Mohs Micrographic Surgery With Immunohistochemistry for the Treatment of Periocular Melanoma In Situ.

PURPOSE: Mohs micrographic surgery with immunohistochemistry allows for same-day comprehensive margin assessment of melanoma in situ prior to subspecialty reconstruction. This study describes the oncologic and reconstructive outcomes of eyelid and periorbital melanoma in situ and identifies risk factors for complex reconstructive demands. METHODS: Retrospective case series of all patients treated with Mohs micrographic surgery with immunohistochemistry for melanoma in situ affecting the eyelids or periorbital region from 2008 to 2018 at a single institution. Tumors were assigned to the eyelid group if the clinically visible tumor involved the skin inside the orbital rim. Reconstructive variables were compared between the eyelid and periorbital cohorts. RESULTS: There were 24 eyelid and 141 periorbital tumors included. The initial surgical margin for all tumors was 5.34 ± 1.54 mm and multiple Mohs stages were required in 24.2% of cases. Eyelid tumors included more recurrences (p = 0.003), and the average defect size was larger (14.0 ± 13.3 cm2 vs. 7.7 ± 5.4 cm2, p = 0.03). Risk factors for complex reconstruction included: initial tumor diameter >2 cm (odds ratio [OR]: 3.84, 95% confidence interval [CI]: 1.95-7.57) and eyelid involved by initial tumor (OR: 4.88, 95% CI: 1.94-12.28). At an average follow-up of 4.8 years, there were no melanoma-related deaths and 1 local recurrence (0.6% recurrence rate). CONCLUSIONS: Mohs micrographic surgery with immunohistochemistry achieves excellent local control rates for periocular melanoma in situ. An initial surgical margin of 5 mm is frequently insufficient to achieve clear margins. The resulting defects are large, and the complexity of reconstruction can be predicted by tumor size and clinical involvement of eyelid skin.

Application of a hybrid zwitterionic hydrophilic interaction liquid chromatography column in metabolic profiling studies.

Metabolic phenotyping studies using mouse liver extracts as a model, performed on a novel zwitterionic HILIC UHPLC column, which is based on ethylene-bridged hybrid organic/inorganic particles bonded with sulfobetaine groups and packed into column hardware modified with hybrid surface technology are reported. Initially the chromatographic performance was evaluated under different mobile phase conditions using selected metabolite standards. Following optimization of the chromatographic conditions for 88 hydrophilic metabolites both targeted and untargeted profiling analyses were performed on tissue extracts using LC-MS/MS and LC-TOF/MS, respectively. Chromatographic efficiency parameters such as peak resolution, peak shapes, selectivity and precision in retention and peak areas as well as characteristics that are critical for metabolic profiling analysis such as metabolite coverage and retention time distribution were assessed. The hybrid zwitterionic column exhibited efficient chromatographic separations providing analysis of ca 80 hydrophilic metabolites from different chemical classes and polarities. Utilizing a one-dimensional separation both targeted and untargeted profiling provided comprehensive metabolic signatures that enabled the acquisition of the metabolic phenotypes of the tissue extracts.

Sensitive and Reproducible Mass Spectrometry-Compatible RP-UHPLC Analysis of Tricarboxylic Acid Cycle and Related Metabolites in Biological Fluids: Application to Human Urine.

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.

The Chemical Characterization of Eleutherococcus senticosus and Ci-wu-jia Tea using UHPLC-UV-QTOF/MS.

Eleutherococcus senticosus Maxim. belongs to the Araliaceae family. Phytochemical studies reveal that E. senticosus leaves contain triterpene glycosides along with organic acid derivatives and flavonoid compounds. It is believed that E. senticosus is similar to ginseng because they come from same family and both contain triterpene saponins. E. senticosus leaves have been developed as a functional beverage called ci-wu-jia tea in recent years. Triterpene glycosides are difficult to identify by ultraviolet (UV) detection and contents of these compounds are low in E. senticosus leaves. In this study, a sensitive ultra-high performance liquid chromatographic (UHPLC) method combining UV and tandem mass spectrometry (MS/MS) was developed to characterize the triterpene glycosides from E. senticosus leaves and related commercial products. Fragmentation patterns of three sub-groups of triterpene glycosides in E. senticosus leaves were investigated. Additionally, fragmentation pathways and UV characteristics of organic acid derivatives and flavonoids were also characterized. A compound screening library, including 241 compounds reported in the literature, was created and used to confirm the compounds in the samples. In this study, a total of 24 samples, including 13 plant samples of E. senticosus and 11 ci-wu-jia tea products, were analyzed. Out of the 11 commercial products, three products were discovered to contain green tea (Camellia sinensis) that was considered to be an adulterant since it was not an ingredient on the labels. The developed UHPLC-UV-MS/MS analytical method combined with the UNIFI processing method can simultaneously characterize organic acid derivatives, flavonoids, and triterpene saponins from E. senticosus. It provides a simple and sensitive way to perform quality control of E. senticosus and related ci-wu-jia tea products.

A kinetic analysis of the inhibition of FOX-4 ß-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic ß-lactams and carbapenems.

OBJECTIVES: Class C ß-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available ß-lactamase inhibitors. In order to find novel scaffolds to inhibit class C ß-lactamases, the comparative efficacy of monocyclic ß-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam ß-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C ß-lactamase (pmAmpC). METHODS: The FOX-4 ß-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the ß-lactam scaffolds described. RESULTS: The K(i) values for the monocyclic ß-lactams against FOX-4 ß-lactamase were 0.04 ± 0.01 µM (aztreonam) and 0.66 ± 0.03 µM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 µM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 µM (ertapenem) to 2.3 ± 0.3 µM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic ß-lactams and carbapenems were reacted with FOX-4 ß-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. CONCLUSIONS: Monocyclic ß-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 ß-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C ß-lactamases.

ß-Lactamase inhibition by 7-alkylidenecephalosporin sulfones: allylic transposition and formation of an unprecedented stabilized acyl-enzyme.

The inhibition of the class A SHV-1 ß-lactamase by 7-(tert-butoxycarbonyl)methylidenecephalosporin sulfone was examined kinetically, spectroscopically, and crystallographically. An 1.14 Å X-ray crystal structure shows that the stable acyl-enzyme, which incorporates an eight-membered ring, is a covalent derivative of Ser70 linked to the 7-carboxy group of 2-H-5,8-dihydro-1,1-dioxo-1,5-thiazocine-4,7-dicarboxylic acid. A cephalosporin-derived enzyme complex of this type is unprecedented, and the rearrangement leading to its formation may offer new possibilities for inhibitor design. The observed acyl-enzyme derives its stability from the resonance stabilization conveyed by the ß-aminoacrylate (i.e., vinylogous urethane) functionality as there is relatively little interaction of the eight-membered ring with active site residues. Two mechanistic schemes are proposed, differing in whether, subsequent to acylation of the active site serine and opening of the ß-lactam, the resultant dihydrothiazine fragments on its own or is assisted by an adjacent nucleophilic atom, in the form of the carbonyl oxygen of the C7 tert-butyloxycarbonyl group. This compound was also found to be a submicromolar inhibitor of the class C ADC-7 and PDC-3 ß-lactamases.

Can inhibitor-resistant substitutions in the Mycobacterium tuberculosis ß-Lactamase BlaC lead to clavulanate resistance?: a biochemical rationale for the use of ß-lactam-ß-lactamase inhibitor combinations.

The current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal ß-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistant M. tuberculosis strains when tested in vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues of M. tuberculosis BlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led to in vitro clavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into an M. tuberculosis H37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to ß-lactam-ß-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen against M. tuberculosis.

Design and exploration of novel boronic acid inhibitors reveals important interactions with a clavulanic acid-resistant sulfhydryl-variable (SHV) ß-lactamase.

Inhibitor resistant (IR) class A ß-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV ß-lactamase, from Klebsiella pneumoniae , results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting ß-lactamases revealed that only -Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM K(i) for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR ß-lactamases.

Exploring the role of a conserved class A residue in the Ω-Loop of KPC-2 ß-lactamase: a mechanism for ceftazidime hydrolysis.

Gram-negative bacteria harboring KPC-2, a class A ß-lactamase, are resistant to all ß-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A ß-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative ß-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015→0.019 µm(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2.

Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276.

ß-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of ß-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A ß-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with ß-lactams and ß-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A ß-lactamase that inactivates carbapenems and ß-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k(cat)/K(m)). In addition, we find that residue 220 is central to the mechanism of ß-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively "inhibitor susceptible" (K(i) of 14 ± 1 µM for clavulanic acid versus K(i) of 25 ± 2 µM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K(m) values for ß-lactamase inhibitors (>100 µM for clavulanic acid versus 25 ± 3 µM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A ß-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the "relatively" clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.

Identification of a class of novel tubulin inhibitors.

We previously developed a series of anticancer agents based on cyclooxygenase-2 (COX-2) inhibitor nimesulide as a lead compound. However, the molecular targets of these agents still remain unclear. In this study, we synthesized a biotinylated probe based on a representative molecule of the compound library and performed protein pull-down assays to purify the anticancer targets of the compound. Via proteomic approaches, the major proteins bound to the probe were identified to be tubulin and heat shock protein 27 (Hsp27), and the compound inhibited tubulin polymerization by binding at the colchicine domain. However, the tubulin inhibitory effect of the compound activated the Hsp27 phosphorylation and possibly overrode the direct Hsp27 inhibitory effects, which made it difficult to solely validate the Hsp27 target. Taken together, the compound was a dual ligand of tubulin and Hsp27, inhibited tubulin polymerization, and had the potential to be a class of new chemotherapeutic agents.

Tissue sample preparation in bioanalytical assays.

Analytical quantitation of compounds in tissue has become a more prevalent addition to biological sample analysis in recent times. This increase will most certainly continue to bring the question of proper analytical method validation to the forefront of discussion. Thoughtful design of sample homogenization, analyte fortification and extraction can ensure a successful analysis. This review presents current trends in tissue sample preparation by harvesting, homogenization techniques, as well as concerns for calibrator and QC preparation. Strategies for consideration and resolution of common pitfalls in method development, for example stability issues and control biomatrices in endogenous analysis, are also presented.

Design and synthesis of a biotinylated probe of COX-2 inhibitor nimesulide analog JCC76.

JCC76 is a derivative of cyclooxygenase-2(COX-2) selective inhibitor nimesulide and exhibits potent anti-breast cancer activity. It selectively induces apoptosis of Her2 positive breast cancer cells. However, the specific molecular targets of JCC76 still remain unclear, which significantly withdraw the further drug development of JCC76. To identify the molecular targets of JCC76, a six carbon linker and biotin conjugated JCC76 probe was designed and synthesized. The anti-proliferation activity of the probe and its analogs was evaluated.

Determination of hexamethylene bisacetamide, an antineoplastic compound, in mouse and human plasma by LC-MS/MS.

Hexamethylene bisacetamide (HMBA) is a polar compound which has recently been discovered to have antineoplastic activity by up-regulating the expression of an endogenous antiproliferative breast cancer protein, HEXIM1 (hexamethylene bisacetamide inducible protein 1) in vivo. HMBA has been shown in the past to induce terminal differentiation in multiple leukemia types at a concentration of 2-5mM, but its phase I and II clinical trials were largely unsuccessful due to serious side effects (notably, thrombocytopenia) with dose escalation. In this work, a sensitive and simple LC-MS/MS method for direct determination of HMBA in mouse and human plasma is described. Plasma samples were prepared by deproteinization with acetonitrile. Separation was achieved on a Waters Atlantis(®) T3 (2.1 mm × 50 mm, 3 µm) column with retention times of 2.2 and 3.7 min for HMBA and 7MBA (internal standard), respectively. The quantitation was carried out by tandem mass spectrometry using positive MRM mode. The linear range of the method was 0.500-100 ng/mL in both mouse and human plasma with injection volume of 5 µL. This method has been validated in accordance with the US Food and Drug Administration (FDA) guidelines for bioanalytical method development and applied to the determination of HMBA concentrations in FVB mice over time after a single dose of HMBA in saline (0.9% NaCl) at 10mg/kg.