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Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs
Dereje D. Jima, Jenny Zhang, Cassandra Jacobs, Kristy L. Richards, Cherie H. Dunphy, William W. L. Choi, Wing Yan Au, Gopesh Srivastava, Magdalena B. Czader, David A. Rizzieri, Anand S. Lagoo, Patricia L. Lugar, Karen P. Mann, Christopher R. Flowers, Leon Bernal-Mizrachi, Kikkeri N. Naresh, Andrew M. Evens, Leo I. Gordon, Micah Luftig, Daphne R. Friedman, J. Brice Weinberg, Michael A. Thompson, Javed I. Gill, Qingquan Liu, Tam How, Vladimir Grubor, Yuan Gao, Amee Patel, Han Wu, Jun Zhu, Gerard C. Blobe, Peter E. Lipsky, Amy Chadburn, Sandeep S. Dave and for the Hematologic Malignancies Research Consortium (HMRC)
Blood 2010 116:e118-e127; doi: https://doi.org/10.1182/blood-2010-05-285403
Dereje D. Jima
Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC;
Jenny Zhang
Cassandra Jacobs
Kristy L. Richards
University of North Carolina, Chapel Hill, NC;
Cherie H. Dunphy
William W. L. Choi
The University of Hong Kong, Queen Mary Hospital, Hong Kong, China;
Wing Yan Au
Gopesh Srivastava
Magdalena B. Czader
Indiana University, Indianapolis, IN;
David A. Rizzieri
Duke University Medical Center, Durham NC;
Anand S. Lagoo
Patricia L. Lugar
Karen P. Mann
Emory University, Atlanta, GA;
Christopher R. Flowers
Leon Bernal-Mizrachi
Kikkeri N. Naresh
Imperial College, London, United Kingdom;
Andrew M. Evens
Northwestern University, Chicago IL;
Leo I. Gordon
Micah Luftig
Daphne R. Friedman
J. Brice Weinberg
Duke University Medical Center, Durham NC; Veteran's Administration Medical Center, Durham, NC;
Michael A. Thompson
ProHealth Care Regional Cancer Center, Waukesha, WI;
Javed I. Gill
Baylor University Medical Center, Dallas, TX;
Qingquan Liu
Tam How
Vladimir Grubor
Yuan Gao
Center for the Study of Biologic Complexity, Virginia Commonwealth University, Richmond, VA; and
Amee Patel
Han Wu
Jun Zhu
Gerard C. Blobe
Peter E. Lipsky
Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD
Amy Chadburn
Sandeep S. Dave
Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC; Duke University Medical Center, Durham NC;
A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.
The role of microRNA (miRNA) is being increasingly recognized in several diverse cellular processes, including oncogenesis1,2 and immune cell function.3,4 Efforts toward a comprehensive characterization of miRNA expression in cellular systems are limited by the continuing discovery of new miRNAs. More than 100 new miRNAs have been identified in humans in the past 2 years.5 Thus, miRNA platforms used to measure miRNA expression in any biologic system are frequently obsolete by the time the studies are published.
Bioinformatic analyses of the genome suggest that there might be thousands of miRNAs encoded in the genome.6 However, thus far only approximately 700 unique miRNAs have been identified in humans.7 Traditional methods for discovering miRNAs have relied on cloning and Sanger sequencing.8,9 The vast dynamic range of miRNAs (5 log or higher) can result in the domination of the data by a few highly expressed miRNAs, limiting the identification of less abundant miRNAs. Massively parallel, high-throughput sequencing (deep sequencing) provides the means to identify the expression of millions of small RNA transcripts in a single tissue type, thereby providing a sensitivity of detection that is several orders of magnitude higher than conventional methods.
Mature B-cell differentiation is of interest, not only for its role in adaptive immunity, but also because malignancies derived from B cells are common. B-cell malignancies reflect defined stages of normal B-cell differentiation and constitute the majority of leukemias and lymphomas. Therefore, we carefully chose a total of 31 samples from normal human B-cell subsets and human B-cell malignancies to represent the spectrum of normal and malignant B cells (Table 1). We reasoned that deep sequencing of a large number of biologically related cases would provide the opportunity to discover new miRNAs and that shared expression of these miRNAs in separate cases would minimize the rate of false discovery.
Normal and malignant B cells used to identify small RNA transcriptome
In this study, we addressed whether deep sequencing could be used to elucidate the small RNA transcriptome of normal human B cells and human B-cell tumors. We analyzed more than 328 934 149 separate sequences (6 billion bases) to identify the expression of small noncoding RNAs including miRNA, transfer RNA (tRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), and small nucleolar RNA (snoRNA). In all, we identified 333 mature miRNAs, a number that is more than twice as high as previously reported in a single tissue type.8 We also identified the expression of 286 candidate novel miRNAs that satisfied the same structural and expression criteria10 that were used to identify known miRNA. Through deep sequencing of biologic replicates, as well as real-time polymerase chain reaction (PCR) of known and candidate novel miRNA, we established deep sequencing as a viable method for reproducibly measuring quantitative miRNA expression. It is anticipated that our work in delineating the expression of small RNAs, including the complete identification of their mature sequences, precursors, genome locations, and conservation patterns of miRNA, will enable a complete exploration of their functional role in normal and malignant B cells. We have thus developed a comprehensive framework that spans the spectrum of identifying new miRNAs using high-throughput sequencing and validating them, and then applying real-time PCR to provide a clear path to clinical utility.
Patient sample processing
Patient samples were obtained using a protocol approved by the Institutional Review Board at Duke University Medical Center and the other collaborating institutions. Total RNA was extracted using the phenol-chloroform method to preserve miRNA, using Ambion reagents.
Selection of B-cell subsets
Tonsils from young patients undergoing routine tonsillectomy were disaggregated and separated by Ficoll. The mononuclear cell layer was harvested, washed in phosphate-buffered saline (PBS), and resuspended in ACK lysing buffer to remove small numbers of red blood cells. After a wash and resuspension with 10 mL of PBS with 10% bovine serum albumin, cells were counted, and 200 million were stained with fluorochrome-tagged monoclonal antibodies to CD19, immunoglobulin D (IgD), CD38, and CD27. The specific monoclonal antibodies used were anti–CD19-phycoerythrin-Cy5.5, anti–IgD-fluorescein isothiocyanate, anti–CD27-phycoerythrin, and anti–CD38-allophycocyanin, all from BD Biosciences and BD Pharmingen. Cells were sorted using the MoFlo Cell sorter (Dako Cytomation) into naive B cells (CD19+IgD+CD27−CD38+), germinal center B cells (CD19+IgD−CD38++), memory B cells (CD19+IgD−CD27+CD38dim), and plasma cells (CD19dimIgD−CD27++CD38+++). Two replicates of each B-cell subset were obtained from separate patients. The sample purity was verified by fluorescence-activated cell sorting and found to be more than 90% in all cases.
miRNA profiling using real-time PCR
miRNA expression profiling was conducted using the Applied Biosystems 384-well multiplexed real-time PCR assay using 400 ng of total RNA. Eight reactions, each containing 50 ng RNA and a multiplex looped primer pool with endogenous snoRNA controls, were used to reverse-transcribe the miRNAs in parallel fashion. The completed reactions were loaded onto the 384-well plate per manufacturer's instructions, and real-time PCR was run on the ABI 7900HT Prism instrument. For each 384-well plate, we used the automatically determined cycle-threshold (CT) using the SDS 2.2.1 software (Applied Biosystems). Consistent with manufacturer recommendations, a CT > 35 was treated as undetected. The probes deemed to be present were normalized to the average expression of a snoRNA control. The expression values were calculated as 2−ΔCT, then median centered to 500 and log2-transformed.
miRNA library preparation and sequencing
Total RNA (typically 5 μg) from each sample was run on denaturing polyacrylamide-urea gels. The approximately 17-25 nucleotide RNAs were excised from the gel, ligated to sequencing adaptors on both ends, and reverse-transcribed. The resulting cDNA library was PCR-amplified for 15 cycles and gel-purified on 6% acrylamide gel. The gel-purified amplicon quality and quantity were analyzed on a 6% acrylamide gel relative to oligonucleotides of known concentration and size. Library (120 μL 1-4pM) was loaded on to the Illumina cluster station, where DNA molecules were attached to high-density universal adaptors in the flow cells and amplified. The DNA clusters generated via this process were sequenced with sequencing-by-synthesis technology, where successive high-resolution images of the 4-color fluorescence excitation dependent on the base incorporated during each cycle were captured. Sequencing reads were generated for each of the 31 samples, and base calls were rendered using Illumina pipeline Version 1.3.2. All the primary sequencing data and gene expression data are publicly available through the Gene Expression Omnibus (GEO) archive through accession GSE22898
Activation of B cells
B cells were selected from peripheral blood of normal individuals using an antibody for CD19 and magnetic beads. These peripheral blood B cells were incubated with CpG and polyclonal antibodies to IgM and CD40 for 24 hours and then harvested.
Transformation of B cells with Epstein-Barr virus
Peripheral blood B cells from normal individuals were incubated with high titers of the Epstein-Barr virus and incubated for 10 days. Those cells that were replicating autonomously were cultured separately for 7 days, and RNA was extracted.
Gene expression profiling was performed on 3 replicates of each B-cell subpopulation using standard Affymetrix protocols, as described previously.11 Briefly, 1 μg of total RNA was reversed-transcribed using an oligodT primer, and cDNA was synthesized. In vitro transcription using a T7 primer was used to generate labeled cRNA, which was fragmented and hybridized to Affymetrix whole-genome U133 plus 2.0 microarrays. The arrays were scanned and data normalized as described previously. The data have been deposited in the publicly available GEO database (GSE12366).
Tumor samples from 101 patients with diffuse large B-cell lymphoma (DLBCL) were obtained at the time of diagnosis and freshly frozen. These cases were profiled using Affymetrix Gene 1.0 ST arrays. The molecular subgroups were distinguished using a Bayesian approach described previously.11
GO term enrichment analysis
TargetScan12 (release 5.0) was used to predict conserved target genes of known miRNAs that were differentially expressed during B-cell differentiation. For each pairwise comparison (naive vs germinal center, germinal center vs plasma cell, and germinal center vs memory), significantly overrepresented miRNA seed sequences were identified (χ2 test, P < .001). Genes that were differentially expressed in these binary distinctions for which ontology data for molecular function existed were analyzed using GOEAST program.13 Gene ontology terms were considered significantly enriched at P < .001.
Comparison of real-time PCR and sequencing data
Sequencing frequency for each miRNA was scaled to the total number of known miRNA sequences identified in the sample. These scaled frequencies were multiplied by 106 and log2-transformed for the purpose of comparison between samples to the normalized real-time PCR data.
Primary germinal center and memory B cells were crosslinked with 1% formaldehyde for 10 minutes at 37°C and terminated with 0.125M glycine for 5 minutes at room temperature. Cells were lysed in hypertonic lysis buffer (5mM Tris-HCl, pH 7.5 [Teknova], 85mM KCl, and 0.5% NP-40) on ice for 10 minutes. After centrifugation at 6000 rpm for 5 minutes at 4°C, the pellets of nuclei were lysed in chromatin immunoprecipitation (ChIP) lysis buffer (0.5% sodium dodecyl sulfate, 1% NP-40, 0.5% sodium deoxycholate in 1× PBS) and sonicated (Ultrasonic CV-18) to shear chromatin DNA to approximately 500-bp fragments. Each nuclear lysate (1/20) was kept for input, and the rest was incubated for 4 hours with Dynabeads (Invitrogen) that were coupled overnight with either the anti-H3K4me3 antibody (07-473; Millipore) or the anti-CD20 antibody (sc-15 361; Santa Cruz Biotechnology). Precipitated chromatin DNA was eluted with elution buffer (1% sodium dodecyl sulfate, 1% NaHCO3) and purified using the QIAGEN MinElute PCR purification kit. The library was subsequently sequenced, and semiquantitative PCR was carried out using primers to amplify the putative promoter region of the miRNA cluster (-226 to +49 relative to the predicted transcription start site, which is 4439 bp upstream of the first miRNA gene in the novel cluster).
Deep sequencing identifies the small RNA transcriptome of normal and malignant B cells
Small RNA libraries from these 31 cases were subjected to massively parallel, high-throughput sequencing using the Illumina platform to generate a total of 328 million separate reads (summarized in supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Our approach to analyzing the sequences and discovering miRNAs broadly follows a previously described method14 and is summarized in Figure 1A. All bioinformatics analyses were performed using a cluster of 1024 Linux computer nodes. Preprocessing was carried out using locally written Shell and Perl scripts.
Identifying miRNAs in normal and malignant B cells. (A) Analytic pipeline for the discovery and validation of known and novel miRNAs in B Cells. Small RNAs from B-cell subsets were subjected to massively parallel sequencing. Raw sequence reads (328 934 149) filtered, mapped to the genome, analyzed by miRDeep, and annotated. Identified miRNA loci were cross-referenced with miRBase13 to distinguish known from candidate novel miRNA. (B) Frequency of reads for miR-20b and their alignment to the genome. The sequence surrounded by a rectangle corresponds to the most abundant and longest mature miRNA sequence that perfectly matched the genome. (C) Outputs of the RNAshapes program that computed the abstract structure of the miRNA precursor, the probability of the predicted structure, and the minimum free energy of folding for the precursor structure for miR-20b. (D) Energetically favorable folding of the miR-20b precursor, with the miRNA mature sequence and the microRNA* sequence highlighted in yellow.
From the raw sequences generated by high-throughput sequencing, the 3′- and 5′- adaptor sequences were trimmed. Low-quality sequences were identified as those sequencing reads that contained stretches of consecutive identical bases or uncalled nucleotides (N) in the first 12 bases and sequencing reads shorter than 17 nucleotides. To minimize redundancy, reads were initially curtailed to the first 22 nucleotides, and identical sequences were represented with a single FASTA entry for analysis. Each unique sequence was mapped to the reference genome (Ensembl, build 50), and reads were filtered such that only perfect alignments (full-length, 100% identity) were retained. Reads that aligned to more than 5 positions in the genome and reads that overlapped with the University of California, Santa Cruz (UCSC) RNA genes were identified and excluded from miRNA analysis. Additional details regarding our methods are included in the supplemental materials.
Sequencing reads that mapped to known noncoding RNAs other than miRNAs15 were annotated separately. For each read, flanking sequences were retrieved from the genome, and potential secondary structures were predicted using RNAfold.16 Predictions of miRNA were made using miRDeep,14 which evaluated the sequences that indicated miRNA precursor processing and the thermodynamic energy of folding these precursors. The predicted probability and the thermodynamic energy of the miRNA precursor structures were consistent with previously described criteria for identifying miRNAs.17 An example of miRNA sequences that met the criteria for identifying a known miRNA (miR-20b) is shown in Figures 1B-D. Sequences that occurred 20 or more times in at least 1 sample were consolidated and annotated for the 31 samples. Genomic loci that overlapped with miRNAs described in miRBase (version 13) were identified as known miRNAs. The remaining genomic loci were identified as encoding candidate novel miRNAs.
To carefully calibrate the performance of our sequencing and bioinformatic approach, we performed multiplexed real-time PCR for 360 known miRNAs on biologic replicates of the same normal B-cell types. We found a high degree of overlap between the detection of miRNAs using both methods. For those 360 miRNAs that we measured using both multiplex real-time PCR and deep sequencing, we found that 64 of the 88 miRNAs were measured using both methods corresponding to a sensitivity of 72%, whereas 230/272 miRNAs were not detected by either method, corresponding to a specificity of 84%. Additional details regarding the calculation of sensitivity and specificity, as well as an alternative method of determining these statistics, which produced similar results, are described in supplemental Text 1 and supplemental Figure 1.
In all, we identified 619 miRNA precursor loci from normal and malignant B cells. The 333 miRNA precursor loci annotated in miRBase5 were identified as known miRNAs (supplemental Table 2), whereas the remaining 286 precursor loci were identified as candidate novel miRNAs (supplemental Table 3). Among these known miRNAs, there were 22 mature sequences (supplemental Table 2) that mapped to the ostensible microRNA* sequences and were expressed at levels that were comparable or, in some cases, higher than their complementary miRNAs. These previously unknown sequences likely reflect the influence of recently described pathways18⇓–20 that allow both the miRNAs and the microRNA* strands to independently exert their regulatory functions.
In addition to miRNAs, we identified the expression of other small RNAs, including tRNA, rRNA, snoRNA, snRNA, piRNA, and a recently described class of small RNAs, tiny RNA (tiRNA)21 in our data. The expression of these RNAs is shown in Figure 2A (primary data in supplemental Table 4).
Distribution of small RNAs in normal and malignant B cells. (A) Expression of small RNAs, including miRNAs, tRNA, piRNA, snRNA, rRNA, snoRNA, and tiRNA in each sample are shown more than a 2-fold range. Samples are listed in the same order as in Table 1. (B) Expression of miRNAs measured by sequencing across 2 biologic replicates for naive, germinal center, memory, and plasma B cells. The P values were computed using a correlation test against the null hypothesis that the data were not correlated; P < 10−6 in all 4 cases. (C) Relative expression of miRNAs differentially expressed in the naive to germinal center B-cell transition as measured by real-time PCR and sequencing. (D) Relative expression of miRNAs differentially expressed in the germinal center B-cell to plasma transition as measured by real-time PCR and sequencing. (E) Relative expression of miRNAs differentially expressed in the germinal center B to memory B-cell transition as measured by real-time PCR and sequencing.
Deep sequencing reproducibly measures miRNA expression in biologic replicates
We compared the expression of miRNAs identified by high-throughput sequencing and our analytic approach. In each of the biologic replicates (Figure 2B), we found that the measured miRNA expression in the biologic replicates were highly correlated (P < 10−6, correlation test for each comparison), confirming that our approach produces robust results. We further examined the expression of 107 miRNAs that we had previously identified as being differentially expressed among B-cell transitions.22 We compared the expression of these miRNAs as measured by real-time PCR and deep sequencing (Figures 2C-E) and found highly concordant results with more than 90% of the differentially expressed miRNAs, demonstrating identical patterns of expression regardless of the method of measurement (P < 10−6, χ2 test).
Therefore, we concluded that our sequencing and analytic approach reproducibly identified miRNA expression in biologic replicates, and the differential miRNA expression measured by deep sequencing and real-time PCR produced similar results.
Hundreds of known and novel miRNAs are expressed in normal and malignant B cells
Roughly 2/3 miRNAs measured in each cell type were known miRNAs (Figure 3A), whereas the rest were candidate novel miRNAs. In general, the expression of candidate novel miRNAs was lower than that of the known miRNAs, although there was considerable overlap between their expression levels. We also found that the correlation of miRNA expression measured by real-time PCR and sequencing was similar for both known and candidate novel miRNAs (supplemental Figure 2). The number of known and novel miRNAs detected in each sample type did not vary significantly between individual sample types or between normal and malignant cases. We examined the known and novel miRNAs present in the different normal B-cell subsets and found that the majority of both known (Figure 3B) and novel miRNAs (Figure 3C) were shared across the B-cell lineage.
Identifying novel miRNAs through high-throughput sequencing. (A) Distribution of known and candidate novel miRNAs in normal and malignant B cells. Samples are listed in the same order as Table 1. (B) Distribution of known miRNA expression among the normal B-cell subsets. The majority of miRNAs are shared between the B-cell types. (C) Distribution of candidate novel miRNA expression among the normal B-cell subsets. The majority of miRNAs are shared between the B-cell types. (D) Proportion of samples in which candidate novel miRNAs were identified. The majority of miRNAs are expressed in more than one sample. (E) Proportion of candidate novel miRNAs identified in 20 additional sequencing datasets. (F) Conservation of novel human miRNAs in chimpanzee, rhesus monkey, mouse, and dog. Ninety percent of the 286 novel miRNAs were found to be conserved across humans and one or more additional species, and more than 80% were found to be conserved across humans and 2 or more additional species. (G) Validation of 86 novel miRNAs candidates by an alternative method of measurement, real-time PCR. Maximum measured values for miRNAs expressed in at least 10% of the samples are shown in log2-scale. Detection threshold is the expression level corresponding to CT ≤ 35. (H) miRNA profiling distinguishes the molecular subgroups of DLCBL that were defined based on gene expression profiles. ABC refers to activated B-cell–like DLBCL. GCB refers to germinal center B-cell–like DLCBL. Unclassified cases are those that did not meet criteria for either group. Black bars indicate candidate novel miRNAs that were differentially expressed between the 2 groups.
The majority (96%) of the candidate novel miRNAs were found in more than one sample, with only a small minority of miRNAs expressed exclusively in a specific B-cell subset or malignancy (Figure 3D). We further examined the expression of these miRNAs in all publicly available small RNA datasets from mammals. The combined number of publicly available samples (N = 20) from 4 separate studies14,23,24 (GEO datasets GSE16579, GSE15190, GSE19473, GPL6583, GSE10829) is considerably smaller than that in our study. Nevertheless, we confirmed the expression of 197 (71%) of the candidate novel miRNAs in those data (Figure 3E and supplemental Table 6). Many of the miRNAs that we identified in normal and malignant B cells were expressed at 10-fold or higher levels in these non B-cell cases. These findings suggest that the miRNAs we have identified are broadly expressed and may have roles in several diverse tissue types.
We also examined the conservation of the novel miRNAs across 4 mammals (chimpanzee, rhesus macaque, mouse, and dog). We found that more than 80% of the identified miRNAs were conserved with 2 or more species (Figure 3F). Our data suggest that there may be species-specific differences in miRNA expression, as has been noted previously.25 In our data, 75% of novel and 80% of known miRNA had more than 100 predicted target genes with a perfectly conserved match in the 3′untranslated region of the gene in the following species: human, mouse, rat, and dog. We found that 11% of novel and 9% of known miRNA had > 50 predicted target genes. The remaining miRNA had < 50 predicted target genes (supplemental Figure 4). Analysis of ontology26 of predicted target genes12 of both known and candidate novel miRNAs expressed during B-cell differentiation strongly implicated transcription factor activity as a regulated function, suggesting a potential role of miRNAs in the regulation of these processes (supplemental Table 5).
Real-time PCR independently confirms the expression of 92% of the novel miRNAs
For further validation, we selected 86 candidate novel miRNAs that were detectably measured in at the sequencing data from least one of 4 DLBCL cases. Using stem-loop reverse transcription27 for quantitative PCR, we tested the expression of the 86 miRNAs in 101 primary tumors from patients with DLBCL and found that 79 (92%) were detectably measured by real-time PCR (Figure 3G) in at least 10% of these cases, suggesting that real-time PCR reproducibly identifies miRNAs that are expressed in lymphomas. We also used real-time PCR to measure the expression of 90 known miRNAs in the same 101 samples and found that more than 90% of these were also detected in at least 10% of the cases using real-time PCR. We found that 6 of the 7 real-time PCR constructs that targeted RNA hairpins that had low probability of being a miRNA resulted in no detectable signal. These results suggest that our assays have high specificity for miRNAs and that the computational predictions based on our sequencing data correctly identified miRNAs.
miRNAs are efficacious in the distinction of clinically relevant groups of lymphoma
Gene expression profiling of patients with DLBCL has demonstrated that the tumors comprise at least 2 distinct diseases with different response rates to standard chemotherapy regimens.28 miRNAs have been shown to be robust biomarkers in malignancies.29 We hypothesized that miRNAs might be used to make this clinically important distinction for which gene expression profiling remains the gold standard. We performed gene expression profiling on 101 DLBCL cases and further subdivided these cases into the molecular subgroups. We found that 25 miRNAs with the highest t statistic were equally efficacious as the gene expression profiling in differentiating the 2 groups of DLBCL with more than 95% overlap between the classifications rendered by the 2 methods, using leaveout one cross-validation (Figure 3H). Interestingly, 6 of these 25 predictor miRNAs (listed in the supplemental materials) were candidate novel miRNAs, suggesting a biologic and clinical relevance for these candidate novel miRNAs in DLBCL tumors. The complete miRNA expression data are included in supplemental Table 8.
Deep sequencing reveals a new cluster of 6 novel miRNAs
Although miRNAs appear to be distributed throughout the genome, several miRNAs have been found in clusters such as miR-17-92 that are transcribed from a single primary transcript and cleaved into the individual miRNAs by the enzyme Drosha. The discovery of the miR-17-92 cluster greatly accelerated the identification of the function of those miRNAs in several biologic systems.3,4 We found 2 separate clusters of candidate novel miRNAs on chromosome 9 and chromosome 14 (within the IgH locus), respectively. The first cluster was previously annotated as a hypothetical gene LOC100130622 and subsequently discarded from Refseq when no associated protein was identified. Our data demonstrate that this cluster, conserved only in primates, encodes 6 separate miRNAs (Figure 4A). To evaluate whether the miRNAs encoded in these clusters originate from the same primary transcript, we took KMS12 multiple myeloma cells that express these miRNAs and used siRNA to knock down the expression of the miRNA processing enzyme Drosha. This enzyme acts at the first step of miRNA processing by cleaving miRNA precursors from the primary transcript. We found that decreased Drosha expression was associated with increased accumulation of primary transcripts of both the miR-17-92 cluster as well as the novel miR-3689 cluster. (Figure 4B). miRNAs from this cluster were found to be expressed more highly in normal germinal center (GC) B cells compared with memory cells (Figure 4C). We computationally predicted the core promoter region at 4 kb upstream from the primary transcript.30 We performed chromatin immunoprecipitation for H3K4Me3, the histone marker associated with gene expression. DNA bound to this histone marker was found to be selectively enriched in primary GC cells which express this miRNA cluster highly, but not in primary memory B cells (Figure 4D). The miRNAs of this cluster all share the same seed sequence, suggesting that they target the same genes. Among the computationally predicted targets of this miRNA cluster,12 we identified SMAD2 and SMAD3, which are well known mediators of the transforming growth factor-β (TGF-β) signaling pathway. We noted that gene expression both SMAD2 and SMAD3 in our set of 101 DLBCLs were inversely correlated with this cluster (P < .01, correlation test). Gene set enrichment analysis revealed that expression of the TGF-β pathway in DLBCL samples varied inversely with the expression of the miRNA cluster, with a higher expression of the miRNA association with a lower expression of the pathway31 (P < 10−6; Figure 4E and supplemental material), which has previously been noted to be important in the biology of these tumors.32
Discovery and functional validation of a novel miRNA cluster. (A) Genomic locus of the mir-2355 cluster (chromosome 9q34.3), conservation across 3 primates and one additional mammal (mouse), and predicted secondary structure of each miRNA hairpin, with the mature sequences highlighted in yellow. The sequencess are conserved only in primates. (B) Knockout of the primary miRNA transcript processing enzyme, Drosha, by RNA interference results in accumulation of a known miRNA cluster primary transcript (pri-hsa-mir-17) and the novel miRNA cluster primary transcript (pri-hsa-mir-2355). (C) Expression of hsa-miR-2355 is higher in GCBs compared with memory B cells. (D) Chromatin immunoprecipitation with an antibody to H3K4me3 of GCBs and memory B cells shows enrichment of DNA from the predicted core promoter region of the miR-2355 cluster in GCBs compared with memory B cells. Three sets are shown: input (positive control for PCR using chromatin before immoprecipitation), immunoprecipitation with an antibody for H3K4me3, and immunoprecipitation with an antibody for CD20 (negative control). (E) Gene set enrichment analysis score and distribution of TGF-β pathway genes along the rank of transcripts differentially expressed in high versus low expressors of the miR-2355 cluster. Expression of the TGF-β pathway genes were inversely correlated with expression of the miRNA.
The advent of widely accessible whole genome sequencing has created a new urgency to annotate the human genome to provide the context for understanding the genetic variation underlying health and disease. Noncoding RNAs have emerged as the "dark matter" of the genome33 that may play an important role in regulating the 1% of the total DNA that is expressed as protein. The ongoing identification of new miRNA genes in different tissue types is analogous to the discovery of protein-coding genes. Our work provides an exhaustive identification of the miRNAs in normal and malignant B cells that is a prerequisite to the delineation of their role. Furthermore, we have developed a comprehensive framework that spans the identification of miRNAs from deep sequencing data to measuring their expression using real-time PCR and validating their expression in primary human tumors.
Several miRNAs have previously been suggested as being tissue-specific. For instance, miR-223 has been suggested as a myeloid-specific miRNA,34 whereas miR-9, miR-124, and miR-128 have been suggested as brain-specific miRNAs.8 However, our study found expression of all of these miRNAs in the B-cell lineage. Our data indicate that the highly increased sensitivity of deep sequencing will challenge currently held notions regarding tissue-specificity of miRNAs and suggest that the same miRNAs could play different context and lineage-specific roles in different cell types.
miRNA expression has a wide dynamic range from a few transcripts to hundreds of thousands of transcripts per cell. Deep sequencing is especially efficacious for the identification of miRNAs expressed at low abundance in a cell. The number of miRNA transcripts needed to exert their effects in a cell is not known, and it is likely to be context-dependent. Further, it has been demonstrated that some miRNAs containing the hexanucleotide motif AGUGUU can relocalize to the nucleus to potentially regulate the transcription of target genes.35 These findings suggest that a few copies of a miRNA in a cell may be adequate to exert profound down-stream effects. We found the same hexanucleotide motif in 5 miRNAs expressed in B cells. It is also conceivable that some of the low-abundance miRNAs that we have identified in our study may be expressed at higher levels in other development stages or in other cell types. This notion is confirmed by our examination of the novel miRNAs in non–B-cell data. For instance, several the miRNAs that we discovered were also present at 10-fold or higher levels in cell lines derived from breast cancer and cervical cancer, suggesting that the miRNAs that we have discovered in B cells have broad biologic significance.
miRNAs have shown promise as biomarkers in several malignancies.22,29,36,37 DLCBL is the most common form of lymphoma and is known to comprise at least 2 molecularly distinct subgroups with different responses to standard therapy. There are 2 important clinical applications for the molecular subgrouping of DLBCL patients. First, the prognostic information can inform the choices and expectations of patients and their physicians. Second, the important molecular differences in these subgroups might dictate the use of different therapies in these patients. For instance, the benefit of receiving a proteosome inhibitor, bortezomib, appears to be predominantly limited to those patients who have activated B-cell (ABC) DLBCL.38 However, the current methods can only distinguish germinal center B cells (GCBs) from non-GCB DLBCL in a limited fashion,39 and have yielded sometimes conflicting results.40,41 Gene expression profiling remains the gold standard for the distinguishing the 2 molecular subgroups; however, this is not routinely performed in clinical laboratories. Our data suggest that miRNAs can render this distinction with equal efficacy. Since miRNAs are stable in tissue processed for routine paraffin sections using standard methods,42 miRNA-based assays would be much easier to implement in the clinical laboratory.
Emerging data have demonstrated a regulatory role for small RNAs in nearly every cellular system examined thus far. A comprehensive delineation of their role in any cellular system must be preceded by a complete identification of the small RNA transcriptome in that system. The miRNAs that we have identified will likely be included in future commercial microarray platforms that would be widely available. The expression and role of individual miRNAs can also be queried using existing methods as we have demonstrated. Thus, we anticipate that our data, which represents the most in-depth examination of small RNA sequences using second-generation methods, will serve as a foundation for the exploration of the role of small RNAs in several cellular systems.
Contribution: D.D.J., J.Z., C.J., K.L.R., C.H.D., W.W.L.C., W.Y.A., G.S., M.B.C., D.A.R., A.S.L., P.L.L., K.P.M., C.R.F., L.B.-M., K.N.N., A.M.E., L.I.G., M.L., D.R.F., J.B.W., M.A.T., J.I.G., Q.L., T.H., V.G., Y.G., A.P., H.W., J.Z., G.C.B., P.E.L., A.C., and S.S.D. performed research; D.J., J.Z., C.J., and S.S.D. conducted data analysis; and S.S.D., J.Z., and D.J. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Sandeep S. Dave, 101 Science Dr, Duke University, Box 3382, Durham, NC 27710; e-mail: sandeep.dave{at}duke.edu.
We thank Drs Joseph Nevins and Jen-Tsan Chi for a critical review of the manuscript. We thank Susan Sunay for assistance with and the Georgia Cancer Coalition for support for sample collection.
S.S.D. was supported by the Doris Duke Charitable Foundation and the National Institutes of Health.
↵* D.D.J. and J.Z. contributed equally to this work.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Submitted May 13, 2010.
Accepted August 10, 2010.
© 2010 by The American Society of Hematology
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Dereje D. Jima, Jenny Zhang, Cassandra Jacobs, Kristy L. Richards, Cherie H. Dunphy, William W. L. Choi, Wing Yan Au, Gopesh Srivastava, Magdalena B. Czader, David A. Rizzieri, Anand S. Lagoo, Patricia L. Lugar, Karen P. Mann, Christopher R. Flowers, Leon Bernal-Mizrachi, Kikkeri N. Naresh, Andrew M. Evens, Leo I. Gordon, Micah Luftig, Daphne R. Friedman, J. Brice Weinberg, Michael A. Thompson, Javed I. Gill, Qingquan Liu, Tam How, Vladimir Grubor, Yuan Gao, Amee Patel, Han Wu, Jun Zhu, Gerard C. Blobe, Peter E. Lipsky, Amy Chadburn, Sandeep S. Dave, for the Hematologic Malignancies Research Consortium (HMRC)
Blood Dec 2010, 116 (23) e118-e127; DOI: 10.1182/blood-2010-05-285403
Jima, D. D., Zhang, J., Jacobs, C., Richards, K. L., Dunphy, C. H., Choi, W. W., Yan Au, W., Srivastava, G., Czader, M. B., Rizzieri, D. A., Lagoo, A. S., Lugar, P. L., Mann, K. P., Flowers, C. R., Bernal-Mizrachi, L., Naresh, K. N., Evens, A. M., Gordon, L. I., Luftig, M., Friedman, D. R., Weinberg, J. B., Thompson, M. A., Gill, J. I., Liu, Q., How, T., Grubor, V., Gao, Y., Patel, A., Wu, H., Zhu, J., Blobe, G. C., Lipsky, P. E., Chadburn, A., Dave, S. S., & , . (2010). Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs. Blood, 116(23), e118-e127. Accessed July 17, 2019. https://doi.org/10.1182/blood-2010-05-285403.


text:
Protection vs. Free Speech: Dan Bishop On His Controversial Proposal
WFAE | By Tom Bullock
The North Carolina General Assembly is now back from its January break. And during this session, one Mecklenburg County Republican senator says he will introduce a bill to protect former elected officials from angry protesters. But opponents say it would infringe on First Amendment rights.
Dan Bishop is no stranger to controversial legislation or the heckling and unkind words that follow.
Bishop was a chief sponsor of House Bill 2 last year. The bill was then quickly signed into law by then Governor Pat McCrory. And that legislation is why McCrory encountered this last weekend in Washington, D.C.:
http://www.youtube.com/watch?v=XVQny1bVxWI
For more than three minutes a knot of protesters followed McCrory chanting "Shame on you! Shame on you!" They called McCrory a bigot, anti-gay and the occasional expletive was thrown. But that was it. No punches, or anything physical.
Eventually McCrory is let into a side door and a uniformed officer tells the crowd to leave.
The video made the news. And that's where Dan Bishop, now a newly elected state senator comes in. "I saw the video. I think the conduct was indecent," says Bishop, "and there was a particular moment when one of the people accosting him said, 'we've got you now.'" There was more. "Because McCrory was pinned against the door trying to get in, that's, that's borderline."
Borderline assault in Bishop's mind. And for those not familiar with legal definitions assault is an intentional act by one person that creates an apprehension in another of an imminent harmful or offensive contact. "And here's a governor who's taken a tremendous amount of personal abuse," explains Bishop, "And this sort of riotous sort of protest is now a technique that is common place by certain folks."
Folks Dan Bishop called "ubiquitous leftist rioters," though he now calls that hyperbole. Still, Bishop says McCrory may have been fearful. "And the line, you never know when the line is going to be crossed and he may be in personal danger and he has no protection."
So Bishop plans to introduce a bill this session to make it a crime, punishable up to five years in prison, to "threaten, intimidate, or retaliate against a present or former North Carolina official in the course of, or on account of, the performance of his or her duties."
And he points to a state law and a DC statute as his inspiration.
First, Bishop explains the North Carolina law. "There is one that protects existing legislative or judicial officials and it also protects people who have been elected but are not yet serving. It does not protect former officials."
But these laws are often on the books to stop strong arm corruption – not protect private citizens.
As for the DC statue? We asked Arthur Spitzer for his opinion on the comparison. He's legal director of the DC chapter of the ACLU.
Spitzer watched the video of McCrory and the protesters and he doesn't believe the DC statute would apply in that case. "It was intended to protect government officials and employees from, I think, serious threats and harassment."
Protests, Spitzer says, is something current and yes former government officials have to deal with. "I think public officials are required to tolerate a higher degree of vituperation than ordinary people including ordinary public employees."
There is, however, another potential issue with what Bishop is calling for. The public's first amendment right to freedom of speech.
Democracy after all is messy. It can be a sometimes vulgar affair. Now, we don't have details of this bill as of yet – Bishop says it will be submitted soon – but free speech advocates including the ACLU say this law could stifle freedom of speech.
TB: This is the kind of law that could easily be challenged in terms of First Amendment rights, your right to protest an elected or formally elected politician. DB: Well, look I'm familiar with the First Amendment (laughs). But assaultive conduct has never been protected under the First Amendment. Whether or not this constituted assault, I don't know. Probably not.
But he adds, there is a potential risk there and it makes sense to address it before it occurs. Senator Dan Bishop is also calling on the General Assembly to "take other appropriate steps to guarantee the personal safety of Gov. McCrory by all means necessary."
PoliticsFirst AmendmentDan BishopPat McCroryNCGANC senateNews Desk
Tom Bullock
Tom Bullock decided to trade the khaki clad masses and traffic of Washington DC for Charlotte in 2014. Before joining WFAE, Tom spent 15 years working for NPR. Over that time he served as everything from an intern to senior producer of NPR's Election Unit. Tom also spent five years as the senior producer of NPR's Foreign Desk where he produced and reported from Iraq, Afghanistan, Yemen, Haiti, Egypt, Libya, Lebanon among others. Tom is looking forward to finally convincing his young daughter, Charlotte, that her new hometown was not, in fact, named after her.
See stories by Tom Bullock
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McCrory Loses On Home Turf; Blame I-77 Tolls, HB2, Shifting Vote Patterns
NC Supreme Court Strikes Down Part Of Cyberbullying Law
Firing Of Charlotte City Employee Over Facebook Post Highlights First Amendment Debate

redpajama_set_name: RedPajamaCommonCrawl


text:
L'hôtel de Boisleroy, ou hôtel Moisant-Girollet, est un hôtel particulier situé à Tours.

Localisation
L'hôtel est situé au 23 rue Briçonnet, à Tours.

Historique

L'hôtel est construit à partir de 1678 par Jean-Baptiste Gaullepied de Boisleroy, baron de Sennevières, sieur de Boisleroy, lieutenant particulier au bailliage et siège présidial de Tours, maire de Tours (1677-1678). En 1745, l'hôtel appartient à François Moisant-Girollet, marchand-fabricant en soierie, juge-consul et administrateur de l'Hôtel-Dieu de Tours.

L'hôtel est inscrit à l'inventaire général du patrimoine culturel depuis 1991.

Références

Édouard Gatian de Clérambault, Note sur trois hôtels de la rue Briçonnet, Bulletin de la Société archéologique de Touraine, t. XVI, 1907, p. 324-329.

Hôtel particulier à Tours

redpajama_set_name: RedPajamaWikipedia


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Your Star, Your World
Wicker's vote showed character, courage, loyalty to the Constitution
Sid Salter Syndicated columnist
STARKVILLE — No real surprise that Mississippi Second District U.S. Rep. Bennie Thompson, D-Bolton, voted to certify the nation's electoral vote. By that act of Congress, the Democratic lawmaker's party returned to power in the executive branch of the U.S. government in the form of President-elect Joe Biden and Vice President-elect Kamala Harris.
Mississippi's senior U.S. Sen. Roger Wicker, R-Tupelo, likewise voted to certify the electoral vote, although his party lost the 2020 presidential election in both the popular vote and the electoral vote. The certification vote, coupled with the results of the U.S. Senate runoff elections in Georgia, signaled a shift from Republican control of the Senate to the Democrats.
Wicker worked as hard to support Trump in that election as Thompson worked to support Biden. The election cycle saw Thompson retain his chairmanship of the House Homeland Security Committee while Wicker will lose his Senate Commerce Committee chairmanship and will be the ranking minority party member in the 117th Congress.
As the only Democrat in the state's congressional delegation, Thompson will wield vast influence over presidential patronage in the state. But that's another discussion for another day.
The rest of Mississippi's congressional delegation chose another path. Republicans U.S. Sen. Cindy Hyde-Smith and U.S. Reps. Trent Kelly, Michael Guest and Steven Palazzo – all Republicans – voted against certifying the electoral vote results even though all six of Mississippi's electoral votes were awarded to President Donald Trump.
They weren't alone. Six senators and 135 GOP congressmen voted the same way.
Each of the Mississippi members issued statements explaining their votes; statements met with praise from Trump's supporters and denunciations from his opponents. The reaction to those votes was set against the backdrop of a stunning and dangerous act of insurrection at the U.S. Capitol that was stoked by Trump hours before at a Washington, D.C., rally and carried out by protestors wearing Trump campaign gear and carrying his flags.
Five people, including a Capitol policeman, lost their lives in that disgraceful display. The riot at the seat of American democracy embarrassed the United States before the rest of the civilized world – or certainly should have. It reflected the political actions and reactions of what was called in the days before political correctness a "banana republic" – a small, politically unstable country where changes of power are often marked by violence and chaos.
Our nation has been plagued by political division and strife – particularly in the province of presidential politics – for the last 20 years. Some congressional Democrats protested the electoral vote certification of Republican presidents elected in 2000, 2004, and Trump's win over Hillary Clinton in 2016.
There was never any significant doubt even by the staunchest Republican that once those legal challenges failed, Biden's election was assured and the certification vote was – as it always is – a constitutional formality. As I wrote back in November, Biden's inauguration in 2021 was as inevitable as was Trump's in 2017.
The difference? There was a distinctly different group of people angry after those two campaigns. Many Democrats never accepted Trump's election in 2016 in the manner they advocated Biden's 2020 election be accepted. And there's the rub, as partisan division, rancor, and in some cases abject hatred, endures now as then.
To be clear, Sen. Wicker's vote to certify the 2020 electoral vote was a vote that showed character, courage, and loyalty to the U.S. Constitution. In casting the vote, Wicker knew the political rain would fall. Thompson likewise followed that path.
What about the other four Mississippi members? Will their vote carry future consequences, or will their votes be like the failed anti-certification votes of Democrats in three presidential elections since 2000?
In the final analysis, politics is and always has been a math exercise. In a state that gave Trump 57.8 percent of the state's 2016 vote for president and 57.6 percent in the 2020 presidential election, the practical political impact on those four GOP congressional members will likely be negligible – regardless the roiling of social media.
Biden in 2020 got 54,267 more votes than did Hillary Clinton in 2016. But Trump in 2020 got 56,050 more votes here than he got in 2016.
Sid Salter is a syndicated columnist. Contact him at sidsalter@sidsalter.com.
Public And Administrative Law
Bennie Thompson
Carla Perkins
Graveside services for Mrs. Carla Perkins will be Wednesday, Jan. 20, 2021, at 11 a.m., at New Prospect Cemetery, Savoy. Arrangements have been entrusted to Berry and Gardner Funeral Home.
STINGILY JR., Karl Oct 26, 1932 - Dec 10, 2020
ABILENE, Texas [ndash] On Thursday, Dec. 10, 2021, Karl Otto Stingily Jr. passed away following complications from COVID-19. He was born on Oct. 26, 1932, in Oxford, Miss., to Karl and Olga Stingily. He was the oldest of two children. He has a sister, Sandra Simpson, of Mountain Brook, Ala. …
Lewis, Nathaniel
Graveside services for Mr. Nathaniel Lewis will be Tuesday, Jan. 19, 2021, at 11 a.m., at Mississippi Veterans Memorial Cemetery, Newton. Viewing: Monday, Jan. 18, 2021, from 5-6 p.m., at Berry and Gardner's OP Chapel.
Perkins, Carla
Arrangements were incomplete at Berry & Gardner Funeral Home for Mrs. Carla Perkins, 45, of Meridian, who died Thursday, Jan. 14, 2021, at her residence.
MORGAN, Glynn Aug 29, 1938 - Jan 8, 2021
Meridian police drop child pornography charges against two suspects
Local medical clinics to offer COVID-19 vaccine
Hard work pays off for Hutcherson, Uithoven as they sign to continue soccer careers
Law enforcement reports for Jan. 16
Trump impeached for second time; Mississippi House Republicans stick with president
Meridian girls embracing national MaxPreps ranking
Mississippians 65 years or older, people with certain conditions eligible for COVID-19 vaccine
Winter storm advisory in Lauderdale County; light snow and ice possible
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text:
Welcome to Jeff Darmanin, Executive Director
We extend a warm welcome to Jeff Darmanin, appointed as Executive Director of the Children's Cancer Foundation from January 2020.
Home News Community Welcome to Jeff Darmanin, Executive Director
In 2020, the Foundation will continue to engage in cost-effectively raising funds to support children and their families today, while also working closely with world-class organisations, learning more about the unknown and making progress towards improving childhood cancer survival rates.
As we work towards our goals, we officially welcome Jeff Darmanin to the team. Jeff who led the Foundation in an interim capacity during late 2019, has with the encouragement of the board, formally accepted the position of Executive Director for the Foundation, for a 3-year term commencing January 1, 2020.
While now working in a formal capacity, Jeff has been generously sharing his commercial skills and a lifetime of experience in running businesses both large and small, as non-executive Director with the Children's Cancer Foundation since 2016.
Jeff's close personal connection to the Foundation began over a decade ago, in 2009; the year the course of his family's lives would change forever. The Darmanin's were a normal family of three children and a geriatric dog. William, Jeff's 15-year-old son was having growing pains. These pains were later diagnosed as Desmoplastic small round tumour cells. A rare cancer.
The family knew nothing of cancer then. As their 'normal' world turned upside down, Will was a tough soldier, facing every new unknown with courage. But after four years including a period of remission, he sadly lost his battle in 2013. As the family began rebuilding their lives, Jeff noticed that he was no longer able to find joy in everyday life.
"I wanted to help, I liked the Foundation's focus on research and family support. There was a strong sense of purpose, integrity, passion and intelligence, wrapping a desire to help families cope now while trying to build a better future."
Jeff's search for meaning led him to the Children's Cancer Foundation.
Today in his new leadership role, the direction of the Foundation is unchanged. "I am delighted to be a custodian of the Foundation," says Jeff. "What I am seeking to do is make the Foundation more sustainable through a broader network of committees, a greater sharing of responsibility and greater stakeholder engagement. With the help of others, I think I can make a difference."
The Foundation welcomes Tracey Danaher to the Board
We extend a warm welcome and congratulations to Tracey Danaher to the Board of the Children's Cancer Foundation.
Because I survived cancer, I'm becoming an Oncologist
Anna survived Acute Lymphoblastic Leukemia (ALL) as a child and is becoming a paediatric oncologist to help others like her. She shares her amazing story.
Tassie town goes gold for childhood cancer awareness month
Mum Melinda brings together Oatlands town residents to raise funds for childhood cancer awareness month in September.
If you'd like to share your story, please contact us with the email below.
hello@childrenscancerfoundation.com.au


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I see that the REF 2014 results were released today, so I was very interested to see how the various departments around the UK performed for Theology and Religious Studies. (For those that are not aware, the UK rates the research output for each of the university departments to help determine funding.) For some reason they don't provide a weighted ranking of the departments, so I've done a quick assessment based on the overall percentages given a 0-4 ranking based on the recognized excellence at a national vs international level (see description at the bottom). I was surprised by some of the overall rankings especially given the previous results (see below), but I'll reserve commentary here and just provide the results. The GPA is the weighted average of the 0-4 ranking. You'll see that Durham is the top ranked department, just as it was for the most recent previous exercise: the 2008 RAE.
4* Quality that is world-leading in terms of originality, significance and rigour.
3* Quality that is internationally excellent in terms of originality, significance and rigour but which falls short of the highest standards of excellence.
2* Quality that is recognised internationally in terms of originality, significance and rigour.
1* Quality that is recognised nationally in terms of originality, significance and rigour.


Vulnerability maps overlay third-party vulnerability information with Illumio ASP's real-time application dependency map for a risk-based approach to patch prioritization.
The East-West workload-to-workload traffic within your data center and cloud environments represents a massive attack surface. Organizations are adopting a Zero Trust strategy—essentially operating as if they have already been breached and taking steps to inhibit bad actors from moving laterally within an environment.
Vulnerability maps incorporate data from third-party vulnerability scanning tools like Qualys to provide insights to the exposure of vulnerabilities and attack pathways within your environment.
Vulnerability maps help security and IT operations teams prioritize security and patching decisions; if you cannot patch, micro-segmentation can be quickly mobilized to act as a compensating control.
Vulnerability maps display connections to vulnerabilities between and within applications, enabling security teams to see which of the workloads' vulnerabilities are highly exposed. In addition, vulnerability maps display an attacker's potential lateral pathways.
IT operations teams can use information from the vulnerability map to prioritize patching strategies.
If patching is not viable because there are no patches available or there is a production freeze, security teams can instantly apply micro-segmentation as a compensating control without breaking your applications.
Vulnerability management solutions use vulnerability scores, which are typically the combined Common Vulnerability Scoring System (CVSS) scores of the vulnerabilities found in a workload. Vulnerability scores, while valuable, do not take into account a workload's connectivity relative to other workloads in an environment.
The Illumio Adaptive Security Platform® (ASP) uses the open industry-standard, community-accepted CVSS, which can be scored by an enterprise, and combines this with information on how many workloads can potentially connect with the vulnerable workload—the actual "reachability" of the vulnerable workload—to calculate a Vulnerability Exposure Score. IT operations and security teams use this information to prioritize patching and implement micro-segmentation as a compensating control if the time is not right to patch the application.
Illumio ASP also allows you to model micro-segmentation as a compensating control in test mode. In this mode, traffic is not blocked, but there will be an alert if it does not conform to policy. If traffic is connecting into a port with a known vulnerability, the vulnerability and its severity is included in the traffic alert to inform the security operations center (SOC) of a traffic violation, and that it is connecting to a port with a known vulnerability on it.


Titus Bocseri is a senior executive with over 20 years of Financial Services experience – both in management consulting and on the industry side – centered on the Insurance Industry. He specializes on formulating and implementing business strategies, devising transformation planning, defining business models as well as driving them to successful implementation.
As Managing Director of Slalom's Insurance Practice, Titus is responsible for Business Development, Thought Leadership, Strategic Alliances, Asset Development, and Client Engagement Delivery.
In addition, he is holding an adjunct faculty position at Columbia University where he is teaching various courses in corporate strategy and business development in the School of General Studies.
He previously worked for Carlisle & Gallagher Consulting Group, where he was responsible for the North American Insurance Practice and for HSBC as a Senior Vice President while heading the strategic initiatives unit in the Global Insurance space responsible for building a profitable life insurance business via direct and third party distribution of manufactured and co-branded products. Prior to that, Titus worked on Wall Street as Vice President in charge of Strategic Development in the Global M&A Department of Willis Group Holdings Ltd. His work focused on deal – sourcing, due diligence, contract negotiations, structuring and execution – with the added task of being in charge of the integration tracking phase.Titus' previous experience includes corporate strategy, M&A, as well as management consulting in both domestic and international markets.
He holds a B.S. in Engineering from the Military Technical Academy in Bucharest, an M.S. in Technology Management from Columbia University and an MBA from Columbia Business School – concentration on Finance and Economics.


Campus kilau will be closed on Monday 7th May to allow our electrical supply to be upgraded. Little Belmont Street will be open as usual tho!
Image Posted on April 26, 2012 by kilaucoffee Tagged Coffeekilauold aberdeen	CommentsNo Comments on TABLES!
Posted on January 1, 2012 by kilaucoffee Tagged CoffeeexcitementNews	Comments4 Comments on Happy New Year!
Hey, just a wee note to say Happy New Year and give everyone a heads up on the impending work at Little Belmont Street. We will be closing from the 8th of January until 10am on the Friday the 13th (kilau's 8th Birthday!!) Whilst we are closed, we will be mostly doing painting and plumbing and trying to smarten up those pesky toilets. We are hoping that 2012 will be a great year for kilau, we got confirmation of our building warrant for the high street shop in mid december, so we are hoping to start work on the upstairs early January too!
I hope this year brings everyone what they hope for, and look forward to seeing you in one of the shops soon.


The impact of preconception maternal stress on fecundability and adverse pregnancy outcomes.
It has been commonly hypothesized (and widely believed) that maternal stress either prior to or during pregnancy can adversely affect fertility and pregnancy outcomes. However, surprisingly few epidemiologic studies have rigorously tested these hypotheses. The current study evaluated the effects of pre-conception self-reported stress on fecundability and spontaneous abortion. The study population was derived from the Mount Sinai Study of Woman Office Workers with 487 women included. Women recorded stress (scale from 1 to 4) and covariate information in a daily diary for 12 cycles or until pregnant. Results indicated a significantly increased risk of spontaneous abortion for women with higher levels of self-reported stress during the cycle of conception, and reduced fecundability when high stress occurred during the ovulatory window. This study reinforces the need for ameliorating stress in the aspiring and expecting mother. Future studies may examine effects of stress-related biomarkers and genetic polymorphisms on pregnancy outcomes.
Akhter, Shekufe, "The impact of preconception maternal stress on fecundability and adverse pregnancy outcomes." (2014). Electronic Theses and Dissertations. Paper 21.


The seal, like the logo of the BASLQC, uses CJK seal script in it's design.
A header image replacing the yande.re one should be used.
There will be a 4 CJK character seal on the right, in pixel art rendition.
The main title will be in Greek pixel font, use a free one from Grixel.gr.
This page was last edited on 30 September 2016, at 15:51.


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News	Archaeology
Ancient South Americans tasted chocolate 1,500 years before anyone else
Archaeological finds are the earliest evidence for cacao use in the world
SWEET ROOTS The earliest known evidence of people domesticating and consuming cacao, the main ingredient in chocolate, has turned up at a South American site, researchers say.
YelenaYemchuk/istock.com
Ancient South Americans domesticated and consumed cacao, the plant from which chocolate is made, long before other people did, a new study finds.
Artifacts with traces of cacao suggest that an Amazonian culture located in what's now Ecuador developed a wide-ranging taste for cacao products between 5,450 and 5,300 years ago, researchers report online October 29 in Nature Ecology & Evolution. Societies in southern Mexico and Central America, such as the Olmec and Maya, didn't start concocting their better known and more intensively studied chocolatey drinks for roughly another 1,500 years.
"This is not only the earliest archaeological evidence so far reported for cacao use in the Americas, but also the only archaeological evidence for cacao use in South America," says study coauthor and anthropological archaeologist Michael Blake of the University of British Columbia in Vancouver.
For more than a decade, reports of heightened genetic diversity among present-day domesticated cacao plants in South America's upper Amazon region — near where the artifacts were found — have suggested that domesticated cacao (Theobroma cacao) originated there. Differences in the genetic makeup of related populations of organisms accumulate gradually, so populations displaying the most DNA diversity are presumed to have evolved first. The new study confirms that genetic scenario for cacao for the first time at an archaeological site.
CHOCO-LOT Jugs, bowls and other items unearthed at a South American site contain clues to the origins of domesticated cacao, for which all chocolate fans should be grateful. S. Zarrillo et al/Nature Ecology & Evolution 2018
The ceramic objects that held the cacao clues were previously unearthed at Santa Ana-La Florida, a settlement occupied by members of an ancient South American culture known as Mayo-Chinchipe. Santa Ana-La Florida excavations began in 2002. Cacao-containing items came from in and around household structures and the tombs of presumably high-status individuals. The researchers suspect that cacao plants likely served as food, drink, medicine, stimulants and perhaps a ceremonial substance for Mayo-Chinchipe people.
Three lines of evidence point to cacao use at Santa Ana-La Florida, the team says. First, starch grains characteristic of domesticated cacao today were recovered from charred food stuck to six pottery shards. Second, traces of theobromine, a bitter chemical compound found in seeds of a domesticated cacao species but not in its wild relatives, were identified in 25 ceramic artifacts and 21 stone artifacts. Finally, three artifacts contained DNA fragments bearing gene variants typical of a domesticated cacao species. Genetic signs of domesticated and wild cacao came from another two artifacts.
It's particularly interesting that ancient South Americans apparently consumed cacao seeds as well as pulp, says Cameron McNeil, an archaeobotanist at the City University of New York who was not involved in the study. Some researchers have speculated that ancient South Americans would have avoided the time-consuming process of preparing cacao seeds and focused mainly on cacao pulp as a readily available stimulant.
Domesticated cacao's status as a prestigious, symbolic plant may have blossomed once it reached Central America and Mexico. Those societies further domesticated cacao, McNeil says, probably to enhance the flavor of seeds that were traded and used as currency (SN: 8/4/18, p. 16).
The new work could have implications for today's chocolate lovers. A recent study found that harmful mutations that lower crop yields in a modern form of cacao accumulated as a result of domestication in Central America and Mexico around 3,600 years ago. Cacao growers can boost the plant's productivity by incorporating the genetically diverse, South American varieties into current crops, Blake suggests.
A version of this article appears in the November 24, 2018 issue of Science News.
S. Zarrillo et al. The use and domestication of Theobroma cacao during the mid-Holocene in the upper Amazon. Nature Ecology & Evolution. Published online October 29, 2018. doi:10.1038/s41559-018-0697-x.
O.E. Cornejo et al. Population genomic analyses of the chocolate tree, Theobroma cacao L., provide insights into its domestication process. Communications Biology. Published online October 16, 2018. doi:10.1038/s42003-018-0168-6.
Chocolate may have arrived early to U.S. Southwest
By Rachel Ehrenberg February 2, 2009
Pueblo traded for chocolate big-time
By Bruce Bower March 17, 2011
If you think the Amazon jungle is completely wild, think again
By Bruce Bower March 3, 2017
More Stories from Science News on Archaeology
Clovis hunters' reputation as mammoth killers takes a hit
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Jane works with couples, families and individuals of all ages, including children, adolescents and adults, who have a variety of mental health and/or substance abuse issues. Her attested specialties include dual diagnosis, eating disorders, ADD/ADHD, and chemical dependency. Jane utilizes play therapy ... Read More techniques when working with children. She has worked at Eastwood Clinics for nearly 15 years.


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Posted on July 11, 2019 July 11, 2019 by reliance
BASED ON THE INSPIRING TRUE STORY OF ANAND KUMAR, ONE OF INDIA'S MOST ADMIRED TEACHERS AND MATHEMATICIANS
Mumbai, 11th July 2019: Anil D. Ambani led Reliance Entertainment is releasing the Hrithik Roshan starrer Super 30 globally on July 12, 2019. The movie is inspired by, and based on, true events from the life of Anand Kumar and his students.
Anand Kumar is one of the most renowned and revered mathematicians in India, who has dedicated his life to coaching underprivileged students for the JEE tests, the gateway to the premier Indian Institutes of Technology.
The film chronicles Anand Kumar's incredible life journey, starting at a very young age, and his battle against the insurmountable odds of India's education system. A brilliant student, Anand Kumar, used to regularly travel 6 hours by train from Patna to Varanasi, to access study materials not available in his home town.
In 1994, at the tender age of 21 years, he secured admission to the prestigious University of Cambridge, but could not go to England as he could not arrange funds to pay for the travel. Owing to his father's untimely demise, and despite being such a brilliant mathematician, Anand was compelled to sell papad (poppadum) on the streets of Patna along with his mother, to make ends meet.
Determined that other brilliant but economically backward children should not have to undergo this fate, in 2002, Anand Kumar initiated his Super 30 educational programme, under which he would prepare 30 underprivileged students for the IIT-JEE, free of cost, every year.
By the year 2018, an unprecedented 422 out of the 480 children, all economically underprivileged, and all coached free of cost by Anand Kumar in his Super 30 classes, had made it to the IITs.
Despite being flooded with offers of financial support for his classes from Governments and private sector giants, Anand Kumar declined all assistance, and instead utilised his own meagre resources to coach his students. Anand's family also played a key role in his endeavours. His mother, Jayanti Devi would cook for the students, and his brother, Pranav Kumar, would take care of the management.
On the eve of the film's release, Anand Kumar, said "I'm very happy with the way Super 30 has shaped up. The team has done an incredible job. I hope this film inspires all future generations."
Commenting on the movie, Hrithik Roshan said, "Anand Kumar's life is one of the most inspiring and moving stories I've ever heard. His story had to be told to the world. I am honoured to have played the on screen role of Anand Kumar."
Group CEO, Reliance Entertainment, Shibasish Sarkar, said, "We are very proud of Super 30 and are looking forward to global audiences showering their love on the film. It's a film we believe all families must watch."
Super 30 film is presently ranked the No. 1 most anticipated movie of 2019 on IMDB India. The trailer of the film has garnered over 45 million views on YouTube alone, so far. Headlined by Hrithik Roshan, the film also features Mrunal Thakur, Pankaj Tripathi, Amit Sadh, Nandish Singh and Aditya Srivastava. The film is directed by Vikas Bahl.
About Reliance Entertainment:
www.relianceentertainment.com
Reliance Entertainment is the media and entertainment arm of Reliance Group and is engaged in the creation and distribution of content across film, television, digital and gaming platforms. Internationally, Reliance Entertainment has partnered since 2009 with iconic film producer and director, Steven Spielberg, in the formation of DreamWorks Studios, and thereafter, Amblin Partners. This relationship has produced several successful films such as The Help, War Horse, Lincoln, The Hundred Foot Journey, The Girl on the Train, A Dog's Purpose, Bridge of Spies, The Post and the most recent Golden Globe and Oscar winning film, Green Book
https://www.facebook.com/RelianceEntertainment
https://twitter.com/RelianceEnt
https://www.instagram.com/reliance.entertainment
For further information contact: Spice PR
Daljeet S Singh(+91 93120 14099) – Daljeet.S.Singh@relianceada.com
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McAlear Named CEO Of Kvaerner Philadelphia Shipyard
Kvaerner, the Anglo-Norwegian engineering and construction Group, has elected Ronald J. McAlear, as CEO of its Philadelphia Shipyard. He replaces Baard Bale, who has been acting CEO of the yard since June, and will be returning to Kvaerner's international headquarters in London in December.
McAlear has spent his entire professional career in the maritime industry, and currently holds a senior executive position with Litton Avondale Industries. He will assume his new position with Kvaerner in Philadelphia at the beginning of November.
McAlear, a well-known figure within the U.S. shipbuilding industry, boasts a solid background in shipbuilding, ship repair, ship-operations and marine equipment marketing and manufacturing.
He has held a number of senior positions with Avondale since 1988, and is currently vice president of Operations, reporting to the president of the company.
McAlear's appointment is expected to make the yard, which has been touted as one of the most state-of-theart shipyards currently in operation, a solid investment for both customers, and future prospective owners.
Trained in naval architecture and marine engineering at the Massachusetts Institute of Technology, and the University of Michigan, McAlear also undertook various management courses at Harvard, Tulane University and the University of New Orleans. He is a member of the American Society of Naval Architects and Marine Engineers and has an affiliation with the Propeller Club, the American Society of Naval Engineers, the Navy League, and the Surface Ship Navy Association.
McAlear is also licensed in the state of Louisiana as a Professional Engineer.


Here at EDinteractive, we deliver the University of Salford Business Schools CPD course called Search and Social Media Marketing (SSMM) CPD course. As part of this, we were asked to put together a short collection of top 10 tips for a printed publication, here is our top list.
1. The right people – If you do not have expertise within your organisation, seek advice and training from reputable sources. Search and Social Media Marketing is not rocket science, but you will save a lot of time and money if you work with experienced people with a successful track record. Seek out recommendations from customers, employers or colleagues and speak to the people that they have worked with previously to gauge how effective they have been with previous projects.
2. The right strategy – Planning is everything. Understand your customers needs – how are they finding your business online and how might they find you in the future? Research a list of search keywords you would like people to find your website on in the search engines and then test, monitor and refine.
Once you understand your customers, formulate a comprehensive plan of what you would like to achieve with your search and social media marketing campaign. What is working or not working to date, how can you improve and where are the gaps? Make sure you factor in staff time, it all costs money.
4. Produce great content – put your plan into action and produce great content. A blog can be an excellent way to create great new text, audio or video that people will want to view and link to. Your content could be produced by someone internally or employ experienced people. Your content should be engaging, informative, interesting and relevant to your company. A great idea for content could lead to a lot of positive interest in your company and products if it 'goes viral' on a service like YouTube.
Make sure you have got access to the data from your website visitor information and that you have good people to evaluate this data to help inform company decisions. Google Analytics is one great free solution to allow you to capture and report on this data.
Setting up a presence on a small number of social networks relevant to your target audience can be an invaluable way to promote your business, gain customer feedback and engagement. Devote time and resource to a small number of networks and make them work for your business. Register your company name for free on Google's social network plus.google.com – it may be useful in the future!
7. Link building – Links from high quality websites to your website are vital to your websites search engine rankings. To gauge the quality and authority of a website, or to see who is linking to your competitors, you can use tools such as Open site Explorer.
8. Local searches – If you provide a local service, make sure people can find you locally, by registering your website and business at services such as Google Places. Make sure you have good pages on your website devoted to the fact that you provide services in the areas that you operate. Local keyword searches can be crucial, particularly in the age of mobile devices. Try experimenting with location based social media services such as Foursquare.com.
9. Go Mobile – It sounds obvious, but make sure your website works on smart phones and tablets. You may not need an app, but your website should work properly and quickly on all the popular mobile devices. Increasingly, more purchases are made on mobile devices than desktops. This means it is vital that your website functions properly on these devices. Feature your website and QR codes on any printed material to help people find your website.
10. Return on Investment – your search and social media strategy should form part of your overall marketing plan. This includes everything you do offline and online. All of your communications should be targeted carefully at different points of the customer journey from their initial awareness of your service, to their final action or purchase. Make sure you track and record this process carefully so that you can gauge which aspects of your strategy lead to return on investment.
Salford University Business School have worked with Manchester Digital marketing companies to develop a Search and Social Media Marketing CPD course which is designed to help you and your organisation. The course is delivered at MediaCityUK in partnership with reputable local agencies and is geared towards helping your company to get return on your Search and Social Media Marketing investment.


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Acid Rain Hypothesis
The effect of acid rain on seedling germination
By Lily and Zeenat
How does acid rain effect seedling germination(bean seeds)
The bean seeds being watered with acid rain will begin to dry up, lose colour, start cracking and stop growing
To test whether acid rain will have an effect on seedling germination
When sulfur oxides and nitrogen oxides incorporate into rain, snow or mist, the result is acid precipitation. Acid rain is particularly deadly to green growing things, impacting forest health, farmers' livelihood and the world's precious food supply.
Diminished Soil Nutrients
As acid rain falls onto soil, it may reduce in acidity because of contact with alkaline or basic substances…show more content…
Step 4 : the next two bean seedlings were watered regularly every 3-4 days with 10ml of water and 3ml of the acidic solution for 17 days, they were referred to as B.
Step 5: The third set of bean seedlings were watered regularly every 3-4 days with 10ml of water and 5ml of the acidic solution for 17 days , they were referred to as C.
Table of results showing the effect of acid rain of seedling germination:
My results showed that an increase in the amount of acidic solution the seedlings were exposed to meant the seedlings becoming increasingly dehydrated and began cracking, the colours of the seedlings began changing to a white colour when exposed to small amount of vinegar and lost colour completely when when exposed to a larger amount of vinegar.Whereas A showed signs of growth by its skin shedding, B and C showed no signs of growth.There was a clear significant difference in height, the control seedlings were 2cm whereas the seedlings exposed to 5ml of vinegar only grew to the height of 7mm showing clearly that an acidic solution would have a very definite affect on the growth of the
Sweet Sorghum Lab Report
There were four rows in each plot, which the row width and length was 0.6 and 6 m, respectively. At third leaf stage, plants were thinned for the appropriate final stand of 160000plants ha-1. The applied factors consist of sweet sorghum varieties (KFS2 and KFS3), top removal treatments (control, mechanical removal and chemical removal with ethephon) and magnesium sulfate rates (0, 4 and 8 mM). Sweet sorghum varieties are new hybrids released in Iran. Magnesium sulfate levels sprayed at the rapid plants growth stage that was approximately 60 days after planting and repeated in 10 days later.
Capssicum Annuum Lab Report
Spray the chilli plants in their fourth leaf growth stage with spore suspension containing 3 × 105 conidia ml−1 (15 ml/pot). Spray the non inoculated plants with distilled water containing same amount of Tween 20 (0.02 %). After spraying, keep both inoculated and non inoculated plants in a moist chamber with 98−100% relative humidity for 24 hours. Seven days after inoculation, evaluation of the wilt disease severity is based on visual assessment of lesions caused R. solani on a nine grade scale according to IRRI standards (2002). Disease severity for each replication was calculated according to the equation below (Cai et al.,
Phosphoric Acid Lab Report
The flask was then vented. The shaking and venting of the flask was further repeated several times to remove as much CO2 from the soft drink sample as possible. Now the 100 mL beaker was taken and 10.0 mL of the soft drink was measured into it. To this 100 mL beaker, 25 mL of distilled was
How Does Acid Rain Affect Plant Growth
Research Questions: 	What effect does acid rain have on the growth rate of plants in the wetland ecosystem? 	What results are expected? 	What is your conclusion? Hypothesis: Acid rain will have a negative impact on the plant growth in the wetland environment, this will result in plants dying and
Essay On Artificial Acid Rain
Every day, the numbers of leaves of the duckweed were counted, which determined if the plant was still alive. The results showed that more plants died in the rainwater with more drops of AAR than the distilled water. Background of the Study Water and atmospheric
Essay On Effects Of Acid Rain
Acid rain makes waters acidic and causes them to absorb the aluminum that makes its way from soil into lakes and streams. This combination makes waters toxic to crayfish, clams, fish, and other aquatic animals. Some species can tolerate acidic waters better than others. However, in an interconnected ecosystem, what impacts some species eventually impacts many more throughout the food chain—including non-aquatic species such as birds. Acid rain also damages forests, especially those at higher elevations.
Essay On Toxic Plants
The mode of action of lantana toxin appears to be by inhibiting the active secretion of bile canaliculi in liver and result secondary photosensitization in all grazing animals (Csurhes and Edwards, 1998). Due to its strong allopathic properties, aggressiveness and its dense impenetrable thorny thickets, Lantana camara has the potential to interrupt the health and regeneration process of other species by decreasing germination, growth of seedlings and biomass production which in turn increases mortality and decline of plant species, pasture and crops (Gentle and Duggin, 1998; Catherine and Russell, 2005; Sharma et al., 2005; Sharma and Raghubanshi, 2006). Lantana camara was introduced to Ethiopia as an ornamental plant due to its beautiful aromatic flowers (Binggeli and Desalegn, 2002). However, because of prolific seed production and easy dispersal, it escaped cultivation and become a pest in the social, ecological and economic concerns. Presently, it has spread almost all over the country, but still it is not much perceived as a chronic environmental problem, except in few parts of Ethiopia, such as Oromia and Somali regions (Binggeli and Desalegn,
The Pros And Cons Of Environmental Pollution On The Environment
Mining requires the removal of topsoil containing valuable organic matters. The removal of topsoil causes the land to be barren which are unsuitable for cultivation and unproductive, which is a kind of land pollution. Due to urbanization, many construction activities are taking place. Waste materials such as wood, metal and plastic often being left at the construction site. When deforestation is committed, the green cover which helps in balancing the atmosphere is reduced and it leads to natural disasters such as global warming, flash floods and green house effect.
Negative Effects Of Water Logging
The adverse effects of waterlogging are creation of anaerobic condition in the crop root-zone, growth of water loving wild plants, impossibility of tillage operations, accumulation of harmful salts, lowering of soil temperature, reduction in time of maturity. We can say that the waterlogging effects in such a way when there is a satisfactory of the aeration of the soil produce the required nitrates from the nitrogenous compounds present in the soil produce by the bacteriological activities that helps the crop growth. Anaerobic condition in the soil creates by extreme moisture content. The plant roots do not get the required nourishing food or nutrients due
Effective Effects Of Environmental Pollution
When it rains, the water droplets integrate with these air pollutants, so it becomes acidic and then drops on the ground in the shape of acid rain ("Environmental pollution," n.d.). Therefore, acid rain causes great harm for greens and crops. The greens and crops are damaged leads to lower production, increase poverty, and provide lung problems. Dropping the sales in the supermarkets and reducing the gain happened by lower production of crops. Consequently, that causes poverty by having no food to people eat.
More about Acid Rain Hypothesis


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