Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Metabolic regulation of gene expression by histone lactylation.

The Warburg effect, which originally described increased production of lactate in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, polarization of macrophages and activation of T cells. This phenomenon is intimately linked to several diseases including neoplasia, sepsis and autoimmune diseases1,2. Lactate, which is converted from pyruvate in tumour cells, is widely known as an energy source and metabolic by-product. However, its non-metabolic functions in physiology and disease remain unknown. Here we show that lactate-derived lactylation of histone lysine residues serves as an epigenetic modification that directly stimulates gene transcription from chromatin. We identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce the production of lactate by glycolysis, and this acts as a precursor that stimulates histone lactylation. Using M1 macrophages that have been exposed to bacteria as a model system, we show that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, increased histone lactylation induces homeostatic genes that are involved in wound healing, including Arg1. Collectively, our results suggest that an endogenous 'lactate clock' in bacterially challenged M1 macrophages turns on gene expression to promote homeostasis. Histone lactylation thus represents an opportunity to improve our understanding of the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.

Characterization of a novel endornavirus isolated from the phytopathogenic fungus Rhizoctonia solani.

Rhizoctonia solani endornavirus 8 (RsEV8) was isolated from strain XY175 of Rhizoctonia solani AG-1 IA. The full-length genome of RsEV8 is 16,147 nucleotides (nt) in length and contains a single open reading frame that encodes a large polyprotein of 5227 amino acids. The polyprotein contains four conserved domains: viral methyltransferase, putative DEAH box helicase, viral helicase, and RNA-dependent RNA polymerase (RdRp). RsEV8 has a shorter 3'-UTR (58 nt) and a longer 5'-UTR (404 nt). A multiple sequence alignment indicated that the RdRp of RsEV8 possesses eight typical RdRp motifs. According to a BLASTp analysis, RsEV8 shares 39.31% sequence identity with Rhizoctonia cerealis endornavirus-1084-7. Phylogenetic analysis demonstrated that RsEV8 clusters with members of the genus Betaendornavirus.

An Adaptive Hybrid Model for Wind Power Prediction Based on the IVMD-FE-Ad-Informer.

Accurate wind power prediction can increase the utilization rate of wind power generation and maintain the stability of the power system. At present, a large number of wind power prediction studies are based on the mean square error (MSE) loss function, which generates many errors when predicting original data with random fluctuation and non-stationarity. Therefore, a hybrid model for wind power prediction named IVMD-FE-Ad-Informer, which is based on Informer with an adaptive loss function and combines improved variational mode decomposition (IVMD) and fuzzy entropy (FE), is proposed. Firstly, the original data are decomposed into K subsequences by IVMD, which possess distinct frequency domain characteristics. Secondly, the sub-series are reconstructed into new elements using FE. Then, the adaptive and robust Ad-Informer model predicts new elements and the predicted values of each element are superimposed to obtain the final results of wind power. Finally, the model is analyzed and evaluated on two real datasets collected from wind farms in China and Spain. The results demonstrate that the proposed model is superior to other models in the performance and accuracy on different datasets, and this model can effectively meet the demand for actual wind power prediction.

Chromosome-scale genome assembly of Rhododendron molle provides insights into its evolution and terpenoid biosynthesis.

BACKGROUND: Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information. RESULTS: In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-ß) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found. CONCLUSIONS: We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle.

Study on the physicochemical properties changes of field aging biochar and its effects on the immobilization mechanism for Cd2+ and Pb2.

It has been widely reported that biochar can be used as a cost-effective amendment to immobilize of heavy metal contaminants in soil. While less research has been conducted on effect of biochar long-term field aging on its properties and the adsorption capability. In this study, the characteristics of aged biochar were investigated by comprehensive characterization to elucidate its mechanism transformation for heavy metal immobilization. Our results showed that, compared to fresh biochar, the relative content of C of aged biochar was reduced by 34.12%, while O was increased by 8.79%. Additionally, the specific surface area, pore volume, pore size and oxygen-containing functional groups of aged biochar were significantly increased compared to the fresh biochar. Batch adsorption experiment indicated that the maximum adsorption for Cd2+ (Qm = 32.157 mg/g) and Pb2+ (Qm = 39.216 mg/g) on aged biochar surface was much larger than that of Cd2+ (Qm = 7.573 mg/g) and Pb2+ (Qm = 8.134 mg/g) on fresh biochar. The underlying adsorption mechanisms for Cd2+ and Pb2+ on fresh biochar were dominated by coprecipitation, cation exchange and cation-π interaction, whereas surface complexation and cation exchange appeared to be more vital for aged biochar, as more active adsorption sites and Oxygen-containing functional groups were formed on its surface during aging, which was well explained by BET, XPS, FTIR and Elemental Analysis. Our study found that the physicochemical properties of biochar changed significantly during field aging. Although these changes increased the adsorption of heavy metals by biochar, the reduced stability of biochar to passivated heavy metal ions.

The effects of short-term, long-term, and reapplication of biochar on the remediation of heavy metal-contaminated soil.

Biochar, a cost-effective amendment, has been reported to play pivotal roles in improving soil fertility and immobilizing soil pollutants due to its well-developed porous structure and tunable functionality. However, the properties of biochar and soils can vary inconsistently after field application. This may affect the remediation of biochar on heavy metal (HM)-contaminated soil being altered. Therefore, we selected lettuce as a model crop to determine the effects of short-term, long-term, and reapplication of biochar on soil physicochemical properties, microbial community, HM bioavailability, and plant toxicity. Our investigation revealed that the long-term application of biochar remarkably improved soil fertility, increased the relative abundance of the phylum Proteobacteria which was highly resistant to HMs, and reduced the abundance of phylum Acidobacteria. These changes in soil properties decreased the accumulation of Cd and Pb in lettuce tissues. The short- and long-term applications of biochar had no substantial effects on biomass, quality, and photosynthesis of lettuce. Moreover, the short-term and reapplication of biochar had no significant effects on soil bacterial communities but decreased the accumulation of Cd and Pb in lettuce tissues. It showed that the changes in the physical, chemical, and biological properties of soil after long-term application of biochar promoted the remediation of HM-contaminated soil. Furthermore, microbial community compositions varied with metal stress and biochar application, while the relative abundance of the phylum Actinobacteria in HM-contaminated soil with long-term biochar application was markedly higher than in HM-contaminated soil without biochar application.

A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome.

Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.

Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation.

BACKGROUND: Ascorbic acid (Vitamin C, AsA) is an antioxidant metabolite involved in plant development and environmental stimuli. AsA biosynthesis has been well studied in plants, and MIOX is a critical enzyme in plants AsA biosynthesis pathway. However, Myo-inositol oxygenase (MIOX) gene family members and their involvement in AsA biosynthesis and response to abiotic stress remain unclear. RESULTS: In this study, five tomato genes encoding MIOX proteins and possessing MIOX motifs were identified. Structural analysis and distribution mapping showed that 5 MIOX genes contain different intron/exon patterns and unevenly distributed among four chromosomes. Besides, expression analyses indicated the remarkable expression of SlMIOX genes in different plant tissues. Furthermore, transgenic lines were obtained by over-expression of the MIOX4 gene in tomato. The overexpression lines showed a significant increase in total ascorbate in leaves and red fruits compared to control. Expression analysis revealed that increased accumulation of AsA in MIOX4 overexpression lines is possible as a consequence of the multiple genes involved in AsA biosynthesis. Myo inositol (MI) feeding in leaf and fruit implied that the Myo-inositol pathway improved the AsA biosynthesis in leaves and fruits. MIOX4 overexpression lines exhibited a better light response, abiotic stress tolerance, and AsA biosynthesis capacity. CONCLUSIONS: These results showed that MIOX4 transgenic lines contribute to AsA biosynthesis, evident as better light response and improved oxidative stress tolerance. This study provides the first comprehensive analysis of the MIOX gene family and their involvement in ascorbate biosynthesis in tomato.

De novo transcriptome sequencing of Rhododendron molle and identification of genes involved in the biosynthesis of secondary metabolites.

BACKGROUND: Rhododendron molle (Ericaceae) is a traditional Chinese medicinal plant, its flower and root have been widely used to treat rheumatism and relieve pain for thousands of years in China. Chemical studies have revealed that R. molle contains abundant secondary metabolites such as terpenoinds, flavonoids and lignans, some of which have exhibited various bioactivities including antioxidant, hypotension and analgesic activity. In spite of immense pharmaceutical importance, the mechanism underlying the biosynthesis of secondary metabolites remains unknown and the genomic information is unavailable. RESULTS: To gain molecular insight into this plant, especially on the information of pharmaceutically important secondary metabolites including grayanane diterpenoids, we conducted deep transcriptome sequencing for R. molle flower and root using the Illumina Hiseq platform. In total, 100,603 unigenes were generated through de novo assembly with mean length of 778 bp, 57.1% of these unigenes were annotated in public databases and 17,906 of those unigenes showed significant match in the KEGG database. Unigenes involved in the biosynthesis of secondary metabolites were annotated, including the TPSs and CYPs that were potentially responsible for the biosynthesis of grayanoids. Moreover, 3376 transcription factors and 10,828 simple sequence repeats (SSRs) were also identified. Additionally, we further performed differential gene expression (DEG) analysis of the flower and root transcriptome libraries and identified numerous genes that were specifically expressed or up-regulated in flower. CONCLUSIONS: To the best of our knowledge, this is the first time to generate and thoroughly analyze the transcriptome data of both R. molle flower and root. This study provided an important genetic resource which will shed light on elucidating various secondary metabolite biosynthetic pathways in R. molle, especially for those with medicinal value and allow for drug development in this plant.

GREEN STRIPE, encoding methylated TOMATO AGAMOUS-LIKE 1, regulates chloroplast development and Chl synthesis in fruit.

Fruit development involves chloroplast development, carotenoid accumulation and fruit coloration. Although genetic regulation of fruit development has been extensively investigated, epigenetic regulation of fruit coloration remains largely unexplored. Here, we report a naturally occurring epigenetic regulation of TAGL1, and its impact on chloroplast development and fruit coloration. We used a genome-wide association study in combination with map-based cloning to identify the GREEN STRIPE (GS) locus, a methylated isoform of TAGL1 regulating diversified chloroplast development and carotenoid accumulation. Nonuniform pigmentation of fruit produced by GS was highly associated with methylation of the TAGL1 promoter, which is linked to a SNP at SL2.50ch07_63842838. High degrees of methylation of the TAGL1 promoter downregulated its expression, leading to green stripes. By contrast, low degrees of methylation led to light green stripes in gs. RNA-seq and ChIP collectively showed that the expression of genes involved with Chl synthesis and chloroplast development were significantly upregulated in green stripes relative to light green stripes. Quantitative PCR and dual luciferase assay confirmed that TAGL1 downregulates expression of SlMPEC, SlPsbQ, and SlCAB, and upregulates expression of PSY1 - genes which are associated with chloroplast development and carotenoid accumulation. Altogether, our findings regarding the GS locus demonstrate that naturally occurring methylation of TAGL1 has diverse effects on plastid development in fruit.

Meta-analysis found that studies may have overestimated Caesarean section risks for attention-deficit hyperactivity disorder by ignoring confounding factors.

AIM: Epidemiological studies on associations between Caesarean sections (C-sections) and attention-deficit hyperactivity disorder (ADHD) have been inconsistent, and we performed a meta-analysis. METHODS: We systematically searched PubMed and Embase to December 2018 and included nine hospital-based and population registry studies published in 2011-2018. These covered a total study cohort of more than 2.5 million people in eight countries: Australia, Brazil, Denmark, Finland, Germany, Sweden, Turkey and the UK. The analysis provided summary odds ratios (ORs) and 95% confidence intervals (CI) while taking heterogeneity into account. RESULTS: We found that that C-sections were associated with a small increase in the risk of ADHD (OR 1.14, 95% CI 1.11, 1.17, I2 0%) in offspring. In subgroup analyses, the association remained for both infants born after elective C-sections (OR, 1.15, 1.11, 1.19, I2 0%) and emergency C-sections (OR, 1.13, 1.1, 1.17, I2 45.4%). However, these were only marginally significant when we pooled data from siblings from other pregnancies (OR, 1.06, 1.00-1.13, I2 0%), implying that the association was due to confounding. CONCLUSION: The statistically significant association between C-sections and ADHD in children can be partially explained by unmeasured confounding. Further research controlling for important confounders is required before firm conclusions can be drawn.

Variation of Electrocardiogram Features Across Sleep Stages in Healthy Controls and in Patients with Sleep Apnea Hypopnea Syndrome.

Sleep apnea hypopnea syndrome (SAHS) is an independent risk factor for various cardiovascular diseases. Electrocardiogram (ECG) features such as the RR, PR, QT, QTc, Tpe intervals and the Tpe/QT, Tpe/QTc ratios are used to predict and study cardiovascular diseases. It is not clear whether regular patterns of PR and Tpe-related features across sleep stages exist in SAHSs or healthy controls nor whether sleep stages affect the short- and long-range influences of respiratory events on ECG indices. We enrolled 36 healthy controls and 35 patients with SAHS in our study and analyzed the abovementioned ECG features. In the healthy controls, a significant regularity existed in these indices across sleep stages, which were weakened or disturbed in the patient group, especially the Tpe-related features. The differences between the patients and healthy controls were generally consistent across all sleep stages: patients had smaller RR, PR, QT and Tpe/QTc values, but larger QTc, Tpe and Tpe/QT values. After filtering the short-range influence of respiratory events, the differences in most features remained highly significant, except the QT interval. In the patient group, respiratory events decreased RR and PR intervals in most sleep stages and increased the Tpe-related features' values in deep sleep stages. These results may aid in the study of the relationships among SAHS, sleep disorders and cardiovascular diseases.

Spatial-temporal differentiation of urban eco-efficiency and its driving factors: A comparison of five major urban agglomerations in China.

This paper utilizes an improved undesirable output DEA model to measure the eco-efficiency of cities in five major urban agglomerations in China during 2006-2020. It employs the Theil Index and Geodetector to investigate the spatial-temporal distribution differentiation characteristics and driving factors of urban eco-efficiency. The main findings are as follows. Firstly, the eco-efficiency of all urban agglomerations showed a fluctuating upward trend, but the eco-efficiency performance of different urban agglomerations in China shows a stratification characteristic. Specifically, the Pearl River Delta urban agglomeration consistently ranks first in China, while the mean values of the Yangtze River Delta urban agglomeration, Beijing-Tianjin-Hebei urban agglomeration, and Chengdu-Chongqing urban agglomeration are lower than the national average. Secondly, the overall differences in the urban eco-efficiency of all sample cities show a consistently fluctuating downward trend. The factor that affects the level differences of eco-efficiency in different cities is the intra-regional differences. Last but not least, the top three factors affecting the spatial distribution difference of urban eco-efficiency in the whole sample are environmental pollution control investments, innovation level, and environmental infrastructure investments. In the end, this paper proposes that reducing the intra-regional differences is the primary task to achieve the coordinated improvement of urban eco-efficiency in urban agglomerations, and then puts forward some policy suggestions to improve eco-efficiency for the five major urban agglomerations.

Pharmacological Effects of Ginseng: Multiple Constituents and Multiple Actions on Humans.

Ginseng is a very commonly used natural product in the world, and its two main species are Asian ginseng and American ginseng. Ginseng is an adaptogenic botanical that reportedly protects the body against stress, stabilizes physiological processes, and restores homeostasis. Previously, different animal models and contemporary research methodologies have been used to reveal ginseng's biomedical activities in different body systems and the linked mechanisms of actions. However, human clinical observation data on ginseng effects have attracted more attention from the general public and medical community. In this paper, after an introduction of the phytochemistry of ginseng species, we review positive ginseng clinical studies, mainly conducted in developed countries, performed over the past 20 years. The reported effects of ginseng are presented in several sections, and conditions impacted by ginseng include diabetes; cardiovascular disorders; cognition, memory, and mood; the common cold and flu; cancer fatigue and well-being; quality of life and social functioning, etc. Administration of ginseng demonstrated a good safety record in humans. Although encouraging beneficial effects obtained from clinical data, using the study treatment regimen, the reported ginseng effects in general only ranged from mild to moderate. Nonetheless, these beneficial effects of ginseng could be a valuable add-on therapy for patients receiving standard drug treatments. Additionally, as a dietary supplement, ginseng possesses an important role in maintaining and promoting human health. We believe that the quality of future ginseng trials should be improved, particularly by providing detailed herbal phytochemistry and quality control information. With solid effectiveness data obtained from a well-designed, carefully executed ginseng clinical trial, this meritoriously herbal medicine will be widely used by consumers and patients.

Isolation and characterization of 13 new polymorphic microsatellite markers in the Phaseolus vulgaris L. (Common Bean) genome.

In this study, 13 polymorphic microsatellite markers were isolated from the Phaseolus vulgaris L. (common bean) by using the Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) protocol. These markers revealed two to seven alleles, with an average of 3.64 alleles per locus. The polymorphic information content (PIC) values ranged from 0.055 to 0.721 over 13 loci, with a mean value of 0.492, and 7 loci having PIC greater than 0.5. The expected heterozygosity (H(E)) and observed heterozygosity (H(O)) levels ranged from 0.057 to 0.814 and from 0.026 to 0.531, respectively. Cross-species amplification of the 13 prime pairs was performed in its related specie of Vigna unguiculata L. Seven out of all these markers showed cross-species transferability. These markers will be useful for future genetic diversity and population genetics studies for this agricultural specie and its related species.

Filgrastim, fibrinolysis, and neovascularization.

Segmental recanalization of chronically occluded arteries was observed in patients with chronic limb-threatening ischemia (CLTI) treated with Filgrastim, a granulocyte colony stimulating factor, every 72 h for up to a month, and an infra-geniculate programmed compression pump (PCP) for 3 h daily. Molecular evidence for fibrinolysis and neovascularization was sought. CLTI patients were treated with PCP alone (N = 19), or with Filgrastim and PCP (N = 8 and N = 6, at two institutions). Enzyme-Linked Immunosorbent Assay was used to measure the plasma concentration of plasmin and of fibrin degradation products (FDP), and the serum concentration of proteins associated with neovascularization. In the PCP-alone group, blood was sampled on Day 1 (baseline) and after 30 days of daily PCP. In the Filgrastim and PCP group, blood was drawn on Day 1, and 1 day after the 5th and the 10th Filgrastim doses. Each blood draw occurred before and after 2 h of supervised PCP. Significant (p < 0.01) PCP independent increases in the plasma concentration of plasmin (>10-fold) and FDP (>5-fold) were observed 1 day after both the 5th and the 10th Filgrastim doses, compared to Day 1. Significant (p < 0.05) increases in the concentration of pro-angiogenic proteins (e.g., HGF, MMP-9, VEGF A) were also observed. Filgrastim at this novel dosimetry induced fibrinolysis without causing acute hemorrhage, in addition to inducing a pro-angiogenic milieu conducive to NV. Further clinical testing is warranted at this novel dosimetry in CLTI, as well as in other chronically ischemic tissue beds. Trial registration. https://clinicaltrials.gov/ct2/show/NCT02802852.

Short-Term Impact of Sleep Apnea/Hypopnea on the Interaction Between Various Cortical Areas.

INTRODUCTION: Sleep apnea/hypopnea syndrome (SAHS) can change brain structure and function. These alterations are related to respiratory event-induced abnormal sleep, however, how brain activity changes during these events is less well understood. METHODS: To study information content and interaction among various cortical regions, we analyzed the variations of permutation entropy (PeEn) and symbolic transfer entropy (STE) of electroencephalography (EEG) activity during respiratory events. In this study, 57 patients with moderate SAHS were enrolled, including 2804 respiratory events. The events terminated with cortical arousal were independently researched. RESULTS: PeEn and STE were lower during apnea/hypopnea, and most of the brain interaction was higher after apnea/hypopnea termination than that before apnea in N2 stage. As indicated by STE, the respiratory events also affected the stability of information transmission mode. In N1, N2, and rapid eye movement (REM) stages, the information flow direction was posterior-to-anterior, but the anterior-to-posterior increased relatively during apnea/hypopnea. The above EEG activity trends maintained in events with cortical arousal. CONCLUSIONS: These results may be related to the intermittent hypoxia during apnea and the cortical response. Furthermore, increased frontal information outflow, which was related to the compensatory activation of frontal neurons, may associate with cognitive function.

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals.

BACKGROUND: NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated. RESULTS: An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using sequence homology and prediction algorithms, four putative nuclear localization signals (NLSs) were identified. NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins confirmed the functionality of three of these NLSs. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the minimal functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The nucleolar occupancy of NOL7 revealed a complex balance of rapid nucleoplasmic shuttling but low nucleolar mobility, suggesting NOL7 may play functional roles in both compartments. In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7. CONCLUSIONS: These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus. Further, this work characterizes the relative contribution of each sequence to NOL7 nuclear and nucleolar dynamics, the subnuclear constituents that participate in this targeting, and suggests a functional role for NOL7 in both compartments. Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

Silencing of B cell receptor signals in human naive B cells.

To identify changes in the regulation of B cell receptor (BCR) signals during the development of human B cells, we generated genome-wide gene expression profiles using the serial analysis of gene expression (SAGE) technique for CD34(+) hematopoietic stem cells (HSCs), pre-B cells, naive, germinal center (GC), and memory B cells. Comparing these SAGE profiles, genes encoding positive regulators of BCR signaling were expressed at consistently lower levels in naive B cells than in all other B cell subsets. Conversely, a large group of inhibitory signaling molecules, mostly belonging to the immunoglobulin superfamily (IgSF), were specifically or predominantly expressed in naive B cells. The quantitative differences observed by SAGE were corroborated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling. Conversely, activation or impairment of the inhibitory IgSF receptor LIRB1 affected BCR-dependent Ca(2+) mobilization only in naive but not memory B cells. Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation. In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.