Correlation of preoperative ROMA scores with clinical stage in epithelial ovarian cancer patients.

PURPOSE: The significance of the Risk of Ovarian Malignancy Algorithm (ROMA) in differentiating benign and malignant ovarian lesions has been evidenced. In our clinical work, we found that advanced ovarian cancer were accompanied commonly with high ROMA scores. Thus, this study aimed to clarify the performance of ROMA in different disease stage of epithelial ovarian cancer (EOC) prior to surgery. METHODS: Carbohydrate antigen (CA125) and human epididymis protein 4 (HE4) levels and ROMA scores in 221 patients with FIGO stage I, II or III/IV stage EOC were analyzed. The positive rates of CA125, HE4 and ROMA at each disease stage were calculated. Their cutoff values, sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) for distinguishing patients with FIGO stage I/II from those with FIGO stage III/IV were estimated via ROC curves. RESULTS: Serum CA125 and HE4 levels and ROMA scores rose significantly with advancing stage. ROMA and CA125 were significantly elevated more frequently in comparing with HE4 in EOC patients at with the same stage. Based on ROC curves, the cutoff values for FIGO stage III/IV EOC were 110 IU/mL, 126 pmol/L, 78 and 68% for CA125, HE4, premenopausal and postmenopausal ROMA, respectively. ROMA was the strongest predictor of FIGO stage, with the highest specificity, accuracy, and PPV, which were 84.4, 82.5, and 87.0% for postmenopausal patients, 89.3, 85.6, and 74.3% for premenopausal patients. CONCLUSIONS: Our data suggest high ROMA scores correlated with advanced ovarian cancer prior to surgery. These observations suggest potential utility of ROMA in the comprehensively preoperative evaluation of EOC patients.

Complete chloroplast genome sequence of cultivated Morus L. species.

The complete chloroplast genome (cpDNA) sequences of two cultivated species of Morus L. (Morus atropurpurea and Morus multicaulis) are reported and reconstructed in this study, and were compared with that of wild Morus mongolica. In M. atropurpurea, the circular genome is 159,113 bp in size and comprises two identical inverted repeat (IR) regions of 25,707 bp each, separated by a large single-copy (LSC) region of 87,824 bp and a small single-copy (SSC) region of 19,875 bp. The cpDNA sequence of M. multicaulis is longer than that of M. atropurpurea (159,154 bp), and consists of two IRs (25,678 bp), a LSC region (87,763 bp), and a SSC region (20,035 bp). Each cpDNA contains 112 unique genes including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes, with a GC content of 36.2%. There were 83 simple sequence repeats (SSRs) with mononucleotides being the most common (60) and di-, tri-, tetra-, and hexanucleotides appearing less frequently in M. atropurpurea. M. multicaulis contains 81 SSRs containing 63 mononucleotide repeats. The genes and SSRs identified in this study may enhance understanding of cpDNA evolution at both intra- and interspecific levels. MEGA 6.0 was used to construct a phylogenetic tree of 27 species, which revealed that M. atropurpurea and M. multicaulis are more related to their congeners than to others. The cpDNA of M. atropurpurea and M. multicaulis and its structural analysis are important for the chloroplast genome project, development of molecular markers for Morus species, and breeding of varieties.

A SNP in the 3'-UTR of HSF1 in dairy cattle affects binding of target bta-miR-484.

The heat shock transcription factor 1 gene (HSF1) plays a key role in the heat stress response. We previously found a single nucleotide polymorphism (SNP) in the 3'-untranslated region (g.4693G>T) of HSF1 that was related to thermo tolerance in Chinese Holstein cattle through association analysis. However, it is not known whether other SNPs also affect thermo tolerance.In this study a novel SNP, g.1451G>T, was identified by DNA sequencing and genotyped using creating restriction site-polymerase chain reaction methodology. The g.1451G>T polymorphic site met Hardy-Weinberg equilibrium (P > 0.05). Association analysis demonstrated that this SNP had no effect on thermo tolerance traits in Holstein cattle. Findings of the study compared to the analysis of g.4693 G>T further indicated that g.4693 G>T may play an important role in thermo tolerance, although the mechanism is not clear. RNA hybrid and Targetscan prediction showed that the minimum free energy hybridization of bta-miR-484 with HSF1 3'-UTR was -31.9 kcal/mol and g.4693 G>T was in the seed sequence of bovine HSF1 that binds to bta-miR-484. Analysis by Luciferase assay indicated that HSF1 expression was directly targeted by bta-miR-484 in HEK 293T cells, and the Rluc/luc ratio of wildtype (GG) was lower than that of the mutant (TT) (P < 0.05). These results suggest that g.4693 G>T affects binding of HSF1 to bta-miR-484.

Identification and characterization of a novel splice variant of the PLCζ1 gene in bull testis tissues.

Phospholipase C zeta 1 (PLCζ1), which transcribes a key protein, has an important function in oocyte activation and embryo development because PLCζ1 can trigger a series of intracellular Ca2+ oscillations in mammals. In this study, a novel splice variant in the testis tissues of adult and fetal Chinese Holstein bulls was characterized by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analysis. The novel splice variant PLCζ1-sv1 was derived from the PLCζ1 complete transcript (PLCζ1-complete) by alternative splicing; the alternative splicing pattern exhibited alternative 5'-splice sites. The full-length transcript, PLCζ1-complete, is the main transcript found in fetal and adult cow testis tissue. Quantitative real-time PCR (qPCR) analysis demonstrated that the expression levels of the PLCζ1-complete transcript were significantly higher than those of the PLCζ1-sv1 splice variant in bovine testis tissues. PLCζ1 protein sequencing analysis showed that the amino acids at positions 453 to 457 were deleted in PLCζ1-sv1, thereby terminating transcription prematurely. In summary, this study provided information to elucidate the structure and function of the bovine PLCζ1 gene.

Novel SNPs of the mannan-binding lectin 2 gene and their association with production traits in Chinese Holsteins.

The mannan-binding lectin gene (MBL) participates as an opsonin in the innate immune system of mammals, and single nucleotide polymorphisms (SNPs) in MBL cause various immune dysfunctions. In this study, we detected SNPs in MBL2 at exon 1 using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing techniques in 825 Chinese Holstein cows. Four new SNPs with various allele frequencies were also found. The g.1164 G>A SNP was predicted to substitute arginine with glutamine at the N-terminus of the cysteine-rich domain. In the collagen-like domain, SNPs g.1197 C>A and g.1198 G>A changed proline to glutamine, whereas SNP g.1207 T>C was identified as a synonymous mutation. Correlation analysis showed that the g.1197 C>A marker was significantly correlated to somatic cell score (SCS), and the g.1164 G>A locus had significant effects on SCS, fat content, and protein content (P < 0.05), suggesting possible roles of these SNPs in the host response against mastitis. Nine haplotypes and nine haplotype pairs corresponding to the loci of the 4 novel SNPs were found in Chinese Holsteins. Haplotype pairs MM, MN, and BQ were correlated with the lowest SCS; MN with the highest protein yield; MM with the highest protein rate, and MN with the highest 305- day milk yield. Thus, MM, MN, and BQ are possible candidates for marker-assisted selection in dairy cattle breeding programs.

Increased expression of a novel splice variant of the complement component 4 (C4A) gene in mastitis-infected dairy cattle.

The complement system helps in the direct lysis of invading pathogens and modulates phagocytic, humoral and cellular immune responses. Complement 4 is a critical component in complement activity and protection against many bacterial pathogens because it is essential to classical and lectin activation pathways. We used reverse transcription and PCR to investigate alternative splicing and expression of the complement component 4 (C4A) gene in Chinese Holstein cattle. The PCR products were cloned and sequenced. A novel splice variant involving intron 10 was identified, which we named C4A-AS. To examine how C4A gene activity is affected by bovine mastitis, six Chinese Holstein cattle were divided into healthy (non-mastitic) and Staphylococcus aureus-induced mastitic groups. Real-time quantitative PCR (qRT-PCR) revealed that the C4A-complete and C4A-AS transcripts are expressed at significantly different levels in healthy cows, while there were no significant differences in the mastitic group (P = 0.257). Expression of C4A-AS increased significantly when mastitis developed. We also examined the expression of C4A-complete and C4A-AS in several tissues (liver, heart, spleen, lung, kidney, tongue, and muscle). The two transcripts were expressed in all of these tissues but there were no significant differences in expression between healthy and mastitic cows. We therefore conclude that the C4A-complete transcript is the main transcript under normal physiological conditions, while C4A-AS is augmented when mastitis develops.

Two splice variants of the bovine lactoferrin gene identified in Staphylococcus aureus isolated from mastitis in dairy cattle.

Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

Three novel SNPs of the bovine Tf gene in Chinese native cattle and their associations with milk production traits.

Transferrin (Tf) is a ß-globulin protein that transports iron ions in mammalian cells. It contributes to innate immunity to microbial pathogens, primarily by limiting microbial access to iron. Thus, polymorphisms present in bovine Tf could potentially underlie inherited differences in mastitis resistance and milk production traits. We detected three novel single-nucleotide polymorphisms of the Tf gene in Chinese native cattle by screening for genetic variation of Tf in 751 individuals of three Chinese cattle breeds, namely China Holstein, Luxi Yellow and Bohai Black, using PCR-RFLP and DNA sequencing techniques. The three new SNPs, g.-1748G>A ss250608649, g.13942T>C ss250608650, and g.14037A>G ss250608651, had allele frequencies of 85.9, 86.3 and 92.5%, 64.5, 73.3 and 65.0%, and 67.6, 73.7 and 60.0%, respectively. SNP g.-1748G>A was located in the 5' flanking region of Tf. SNP g.14037A>G was located in intron 8 of Tf. SNP g.13942T>C, located in exon 8 of Tf, was a synonymous mutation (TTA > CTA), encoding a leucine (326 aa) in the Tf protein. Associations of the Tf SNPs with milk traits were also analyzed. Significant (P < 0.05) relationships among the Tf polymorphisms, somatic cell scores (SCS), and milk productive traits were observed. Cows with genotypes TT (g.13942T>C), GG (g.-1748G>A) and AG (g.14037A>G) had a lower SCS and higher protein levels and 305-day milk yield. Nineteen combinations of different haplotypes from the three SNPs were identified in Chinese Holstein cattle. The haplotype combination ATA/GCA, GCA/GCA and GCG/ GTA was dominant in cows with a lower SCS, a higher protein level and a higher 305-day milk yield, respectively. Moreover, the gene expression level of Tf was higher in mastitis-affected mammary tissues than in normal mammary tissues. These results suggest that the Tf gene affects milk production, as well as mastitis-resistance traits, in Chinese Holsteins.