Predominant Nucleotide Research Articles

Characterization of the mitochondrial genomes of the Mexican endemic bats Corynorhinus mexicanus and Corynorhinus leonpaniaguae (Chiroptera: Vespertilionidae).

The genus Corynorhinus is composed of four recognized species: C. rafinesquii, C. townsendii, C. mexicanus, and C. leonpaniaguae, the latter two being endemic to Mexico. According to the IUCN, C. mexicanus is considered "Near Threatened", as its populations are dwindling and habitats are affected by anthropogenic disturbance. Corynorhinus leonpaniaguae has not been assigned to an IUCN Red List risk category due to its recent description. In this study, the mitochondrial genomes of C. mexicanus and C. leonpaniaguae were assembled and characterized in detail. The mitochondrial genomes (mtDNA) of C. mexicanus and C. leonpaniaguae have lengths of 16,470 and 16,581bp respectively, with a predominant nucleotide usage of adenine (31.670% and 31.729%, respectively) and thymine (26.15% and 26.18%, respectively). The mtDNA of C. mexicanus and C. leonpaniaguae is composed of 37 coding and non-coding elements: 22 transfer RNAs (tRNA), 13 protein-coding genes (PCGs), two ribosomal RNAs and a non-coding region, the control region, which has a length of 933bp and 1,149bp, respectively. All tRNAs exhibited a cloverleaf secondary structure, with the exception of trn-Ser1 which showed a deletion of the dihydrouridine arm in the two species. All PCGs are subjected to purifying selection, with atp8 being the gene showing the highest Ka/Ks value. These are the first whole mitogenomic resources developed for C. mexicanus and C. leonpaniaguae and enhance our knowledge of the ecology of these species and aid in their conservation.

Profile of Nucleotides in Chinese Mature Breast Milk from Six Regions.

This study measured the total potentially available nucleoside (TPAN) content in breast milk from six different regions of China as a part of the Maternal Nutrition and Infant Investigation (MUAI) study. A total of 631 breast milk samples were collected from healthy, lactating women with singleton, full-term pregnancies between 40 and 45 days postpartum in Changchun, Chengdu, Lanzhou, Shanghai, Tianjin, and Guangzhou. TPAN and free 5′-monophosphate nucleotide (5′-MNT) contents were determined by high-performance liquid chromatography. The TPAN content of the Chinese mature milk ranged from 11.61 mg/L to 111.09 mg/L, with a median level of 43.26 mg/L. Four types of nucleotides were identified, and the median levels of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), and adenosine monophosphate (AMP) were 22.84 mg/L, 9.37 mg/L, 4.86 mg/L, and 4.80 mg/L, respectively. CMP was the predominant nucleotide, accounting for 52.9% of the TPAN content, while free 5′-MNT accounted for 18.38% of the TPAN content. The distribution pattern of the TPAN content and level of the individual nucleotides were significantly different among the selected regions (p < 0.05), but the result showed no significant differences in the TPAN level in breast milk (p > 0.05). In addition, no correlation was reported between the geographic distribution and TPAN levels. This result showed that TPAN better reflects the level of total potential nucleosides in Chinese breast milk rather than 5′-MNT in free form. CMP, UMP, GMP, and AMP are the only 4 types of nucleotides reported in all detections. In addition, results revealed a large variation of TPAN levels in Chinese breast milk across six regions, so that the median value may not be the optimal fortification level of TPAN for Chinese infant populations.

The Quality and Functional Improvement of Retorted Korean Ginseng Chicken Soup (Samgyetang) by Enzymolysis Pre-Treatment with Cordyceps militaris Mushroom Extract.

This study aimed to investigate the functional and quality improvement of retorted Korean ginseng chicken soup that was hydrolyzed using a single extract from Cordyceps militaris (CM) mushroom, or in combination with bromelain, flavorzyme, or a mix of both. A total of 36 fat-trimmed breast meat from commercial broilers were hydrolyzed with one of six treatments, (1) flavorzyme as a positive control (PC), (2) no addition as negative control (NC), (3) crude CM extract (CME), CM extract prepared with either (4) bromelain (CMB), (5) flavorzyme (CMF), or (6) bromelain:flavorzyme mixture (CMBF) in a water bath at 55 °C for 2.5 h, and subsequently retorted at 121.1 °C, 147.1 kPa for 1 h. The highest antioxidant activity was observed in the CMB treatment (40.32%), followed by CMBF (34.20%), and CME (32.97%). The suppression of malondialdehyde ranged between 28 and 83%. The water-holding-capacity of the treated samples increased, ranging between 59.69 and 62.98%, and significantly tenderized the meat. The shear force decreased from 23.05 N in negative control to 11.67 N in the CMB samples. The predominant nucleotides across the samples were 5′-IMP and hypoxanthine, and the lowest was adenosine. The intensification of the taste properties was due to the increase of umami substances, both by 5′-nucleotides (5′-IMP, 5′-GMP) and free amino acids (FAAs), whereas the highest improvement was observed in the CMB group. Therefore, the hydrolyzation of Korean ginseng chicken soup using CM extract, prepared using bromelain, improves functional and quality profiles.

Effects of superchilling on quality of crayfish (Procambarus clarkii): water migration, biogenic amines accumulation, and nucleotides catabolism

SummaryEffects of refrigerated (3.5 ± 0.5 °C), iced (0 ± 0.5 °C) and superchilled (−3.0 ± 1.0 °C) storage on quality of crayfish (Procambarus clarkii) were evaluated by monitoring the water migration, microbial contamination, biogenic amines accumulation, and nucleotides catabolism during 21 days. The results indicated that superchilling was effective in accelerating the rate of water migration and reducing total volatile basic nitrogen, trimethylamine, total viable bacteria, Enterobacteriaceae and psychrotrophic bacteria of crayfish. Meanwhile, significant (P &lt; 0.05) lower putrescine, cadaverine and tyramine contents were observed in superchilled sample (SS), whereas histamine concentrations were at lower levels (0.76–2.31 μmol g−1) in all treatments. In addition, the predominant nucleotides degradation pathway in crayfish was the conversion of inosine monophosphate, which was accumulated massively (3.74 µmol g−1) in SS at the end of storage. According to the dynamic changes of inosine and hypoxanthine contents, the shelf life of SS was prolonged to 15 days.

Benzo[a]pyrene promotes progression in tongue squamous cell carcinoma.

Benzo[a]pyrene (B[a]P) is a member of the polycyclic aromatic hydrocarbon (PAH) family. Although the potent carcinogenicity of high-dose B[a]P has been extensively reported, the effects of long-term exposure to B[a]P on the progression of tongue squamous cell carcinoma (TSCC) are poorly understood. In the present study, TSCC cells were treated with 5 or 50nM of B[a]P for three months. The proliferation and chemoresistance of B[a]P-treated cells to 5-fluorouracil or cisplatin were detected by CCK8. The motility of the B[a]P-treated cells was evaluated with wound healing analysis, invasion assay, and three-dimensional culture in decellularized mouse tongue matrix. Xenograft assay was used to investigate the aggressiveness of B[a]P-treated cells. Immunofluorescence staining, terminal restriction fragment assay, and whole-genome sequence were used to determine the mutation spectrums. Long-term 50nM B[a]P-treated cells exhibited increased aggressiveness and chemoresistance to 5-fluorouracil or cisplatin. In addition, data from whole-genome sequencing demonstrated that C:T to A:T transitions were the predominant nucleotide substitutions occurred in 50nM B[a]P-treated CAL27 cells. Furthermore, 102 non-synonymous or stop-gain mutations were enriched in the extracellular-matrix-receptor interactive pathway. B[a]P exposure may contribute to genomic instability, and therefore, B[a]P may promote the progression of TSCC.

ARHGEF7 (β-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function.

Previous studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown. We used a proximity-based ligation assay, BioID, to identify guanine nucleotide exchange factors that activate Cdc42 in immortalized human podocytes. We generated podocyte-specific ARHGEF7 (commonly known as β-PIX) knockout mice by crossing β-PIX floxed mice with Podocin-Cre mice. Using shRNA, we established cultured mouse podocytes with β-PIX knockdown and their controls. We identified β-PIX as a predominant guanine nucleotide exchange factor that interacts with Cdc42 in human podocytes. Podocyte-specific β-PIX knockout mice developed progressive proteinuria and kidney failure with global or segmental glomerulosclerosis in adulthood. Glomerular podocyte density gradually decreased in podocyte-specific β-PIX knockout mice, indicating podocyte loss. Compared with controls, glomeruli from podocyte-specific β-PIX knockout mice and cultured mouse podocytes with β-PIX knockdown exhibited significant reduction in Cdc42 activity. Loss of β-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the prosurvival transcriptional regulator Yes-associated protein. These findings indicate that β-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream Yes-associated protein activities. This appears to be the first evidence that a Rho-guanine nucleotide exchange factor plays a critical role in podocytes.

Evolution of self-compatibility by a mutant Sm-RNase in citrus.

Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and avoids inbreeding in flowering plants. The most widespread SI system utilizes S-ribonucleases (S-RNases) and S-locus F-boxes (SLFs) as S-determinants. In citrus, SI is ancestral; Citrus maxima (pummelo) is self-incompatible, while Citrus reticulata (mandarin) and its hybrids are self-compatible (SC). Here we identified nine highly polymorphic pistil-specific, developmentally expressed S-RNases from pummelo that segregate with S-haplotypes in a gametophytic manner and cluster with authentic S-RNases. We provide evidence that these S-RNases function as the female S-determinants in citrus. Moreover, we found that each S-RNase is linked to ~nine SLFs. Analysis of 117 citrus SLF/SLFL genes revealed clustering into 12 types and evidence that the S-RNases and intra-haplotypic SLFs/SLFLs co-evolved. Our data are consistent with citrus having an S-locus comprising a S-RNase and several SLFs that fit the non-self-recognition model. We identified a predominant single nucleotide mutation, Sm-RNase, in SC citrus, which provides a ‘natural’ loss of function. We present evidence that SI-SC transitions due to the Sm-RNase, initially arose in mandarin, spreading to its hybrids and became fixed. Identification of an evolutionarily distant new genus utilizing the S-RNase-based SI system, >100 million years separated from the nearest S-RNase family, is a milestone for evolutionary comparative studies.

Genomic variations in the counterpart normal controls of lung squamous cell carcinomas.

Lung squamous cell carcinoma (LUSC) causes approximately 400 000 deaths each year worldwide. The occurrence of LUSC is attributed to exposure to cigarette smoke, which induces the development of numerous genomic abnormalities. However, few studies have investigated the genomic variations that occur only in normal tissues that have been similarly exposed to tobacco smoke as tumor tissues. In this study, we sequenced the whole genomes of three normal lung tissue samples and their paired adjacent squamous cell carcinomas.We then called genomic variations specific to the normal lung tissues through filtering the genomic sequence of the normal lung tissues against that of the paired tumors, the reference human genome, the dbSNP138 common germline variants, and the variations derived from sequencing artifacts. To expand these observations, the whole exome sequences of 478 counterpart normal controls (CNCs) and paired LUSCs of The Cancer Genome Atlas (TCGA) dataset were analyzed. Sixteen genomic variations were called in the three normal lung tissues. These variations were confirmed by Sanger capillary sequencing. A mean of 0.5661 exonic variations/Mb and 7.7887 altered genes per sample were identified in the CNC genome sequences of TCGA. In these CNCs, C:G→T:A transitions, which are the genomic signatures of tobacco carcinogen N-methyl-N-nitro-N-nitrosoguanidine, were the predominant nucleotide changes. Twenty five genes in CNCs had a variation rate that exceeded 2%, including ARSD (18.62%), MUC4 (8.79%), and RBMX (7.11%). CNC variations in CTAGE5 and USP17L7 were associated with the poor prognosis of patients with LUSC. Our results uncovered previously unreported genomic variations in CNCs, rather than LUSCs, that may be involved in the development of LUSC.

The sequence preference of DNA cleavage by T4 endonuclease VII

The enzyme T4 endonuclease VII is a resolvase that acts on branched DNA intermediates during genetic recombination, by cleaving DNA with staggered cuts approximately 3–6 bp apart. In this paper, we investigated the sequence preference of this cleavage reaction utilising two different DNA sequences. For the first time, the DNA sequence preference of T4 endonuclease VII cleavage sites has been examined without the presence of a known DNA substrate to mask any inherent nucleotide preference. The use of the ABI3730 platform enables the cleavage site to be determined at nucleotide resolution. We found that T4 endonuclease VII cleaves DNA with a sequence preference. We calculated the frequency of nucleotides surrounding the cleavage sites and found that following nucleotides had the highest incidence: AWTAN*STC, where N* indicates the cleavage site between positions 0 and 1, N is any base, W is A or T, and S is G or C. An A at position −1 and T at position +2 were the most predominant nucleotides at the cleavage site. Using a Sequence Logo method, the sequence TATTAN*CT was derived at the cleavage site. Note that A and T nucleotides were highly preferred 5′ to the cleavage sites in both methods of analysis. It was proposed that the enzyme recognises the narrower minor groove of these consecutive AT base pairs and cleaves DNA 3′ to this feature.

Genomic variations in paired normal controls for lung adenocarcinomas.

Somatic genomic mutations in lung adenocarcinomas (LUADs) have been extensively dissected, but whether the counterpart normal lung tissues that are exposed to ambient air or tobacco smoke as the tumor tissues do, harbor genomic variations, remains unclear. Here, the genome of normal lung tissues and paired tumors of 11 patients with LUAD were sequenced, the genome sequences of counterpart normal controls (CNCs) and tumor tissues of 513 patients were downloaded from TCGA database and analyzed. In the initial screening, genomic alterations were identified in the “normal” lung tissues and verified by Sanger capillary sequencing. In CNCs of TCGA datasets, a mean of 0.2721 exonic variations/Mb and 5.2885 altered genes per sample were uncovered. The C:G→T:A transitions, a signature of tobacco carcinogen N-methyl-N-nitro-N-nitrosoguanidine, were the predominant nucleotide changes in CNCs. 16 genes had a variant rate of more than 2%, and CNC variations in MUC5B, ZXDB, PLIN4, CCDC144NL, CNTNAP3B, and CCDC180 were associated with poor prognosis whereas alterations in CHD3 and KRTAP5-5 were associated with favorable clinical outcome of the patients. This study identified the genomic alterations in CNC samples of LUADs, and further highlighted the DNA damage effect of tobacco on lung epithelial cells.

A Single Nucleotide Polymorphism Is Involved in Regulation of Growth and Spore Formation of Bacillus anthracis Pasteur II Strain

Anthrax toxins and capsules, which are encoded by genes located on pXO1 and pXO2, respectively, are major virulence factors of Bacillus anthracis. Our previous studies demonstrated that exposure to high-temperatures is unable to abolish the pXO1 plasmid of the Pasteur II strain, but the growth of the strain was obviously slower than that of the Sterne strain and wild-type virulent strain. To elucidate a potential regulatory mechanism of slowing growth, we employed comparative genome and bioinformatic analysis and revealed a unique SNP (G to T) at the 143135 bp position in pXO1 that is possibly involved in the mediation of growth of Pasteur II. However, the T to G mutation in groR did not result in any change of the amino acid sequence. A predominant nucleotide G existed at the 143135 bp in pXO1 of 100 wild-type B. anthracis isolates and 9 isolates documented in GenBank, whereas T replaced G in pXO1 of the Pasteur II strain. Further analysis indicate that the SNP is located in a gene between 143042 and 143173 bp, and that it encodes a small protein of 43 amino acids and is termed as a growth regulator (GroR). Site-directed mutagenesis and gene deletion demonstrates that groR regulates the growth and spore formation of B. anthracis. Our results indicate that the pXO1 plasmid is involved in the regulation of growth and spore formation in B. anthracis.

Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction.

HIV-1 evades host defense through mutations and recombination events, generating numerous variants in an infected patient. These variants with an undiminished virulence can multiply rapidly in order to progress to AIDS. One of the targets to intervene in HIV-1 replication is the trans-activator of transcription (Tat), a major regulatory protein that transactivates the long terminal repeat promoter through its interaction with trans-activation response (TAR) RNA. In this study, HIV-1 infected patients (n = 120) from North India revealed Ser46Phe (20%) and Ser61Arg (2%) mutations in the Tat variants with a strong interaction toward TAR leading to enhanced transactivation activities. Molecular dynamics simulation data verified that the variants with this mutation had a higher binding affinity for TAR than both the wild-type Tat and other variants that lacked Ser46Phe and Ser61Arg. Other mutations in Tat conferred varying affinities for TAR interaction leading to differential transactivation abilities. This is the first report from North India with a clinical validation of CD4 counts to demonstrate the influence of Tat genetic variations affecting the stability of Tat and its interaction with TAR. This study highlights the co-evolution pattern of Tat and predominant nucleotides for Tat activity, facilitating the identification of genetic determinants for the attenuation of viral gene expression.

Different distribution of the common HFE variants between western and eastern Spanish populations evidences an independent haplotype gene flow

IntroductionHereditary hemochromatosis is a disease responsible for excess blood iron. The hemochromatosis gene has two predominant variants, H63D and C282Y single nucleotide polymorphisms. Our study aims to analyze the diagnostic utility of genotyping the 63 and 282 loci, and examine the geographic distribution of these mutations in Spain. Methods and materialsGenotyping was performed on 94 healthy control individuals and 324 patients suspected of hereditary hemochromatosis, and also biochemical test to 313 individuals in the patients group. ResultsThe comparison of allelic frequencies between East and West of Spain, as well as other countries located at a similar longitude, evidenced a west-east distribution gradient of the C282Y allele. In addition, heterogeneous distribution of the H63D mutation in Spain was observed. Patients who carried the 282YY genotype showed significantly higher biochemical parameters (ferritin>300μg/L, Fe>180μg/L, IST>60%, UIBC>355μg/L and CTFH>370μg/dL), which confirmed the correlation between the mutated homozygous genotype and the associated hemochromatosis phenotype. ConclusionOur results strengthen the importance of executing genetic tests to increase the efficiency of hereditary hemochromatosis diagnosis, which reveal an interesting variability among geographical regions.

A snapshot of the predominant single nucleotide polymorphism cluster groups of Mycobacterium tuberculosis clinical isolates in Delhi, India

Several attempts have been made to associate phylogenetic differences among Mycobacterium tuberculosis strains to variations in the clinical outcome of the disease and to drug resistance. We genotyped 139 clinical isolates of M.tuberculosis obtained from patients of pulmonary tuberculosis in North Delhi region. The isolates were analyzed using nine Single nucleotide polymorphism (SNP) markers, spoligotyping and MIRU-VNTRs; and the results were correlated with their drug susceptibility profile. Results of SNP cluster group (SCG) analysis (available for 138 isolates) showed that the most predominant cluster was SCG 3a, observed in 58.7% (81/138) of the isolates with 44.4% (36/81) of these being drug susceptible, while 16% (13/81) were multidrug resistant (MDR). Of the ancestral cluster SCG 1 observed in 19.5% (27/138) of the isolates, 14.8% (4/27) were MDR while 44.4% (12/27) were drug susceptible. SCG 2 formed 5.79% (8/138) of the isolates and 50% (4/8) of these were multidrug resistant (MDR). Spoligotyping subdivided the strains into 45 shared types (n=125) and 14 orphan strains. The orphan strains were mostly associated with SCG 3a or SCG 1, reflecting the principal SCGs found in the Indian population. SCG 1 and SCG 2 genotypes were concordant with the East African Indian (EAI) and Beijing families respectively. Central Asian (CAS) clade and its sublineages were predominantly associated with SCG 3a. No consistent association was seen between the SCGs and Harlem, T or X clades. The 15 loci MIRU-VNTR typing revealed 123/136 isolates to be unclustered, while 13 isolates were present in 6 clusters of 2-3 isolates each. However, correlating the cluster analysis with patient details did not suggest any evidence of recent transmission. In conclusion, though our study revealed the preponderance of SCG 1 and 3a in the M.tuberculosis population circulating in the region, the diversity of strains highlights the changes occurring within lineages and reemphasizes the importance of cluster investigations in extended studies.

Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: NEW AP-1 TRANSCRIPTION FACTOR SITE AND NEGATIVE REGULATOR

We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at -1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.

GENO-27GENOMIC CHARACTERIZATION OF PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA REVEALS PREDOMINANT NONSYNONYMOUS SOMATIC MUTATIONS OF MYD88 AND PIM1 GENES

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to CNS. Despite low tendency of systemic dissemination, its prognosis is poor with median overall survival of 4 years when treated with standard of care high-dose methotrexate-based chemoradiotherapy. Despite majority of PCNSL is diffuse large B-cell lymphoma (DLBCL), its response to therapy and biological behavior differ from the systemic disease, necessitating detailed investigation of its pathogenesis and genetic alterations. Here we performed whole-exome sequencing (WES) on 41 PCNSL and paired normal specimens as well as RNA sequencing. Seven cases were classified as germinal center B-cell-like (GCB) subtype (17%), while 34 cases were non-GCB. Progression-free survival was significantly longer in cases with GCB subtype as in systemic DLBCL (p = 0.027). WES yielded sequencing data with a mean coverage of 158Χ with 98% of the targeted bases covered by ≥20 independent reads. We identified a total of 17,385 somatic mutations, of which 62% were nonsynonymous single nucleotide variations. The predominant nucleotide substitution was a C > T transition (57.8%), especially in the context of the sequence GCG. WES revealed a substantial extent of aberrant somatic hypermutation (aSHM) with a high frequency of nonsynonymous somatic mutations especially in PIM1 (90.2%) and BTG2 (65.9%). Genes mutated other than aSHM include MYD88 (85.4%), most of which being L265P activating mutation. These mutations were not associated with GCB/non-GCB subtypes. Notably, mutations in a number of genes related to the NF-κB signaling pathway were also prominent. Copy number aberration analysis identified gains in chromosomes 1q, 7, 12, and 18q, and a loss of 6q. These results suggest that MYD88 and PIM1 mutations may play an important role in PCNSL development regardless of GCB subtypes, and NF-κB pathway could be the therapeutic targets of this intractable disease.

Abstract 3893: Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations

Abstract Primary central nervous system lymphoma (PCNSL) is defined as diffuse large B-cell lymphoma (DLBCL) confined to the central nervous system. Although PCNSL cannot be histologically distinguished from extracerebral DLBCL, its prognosis is quite poor. To gain insights into the transforming mechanism of PCNSL, we conducted whole-exome sequencing for 44 PCNSL specimens. Genomic DNA was extracted from tumors and their matched normal samples. Exome fragments were captured by using SureSelectXT Human All ExonV5+lncRNA, and subjected to deep sequencing with the HiSeq 2000 system. There were 8 cases with germinal center B-cell-like (GCB) subtype and 34 cases with non-GCB subtype. Whole-exome sequencing of tumor and paired normal yielded sequencing data with a mean coverage of 156X and 80X, with 98% and 99% of the targeted bases covered by ≥20 independent reads, respectively. Of 17,832 somatic mutations identified in the PCNSL specimens, 11,611 (65%) were nonsynonymous single nucleotide variations. Predominant nucleotide substitution was a C&amp;gt;T transition (57%) especially in the context of the sequence GCG. We also identified 798 insertions/deletions. Interestingly, MYD88 mutations were detected in 35 of 44 patients (79.5%), and PIM1 mutations were detected in 42 patients (95.5%). Also, a number of genetic aberrations were shown to activate the NF-κB pathway. These results suggest that MYD88 and PIM1 mutations have important roles in the development of PCNSL and could be the therapeutic targets of this intractable disorder. Citation Format: Motoo Nagane, Kazutaka Fukumura, Toshihide Ueno, Jeunghun Lee, Yukiko Shishido-Hara, Mitsuaki Shirahata, Kazuhiko Mishima, Koichi Ichimura, Akitake Mukasa, Yoshitaka Narita, Ryo Nishikawa, Hiroyuki Mano. Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3893. doi:10.1158/1538-7445.AM2015-3893

P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP‐1 Site and Negative Regulator

Recently we reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up‐regulation of tissue factor (TF), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R‐TF axis remains unclear. Here we report a role of a newly identified AP‐1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP‐1 site at ‐1363 bp of human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion and mutation of this new distal AP‐1 site all significantly supressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c‐Jun, ATF‐2 and Fra‐1, but not the typical c‐Fos, bound to the new AP‐1 site. In addition, loss‐of‐function studies using siRNAs confirmed a positive transactivation role of c‐Jun and ATF‐2, but unexpectedly revealed a strong negative role of Fra‐1 in P2Y2R‐induced TF up‐regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation, leading to Fra‐1 activation while JNK activated c‐Jun and ATF‐2. These findings reveal the basis for P2Y nucleotide receptor control of endothelial TF expression and indicate that targeting the P2Y2R‐Fra‐1‐TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.

Rifampicin resistance among multi-resistant MRSA clinical isolates from Chennai, India, and their molecular characterization.

High-level methicillin-resistant Staphylococcus aureus (MRSA) isolates show rapid evolution of rifampicin resistance, forcing reliance upon expensive and often inferior antibiotics to manage these infections. Accordingly, this study was conducted to: 1) evaluate the level of multidrug resistance among hospital-associated MRSA isolates from Chennai, India; 2) determine their rifampicin resistance and molecular characterization; and 3) analyze the identified rpoB gene mutations for predominant high-level rifampicin resistance-associated nucleotide changes. Conventional laboratory techniques and antibiogram evaluation by disc diffusion were utilized for isolate phenotypic identification. Among the 54 isolates obtained, 74% (42) were found to be MRSA. All the isolates exhibited complete susceptibility to linezolid and vancomycin, and variable resistance to conventional antibiotics; the MAR index value calculated was 0.64. Genotypic identification of the high-level rifampicin-resistant isolate S. aureus KM05 was established through rpoB amplification and sequencing. The evolutionary relationship of KM05 to other isolates and its rpoB gene mutation status was determined to understand the genetic basis of its high rifampicin resistance. The S. aureus KM05 rpoB gene yielded ≥ 98% sequence similarity and a close phylogenetic relationship with other known reference database organisms. It also showed mutational changes in three rpoB codon positions encoding amino acids at positions 455, 481, and 529. These results have established the prevalence of rifampicin resistance among Chennai hospital MRSA isolates, and suggest that the predominant high-level resistance nucleotide changes would serve to form a basis for their diagnosis and control of rifampicin-resistant MRSA.

Characterization of Hepatitis C Virus Genotypes by Direct Sequencing of HCV 5′UTR Region of Isolates from Saudi Arabia

The current study was designed to determine the Hepatitis C Virus (HCV) genotypes in a representative sample of HCV chronically infected patients in Saudi Arabia. All HCV isolates were genotyped by sequencing of the 5′UTR region and newly identified HCV isolates were identified. Specific universal primers targeting 5′UTR region were used for both amplification and sequencing of all isolates that resulted in 244 bp fragment which represent about 80% of 5′UTR region. Most of HCV isolates in this study were genotype 4 (76.4%) where only few isolates were recognized as genotype 1 (19.6%). All results were compared to HCV reference sequences from LOS ALAMOS HCV database, considering only the complete full genomes for the main phylogenetic analysis. Sequences that showed maximum identity (98% –100%) were selected. Most isolates were identical with HCV genotype 4 references. Some isolates were similar to different subtypes of HCV genotypes 4, 1 and 6. Phylogenetic analysis showed resemblance of most isolates to similar ones from the Far East, North America and Egypt. Using sequence Weblogo, Alignment analysis of isolated HCV genotypes 4 and 1 showed 92% and 95.5% nucleotide conservation, respectively. There was no predominant nucleotide in the varied sites, in both genotypes. All isolated sequences were submitted to GenBank database.