]) and are mostly located in the functional domains related to RNP-RNP interactions that are important for viral replication. Upon inspecting 21 human-isolated avian influenza viral genomes from NCBI, we found 19 that exhibited > or =1 species-associated residue changes; 7 of them contained > or =2 substitutions. Histograms based on pairwise sequence comparison showed that NP disjointed most between human and avian influenza viruses, followed by PA and PB2.

Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.

Discovering target and off-target effects of specific compounds is critical to drug discovery and development. We generated a compendium of "chemical-genetic interaction" profiles by testing the collection of viable yeast haploid deletion mutants for hypersensitivity to 82 compounds and natural product extracts. To cluster compounds with a similar mode-of-action and to reveal insights into the cellular pathways and proteins affected, we applied both a hierarchical clustering and a factorgram method, which allows a gene or compound to be associated with more than one group. In particular, tamoxifen, a breast cancer therapeutic, was found to disrupt calcium homeostasis and phosphatidylserine (PS) was recognized as a target for papuamide B, a cytotoxic lipopeptide with anti-HIV activity. Further, the profile of crude extracts resembled that of its constituent purified natural product, enabling detailed classification of extract activity prior to purification. This compendium should serve as a valuable key for interpreting cellular effects of novel compounds with similar activities.

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function.

Aminophospholipid translocases (APLTs) are defined primarily by their ability to flip fluorescent or spin-labeled derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external leaflet of a membrane bilayer to the cytosolic leaflet and are thought to establish phospholipid asymmetry in biological membranes. The identities of APLTs remain unknown, although candidate proteins include the Drs2p/ATPase II subfamily of P-type ATPases. Drs2p from budding yeast localizes to the trans-Golgi network (TGN), and here we show that this membrane contains an ATP-dependent APLT that flips 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) PS and PE derivatives from the luminal to the cytosolic leaflet. To assess the contribution of Drs2p to this activity, TGN membranes were prepared from strains harboring WT or temperature-sensitive alleles of DRS2 and null alleles of three other potential APLT genes (DNF1, DNF2, and DNF3). Assay of these membranes indicated that Drs2p was required for the ATP-dependent translocation of NBD-PS, whereas no active translocation of NBD-PE or NBD-phosphatidylcholine was detected. The specificity of Drs2p for NBD-PS suggested that translocation of PS would be required for the function of Drs2p in protein transport from the TGN. However, cho1 yeast strains that are unable to synthesize PS do not phenocopy drs2 but instead transport proteins normally via the secretory pathway. In addition, a drs2 cho1 double mutant retains drs2 transport defects. Therefore, whereas NBD-PS is a preferred substrate for Drs2p in vitro, endogenous PS is not an obligatory substrate in vivo for the role Drs2p plays in protein transport.