A Randomized Single-Blinded Study Comparing Preoperative with Post-Mastectomy PECS Block for Post-operative Pain Management in Bilateral Mastectomy with Immediate Reconstruction.

BACKGROUND: Ultrasound-guided pectoralis muscle blocks (PECS I/II) are well established for postoperative pain control after mastectomy with reconstruction. However, optimal timing is unclear. We conducted a randomized controlled single-blinded single-institution trial comparing outcomes of block performed pre-incision versus post-mastectomy. METHOD: Patients with breast cancer undergoing bilateral mastectomy with immediate expander/implant reconstruction were randomized to receive ultrasound-guided PECS I/II either pre-incision (PreM, n = 17) or post-mastectomy and before reconstruction (PostM, n = 17). The primary outcome was the average pain score using the Numerical Rating Score during post-anesthesia care unit (PACU) and inpatient stay, with the study powered to detect a difference in mean pain score of 2. Secondary outcomes included mean pain scores on postoperative day (POD) 2, 3, 7, 14, 90, and 180; pain catastrophizing scores; narcotic requirements; PACU/inpatient length of stay; block procedure time; and complications. RESULT: No significant differences between the two groups were noted in average pain score during PACU (p = 0.57) and 24-h inpatient stay (p = 0.33), in the 2 weeks after surgery at rest (p = 0.90) or during movement (p = 0.30), or at POD 90 and 180 at rest (p = 0.42) or during movement (p = 0.31). Median duration of block procedure (PreM 7 min versus PostM 6 min, p = 0.21) did not differ. Median PACU and inpatient length of stay were the same in each group. Inpatient narcotic requirements were similar, as were length of stay and post-surgical complication rates. CONCLUSION: Intraoperative ultrasound-guided PECS I/II block administered by surgeons following mastectomy had similar outcomes to preoperative blocks. TRIAL REGISTRATION: This trial is registered with Clinical Research Information Service (NCT03653988).

Inhibitors of hydroperoxide metabolism enhance ascorbate-induced cytotoxicity.

Pharmacological ascorbate, via its oxidation, has been proposed as a pro-drug for the delivery of H(2)O(2) to tumors. Pharmacological ascorbate decreases clonogenic survival of pancreatic cancer cells, which can be reversed by treatment with scavengers of H(2)O(2). The goal of this study was to determine if inhibitors of intracellular hydroperoxide detoxification could enhance the cytotoxic effects of ascorbate. Human pancreatic cancer cells were treated with ascorbate alone or in combination with inhibitors of hydroperoxide removal including the glutathione disulfide reductase inhibitor 1,3 bis (2-chloroethyl)-1-nitrosurea (BCNU), siRNA targeted to glutathione disulfide reductase (siGR), and 2-deoxy-D-glucose (2DG), which inhibits glucose metabolism. Changes in the intracellular concentration of H(2)O(2) were determined by analysis of the rate of aminotriazole-mediated inactivation of endogenous catalase activity. Pharmacological ascorbate increased intracellular H(2)O(2) and depleted intracellular glutathione. When inhibitors of H(2)O(2) metabolism were combined with pharmacological ascorbate the increase in intracellular H(2)O(2) was amplified and cytotoxicity was enhanced. We conclude that inclusion of agents that inhibit cellular peroxide removal produced by pharmacological ascorbate leads to changes in the intracellular redox state resulting in enhanced cytotoxicity.

Specific down-regulation of annexin II expression in human cells interferes with cell proliferation.

The protein-tyrosine kinase substrate annexin II is a growth regulated gene whose expression is increased in several human cancers. While the precise function of this protein is not understood, annexin II is proposed to be involved in multiple physiological activities, including DNA synthesis and cell proliferation. Targeted disruption of the annexin II gene affects calcium signaling, tyrosine phosphorylation and apoptosis, indicating the important physiological role of this protein. We used a transient co-transfection assay to regulate annexin II expression in human HeLa, 293 and 293T cells, and measured the effects of annexin II down regulation on DNA synthesis and proliferation. Transfection of cells with an antisense annexin II vector results in inhibition of cell division and proliferation, with concomitant reduction in annexin II message and protein levels. Cellular DNA synthesis is significantly reduced in antisense transfected cells. Replication extracts made from antisense transfected cells have significantly reduced efficiency to support SV40 in vitro DNA replication, while the extracts made from sense transfected cells are fully capable of replication. Our results indicate an important role of annexin II in cellular DNA synthesis and cell proliferation.

The 32- and 14-kilodalton subunits of replication protein A are responsible for species-specific interactions with single-stranded DNA.

Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa. While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low cooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins.

Identification of DNA-binding proteins that recognize a conserved type I repeat sequence in the replication origin region of Tetrahymena rDNA.

An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(II)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssA-TIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5' or 3' borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssA-TIBF, coupled with genetic evidence that Type I sequences function as cis-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.