Toxic effects of the emerging Alexandrium pseudogonyaulax (Dinophyceae) on multiple trophic levels of the pelagic food web.

The dinoflagellate Alexandrium pseudogonyaulax, a harmful algal bloom species, is currently appearing in increasing frequency and abundance across Northern European waters, displacing other Alexandrium species. This mixotrophic alga produces goniodomins (GDs) and bioactive extracellular substances (BECs) that may pose a threat to coastal ecosystems and other marine resources. This study demonstrated the adverse effects of A. pseudogonyaulax on four marine trophic levels, including microalgae (Rhodomonas salina), microzooplankton (Polykrikos kofoidii) and mesozooplankton (Acartia tonsa), as well as fish gill cells (RTgill-W1, Oncorhynchus mykiss), ultimately leading to enhanced mortality and cell lysis. Furthermore, cell-free supernatants collected from A. pseudogonyaulax cultures caused complete loss of metabolic activity in the RTgill-W1 cell line, indicating ichthyotoxic properties, while all tested GDs were much less toxic. In addition, cell-free supernatants of A. pseudogonyaulax led to cell lysis of R. salina, while all tested GDs were non-lytic. Finally, reduced egg hatching rates of A. tonsa eggs exposed to cell-free supernatants of A. pseudogonyaulax and impaired mobility of P. kofoidii and A. tonsa exposed to A. pseudogonyaulax were also observed. Altogether, bioassay results suggest that the toxicity of A. pseudogonyaulax is mainly driven by BECs and not by GDs, although further research into factors modulating the lytic activity of Alexandrium spp. are needed.

Thecal plate morphology, molecular phylogeny, and toxin analyses reveal two novel species of Alexandrium (Dinophyceae) and their potential for toxin production.

This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2'/4' suture length, narrower plates 1' and 6'', with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1' having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6-14 pg cell-1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4-7.3 pg cell-1). None of the strains contained detectable levels of PSTs and cycloimines.

Mass spectrometric characterization of the seco acid formed by cleavage of the macrolide ring of the algal metabolite goniodomin A.

Goniodomin A (GDA) is a polyketide macrolide produced by multiple species of the marine dinoflagellate genus Alexandrium. GDA is unusual in that it undergoes cleavage of the ester linkage under mild conditions to give mixtures of seco acids (GDA-sa). Ring-opening occurs even in pure water although the rate of cleavage accelerates with increasing pH. The seco acids exist as a dynamic mixture of structural and stereo isomers which is only partially separable by chromatography. Freshly prepared seco acids show only end absorption in the UV spectrum but a gradual bathochromic change occurs, which is consistent with formation of α,ß-unsaturated ketones. Use of NMR and crystallography is precluded for structure elucidation. Nevertheless, structural assignments can be made by mass spectrometric techniques. Retro-Diels-Alder fragmentation has been of value for independently characterizing the head and tail regions of the seco acids. The chemical transformations of GDA revealed in the current studies help clarify observations made on laboratory cultures and in the natural environment. GDA has been found to reside mainly within the algal cells while the seco acids are mainly external with the transformation of GDA to the seco acids occurring largely outside the cells. This relationship, plus the fact that GDA is short-lived in growth medium whereas GDA-sa is long-lived, suggests that the toxicological properties of GDA-sa in its natural environment are more important for the survival of the Alexandrium spp. than those of GDA. The structural similarity of GDA-sa to that of monensin is noted. Monensin has strong antimicrobial properties, attributed to its ability to transport sodium ions across cell membranes. We propose that toxic properties of GDA may primarily be due to the ability of GDA-sa to mediate metal ion transport across cell membranes of predator organisms.

Synthesis, characterization and absolute configurations of methyl ladderanoates.

Methyl esters of [5]-ladderanoic acid and [3]-ladderanoic acid were prepared by esterification of the acids isolated from biomass at a wastewater treatment plant. Optical rotations at six different wavelengths (633, 589, 546, 436, 405 and 365 nm) and vibrational circular dichroism (VCD) spectra in the 1800-900 cm-1 region were measured in CDCl3 solvent and compared with quantum chemical (QC) predictions using B3LYP functional and 6-311++G(2d,2p) basis set with polarizing continuum model representing the solvent. QC predictions gave negative optical rotations at all six wavelengths for (R)-methyl [5]-ladderanoate and positive optical rotations for (R)-methyl [3]-ladderanoate, the same signs as previously reported for the corresponding acids. The crystal structure of (-)-methyl [5]-ladderanoate independently confirmed (R) configuration. The QC-predicted VCD spectra using Boltzmann population weighted spectra of individual conformers did not provide satisfactory quantitative agreement with the experimental VCD spectra. An improved quantitative agreement for VCD spectra could be obtained when conformer populations were optimized to maximize the similarity between experimental and predicted VCD spectra, but more improvements in VCD predictions are needed.

Alkali Metal- and Acid-Catalyzed Interconversion of Goniodomin A with Congeners B and C.

Goniodomin A (GDA, 1) is a phycotoxin produced by at least four species of Alexandrium dinoflagellates that are found globally in brackish estuaries and lagoons. It is a linear polyketide with six oxygen heterocyclic rings that is cyclized into a macrocyclic structure via lactone formation. Two of the oxygen heterocycles in 1 comprise a spiro-bis-pyran, whereas goniodomin B (GDB) contains a 2,7-dioxabicyclo[3.3.1]nonane ring system fused to a pyran. When H2O is present, 1 undergoes facile conversion to isomer GDB and to an α,ß-unsaturated ketone, goniodomin C (GDC, 7). GDB and GDC can be formed from GDA by cleavage of the spiro-bis-pyran ring system. GDA, but not GDB or GDC, forms a crown ether-type complex with K+. Equilibration of GDA with GDB and GDC is observed in the presence of H+ and of Na+, but the equilibrated mixtures revert to GDA upon addition of K+. Structural differences have been found between the K+ and Na+ complexes. The association of GDA with K+ is strong, while that with Na+ is weak. The K+ complex has a compact, well-defined structure, whereas Na+ complexes are an ill-defined mixture of species. Analyses of in vitro A. monilatum and A. hiranoi cultures indicate that only GDA is present in the cells; GDB and GDC appear to be postharvest transformation products.

Revisiting the toxin profile of Alexandrium pseudogonyaulax; Formation of a desmethyl congener of goniodomin A.

During a survey of the production of goniodomin A (GDA) by Alexandrium pseudogonyaulax in Danish coastal waters, Krock et al. (2018) obtained mass spectral evidence for the presence of a truncated congener, herein termed GD754, having a molecular weight 14 Da lower than GDA and assigned it as goniodomin B (GDB). An erroneous structure of GDB involving deletion of a methylene group between rings B and D had previously been reported by Espiña et al. (2016) but without experimental details. HPLC properties reported by Krock for GD754 point to it being a homolog of GDA. Comparison of mass spectral fragmentation data reported for GD754 with fragmentation data for GDA, show it to be a truncated form of GDA with the deletion involving a CH2 group from ring F or one of the two methyl substituents on ring F, not elsewhere on the molecule. On biosynthetic grounds, the GD754 congener is proposed to be 34-desmethyl-GDA. Further experimental work will be required to confirm this hypothesis.

The toxin goniodomin, produced by Alexandrium spp., is identical to goniodomin A.

In 1968 Burkholder and associates (J. Antibiot. (Tokyo)1968, 21, 659-664) isolated the antifungal toxin goniodomin from an unidentified Puerto Rican dinoflagellate and partially characterized its structure. Subsequently, a metabolite of Alexandrium hiranoi was isolated by Murakami et al. from a bloom in Japan and its structure was established (Tetrahedron Lett.1988, 29, 1149-1152). The Japanese substance had strong similarities to Burkholder's but due to uncertainty as to whether it was identical or only similar, Murakami named his toxin goniodomin A. A detailed study of this question now provides compelling evidence that Burkholder's goniodomin is identical to goniodomin A. Morphological characterization of the dinoflagellate suggests that it was the genus Alexandrium but insufficient evidence is available to make a definite identification of the species. This is the only report of goniodomin in the Caribbean region.

Algal Toxin Goniodomin A Binds Potassium Ion Selectively to Yield a Conformationally Altered Complex with Potential Biological Consequences.

The marine toxin goniodomin A (GDA) is a polycyclic macrolide containing a spiroacetal and three cyclic ethers as part of the macrocycle backbone. GDA is produced by three species of the Alexandrium genus of dinoflagellates, blooms of which are associated with "red tides", which are widely dispersed and can cause significant harm to marine life. The toxicity of GDA has been attributed to stabilization of the filamentous form of the actin group of structural proteins, but the structural basis for its binding is not known. Japanese workers, capitalizing on the assumed rigidity of the heavily substituted macrolide ring, assigned the relative configuration and conformation by relying on NMR coupling constants and NOEs; the absolute configuration was assigned by degradation to a fragment that was compared with synthetic material. We have confirmed the absolute structure and broad features of the conformation by X-ray crystallography but have found GDA to complex with alkali metal ions in spite of two of the heterocyclic rings facing outward. Such an arrangement would have been expected to impair the ability of GDA to form a crown-ether-type multidentate complex. GDA shows preference for K+, Rb+, and Cs+ over Li+ and Na+ in determinations of relative affinities by TLC on metal-ion-impregnated silica gel plates and by electrospray mass spectrometry. NMR studies employing the K+ complex of GDA, formed from potassium tetrakis[pentafluorophenyl]borate (KBArF20), reveal a major alteration of the conformation of the macrolide ring. These observations argue against the prior assumption of rigidity of the ring. Alterations in chemical shifts, coupling constants, and NOEs indicate the involvement of most of the molecule other than ring F. Molecular mechanics simulations suggest K+ forms a heptacoordinate complex involving OA, OB, OC, OD, OE, and the C-26 and C-27 hydroxy groups. We speculate that complexation of K+ with GDA electrostatically stabilizes the complex of GDA with filamentous actin in marine animals due to the protein being negatively charged at physiological pH. GDA may also cause potassium leakage through cell membranes. This study provides insight into the structural features and chemistry of GDA that may be responsible for significant ecological damage associated with the GDA-producing algal blooms.

Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma.

OBJECTIVES: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. METHODS: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. RESULTS: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. CONCLUSION: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2643-2649, 2020.

Integrated Genomic and Functional microRNA Analysis Identifies miR-30-5p as a Tumor Suppressor and Potential Therapeutic Nanomedicine in Head and Neck Cancer.

PURPOSE: To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). EXPERIMENTAL DESIGN: We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. RESULTS: We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. CONCLUSIONS: We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.

Optimal targeting of BCL-family proteins in head and neck squamous cell carcinoma requires inhibition of both BCL-xL and MCL-1.

Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares - Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9µM (range 6.6µM - 13.9µM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7µM (range, 8.8µM to 12.7µM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.

Absolute Configurations of Naturally Occurring [5]- and [3]-Ladderanoic Acids: Isolation, Chiroptical Spectroscopy, and Crystallography.

We have isolated mixtures of [5]- and [3]-ladderanoic acids 1a and 2a from the biomass of an anammox bioreactor and have separated the acids and their phenacyl esters for the first time by HPLC. The absolute configurations of the naturally occurring acids and their phenacyl esters are assigned as R at the site of side-chain attachment by comparison of experimental specific rotations with corresponding values predicted using quantum chemical (QC) methods. The absolute configurations for 1a and 2a were independently verified by comparison of experimental Raman optical activity spectra with corresponding spectra predicted using QC methods. The configurational assignments of 1a and 2a and of the phenacyl ester of 1a were also confirmed by X-ray crystallography.

Identification of N-{cis-3-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases.

Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.

Apolipoprotein E Promotes Invasion in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) patients generally have a poor prognosis, because of the invasive nature of these tumors. In comparing transcription profiles between OSCC tumors with a more invasive (worst pattern of tumor invasion 5) versus a less invasive (worst pattern of tumor invasion 3) pattern of invasion, we identified a total of 97 genes that were overexpressed at least 1.5-fold in the more invasive tumor subtype. The most functionally relevant genes were assessed using in vitro invasion assays with an OSCC cell line (UM-SCC-1). Individual siRNA knockdown of 15 of these 45 genes resulted in significant reductions in tumor cell invasion compared to a nontargeting siRNA control. One gene whose knockdown had a strong effect on invasion corresponded to apolipoprotein E (APOE). Both matrix degradation and the number of mature invadopodia were significantly decreased with APOE knockdown. APOE knockdown also resulted in increased cellular cholesterol, consistent with APOE's role in regulating cholesterol efflux. APOE knockdown resulted in decreased levels of phospho-extracellular signal-regulated kinase 1/2, phospho-c-Jun N-terminal kinase, and phospho-cJun, as well as decreased activator protein 1 (AP-1) activity. Expression of matrix metalloproteinase 7 (MMP7), an AP-1 target, was also significantly decreased. Our findings suggest that APOE protein plays a significant role in OSCC tumor invasion because of its effects on cellular cholesterol and subsequent effects on cell signaling and AP-1 activity, leading to changes in the expression of invasion-related proteins, including MMP7.

miR-375 Regulates Invasion-Related Proteins Vimentin and L-Plastin.

Invasion is a hallmark of advanced head and neck squamous cell carcinoma (HNSCC). We previously determined that low relative miR-375 expression was associated with poor patient prognosis. HNSCC cells with increased miR-375 expression have lower invasive properties and impaired invadopodium activity. Using stable isotope labeling with amino acids in cell culture and reverse-phase liquid chromatography mass spectrometry, we assessed the impact of miR-375 expression on protein levels in UM-SCC-1 cells. Increased miR-375 expression was associated with down-regulation of proteins involved in cellular assembly and organization, death and survival, and movement. Two invasion-associated proteins, vimentin and L-plastin, were strongly down-regulated by miR-375. Luciferase reporter assays demonstrated that high miR-375 expression reduced vimentin promoter activity, suggesting that vimentin is an indirect target of miR-375. Runt-related transcription factor 1 (RUNX1) is a potential miR-375 direct target, and its knockdown reduced vimentin and L-plastin expression. Data in The Cancer Genome Atlas HNSCC database showed a significant inverse correlation between miR-375 expression and RUNX1, vimentin, and L-plastin RNA expression. These clinical correlations validate our in vitro model findings and support a mechanism in which miR-375 suppresses RUNX1 levels, resulting in reduced vimentin and L-plastin expression. Furthermore, knockdown of RUNX1, L-plastin, and vimentin resulted in significant reductions in cell invasion in vitro, indicating the functional significance of miR-375 regulation of specific proteins involved in HNSCC invasion.

Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites.

Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a ß- or ß,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both ß- and ß,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring.

Epigenetic changes in the CDKN2A locus are associated with differential expression of P16INK4A and P14ARF in HPV-positive oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease entity associated with improved survival. DNA hypermethylation profiles differ significantly by HPV status suggesting that a specific subset of methylated CpG loci could give mechanistic insight into HPV-driven OPSCC. We analyzed genome-wide DNA methylation of primary tumor samples and adjacent normal mucosa from 46 OPSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M value). From these analyses, we identified a 22 CpG loci panel for HPV+ OPSCC that included four CDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites. This panel was significantly associated with overall HPV detection (P < 0.05; ROC area under the curve = 0.96, 95% CI: 0.91-1.0) similar to the subset of four CDKN2A-specific CpG loci (0.90, 95% CI: 0.82-0.99) with equivalence to the full 22 CpG panel. DNA hypermethylation correlated with a significant increase in alternative open reading frame (ARF) expression in HPV+ OPSCC primary tumors, but not to the other transcript variant encoded by the CDKN2A locus. Overall, this study provides evidence of epigenetic changes to the downstream region of the CDKN2A locus in HPV+ oropharyngeal cancer that are associated with changes in expression of the coded protein products.

Proteomic analysis of oral cavity squamous cell carcinoma specimens identifies patient outcome-associated proteins.

CONTEXT: Global proteomic analysis of oral cavity squamous cell carcinoma was performed to identify changes that reflect patient outcomes. OBJECTIVES: To identify differentially expressed proteins associated with patient outcomes and to explore the use of imaging mass spectrometry as a clinical tool to identify clinically relevant proteins. DESIGN: Two-dimensional separation of digested peptides generated from 43 specimens with high-resolution mass spectrometry identified proteins associated with disease-specific death, distant metastasis, and loco-regional recurrence. RNA expressions had been correlated to protein levels to test transcriptional regulation of clinically relevant proteins. Imaging mass spectrometry explored an alternative platform for assessing clinically relevant proteins that would complement surgical pathologic diagnosis. RESULTS: Seventy-two peptide features were found to be associated with 3 patient outcomes: disease-specific death (9), distant metastasis (16), and loco-regional recurrence (39); 8 of them were associated with multiple outcomes. Functional ontology revealed major changes in cell adhesion and calcium binding. Thirteen RNAs showed strong correlation with their encoded proteins, implying transcriptional control. Reduction of DSP, PKP1, and TRIM29 was associated with significantly shorter time to onset of distant metastasis. Reduction of PKP1 and TRIM29 correlated with poorer disease-specific survival. Additionally, S100A8 and S100A9 reductions were verified for their association with poor prognosis using imaging mass spectrometry, a platform more adaptable for use with surgical pathology. CONCLUSIONS: Using global proteomic analysis, we have identified proteins associated with clinical outcomes. The list of clinically relevant proteins observed will provide a means to develop clinical assays for prognosis and optimizing treatment selection.

Investigation of the mechanism of racemization of litronesib in aqueous solution: unexpected base-catalyzed inversion of a fully substituted carbon chiral center.

Mitosis inhibitor (R)-litronesib (LY2523355) is a 1,3,4-thiadiazoline-bearing phenyl and N-(2-ethylamino)ethanesulfonamido-methyl substituents on tetrahedral C5. Chiral instability has been observed at pH 6 and above with the rate of racemization increasing with pH. A positively charged trigonal intermediate is inferred from the fact that p-methoxy substituent on the phenyl accelerated racemization, whereas a p-trifluoromethyl substituent had the opposite effect. Racemization is proposed to occur through a relay mechanism involving intramolecular deprotonation of the sulfonamide by the side chain amino group and attack of the sulfonamide anion on C5, cleaving the C5S bond, to form an aziridine; heterolytic dissociation of the aziridine yields an ylide. This pathway is supported by (1) a crystal structure providing evidence for a hydrogen bond between the sulfonamide NH and the amino group, (2) effects of substituents on the rate of racemization, and (3) computational studies. This racemization mechanism results from neighboring group effects in this densely functionalized molecule. Of particular novelty is the involvement of the side-chain secondary amino group, which overcomes the weak acidity of the sulfonamide by anchimeric assistance.