The use of bilateral transversus abdominis plane blocks with liposomal bupivacaine on postoperative cesarean delivery patients during COVID-19 pandemic is associated with reduced narcotics use and reduced length of stay.

BACKGROUND: The use of transversus abdominis plane blocks has been previously shown in both large-scale studies and our own institution to significantly reduce postoperative pain and opioid use. In addition, the use of bilateral transversus abdominis plane blocks using liposomal bupivacaine in combination with neuraxial morphine significantly reduced post-cesarean-delivery pain and opioid use. During the COVID-19 crisis, our anesthesia department in a collaborative effort with our obstetric colleagues thought that the use of bilateral transversus abdominis plane blocks with liposomal bupivacaine could reduce the use of opioids to treat postoperative pain and might result in decreased length of stay. METHODS: After institutional review board approval, a retrospective study of 288 patients who underwent cesarean delivery under spinal or epidural (neuraxial) anesthesia at Maimonides Medical Center in Brooklyn, NY was conducted. Historical controls were from 142 consecutive patients from 1 January 2012 through 12 May 2012. An additional set of controls consisted of 30 consecutive patients from 10 March 2020 through 13 April 2020. The primary outcome data analyzed were the use of opioids and length of stay. RESULTS: Post cesarean delivery, patients who received both bilateral transversus abdominis plane blocks with liposomal bupivacaine and neuraxial morphine was associated with a significant decrease in the number of patients using post operative opioids, 54%-60% decreased to 18% (p < 0.001), and a decreased length of stay; 3.1 days was reduced to 2.39 (p < 0.001). CONCLUSION: Neuraxial opioids combined with liposomal bupivacaine transversus abdominis plane blocks provided significant pain relief for patients post cesarean delivery, required less post operative opioids, and facilitated earlier discharge that may aid in reducing patient exposure and hospital burden secondary to COVID-19.

A novel mutation detection approach of hMLH1 and hMSH2 genes for screening of colorectal cancer.

BACKGROUND: Recently, Denaturing High-Performance Liquid Chromatography (DHPLC) has been widely used for mutation detection hMLH1 and hMSH2 genes due to reduced cost of analysis, accuracy, and high sample throughput. Unfortunately, one major drawback in screening the hMLH1 and hMSH2 genes with any analysis technique involves sample preparation. Additionally, there are limitations to this technique which include: (1) amplicons for hMLH1 and hMSH2 exons cannot be generated under the same PCR thermal cycler condition due to differences in the annealing temperatures of the traditional primer sets which drastically increases sample preparation time; (2) due to minimal changes in the DHPLC chromatogram when compared to the corresponding wild-type amplicon, there is a possibility to not detect a homozygous mutation; and (3) lack of specialized mutation analysis software for automated screening of the hMLH1 and hMSH2 genes with the Transgenomic Wave system. METHODS: To overcome these limitations, the hMLH1 and hMSH2 condition-oriented-PCR primer-embedded-reactor (COPPER) plate was developed to reduce the sample preparation time and technological skill required for analysis, as well as standardize a mutation screening technique for hMLH1 and hMSH2 analysis using the Transgenomic Wave system for research and clinical genetics investigations. In this study, we validated the COPPER plate for simultaneous amplification of the exons of the hMLH1 and hMSH2 genes coupled to DHPLC detection for colorectal cancer (CRC) patients. RESULTS AND CONCLUSIONS: Our results suggest that the COPPER plate DHPLC approach is a simple, cost effective, accurate, universal, and reproducible technology for screening hMLH1 and hMSH2 genes which are associated with human CRC. We also believe that the COPPER plate DHPLC approach is amenable for characterization of other germline alterations in clinical genetics and pharmacogenetics.

Modulation of CDK2-AP1 (p12(DOC-1)) expression in human colorectal cancer.

We have previously demonstrated an association between microsatellite instability and decreased CDK2-AP1 (p12(DOC-1)) expression in human colorectal cancer (CRC) cell lines. In those same studies, induction of CDK2-AP1 expression promoted both cell cycle arrest and apoptosis. The goals of our present study were to better understand the mechanisms leading to reduced CDK2-AP1 expression in microsatellite unstable (MSI) CRC and to study further the effect of CDK2-AP1 modulation on cell proliferation and apoptosis utilizing RNA interference (RNAi) techniques. We used direct sequencing to screen for mutations of the poly (T)8 microsatellite-like region in the 3' end of the CDK2-AP1 gene in 24 CRC cell lines. We then utilized an in vitro human mismatch repair (MMR) recombinant system to assess for correction of the mutation and changes in CDK2-AP1 expression secondary to hMLH1 transfection. We also investigated the effect of CDK2-AP1 modulation in four settings: (1) native CDK2-AP1 absence, (2) endogenous CDK2-AP1 expression, (3) RNAi-induced CDK2-AP1 inhibition and (4) induced CDK2-AP1 over expression. The mutation - del T poly (T)8 - at the 3' end of the CDK2-AP1 gene was found in 3/12 (25%) of MSI CRC cell lines, but in none of the microsatellite stable samples (0/12). Interestingly, when wild-type MMR protein - MLH1 - was induced in an in vitro human recombinant system, the del T poly (T)8 mutation was reversed and CDK2-AP1 expression increased. RNAi-mediated CDK2-AP1 inhibition was associated with decreased apoptosis and increased cell proliferation in CDK2-AP1-non deficient CRC cell lines. We conclude that mutations in the microsatellite-like sequence of the CDK2-AP1 gene in MSI CRC are associated with decreased CDK2-AP1 expression. In addition, modulation of CDK2-AP1 expression in human CRC alters cell proliferation and apoptosis.

Deleted in oral cancer-1 expression upregulates proapoptosis elements in microsatellite-unstable human colorectal cancer.

BACKGROUND: We previously reported differential expression of the growth suppressor, deleted in oral cancer-1 (DOC-1), in microsatellite-unstable (MSI+) versus microsatellite-stable colorectal cancer (CRC) cell lines. MSI+ CRC cell lines demonstrated decreased DOC-1 expression and decreased apoptosis. Transfection of wild-type DOC-1 into an MSI+ cell line (SW48) resulted in increased apoptosis. We undertook our current experiment to identify specific elements modulated by DOC-1 expression that result in increased apoptosis. METHODS: SW48 is an MSI+ CRC cell line that does not constitutively express DOC-1. SW48 was suspended in culture medium and incubated to 60% confluence. Half the plates were transfected with cytomegalovirus (CMV)-DOC-1. At 30 hours, RNA and protein were isolated with Trizol. Complementary DNA microarray was performed to compare SW48(CMV-DOC-1) with SW48, which lacks DOC-1. Signal intensity was analyzed by GenePix Pro 3.0 software. Expression ratios / 1.5 were considered significant. Poor-quality spots were flagged and excluded from analysis. Real-time polymerase chain reaction was performed to determine DOC-1 levels in both cell lines. RESULTS: Successful transfection of DOC-1 was confirmed by real-time polymerase chain reaction and by Western blot. Microarray revealed significant differential expression of DOC-1, as expected. Increased DOC-1 expression in SW48(CMV-DOC-1) was associated with significantly increased expression of proapoptosis components of the caspase cascade (CASP7, CASP9) and bcl2/bax pathway (BNIP3, BNIP3L, BID). CONCLUSIONS: DOC-1 expression promotes apoptosis by upregulation of specific elements of the caspase cascade and bcl2/bax pathways. DOC-1 therefore deserves further study as a candidate for the therapeutic modulation of apoptosis in MSI+ CRC.

The Caenorhabditis elegans innexin INX-3 is localized to gap junctions and is essential for embryonic development.

Innexins are the proposed structural components of gap junctions in invertebrates. Antibodies that specifically recognize the Caenorhabditis elegans innexin protein INX-3 were generated and used to examine the patterns of inx-3 gene expression and the subcellular sites of INX-3 localization. INX-3 is first detected in two-cell embryos, concentrated at the intercellular interface, and is expressed ubiquitously throughout the cellular proliferation phase of embryogenesis. During embryonic morphogenesis, INX-3 expression becomes more restricted. Postembryonically, INX-3 is expressed transiently in several cell types, while expression in the posterior pharynx persists throughout development. Through immuno-EM techniques, INX-3 was observed at gap junctions in the adult pharynx, providing supporting evidence that innexins are components of gap junctions. An inx-3 mutant was isolated through a combined genetic and immunocytochemical screen. Homozygous inx-3 mutants exhibit defects during embryonic morphogenesis. At the comma stage of early morphogenesis, variable numbers of cells are lost from the anterior of inx-3(lw68) mutants. A range of terminal defects is seen later in embryogenesis, including localized rupture of the hypodermis, failure of the midbody to elongate properly, abnormal contacts between hypodermal cells, and failure of the pharynx to attach to the anterior of the animal.