Human killer immunoglobulin-like receptors (KIRs) play a critical role in governing the immune response to neoplastic and infectious disease. Rhesus macaques serve as important animal models for many human diseases in which KIRs are implicated; however, the study of KIR activity in this model is hindered by incomplete characterization of KIR genetics.
Here we present a characterization of KIR genetics in rhesus macaques (Macaca mulatta). We conducted a survey of KIRs in this species, identifying 47 novel full-length KIR sequences. Using this expanded sequence library to build upon previous work, we present evidence supporting the existence of 22 Mamu-KIR genes, providing a framework within which to describe macaque KIRs. We also developed a novel pyrosequencing-based technique for KIR genotyping. This method provides both comprehensive KIR genotype and frequency estimates of transcript level, with implications for the study of KIRs in all species.
The results of this study significantly improve our understanding of macaque KIR genetic organization and diversity, with implications for the study of many human diseases that use macaques as a model. The ability to obtain comprehensive KIR genotypes is of basic importance for the study of KIRs, and can easily be adapted to other species. Together these findings both advance the field of macaque KIRs and facilitate future research into the role of KIRs in human disease.
Killer immunoglobulin-like receptors (KIRs) are a highly polymorphic family of cell surface receptors expressed on natural killer (NK) cells and a subset of T-lymphocytes [1–3]. KIR mediated signaling plays a key role in the identification of foreign cells and the antiviral response [4–9]. The best characterized KIR ligands are major histocompatibility complex class I (MHC-I) molecules, although ligands have not been identified for all KIRs [10, 11]. Because both KIRs and MHC-I are highly polymorphic, host genotype plays an important role in KIR function.
KIR genetic diversity can be described in terms of polymorphism and polygenicity. To date, there are 15 KIR genes described in humans . The number of KIR genes varies between individuals, with 7-12 genes per haplotype [13, 14]. Because the protein product of each KIR gene generally binds a unique set of ligands, the subset of KIRs encoded by an individual dictates the potential KIR interactions that can occur. In addition to variation in gene content between haplotypes, there is allelic polymorphism within each KIR gene . Broadly speaking, the allotypic variants encoded by a KIR gene bind the same subset of MHC-I ligands, although exceptions do exist . Distinct KIR allotypes can have differing binding affinities for particular MHC-I allotypes. These differences in KIR/MHC-I binding affinity can alter KIR signaling and NK cell activity . In addition to KIR genotype, MHC-I genotype must be considered since it determines the set of available KIR ligands and since it is possible to express a KIR with or without its cognate MHC-I molecule [10, 17].
Specific KIR/MHC-I genotypes have been implicated as a factor contributing to the immune control of multiple human diseases including hepatitis C virus, human papilloma virus, malaria, and human immunodeficiency virus (HIV) [6–9, 18]. One of the best-studied examples of KIR/MHC-I genetics and disease is that of KIR3DL1/KIR3DS1 and HLA-Bw4 in HIV infection. Individuals who express specific KIR3DL1/KIR3DS1 alleles in combination with certain HLA-B alleles containing the Bw4 motif show slower progression to AIDS [8, 9]. This genetic association has more recently been supported by functional data demonstrating that NK cells expressing KIR3DS1 have increased anti-HIV activity against target cells expressing HLA-Bw4, although the underlying mechanism remains to be elucidated .
Despite advances in our understanding of KIR biology, the mechanisms through which specific KIR/MHC-I combinations influence disease progression are not fully understood. This is at least partially due to the complexity of KIR/MHC-I genotypes and difficulty in identifying KIR/MHC-I matched cohorts. Rhesus macaques (Macaca mulatta) are an established and widely used experimental model system for many human diseases, including immunodeficiency virus . The advantages of studying infectious disease in rhesus macaques include the ability to manipulate the dose, route, and strain of the infectious agent, as well as the ability to analyze specimens from defined time points. For the study of KIR activity, perhaps the most important advantage is the ability to select subjects based on genetics. This benefit is evidenced by the work in macaques to elucidate the role of the cytotoxic T-lymphocyte (CTL) response in immunodeficiency viral infection, which is also heavily dependent on host genetics [21, 22].
Macaque KIRs have received less study than human KIRs. While previous work shows that macaque KIRs have structure and genomic organization similar to human KIRs, and suggests that they play a similar functional role, these studies also demonstrate that there has been considerable evolution within macaque KIRs since the species diverged . While 15 genes have been described in humans, the number and identity of the KIR genes present in macaques is distinct. Developing an understanding of the KIR genes present in this species and an overall assessment of KIR genetic diversity is a matter of practical importance for the use of macaques as a model for KIR function. Using cDNA sequences, an initial model for macaque KIR genetic organization was formed containing 18 putative KIR groups [23, 24]. In addition to the sequence of KIR transcripts, the genomic sequence of one rhesus macaque KIR haplotype has been described . More recent studies have added to the total number of described macaque KIR sequences [23, 24, 26–28]. With more sequence data available, phylogenetic relationships became clearer, and the model of macaque KIR genetics has been refined. This body of work has been used to create a model for macaque KIR genetic organization and to develop a formal system of nomenclature (Guethlein et al, in preparation).
Here we present the results of a survey of rhesus macaque KIR genetics. Using full-length cloning, we identified 47 novel full-length rhesus macaque KIRs, substantially increasing the library of known sequences. Using this expanded library, we performed phylogenetic analysis supporting the existence of 22 rhesus macaque KIR genes. Together with previously published KIR sequences, this provides a framework with which to describe KIR genetics in this species. In addition to improving our understanding of macaque KIRs at the population level, we developed a novel pyrosequencing-based approach for KIR genotyping. This technique provides both comprehensive KIR genotyping and frequency estimates for expression of each KIR transcript. The findings presented here, along with the novel techniques set forth, should serve as a foundation for further research on rhesus KIR genetics and for defining KIR function in this important animal model.
Animals used in this study were housed and cared for by the trained veterinary staff at the Wisconsin National Primate Research Center (WNPRC) or the New England Primate Research Center (NEPRC). All procedures were approved by the host institution's Animal Care and Use Committee. Nucleic acid was obtained from peripheral blood mononuclear cells (PBMC) or purified natural killer (NK) cells, as indicated. RNA purification was accomplished using either the MagnaPure LC Total Nucleic Acid Purification kit (Roche, Branford, CT) or the DNA/RNA Allprep Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions.
NK cells were isolated from whole PBMC by negative magnetic bead fractionation. First, PBMC were incubated for 20 minutes at room temperature in 0.1% BSA/PBS with a cocktail of cross-reactive human monoclonal IgG antibodies composed of the following: anti-CD3 (clone SP34-2, BD Biosciences, La Jolla, CA), anti-CD14 (clone M5E2, BD Biosciences), anti-CD40 (clone 5C3, BD Biosciences), and anti-CD66/CEACAM (clone TET2, AbCam, Cambridge, MA or Santa Cruz Biotechnology, Santa Cruz, CA). Next, antibody-coated PBMC were washed and resuspended in 0.1% BSA/PBS then incubated for 35 minutes at room temperature with Pan-IgG Dynabeads (Dynal Biotech, Norway) at a 4:1 bead-to-cell ratio. The suspension was then placed on a Dynal magnet and the unbound cells were collected. Purity was assessed by flow cytometry with >90% of collected cells routinely bearing an NK cell phenotype of NKG2A+CD8+CD3-. All acquisitions were made on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR).
First-strand cDNA was synthesized using the Superscript III First-Strand One-Step RT-PCR kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. PCR amplification was performed using Phusion high-fidelity polymerase (New England Biolabs, Ipswich, MA) and the following external primers: 5'-CAGCACCATGTCGCTCAT-3' and 5'-GGGGTCAAGTGAAGTGGAGA-3'. PCR conditions were: 98°C for 30 s, 28 cycles of 98°C for 5 s, 63°C for 1 s, 72°C for 20 s, and a final extension at 72°C for 5 min. PCR products were cloned into pCR-Blunt TOPO (Invitrogen, Carlsbad, CA) and bidirectionally sequenced using DYEnamic ET Terminator cycle sequencing kit (GE Healthcare, Piscataway, NJ). Internal primers used in sequencing were 5'-AACCTTCCCTCCTGGCC-3 and 5'-TTGGTTCAGTGGGTGAAGGCCAA-3.' CodonCode Aligner (CodonCode Corporation, Dedham, MA) was used for sequence analysis, and in order to minimize error introduced by PCR artifacts, novel alleles were only included when three or more identical full-length cDNA clones were observed. Novel sequences have been deposited in Genbank (Additional file 1, Table S1). Novel full-length sequences were assigned formal names through the Immuno Polymorphism Database .
The cDNA sequences obtained in this analysis and from previous studies [23, 25, 27, 28, 30] were aligned using Clustal X  and manually corrected in BioEdit (Ibis Therapeutics, Carlsbad, CA). Phylogenetic trees for the complete dataset were made using both neighbor-joining (1000 replicates, pairwise deletion, Tamura-Nei, in MEGA4) and parsimony (1000 replicates in Paup). These trees were used to divide the sequences into Mamu-KIR3DL20, -KIR2DL04, -KIR1D, and -KIR3D (lineage II) groups. Subsequent analysis was restricted to the lineage II dataset using the same methods. Alleles of Mamu-KIR3DL11, -KIR3DS01, -KIR3DS03, -KIR3DSW07, and -KIR3DSW08 were not obtained in this study and sequences for these genes were taken from GenBank [23, 27, 30, 32].
A dataset containing consensus sequences for each of the lineage II KIR genes was constructed using all known sequences to compute the consensus sequence. For groups containing only two allotypes, one of the allotypes was chosen to represent the consensus. The sequences used are indicated in Additional file 1, Figure S1. The individual extracellular Ig domains (D0, D1, and D2) were analyzed separately using the methods described above. The stem, transmembrane and cytoplasmic tails were not included in this analysis as previous reports have shown that the stem, transmembrane, and tail of macaque activating KIRs share similarity to KIR2DL4 and are distinct .
As with our full-length products, cDNA was synthesized using the Superscript III First-Strand One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). cDNA-PCR amplicons spanning 623 base pairs of the D1 and D2 domains were synthesized using Phusion high-fidelity polymerase (New England Biolabs, Ipswich, MA). Each PCR primer contained a target-specific sequence, an MID tag, and an adapter sequence (Additional file 1, Table S2). PCR conditions were: 98°C for 30 s, 28-33 cycles of 98°C for 5 s, 61°C for 1 s, 72°C for 20 s, and a final extension at 72°C for 5 min. cDNA-PCR product purification was accomplished using Ampure XP beads (Agencourt, Beverly, MA) according to the manufacturer's instructions. Amplicons were then normalized to equimolar concentrations and grouped into pools of twelve samples for Titanium amplicon pyrosequencing. Emulsion PCR, Roche/454 Titanium amplicon pyrosequencing, image processing, and base calling were performed according to the manufacturer's instructions (Roche/454 Life Sciences, Branford, CT) at the University of Illinois at Urbana-Champaign High-Throughput Sequencing Center. Each pool of twelve samples was sequenced in one-sixteenth of a 70 × 75 PicoTiterPlate.
Pyrosequencing flowgram data was processed using a custom analysis pipeline. Briefly, data were trimmed by sequence quality and aligned against a reference database of all known macaque KIR sequences using the Mosaik aligner (http://code.google.com/p/mosaik-aligner/). The reference library of KIR sequences was obtained from the Immuno Polymorphism Database . Polymorphisms between reads and the reference sequences were scored with custom scripts that utilized Samtools and BioPerl [33, 34]. The source code for this pipeline can be obtained from a subversion repository (https://hedgehog.fhcrc.org/tor/stedi/trunk/server/customModules/SequenceAnalysis). The pipeline itself has been integrated into the LabKey Software platform as the SequenceAnalysis module, which provides a graphical, web-based platform to initiate analysis pipelines and view results. LabKey is a free, open source software package available at http://www.labkey.org. Sanger sequence data was analyzed using CodonCode Aligner (CodonCode Corporation, Dedham, MA).
In order to reduce errors introduced by PCR artifacts, KIR sequences were only considered to be present in an animal if they represented one percent or more of total KIR sequence reads from that animal. In order to identify novel alleles not present in the reference library, unaligned sequences were then assembled in CodonCode Aligner at 100% identity. BLAST analysis was performed for the resulting contigs against a database of published Mamu-KIR sequences. Unaligned sequences were deemed novel when they represented at least one percent of total sequence reads from at least one animal and did not represent a potential insertion/deletion error in the pyrosequencing base-calling.
KIR genotype has a significant influence on NK cell activity. Studying the role of macaque KIR genetics in disease pathogenesis first requires an understanding of total diversity within the population and a framework within which to describe it. To date, 149 distinct full-length Mamu-KIR sequences have been deposited in Genbank. By comparison, there are more than 615 distinct KIR alleles identified in humans--more than four times as many alleles as have been identified to date in rhesus macaques . Thus, the previously catalogued alleles likely represent only a fraction of total Mamu-KIR diversity. In order to expand the repertoire of known Mamu-KIR alleles, we performed full-length cloning and sequencing of KIR alleles from 17 Indian rhesus macaques. These animals yielded 47 novel KIR alleles, a substantial expansion of documented rhesus macaque KIR diversity (Additional file 1, Table S1). The novel sequences have been assigned names following the KIR genes and nomenclature conventions established in the Non-human Primate KIR Nomenclature Report (Guethlein et al, in preparation)1.
As a first step in the analysis of the rhesus macaque sequences recovered from cloning, phylogenetic trees based on the full-length sequences were constructed. Human KIRs have been divided by phylogenetic analyses into four lineages2. Macaque KIRs assorted into groups matching these lineages. The sequences assorted into Mamu-KIR2DL04 (lineage I), -KIR3D (lineage II), -KIR1D (lineage III), and -KIR3DL20 (lineage V), with the majority of sequences resembling human lineage II, in accordance with previous publications [25, 30, 36]. Relatively little allelic variation was observed within the Mamu-KIR2DL04, -KIR3DL20, and -KIR1D groups (data not shown). In contrast, the Mamu-KIR3D lineage II sequences were highly diverse. Our sequences assorted into 19 distinct groups corresponding to 19 of the rhesus macaque lineage II genes (Figure 1 and Additional file 1, Figure S1). An important point to note is that rather than increasing the number of detected KIR genes, most novel sequences share strong similarity with established macaque KIR genes. Therefore, while there are likely a large number of undiscovered macaque KIR alleles, the most common macaque KIR genes may now have been identified.
Rhesus macaque lineage II KIR alleles form 19 distinct groups. Neighbor-joining and parsimony analysis was performed using predicted amino-acid sequences for the dataset comprising the lineage II sequences obtained in this study. The neighbor-joining tree is shown, which was not significantly different from the parsimony tree [data not shown]. Bootstrap support of greater than 50% is indicated. Representative hominoid lineage II KIR were used as an outgroup. For each gene, the numbers in parentheses indicate the total number of alleles discovered in this study compared with the total number known.
There is evidence for extensive recombination among the Mamu-KIR sequences described here. To identify recombination events affecting individual alleles, sequences were analyzed with the RDP (recombinant detection program) package and inspected manually (data not shown). In addition, domain-by-domain phylogenetic analysis using consensus sequences for each gene revealed extensive domain sharing between macaque KIR genes (Figure 2). This suggests that the genes are themselves products of ancient duplication and recombination events. This mechanism for the generation of KIR genes is consistent with observations of KIRs in other species . Pairs of KIRs with similar extracellular domains but differing cytoplasmic tails have been observed in other species, with human KIR3DL1/3DS1 being a notable example . In this analysis we also found such pairing, with the best-matched pair being Mamu-KIR3DLW03 and Mamu-KIR3DS05. Other pairs were identified that were similar in two of the three extracellular Ig-like domains, for example Mamu-KIR3DL07 and Mamu-KIR3DSW09. Cytoplasmic domains were not included in this analysis, although it should be noted that macaque long and short cytoplasmic tails are phylogenetically distinct, with the latter resembling the tail of human KIR2DL4 .
Domain shuffling has acted to form rhesus macaque KIR genes. The results of a domain-by-domain phylogenetic analysis are shown schematically. Predicted amino-acid consensus sequences for each of the genes were used to form both neighbor-joining and parsimony trees as described in the Methods section. The sequences encoding the three extracellular Ig-like domains were analyzed separately. Boxes are colored for the D0, D1, and D2 domains where the grouping was supported by >50% bootstrap support in the phylogenetic analyses. Boxes that are not completely colored represent cases where support of >50% was only found in the neighbor-joining analysis. Long cytoplasmic tails are colored red and short tails are colored green. The domains colored white were not resolved into any group in the analysis and should not be interpreted as being closely related to each other. The stem, transmembrane and cytoplasmic tails were grouped as either long or short by inspection of the alignment. D0, D1, and D2 denote the Ig-like domains. ST/TM/CYT denotes the stem, transmembrane, and cytoplasmic tail domains.
The expanded library of macaque KIR sequences described in this publication and others has enabled the creation of a framework to describe macaque KIRs, and its phylogenetic analysis suggests that the most common KIR genes have been identified [24–28, 30, 32]. For the study of KIRs in disease pathogenesis, one of the most basic requirements is the ability to identify the KIRs expressed by individual subjects. KIRs pose many challenges for genotyping: each subject expresses a distinct number of KIRs, there is extensive sequence homology between KIR transcripts, splice variants may be functionally significant, and pseudogenes are common. The cloning-based strategy we employed for allele discovery can be used to provide genotypes of individual subjects; however, this method is labor intensive and will frequently miss low-abundance transcripts due to the limited number of clones examined. Techniques such as sequence-specific PCR (SSP) can be used to identify the presence or absence of a particular gene or allele; however, sequence homology between KIRs complicates primer design, and a large number of primer pairs would be required for comprehensive genotyping.
To overcome these limitations, we developed a novel sequence-based typing approach. Recent developments in Roche/454 Titanium pyrosequencing technology have enabled sequencing of cDNA-PCR amplicons with high sensitivity and throughput. To adapt this approach for KIR genotyping, we first identified primer sites that are highly conserved across all published Mamu-KIR3D sequences (Figure 3). These primers amplify a region of 623 bp spanning the majority of D1, all of D2, and part of the stem region. This amplicon was selected to span a polymorphic region of the transcript and to maximize conservation of primers. These primer sites are also conserved amongst most published Mamu-KIR1D alleles--although they will not amplify Mamu-KIR2DL04 sequences, as these lack the D1 region and therefore the 5' primer-binding site. These primers were used for PCR on cDNA derived from total RNA, producing amplicons representing the distinct Mamu-KIR3D and -KIR1D transcripts expressed by each subject. Pyrosequencing of these amplicons produces clonal sequence reads corresponding to individual input transcripts. Collectively, these reads represent the KIRs expressed by the subject, with the exception of Mamu-KIR2DL04. A schematic of this process is shown in Figure 4.
KIR 454 Titanium Amplicon Primer Design. A) Schematic representation of macaque KIR3D and KIR2D molecules showing domain structure. B) Variability plot for a cDNA sequence alignment of all published KIR3DL alleles. PCR primer sites are indicated along with the region amplified by PCR. Note that 454 sequencing reads span the D1, D2, and stem (ST) regions. The signal sequence (SS), D0, transmembrane region (T), and cytoplasmic region (C) are not amplified by our PCR primers.
Outline of KIR genotyping strategy. The input material is isolated RNA, which will represent all KIRs transcribed by the subject. PCR is performed using conserved KIR-specific primers that add adapter tags. The PCR products are pyrosequenced, producing 1000s of reads, with each read representing a single input molecule. These reads are compared against a reference database of macaque KIR sequences. This analysis produces a list of all KIRs detected per subject, including the relative frequency of each KIR.
Using this approach, we genotyped PBMC samples from 61 animals. We detected an average of 1836 total sequence reads per animal, representing an average of 10.7 distinct transcripts per animal. The KIR genotypes of three half-siblings are shown in Figure 5. For each animal, the distinct KIRs detected are shown, along with the relative frequency of each KIR. In most cases, each sequence read unambiguously matched a single KIR allele, providing typing resolution to the allele level. When reads were identical to more than one known KIR, the result is presented either as a set of alleles (ie. Mamu-KIR3DL10*001/004, meaning the animal is positive for either Mamu-KIR3DL10*001 or *004) or as positive for a gene (ie. Mamu-KIR1Dg, meaning the animal has an allele of this gene). The latter is comparable to the level of resolution commonly provided by sequence specific PCR. To validate this technique, we compared the results of pyrosequencing with cloning data (Figure 5 and Additional file 1, Table S3). Because pyrosequencing examines hundreds or thousands of clones, as opposed to the tens of clones generally examined in conventional cloning, the former is able to detect more transcripts per animal, providing a more comprehensive genotype. An example is seen in r95061 with Mamu-KIR3DS06, which was detected by pyrosequencing, but was not detected by cloning. While the 623bp amplicon used in pyrosequencing was frequently able to provide allele-level typing, there are examples for which full-length cloning provided higher resolution typing, as we observe for KIR3DL01*001 and KIR3DL10*004 in r95061 (Figure 5).
Differential KIR expression between subjects with a common KIR haplotype. A) KIR genotypes obtained by pyrosequencing are shown for three rhesus macaque half-siblings. The relative expression level of each detected KIR allele is shown. Striped bars indicate alleles present on their shared KIR haplotype. The number of pyrosequencing reads is shown to the left of each graph. B) For r95061, the KIR alleles detected by cDNA cloning are shown. A total of 101 clones were examined. If indicated, the allele detected is listed. A plus sign indicates the genotyping resolution obtained by cloning is identical to the resolution obtained by pyrosequencing.
A key advantage of pyrosequencing is that it provides estimates of the relative transcript frequency for each KIR. This is a dimension not usually captured by genotyping techniques. The three animals in Figure 5 share a KIR haplotype containing Mamu-KIR1D, Mamu-KIR3DL1*001, Mamu-KIR3DL10*004 and Mamu-KIR3DS04*002 (Figure 5, striped bars). KIR expression is influenced by the complete KIR and MHC genotype of the subject [10, 17, 39]. Work in humans has shown that KIR genotype is only loosely predictive of KIR RNA expression . While the animals in Figure 5 share one KIR haplotype, their second KIR haplotypes are distinct, and none are MHC identical (data not shown). We observe considerable differences in the expression level of the KIRs on the shared haplotype, which is likely attributable to differences in KIR/MHC genotype. While these findings are not unexpected given the complex regulation of KIR expression, they do underscore the need to examine factors beyond the simple presence or absence of a given KIR.
To validate this technique, we examined the reproducibility of KIR expression levels. We performed two independent NK cell preparations from four animals, and two independent PCRs from each cell preparation. The resulting PCR amplicons were pyrosequenced. A graph of the relative expression level of each KIR is shown in Figure 6, with the full data in Additional file 1, Table S4. The frequency of each KIR was highly reproducible between sample preparations and PCR replicates. In every instance for which the average expression of a KIR was greater than 3% of total reads, that KIR transcript was detected in all replicates, demonstrating high sensitivity. It should be noted that transcripts present in less than 3% of total transcripts were missed in some reactions. There was also greater variability in detected expression levels among lower frequency transcripts. Of interest, in three of four animals, a single KIR transcript was dominant, representing greater than 60% of reads. Animal 225-97 was an exception, where four distinct KIRs comprise between 15-20% of the population each.
Pyrosequencing produces reproducible frequency estimates for KIR transcripts. Pyrosequencing results from four animals are shown. Per animal, two independent cell preparations were performed, and two independent PCRs were performed per cell preparation. For each replicate, the expression of each KIR is graphed as a percentage of total reads. Bars represent the average and standard deviation among replicates. For animal 225-97 (asterisk), only one cell pellet was available, so only two data points are shown.
Another advantage of sequence-based typing, as opposed to techniques such as sequence-specific PCR, is that it is not limited to previously described alleles. From this data we were able to characterize an additional 44 novel partial-length rhesus macaque KIR sequences, 32 of which were found in multiple animals. As was the case with our novel full-length sequences, most novel KIR sequences showed significant sequence homology to the phylogenetic groups defined in Figure 1 (Additional file 1, Table S5), further supporting these putative gene groupings. While the majority of novel sequences were similar to these groups, we also identified two novel sequences, KIRnov03 and KIRnov04, that are similar to Mamu-KIR3DS03, but with a distinctive sequence motif in D1 (Figure 7). These sequences are of interest because they share this motif with a sequence previously found only in cynomolgus macaques (Macaca fascicularis) (EU419113). The unique residues for these sequences (186-193) correspond to predicted MHC class I binding sites . These sequences may represent an additional macaque KIR gene, or a lineage within KIR3DS03.
Novel rhesus macaque KIR sequences share homology with cynomolgus macaque KIRs. Amino acid differences between the consensus sequences for the lineage II Mamu-KIR genes, KIRnov03, KIRnov04 and the Mafa-KIR sequence EU419113 are shown. The amino-acid position is indicated above the amino-acid sequences. The positions in the novel sequences that vary from the consensus are highlighted. Residues predicted to be involved in MHC-binding or alpha-helix loops are indicated below the sequences with B or L respectively. Black shading indicates residues for which no sequence coverage was available.
Obtaining comprehensive KIR genotypes from this large cohort allowed us to examine the relative frequency of each KIR gene at the population level (Figure 8A). We combined the pyrosequencing data with full-length cloning data to form a 69-animal cohort. While no KIR gene was present in every animal, Mamu-KIR3DL01 was present in approximately 84% of the cohort, making it the closest approximation to a framework gene in this population. While Mamu-KIR3DL20, -KIR3DL11, and -KIR3DSW08 have been proposed as framework genes for this species, each was present in less than 25% of this cohort . It should be noted that our approach will identify transcribed KIRs only, while Kruse et al. employed sequence-specific PCR from genomic DNA. Our previous work in cynomolgus macaques suggests that Mafa-KIR3DL20 is commonly found as a pseudogene , which would be detected from gDNA, but not mRNA. It is possible some rhesus macaque haplotypes also have Mamu-KIR3DL20 as a pseudogene. While differences in technique may explain some discrepancies, it is also possible that the distinct breeding populations of rhesus macaques have distinctive genetic compositions. Mamu- KIR2DL04 has also been suggested as a framework gene in macaques , although -KIR2DL04 was only identified in 2 of 8 published cynomolgus macaque haplotypes . As noted previously, the primers used in this study select against Mamu-KIR2DL04 transcripts, so we could not determine the presence or absence of this gene.
Frequency and relative expression of KIR genes in the rhesus macaque cohort. A) Y-axis indicates the percentage of animals within the cohort (n = 69) that express the indicated KIR gene. Genes not listed were not present in any animal within our cohort. Mamu-KIR2DL04 was excluded since it is not amplified by our pyrosequencing amplicon. B) Graph illustrates the percent of total pyrosequencing reads per animal (n = 61) for each KIR gene. Averages and SEM are represented by error bars. Each animal included had at least 100 sequencing reads. Genotyping results representing less than 1% of total reads in an animal were excluded to mitigate the influence of PCR artifacts. Ambiguous reads matching more than one KIR gene and splice variants were also excluded.
Using the pyrosequencing data generated from our cohort of 61 animals, we compared the relative contribution of each KIR gene to the total KIR transcripts identified in each animal (Figure 8B). Although pyrosequencing provides information about the KIR allele, genotyping results were condensed to the gene level for this comparison. The error bars representing the mean and SEM for each gene demonstrate highly variable expression levels for most genes between subjects, with a few KIRs consistently expressed at either high (i.e. Mamu-KIR3DL08) or low (i.e. Mamu-KIR3DSw09) levels. As observed in Figure 5, the expression level of a given KIR gene can differ even between genetically similar subjects and is likely influenced by the complete KIR/MHC genotype of that subject. While it would be interesting to examine the relative expression of each KIR in the context of MHC genotype, macaques can express up to 20 distinct MHC class I transcripts and only a handful of macaque KIR/MHC binding partners have been identified [42, 43]. Therefore this analysis is not practical until physiological macaque KIR/MHC interactions are better understood.
While not all members of this cohort had pedigree information available, there were five distinct pedigreed families within the cohort for which segregation analysis was possible, with a total of 46 animals. By performing segregation analysis using genotypes obtained from cloning and pyrosequencing data, we were able to infer the KIR gene content of 9 haplotypes (Figure 9). Because these haplotypes were defined using segregation analyses, the linear order of KIR genes cannot be conclusively determined and the physical map of gene order shown in Figure 9 is arbitrary. It should be noted that this analysis allows only for definition of the minimum number of genes on a given haplotype, as heterozygosity versus homozygosity is sometimes not possible to rigorously determine, and low-level transcripts may not be detected in all animals with a given haplotype . Among our 9 haplotypes, each was unique as compared to previously published rhesus macaque KIR haplotypes, with an average of 4.6 genes present per haplotype (Range: 3-7) [25, 27, 28, 30]. In accordance with previous studies, we noted one incidence of a duplicated KIR3DL gene (Mamu-KIR3DL10) [27, 30]. As described above, there is a notable absence of framework genes among these haplotypes. Mamu-KIR3DL01 represents the most common KIR in our cohort, yet it is present in only 7 of the 9 haplotypes.
Novel rhesus macaque KIR haplotypes. KIR genes are indicated along the top axis. The identity of the allele is indicated within the schematic boxes if it was determined. Because data were generated from cDNA expression, only expressed KIRs are shown, and the physical map of gene order is arbitrary. Brackets indicate gene duplication. Since Mamu-KIR2DL04 cannot be amplified by our pyrosequencing amplicon, dotted boxes indicate haplotypes in which a false negative typing for this locus is possible.
Killer immunoglobulin-like receptor signaling is implicated in the immune response to numerous human pathogens, yet elucidating the role of KIR mediated signaling in disease pathogenesis is difficult in human subjects. The results of this study advance our understanding of macaque KIRs, enabling the study of KIR activity in this important non-human primate model for many human diseases. This study identified 47 novel full-length rhesus macaque KIRs, a substantial increase in the number of published sequences. Combined with previously published data, our results confirm the existence of 22 common macaque KIR genes, with extensive allelic variation within each gene (Guethlein et al, in preparation). This model of macaque KIR genetics provides an essential framework within which to describe and characterize macaque KIRs. In addition, among the novel sequences found using pyrosequencing, two sequences provide evidence for the existence of either a divergent lineage of Mamu-KIR3DS03 or an additional gene. While no KIR gene was present in all subjects, Mamu-KIR3DL01 was expressed in 84% of subjects and 8 genes were present in >50% of the cohort. This frequency information may be useful to prioritize KIRs for further functional characterization or the design of cohorts. The comparative lack of expressed framework genes is a distinction from human KIR haplotypes, which may be related to differences in MHC-I genetics. Humans express a maximum of six distinct HLA molecules, from 3 genomic loci. Macaques lack MHC-I C, but can express as many as 20 distinct MHC-I A/B alleles . The expansion of macaque lineage II KIR3D loci, with a larger number of loci and few dominant genes, likely mirrors the expansion of macaque MHC-I A/B.
We also present a novel pyrosequencing-based approach for KIR genotyping. While this technique was developed in macaques, it could easily be applied to other species, including humans. The ability to characterize the KIR genotype of individual subjects is a basic requirement for the study of KIR function; however, the KIR region presents many challenges for genotyping. The approach we present comprehensively identifies the expressed KIR transcripts and provides a semi-quantitative measure of the relative expression level for each KIR. Our data demonstrate that even subjects sharing a KIR haplotype can have widely different expression levels of these shared KIRs. NK cells undergo complex regulation of KIR expression [10, 17]. NK clones expressing fewer distinct KIRs can have a reduced activation threshold under certain conditions, resulting in enhanced NK cell activity . The relative expression of each KIR therefore provides potentially important functional information.
While this and other recent studies have advanced our understanding of the genetic organization and diversity of macaque KIRs, KIR activity must be understood in the context of MHC genotype. The limited knowledge of functional KIR/MHC binding partners remains a key obstacle. Recent work has identified multiple macaque KIR/MHC-I interactions, including allotypes of Mamu-KIR3DL05, -KIR3DLW03, -KIR3DL11 and -KIR3DS05 [43, 46]. Preliminary evidence suggests the residues of the Bw4/Bw6 motifs influence KIR/MHC binding in macaques, although the important residues differ from those in humans. Expanding the number of identified KIR/MHC partners and further defining the motifs necessary for macaque KIR/MHC interaction remains essential for the advancement of the field.
The data and techniques presented here have implications for the study of many diseases using rhesus macaques as a model system and for the KIR field as a whole. The ability to study KIRs in a nonhuman primate model creates an opportunity for significant advancement in our understanding of KIRs in human disease. The novel high-throughput KIR genotyping method we developed has implications for many species, including potential application in humans, and the approach could be adapted for other polymorphic immune loci.
1 A note on nomenclature: KIR nomenclature reflects both the domain structure and gene designation of the sequence. For example, an allele named "KIR3DL01*001" denotes a KIR with 3 extracellular immunoglobulin-like domains (3D). The "L" indicates that this allele's protein product has a long, inhibitory cytoplasmic tail, while an "S" would instead indicate that the protein product had a short, activating cytoplasmic tail. The next two digits refer to the gene, in this case KIR3DL01. Finally, the digits after the asterisk, "001" in this example, refer to the specific allele.
2 Human KIRs are divided into four lineages. Lineage I includes KIR2D genes having a D0 (immunoglobulin-like domain 0) plus D2 configuration such as KIR2DL4 and KIR2DL5; lineage II includes the KIR3DL genes that bind MHC-A and -B epitopes; lineage III includes KIR3D or KIR2D genes with a D1 plus D2 domain configuration that bind MHC-C epitopes, and lineage V is represented by KIR3DL3. Lineage IV, which included Mamu-KIR3DL01 and Mamu-KIR3DL10, was originally described in Rajalingam, et al. . Further work has instead grouped these KIRs into lineage II and removed lineage IV .
We thank Roger Wiseman and Simon Lank for advising on technical aspects of this study and for thoughtful comments on this manuscript. We also thank Chris Wright and the staff of the University of Illinois High Throughput Sequencing Center. Finally, we thank Jon Warren and the National Institute of Allergy and Infectious Diseases (NIAID), a component of the National Institutes of Health (NIH), for support. The contents of this manuscript are solely attributed to the authors and do not necessarily represent the official views of the research sponsors. This publication was made possible in part by grants P51 RR000167 and P51 RR000168 from the National Center for Research Resources (NCRR), a component of the NIH, to the Wisconsin National Primate Research Center and the New England Primate Research Center, respectively along with R24 RR021745 to D.H.O., AI071306 to R.P.J. and AI24258 to PP. Development of KIR pyrosequencing was also supported by the NIH/NIAID Reagent Resource Support Program for AIDS Vaccine Development, Quality Biological, Inc., Gaithersburg, MD. The project was also supported by CHAVI/HVTN Early Career Investigator award, grant number U19 AI 067854-04, to R.K.R.
AJM was involved in data collection, data analysis and writing the manuscript. LAG and PP performed data analysis and assisted with manuscript writing. RKR performed NK cell sample preparations and was involved in the experimental design. KWB performed statistical analysis of pyrosequencing data. RPJ and DHO provided grant support and were involved in the experimental design. BNB was involved in experimental design, data collection, data analysis and manuscript writing. All authors have read and approve the final manuscript.


Social media such as Facebook and LinkedIn allows you to build awareness of your brand and interact with people likely interested in your products and services. Instead of reaching people as they search for your service social media creates brand awareness prior to the need effectively positioning your company to win new business.
Does your company rely on word of mouth referrals to grow your business? Social media done effectively is digital word of mouth advertising leading to more customers.


I decided to brew ginger beer the other weekend. For one, it's an easy way to debut in brewing (ginger beer has a negligible amount of alcohol — 0.5% — which makes it easier to brew) and establishing one's presence in the kitchen in general. Ginger beer also happens to be one of my favorite drinks (if you think you've tried ginger beer before, try Fentimans Ginger Beer and prepare to be gobsmacked).
I found very helpful recipes online and adapted them slightly to make the beer taste a little more like Fentiman's: ensure a distinctive "kick" of ginger with hints of juniper. I did two batches — I wanted to try the first, then improve my process for the second one. The first batch took forever and the beer was too lemonade-y and not ginger-y enough. I improve my recipe and the second batch turned out to be a success.
I bought some fresh ginger root, peeled it and juiced it with a garlic press, chunk by chunk (each chunk was big enough to fit in the press).
I brought the water to a boil, added the ginger juice, the sugar, the lemon juice, cream of tartar and the juniper berries. I let the water boil for 10 minutes.
At the same time I reconstructed the yeast — I put it in a cup of lukewarm water with a teaspoon of sugar and let sit for 10 minutes.
I let the compote cool down, added yeast and poured into a large bottle (I used two large orange juice bottles). I covered the bottles with cloth and let sit for 16 hours, undisturbed in a dark, warm place.
Finally I poured the mixture into bottles (I used those flip-top bottles, they are great), sealed the bottles and let them sit in a dark, warm place for 36 hours.
I was very pleased with the result — the beer had the kick I was looking for, and the lemon wasn't overpowering. Mind you, I was probably biased since I wanted the beer to work out, but overall it was a fun experiment. I think what would make it even better would be to use actual ginger beer plant.


text:
Q: Retrieve a ListView from Firebase I want to get data from a Firebase database in a list view. When I open the page, it appears as follows:

When I go back and open it again, it now appears like this:

The database contains the following data:

My two problems are:

*

*It takes a lot of time to retrieve the data

*More data is retrieved than expected - it is repeated in the listview.

My code:
public class CustomAdapter extends BaseAdapter{
Context c;
ArrayList<Doctor> doctors;

public CustomAdapter(Context c, ArrayList<Doctor> doctors) {
    this.c = c;
    this.doctors = doctors;
}

@Override
public int getCount() {
    return doctors.size();
}

@Override
public Object getItem(int position) {
    return doctors.get(position);
}

@Override
public long getItemId(int position) {
    return position;
}

@Override
public View getView(int position, View convertView, ViewGroup parent) {
    if(convertView==null)
    {
        convertView= LayoutInflater.from(c).inflate(R.layout.model,parent,false);
    }

    TextView nameTxt= (TextView) convertView.findViewById(R.id.nameTxt);
    TextView propTxt= (TextView) convertView.findViewById(R.id.propellantTxt);
    TextView descTxt= (TextView) convertView.findViewById(R.id.descTxt);

    final Doctor s= (Doctor) this.getItem(position);

    nameTxt.setText(s.getDoctorName());
    propTxt.setText(s.getSpecialist());
    descTxt.setText(s.getAdress());

    //ONITECLICK
    convertView.setOnClickListener(new View.OnClickListener() {
        @Override
        public void onClick(View v) {
            Toast.makeText(c,s.getDoctorName(),Toast.LENGTH_SHORT).show();
        }
    });
    return convertView;
}

and this for model class
public class Doctor {
String doctorName,specialist,adress;

public Doctor() {
}

public String getDoctorName() {
    return doctorName;
}

public String getSpecialist() {
    return specialist;
}

public String getAdress() {
    return adress;
}

The Activity:
public class DoctorsActivity extends AppCompatActivity {
DatabaseReference db;
FirebaseHelper helper;
CustomAdapter adapter;
ListView lv;
@Override
protected void onCreate(Bundle savedInstanceState) {
    super.onCreate(savedInstanceState);
    setContentView(R.layout.activity_doctors);
    lv = (ListView) findViewById(R.id.lv);
    db = FirebaseDatabase.getInstance().getReference().child("Doctor");
    helper = new FirebaseHelper(db);

    adapter = new CustomAdapter(this, helper.retrieve());
    lv.setAdapter(adapter);

}

Firebase Helper:
public class FirebaseHelper {

DatabaseReference db;
Boolean saved;
ArrayList<Doctor> doctors = new ArrayList<>();

public FirebaseHelper(DatabaseReference db) {
    this.db = db;
}

//IMPLEMENT FETCH DATA AND FILL ARRAYLIST
private void fetchData(DataSnapshot dataSnapshot) {
    doctors.clear();

    for (DataSnapshot ds : dataSnapshot.getChildren()) {
        Doctor doctor = dataSnapshot.getValue(Doctor.class);
        doctors.add(doctor);
    }
}

//RETRIEVE
public ArrayList<Doctor> retrieve() {
    db.addChildEventListener(new ChildEventListener() {
        @Override
        public void onChildAdded(DataSnapshot dataSnapshot, String s) {
            fetchData(dataSnapshot);
        }

        @Override
        public void onChildChanged(DataSnapshot dataSnapshot, String s) {
            fetchData(dataSnapshot);

        }

        @Override
        public void onChildRemoved(DataSnapshot dataSnapshot) {

        }

        @Override
        public void onChildMoved(DataSnapshot dataSnapshot, String s) {

        }

        @Override
        public void onCancelled(DatabaseError databaseError) {

        }
    });

    return doctors;
}

redpajama_set_name: RedPajamaStackExchange


Heat the olive oil in a sauté pan placed over a medium heat. Season the chicken thighs with salt and pepper and add them to the pan. Cook until browned all over. Remove the chicken and set aside.
Add the onions, garlic and peppers to the pan and gently fry for 10 minutes. Stir in the paprikas and flour. Cook, stirring all the time, for a few minutes. Stir in the tomatoes and stock, then return the chicken thighs to the pan. Simmer gently for 30–40 minutes, or until the chicken is cooked through.
Meanwhile, to make the dumplings, mix together the flour, suet, herbs and seasoning in a bowl. Add 100ml/3½fl oz water and mix to form a sticky dough.
Roll the dough into small balls and drop into a shallow baking dish. Pour over the hot chicken stock and bake for 30 minutes.
When ready to serve, stir the soured cream into the chicken and serve with the dumplings.


While on vacation with your vehicle or after relocating to the State of New York, you will have to worry about winter parking in New York as alternate side parking and street cleaning regulations will be suspended during a snow emergency making spots harder to find as these will be taken up by locals who prepared for storms and are familiar on where to park.
As for winter parking, you will need to avoid parking at all times on any of the 250 snow emergency routes in New York as if a snow storm hits the New York area by surprise, you may discover your vehicle later on with a parking ticket or two for failing to move it in time and will have to waste precious time later on to find parking at alternate areas along with making time to appear in court if you plan to fight the tickets at Finance Business Centers. In some instances, your vehicle will qualify for the city's valet service and will be towed at your expense to one of four (4) in impound lots in New York City.
By knowing and following the rules, you can help reduce black ice & manhole fires in New York by helping the city to be able to clear the streets quickly and safely.
3 Ave. 3 Ave. Bridge – 161 St.
3 Ave. East 163rd St. – East 168th St.
Baychester Ave. Co-op City Blvd. – Bartow Ave.
Broadway City Line – W. 230 St.
Bruckner Blvd. N & S Service. Rds Bronx River Ave. – Zerega Ave.
Bruckner Blvd. Whitlock Ave. – Lincoln Ave.
Burr Ave. Pelham Pkwy South – Westchester Ave.
Co-op City Blvd. Baychester Ave. – Bartow Ave.
Concourse Village E E. 156 St. – E. 161 St.
Crosby Ave. Bruckner Blvd. – Westchester Ave.
Cross Bx Expwy N & S Service.Rds. 3 Ave. – Park Ave.
Cross Bx Expwy N & S Service.Rds. Castle Hill Ave. – Westchester Ave.
E 138 St. Major Deegan Exp. – Bruckner Blvd.
E 149 St. Exterior St. – Bruckner Blvd.
E 161 St. Jerome Ave. – 3 Ave.
E 167 St. Jerome Ave. – Webster Ave.
E 175 St. Grand Concourse – Jerome Ave.
E L Grant Highway Washington Pl. – Jerome Ave.
E/W Tremont Ave. Andrews Ave. – Schurz Ave.
Henry Hudson Pkwy (E/S Rd) Kappock St. – W 254 St.
Henry Hudson Pkwy (W/S Rd) W 254 St. – Kappock St.
Hunts Point Ave. Southern Blvd. – Oak Point Ave.
Hutchinson River Pkwy E Palmer Ave. – Bartow Ave.
Jerome Ave. Sedgwick Ave. – E 233 St.
Johnson Ave. Riverdale Ave. – Kappock St.
Kappock St. Johnson Ave. – Kappock St.
Laconia Ave. E 224 St. – E 225 St.
Melrose Ave. E 149 St. – Webster Ave.
Metcalf Ave. Westchester Ave. – Watson Ave.
Pelham Pkwy N & S White Plains Rd – Stillwell Ave.
Pelham Pkwy S Hutchinson River Pkwy – Burr Ave.
Riverdale Ave. Johnson Ave. – W 239 St.
Sedgwick Ave. Jerome Ave. – West 167 St.
Soundview Ave. Bruckner Blvd. – Metcalf Ave.
Soundview Ave. Bruckner Blvd. – Cornell Ave.
Southern Blvd. Bruckner Blvd. – Westchester Ave.
Tremont Ave. Andrews Ave. – Schurz Ave.
Van Cortlandt Ave. S Broadway – Bailey Ave.
Van Cortlandt Ave. W Bailey Ave. – Sedgwick Ave.
Washington Ave. Fordham Rd – 161 St.
Willis Ave. Major Deegan Exp. – E 149 St.
3 Ave. Shore Rd – Livingston St.
4 Ave. Belt Pkwy – Flatbush Ave.
11 Ave. New Utrecht Ave. – 86 St.
18 Ave. 52 St. – Cropsey Ave.
Adams St. Atlantic Ave. – Tillary St.
Avenue L Flatbush Ave. – Nostrand Ave.
Avenue N McDonald Ave. – Coney Island Ave.
Avenue R Nostrand Ave. – E 36 St.
Avenue S Flatbush Ave. – E 36 St.
Avenue U 86 St. – E 74 St.
Bay 8 St. Belt Pkwy – Cropsey Ave.
Bedford Ave. Taylor St. – Rogers Ave.
Berry St. Broadway – Nassau Ave.
Borinquen Pl. Havemeyer St. – Rodney St.
Broadway Wythe Ave. – Flushing Ave.
Buffalo Ave. Eastern Pkwy – E New York Ave.
Bushwick Ave. Flushing Ave. – Jamaica Ave.
Cadman Plaza West Prospect St. – Joralemon St.
Church Ave. Coney Island Ave. – 37 St.
Conduit Blvd. Atlantic Ave. – Sutter Ave.
Court St. Atlantic Ave. – Hamilton Ave.
Cropsey Ave. 14 Ave. – W 17 St.
Dekalb Ave. Fulton St. – Bushwick Ave.
Driggs Ave. Broadway – Lorimer St.
Eastern Pkwy Grand Army Plaza – Atlantic Ave.
Eastern Pkwy Ext. Atlantic Ave. – Bushwick Ave.
Flatlands Ave. Flatbush Ave. – Pennsylvania Ave.
Flushing Ave. Navy St. – Cypress Ave.
Foster Ave. Kings Highway – Remsen Ave.
Foster Ave. Rockaway Pkwy – Bank St.
Franklin Ave. Wallabout Ave. – Washington Ave.
Fulton St. Flatbush Ave. – Jamaica Ave.
Glenwood Rd Avenue H – Ralph Ave.
Greenpoint Ave. McGuinness Blvd. – Kingsland Ave.
Hamilton Ave. Van Brunt St. – 3 Ave./18 St.
Jamaica Ave. Atlantic Ave. – Bushwick Ave.
Jay St. Atlantic Ave. – Sands St.
Kings Highway Bay Pkwy – Howard Ave.
Lafayette Ave. Flatbush Ave. – Bushwick Ave.
Lee Ave. Flushing Ave. – Taylor St.
Linden Blvd. Flatbush Ave. – Conduit Blvd.
McGuinness Blvd. Meeker Ave. – Freeman St.
Metropolitan Ave. Rodney St. – Onderdonk Ave.
Mill Ave. Ralph Ave. – Strickland Ave.
Myrtle Ave. Flatbush Ave. – Wyckoff Ave.
Nassau Ave. Berry St. – Varick St.
Nassau St. Navy St. – Flatbush Ave. Ext.
Nostrand Ave. Emmons Ave. – Flushing Ave.
Ocean Pkwy Parkside Ave. – Surf Ave.
Old Fulton St. Prospect St. – Furman St.
Parkside Ave. Flatbush Ave. – Coney Island Ave.
Remsen Ave. Linden Blvd. – Seaview Ave.
Rockaway Ave. Rockaway Pkwy – Atlantic Ave.
Rockaway Pkwy Belt Pkwy – Rockaway Ave.
Rodney St. Borinquen Pl. – Metropolitan Ave.
Rogers Ave. Bedford Ave. – Flatbush Ave.
Seaview Ave. Remsen Ave. – E 108 St.
Shore Pkwy N & S Service. Rds Ocean Ave. – Knapp St.
Stillwell Ave. Bay Pkwy – Surf Ave.
Strickland Ave. Mill Ave. – E 56 Dr.
Tillary St. Cadman Plaza West – Navy St.
Utica Ave. Fulton St. – Flatbush Ave.
Vanderbilt Ave. Plaza St. East – Atlantic Ave.
W 17 St. Cropsey Ave. – Surf Ave.
Washington Ave. Franklin Ave. – Flatbush Ave.
Wythe Ave. Broadway – Wallabout Ave.
1st Ave. Houston St. – E 125 St.
2nd Ave. Houston St. – E 128 St.
3rd Ave. Bowery – E 128 St.
5th Ave. 25 St. – 120 St.
7th Ave. Varick St. – W 14 St.
8th Ave. Hudson St. – W 14 St.
9th Ave. W 14 St. – W 59 St.
10th Ave. Nagle Ave. – W 207 St. W 14 St. – W59 St.
14th St. FDR Dr. – 11 Ave.
23rd St. FDR Dr. – 12 Ave.
34th St. FDR Dr. – 12 Ave.
42nd St. 1st Ave. – 12 Ave.
55th St. Sutton Pl. – 12 Ave.
56th St. Sutton Pl. – 12 Ave.
57th St. Sutton Pl. – 12 Ave.
59th St. 2nd Ave. – 5 Ave.
60th St. 2nd Ave. – 5 Ave.
86th St. York Ave.-5 Ave. Central Pk W-Riverside Dr.
120th St. 5 Ave. – Riverside Dr.
124th St. Mt. Morris Park W – 5 Ave.
125th St. 1st Ave. – Riverside Dr.
Allen St. Houston St. – Division St.
Amsterdam Ave. Ft. George Ave. – W 59 St.
Avenue A Houston St. – E 14th St.
Avenue B Houston St. – E 14th St.
Broadway W 181 St. – W 207 St.
Broadway Canal St. – E 14th St.
Broadway W 110th St. – W 155th St.
Broome St. Lafayette St. – Varick St.
Chambers St. West St. – Centre St.
Chrystie St. Houston St. – Canal St.
Clinton St. E. Broadway – Houston St.
Columbus Ave. Cathedral Parkway – W 59 St.
Delancey St. Bowery – FDR Dr.
E. Broadway Bowery – Clinton St.
Essex St. Houston St.- Canal St.
Ft. George Ave. Amsterdam Ave. – St. Nicholas Ave.
Frederick Douglass Blvd. W 145 St. – Macombs Pl.
Grand St. Varick St. – FDR Dr.
Hudson St. Chambers St. – 8 Ave.
Lafayette St. Kenmare St. – Broome St.
Lenox Ave. W 110 St. – W 145 St.
Macombs Pl. Frederick Douglass Blvd. – W 154 St.
Madison St. Pearl St. – Grand St.
Mt. Morris Park W 120 St. – 124 St.
Nagle Ave. Broadway – 10 Ave.
Pearl St. St. James Pl. – Fulton St.
Pike St. Division St. – South St.
Water St. State St. – Pearl St.
15 Ave. 110 St. – 127 St.
20 Ave. Parsons Blvd. – 119 St.
23 Ave. 119 St. – Linden Pl.
43 Ave. Whitney Ave. – 108 St.
73 Ave. Main St. – 230 St.
21 St. Ditmars Blvd. – 49 Ave.
82 Ave. Roosevelt Ave. – Baxter Ave.
108 St. Roosevelt Ave. – Horace Harding Exp.
154 St. Northern Blvd. – Clintonville St.
162 St. Northern Blvd. – 46 Ave.
164 St. 46 Ave. – Jamaica Ave.
187 Pl. Hillside Ave. – Jamaica Ave.
188 St. Utopia Pkwy – Jamaica Ave.
222 St. Hempstead Tpke – Jamaica Ave.
223 St. Northern Blvd. – 46 Ave.
Astoria Blvd. 31 St. – Northern Blvd.
Atlantic Ave. Eldert La – Van Wyck Exp.
B 25 St. Brookhaven Ave. – Beach Channel Dr.
Baxter Ave. Broadway – Roosevelt Ave.
Bell Blvd. 208 St. – Northern Blvd.
Booth Memorial Ave. College Pt Blvd. – Horace Harding Exp.
Braddock Ave. Springfield Blvd. – Jamaica Ave.
Broadway Roosevelt Ave. – Queens Blvd.
Brookhaven Ave. B 30 St. – B 25 St.
Cloverdale Blvd. 46 Ave. – Horace Harding Exp.
College Pt Blvd. Northern Blvd. – 5 Ave.
Commonwealth Blvd. Marathon Pkwy – Jamaica Ave.
Cross Island Pkwy S/S Rd 208 St. – Clearview Exp.
Cypress Hills St. Fresh Pond Rd – Jamaica Ave.
Ditmars Blvd. 21 St. – 48 St.
Douglaston Pkwy 38 Rd – Horace Harding Exp.
Edgemere Ave. B 59 St. – B 32 St.
Farmers Blvd. 99 Ave. – Rockaway Blvd.
Flushing Ave. Cypress Ave. – Grand Ave.
Francis Lewis Blvd. Cross Island Pkwy – Linden Blvd.
Fresh Pond Rd Flushing Ave. Myrtle Ave.
Grand Ave. Flushing Ave. – Queens Blvd.
Greenpoint Ave. Review Ave. – Queens Blvd.
Guy R. Brewer Blvd. Jamaica Ave. – Rockaway Blvd.
Hollis Ave. Jamaica Ave. – Springfield Blvd.
Homelawn St. Grand Central Pkwy – Hillside Ave.
Jackson Ave. Vernon Blvd. – Queens Blvd.
Junction Blvd. Queens Blvd. – 32 Ave.
Kissena Blvd. Main St. – Parsons Blvd.
Liberty Ave. Rockaway Blvd. – Farmers Blvd.
Linden Pl. 23 Ave. – Northern Blvd.
Little Neck Pkwy 40 Ave. – Jamaica Ave.
Marathon Pkwy Little Neck Pkwy – Commonwealth Blvd.
Metropolitan Ave. Onderonk Ave. – Jamaica Ave.
Mott Ave. Beach Channel Dr. – Central Ave.
Mrytle Ave. Wyckoff Ave. – Jamaica Ave.
North Conduit Ave. Cross Bay Blvd. – Sutter Ave.
Queens Blvd. Jackson Ave. – Hillside Ave.
Queens Plaza Queensboro Bridge – Jackson Ave. &Northern Blvd.
Rockaway Beach Blvd. B 55 St. – B 150 St.
Roosevelt Ave. Queens Blvd. – Northern Blvd.
Seagirt Blvd. Beach Channel Dr. – B 3 St.
South Conduit Ave. Sutter Ave. – Cross Bay Blvd.
Springfield Blvd. Jamaica Ave. – Rockaway Blvd.
Sutphin Blvd. Hillside Ave. – Rockaway Blvd.
Thomson Ave. Jackson Ave. – Van Dam St.
Woodhaven Blvd. Queens Blvd. – Rockaway Blvd.
Amboy Rd Richmond Ave. – Craig Ave.
Castleton Ave. Richmond Ave. – Jersey St.
Clove Rd Richmond Rd – Hylan Blvd.
Ferndale Ave. Rockland Ave. – Travis Ave.
Father Capodanno Blvd. Lily Pond Ave. – Greeley Ave.
Forest Ave. Victory Blvd. – Gulf Ave.
Forest Hill Rd Willow Brook Rd – Platinum Ave.
Forest Hill Rd Richmond Ave. – Yukon Ave.
Huguenot Ave. Arthur Kill Rd – Hylan Blvd.
Hylan Blvd. Bay St. – Saterlee St.
Lamberts La Fahy Ave. – Richmond Ave.
Lily Pond Ave./ School Rd Father Capodanno Blvd. – Bay St.
Manor Rd Brielle Ave. – Delafield Ave.
Mason Ave. Seaview Ave. – Cromwell Ave.
McClean Ave. Tompkins Ave. – Norway Ave.
Merry Mount Ave. Richmond Hill Rd – Travis Ave.
Midland Ave. Father Capodanno Blvd. – Hylan Blvd.
Narrows Rd South Richmond Rd – Lily Pond Ave.
New Dorp La Hylan Blvd. – Cedargrove Ave.
North Gannon Ave. Slosson Ave. – Victory Blvd.
Reid Ave. Norway Ave. – South Railroad Ave.
Richmond Ave. Hylan Blvd. – Forest Ave.
Richmond Hill Rd Richmond Rd – Richmond Ave.
Richmond Rd Richmond Hill Rd – Vanderbilt Ave.
Rockland Ave. Richmond Rd – Brielle Ave.
Sand La Father Capodanno Blvd. – Hylan Blvd.
Seaview Ave. Hylan Blvd. – Father Capodanno Blvd.
Seguine Ave Amboy Rd – Hylan Blvd.
Slosson Ave. Todt Hill Rd – Martling Ave.
Targee St. Richmond Rd – Van Duzer St.
Todt Hill Rd Richmond Rd – Slosson Ave.
Trantor Pl. Forest Ave. – Innis St.
Travis Ave. Draper Pl. – South Ave.
Travis Ave. Forest Hill Rd – Richmond Ave.
Willow Brook Rd South Gannon Ave. – Bradley Ave.


text:
Vyapam scam: Whiff of a cover-up in accused's mystery death
Modi to Xi: Backing Pak on Lakhvi unacceptable
Indian business dreads lifting of Iran sanctions
Root ton makes it England's day
Railways launch ticketing app for those without reserved seats in Mumbai
All Sections City news Sports Business Editorial Techno Stuff Nation Television Bollywood Hollywood Town & About Film Review Special Report Book Review Spectrum Feature TV Guide Coffee Break Classified Brush With Art Interview Navi Mumbai Notes From New Delhi Countdown To SSC Culture World
Yearly Forecast 2015
Television should not be kept in bedroom.
Home > 48 Hrs > Easter Excitement
Easter Excitement
Easter Sunday is this weekend, but we're sure you've already indulged in a few chocolate bunnies and marzipan eggs! The treasure hunts, Easter brunches and festivities make it a celebration that children and adults love in equal part, but that's not all there is to it. Dev Goswami & Yamini Walia tell you about the history and significance of the festival, bring you some fun facts and also tell you how to throw your own Easter hunt
Seeing its significance
Easter celebrates Jesus Christ's resurrection from the dead, but unlike other festivals, there's no specific date set for it. Easter is observed on the first full moon after the spring equinox (one of the two days of the year when the sun crosses the plane of earth's equator; on this occasion, day and night are of almost equal length), which usually occurs somewhere around March 20-21. So, Easter can be observed on any day between March 22 to April 25. Here are a few facts that you should know about the festival:
Easter is about more than just the resurrection of Christ. In fact, it's really an entire season that celebrates different aspects of the life of Jesus Christ, instead of a one-day celebration.
Lent, the 40-day fasting period that is observed in the weeks leading up to Easter, is a time of reflection and penance, while the Friday preceding Easter, known as Good Friday, marks the crucifixion of Jesus Christ.
The Holy Saturday, the day after Good Friday, celebrates Christ's transition from crucifixion to resurrection, while a 50-day period after Easter, known as Eastertide, is a celebration of his ascension into Heaven.
The exact origins of this festival are unknown — however, a few historians have managed to trace the symbols and traditions that are associated with the festival to a pre-Christian era.
A popular theory is that Easter is named after a Teutonic (a word used for ancient Germans) goddess of spring and fertility named Oestre. Interestingly, the word for female hormones, oestrogen, is also believed to be derived from her name.
Another theory traces the origins to the Spanish word Pascua, derived from the Greek and Latin Pascha or Pasch. Pascaha is the Jewish festival Passover, which commemorates the exodus of ancient Israelites' from Egyptian slavery. The story goes that Jesus' crucifixion and resurrection followed his visit to Jerusalem, where he celebrated Passover.
What came first — the bunny or the egg?
Have you wondered what the bunny and egg have to do with Easter? If you've read The Holy Bible, you will known that there is no mention of an Easter bunny or painted eggs. Also, bunnies do not lay eggs — even though several legends argue otherwise. So, what do these two popular Easter symbols have to do with the festival? Well, not much. The connection comes from what both the rabbit and the egg symbolise — fertility. Rabbits are known to be prolific procreators and they were also the symbol of the ancient, pre-Christian, Teutonic goddess, Oestre. Spring (Easter can be seen as a spring festival) is a period that symbolises new life and birth, and this is where the rabbit and the egg come in.
Easter bunnies and eggs were first documented in the 1600s. Some time during this century, the first story about rabbits who lay eggs and hide them in gardens was published. These legends were then taken to the United States when German immigrants settled in the country. The legend of egg-laying bunnies is what gave rise to the tradition of making nests for rabbits to lay eggs in — and, as you may have guessed, these nests are what we see today as decorated baskets full of colourful eggs.
While most theories about the Easter bunny are related to it being a symbol of goddess Oestre, there is another reason that eggs are connected to this festival. Apart from signifying Jesus' resurrection, eggs are also believed to be one of the foods that were forbidden during Lent. Because of this, the theory goes, people used to decorate and paint eggs in order to mark the end of the season of penance and to celebrate Easter.
Throw your own Easter egg hunt
Easter Egg treasure hunts may have been created for children, but we all know that adults love them too! While most establishments will organise hunts this time of year, there's nothing quite like organising your own. It's a great way to add a personalised touch and a fun element to your weekend, whether you celebrate the festival or not. After all, who doesn't want to find a handful (or basketful in this case) of goodies?! If you want to throw your own Easter egg hunt, but are at a loss about what to do, use this simple guide.
Before throwing any kind of party, the time and date are the most essential things to consider. Remember that on Easter, most of your Christian friends will have their own family dinners and gatherings to attend. So, selecting a day leading up to Easter (or one after) is a better idea. While you don't have to follow this, the most common time for hunts is late morning or afternoon — however, for a city like ours, we think that a late afternoon or early evening egg hunt makes more sense.
Since Easter hunt is a family event, you can expect people of different ages to participate. So, when you're planning the hunt, keep the ages of your guests in mind. If a majority of your guest list includes young children, you need to make sure that you hide the goodies in places that they can reach easily. For toddlers, spreading the eggs out on the ground in a lawn makes more sense. However, if your guests are predominately adults or teenagers, the entire world is a hiding place — think, trees, bushes, or even car tyres!
Location of the hunt
Your Easter hunt will only be a success if you choose the perfect location. Considering the space issues in our city, it's a good idea to organise the hunt in a park near your home — unless of course, you're blessed with an expansive garden or lawn in your building or society. If you're determined to organise a hunt inside your house, make sure that you move all your furniture out of the way in order to minimise the risk of injuries. Also, remember to hide the eggs in a way that enables your guests to find them without messing up your home.
Irrespective of your location, is it's indoors or outdoors, setting boundaries is essential. This is important, not only to ensure safety, but also to keep everyone in the same place. For example, you don't want children destroying your bedroom, when the eggs are only hidden in your living room.
Imagine being left wondering where you hid all the eggs just half an hour into the hunt — it's quite possible! So, you will be better off if you make a list of all the locations where you've placed the eggs. Not only will this list help you to create better clues, it will also come in handy if you need to direct children to some of the really well-hidden eggs.
Make sure there are prizes
No one likes to participate in a competition that has no rewards. So, make sure that you arrange for a few prizes for your participants. For example, you can paint a bunny on a small jar, place a few chocolates inside it and hand it to the winner. But, don't forget that the person who finds the least number of eggs will require motivation too! Make sure there is a consolation prize for them.
Before preparing for your Easter celebrations, we have some interesting facts about Easter that you should know about. Take a look.
Expensive deal
What would you say about an egg that was covered in diamonds? In 2007, one such egg was sold for a whopping `60 crore. Wonder what's so special about it? Every hour, a cockerel made of jewels pops up over the top of the egg, flaps its wings four times, nods its head three times and makes a crowing noise! Wow!
Elephant egg
No we're not referring to an egg laid by an elephant, but to an Easter chocolate egg that is even bigger than one! Needless to say, it's the tallest Easter egg ever made — it's 10.39 metres tall and weighs a whopping 7,200kg! It was created in Italy, in 2011.
Easter pretzels
If you enjoy eating pretzels, you will be thrilled to know that pretzels were associated with Easter because the twists of the pretzel were thought to resemble human arms crossed prayer. Interesting or just plain weird? We're a little confused too.
Throw the egg
In medieval times, an egg-throwing festival was held in churches. The priest would throw a hard-boiled egg at one of the choir boys, from where it would be tossed from one choir boy to the next. When the clock struck 12, whoever held the egg was the winner and got to keep the egg. Talk about medieval entertainment!
Historic jellybeans
Jellybeans were first made in the United States of America by Boston candy maker William Schrafft, who ran advertisements urging people to send jellybeans to soldiers fighting in the Civil War. Today, Americans consume over 16 million jellybeans on Easter — the amount is enough to circle the globe three times over!
Something's Brewing!
The craft beer trend has been growing in India, a
Camp to Ladakh
Anindra Siqueira tells you about the BNHS's ten-d
Event SCAVENGER HUNT AT BANDRA
Some Place Else is gearing up for another scaveng
HOME | City news | Sports | Business | Editorial | Techno Stuff | Nation | Television | Bollywood | Hollywood | Town & About | Film Review | Leisure | Special Report | Book Review | Spectrum | Feature | TV Guide | Coffee Break | Classified | Health & Fitness | Brush With Art | Education & Career | Woman's World | 48 Hrs | Property & Interior | Letters | Opinion Poll | Diary | Rajan Bhonsle | Impact Shopping | ADC Initiative | Business & Investment | Interview | Jamshedi Navroze | Gigantika | RTI | Vastu Tip | Navi Mumbai | Notes From New Delhi | Countdown To SSC | Simhasan | Culture | World | Maha Poll

redpajama_set_name: RedPajamaCommonCrawl


text:
McGLINCHEY, Nancy
Sept 4, 2022
May 28, 1951 - August 1, 2022
Nancy Irene McGlinchey, (71) born May 28, 1951, died Aug. 1, 2022. Nan was born in Brooklyn, NY, to Arthur and Hilda McGlinchey and grew up in Belmont, Dayton, in the pleasant, safe 1950's. Raised by Hilda (best mom ever) with backup parents Bob and Fran Whitworth she attended Cleveland and Belmont (1969) making lifelong friends along the way.
She worked for many years at Community Blood Center where the shipping dept. was her extended family. Nan and Hilda did some traveling in the U.S. and Italy, viewing Halley's Comet from aboard a ship passing through the Panama Canal. She was a happy, kind and generous person who made the world a better place.
Politics and cruelty horrified her but she would step out of her comfort zone to work for change. She marched in Washington, D.C., for animals, in NYC for women and in Dayton for local issues. She led a quiet well deserved retirement enjoying books, music, art and her cats (first Glowie, then Ruby). Nan is preceded in death by parents, the Whitworths, ex-husband Neil Junker and pets Buffy, Celine and Glowie. She is survived by sister Diane, nephew Carlos (Carrie), longtime friend Tim Vest and New Jersey cousins.
To remember Nan please do something kind for an animal or plant something that blooms first in the spring. She would like that.
Sign the guestbook at Legacy.com
View the obituary on Legacy.com
BLACKSHEAR, Steven
YURKOWSKI, Christopher
SIZEMORE, James
ERISMAN, Martha
HURLEY, James


As a premier company specializing in residential roofing and home improvement services, we are dedicated to providing customer service that is second to none. Whether you have a new construction project or need to replace an old or damaged roof, we offer roofing repair and replacement services. Our roofing personnel has years of experience in dealing with all types of storm, hail, and wind damage. We will provide you with a full consultation to explain all of your available options so that you can choose the roofing plan that's right for your home or business. We will work with you and adhere to your personal needs.


This hair hasn't seen a comb in 3 weeks. Practicing finger combing and shaping to reduce manipulation.
Regimen: L.O.C twice a day to keep dryness at bay (takes 3mins). My hair loves water. Silk cover at bedtime. Fingercombing. Cowash weekly.
Favorite products: water+oil mix, Shea moisture curling gel souffle, Camille rose pudding.


Easy to apply, SPRAY grade pre-mixed single component, liquid, cold applied elastomeric waterproofing membrane. MasterSeal HLM 5000 is a moisture-curing, bitumen-modified polyurethane elastomeric waterproofing membrane for exterior below-grade or between-slab applications.
HLM 5000® SPRAY grade is a one-component, moisture-curing, bitumen-modified polyurethane elastomeric waterproofing membrane for exterior below-grade or between-slab applications.