Alterations in Sarcopenia Index are Associated with Inflammation, Gut and Oral Microbiota among Heart Failure, Left Ventricular Assist Device and Heart Transplant Patients.

BACKGROUND: Sarcopenia, characterized by loss of muscle mass and function, is prevalent in heart failure (HF) and predicts poor outcomes. We investigated alterations in sarcopenia index (SI), a surrogate for skeletal muscle mass, in HF, left ventricular assist device (LVAD) and heart transplant (HT), and assessed its relationship with inflammation and digestive tract (gut and oral) microbiota. METHODS: We enrolled 460 HF, LVAD and HT patients. Repeated measures pre/post procedures were obtained prospectively in a subset of LVAD and HT patients. SI (serum Creatinine/Cystatin C) and inflammatory biomarkers (C-reactive protein, interleukin-6, tumor necrosis factor-alpha) were measured in 271 and 622 blood samples, respectively. Gut and saliva microbiota were assessed via 16S rRNA sequencing among 335 stool and 341 saliva samples. Multivariable regression assessed the relationship between SI and i) New York Heart Association class; ii) pre- vs. post-LVAD or HT; iii) biomarkers of inflammation and microbial diversity. RESULTS: Median (interquartile range) natural logarithm (ln)-SI was -0.13 (-0.32,0.05). Ln-SI decreased across worsening HF class, further declined at 1-month after LVAD and HT and rebounded over time. Ln-SI was correlated with inflammation (r=-0.28, p<0.01), and gut (r=0.28, p<0.01) and oral microbial diversity (r=0.24, p<0.01), these associations remained significant after multivariable adjustment in the combined cohort but not for all individual cohorts. Presence of the gut taxa Roseburia inulinivorans was associated with increased SI. CONCLUSIONS: SI levels decreased in symptomatic HF and remained decreased long-term after LVAD and HT. In the combined cohort, SI levels covaried with inflammation in a similar fashion and significantly related to overall microbial (gut and oral) diversity, including specific taxa compositional changes.

Vertical Sleeve Gastrectomy Reduces Gut Luminal Deoxycholic Acid Concentrations in Mice.

BACKGROUND: Bariatric surgery alters bile acid metabolism, which contributes to post-operative improvements in metabolic health. However, the mechanisms by which bariatric surgery alters bile acid metabolism are incompletely defined. In particular, the role of the gut microbiome in the effects of bariatric surgery on bile acid metabolism is incompletely understood. Therefore, we sought to define the changes in gut luminal bile acid composition after vertical sleeve gastrectomy (VSG). METHODS: Bile acid profile was determined by UPLC-MS/MS in serum and gut luminal samples from VSG and sham-operated mice. Sham-operated mice were divided into two groups: one was fed ad libitum, while the other was food-restricted to match their body weight to the VSG-operated mice. RESULTS: VSG decreased gut luminal secondary bile acids, which was driven by a decrease in gut luminal deoxycholic acid concentrations and abundance. However, gut luminal cholic acid (precursor for deoxycholic acid) concentration and abundance did not differ between groups. Therefore, the observed decrease in gut luminal deoxycholic acid abundance after VSG was not due to a reduction in substrate availability. CONCLUSION: VSG decreased gut luminal deoxycholic acid abundance independently of body weight, which may be driven by a decrease in gut bacterial bile acid metabolism.

Plasma cytokine and growth factor response to acute psychosocial stress in major depressive disorder.

BACKGROUND: Pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α are elevated in response to psychosocial stress; however, less is known about other inflammatory markers. METHODS: We explored response to the Trier Social Stress Test (TSST) of 16 cytokines and growth factors in patients with major depressive disorder (MDD, n = 12) vs. healthy volunteers (HV, n = 16). Outcomes were baseline and post-stress levels estimated by area under the curve (AUCi) and peak change over 3 timepoints. We also explored correlations between biomarkers and clinical characteristics. RESULTS: Baseline concentrations were higher in MDD for platelet-derived growth factor (PDGF)-AB/BB (p = 0.037, d = 0.70), granulocyte-macrophage colony-stimulating factor (GM-CSF, p = 0.033, d = 0.52), and IL-8 (p = 0.046, d = 0.74). After TSST, AUCi was higher in MDD for GM-CSF (p = 0.003, d = 1.21), IL-5 (p = 0.014, d = 1.62), and IL-27 (p = 0.041, d = 0.74). In MDD, depression severity correlated positively with soluble CD40L (sCD40L) for AUCi (Spearman's ρ = 0.76, p = 0.004) and with baseline vascular endothelial growth factor A (VEGFA, r = 0.85, p < 0.001), but negatively with baseline monokine induced by gamma interferon (MIG, aka CXCL9; r = -0.77, p = 0.003). CONCLUSIONS: Effect sizes were robust in this exploratory study, although interpretation of the results must be cautious, given small sample size and multiple comparisons. Differential study of stress-induced biomarkers may have important ramifications for MDD treatment.

Inflammation and Mortality in COVID-19 Hospitalized Patients With and Without Type 2 Diabetes.

CONTEXT: COVID-19 mortality is increased in patients with diabetes. A common hypothesis is that the relationship of inflammation with COVID-19 mortality differs by diabetes status. OBJECTIVE: The aim of this study was to determine the relationship of inflammation with mortality in COVID-19 hospitalized patients and to assess if the relationship differs by strata of type 2 diabetes status. METHODS: A case-control (died-survived) study of 538 COVID-19 hospitalized patients, stratified by diabetes status, was conducted at Columbia University Irving Medical Center. We quantified the levels of 8 cytokines and chemokines in serum, including interferon (IFN)-α2, IFN-γ, interleukin (IL)-1α, IL-1ß, IL-6, IL-8/CXCL8, IFNγ-induced protein 10 (IP10)/CXCL10 and tumor necrosis factor α (TNF-α) using immunoassays. Logistic regression models were used to model the relationships of log-transformed inflammatory markers (or their principal components) and mortality. RESULTS: In multiple logistic regression models, higher serum levels of IL-6 (adjusted odds ratio [aOR]:1.74, 95% CI [1.48, 2.06]), IL-8 (aOR: 1.75 [1.41, 2.19]) and IP10 (aOR: 1.36 [1.24, 1.51]), were significantly associated with mortality. This association was also seen in second principal component with loadings reflecting similarities among these 3 markers (aOR: 1.88 [1.54-2.31]). Significant positive association of these same inflammatory markers with mortality was also observed within each strata of diabetes. CONCLUSION: We show that mortality in COVID-19 patients is associated with elevated serum levels of innate inflammatory cytokine IL-6 and inflammatory chemokines IL-8 and IP10. This relationship is consistent across strata of diabetes, suggesting interventions targeting these innate immune pathways could potentially also benefit patients with diabetes.

Levels of Trimethylamine N-Oxide Remain Elevated Long Term After Left Ventricular Assist Device and Heart Transplantation and Are Independent From Measures of Inflammation and Gut Dysbiosis.

BACKGROUND: Trimethylamine N-oxide (TMAO)-a gut-derived metabolite-is elevated in heart failure (HF) and linked to poor prognosis. We investigated variations in TMAO in HF, left ventricular assist device (LVAD), and heart transplant (HT) and assessed its relation with inflammation, endotoxemia, oxidative stress, and gut dysbiosis. METHODS: We enrolled 341 patients. TMAO, CRP (C-reactive protein), IL (interleukin)-6, TNF-α (tumor necrosis factor alpha), ET-1 (endothelin-1), adiponectin, lipopolysaccharide, soluble CD14, and isoprostane were measured in 611 blood samples in HF (New York Heart Association class I-IV) and at multiple time points post-LVAD and post-HT. Gut microbiota were assessed via 16S rRNA sequencing among 327 stool samples. Multivariable regression models were used to assess the relationship between TMAO and (1) New York Heart Association class; (2) pre- versus post-LVAD or post-HT; (3) biomarkers of inflammation, endotoxemia, oxidative stress, and microbial diversity. RESULTS: ln-TMAO was lower among HF New York Heart Association class I (1.23 [95% CI, 0.52-1.94] µM) versus either class II, III, or IV (1.99 [95% CI, 1.68-2.30], 1.97 [95% CI, 1.71-2.24], and 2.09 [95% CI, 1.83-2.34] µM, respectively; all P<0.05). In comparison to class II-IV, ln-TMAO was lower 1 month post-LVAD (1.58 [95% CI, 1.32-1.83] µM) and 1 week and 1 month post-HT (0.97 [95% CI, 0.60-1.35] and 1.36 [95% CI, 1.01-1.70] µM). ln-TMAO levels in long-term LVAD (>6 months: 1.99 [95% CI, 1.76-2.22] µM) and HT (>6 months: 1.86 [95% CI, 1.66-2.05] µM) were not different from symptomatic HF. After multivariable adjustments, TMAO was not associated with biomarkers of inflammation, endotoxemia, oxidative stress, or microbial diversity. CONCLUSIONS: TMAO levels are increased in symptomatic HF patients and remain elevated long term after LVAD and HT. TMAO levels were independent from measures of inflammation, endotoxemia, oxidative stress, and gut dysbiosis.

Combined oral 5-azacytidine and romidepsin are highly effective in patients with PTCL: a multicenter phase 2 study.

Peripheral T-cell lymphomas (PTCLs) are uniquely vulnerable to epigenetic modifiers. We demonstrated in vitro synergism between histone deacetylase inhibitors and DNA methyltransferase inhibitors in preclinical models of T-cell lymphoma. In a phase 1 trial, we found oral 5-azacytidine and romidepsin to be safe and effective, with lineage-selective activity among patients with relapsed/refractory (R/R) PTCL. Patients who were treatment naïve or who had R/R PTCL received azacytidine 300 mg once per day on days 1 to 14, and romidepsin 14 mg/m2 on days 8, 15, and 22 every 35 days. The primary objective was overall response rate (ORR). Targeted next-generation sequencing was performed on tumor samples to correlate mutational profiles and response. Among 25 enrolled patients, the ORR and complete response rates were 61% and 48%, respectively. However, patients with T-follicular helper cell (tTFH) phenotype exhibited higher ORR (80%) and complete remission rate (67%). The most frequent grade 3 to 4 adverse events were thrombocytopenia (48%), neutropenia (40%), lymphopenia (32%), and anemia (16%). At a median follow-up of 13.5 months, the median progression-free survival, duration of response, and overall survival were 8.0 months, 20.3 months, and not reached, respectively. The median progression-free survival and overall survival were 8.0 months and 20.6 months, respectively, in patients with R/R disease. Patients with tTFH enjoyed a particularly long median survival (median not reached). Responders harbored a higher average number of mutations in genes involved in DNA methylation and histone deacetylation. Combined azacytidine and romidepsin are highly active in PTCL patients and could serve as a platform for novel regimens in this disease. This trial was registered at www.clinicaltrials.gov as #NCT01998035.

Arsenic exposure associated T cell proliferation, smoking, and vitamin D in Bangladeshi men and women.

There are limited data examining the consequences of environmental exposure to arsenic on the immune system in adults, particularly among smokers. Smoking has been shown to exacerbate or contribute to impaired immune function in men chronically exposed to arsenic. In contrast, vitamin D (VitD) is known to have a positive influence on innate and adaptive immune responses. The effect of circulating VitD on arsenic-associated immune dysfunction is not known. Here we examine the relationship of arsenic exposure and T cell proliferation (TCP), a measure of immune responsiveness, and circulating VitD among adult men and women in Bangladesh. Arsenic exposure was assessed using total urinary arsenic as well as urinary arsenic metabolites all adjusted for urinary creatinine. TCP was measured ex vivo in cryopreserved peripheral blood mononuclear cells from 614 adult participants enrolled in the Bangladesh Health Effects of Arsenic Longitudinal Study; serum VitD was also evaluated. The influence of cigarette smoking on arsenic-induced TCP modulation was assessed only in males as there was an inadequate number of female smokers. These studies show that arsenic suppressed TCP in males. The association was significantly strong in male smokers and to a lesser extent in male non-smokers. Interestingly, we found a strong protective effect of high/sufficient serum VitD levels on TCP among non-smoking males. Furthermore, among male smokers with low serum VitD (⊔20 ng/ml), we found a strong suppression of TCP by arsenic. On the other hand, high VitD (>20 ng/ml) was found to attenuate effects of arsenic on TCP among male-smokers. Overall, we found a strong protective effect of VitD, when serum levels were >20 ng/ml, on arsenic-induced inhibition of TCP in men, irrespective of smoking status. To our knowledge this is the first large study of immune function in healthy adult males and females with a history of chronic arsenic exposure.

Gut microbial diversity, inflammation, and oxidative stress are associated with tacrolimus dosing requirements early after heart transplantation.

INTRODUCTION: Effective tacrolimus (TAC) dosing is hampered by complex pharmacokinetics and significant patient variability. The gut microbiome, a key mediator of endotoxemia, inflammation and oxidative stress in advanced heart failure (HF) patients, is a possible contributor to interindividual variations in drug efficacy. The effect of alterations in the gut microbiome on TAC dosing requirements after heart transplant (HT) has not been explored. METHODS: We enrolled 24 patients (mean age = 55.8 ±2.3 years) within 3 months post-HT. Biomarkers of endotoxemia ((lipopolysaccharide (LPS)), inflammation (tumor necrosis factor-α (TNF-α)) and oxidative stress (8,12-iso-Isoprostane F-2alpha-VI) were measured in 16 blood samples. 22 stool samples were analyzed using 16S rRNA sequencing. TAC dose and serum trough level were measured at the time of stool and blood collection. TAC doses were reported in mg/kg/day and as level-to-dose (L/D) ratio, and categorized as ≤ vs. > median. RESULTS: The median TAC dose was 0.1 mg/kg/day and L/D ratio was 100.01. Above the median daily weight-based TAC dose was associated with higher gut microbial alpha diversity (p = 0.03); similarly, TNF-α and 8,12-iso-Isoprostane F-2alpha-VI levels were lower and LPS levels were higher in the above median TAC group, although these findings were only marginally statistically significant and dependent on BMI adjustment. We observed n = 37 taxa to be significantly enriched among patients with > median TAC dose (all FDR<0.05), several of which are potential short-chain fatty acid producers with anti-inflammatory properties, including taxa from the family Subdoligranulum. CONCLUSIONS: Our pilot study observed gut microbial alpha diversity to be increased while inflammation and oxidative stress were reduced among patients requiring higher TAC doses early after HT.

Gut microbiota, endotoxemia, inflammation, and oxidative stress in patients with heart failure, left ventricular assist device, and transplant.

BACKGROUND: Gut microbial imbalance may contribute to endotoxemia, inflammation, and oxidative stress in heart failure (HF). Changes occurring in the intestinal microbiota and inflammatory/oxidative milieu during HF progression and following left ventricular assist device (LVAD) or heart transplantation (HT) are unknown. We aimed to investigate variation in gut microbiota and circulating biomarkers of endotoxemia, inflammation, and oxidative stress in patients with HF (New York Heart Association, Class I-IV), LVAD, and HT. METHODS: We enrolled 452 patients. Biomarkers of endotoxemia (lipopolysaccharide and soluble [sCD14]), inflammation (C-reactive protein, interleukin-6, tumor necrosis factor-α, and endothelin-1 adiponectin), and oxidative stress (isoprostane) were measured in 644 blood samples. A total of 304 stool samples were analyzed using 16S rRNA sequencing. RESULTS: Gut microbial community measures of alpha diversity were progressively lower across worsening HF class and were similarly reduced in patients with LVAD and HT (p < 0.05). Inflammation and oxidative stress were elevated in patients with Class IV HF vs all other groups (all p < 0.05). Lipopolysaccharide was elevated in patients with Class IV HF (vs Class I-III) as well as in patients with LVAD and HT (p < 0.05). sCD14 was elevated in patients with Class IV HF and LVAD (vs Class I-III, p < 0.05) but not in patients with HT. CONCLUSIONS: Reduced gut microbial diversity and increased endotoxemia, inflammation, and oxidative stress are present in patients with Class IV HF. Inflammation and oxidative stress are lower among patients with LVAD and HT relative to patients with Class IV HF, whereas reduced gut diversity and endotoxemia persist in LVAD and HT.

Oral 5-azacytidine and romidepsin exhibit marked activity in patients with PTCL: a multicenter phase 1 study.

The peripheral T-cell lymphomas (PTCLs) are uniquely sensitive to epigenetic modifiers. Based on the synergism between histone deacetylase inhibitors and hypomethylating agents that we established in preclinical PTCL models, we conducted a phase 1 study of oral 5-azacytidine (AZA) and romidepsin (ROMI) in patients with advanced lymphoid malignancies, with emphasis on PTCL. According to a 3 + 3 design, patients were assigned to 1 of 7 cohorts with AZA doses ranging from 100 mg daily on days 1 to 14 to 300 mg daily on days 1 to 21, ROMI doses ranging from 10 mg/m2 on days 8 and 15 to 14 mg/m2 on days 8, 15, and 22, with cycles of 21 to 35 days. Coprimary end points included maximum tolerated dose (MTD) and dose-limiting toxicity (DLT). We treated a total of 31 patients. The MTD was AZA 300 mg on days 1 to 14 and ROMI 14 mg/m2 on days 8, 15, and 22 on a 35-day cycle. DLTs included grade 4 thrombocytopenia, prolonged grade 3 thrombocytopenia, grade 4 neutropenia, and pleural effusion. There were no treatment-related deaths. The combination was substantially more active in patients with PTCL than in those with non-T-cell lymphoma. The overall response rate in all, non-T-cell, and T-cell lymphoma patients was 32%, 10%, and 73%, respectively, and the complete response rates were 23%, 5%, and 55%, respectively. We did not find an association between response and level of demethylation or tumor mutational profile. This study establishes that combined epigenetic modifiers are potently active in PTCL patients. This trial was registered at www.clinicaltrials.gov as NCT01998035.

Precision Targeting with EZH2 and HDAC Inhibitors in Epigenetically Dysregulated Lymphomas.

PURPOSE: Both gain-of-function enhancer of zeste homolog 2 (EZH2) mutations and inactivating histone acetyltransferases mutations, such as CREBBP and EP300, have been implicated in the pathogenesis of germinal center (GC)-derived lymphomas. We hypothesized that direct inhibition of EZH2 and histone deacetyltransferase (HDAC) would be synergistic in GC-derived lymphomas. EXPERIMENTAL DESIGN: Lymphoma cell lines (n = 21) were exposed to GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor. Synergy was assessed by excess over bliss. Western blot, mass spectrometry, and coimmunoprecipitation were performed. A SU-DHL-10 xenograft model was utilized to validate in vitro findings. Pretreatment RNA-sequencing of cell lines was performed. MetaVIPER analysis was used to infer protein activity. RESULTS: Exposure to GSK126 and romidepsin demonstrated potent synergy in lymphoma cell lines with EZH2 dysregulation. Combination of romidepsin with other EZH2 inhibitors also demonstrated synergy suggesting a class effect of EZH2 inhibition with romidepsin. Dual inhibition of EZH2 and HDAC led to modulation of acetylation and methylation of H3K27. The synergistic effects of the combination were due to disruption of the PRC2 complex secondary to acetylation of RbAP 46/48. A common basal gene signature was shared among synergistic lymphoma cell lines and was characterized by upregulation in chromatin remodeling genes and transcriptional regulators. This finding was supported by metaVIPER analysis which also revealed that HDAC 1/2 and DNA methyltransferase were associated with EZH2 activation. CONCLUSIONS: Inhibition of EZH2 and HDAC is synergistic and leads to the dissociation of PRC2 complex. Our findings support the clinical translation of the combination of EZH2 and HDAC inhibition in EZH2 dysregulated lymphomas.

Multivalent Small-Molecule Pan-RAS Inhibitors.

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Estimating systemic exposure to levonorgestrel from an oral contraceptive.

OBJECTIVE: The gold standard for measuring oral contraceptive (OC) pharmacokinetics is the 24-h steady-state area under the curve (AUC). We conducted this study to assess whether limited sampling at steady state or measurements following use of one or two OCs could provide an adequate proxy in epidemiological studies for the progestin 24-h steady-state AUC of a particular OC. STUDY DESIGN: We conducted a 13-sample, 24-h pharmacokinetic study on both day 1 and day 21 of the first cycle of a monophasic OC containing 30-mcg ethinyl estradiol and 150-mcg levonorgestrel (LNG) in 17 normal-weight healthy White women and a single-dose 9-sample study of the same OC after a 1-month washout. We compared the 13-sample steady-state results with several steady-state and single-dose results calculated using parsimonious sampling schemes. RESULTS: The 13-sample steady-state 24-h LNG AUC was highly correlated with the steady-state 24-h trough value [r=0.95; 95% confidence interval (0.85, 0.98)] and with the steady-state 6-, 8-, 12- and 16-h values (0.92≤r≤0.95). The trough values after one or two doses were moderately correlated with the steady-state 24-h AUC value [r=0.70; 95% CI (0.27, 0.90) and 0.77; 95% CI (0.40, 0.92), respectively]. CONCLUSIONS: Single time-point concentrations at steady state and after administration of one or two OCs gave highly to moderately correlated estimates of steady-state LNG AUC. Using such measures could facilitate prospective pharmaco-epidemiologic studies of the OC and its side effects. IMPLICATIONS: A single time-point LNG concentration at steady state is an excellent proxy for complete and resource-intensive steady-state AUC measurement. The trough level after two single doses is a fair proxy for steady-state AUC. These results provide practical tools to facilitate large studies to investigate the relationship between systemic LNG exposure and side effects in a real-life setting.

Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib.

Purpose: Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215.Experimental Design: The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model.Results: Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC50 values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL.Conclusions: The development of this ACY-1215-resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. Clin Cancer Res; 23(12); 3084-96. ©2016 AACR.

Aurora A Kinase Inhibition Selectively Synergizes with Histone Deacetylase Inhibitor through Cytokinesis Failure in T-cell Lymphoma.

PURPOSE: Aurora A kinase (AAK) is expressed exclusively during mitosis, and plays a critical role in centrosome duplication and spindle formation. Alisertib is a highly selective AAK inhibitor that has demonstrated marked clinical activity of alisertib across a spectrum of lymphomas, though particularly in patients with T-cell lymphoma (TCL). We sought to compare and contrast the activity of alisertib in preclinical models of B-cell lymphoma (BCL) and TCL, and identify combinations worthy of clinical study. High-throughput screening of pralatrexate, the proteasome inhibitor (ixazomib), and the histone deacetylase (HDAC) inhibitor (romidepsin) revealed that only romidepsin synergized with alisertib, and only in models of TCL. We discovered that the mechanism of synergy between AAK inhibitors and HDAC inhibitors appears to be mediated through cytokinesis failure. EXPERIMENTAL DESIGN: A high-throughput screening approach was used to identify drugs that were potentially synergistic in combination with alisertib. Live-cell imaging was used to explore the mechanistic basis for the drug: drug interaction between alisertib and romidepsin. An in vivo xenograft TCL model was used to confirm in vitro results. RESULTS: In vitro, alisertib exhibited concentration-dependent cytotoxicity in BCL and TCL cell lines. Alisertib was synergistic with romidepsin in a T-cell-specific fashion that was confirmed in vivo. Live-cell imaging demonstrated that the combination treatment resulted in profound cytokinesis failure. CONCLUSIONS: These data strongly suggest that the combination of alisertib and romidepsin is highly synergistic in TCL through modulation of cytokinesis and merits clinical development.

Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity.

Parkinson's disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat "hijacks" the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression.

Proteomic analysis of 17ß-estradiol degradation by Stenotrophomonas maltophilia.

Microbial degradation plays a critical role in determining the environmental fate of steroid hormones, such as 17ß-estradiol (E2). The molecular mechanisms governing the microbial transformation of E2 and its primary degradation intermediate, estrone (E1), are largely unknown. The objective of this study was to identify metabolism pathways that might be involved in microbial estrogen degradation. To achieve the objective, Stenotrophomonas maltophilia strain ZL1 was used as a model estrogen degrading bacterium and its protein expression level during E2/E1 degradation was studied using quantitative proteomics. During an E2 degradation experiment, strain ZL1 first converted E2 to E1 stoichiometrically. At 16 h E1 reached its peak concentration, and microbial growth started. At the same time, enzymes involved in certain catabolic and anabolic pathways were most highly expressed compared to the other time points tested. Among those enzymes, the ones involved in protein and lipid biosyntheses were observed to be particularly active. Based on the metabolite information from a previous study and the proteomic data from this study, we hypothesized that S. maltophilia strain ZL1 was able to convert E1 to amino acid tyrosine through ring cleavage on a saturated ring of the E1 molecule and then utilize tyrosine in protein biosynthesis.

Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery.

Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA([Ser]Sec) and its insertion was dependent on the Sec insertion sequence element in the 3'UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA([Ser]Sec) to generate Cys-tRNA([Ser]Sec). Cys was inserted in vivo at UGA codons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinery-based mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.

Identification of selected respiratory pathogens in endodontic infections.

OBJECTIVE: To determine whether endodontic infections could harbor common etiologic agents of respiratory infections such as Streptococcus pneumoniae and Chlamydia pneumoniae. STUDY DESIGN: Specimens were aseptically obtained from 40 patients with endodontic infections. For the detection of C. pneumoniae, single-step 16S rRNA-based polymerase chain reaction (PCR) and nested PCR targeting aromatic amino acid hydroxylase were used. For the identification of S. pneumoniae, primers targeting 16S rRNA gene and autolysin (lytA) were used. RESULTS: Of 21 patient samples tested with the 16S rRNA-based PCR for S. pneumoniae, positive amplification was observed in all except 3 specimens. However, sequencing and phylogenetic analysis revealed that the product belonged to other bacterial phylotypes. The lytA-based PCR for S. pneumoniae and both PCR assays for C. pneumoniae failed to detect these organisms in all of the specimens tested. CONCLUSIONS: Streptococcus pneumoniae and C. pneumoniae were not present in endodontic infections. PCR primers with less stringent specificity will inaccurately identify respiratory pathogens.

Primer sensitivity: can it influence the results in Enterococcus faecalis prevalence studies?

BACKGROUND/OBJECTIVE: Recent polymerase chain reaction (PCR)-based studies have shown significant variability in the prevalence of Enterococcus faecalis cases with nonhealing endodontic infections. This variability may be, at least in part, due to the differences in sensitivities of the primers used. The purpose of this study was to compare the sensitivity of 3 sets of PCR primers which have been reported in the endodontic literature. METHODS: The 3 primers sets used were: group 1) tuf gene-based primers with genus-level specificity; and groups 2 and 3) 16S rDNA-based primers that were E. faecalis specific. Three strains of E. faecalis at concentrations of 10(2)-10(8) cells/mL were included in this study. RESULTS: The PCR amplification of E. faecalis strains with the 3 primer pairs showed that group 1 primers consistently had the highest sensitivity, followed by group 2 and group 3 (P<.0001). CONCLUSION: A tuf-based PCR identification assay followed by direct sequencing would yield accurate and consistent prevalence rates of E. faecalis in endodontic infections.