Small Bowel Obstruction Secondary to Endometriosis and Hydrosalpinx.

We describe the case of a 32-year-old female who presented to the emergency department (ED) with a 3-day history of severe epigastric abdominal pain accompanied by nausea, vomiting, and constipation. Past medical history was significant for known right hydrosalpinx and previous pelvic inflammatory disease (PID), without past surgical history. Clinical examination revealed a hemodynamically stable patient with a soft but distended abdomen, tenderness in the epigastric region, without signs of peritonitis. Bloodwork including white blood cell count, electrolytes, and lactic acid was unremarkable. Initial computed tomography (CT) scan of the abdomen and pelvis with contrast demonstrated a small bowel obstruction (SBO) with a transition point in the right lower quadrant, accompanied by mesenteric edema and free fluid. Exploratory laparotomy was performed and revealed obstruction secondary to dense adhesions involving the terminal ileum, appendix, sigmoid colon, and right ovary. Lysis of adhesions, appendectomy, and excision of a right paratubal cyst were performed. Histopathology demonstrated endometriosis of the appendix and a benign paratubal cyst.

Surgical residents' interpretation of diagnostic radiologic imaging in the traumatically injured patient.

BACKGROUND: Senior surgical residents are of paramount importance in directing further therapeutic modalities based on their interpretation of critical diagnostic imaging. We propose that senior surgical residents are proficient with interpreting radiologic imaging studies in the trauma patient. METHODS: A prospective cohort study was performed comparing surgery resident interpretations of computed tomography (CT) scans of the head, maxillofacial bones, spine (cervical, thoracic, lumbar), chest, abdomen, pelvis, and chest X-rays versus final radiologists' reports at a Level II trauma center from September 2014 to May 2015. A Cohen κ coefficient and a statistical analysis of variance testing were used to make multiple comparisons of the data. RESULTS: There were 951 trauma alerts activated in the period stated. Of these, 860 met our age inclusion criteria (age, > 18 years). There were 204 images included with an overall accuracy of 81.3%. Residents were more than 70% successful interpreting seven of nine categories. They achieved an accuracy of 84.6%, 62.5%, and 75% in the cervical, thoracic, and lumbar spine categories, respectively. Forty-one of 50 CT scans of the head were interpreted correctly. Maxillofacial CT scans proved to be the weakest category, with only 50% read accurately. In regard to CT scans of the abdomen and pelvis, 80% proficiency was achieved. Abdominal x-rays were read correctly in all instances and chest x-rays 83.3%. On κ analysis, there was an overall moderate agreement between the two groups with K = 0.449, and an overall p less than 0.0005 (Table 1). A perfect agreement existed with abdominal x-rays. CONCLUSION: Senior surgical residents are capable of interpreting critical images obtained in the trauma setting. When discordance existed with attending radiologists' interpretation, it did not change the clinical outcome or result in any critically missed findings. LEVEL OF EVIDENCE: Therapeutic/Care Management study, Level IV.

Identification of Natural Products That Inhibit the Catalytic Function of Human Tyrosyl-DNA Phosphodiesterase (TDP1).

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an enzyme crucial for cleavage of the covalent topoisomerase 1-DNA complex, an intermediate in DNA repair. TDP1 plays a role in reversing inhibition of topoisomerase I by camptothecins, a series of potent and effective inhibitors used in the treatment of colorectal, ovarian, and small-cell lung cancers. It is hypothesized that inhibition of TDP1 activity may enhance camptothecin sensitivity in tumors. Here, we describe the design, development, and execution of a novel assay to identify inhibitors of TDP1 present in natural product extracts. The assay was designed to address issues with fluorescent "nuisance" molecules and to minimize the detection of false-positives caused by polyphenolic molecules known to nonspecifically inhibit enzyme activity. A total of 227,905 purified molecules, prefractionated extracts, and crude natural product extracts were screened. This yielded 534 initial positives (0.23%). Secondary prioritization reduced this number to 117 (0.05% final hit rate). Several novel inhibitors have been identified showing micromolar affinity for human TDP1, including halenaquinol sulfate, a pentacyclic hydroquinone from the sponge Xestospongia sp.

Biochemical assays for the discovery of TDP1 inhibitors.

Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I-, alkylation-, and chain terminator-induced DNA damage, whereas TDP2 repairs topoisomerase II-induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors.

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs.

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs-acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)-we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3'-ends. We also show that Tdp1-/- cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1-/- cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.

5-Arylidenethioxothiazolidinones as inhibitors of tyrosyl-DNA phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a cellular enzyme that repairs the irreversible topoisomerase I (Top1)-DNA complexes and confers chemotherapeutic resistance to Top1 inhibitors. Inhibiting Tdp1 provides an attractive approach to potentiating clinically used Top1 inhibitors. However, despite recent efforts in studying Tdp1 as a therapeutic target, its inhibition remains poorly understood and largely underexplored. We describe herein the discovery of arylidene thioxothiazolidinone as a scaffold for potent Tdp1 inhibitors based on an initial tyrphostin lead compound 8. Through structure-activity relationship (SAR) studies we demonstrated that arylidene thioxothiazolidinones inhibit Tdp1 and identified compound 50 as a submicromolar inhibitor of Tdp1 (IC50 = 0.87 µM). Molecular modeling provided insight into key interactions essential for observed activities. Some derivatives were also active against endogenous Tdp1 in whole cell extracts. These findings contribute to advancing the understanding on Tdp1 inhibition.

Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase I (Tdp1)-topoisomerase I (Top1) inhibitors.

Substances with dual tyrosyl-DNA phosphodiesterase I-topoisomerase I inhibitory activity in one low molecular weight compound would constitute a unique class of anticancer agents that could potentially have significant advantages over drugs that work against the individual enzymes. The present study demonstrates the successful synthesis and evaluation of the first dual Top1-Tdp1 inhibitors, which are based on the indenoisoquinoline chemotype. One bis(indenoisoquinoline) had significant activity against human Tdp1 (IC(50) = 1.52 ± 0.05 µM), and it was also equipotent to camptothecin as a Top1 inhibitor. Significant insights into enzyme-drug interactions were gained via structure-activity relationship studies of the series. The present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in this indenoisoquinoline class of inhibitors even though it was demonstrated to work well for the steroid NSC 88915 (7). The current study will facilitate future efforts to optimize dual Top1-Tdp1 inhibitors.

Elvitegravir overcomes resistance to raltegravir induced by integrase mutation Y143.

OBJECTIVE: In this study, we characterized elvitegravir activity in the context of raltegravir resistance mutations. DESIGN: Using site-directed mutagenesis, we generated recombinant integrase proteins and viruses harboring raltegravir resistance mutation to assess the biochemical and cellular activity of elvitegravir in the presence of such mutants. METHODS: Recombinant proteins were used in gel-based assays. Antiviral data were obtained with reporter viruses in a single-round infection using a luciferase-based assay. RESULTS: Although main raltegravir resistance pathways involving mutations at integrase position 148 and 155 confer cross-resistance to elvitegravir, elvitegravir remains fully active against the Y143R mutant integrase and virus particles. CONCLUSION: In addition to favorable pharmacokinetics compared to raltegravir, our findings provide the rationale for using elvitegravir in patients failing raltegravir because of the integrase mutation Y143.

Biochemical and pharmacological analyses of HIV-1 integrase flexible loop mutants resistant to raltegravir.

Resistance to raltegravir (RAL), the first HIV-1 integrase (IN) inhibitor approved by the FDA, involves three genetic pathways: IN mutations N155H, Q148H/R/K, and Y143H/R/C. Those mutations are generally associated with secondary point mutations. The resulting mutant viruses show a high degree of resistance against RAL but somehow are affected in their replication capacity. Clinical and virological data indicate the high relevance of the combination G140S + Q148H because of its limited impact on HIV replication and very high resistance to RAL. Here, we report how mutations at the amino acid residues 140, 148, and 155 affect IN enzymatic activity and RAL resistance. We show that single mutations at position 140 have limited impact on 3'-processing (3'-P) but severely inactivate strand transfer (ST). On the other hand, single mutations at position 148 have a more profound effect and inactivate both 3'-P and ST. By examining systematically all of the double mutants at the 140 and 148 positions, we demonstrate that only the combination G140S + Q148H is able to restore the catalytic properties of IN. This rescue only operates in cis when both the 140S and 148H mutations are in the same IN polypeptide flexible loop. Finally, we show that the G140S-Q148H double mutant exhibits the highest resistance to RAL. It also confers cross-resistance to elvitegravir but less to G-quadraduplex inhibitors such as zintevir. Our results demonstrate that IN mutations at positions 140 and 148 in the IN flexible loop can account for the phenotype of RAL-resistant viruses.

Modulation of tumor induction and progression of oncogenic K-ras-positive tumors in the presence of TGF- b1 haploinsufficiency.

Oncogenic K-ras is one of the most common genetic alterations in human lung adenocarcinomas. In addition, inactivation of clusters of tumor suppressor genes is required to bring about classical characteristics of cancer including angiogenesis as a prelude to invasion and metastasis. Transforming growth factor-beta (TGF-beta) 1 is a tumor suppressor gene that is implicated in lung cancer progression. Although in vitro studies have shown that TGF-beta1 and Ras pathways cooperate during tumorigenesis, the biology of interaction of TGF-beta1 and Ras has not been studied in in vivo tumorigenesis. We hypothesized that inactivation of TGF-beta1 in addition to oncogeneic activation of K-ras would lead to early initiation and faster progression to lung adenocarcinoma and invasion and metastasis. Heterozygous (HT) TGF-beta1 mice were mated with latent activatable (LA) mutated K-ras mice to generate TGF-beta1(+/+), K-ras LA (wild-type (WT)/LA) and TGF-beta1(+/-), K-ras LA (HT/LA) mice. Both HT/LA and WT/LA mice developed spontaneous lung tumors, but HT/LA mice progressed to adenocarcinomas significantly earlier compared with WT/LA mice. In addition, WT/LA adenocarcinomas had significantly higher angiogenic activity compared with HT/LA adenocarcinomas. Thus, while oncogenic K-ras mutation and insensitivity to the growth regulatory effects of TGF-beta1 is essential for initiation and progression of mouse lung tumors to adenocarcinoma, a full gene dosage of TGF-beta1 is required for tumor-induced angiogenesis and invasive potential. This study identifies a number of genes not previously associated with lung cancer that are involved in tumor induction and progression. In addition, we provide evidence that progression to invasive angiogenic lesions requires TGF-beta1 responsiveness in addition to Ras mutation.