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Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies
- Andreas Agathangelidis1,
- Nikos Darzentas1,
- Anastasia Hadzidimitriou1,
- Xavier Brochet2,
- Fiona Murray3,
- Xiao-Jie Yan4,
- Zadie Davis5,
- Ellen J. van Gastel-Mol6,
- Cristina Tresoldi7,
- Charles C. Chu4,
- Nicola Cahill8,
- Veronique Giudicelli2,
- Boris Tichy9,
- Lone Bredo Pedersen10,
- Letizia Foroni11,
- Lisa Bonello12,
- Agnieszka Janus13,
- Karin Smedby14,
- Achilles Anagnostopoulos15,
- Helene Merle-Beral16,
- Nikolaos Laoutaris17,
- Gunnar Juliusson18,
- Paola Francia di Celle12,
- Sarka Pospisilova9,
- Jesper Jurlander10,
- Christian Geisler10,
- Athanasios Tsaftaris1,
- Marie-Paule Lefranc2,
- Anton W. Langerak6,
- David Graham Oscier5,
- Nicholas Chiorazzi4,
- Chrysoula Belessi17,
- Frederic Davi16,
- Richard Rosenquist8,
- Paolo Ghia13, and
- Kostas Stamatopoulos1,15
- 1Institute of Agrobiotechnology, Center for Research and Technology Hellas, Thessaloniki, Greece;
- 2IMGT, Université Montpellier 2, Laboratoire d'ImmunoGénétique Moléculaire, Institut de Génétique Humaine, UPR Centre National de la Recherche Scientifique 1142, Montpellier, France;
- 3Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden;
- 4The Feinstein Institute for Medical Research, North Shore–Long Island Jewish Health System, Manhasset, NY;
- 5Department of Haematology, Royal Bournemouth Hospital, Bournemouth, United Kingdom;
- 6Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands;
- 7Unit of Molecular Hematology, Istituto Scientifico San Raffaele, Milan, Italy;
- 8Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden;
- 9Department of Hematooncology, University Hospital Brno and Central European Institute of Technology, Masaryk University, Brno, Czech Republic;
- 10Department of Hematology, Rigshospitalet, Copenhagen, Denmark;
- 11Department of Haematology, Imperial College Academic Health Science Centre, Hammersmith Hospital, London, United Kingdom;
- 12Center for Experimental Research and Medical Studies, S. Giovanni Battista Hospital, Turin, Italy;
- 13Laboratory of B Cell Neoplasia and Unit of Lymphoid Malignancies, Università Vita-Salute San Raffaele and Istituto Scientifico San Raffaele, Milan, Italy;
- 14Department of Medicine, Clinical Epidemiology Unit, Karolinska Institutet, Stockholm, Sweden;
- 15Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece;
- 16Hematology Department and University Pierre et Marie Curie, Hôpital Pitié-Salpètrière, Paris, France;
- 17Hematology Department, Nikea General Hospital, Piraeus, Greece; and
- 18Lund University and Hospital Department of Hematology, Lund Stem Cell Center, Lund, Sweden
Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.
The analysis of the Ig genes in chronic lymphocytic leukemia (CLL) has contributed significantly toward deciphering the molecular pathogenesis of the disease. Studies from the 1990s provided the first indications for a possible role of Ag(s) in selecting the CLL progenitor cells, through the discovery of a biased Ig heavy variable (IGHV) gene repertoire, different from that of normal B cells, as well as distinctive Ag-binding sites among unrelated cases.1⇓⇓⇓–5
By the late 1990s, it emerged that the mutational status of the rearranged IGHV genes directly correlated with patient survival. In particular, patients with unmutated IGHV genes were found to follow a more aggressive clinical course and have significantly shorter survival than patients carrying mutated IGHV genes.6,7 Yet, there were exceptions to this rule: cases using the IGHV3-21 gene, although mostly expressing mutated Ig, had a survival similar to that of unmutated cases.8 Intriguingly, approximately half of the IGHV3-21 cases were found to display restricted and, in some instances, essentially identical variable heavy complementarity determining region 3 (VH CDR3) sequences and identical light chains, strongly suggesting recognition of a common antigenic determinant.9
Soon thereafter, the study of Ig sequences in CLL by groups in both Europe and the United States led to the identification of several other subsets of cases carrying highly similar BCR Igs among both mutated and unmutated cases (stereotyped BCR).10⇓⇓⇓–14 The identification of stereotypy among unrelated and geographically distant cases was widely accepted as evidence for the recognition of individual, discrete Ags or classes of structurally similar epitopes, likely selecting the leukemic clones.10⇓⇓–13
Subsequently, the study of stereotypy in large cohorts revealed that a significant fraction of CLL cases (20%-28%) carried stereotyped VH CDR3 sequences within their BCR Ig,14⇓⇓–17 and, more importantly, that stereotypy may extend from restricted sequence patterns within the Ig to shared biologic and clinical characteristics and, perhaps, outcome.10,14,16 In addition, it was conclusively demonstrated that the IGHV gene repertoire restrictions typical of CLL were in essence a property confined to stereotyped cases, clearly segregating them from the "heterogeneous" (nonstereotyped) cases.17 Hence, it was proposed that ontogenies of the 2 CLL groups (stereotyped vs heterogeneous) as well as the selective forces shaping the Ig repertoire of the CLL precursor cell population(s) might differ.17
The nature of the selecting Ags and the functional consequences of their recognition cannot be directly deduced from primary Ig rearrangement features and have so far remained largely unknown. However, recent studies on BCR reactivity using CLL clones from stereotyped cases suggest that the expression of a stereotyped BCR may be linked to the reactivity profile of the CLL clone,18⇓⇓–21 and, eventually, to disease outcome.22
Against this background, several issues remain to be further addressed: (1) the criteria for the discovery of stereotypy and subset assignment; (2) the question as to whether stereotypy may be found for each BCR Ig provided enough cases are analyzed; (3) the number and size of different subsets; and (4) the identification of "CLL-biased" features among BCR Ig stereotypes with potential implications for disease ontogeny.
To address these issues, we systematically explored stereotypy based on VH CDR3 in a series of > 7000 VH (IGHV-IGHD-IGHJ) sequences from patients with CLL, 3 times the size of the largest published study. The results reported herein provide definitive evidence to our previous hypothesis17 that not all CLL cases will end up being part of stereotyped subsets or, in other words, that CLL indeed comprises 2 distinct categories: one with stereotyped and the other with heterogeneous Ig, in an approximate ratio of 1:2. The major stereotyped subsets collectively represent a substantial proportion of the respective category, with "CLL-biased" and often highly distinctive molecular features. Consequently, this deeper and compartmentalized view of BCR Ig primary structures in conjunction with biologic and clinical information may not only lead to a better basic understanding of the disease, but also potentially pave the way for tailored treatment strategies applicable to each major stereotyped subset.
Patient group
A total of 7424 patients diagnosed with CLL from collaborating institutions in Europe and the United States were included in the present study. All cases met the recently revised diagnostic criteria of the National Cancer Institute Working Group.23 The study was approved by the local ethics review committee of each institution.
PCR amplification of IGHV-IGHD-IGHJ rearrangements
PCR amplification and sequence analysis of IGHV-IGHD-IGHJ rearrangements were performed on either genomic DNA (gDNA) or cDNA, as previously described,5,10–11,15 or after the BIOMED-2 protocol.24
Sequence analysis and data mining
PCR amplicons were subjected to direct sequencing on both strands. Sequence data were analyzed using the IMGT databases25 and the IMGT/V-QUEST tool (http://www.imgt.org).26 Codons and amino acid positions are according to the IMGT unique numbering for V domain.27 Only productive rearrangements were evaluated.
Output data from IMGT/V-QUEST for all productive IGHV-IGHD-IGHJ rearrangements were parsed, reorganized, and exported to a spreadsheet through the use of computer programming. Information was extracted regarding Ig gene repertoires, VH CDR3 length and amino acid sequence and somatic hypermutation (SHM), as previously described.15
Identification and clustering of IGHV-IGHD-IGHJ rearrangements based on stereotypy within VH CDR3 sequences
To identify and cluster stereotyped rearrangements, we used a purpose-built bioinformatics method, which has been previously described and applied with efficacy.17 As shown previously,17 VH CDR3 sequences were connected if sharing at least 50% amino acid identity and 70% similarity calculated through common sequence patterns. For the present study though, a novel parameter and more stringent criteria were introduced.
In particular, the application of the novel parameter was based on evidence that IGHV genes are phylogenetically related,28 that is, they originate from 3 ancestral phylogenetic clans: IGHV1/5/7 subgroup genes from clan I, IGHV2/4/6 from clan II, and, finally, IGHV3 genes from clan III.28 Consequently, we required that only sequences carrying IGHV genes of the same clan could be assigned to the same cluster, thus taking into account the role of the IGHV region in Ag recognition.
Furthermore, more stringent criteria were related to the 3-dimensional structure of the BCR and included the requirement for identical VH CDR3 lengths and identical offsets (ie, exact locations within the VH CDR3 region) of shared patterns between connected sequences.
The clustering process starts by building clusters on a first (ground) level, called level 0. Members of each level 0 cluster are guaranteed to meet all the aforementioned criteria for cluster assignment and can appear in more than one level 0 cluster, highlighting complex relationships. Finally, the existence of common amino acid patterns between sequences belonging to different level 0 clusters leads to their grouping in clusters at progressively higher levels of hierarchy describing more distant, and thus relaxed, sequence relationships with more widely shared sequence patterns. A schematic example of the hierarchical clustering process is given in supplemental Figure 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).
Data visualization tools
VH CDR3 amino acid patterns of different subsets were visualized using WebLogo (http://weblogo.berkeley.edu/). Each logo consists of stacks of symbols, one stack for each position of the sequence. CDR3 are shown from IMGT positions 105 to 117.27
A synopsis of IGH gene repertoires, somatic hypermutation status, and VH CDR3 features
A total of 7596 VH (IGHV-IGHD-IGHJ) productive rearrangements from 7424 cases with CLL were evaluated; 172 cases appeared to carry double productive rearrangements. The IGHV, IGHD, and IGHJ subgroup and gene repertoires were generally similar to those reported in previous studies5,11⇓⇓⇓⇓⇓–17 (listed in detail in supplemental Tables 1-3). Following the 98% germline identity (GI) cutoff value, 4171 (55%) of 7596 rearrangements were assigned to the mutated subgroup, whereas the remainder (3425 [45%] of 7596,) were classified as unmutated, of which 2516 (73.5%) of 3425 displayed 100% sequence identity against the corresponding germline IGHV gene sequences.
Focusing on rearrangements of the predominant IGHV genes, specific associations with certain IGHD and IGHJ genes were identified, often accounting for sizeable fractions of cases. This is best depicted by rearrangements using the IGHV1-69 and IGHV1-2 genes, which were strongly biased toward the usage of the IGHD3-3 gene (298 [30.6%] of 973) and the IGHD6-19 gene (80 [23%] of 348), respectively. On the contrary, less pronounced or no biases were noted for rearrangements using other frequent IGHV genes, namely IGHV4-34, IGHV3-7, and IGHV3-23. Specific combinations of IGHV and IGHJ genes were also identified: for example, IGHV1-69 and IGHV3-21 with IGHJ6 (542 [55.7%] of 973, and 248 [70.3%] of 353 cases, respectively). VH CDR3 median length was 17 aa (range, 5-37).
One-third of CLL cases were assigned to subsets with stereotyped VH CDR3
Through the use of our recently established bioinformatics method based on shared VH CDR3 amino acid sequence patterns17 herein updated with more stringent and novel criteria, 2308 (30.4%) of 7596 CLL VH CDR3 sequences were placed in 952 subsets at the ground level (level 0) that included 2-56 sequences each, that is, considered as stereotyped.
VH CDR3 sequences may contain multiple shared patterns and this allowed their concurrent assignment to different ground-level subsets. This overlap enabled the discovery of subsets at 3 successive hierarchically higher levels (supplemental Figure 1), characterized by more broadly shared sequence patterns and, hence, greater size. At the highest level, 19 different subsets contained 20 or more (up to 213) sequences and were defined as major (supplemental Tables 4-5): collectively, major subsets included 943 cases and, thus, accounted for 41% of the stereotyped cases and 12% of the cohort, respectively. In other words, 1 in 8 of all CLL patients belonged to a major subset (Figure 1).
Aside from unique lengths, the widely shared sequence patterns characteristic of major subsets could comprise the entire VH CDR3 or a few, even a single, strategically positioned residue. A prime example of the former case is subset 6 that comprises cases with stereotyped, unmutated IGHV1-69/IGHD3-16/IGHJ3 rearrangements.12,21,22 In the present series, subset 6 included 68 rearrangements, thus accounting for 0.9% of the cohort, in keeping with the literature.12 Except for 4 CDR3 positions, which were characterized by variability, all other 17 positions were extremely, if not entirely, conserved. Notably, the first 2 aa (Gly G107, Gly G108) of the N1 region at the IGHV-IGHD junction were identical in 67 of 68 subset 6 cases (Figure 2A).
At the opposite end stands subset 29,14 with 213 IGHV3-21–expressing CLL cases (2.8% of the cohort) characterized by a very short VH CDR3 sequence (9 aa) involving the IGHJ6 gene and no identifiable IGHD gene. Interestingly, at the center of the short VH CDR3, virtually all subset 2 cases (211 [99%] of 213) carried a landmark amino acid (aspartic acid, D) at position 107, between the amino acids qualified as IGHV- or IGHJ-encoded; 2 cases lacking an aspartic acid were found to carry a glutamic acid (Glu E) at the same position, that is, an amino acid of the same IMGT physicochemical class29 (both acidic; Figure 2B). On these grounds, CLL IGHV3-21 rearrangements can be assigned to subset 2 if meeting only 2 criteria: (1) restricted VH CDR3 length of 9 aa, and (2) a "landmark" acidic amino acid (D or E) at VH CDR3 position 107.
Two types of CDR3 sequence patterns define CLL subsets
Shared sequence patterns defining subsets were broadly divided into 2 types: (1) "mainly combinatorial," that is, largely encoded by unmutated sequences of the D region and 5′J-region of specific combinations of IGHD-IGHJ genes, and (2) "combinatorial + junctional," that is, encoded in part by the N-diversity regions (N1 and N2) generated by junctional diversity mechanisms (as a result of P nucleotide addition, exonuclease trimming of the 3′ IGHV, 5′ and 3′ IGHD, and 5′ IGHJ gene ends and N nucleotide addition) leading to restricted motifs at the IGHV-IGHD and/or IGHD-IGHJ gene junctions.
Subset 810,14 epitomizes the mainly combinatorial type of VH CDR3 sequence patterns. The stereotyped unmutated heavy chains are encoded by the specific combination of the IGHV4-39/IGHD6-13/IGHJ5 genes (IGHD gene in reading frame 1) and carry VH CDR3s with a length of 19 aa (Figure 3A). The junctional N1 (IGHV-IGHD) and N2 (IGHD-IGHJ) regions were heterogeneous, yet this restricted combination of IGHV, IGHD, and IGHJ genes proved to be "subset 8–specific" and "CLL-specific, as revealed by the extensive examination of IGH rearrangements deposited in public generalist and specialist (IMGT/LIGM-DB30) databases.
Exemplifying the combinatorial + junctional type of VH CDR3 patterns is subset 4,11,14 represented by 74 cases (0.97% of the cohort sequences). The stereotyped mutated IGH chains are generated through the combination of the IGHV4-34 and IGHJ6 genes (reliable IGHD gene assignment was not possible because of heterogeneity at the tip of the VH CDR3 loop, possibly because of SHM). Interestingly, in subset 4, conserved amino acids characterize the N regions. Thus, the N1 region comprises glycine G107 and tyrosine Y108 (or the other aromatic amino acids, phenylalanine F or tryptophan W) whereas the N2 region has 2 arginine R at positions 112.4 and 112.3 (in 13 of 74 sequences, the arginine R112.4 located at the tip of the CDR3 is replaced by a lysine K, another basic amino acid; Figure 3B). Public database examinations revealed the [RK]RYYYY pattern that reflects the junctional N2 conserved amino acids and the IGHJ6 use to be "subset 4–specific" and "CLL-specific." Other examples of major subsets defined by mainly combinatorial and combinatorial + junctional patterns are given in supplemental Table 6.
Subset-defining sequence patterns in CLL differ from those underlying VH CDR3 stereotypy in other B-cell malignancies
Recent studies by us and others have documented the existence of stereotyped BCR Ig in mantle cell lymphoma (MCL)31 and splenic marginal-zone lymphoma (SMZL),32,33 albeit at significantly lower frequencies than CLL. We therefore sought to investigate whether the stereotypes observed in CLL are disease-biased or common by performing cross-entity comparisons.
In the case of MCL, stereotyped IGHV-IGHD-IGHJ rearrangements collectively represent ∼ 10% of the cohort31 and display extreme restrictions in IGHV gene utilization, with the IGHV4-34 and IGHV3-21 genes accounting for 67% of all such cases. Stereotyped MCL subsets were compared with major CLL subsets using the same IGHV genes, namely subsets 4, 16 (both IGHV4-34), and 2 (IGHV3-21). In all comparisons, significant differences were identified in terms of IGHD and IGHJ gene usage, VH CDR3 sequence length and amino acid composition (Figure 4).
Differences were even more fundamental when CLL stereotypes were compared with those identified in SMZL, in this case comparing stereotypes using the IGHV1-2 gene, which by far predominates in SMZL.32,33 Hence, BCR Ig VH stereotypes in CLL were significantly different from "related" ones in MCL or SMZL, even though they expressed the same IGHV gene, and can thus be considered as disease-biased (supplemental Table 7).
Even if numbers increase significantly, not all CLL cases can be classified as stereotyped
From this large study involving > 7000 sequences, it became evident that the frequency of stereotyped rearrangements in CLL is not directly correlated to cohort size. Despite a significant increase in sample size between the present series and previous studies (including up to 2662 sequences),11,13⇓⇓⇓–17 the increase in the frequency of stereotypy is only marginal (30.4% vs 28%, respectively). In other words, although the proportion of stereotyped BCR Ig VH in CLL seems to continuously increase with larger cohort sizes, the rate of this increase starts to drop after a critical value of cohort size until the frequency of stereotypy plateaus at ∼ 30% of the cohort, independent of cohort size (Figure 5).
To corroborate this finding, while avoiding potential bias because of slightly different criteria to define stereotypy across the published studies, we performed random set simulations using the latest version of the algorithm and the current cohort of VH CDR3 sequences. Indeed, we found that above a critical value of cohort size of 2000 sequences, each addition of a thousand sequences resulted in an increase in the frequency of stereotypy by a steadily decreasing and disproportional rate (supplemental Table 8).
Distinctive immunogenetic characteristics and CLL ontogenetic implications
The present analysis led to the discovery of several noteworthy immunogenetic features, with implications for CLL ontogeny and Ag selection.
Subset 10: virtually identical VH CDR3 sequences.
VH CDR3 sequence homogeneity reached its maximum potential for cases assigned to subset 10. This subset included 18 sequences expressing stereotyped, unmutated IGHV4-39/IGHD2-2/IGHJ6 Ig and is quite remarkable for the fact that the defining sequence pattern covered all 22 aa of the VH CDR3. In fact, only 10 amino acid differences were identified in a total of 396 VH CDR3 amino acid residues (2.5%) of subset 10 cases, rendering it the most homogeneous high-level subset thus far described (supplemental Figure 2).
Subsets with different IGHV genes of the same clan.
Subset 59 comprised 21 unmutated cases with stereotyped VH CDR3 using one of the IGHV1-2, IGHV1-58, or IGHV1-69 genes (2, 8 and 12 sequences, respectively), which are all members of the IGHV1 subgroup. Further exemplifying and extending this principle, subset 12 consisted of 22 cases with stereotyped VH CDR3 using one of the IGHV1-2, IGHV1-46, or IGHV5-a genes (11, 10, and 1 sequences, respectively), which, though belonging to different IGHV subgroups (namely, IGHV1 and IGHV5), are members of the same clan of IGHV genes (clan I), that is, they still exhibit significant sequence similarity. This may indicate that amino acids of the CDR1 and/or CDR2 may contribute, in addition to the CDR3, to the Ig specificity. Subsets 59 and 12, along with additional examples, are presented in supplemental Table 9.
Closely related subsets using different IGHV genes.
The IGHV3-48 gene is very similar to the IGHV3-21 gene (overall amino acid identity 97%). Thirteen rearrangements of the cohort using the IGHV3-48 gene were assigned to a novel subset (169) with a short, 9-aa long VH CDR3, that is, identical in size to that of subset 2, which includes stereotyped rearrangements of the IGHV3-21 gene. Intriguingly, as in subset 2, all subset 169 rearrangements carried an aspartic acid D107. The fact that sequences from these 2 subsets were not finally grouped together indicates that they differ to an extent that did not satisfy the algorithmic criteria. However, the similarity of the IGHV3-21 and IGHV3-48 genes along with identical VH CDR3 length and the presence of D107, suggests that the 2 subsets 169 and 2 may be closely related (Figure 6).
Sequence convergence induced by somatic hypermutation.
We and others have previously shown that sequences assigned to subsets with stereotyped VH CDR3 can also exhibit identical amino acid changes (as a result of SHM across the IGHV region11,15; supplemental Table 10), clearly indicating that certain positions throughout the VH domain of cases belonging to the same subset seem to be under selective pressure for specific changes.
We here extend this notion showing that certain subsets with stereotyped VH CDR3 expressing different, yet related, IGHV genes can exhibit SHM-derived sequence convergence. This is illustrated by subset 77, which consists of 25 mutated stereotyped rearrangements using either the IGHV4-4 or the IGHV4-59 gene. In selected positions where the germline sequences of the above IGHV genes varied, we noted that the changes introduced by SHM could lead to higher sequence identity, hence eliminating germline-encoded variation: for instance, 4 of 13 IGHV4-59 cases carried a tyrosine (Y) to histidine (H) change at VH CDR2 codon 58 (Y58 > H), resembling the IGHV4-4*02 and IGHV4-4*07 alleles; moreover, 16 (67%) of 24 of all subset 77 cases carried an isoleucine (I) to methionine (Met M) change at VH FR3 codon 78 (I78 > M), regardless of whether they expressed the IGHV4-4 or the IGHV4-59 gene (supplemental Figure 3). These findings could not be attributed to ambiguous IGHV germline gene assignment because rearrangements using one or the other gene retained "gene-specific" features (supplemental Table 11).
Emerging data suggest that grouping CLL cases into subsets based on BCR Ig stereotypy can reflect similar Ag-reactivity profiles22 and similar clinical outcomes,14,16 at least for certain subsets. Therefore, the study of stereotypy in CLL has important implications for both understanding the pathogenesis of the disease34 and, most likely, for improving patient stratification toward personalized therapeutic applications.
Here, we analyzed the Ig gene repertoire of 7424 patients with CLL to address several open issues related to: (1) the proper criteria for stereotypy detection and eventual subset assignment; (2) the exact magnitude of and future projections about BCR Ig stereotypy; (3) the identification of CLL-biased features in BCR Ig stereotypes. This latter issue is particularly timely given recent reports by several groups, including ours, regarding the existence of BCR Ig stereotypes in the normal repertoire35 as well as in other B-cell malignancies, namely MCL31 and SMZL.32,33
To answer these questions, we applied an updated version of our published clustering method,17 purpose-built for the identification of shared amino acid sequence patterns within VH CDR3 sequences and their subsequent clustering. Accumulating evidence from the recent literature,35,36 combined with our experience from similar analyses, led us to adopt more stringent subset-defining criteria than we did previously, so that the identified primary amino acid sequence relatedness may correspond to actual structural similarity.37,38 The revised criteria mainly related to (1) VH CDR3 length, known to be a critical determinant of the structure of the Ag recognition loop,39 and (2) the exact location of the shared pattern within the VH CDR3 region, given ample evidence that the positioning of certain amino acids may affect Ig structure stabilization.40,41 For these reasons and to ensure maximum accuracy, we followed a conservative approach and required for same-subset assignment at the ground level no less than total identity in VH CDR3 length and offset of shared patterns. Through this approach, groups of rearrangements using the same IGHV, IGHD, and IGHJ genes with highly similar VH CDR3 sequences, yet slightly dissimilar VH CDR3 lengths, were eventually clustered in related yet different subsets (supplemental Table 12). This maximizes homogeneity and is now our preferred strategy for subset assignment, at least until other types of evidence (functional, structural, clinical) become available to support that rearrangements should be placed in a single subset irrespective of slight VH CDR3 length variation, as has been the case so far for subset 1.11,14,16
The novel parameter introduced in our clustering algorithm required that only sequences carrying IGHV genes of the same clan could be placed in the same subset. This development takes into account the role of the CDR1 or CDR2 amino acids in classic Ag recognition as well as the binding of specific classes of Ags41 (ie, superantigens, bacterial polysaccharides) to amino acids of the framework regions. Its implementation highlighted several subsets of cases that express different yet phylogenetically related IGHV genes, including the well-established subset 111,14,16 (IGHV genes belonging to clan I) as well as other cases, for example, subsets 12 (IGHV1-2 and IGHV1-46), 59 (IGHV1-58 and IGHV1-69), and 77 (IGHV4-4 and IGHV4-59). The latter, including cases with mutated Ig, is also remarkable for the fact that distinct yet related IGHV genes, when rearranged, can eventually become more similar through functionally selected SHM. In other words, starting from different germline-encoded genes, the affinity maturation process can reduce germline sequence disparities leading to sequence convergence.
Altogether, it now becomes clear that the existence of CLL subsets with stereotyped VH CDR3s using different, yet related, IGHV genes is not a peculiarity but a rather general phenomenon. Potentially, these related genes might also be prefavored for selection in response to specific Ags and amenable to stereotypy. In addition, it may mean that a few critical IGHV-encoded positions are important for defining the specificity of certain CLL Ig and, hence, their ability to recognize Ag and therefore to be selected. This is similar to what has been described for recombinant mAbs using CDR shuffling approaches42 and, very recently, for potent broadly neutralizing CD4-binding site anti-HIV Abs that mimic binding to CD4.43 Intriguingly, in the latter case, despite extensive SHM, the anti-HIV Abs shared a consensus motif of 68 aa in the VH domain and arose independently from 2 distinct yet related IGHV genes (IGHV1-2 and IGHV1-46, also used in major CLL stereotyped subsets, eg, 1 and 12).
Reviewing previous studies of BCR Ig stereotypy in CLL reporting on incrementally larger series of patients (starting from 255 and peaking at 2662 sequences),11⇓⇓⇓⇓⇓–17 a trend was apparent toward a rise in the percentage of stereotyped cases (from 8.6% up to 28%) along with the increase in the size of the series. Although not proportional to the increase of the cohort, in principle, this upward trend might continue to rise to an unknown figure if the series of cases available for analysis would continue to expand. The large size of our cohort—3 times the size of the largest published study17—enabled us to reliably investigate this issue and ascertain the mathematical relationship of BCR Ig stereotypy with cohort size. Our findings support the notion that not all CLL cases will eventually be clustered: in fact, only a fraction of CLL—approximately one-third of cases—could be assigned to subsets defined by stereotyped VH CDR3 with the most recent criteria, a ratio also supported by random simulations. Hence, overall, the present study reinforces our earlier claim that CLL can be subdivided into 2 broad categories: one with stereotyped BCR Ig and the other displaying heterogeneity in terms of BCR Ig sequence features.17
Given that some of the participating institutions of the present study are secondary referral centers and, thus, have a larger proportion of cases at advanced stages, the relative sizes of the IGHV-mutated and IGHV-unmutated subgroups reported here appear to differ somewhat from those found in everyday clinical practice (community CLL). Therefore, the relative frequencies of individual subsets may be slightly different from those expected in community CLL. That notwithstanding, considering that similar frequencies have been reported previously by us and others for several major subsets,12,14⇓⇓–17 it is reasonable to claim that, collectively, just a few subsets account for a sizeable proportion of the CLL cohort.
The recent identification of BCR VH CDR3 stereotypes in other B-cell malignancies, namely MCL31 and SMZL,32,33 abrogated the notion that stereotypy by itself is CLL-unique. Furthermore, it raised questions regarding the possible existence of common immune-mediated mechanisms of lymphomagenesis among different entities, reflected in shared Ag-binding sites.44 To address these questions, we took advantage of our recently published, large Ig sequence datasets from MCL31 and SMZL33 and performed cross-entity comparisons to search for commonalities between their Ag-binding sites that might allude to common immune pathways to lymphoma development. Through this approach, it became apparent that differences were fundamental regarding both VH CDR3 length and composition. We interpret this finding as an indication that distinct functional and/or developmental processes act to shape the respective Ig repertoires. A corollary of these comparisons is that the characteristics of VH CDR3 stereotypes in CLL can be considered as truly disease-biased. Needless to say, these rearrangements are expected to be present within the repertoire of normal B cells, as reported in the literature,35 however it remains scientifically interesting and unique that few specific ones are expanded repeatedly and consistently in the same lymphoma subtype.
A major such characteristic is the VH CDR3 sequence itself. In certain instances, CLL-biased sequence patterns extended over the entire VH CDR3 sequence (eg, in subset 10); in contrast, in other instances, only a few VH CDR3 amino acids seemed to be sufficient for defining a subset. The most impressive example of the latter case is subset 2, where subset assignment is reliable provided the following 3 requirements are met: (1) IGHV3-21/IGHJ6 gene association; (2) 9-aa long VH CDR3; and, most intriguingly (3) an acidic residue at VH CDR3 position 107. In other words, the defining features of subset 2 IGHV3-21 BCR are VH CDR3 length restriction and sequence restriction at a single landmark position. In keeping with previous reports for certain mAbs with defined specificities,45 these 2 features might be linked, in that VH CDR3 length restriction could ensure proper positioning of a specificity determinant within the VH CDR3 itself or of critical contacts between the heavy and light chains.
The finding of this and several other examples of CLL BCR stereotypes which are defined by constraints in VH CDR3 length and composition in a few critical positions, should in no way be taken to imply that the actual sequence of the entire VH CDR3 is less important for defining specificity toward the cognate Ag. However, it leaves open the possibility of selection of a paratope endowed with adequate plasticity for binding a range of diverse ligands, recalling what has been reported in the mouse for Abs against small haptens or carbohydrates, where a preferred length usually dominates for a given specificity.46
What could be the biologic and practical implications of these findings? From an ontogenetic perspective, it is worth underscoring the molecular analogies of the Ag-binding sites in CLL with stereotyped BCR Ig with those of murine B-1 cells. These cells have been reported to exhibit a different and considerably more restricted Ig repertoire than conventional B cells, distinctive for the frequent occurrence of identical Ig heavy and light chain rearrangement.47 Their repertoire and reactivity pattern seems to be stable within each species and even between species, likely reflecting evolutionary selection for specificities which would afford protection against pathogen invasion and also serve housekeeping functions of recognizing and removing everyday apoptotic material.47 Interestingly, a similar reactivity profile has been identified for CLL mAbs that can be polyreactive18,19 (in particular when unmutated) and have been shown to react with molecular structures present on apoptotic cells and bacteria.19⇓–21,48 Whether these observations and facts are relevant for CLL ontogeny, especially in view of a recent report describing human fundamental cells with functional properties of B-1 cells,49 is currently unknown and will require further multidisciplinary research.
From a practical perspective, the sheer size of the stereotyped CLL fraction has important potential clinical implications. This is supported by independent recent studies which have shown striking correlations between certain CLL stereotypes and disease presentation, course, or outcome, for example, young age at diagnosis and remarkably indolent course for subset 4,14⇓–16 significantly increased risk for Richter transformation for subset 8,50 or adverse clinical course for subsets 1 and 2,14,16 even compared with cases with similar IGHV gene mutational status. Hence, it seems reasonable to propose that reliable recognition of stereotypy may soon be instrumental in patient risk stratification, perhaps even beyond IGHV gene mutational status, and, eventually, form the basis for the development and implementation of "subset-specific" therapeutic protocols.
Contribution: A. Agathangelidis performed research, analyzed data, and wrote the manuscript; N.D., A.W.L., D.G.O., N. Chiorazzi, C.B., F.D., R.R., and P.G. designed the study and supervised research; A.H. performed research and analyzed data; X.B. and V.G. were responsible for the curation of IGHV-IGHD-IGHJ sequences; F.M., X.-J.Y., Z.D., E.J.v.G.-M., C.T., C.C.C., N. Cahill, B.T., L.B.P., L.F., L.B., A.J., and P.F.d.C. performed research; K. Smedby, A.T., and M.-P.L. supervised research; A. Anagnostopoulos, H.M.-B., N.L., G.J., S.P., and J.J. provided samples and associated clinicopathologic data and supervised research; and K. Stamatopoulos designed the study, supervised research, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Jesper Jurlander, one of the co-authors of the present contribution, unexpectedly passed away during the preparation of the manuscript. This work is dedicated to his memory.
Correspondence: Paolo Ghia, MD, PhD, Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milano, Italy; e-mail: ghia.paolo{at}hsr.it.
The authors thank the following members of the collaborating institutions for their help with sample processing: Vasiliki Douka, Hana Skuhrova Francova, Evangelia Stalika, Dennis Tielemans, Ashley van der Spek, Brenda Verhaaf, Joyce Vermeulen, and Ingrid L. M. Wolvers Tettero.
This work was supported in part by the ENosAI project (code 09SYN-13-880) co-funded by the European Union and the Hellenic General Secretariat for Research and Technology (N.D., K. Stamatopoulos); the Cariplo Foundation, Milan, Italy (P.G., K. Stamatopoulos); Program Molecular Clinical Oncology-5 per mille no. 9965 and Investigator grant, Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano, Italy (P.G.); the National Cancer Institute/National Institutes of Health RO1 CA81554 (N. Chiorazzi); the Nordic Cancer Union, the Swedish Cancer Society, the Swedish Research Council, the Uppsala University Hospital, and the Lion's Cancer Research Foundations in Uppsala (R.R.) for the analysis of the Swedish cases; and grant IGA MZ CZ NS10439-3/2009 by the Ministry of Health of the Czech Republic (S.P.) for the analysis of the Czech cases. A. Agathangelidis is the recipient of a grant from the A. G. Leventis Foundation (http://www.leventisfoundation.org/).
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 November 26, 2011.
- Accepted February 26, 2012.
- © 2012 by The American Society of Hematology


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During a recent visit to central Michigan I decided to stay a couple of extra days and poke around, see if there might be any fishing opportunities, and just hang out. By Alan Clemons, Southeast Managing Editor I'd also thought about doing a little hunting recon in the area. Found out there may be...
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SANTA ROSA -- To help strengthen local business access to global markets, keep jobs at home and provide North Bay companies with information they can use to expand and facilitate foreign trade, the Sonoma County Economic Development Board and the U.S. Department of Commerce plan to co-host a first-of-its-kind export seminar in Petaluma on May 9.
Using an "Export University" format, this event will take place at the Rooster Run Golf Course located at 2301 E. Washington St. in Petaluma from 8 to 11 a.m.
Key topics covered in the curriculum will include export logistics, trade financing, global marketing, as well as federal government export resources.
[caption id="attachment_72276" align="alignright" width="200"] Ben Stone[/caption]
"North Bay companies must have the tools they need to expand their sales into international markets," said Ben Stone, executive director of the EDB.
"Through this event those attending will receive practical export training and education provided by U.S. Commercial Service, the trade promotion arm of the U.S. Department of Commerce's International Trade Administration, on how to market to Asian, African and South American markets, among others."
U.S. Commercial Service trade professionals in over 100 U.S. cities and in more than 75 countries help U.S. companies get started in exporting or increase sales to new global markets.
[caption id="attachment_72277" align="alignleft" width="200"] David Rabbitt[/caption]


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Pokemon Riddles
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June 25th, 2011 (6:35 PM).
Team Rocket's Rockin'
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Okay, this is the last time I'm reminding you guys. ONE GUESS AT A TIME. I see it again, I'm just going to say your wrong, okay?
But yes, Beldum, next riddle:
I rattle and echo loudly in the mountains.
But only when my species becomes my position.
I have a tail, arms, legs. a head...
...and perhaps fangs but few know and even those knowing don't really know.
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Ultimate Honeymoons
: Madikwe Hills, South Africa
Today we're kicking off a new series of recommendations for ultimate honeymoon escapes in Virgin Atlantic destinations. First up, an unforgettable and truly romantic safari adventure at Madikwe Hills Private Game Lodge in South Africa...
Where in the world
Madikwe Game Reserve is 75,000 hectares of untamed African bushveld, nudging up against the border with Botswana in North West Province. Teeming with wildlife, it's home to the Big Five and dozens of other mammals including black and white rhino and the extremely rare African wild dog, as well as more than 300 species of bird.
Despite being one of the nation's largest reserves, development has been closely monitored. Guests of Madikwe's high-end lodges have the place to themselves; day visitors are largely absent due to the distance from any major population centre, and the relative remoteness and sense of escape is a huge part of Madikwe's appeal.
The lodge
Madikwe Hills is built upon a rocky outcrop with sweeping views over miles of open plain. The lodge was discreetly designed to blend almost imperceptibly into the surroundings - huge granite boulders are a major feature of the property, all of which were left completely undisturbed, even if it meant having to build around them as was the case in the double-height bar and some of the bathrooms.
Vast, well-spaced and utterly private glass-fronted suites are scattered across the hillside, camouflaged by tamboti-beamed thatch roofs. Wooden walkways lit by hurricane lamps criss-cross the grounds and connect the secluded suites to the central open-plan lounge, bar, wine cellar, library and dining area up the slope, where cooling fans make slow rotations and the giant twisted trunks of leadwood trees hold up the vaulted ceiling.
This is the beating heart of the lodge, selectively decked out with paintings and pottery, maps and magazines, telescopes, textiles, tea chests and travel trunks. Calabash-shaped lanterns hang from the rafters over the bar and low-lit table lamps brighten corner nooks. It's where we gather for turbo-charged coffees in the pre-dawn light before setting out on the early morning game drive, and reconvene for laid-back cooked breakfasts afterwards.
A few steps below, a large shady verandah looks out over the nearby watering hole. After a lazy morning we're back here for lunch, accompanied by bright yellow weaverbirds who swoop low over the small infinity pool and land in the trees above us. Elephants lift their trunks and blow sand across their scorching backs just a few dozen feet beyond our table. Later in the day we're back again, for a wine-fuelled, post-sunset dinner illuminated by nothing other than candelight and the stars above.
Accommodating just 24 guests, the lodge feels both exclusive and intimate; service is friendly rather than fawning, staff address you by name and you can be as sociable or as cut-off as you wish. The morning and evening game drives are the highlight of most people's stay at Madikwe but if you choose to stay rooted to your suite for the day, no-one will bat an eyelid. This is your honeymoon, after all.
What makes it special?
We love the suites, which are all supremely spacious and very hard to leave. A cosy living room with huge slate-clad chimney (snuggle up in front of an open fire on cooler nights) leads into a peaceful bedroom with gigantic pillow-strewn four-poster bed draped in generous layers of muslin, from where it's possible to lie and gaze in wonder as herds of wandering animals make their way to the watering hole.
Better still, throw open all the floor-to-ceiling windows and retreat to a sunbed in the shade of an acacia tree, or grab a towel and jump into your own personal plunge pool for the ultimate in relaxed game viewing - you'll be amazed at what passes by just a hundred feet or so from your deck; in the space of an hour we watched large groups of wildebeest, elephants and zebra come and go.
The candle-filled, cathedral-like bathrooms are ultra luxurious. A large deep tub takes centre stage in front of fold-back windows that reveal the same wilderness views and a cavernous step-up circular rain shower is built into the opposite wall - but that's not all. Just outside the far door is an open-air extension with a private, bamboo-enclosed outdoor shower (with hot water) perfect after prolonged plunge pool dips.
As the sun goes down, there's nothing better than a candelit bath open to the elements, accompanied by a soundtrack of whirring cicadas and the plaintive call of some unfamiliar bush bird. Some nights, you may even find a freshly run bath when you return to your room, a trail of rose petals leading the way. If you manage to nab Madikwe's honeymoon suite, there's an extra special treat in store - a claw-foot bath tub built into a well-concealed outdoor corner for secluded soaks under African skies. There is absolute privacy here, and it really is quite a feat to tear yourself away for the game drives...
Beyond the lodge
Aside from the pleasures of the lodge itself, the game drives are the undisputed highlight of any visit to Madikwe Hills. Even being woken at 4.30am can't quell the enthusiasm and anticipation. Jumping into the back of the 4 x 4 and heading out the gates as the first streaks of pale pink appear on the horizon is a magical experience, made even more exciting by sudden stops and off-road turns when your ranger or tracker spots something interesting.
If you're lucky you'll see everything you came here to see, and your ranger and tracker will go out of their way to make it happen. Close to the end of our drive one morning, we got word of a leopard far away on the other side of the reserve. Did we want to go for it? Of course we did, and moments later we were pelting through the bush in hot pursuit, bumping over rocks and leaning inwards to avoid thorn trees, hanging on for dear life.
As the sun set on another drive we sat, spellbound, as a lioness lazed in the fading light with her five cubs playing around her. We saw a four-hour old elephant cross its first road and pursued a pack of wild dogs on a kill. We watched a pack of vultures attack the remains of a recently-deceased hippo, learned about the complexities of a termite mound and discovered why red-billed oxpeckers feed off the ticks on a kudu. This is the real deal; no-holds-barred safari country where nothing is laid on - or taken away - for tourists. Because of this, just being here is an incredibly stirring, moving experience.
Madikwe Game Reserve is a straightforward 4-hour drive from Johannesburg, though many opt for the quick domestic flight to Madikwe's airstrip, where you'll be met and escorted to the lodge. Madikwe is a malaria-free reserve so there's no need for pills.
All meals are included in your stay, as are two game drives per day and the services and expertise of a designated ranger and tracker.
Evening meals are generally taken as a group with your ranger and tracker, though you are welcome to dine in private on your deck. Prices start at around £360 pp per night in low season, though various free night offers are available - contact Virgin Holidays for details.
And don't miss our interview with Madikwe Hills ranger Jacques Snyman for the lowdown on life in the bush.
Virgin Atlantic operates a daily flight to Johannesburg from London Heathrow. Virgin Holidays can organise the ultimate Madikwe Hills honeymoon stay and any other arrangements you require.
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About the author: MaxineMaxine Sheppard
Maxine is the editor of the Virgin Atlantic blog. Travel and music are her joint first loves, and despite having written for Virgin for more years than she cares to remember she still loves nothing more than jumping on a plane in search of new sights and new sounds.


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US law firm teams up with Kuwait's MM Al-Ghazali & Partners
US-based law firm Squire, Sanders & Dempsey is enhancing its ability to serve clients with interests in post-conflict Iraq by partnering with a Kuwaiti law firm and adding a lawyer who has Middle East experience.
Squire Sanders, which currently represents companies rebuilding Iraq, is partnering up with Kuwait's MM Al-Ghazali & Partners, a full-service international law firm established in 1992. In addition, Daniel Q. Callister, legal counsel for the Kuwait-America Foundation, has joined Squire Sanders as of counsel.
"Mishari M. Al-Ghazali and his multi-national team of lawyers from the Middle East will enable us to provide legal counsel, and equally important, make introductions for clients entering Iraq or otherwise doing business in the Gulf Region," said Squire Sanders', Joseph P. Markoski.
Squire, Sanders & Dempsey, founded in 1890, is a US-based international law firm with more than 750 attorneys and 27 offices strategically located around the world. The has firm offices in the Americas, Europe and Asia. The firm also has associations with legal firms in Bucharest and Dublin. — (menareport.com)
© 2003 Mena Report (www.menareport.com)
Top Headlines
- ECC teams up with US engineering firm to aid Iraqi children
- Madouni to join Algeria National Team
- Delta Partners – the regions first top management advisory and investment firm in telecommunications opens its doors in Dubai Internet City
- Oxford business group signs agreement with al-Sarraf & al-Ruwayeh as its official legal research partner
- Al Tamimi & Company Wins International Law Office 'Client Choice Awards 2007'


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Results 1 to 5 of 22
Thread: Joss as a nickname for Joseph?
February 15th, 2014 09:11 PM #1Senior Member
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Joss as a nickname for Joseph?
Still debating between a few names for our baby boy, who may arrive any day. We like the name Joseph, but aren't crazy about the nicknames Joe or Joey. I know we could just call him Joseph, like we call Andrew by his full name, but would like a nickname alternative. We like Jay, so that might work, but recently heard Joss for Joseph, and really like it.
What do you all think of Joss as a nickname for Joseph?Mom to: Andrew and Benjamin (identical twins), born April 16, 2012; and Alexander, born February 25, 2014.
February 15th, 2014 09:19 PM #3Senior Member
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February 15th, 2014 09:20 PM #5Senior Member
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February 15th, 2014 09:29 PM #7
Yes!!! Joss is so much better than Joe as a nn for Joseph. I dissagre with the poster above, the only Jocelyns I know just go by their full name, and I would not assume Joss is a girl (I do like Joss as a nn for Jocelyn, but I consider Jocelyn unisex anyway, so I wouldn't assume it was a girl either way). Joss Whedon is a perfect example. His full name is Joseph. Joss is awesome. I'm in love with this nn. Go for it. I love Joseph with Andrew and Benjamin btw, a fabulous sibset. Congrats and good luck!Just another teenberry and writer, a lover of names
Current Favorites:| Zoe | Jane | Cecily | Marta | Ariadne | Petra | Elizabeth | Eulalie | Susannah | Margo |
| Linus | Jonas | Basil | Jasper | Augustine | Jude | Thomas | Ezra | Gus | Augustus |
Pondering: | Hermione | Miriam |
Have a fabulous day!
February 16th, 2014 12:55 AM #9


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Murder in the Collective
Introducing Pam Nilsen, her twin sister Penny and the Best Printing collective, a print-shop hovering on the brink of a merger with lesbian-owned B. Violet Typesetting. When sabotage and murder erupt, Pam follows a trail of suspects. The author won the Crime Writer's Association Award in 1992.
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Google didn't help finding a picture of a type of public bike rack that I saw in Holland, where every other bike is raised a bit so that the handlebars don't clash and so, more bikes can be parked in the same amount of space.
Here's what happens when all the bikes are parked at the same height:
Here's what happens when every other bike is parked just slightly higher so that the handlebars aren't at the same height:
Do you know where I could find a picture of that kind of public parking rack so that I could show it to someone and make myself understood?
Thank you.


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Suburban, suburban
Mexican Cuisine
Specialty Hamburgers
International House of Pancakes
Pancakes, Sandwiches, Dinner Entrees
Olive Garden
National chain Italian restaurant
Rates quoted in currencies other than hotel's base currency are subject to the rate of international exchange.Rates may change throughout the stay. Rate may reflect average nighly rate. Extra charges may apply for children. Incidental charges, such as room service, are not included in the rate.
The ameneties and/or facilities may not be located at this hotel. but within a reasonable distance and additional charges may apply for use. All information has been provided by this hotel.


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This mixtape was recorded, mixed and mastered over the course of three weeks at the Brim Dog Studios here in Honolulu, Hawaii. P.O.M.E.R came through all the way from Brooklyn, NYC and delivered dynamic and energetic performances over both new school and classic boom bap beats. Alot oh hard work went into this project so we hope you take a listen and enjoy!
BrewKon – Bad Art Vol. 2
BrewKon is back with the second installment in the "Bad Art" series of beat tapes. Volume 2 features a unique blend of synth based and sample based productions. Click the cover art for a free HQ download of the tape, or check out the embedded SoundCloud links below. Feel free to use any of these instrumentals for your own productions. Enjoy!
I just heard about these dudes out of NYC and they are definitely doing their thing. This track delivers solid bars and a good dose of hype; I'm feeling it for sure. I think its dope how we're starting to see the resurgence of the hip hop group because its just more interesting to hear how different flows and vocal tones can come together. Keep your eyes on these dudes, I think they're starting to bubble and for good reason. Subscribe to their YouTube channel for more videos and cop their mixtape on BandCamp.


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You've been invited to a fancy ball but the only thing you have to wear is an orange wooly jumper. What shoes do you wear?
I think I will skip the ball. Not my kind of scene, anyway and I look like a beach ball in orange wooly jumpers.


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Project-based learning is a mainstay at Patrick F. Daly School of the Arts, located in Red Hook, Brooklyn. STEM educator Jenna Utter engages students from the 2nd to 5th grades and enhances learning with her classroom Makerspace. Using five MakerBot 3D printers and Tinkercad, she's able to reinforce content from lessons and incorporate concepts from all areas of STEM. Learn how a MakerSpace in the classroom can make STEM learning interactive, interesting, and enjoyable for students across grade levels by downloading this case study.
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The latest is "hosted" solutions. Hosted or cloud computing and hosted VoIP telephony services are being touted as the best thing since sliced bread.
From a telephony perspective, I find it interesting because the concept of a hosting in the telephony world is not new. The phone company has been offering Centrex service for decades, in fact they still do. With Centrex, customers would in essence, rent space in the Central office PBX. Each user would have their own 10 digit telephone number that would ring directly on their telephone. Users could "intercom" each other by dialing the last 4 digits of the desired telephone number in the network. Calls could even be transferred to off-site locations.
In its day Centrex was quite advanced. It offered features that were not available anywhere else. However, it did come at a cost. Like hosted VoIP, Centrex was basically a rental. Each telephone number cost subscribers a monthly fee and did not include long distance toll charges, which back then were quite significant.
On the plus side, there was no equipment on-site, which in theearly days of PBX's took up quite a bit of space and required significant air conditioning and electricity to operate. But, a small company with say 20 to 40 employees would spend the equivalent of a new phone system every few years for the cost of Centrex service.
With the advent of digital PBX telephone systems, however, with their small footprint, built-in features and lower operating costs the Centrex model of a telephone number for each employee didn't seem to make sense.
That brings me back to hosted VoIP service. It is basically Centrex over the Internet. In my opinion, the economic model is the same, a monthly rental for equipment and now included telephone service for each employee. However, on-site digital PBX equipment can offer the same or more features, with significantly lower operating costs.
But to me, the biggest misconception is the implementation of hosted VoIP over existing Internet connections. There are some companies claiming to save users thousands of dollars a year by using their existing broadband Internet connection to also provide hosted VoIP service.
Remember the old adage: "If it seems too good to be true….. it probably is". In my opinion, hosted VoIP service should be delivered over a dedicated T1, use quality routers from name manufacturers, have quality IP phones, not low cost knock offs and have a hosting company that can prove redundant capability and offer quality support staff.
Telephones are a mission critical system to any business. Yes, we all want to save money wherever possible. But when there is a problem with the telephone, the savings seem less important. So, save money prudently, not foolishly.
Stevens Communications, Inc. has been serving the Connecticut business community since 1983. For over 32 years, we have been helping local businesses grow and prosper. In today's ever-changing world, it's important to know your vendor has a long and stable track record.


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I have a 30 year old ranch house with three bathrooms. Bathroom #1 is in the basement and has new plumbing and a new single lever shower facet. Bathroom # 2 has a powers Hydrogaurd handicap single lever shower faucet. These two bathrooms are about 8-10 ft. from the hot water tank and water supply. Bathroom #3 has been newly remodeled. There is not problem with bath #3.It is approx 20ft. from the water source and hot water tank and has a new shower faucet in it. The reason I am writing to you is that I have a problem with bath #1 and #2. When I use the cold water I get great water pressure in both the tub and shower option, but when I turn to add hot water the pressure drops to about half. I have taken apart the insides of the faucet and have not found any debris. I don't believe the pipes have corrosion in them because I still have the problem in the #3 bath and this bath is new and the pipes are clean. The water pressure in all the bathroom sink faucets and kitchen sink are good. It seems that the hot side of the two bath showers are the problem. I have owned the home for 4 years, in the past bathroom #2 had good pressure, the pressure problem was there before bathroom #2 was put in. I have normal city water, no water softener is involved. I would like your help in solving this problem..
Sounds like corroded hot water pipes. I have the same problem in my house with the original bathrooms. When Im in the shower and someone turns on the water anywhere in the house, doesnt matter if its the hot or cold, the shower turns cold. In this case I think its the faucet which may be first generation anti scald. I know for a fact that the galvinized pipes are about half the diameter they were when new from all the rust and crap in our water.
Pedestal sink plumbing....UUGGHH!!
Plumbing - My hot water is not working
plumbing moans when the water is turned off
plumbing in 1910 farmhouse
- 15 Old House Features We Shouldn't Abandon
- 13 Lazy Cleaning Tricks for a Spotless Home
- Laundry Room Ideas to Knock Your Socks Off
- Insanely Easy 60-Minute Home Improvements
- 12 Sheds You Could Live (or Work) In
- Assembly Required: 15 DIY Kit Homes
- 7 House Sounds You Never Want to Ignore
- 10 Surprisingly Simple Woodworking Projects
- Worth It: 8 Renovations That Pay You Back
- Organize Your Life with 12 Dollar-Store Buys
- 9 Totally Amazing Mobile Home Makeovers
- Don't Make These 7 Mistakes in Small Spaces
- 20 Sneaky Storage Ideas
- 15 Totally Unexpected DIY Flooring Alternatives
- 7 Easy Budget-Friendly Backyard Makeovers
- 10 Closet Cures That Cost Less Than $100
- 11 Easy DIY Projects to Declutter Your Home
- 10 "Zero Dollar" Garden Hacks
- 10 Killproof Plants for a No-Effort Landscape
- 9 Insanely Easy 1-Hour Backyard Projects


text: Can anyone tell me where can i download achievements on Xbox 360? I have tried going online on my xbox 360 and tried to get the new achievements that are available but cannot get it? So, please tell me whether is there any tips of tricks to get the achievements? It should be available in the download content manager but it isnt? Thanks for any information.


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Prior to having our ignition switch replaced, our Tahoe horn would honk when attempting to turn key unless lights were activated before turning key. Shop is saying that this is after market and feature not offered. We purchased new with Starter Interrupt System and Passlock Theft deterrent. How can I verify that this feature exists with horn honking when trying to turn key on?
by maxeyguy in Cypress, TX on May 04, 2011


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Call opens August 2016
The Research Innovation Awards Scheme has replaced our previous Project & Pilots grant round to drive us forward in our bold new strategy. This call is for bold, innovative research that has the real potential to impact the way prostate cancer is diagnosed, treated or even prevented.
All proposals must align with our new research strategy and must clearly demonstrate the innovative nature of the research and how it will lead to improved health and wellbeing of men affected by, or at high risk of, prostate cancer. The application process and assessment of your proposal will reflect our increased focus on innovativeness and potential impact and you should make every effort to detail how your proposal meets those criteria.Guidance notes


text: Posted 19 Aug 2012 This is just out and a good article about Panthers! "It's the equivalent of playing 'Madden,'" Williams recently said at Panthers training camp. "It was like having a running quarterback when (Michael) Vick was with Atlanta. Everybody would just go five wide (receivers) and run it with the quarterback. Good article hope it gives little insight into team didn't see it postedit not posted http://msn.foxsports.com/nfl/story/carolina-panthers-cam-newton-sophomore-slump-big-expectations-081412 2 people like this Share this post Link to post Share on other sites


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Recognizing Jerusalem at Issue in Passport Case
Recognizing Jerusalem at Issue in Passport Case
428 results for ' obama administration'. Showing results 289 - 306
Recognizing Jerusalem at Issue in Passport Case
Obama Admin.: No FDR Prayer on WWII Memorial
The Tea Party: Extreme or Mainstream in 2012?
US Tunnel Vision Strengthening Jihad against America
The Tehran Initiative: Destroying the 'Great Satan'
White House Calling for Global Support against Iran
Top US Envoy Attacked by Mob in Syria
Saudi King: Women Can Vote in Four Years
Obama: Peace Talks Only Road for Statehood
Emails Show White House Warned About Solyndra
White House in Hot Spot over Solar Company Loan
Texas Crews Make Gains Against Wildfire Near Austin
White House: NATO Must Search for Libya WMDs
White House Calls for Syria's Assad to Resign
Obama Admin: Healthcare Ruling Won't Stand
U.S. Hits Syrian Bank, Cellular Firm with Sanctions
Catholic Hospitals Object to Law's Free Birth Control
Economic Growth Slower than Expected


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A Rocky Mount city councilman has been charged with assault stemming from a scuffle after a city council meeting Monday night.
Andre Knight was served with a criminal summons Tuesday and has a January 14th court date.
Police chief John Manley says he had to step in between Knight and Herbert Moore after the meeting.
Manley says he did not arrest the councilman at the time because he felt it was more appropriate to simply mitigate the situation. Moore later pressed charges at the magistrate's office.


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Friends of Grand Rapids Parks Presents
For the Greenest Bash of the year
August 27, 2015 • 7 o'clock
Plante Moran/Christman Building at
Fish Ladder Park
Indulge in a Green Gala cocktail, local beer, or Michigan wine while visiting the photo wall. Check out the silent auction and bid on unique items and experiences.
Learn more about our community's amazing parks and projects while feasting on delicious locally-sourced food during the peak of Michigan's harvest season.
Get your final auction bids in, then head to the dance floor to let loose to the amazing beats of DJ Adrian "AB" Butler.
The Green Gala is a fun cocktail party so dress to impress. See the photos and video to get inspired. Also, check out what local vendors have to offer.
Parking is available on the two lots between 4th and 6th Streets in front of Riverview Center (678 Front NW) and the Plante & Moran/Chistman Buildings (634 Front NW).
BY BUS Route 7 is the best option for this destination. Service stops at 11.00 PM so plan your trip accordingly.
By BIKE Use the Riverfront Trail System. There is covered bike parking available at the event.
Adtegrity, Amway Grand Plaza Hotel, Rosalynn Bliss, CWS, Doorganics, Erhardt Construction, Experience Grand Rapids, Franklin Partners, Laurie & Don Gardner, Grand Rapids Public Museum, Grand Rapids Public Schools, John Hunting, Long Road Distilllers, Miller Johnson, Padnos Iron & Metal Co., Rockford Construction, Milt & Barbara Rohwer, Tower Pinkster, TypeTerrance, United Bank of Michigan, Williams Kitchen & Bath


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Click on a thumbnail to go to Google Books.
Home to Harmony (2002)
by Philip Gulley
No current Talk conversations about this book.
The author, like his narrator, is a Quaker minister in a small midwestern town. The writing is lovely. Each chapter has a moral, but it is very gently delivered. Lots of humor and situations that are universal.
This is a series a friend suggested I read and I am glad I did!
I must say it's refreshing to read comedy that is intelligent. Yes ladies and gentlemen, it IS possible to be funny without the use of profanity.
Mr. Gulley brings his characters to life in bold colors and the town has something that should appeal to everyone.
And...as much trouble as his congregation gives him, I find myself wishing I could move to Harmony.
The world needs more communities like this!
Wholesome, heartwarming with a sense of humor you wouldn't expect from a Quaker minister. It was just what I needed after the last book. It's a simple quick read. Gulley's writing style is very easy to like, as is his personality which shines through it.
"Church would be a wonderful thing, if it weren't for all the people." This is a common quip among pastors and church members alike sometimes, when the quest for spiritual growth runs up against the bureaucracy of the church and the stubbornness of some church members.
Sam Gardner, the Quaker pastor in Philip Gulley's charming and very funny Home to Harmony (the first of Gulley's "Harmony Novels"), would certainly agree. By an unlucky coincidence, Sam finds himself called to pastor the church in which he grew up, located in small town Harmony, Indiana. In his first year, he adjusts to life in his hometown, dealing with an assortment of odd, yet endearing, church folk.
This book at once celebrates many components of American small town life: the local diner, the local newspaper, the church, the special annual events. It gently presents the challenge any newcomers face when they try to move into a small town. It shows the power of small town gossip and the pervasive watchfulness, and subsequent discussion, that follows almost every action in a small town.
But Home to Harmony is squarely focused on the eccentricities of the local Quaker congregation. Go to any church long enough, and you will recognize the denizens of the Harmony Friends Meeting. The unspoken seating chart in the sanctuary. The ill-advised but earnest plans of a couple of the elders. The frustrations of church meetings. The underlying power of the women in determining what's really important on the church calendar.
Gulley is a pastor (as am I) and he knows these people and these situations. It is clear that they have frustrated him over his career. But it is also clear that he loves them: he loves them for their earnest, if misdirected passion, loves them for their subconscious assumptions, loves them for their quiet moments of honor and love.
The book is filled with gentle humor, and is a pleasure to read. In fact, it is such an easy and enjoyable read that Gulley's skill is not immediately apparent. In the midst of these almost self-contained chapters, each of which ends with a delicate moral (sometimes posed more as a question than as an assertion), Gulley gives life to the fictitious town of Harmony and many of its citizens.
References to this work on external resources.
Wikipedia in English
Amazon.com Product Description (ISBN 0060858370, Mass Market Paperback)
Welcome to Harmony ...
In this acclaimed inaugural volume in the Harmony series, master American storyteller Philip Gulley draws us into the charming world of minister Sam Gardner in his first year back in his hometown, capturing the essence of small-town life with humor and wisdom.
(retrieved from Amazon Thu, 12 Mar 2015 18:23:58 -0400)
Sam Gardner, pastor of Harmony Friends Meeting, recounts the moving and humorous adventures of his first year leading the hometown Indiana church.
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How to Steer a Boat on a Straight Line Course in Fog Without a Compass or GPS
It is well known that when steering a boat in a fog without instruments one tends to steer in a circle. Years ago I hired a water taxi to ferry me to my cabin located on a directly opposite shore about three miles across the water, Once underway in a dense fog I determined the skipper had neither compass nor GPS. "No mater" he said, "I know my way around these waters." After about 10 minutes of cruising, we arrived back at our departure point. Here is how we solved the problem.
This article has been tagged as a stub.
That means it's off to a good start, but still has room to grow into a more helpful resource. Until the article reaches its full potential, it will be hidden from search results. Can you help it flourish? If you think the article offers complete and accurate instructions, feel free to remove this tag.
Community Q&A
Ask a Question
- Have a spare 10 feet (3.0 m) or longer line aboard your boat
- Equip your boat with the proper instruments such as compass and GPS
Things You'll Need
- A 10–20 feet (3.0–6.1 m). line (or longer as it can be attached to the stern and trailed at the desired length


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The 2009 Washington Homecoming Parade was held Friday afternoon in downtown Washington.
The Washington High School band, class floats, school clubs, local dance teams, a fire truck and the homecoming court moved down Main Street.
Homecoming's scheduled for Friday evening at Washington High School, as the Pam Pack takes on White Oak.


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Fragile Things CD: Stories (Compact Disc)
A mysterious circus terrifies an audience for one extraordinary performance before disappearing into the night, taking one of the spectators along with it . . . In a novella set two years after the events of American Gods, Shadow pays a visit to an ancient Scottish mansion, and finds himself trapped in a game of murder and monsters . . . In a Hugo Award-winning short story set in a strangely altered Victorian England, the great detective Sherlock Holmes must solve a most unsettling royal murder . . . Two teenage boys crash a party and meet the girls of their dreams—and nightmares . . . In a Locus Award-winning tale, the members of an exclusive epicurean club lament that they've eaten everything that can be eaten, with the exception of a legendary, rare, and exceedingly dangerous Egyptian bird . . . Such marvelous creations and more—including a short story set in the world of The Matrix, and others set in the worlds of gothic fiction and children's fiction—can be found in this extraordinary collection, which showcases Gaiman's storytelling brilliance as well as his terrifyingly entertaining dark sense of humor. By turns delightful, disturbing, and diverting, Fragile Things is a gift of literary enchantment from one of the most unique writers of our time.
About the Author
Neil Gaiman is the New York Times bestselling author of the novels Neverwhere, Stardust, American Gods, Coraline, Anansi Boys, The Graveyard Book, and Good Omens (with Terry Pratchett); the Sandman series of graphic novels; and the story collections Smoke and Mirrors and Fragile Things. He is the winner of numerous literary honors, including the Hugo, Bram Stoker, and World Fantasy Awards, and the Newbery Medal. Originally from England, he now lives in America.


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ACCOUNTING: A leading insurance brokerage company located on Hilton Head Island is seeking a full-time staff accMore info
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CONSTRUCTION: KITCHEN/BATH Remodeling Firm Seeking Experienced Individuals or Subs: Dry Wall, Paint, Tile, CabinetMore info
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SKILLED LABOR - TRADES: Local Heating and Air Company is looking for a TECHNICIAN. Honest hard working individual with a ValMore info
HEALTH CARE: Summit of Beaufort Assisted Living 843-770-0105 Full time MEMORY CARE DIRECTOR Must have a basicMore info
ADMIN - CLERICAL: EXPERIENCED RECEPTIONIST Full-Time needed for Veterinary Medical Practice in Beaufort. Email resumeMore info
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ADMIN - CLERICAL: Part-time legal assistant needed from 9am-3pm, Monday-Friday. Must be able to type and transcribe usMore info
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UPDATE (03/04): Due to inclement weather, the construction work on KY 480 in Bullitt County for this weekend has been postponed.
This work and the temporary closure of KY 480 is scheduled from 6 a.m. Saturday, Mar. 12, until 5 a.m. Monday, Mar. 14.
Motorists who usually travel Cedar Grove Road on the weekends should be warned.
Harlyn Edward Nall, 92, of Pioneer Village, died on Tuesday, March 1, 2011.
SHEPHERDSVILLE - When John Schmidt graduated law school, Burlyn Pike was there to hire him at his Shepherdsville legal practice.
A list of hot topics posted by the Better Business Bureau for the month of March. For more information visit www.bbb.org.
BROOKS - Each year the students at Brooks Elementary show more heart towards charitable donations.
SHEPHERDSVILLE - A California company may soon be distributing its line of swimwear from a distribution facility in Bullitt County.
LOUISVILLE - Facing some major adversity, the Bullitt Central Lady Cougars put up a strong effort on Tuesday before falling 48-46 to Holy Cross in the opening round of the Girls' Sixth Region Tournament at Atherton High School.
SHEPHERDSVILLE - The Shepherdsville Police Department is continuing an investigation of a fatal accident in the 2800 block of Coral Ridge Road.
At approximately 9:10 p.m. yesterday, Vernon Jewell, 40, of Louisville, was struck and fatally injured as he walked along Coral Ridge Road by a vehicle driven by Michael Jewell, brother of the deceased.
None of the passengers in the vehicle were injured.
SHEPHERDSVILLE - Last Friday turned out to be a good day for many in the Bullitt County hoops community as the Coaches' Sixth Region awards were announced in the state's largest newspaper.
LOUISVILLE - It wasn't the state title that she had dreamed about, but North Bullitt senior Caroline Lee closed out a stellar high school swimming career on Saturday at the Kentucky State Swimming and Diving Championships with a pair of top six finishes.
Copyright © 2016
The Pioneer News Online is your source for local news, sports, events and information in Bullitt County and Shepherdsville, KY, and the surrounding area.


text: If art, architecture and 1000 years of history are drawing you to Prague, you'll love our Old Town hotels. A settlement from medieval days, once surrounded by a moat, it's one of the quaintest and most impressive-looking areas of Prague. Wander the narrow cobblestone lanes around the Old Town Square and see the Astronomical Clock and the Old New Synagogue. Book one of our hotels in Old Town Prague and get a real feel for this ancient city's oldest region.Read More


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Other hotels nearby
A bit too far from the city center. The Internet in the room was weak. Breakfast is common.See all 52 Hotels.com verified guest reviews
Too much noise in The night. Due to very loud music played at The on deck bar. I warn all travellers of that hotel.See all 53 Hotels.com verified guest reviews
Situated in Malacca, Sunshine Inn Malacca is in the business district and close to Portuguese Settlement, A Famosa, and St Paul's Church. Also nearby are St. John's Fort and Hatten Square.
Hotel Features.
Complimentary wireless Internet access is available in public areas. This is a smoke-free property.
The 17 air-conditioned guestrooms at Sunshine Inn Malacca include coffee/tea makers and hair dryers. Complimentary wireless high-speed Internet access is provided. Rooms are equipped with LCD TVs with premium satellite channels.
hotel is filthy and is not a very pleasant stay for me and my friends.See all 3 Hotels.com verified guest reviews
This hotel location is great as it is in central location to shopping malls n Jonker street. Room size is small but it is clean . Breakfast variety is...See all 26 Hotels.com verified guest reviews
It was a pleasant stay, but my room toilet was without the toilet seat. Location of hotel was good as it was outside the traffic jam area. Too expensi...See all 21 Hotels.com verified guest reviews


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Although Transfer and Off-Campus Student Life does not deal directly with transportation issues, here is some helpful information. Please direct transportation inquiries to The Department of Transportation Services at http://www.dots.umd.edu.
Looking for convenient ways to get to and from the University of Maryland? Check out the following links based on your transportation needs.
Getting to University of Maryland
By Metro
The closest metro to College Park is the College Park Metro. People may also stop at Greenbelt Metro station and take a metrobus to campus, or the Prince George's Metro, which also has a Shutte UM bus.
Greyhound Bus Lines offers long distance bus travel throughout the United States. Just as with Amtrak, there are student discounts with Student Advantage card holders.
Washington Deluxe provides direct bus service to and from DC and New York City.
Apex Bus offers direct bus service between DC, New York, Philadelphia, Atlanta, and Richmond, Virginia.
There are three international airports serving the area: Baltimore/Washington International Airport(Airport Code:BWI), Washington Dulles International Airport (Airport Code: IAD), and Ronald Reagan National Airport(Airport Code: DCA).
Check out the information below for transportation options and information for all three airports
Baltimore/Washington International Airport (1-800-IFLYBWI)
You can access the BWI Airport in a variety of ways. You can take the B30 Metrobus from Metrorail's Greenbelt station on the green line. The easiest way to access the B30 Metrobus from campus is by taking the College Park Park Metro Shuttle bus.
There is also rail transportation with Amtrak and Marc between BWI Airport and the Union Station on Metrorail's red line. You can take to Metrorail to the red line and get off at Union Station. From there, board and transfer to the Marc train and get off at the BWI Rail Station. There is a FREE shuttle from the Marc station to access the airport. The Union Station Ticket Office number is (202) 906-3104 and the BWI Rail Station number is (410) 672-6169.
If you live in or near Howard County, Howard County Transit has the red express route that provides bus service to BWI Airport via the Marc/Amtrak station and the airport terminals.
If you already live in the Baltimore area, you can get to BWI by taking the Penn Station Marc train.
Washington Dulles International Airport (1-703-572-2700)
To access Dulles Airport from Metrorail, you can take the West Fall Church-VT/UVA station on the orange line and transfer to the Washington Flyer bus service. You can also call (888) WASHFLY.
Metrobus 5A serves Dulles Airport, operating between Metrorail's L'Enfant Plaza Station on the yellow line and Dulles. There are stops at Metrorail's Rosslyn station on the orange line and the Herndon-Monroe Park and Ride.
Ronald Reagan National Airport (1-703-417-8000)
Metrorail goes directly to National Airport. There is a Ronald Reagan Washington National Airport station on the yellow line.
Ground Transportation
All three airports provide shuttle service. Contact the airport you plan to use for further information.
Your browser does not support inline frames or is currently configured not to display inline frames
Carpooling and Ridesharing
Forming a Carpool
The Maryland Transit Administration (MTA) (800-492-3757) and Commuter Connections at 800-745-RIDE also provide information on carpooling and can assist in matching people interested in carpooling.
Regional Park and Ride Lots
If you are looking for a convenient place to meet others for a carpool or canpool, you may want to locate a nearby Park and Ride lot. For a list of lots and locations, visit the Commuter Connections web site.
Guaranteed Ride Home
For those who utilize ridesharing programs or public transportation, the Guaranteed Ride Home program through Commuter Connections can provide you with a reliable ride home when an unexpected emergency arises. Through this program, commuters can get home for unexpected personal emergencies or unscheduled overtime up to four times per year. Best of all, the ride home by taxi, rental car, bus, or train is FREE. Participants register in advance and must reside and work within the designated regions of the program.
Student Parking Appeals
If you receive a campus parking ticket, you have the opportunity to appeal it with either Prince George's District Court (oral review) or the University of Maryland (written appeal). By marking "University of Maryland, College Park" on your ticket, your appeal will be heard by the Student Parking Appeals Office, a part of the Office of Student Conduct staffed by graduate and undergraduate students and is not a part of the Department of Transportation Services (DOTS).
your appeal must be filed with DOTS within 15 days of the violation. Do not send appeals directly to the Student Parking Appeals Office. You may mail your appeal in the envelope provided with your ticket or drop it off in the DOTS lobby on the first level of the Regents Drive Parking Garage.
Out-of-State Vehicle Registration
Commuter students residing in Maryland and are from out of state with vehicles must do one of two things to comply with Maryland motor vehicle law:
Register their vehicles with the State of Maryland Motor Vehicle Administration (MVA) OR Obtain a non-resident permit.
To register a motor vehicle in Maryland: one must go to any full-service MVA branch and ask for a title application. The applicant must have the following vehicle documentation: (1) a certificate of title from the state that the vehicle is currently registered in, (2) a certificate of inspection, (3) insurance information from a Maryland licensed insurance company, (4) title fee of $23, (5) lien fee of $20, (6) registration fee that varies depending on type and weight of vehicle, and (7) excise tax that also varies.
To obtain a non-resident permit: Students may obtain an application for a non-resident permit by visiting any full-service MVA branch office or by calling 1-800-950-1682. The following items are necessary to obtain a non-resident permit: copy of student ID or student registration to prove student status, vehicle insurance information, and a copy of the current vehicle registration. The fee for obtaining a non-resident permit is $20. Students may also purchase a permit from the College Park Municipal Center for $31.
For further information and location of MVA offices, call 1-800-950-1682 or visit MVA.
For more information on Maryland Vehicle Registration please visit:
For information on Washington, DC Vehicle Registration please visit:
For information on Virginia Vehicle Registration please visit:
Motor Assistance Vehicle
The Motor Assistance Vehicle is a free service offered by the Department of Transportation Services for current students, faculty, staff, or visitor on the campus of the University of Maryland at College Park experiencing automobile difficulties. Services which include jump starts, lockouts, tire changes, inflations, and gas transports are available for cars located on University property only.
For service, call 301-314-4CAR. Be ready to give the dispatcher your exact location within the lot on campus, a detailed description of your car including make, model, color, license plate number, and type of problem experienced. Raise your hood if possible and stay with your vehicle until the Motor Assistance Vehicle arrives.
About The Stamp
Food, Shops, and Services
Maps Directions Parking
Hours of Operation
University Book Center
Calendar of Events
Green Choices
Student Organizations
3972 Campus DriveCollege Park, MD 20742301-314-3375Comments and Feedback


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Also Known as: banknewport.com
doxo is not an affiliate of BankNewport. Logos and other trademarks within this site are the property of their respective owners. Learn how doxo works.
BankNewport FAQ
- Does BankNewport offer online bill pay?
- Where can I login to my online BankNewport account?
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- Does BankNewport offer a mobile app?
- What's the best way to contact customer support for BankNewport?
Contacting BankNewport
Options for reaching BankNewport customer service.
Online Links
Phone Numbers
|Contact Phone
|Support Phone
|(877) 709-2265
|Bristol Branch
Email Addresses
|Contact Email
|Support Email
Locations and Addresses
|HQ Address
|330 County Rd
Barrington, RI 02806
|Bristol Branch
|6 Gooding AvE
Bristol, RI 02809
General information about BankNewport
|Checking & Savings, Mortgage, Home Equity Loan, Auto Loan, Loan (Other), and Credit Card
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- BankNewport can be paid online at their website. Click here to make a payment to BankNewport for BankNewport bill pay, or visit the BankNewport website at www.banknewport.com
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- You can login to your BankNewport account online on their site at: www.banknewport.com.
- I need help logging in to my BankNewport account. Where's the best place to get help?
- The support team for BankNewport can assist with your account access questions. View their contact information here: banknewport.com.
- Does BankNewport offer a mobile app?
- BankNewport customers with an iPhone or iPad can install the BankNewport for iOS app to login to their account via a mobile phone or tablet. There is no BankNewport app for Android available.
- What's the best way to contact customer support for BankNewport?
- Customer support for BankNewport can be reached online at banknewport.com. Reach BankNewport via phone at (877) 709-2265 or email at email@example.com
- Does BankNewport have any nicknames or is it known as anything else?
- No, BankNewport only seems to be known as "BankNewport".
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'Paranormal Activity' to Be Turned Into Virtual Reality Game
- Tue Jul 7, 2015, 7:26 AM GMT
Beast Media Group is developing a still-unnamed virtual reality game of the horror movie with a whole new storyline.READ MORE
After a young, middle class couple moves into what seems like a typical suburban "starter" tract house, they become increasingly disturbed by a presence that may or may not be somehow demonic but is certainly most active in the middle of the night. Especially when they sleep. Or try to.
Beast Media Group is developing a still-unnamed virtual reality game of the horror movie with a whole new storyline.READ MORE
Jason Blum, who executive produced 'The River' that featured a similar style, teams up with Naomi Despres to produce the new 'found-footage mystery' project.READ MORE
The movie star admits he's always nervous before scenes with ouija boards, crucifixes and other symbols of black magic and demon worship.READ MORE
'Twilight' franchise wins big at the Sunday, August 8 ceremony, grabbing eight gongs from 'New Moon' and additional three from 'Eclipse'.READ MORE
In Best Fantasy Film category, 'The Twilight Saga's New Moon' is up against 'Avatar', 'Alice in Wonderland' and 'Clash of the Titans'.READ MORE
The year of 2009 comes to a close as 'Paranormal Activity' became a phenomenon, rights to 'Terminator' franchise was put up for auction, and 'Up in the Air' won critics' hearts.READ MORE


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Browsing by Subject "Racial Identity"
Now showing items 1-2 of 2
Racial(ized) Identity, Ethnic Identity, and Afrocentric Values-Conceptual and Methodological Challenges in Understanding African American Identity (Journal of Counseling Psychology, Vol. 52, No. 4, 2009-03-30)
To Be or Not to Be Black: Problematics in Racial Identity ("To Be or Not to Be Black: Problematics in Racial Identity" In The Quest For Community And Identity: Critical Essays In Africana Social Philosophy, edited by R. Birt, 29-44. Lanham: Rowman and Littlefield Publishers Inc., 2009-03-30)


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Other projects made by So Sew Easy:
Offer some kudos...
Join the conversation...
Ask a question...
Add your comment...
What was your source for this sewing project?
Online Photo or Website
Name of website or photo McCalls 6074
What materials did you use? A black and white birds nest soft knit from Fabric Mart and a tissue paper thin Roxy cotton knit from Girl Charlee
What are you most proud of? I made a lot of alterations to the first dress, copied over to my pattern and then made it again. Do pop over to my blog post to read more about it here; http://so-sew-easy.com/mccalls-m6074-the-one-to-four-hours-dress/ It was really surprising how much difference the two fabrics made when it came to the fit and style of the finished dresses - more on the blog.
What advice would you give someone starting this project? I would suggest measuring the pattern pieces before deciding on what size to cut. I cut according to the pattern envelope sizing and it was VERY big. The skirt is also not as flared as the pattern envelope suggests, at least not in my fabrics.
Thanks for the inspiration!! I need some easy summer dresses for our Negril trip this year!!
very nice summer dress..love the style and fit
I wear this one all the time!
So very cute. Would love to try and make one.
will it work with peach printed silk fabric..no choice cause its hard to find knit fabric here in PNG.
No, this pattern has to be made with a stretch fabric, sorry.
Where can I find this pattern?


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