Surgery is the first-line treatment for early, localized, or operable breast cancer. Regional anesthesia during mastectomy may offer the prevention of postoperative pain. One potential protocol is the combination of serratus anterior plane block (SAM block) with pectoral nerve block I (PECS I), but the results and potential benefits are limited. Our study compared general anesthesia with or without SAM block + PECS I during radical mastectomy with axillary node dissection and breast reconstruction using evaluations of pain, opioid consumption, side effects and serum levels of interleukin (IL)-1beta, IL-6 and IL-10. This is a prospective, randomized controlled trial. Fifty patients were randomized to general anesthesia only or general anesthesia associated with SAM block + PECS I (25 per group). The association of SAM block + PECS I with general anesthesia reduced intraoperative fentanyl consumption, morphine use and visual analog pain scale scores in the post-anesthetic care unit (PACU) and at 24 h after surgery. In addition, the anesthetic protocol decreased side effects and sedation 24 h after surgery compared to patients who underwent general anesthesia only. IL-6 levels increased after the surgery compared to baseline levels in both groups, and no differences in IL-10 and IL-1 beta levels were observed. Our protocol improved the outcomes of mastectomy, which highlight the importance of improving mastectomy protocols and focusing on the benefits of regional anesthesia.

Sequencing technologies and new bioinformatics tools have led to the complete sequencing of various genomes. However, information regarding the human transcriptome and its annotation is yet to be completed. The Human Cancer Genome Project, using ORESTES (open reading frame EST sequences) methodology, contributed to this objective by generating data from about 1.2 million expressed sequence tags. Approximately 30% of these sequences did not align to ESTs in the public databases and were considered no-match ORESTES. On the basis that a set of these ESTs could represent new transcripts, we constructed a cDNA microarray. This platform was used to hybridize against 12 different normal or tumor tissues. We identified 3421 transcribed regions not associated with annotated transcripts, representing 83.3% of the platform. The total number of differentially expressed sequences was 1007. Also, 28% of analyzed sequences could represent noncoding RNAs. Our data reinforces the knowledge of the human genome being pervasively transcribed, and point out molecular marker candidates for different cancers. To reinforce our data, we confirmed, by real-time PCR, the differential expression of three out of eight potentially tumor markers in prostate tissues. Lists of 1007 differentially expressed sequences, and the 291 potentially noncoding tumor markers were provided.

We report the first quantitative and qualitative analysis of the poly (A)+ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

Using DDRT-PCR, we compared the mRNA content of untreated and TNF-treated mouse embryonic fibroblasts (MEFs). Among differentially represented fragments, we identified and cloned a novel TNF-stimulated gene named Tsg-5. This gene, mapped to mouse chromosome 14, has three exons that can be alternatively spliced giving rise to two mRNA species, one spanning three exons and another that skips the second exon. Analysis of full-length Tsg-5 cDNA revealed a potential start codon within exon 2 encoding an ORF of 40 amino-acids. No homology with known mouse or human sequences, neither at the nucleotide nor at the amino-acid level could be found in public databases. In MEFs, Tsg-5 is induced by tumor necrossis factora (TNF) and IL-1b, albeit with distinct kinetics. TNF-induced Tsg-5 expression is NF-kB-dependent as it was inhibited by MG132, lactacystin, Bay 11-7083, and Bay 11-7085. Analysis of Tsg-5 expression in vivo revealed that the gene and its encoded polypeptide are constitutively expressed in the thymus and ovary, whereas, in LPS-treated mice, Tsg-5 mRNA can be detected in the spleen, lung, and brain. Our data suggest that Tsg-5 encodes a new, rare transcript, with a very tight regulation of expression and differential splicing.