Carnosine Inhibits Growth of Cells Isolated from Human Glioblastoma Multiforme

The present study evaluates the effect of the naturally occurring dipeptide carnosine on primary cell cultures established from patients with glioblastoma multiforme. Surgically removed tumors were used to establish primary cell cultures that were incubated for 96 h with medium supplemented with carnosine at concentrations of 20, 40 and 50 mM. Following incubation, dehydrogenase activity, cellular adenosine triphosphate concentration (ATP), caspase activity, lactate dehydrogenase (LDH) release and the rate of DNA synthesis were determined. After 96 h of carnosine treatment a significant reduction in cellular ATP and dehydrogenase activity was detected already at a concentration of 20 mM carnosine. Carnosine (50 mM) reduced ATP concentration to 42.7 ± 13.5% (n = 6) and dehydrogenase activity to 41.0 ± 19.3% (n = 6) compared to untreated cells. Additional experiments revealed no sign of enhanced apoptosis or necrosis in the presence of carnosine. However, a quantitative bromo-desoxy-uridine-based proliferation assay demonstrated a clear effect of carnosine on DNA synthesis reducing its rate down to 50% (2 cultures) and 10% (4 cultures). Therefore, it can be concluded that carnosine is obviously able to inhibit proliferation of cells derived from glioblastoma. Since it is a naturally occurring substance that appears to be non-toxic to normal tissue and is able to penetrate the blood–brain barrier it may be a candidate for a therapeutic agent that may reduce proliferation of neoplastic cells even in vivo and especially in cases of glioblastoma multiforme.     

Correlation between Human Leukocyte Antigen Class II Alleles and HAI Titers Detected Post-Influenza Vaccination

Influenza is a major cause of morbidity and mortality. Despite vaccination, many elderly recipients do not develop a protective antibody response. To determine whether Human Leukocyte Antigen (HLA) alleles modulate seroprotection to influenza, a cohort of HLA class II-typed high-risk vaccine recipients was investigated. Haemagglutinin inhibition (HAI) titres were measured 14–40 days post-subunit vaccination. Seroprotection was defined as HAI titres reaching 40 or greater for all three vaccine strains. HLA-DRB1*04∶01 and HLA-DPB1*04∶01 alleles were detected at higher frequencies in seroprotected compared with non-seroprotected individuals. Thus, the presence of certain HLA class II alleles may determine the magnitude of antibody responses to influenza vaccination.     

Genetic Analyses of the Polarity Alleles in Recombinants from Mitochondrial Genetic Crosses

A number of minority recombinant and parental types from a heterosexual cross were analyzed for the omega allele they carry. It was found that recombinant progeny can be omega(-), that minority parental types among the progeny can be omega(+) rather than omega(-), and, finally, that certain of the results suggest that the omega locus may not be at the proximal end of the mitochondrial genetic map (Bolotin et al., 1971; Grivell et al., 1973) but rather may lie between the [cap1-r/cap-s] and [ery1-r/ery-s] loci.     

Construction and Application of a Korean Reference Panel for Imputing Classical Alleles and Amino Acids of Human Leukocyte Antigen Genes

Genetic variations of human leukocyte antigen (HLA) genes within the major histocompatibility complex (MHC) locus are strongly associated with disease susceptibility and prognosis for many diseases, including many autoimmune diseases. In this study, we developed a Korean HLA reference panel for imputing classical alleles and amino acid residues of several HLA genes. An HLA reference panel has potential for use in identifying and fine-mapping disease associations with the MHC locus in East Asian populations, including Koreans. A total of 413 unrelated Korean subjects were analyzed for single nucleotide polymorphisms (SNPs) at the MHC locus and six HLA genes, including HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1. The HLA reference panel was constructed by phasing the 5,858 MHC SNPs, 233 classical HLA alleles, and 1,387 amino acid residue markers from 1,025 amino acid positions as binary variables. The imputation accuracy of the HLA reference panel was assessed by measuring concordance rates between imputed and genotyped alleles of the HLA genes from a subset of the study subjects and East Asian HapMap individuals. Average concordance rates were 95.6% and 91.1% at 2-digit and 4-digit allele resolutions, respectively. The imputation accuracy was minimally affected by SNP density of a test dataset for imputation. In conclusion, the Korean HLA reference panel we developed was highly suitable for imputing HLA alleles and amino acids from MHC SNPs in East Asians, including Koreans.     

Establishment of a New Human Glioblastoma Multiforme Cell Line (WJ1) and Its Partial Characterization

(1) A new human glioblastoma multiforme (GBM) cell line, WJ1, was established from the tissue derived from a 29-year-old patient diagnosed with a grade IV GBM. (2) The WJ1 cell line has been subcultured for more than 80 passages in standard culture media without feeder layer or collagen coatings. (3) GBM cells grow in vitro with distinct morphological appearance. Ultrastructural examination revealed large irregular nuclei and pseudo-inclusion bodies in nuclei. The cytoplasm contained numerous immature organelles and a few glia filaments. Growth kinetic studies demonstrated an approximate population doubling time of 60 h and a colony forming efficiency of 4.04%. The karyotype of the cells was hyperdiploid, with a large subpopulation of polyploid cells. Drug sensitivities of DDP, VP-16, tanshinone IIA of this cell line were assayed. They showed a dose- and time-dependent growth inhibition effect on the cells. (4) Orthotopic transplantation of GBM cells into athymic nude mice induced the formation of solid tumor masses about 6 weeks. The cells obtained from mouse tumor masses when cultivated in vitro had the same morphology and ultrastructure as those of the initial cultures. (5) This cell line may provide a useful model in vitro and in vivo in the cellular and molecular studies as well as in testing novel therapies for human glioblastoma multiforme.     

Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus

Proteus species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The Proteus O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of Proteus isolates. Even though most Proteus O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative Proteus O antigen locus and demonstrated this region''s high degree of heterogeneity by comparing sequences of 40 Proteus isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative Proteus O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally, Proteus-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68 Proteus O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative Proteus O antigen locus, and we describe a novel molecular classification method for the identification of different Proteus serogroups.Proteus species are usually found in soil, water, and sewage and are well-known opportunistic pathogens that most commonly cause urinary tract infections (UTIs) in persons with anatomical and physiological defects of their urinary tracts (15, 28). This genus includes the five named species P. mirabilis, P. vulgaris, P. myxofaciens, P. penneri, and P. hauseri and the three unnamed Proteus genomospecies 4, 5, and 6 (20, 21). Among these, P. mirabilis, P. vulgaris, and P. penneri are the most common human pathogens (28). Among Proteus species, P. mirabilis is most frequently associated with UTIs and is a common cause of catheter-associated UTIs (12).Potential virulence factors and bacterial behaviors associated with the infection processes and disease, including swarming, growth rates, fimbria expression, flagella, and the production of hemolysins, ureases, proteases, and amino acid deaminases, in addition to the expression of lipopolysaccharide (LPS) antigens and capsular polysaccharides (CPSs), have been described in many studies (11, 18, 28). Both LPSs and CPSs have been considered to play an important role in the progression of UTIs, in addition to affecting both kidney and bladder stone formation (7, 25, 35). Furthermore, the LPS O antigen confers protection against serum-mediated bactericidal activity (13, 27), and bacterial LPS released from bacteria is a biologically active endotoxin that causes a broad spectrum of pathophysiological conditions, including septic shock (26). Recently, two additional virulence factors with cytotoxic and agglutination properties, the high-affinity phosphate transporter (Pst) and the autotransporter (Pta), have been described (1, 11).The O antigen located on the cell surface of Gram-negative bacteria consists of oligosaccharide repeats (O unit) that normally contain 2 to 8 sugar residues. The O antigen is one of the most variable constituents on the cell surface, due to variations in the types of sugars present and their arrangements and respective linkages, and is subject to intense selection by the host immune system and bacteriophages. The serological classification scheme established by Kauffman and Perch defines 49 different P. mirabilis and P. vulgaris O serogroups (10), and an additional 11 serogroups were later proposed (23). In the case of P. penneri, an additional 15 O antigen serogroups were described (16, 42; Z. Sidorczyk, K. Zych, K. Kolodziejska, D. Drzewiecka, and A. Zablotni, presented at the Second German-Polish-Russian Meeting on Bacterial Carbohydrates, Moscow, Russia, 10 to 12 September 2002). To date, the O antigen structures of 78 Proteus species have been described (unpublished data), and uronic acid, which can sometimes be substituted for amino acids, was identified as a component of the Proteus O antigen. Although acidic O-specific polysaccharides have been identified in most Proteus O antigens, a study of the genetic locus associated with Proteus O antigens has never been carried out.The genome sequence of P. mirabilis was published for the first time in 2008 (22). In this study, we characterized the putative O antigen locus by analyzing genomic sequences and confirming the locus heterogeneity by carrying out PCR-restriction fragment length polymorphism (RFLP) on 40 strains. Four putative O antigen gene clusters were sequenced and analyzed, and specific primers were identified for Proteus species by screening 79 Proteus strains, confirming that the identified loci were specific to particular serogroups.     

Mass Spectrometry of Human Leukocyte Antigen Class I Peptidomes Reveals Strong Effects of Protein Abundance and Turnover on Antigen Presentation

HLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins – those with at least fivefold higher density score than expected for their abundance – we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use.The highly polymorphic Human Leukocyte Antigen class I (HLA-I)¹ genes are encoded by three loci (HLA-A, B, and C) in a gene-rich region on chromosome 6. They produce up to six unique cell surface receptors that bind and present the so-called HLA class I peptidome, which consists of peptides derived from proteolysis of intracellular proteins. Their function is to reflect the health state of the body''s cells to CD8+ cytotoxic T cells. During thymic maturation T cells that react to self-peptides are eliminated (1), leaving T cells with the capability to recognize peptides from viruses and bacteria. This recognition is interpreted as a danger signal, leading to removal of infected cells. Transformed, preneoplastic and cancer cells also tend to display atypical self-peptides from mutated or excessively expressed self-proteins, known as tumor associated antigens (TAAs). Although HLA-I molecules are indispensable in prevention of disease, they also pose a substantial health problem by causing allergies (2), life-threatening autoimmune diseases (3), and the often fatal rejection of donor organs because of recognition of both major and minor histocompatibility antigens (4).Finding the rules for peptide generation and selection is regarded as the most important open issue in the field of HLA-I biology by leading experts (5). Although the antigen presentation pathway is well characterized, it is still unclear how basic properties such as protein abundance, turnover, and subcellular localization influence and shape the HLA-I presented peptidome (6–10). One expectation is that protein abundance should correlate with presentation (11), but previous studies have reported conflicting and contradicting results that mostly argue against a strong link (6, 7, 10, 12, 13). It is also not fully understood why only some HLA-sampled self-peptides from cancer antigens spontaneously activate T cells, whereas others do not.The majority of HLA-I peptides are derived from proteasomal degradation (5). Although the proteasome generates an excess of peptides, only some have the required sequence motifs for HLA binding, resulting in a selective sampling of available peptides (14). The presented peptides are typically nine amino acids long, but the length can range from eight to 15. The high degree of genetic variance of HLA-I receptors translates into allele-specific peptide-binding motifs defined by anchor positions, which are usually the second and the last positions in a peptide (15). Each cell has around 200,000 cell-surface-expressed HLA complexes, which bind about 10,000 unique peptide sequences (16). The affinity of a peptide toward the presenting HLA molecule does not correlate strongly with its immunogenicity, and neither does the number of presented HLA complexes (17). Instead, the most robust predictor of peptide immunogenicity appears to be the number of potential reactive T-cell clones (17–19).The longer the source protein, the higher the chances it will contain sequences that fit to a certain HLA motif, which would inflate the representation of longer proteins regardless of biological role. Furthermore, some HLA-I peptide sequences can be mapped to multiple proteins, potentially causing a problem in determining the number of observed HLA peptides per protein (13). This illustrates that careful accounting of the potentially and actually presented HLA peptides is important in properly delineating trends in propensity of peptide presentation.In cancer immunotherapy, T cells can be directed against tumors, based on the pattern of cancer associated HLA peptides. Therefore, there is great interest in determining the identity of these immunogenic peptides. Bioinformatic methods that attempt to predict HLA peptides of cancer proteins of interest are easily accessible and most commonly used. They typically score sequences with respect to proteasomal degradation, transport into the ER via the transporter associate with antigen processing (TAP) and binding to different HLA-I alleles (20). However, their precision success is modest (21, 22). The second approach is to directly capture the naturally presented peptides using mass spectrometry; however, this requires the relevant biological sample and sophisticated instruments and workflows, which have become accessible only recently for large-scale work (23–28). Although identification of cancer associated HLA peptides by MS, if performed stringently, establish the in vivo existence of the peptide, it still does not guarantee that it will elicit a potent T-cell response, which is required for further development into therapeutics (29). Therefore, like in the case of in silico predicted peptides, the immunogenicity of the peptides must in any case be tested empirically.We here present a rich and high confidence HLA-I peptidome, established by applying state-of-the-art mass-spectrometric techniques on a collection of seven cell lines. We investigate how abundance affects the propensity of proteins to be presented as measurable HLA peptides and whether or not there are specific protein classes that are overrepresented even independent of abundance. Likewise, we explore how to use in silico immunogenicity tools on the set of identified HLA peptides from cancer-associated proteins, with a view to select vaccine candidates.     

Descriptive Epidemiology of Somatising Tendency: Findings from the CUPID Study

Somatising tendency, defined as a predisposition to worry about common somatic symptoms, is importantly associated with various aspects of health and health-related behaviour, including musculoskeletal pain and associated disability. To explore its epidemiological characteristics, and how it can be specified most efficiently, we analysed data from an international longitudinal study. A baseline questionnaire, which included questions from the Brief Symptom Inventory about seven common symptoms, was completed by 12,072 participants aged 20–59 from 46 occupational groups in 18 countries (response rate 70%). The seven symptoms were all mutually associated (odds ratios for pairwise associations 3.4 to 9.3), and each contributed to a measure of somatising tendency that exhibited an exposure-response relationship both with multi-site pain (prevalence rate ratios up to six), and also with sickness absence for non-musculoskeletal reasons. In most participants, the level of somatising tendency was little changed when reassessed after a mean interval of 14 months (75% having a change of 0 or 1 in their symptom count), although the specific symptoms reported at follow-up often differed from those at baseline. Somatising tendency was more common in women than men, especially at older ages, and varied markedly across the 46 occupational groups studied, with higher rates in South and Central America. It was weakly associated with smoking, but not with level of education. Our study supports the use of questions from the Brief Symptom Inventory as a method for measuring somatising tendency, and suggests that in adults of working age, it is a fairly stable trait.     

Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low‐ and high‐grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low‐grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma‐specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC‐MS/MS. The identified proteins from the two cell types were quantified using label‐free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin‐based immunosorbent assay.     

Immunophenotypic and Ultrastructural Validation of a New Human Glioblastoma Cell Line

1. A human glioma cell line, NG97, was established by Grippo et al. in 2001 from tissue obtained from a grade III astrocytoma (WHO, 2000). In this first study, the cell line grew as two morphologically distinct subpopulations: dendritic/spindle cells and small round cells. The injection of NG97 cells into nude mice induced an aggressive tumor characterized by: severe cytological atypia, vascular proliferation and pseudopalisading necrosis (glioblastoma multiforme features). 2. The purpose of the present study was to characterize the immunophenotype and ultrastructural aspects of this cell line, using the parental tumor, cultured cells and the xenotransplant, in order to assess its glial nature and possible divergent differentiation. 3. NG97 cells and xenotransplant expressed the main neuroglial markers (GFAP, S-100 protein, NSE and Leu-7) and showed no aberrant expression of other histogenetic markers. GFAP was similarly expressed in the parental tumor and in the cells in culture, but decreased in the xenotransplant. NSE expression was reduced in NG97 cells, but substantially recovered in the xenotransplant. This variability in expression of GFAP and NSE was interpreted as either a phenomenon of dedifferentiation or to microenvironmental selection of specific subclones. S-100 was equally expressed in the three contexts. The xenotransplant's ultrastructural features were those of a highly undifferentiated tumor. No significant immunophenotypic or ultrastructural differences between the two morphologically distinct populations were found. 4. Thus, our data demonstrate that NG97 cells constitute a pure glial-committed cell line, which may prove useful as a malignant glioma model in studies addressing pathophysiological, diagnostic and therapeutic issues.     

Genetic Mechanism of Human Neutrophil Antigen 2 Deficiency and Expression Variations

Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3–5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases.     

Association of Human Leukocyte Antigen Class II with Susceptibility to Primary Biliary Cirrhosis: A Systematic Review and Meta-Analysis

Purpose

Several previous studies suggested that HLA-Class II may be associated with susceptibility to primary biliary cirrhosis (PBC), but data from individual studies remain controversial. Therefore, a systematic review and meta-analysis is needed to comprehensively evaluate the association between HLA-Class II and PBC risk.

Methods

All published reports of an association between HLA class II and PBC risk were searched in PubMed, EMBASE (updated to 22 May 2012). ORs with 95% confidence intervals (CIs) were extracted from each included study and the meta-analysis was performed using the fixed- or random-effects model.

Results

A total of 3,732 PBC patients and 11,031 controls from 34 studies were included in the meta-analysis. An assessment of study quality revealed that the majority of studies included (18 studies) were of high quality. The serological group DR8 was found to be a risk factor for PBC (OR = 2.82, 95%CI: 1.84–4.30). At the allelic level, HLA-DR*08 and HLA-DR*0801 were identified as risk factors for PBC (OR = 2.30, 95%CI: 1.76-3.00; OR = 3.23, 95%CI: 2.22–4.70, respectively), whereas HLA-DR*11 and HLA-DR*13 were potent protective factors (OR = 0.31, 95%CI: 0.27-0.38; OR = 0.62, 95%CI: 0.48-0.81, respectively). HLA-DQB1 and HLA-DQB1*0402 conferred a predisposition to PBC development (OR = 3.47, 95%CI: 2.35–5.13), whereas HLA-DQB1*0604 was protective against PBC (OR = 0.3, 95%CI: 0.18–0.58). No HLA-DPB1 allele was observed to be associated with PBC susceptibility (P > 0.05).

Conclusions

The present study revealed that HLA-Class II components are closely associated with the development of PBC.     

New Genetic and Linguistic Analyses Show Ancient Human Influence on Baobab Evolution and Distribution in Australia

This study investigates the role of human agency in the gene flow and geographical distribution of the Australian baobab, Adansonia gregorii. The genus Adansonia is a charismatic tree endemic to Africa, Madagascar, and northwest Australia that has long been valued by humans for its multiple uses. The distribution of genetic variation in baobabs in Africa has been partially attributed to human-mediated dispersal over millennia, but this relationship has never been investigated for the Australian species. We combined genetic and linguistic data to analyse geographic patterns of gene flow and movement of word-forms for A. gregorii in the Aboriginal languages of northwest Australia. Comprehensive assessment of genetic diversity showed weak geographic structure and high gene flow. Of potential dispersal vectors, humans were identified as most likely to have enabled gene flow across biogeographic barriers in northwest Australia. Genetic-linguistic analysis demonstrated congruence of gene flow patterns and directional movement of Aboriginal loanwords for A. gregorii. These findings, along with previous archaeobotanical evidence from the Late Pleistocene and Holocene, suggest that ancient humans significantly influenced the geographic distribution of Adansonia in northwest Australia.     

A Systematic Review of MicroRNA in Glioblastoma Multiforme: Micro-modulators in the Mesenchymal Mode of Migration and Invasion

Glioblastoma multiforme (GBM) is an incurable form of brain cancer with a very poor prognosis. Because of its highly invasive nature, it is impossible to remove all tumor cells during surgical resection, making relapse inevitable. Further research into the regulatory mechanism underpinning GBM pathogenesis is therefore warranted, and over the past decade, there has been an increased focus on the functional role of microRNA (miRNA). This systematic review aims to present a comprehensive overview of all the available literature on the expression profiles and function of miRNA in GBM. Here, we have reviewed 163 papers and identified 253 upregulated, 95 downregulated, and 17 disputed miRNAs with respect to expression levels; 85 % of these miRNAs have not yet been functionally characterized. A focus in this study has been 26 interesting miRNAs involved in the mesenchymal mode of migration and invasion, demonstrating the importance of miRNAs in the context of the cellular niche. Both oncogenic and tumor-suppressive miRNAs were found to affect target genes involved in cell migration, cytoskeletal rearrangement, invasiveness, and angiogenesis. Clearly, the distinct functional properties of these miRNAs need further investigation and might hold a great potential in future molecular therapies targeting GBM.