Distinguishing thymic cysts from low-risk thymomas via [18F]FDG PET/CT.

BACKGROUND: Thymic cysts are a rare benign disease that needs to be distinguished from low-risk thymoma. [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) is a non-invasive imaging technique used in the differential diagnosis of thymic epithelial tumours, but its usefulness for thymic cysts remains unclear. Our study evaluated the utility of visual findings and quantitative parameters of [18F]FDG PET/CT for differentiating between thymic cysts and low-risk thymomas. METHODS: Patients who underwent preoperative [18F]FDG PET/CT followed by thymectomy for a thymic mass were retrospectively analyzed. The visual [18F]FDG PET/CT findings evaluated were PET visual grade, PET central metabolic defect, and CT shape. The quantitative [18F]FDG PET/CT parameters evaluated were PET maximum standardized uptake value (SUVmax), CT diameter (cm), and CT attenuation in Hounsfield units (HU). Findings and parameters for differentiating thymic cysts from low-risk thymomas were assessed using Pearson's chi-square test, the Mann-Whitney U-test, and receiver operating characteristics (ROC) curve analysis. RESULTS: Seventy patients (18 thymic cysts and 52 low-risk thymomas) were finally included. Visual findings of PET visual grade (P < 0.001) and PET central metabolic defect (P < 0.001) showed significant differences between thymic cysts and low-risk thymomas, but CT shape did not. Among the quantitative parameters, PET SUVmax (P < 0.001), CT diameter (P < 0.001), and CT HU (P = 0.004) showed significant differences. In ROC analysis, PET SUVmax demonstrated the highest area under the curve (AUC) of 0.996 (P < 0.001), with a cut-off of equal to or less than 2.1 having a sensitivity of 100.0% and specificity of 94.2%. The AUC of PET SUVmax was significantly larger than that of CT diameter (P = 0.009) and CT HU (P = 0.004). CONCLUSIONS: Among the [18F]FDG PET/CT parameters examined, low FDG uptake (SUVmax ≤ 2.1, equal to or less than the mediastinum) is a strong diagnostic marker for a thymic cyst. PET visual grade and central metabolic defect are easily accessible findings.

Comparison of SUVA/V and SUVA-V for Evaluating Atherosclerotic Inflammation in 18F-FDG PET/CT.

Purpose: This study aimed to compare the clinical significance of two parameters, division of standardized uptake value (SUV) of target arterial activity by background venous blood pool activity (SUVA/V) and subtraction of background venous blood pool activity from SUV of target arterial activity (SUVA-V) of carotid arteries with atherosclerotic plaques using 18F-fluorodeoxyglucose (FDG) positron emission tomography and computed tomography (PET/CT). Methods: Patients aged 50 years or more who were diagnosed with carotid artery stenosis of 50% or more with carotid Doppler ultrasonography and had torso 18F-FDG PET/CT were enrolled retrospectively and classified patients who developed cerebrovascular events (CVEs) within 5 years after 18F-FDG PET/CT scan as the active group and patients who did not experience the CVE within 5 years as an inactive group. We calculated SUVA/V and SUVA-V using measurements of SUVmax of carotid arteries and mean SUV of superior vena cava (SVC). Results: SUVA-V, SUVA-V_high, and SUVA-V_low were significantly higher in the active group than in the inactive group, but neither SUVA/V, SUVA/V_high, nor SUVA/V_low showed significant differences between the active and inactive groups. The difference in rank between groups of SUVA/V_high and SUVA/V_low was greater than the difference in rank between groups of SUVA-V_high and SUVA-V_low. The CVE incidence differed between SUVA/V_high and SUVA/V_low of high carotid FDG uptake, but the CVE incidence did not differ between SUVA-V_high and SUVA-V_low of high carotid FDG uptake. Conclusion: SUVA-V may be a more rational solution than SUVA/V for evaluating atherosclerotic plaque inflammation on 18F-FDG PET/CT.

Diagnostic value of 18F-FDG PET/CT in discriminating between benign and malignant lesions of the ribs.

PURPOSE: Imaging biomarkers for rib mass are needed to optimize treatment plan. We investigated the diagnostic value of metabolic and volumetric parameters from 18F-fluorodeoxyglucose (FDG) positron-emission tomography/computed tomography (PET/CT) in discriminating between benign and malignant lesions of the ribs. PATIENTS AND METHODS: Fifty-seven patients with pathologically proven diagnosis of rib lesions were retrospectively enrolled. The size of rib lesions, the maximum, mean, and peak standardized uptake value (SUVmax, SUVmean, SUVpeak), tumor-to-background ratio (TBR), metabolic tumor volume (MTV), and total lesions glycolysis (TLG) were measured. The FDG uptake patterns (segmental and discrete) and CT findings (soft tissue involvement and fracture) were also reviewed. RESULTS: Among the multiple parameters extracted from PET/CT, the MTV of malignant lesions was significantly higher than that of benign lesions (median; 4.7 vs 0.2, respectively, P = .041). In receiver operating characteristics curve analysis, MTV had the largest area under curve of 0.672 for differentiating malignant from benign lesions. For identifying malignant lesions, an MTV threshold of 0.5 had a sensitivity of 85.0%, specificity of 47.1%, positive predictive value of 79.1%, negative predictive value of 57.1%, and accuracy of 73.7%. The presence of adjacent soft tissue involvement around rib lesions showed a significant association with malignancy (odds ratio = 6.750; 95% CI, 1.837-24.802, P = .003). CONCLUSIONS: The MTV is a useful PET/CT parameter for assisting in the differential diagnosis of suspected malignant lesions of the ribs. CT finding of adjacent soft tissue involvement around rib was significantly associated with malignant lesions of the ribs.

Isolation of a Melanoblast Stimulator from Dimocarpus longan, Its Structural Modification, and Structure-Activity Relationships for Vitiligo.

A novel melanoblast stimulator (1) was isolated from Dimocarpus longan. Its analogs were also synthesized to support a new furan-based melanoblast stimulator scaffold for treating vitiligo. Isolated 5-(hydroxymethyl)furfural (HMF, 1) is a well-known compound in the food industry. Surprisingly, the melanogenic activity of HMF (1) was discovered here for the first time. Both HMF and its synthetic analog (16) promote the differentiation and migration of melanoblasts in vitro. Typically, stimulator (1) upregulated MMP2 expression, which promoted the migration of melanoblasts in vitro.

A novel thermostable beetle luciferase based cytotoxicity assay.

Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed 'Matador-Glo assay' which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

Narciclasine, an isocarbostyril alkaloid, has preferential activity against primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin lymphoma associated with infection by Kaposi sarcoma-associated herpes virus (KSHV). PEL is an aggressive disease with extremely poor prognosis when treated with conventional chemotherapy. Narciclasine, a natural product present in Amaryllidaceae family of flowering plants including daffodils, belongs to a class of molecules termed 'isocarbostyril alkaloid'. We have found that narciclasine displays preferential cytotoxicity towards PEL at low nanomolar concentrations and is approximately 10 and 100-fold more potent than its structural analogs lycoricidine and lycorine, respectively. Narciclasine arrested cell-cycle progression at the G1 phase and induced apoptosis in PEL, which is accompanied by activation of caspase-3/7, cleavage of PARP and increase in the surface expression of Annexin-V. Although narciclasine treatment resulted in a marked decrease in the expression of MYC and its direct target genes,time-course experiments revealed that MYC is not a direct target of narciclasine. Narciclasine treatment neither induces the expression of KSHV-RTA/ORF50 nor the production of infectious KSHV virions in PEL. Finally, narciclasine provides dramatic survival advantages to mice in two distinct mouse xenograft models of PEL. In conclusion, our results suggest that narciclasine could be a promising agent for the treatment of PEL.

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors.

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modified with chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferase-based assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

A novel luciferase-based assay for the detection of Chimeric Antigen Receptors.

Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy has produced dramatic responses in hematologic malignancies. One of the challenges in the field is the lack of a simple assay for the detection of CARs on the surface of immune effector cells. In this study, we describe a novel luciferase-based assay, termed Topanga Assay, for the detection of CAR expression. The assay utilizes a recombinant fusion protein, called Topanga reagent, generated by joining the extra-cellular domain of a CAR-target in frame with one of the marine luciferases or their engineered derivatives. The assay involves incubation of CAR expressing cells with the Topanga reagent, a few washes and measurement of luminescence. The assay can detect CARs comprising either immunoglobulin- or non-immunoglobulin-based antigen binding domains. We further demonstrate that addition of epitope tags to the Topanga reagent not only allows its convenient one step purification but also extends its use for detection of CAR cells using flow cytometry. However, crude supernatant containing the secreted Topanga reagent can be directly used in both luminescence and flow-cytometry based assays without prior protein purification. Our results demonstrate that the Topanga assay is a highly sensitive, specific, convenient, economical and versatile assay for the detection of CARs.

Predictive Value of Preoperative Volume-Based 18F-2-Fluoro-2-Deoxy-d-Glucose Positron Emission Tomography/Computed Tomography Parameters in Patients with Resectable Lung Adenocarcinoma.

PURPOSE: This study aimed to investigate the prognostic value of metabolic tumor volume (MTV) and total lesion glycolysis (TLG), which are volume-based PET parameters, using 18F-2-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) in patients with surgically resectable lung adenocarcinoma. METHODS: We retrospectively evaluated 149 patients with lung adenocarcinoma who underwent 18F-FDG PET/CT before surgical resection. Maximum standardized uptake value (SUVmax), MTV, and TLG of the primary tumor with threshold value of SUVmax 30, 40, and 50% were calculated, respectively. To compare the predictive performance of volume-based PET parameters, recurrence-free survival was assessed using the Kaplan-Meier method. RESULTS: The study included 70 males and 79 females with an average age of 65.8 years. The median follow-up time was 45.4 months. Recurrence was observed in 53 patients (35.6%). The mean ± SD SUVmax, MTV30%, and TLG30% of the entire cohort were 4.79 ± 2.94, 19.45 ± 24.85, and 56.43 ± 101.88, respectively. The cut-off values of MTV30% and TLG30% for recurrence were 11.07 ad 30.56, respectively. The 1-year recurrence-free survival (RFS) rate was 96.5% in low-MTV30% patients compared with 86.2% in high-MTV30% patients (p = 0.018) and 96.0% in low-TLG30% patients compared with 88.5% in high-TLG30% patients (p < 0.001). On univariate and multivariate analysis, TLG30% (HR, 2.828, p < 0.001; HR, 2.738, p < 0.001, respectively) was an independent prognostic factor for predicting recurrence-free survival (RFS). CONCLUSION: TLG30% value was observed to be a significant prognostic factor for RFS in patients with lung adenocarcinoma treated by surgical resection.

Femoral versus jugular access for Denali Vena Cava Filter placement: Analysis of fluoroscopic time, filter tilt and retrieval outcomes.

PURPOSE: To analyze relevant metrics involved in Denali Vena Cava Filter placement via different venous access sites. MATERIALS AND METHODS: Patients with Denali filters inserted between March 2017 and February 2018 were retrospectively analyzed. Pre-procedural and pre-retrieval computed tomography (CT) were reviewed. We compared inferior vena cava (IVC) diameter, filter tilt angle, filter tip IVC wall abutment, fluoroscopy time, and retrieval outcomes by venous access site. Filter tip abutment/limb penetration and procedure-related complications were investigated. RESULTS: Seventy-eight patients had successfully-placed Denali filters. Seventy-one of 78 (91%) patients had both pre-procedural and pre-retrieval CT. The majority (35 [49%]) were placed via the right femoral vein (left femoral vein: 22 [31%]; right internal jugular vein: 14 [20%]). The jugular approach involved a longer fluoroscopy time (mean 117 ±â€¯37 s [s]) than the right and left femoral approaches (mean 64 ±â€¯21 s, mean 67 ±â€¯15 s, respectively [p < 0.05]). Filter tilt and filter tip abutment were not significantly different between the 3 access routes. Filter tip abutment and limb penetration were observed in 8/71 (11%) and 2/71 (3%) patients, respectively. Filter retrieval was attempted in 68 of 78 (87%) cases, and all filters were successfully retrieved. One filter arm fractured during advanced retrieval; no other procedure related complications were recorded. CONCLUSIONS: Both femoral venous approaches can be safely used for placement of the Denali filter. Femoral venous access involved a shorter fluoroscopy time without any differences in filter tilt and filter tip abutment compared to transjugular access.

Simultaneous and rapid analysis of chemical preservatives in processed animal products by ultra-performance liquid chromatography.

An ultra-performance liquid chromatography-tunable ultraviolet method was optimized and validated for the simultaneous analysis of nine chemical preservatives in processed animal products. The limits of detection and quantification for the preservatives were within the ranges of 0.02-0.23 and 0.07-0.76 µg/mL, respectively. The relative standard deviations for intraday analyses of retention time and peak area were 0.00-0.23 and 0.03-2.93%, respectively, whereas, those for interday analyses were 0.67-2.30 and 2.12-5.37%, respectively. Of the nine preservatives spiked into six different animal products, dehydroacetic acid spiked into soft cheese exhibited the lowest recovery rate of 72.1 ± 0.36% at the lowest concentration (0.25 g/kg). Comparing data between UPLC and high-performance liquid chromatography with a 5% significance level, the t-statistic was 1.42. Moreover, sorbic acid was detected in 16 animal products (0.11-2.49 g/kg) when 278 products were analyzed for preservatives.

Functional visceral fat activity evaluated by preoperative F-18 FDG PET/CT predicts regional lymph node metastasis in differentiated thyroid cancer.

OBJECTIVE: We investigated the predictive role of functional visceral fat activity evaluated by preoperative F-18 FDG PET/CT for regional lymph node metastasis in patients with differentiated thyroid cancer. DESIGN AND PATIENTS: Fifty-eight patients with newly diagnosed differentiated thyroid cancer were enrolled in this study, retrospectively (men 14; women 44; age 53 ± 14.5). They all received preoperative F-18 FDG PET/CT and surgery. Functional visceral fat activity was defined as maximum standardized uptake value (SUVmax) of visceral fat divided by SUVmax of subcutaneous fat (V/S ratio). Pathology results were confirmed through the surgical specimens. RESULTS: The patients with regional lymph node metastasis showed higher functional visceral fat activity (V/S ratio) than the patients without regional lymph node metastasis (2.12 ± 0.6 vs 1.62 ± 0.34, P = .001). V/S ratio of 1.78 was suggested as an optimal cut-off value for predicting regional lymph node metastasis (sensitivity; 70.3%, specificity; 83.3%, area under the curve; 0.778, P < .0001). Furthermore, functional visceral fat activity was significantly associated with regional lymph node metastasis in patients with differentiated thyroid cancer by univariate and multivariate analyses. CONCLUSIONS: Functional visceral fat activity significantly affects the regional lymph node metastasis status in patients with differentiated thyroid cancer. Furthermore, it may also be useful to predict regional lymph node metastasis.

Central venous catheterization-induced right brachiocephalic vein pseudoaneurysm: Successfully treated with stent-assisted coiling.

INTRODUCTION: Central venous catheterization-induced central vein pseudoaneurysm is rare. Several treatment options have been recommended. We describe a case of central venous catheterization-induced right brachiocephalic vein pseudoaneurysm successfully treated with an uncovered self-expandable stent-assisted coil embolization and discuss the imaging findings, treatment strategy, and review of literature associated with thoracic venous pseudoaneurysm. CASE REPORT: A 77-year-old woman was referred to our trauma center to undergo treatment for central venous catheterization-induced central vein pseudoaneurysm. The initial contrast-enhanced chest computed tomography revealed a 3.4-cm pseudoaneurysm arising from the right brachiocephalic vein and a surrounding mediastinal hematoma. The pseudoaneurysm was successfully embolized with stent-assisted coiling. Computed tomography angiography was performed 10 days after the procedure and demonstrated a completely embolized pseudoaneurysm and resolved mediastinal hematoma. Blood flow from the right subclavian and left innominate veins was not disturbed by the stent-assisted coils. CONCLUSION: To our knowledge, this is the first report of treatment of a right brachiocephalic vein pseudoaneurysm with stent-assisted coil embolization. We think that uncovered stent-assisted coil embolization is the safest and most fundamental treatment for wide-neck venous pseudoaneurysm especially in a hemodynamically unstable setting.

Development and characterization of a novel luciferase based cytotoxicity assay.

A simple, accurate, sensitive and robust assay that can rapidly and specifically measure the death of target cells would have applications in many areas of biomedicine and particularly for the development of novel cellular- and immune-therapeutics. In this study, we describe a novel cytotoxicity assay, termed the Matador assay, which takes advantage of the extreme brightness, stability and glow-like characteristics of recently discovered novel marine luciferases and their engineered derivatives. The assay involves expression of a luciferase of interest in target cells in a manner so that it is preferentially retained within the healthy cells but is either released from dead and dying cells or whose activity can be preferentially measured in dead and dying cells. We demonstrate that this assay is highly sensitive, specific, rapid, and can be performed in a single-step manner without the need for any expensive equipment. We further validate this assay by demonstrating its ability to detect cytotoxicity induced by several cellular and immune-therapeutic agents including antibodies, natural killer cells, chimeric antigen receptor expressing T cells and a bispecific T cell engager.

Ratio of Mediastinal Lymph Node SUV to Primary Tumor SUV in 18F-FDG PET/CT for Nodal Staging in Non-Small-Cell Lung Cancer.

PURPOSE: Following determination of the maximum standardized uptake values (SUVmax) of the mediastinal lymph nodes (SUV-LN) and of the primary tumor (SUV-T) on 18F-FDG PET/CT in patients with non-small-cell lung cancer (NSCLC), the aim of the study was to determine the value of the SUV-LN/SUV-T ratio in lymph node staging in comparison with that of SUV-LN. METHODS: We retrospectively reviewed a total of 289 mediastinal lymph node stations from 98 patients with NSCLC who were examined preoperatively for staging and subsequently underwent pathologic studies of the mediastinal lymph nodes. We determined SUV-LN and SUV-R for each lymph node station on 18F-FDG PET/CT and then classified each station into one of three groups based on SUV-T (low, medium and high SUV-T groups). Diagnostic performance was assessed based on receiver operating characteristic (ROC) curve analysis, and the optimal cut-off values that would best discriminate metastatic from benign lymph nodes were determined for each method. RESULTS: The average of SUV-R of malignant lymph nodes was significantly higher than that of benign lymph nodes (0.79 ± 0.45 vs. 0.36 ± 0.23, P < 0.0001). In the ROC curve analysis, the area under the curve (AUC) of SUV-R was significantly higher than that of SUV-LN in the low SUV-T group (0.885 vs. 0.810, P = 0.019). There were no significant differences between the AUCs of SUV-LN and of SUV-R in the medium and high SUV-T groups. The optimal cut-off value for SUV-R in the low SUV-T group was 0.71 (sensitivity 87.5 %, specificity 85.9 %). CONCLUSIONS: The SUV-R performed well in distinguishing between metastatic and benign lymph nodes. In particular, SUV-R was found to have a better diagnostic performance than SUV-LN in the low SUV-T group.

Correlation between Semi-Quantitative (18)F-FDG PET/CT Parameters and Ki-67 Expression in Small Cell Lung Cancer.

PURPOSE: The aim of this study was to evaluate the relationship between semiquantitative parameters on (18)F-FDG PET/CT including maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) and the expression level of Ki-67 in small-cell lung cancer (SCLC). METHODS: Ninety-four consecutive patients with SCLC were enrolled in this study. They underwent (18)F-FDG PET/CT for initial evaluation of SCLC, and we measured SUVmax, avgSUVmean, MTVsum, and TLGtotal on (18)F-FDG PET/CT images. The protein expression of Ki-67 was examined by immunohistochemical staining. RESULTS: Significant correlations were found between the MTVsum and Ki-67 labeling index (r = 0.254, p = 0.014) and the TLGtotal and Ki-67 labeling index (r = 0.239, p = 0.020). No correlation was found between the SUVmax and Ki-67 labeling index (r = 0.116, p = 0.264) and the avgSUVmean and Ki-67 labeling index (r = 0.031, p = 0.770). Dividing the Ki-67 expression level into three categories, it was suggested that increasing Ki-67 expression level caused a stepwise increase in the MTVsum and TLGtotal. (p = 0.028 and 0.039, respectively), but not the SUVmax and avgSUVmean (p = 0.526 and 0.729, respectively). CONCLUSION: In conclusion, the volume-based parameters of (18)F-FDG PET/CT correlate with immunohistochemical staining of Ki-67 in SCLC. Measurement of the MTVsum and TLGtotal by (18)F-FDG PET/CT might be a simple, noninvasive, and useful method to determine the proliferative potential of cancer cells.

Cell-Autonomous Gß Signaling Defines Neuron-Specific Steady State Serotonin Synthesis in Caenorhabditis elegans.

Heterotrimeric G proteins regulate a vast array of cellular functions via specific intracellular effectors. Accumulating pharmacological and biochemical studies implicate Gß subunits as signaling molecules interacting directly with a wide range of effectors to modulate downstream cellular responses, in addition to their role in regulating Gα subunit activities. However, the native biological roles of Gß-mediated signaling pathways in vivo have been characterized only in a few cases. Here, we identified a Gß GPB-1 signaling pathway operating in specific serotonergic neurons to the define steady state serotonin (5-HT) synthesis, through a genetic screen for 5-HT synthesis mutants in Caenorhabditis elegans. We found that signaling through cell autonomous GPB-1 to the OCR-2 TRPV channel defines the baseline expression of 5-HT synthesis enzyme tryptophan hydroxylase tph-1 in ADF chemosensory neurons. This Gß signaling pathway is not essential for establishing the serotonergic cell fates and is mechanistically separated from stress-induced tph-1 upregulation. We identified that ADF-produced 5-HT controls specific innate rhythmic behaviors. These results revealed a Gß-mediated signaling operating in differentiated cells to specify intrinsic functional properties, and indicate that baseline TPH expression is not a default generic serotonergic fate, but is programmed in a cell-specific manner in the mature nervous system. Cell-specific regulation of TPH expression could be a general principle for tailored steady state 5-HT synthesis in functionally distinct neurons and their regulation of innate behavior.

Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing, Butyrate-Forming Acetogen, Eubacterium limosum KIST612.

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na(+) dependent. Consistent with the finding of a Na(+)-dependent Rnf complex is the presence of a conserved Na(+)-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H2 + CO2. The energetic benefit may be one reason that butyrate is formed only from CO but not from H2 + CO2.