Genomic potential and physiological characteristics of C1 metabolism in novel acetogenic bacteria.

Acetogenic bacteria can utilize C1 compounds, such as carbon monoxide (CO), formate, and methanol, via the Wood-Ljungdahl pathway (WLP) to produce biofuels and biochemicals. Two novel acetogenic bacteria of the family Eubacteriaceae ES2 and ES3 were isolated from Eulsukdo, a delta island in South Korea. We conducted whole genome sequencing of the ES strains and comparative genome analysis on the core clusters of WLP with Acetobacterium woodii DSM1030T and Eubacterium limosum ATCC8486T. The methyl-branch cluster included a formate transporter and duplicates or triplicates copies of the fhs gene, which encodes formyl-tetrahydrofolate synthetase. The formate dehydrogenase cluster did not include the hydrogenase gene, which might be replaced by a functional complex with a separate electron bifurcating hydrogenase (HytABCDE). Additionally, duplicated copies of the acsB gene, encoding acetyl-CoA synthase, are located within or close to the carbonyl-branch cluster. The serum bottle culture showed that ES strains can utilize a diverse range of C1 compounds, including CO, formate, and methanol, as well as CO2. Notably, ES2 exhibited remarkable resistance to high concentrations of C1 substrates, such as 100% CO (200 kPa), 700 mM formate, and 500 mM methanol. Moreover, ES2 demonstrated remarkable growth rates under 50% CO (0.45 h-1) and 200 mM formate (0.34 h-1). These growth rates are comparable to or surpassing those previously reported in other acetogenic bacteria. Our study introduces novel acetogenic ES strains and describes their genetic and physiological characteristics, which can be utilized in C1-based biomanufacturing.

Thermococcus argininiproducens sp. nov., an arginine biosynthesis archaeal species isolated from the Central Indian Ocean ridge.

A strictly anaerobic hyperthermophilic archaeon, designated strain IOH2T, was isolated from a deep-sea hydrothermal vent (Onnuri vent field) area on the Central Indian Ocean Ridge. Strain IOH2T showed high 16S rRNA gene sequence similarity to Thermococcus sibiricus MM 739T (99.42 %), Thermococcus alcaliphilus DSM 10322T (99.28 %), Thermococcus aegaeus P5T (99.21 %), Thermococcus litoralis DSM 5473T (99.13 %), 'Thermococcus bergensis' T7324T (99.13 %), Thermococcus aggregans TYT (98.92 %) and Thermococcus prieurii Bio-pl-0405IT2T (98.01 %), with all other strains showing lower than 98 % similarity. The average nucleotide identity and in silico DNA-DNA hybridization values were highest between strain IOH2T and T. sibiricus MM 739T (79.33 and 15.00 %, respectively); these values are much lower than the species delineation cut-offs. Cells of strain IOH2T were coccoid, 1.0-1.2 µm in diameter and had no flagella. Growth ranges were 60-85 °C (optimum at 80 °C), pH 4.5-8.5 (optimum at pH 6.3) and 2.0-6.0 % (optimum at 4.0 %) NaCl. Growth of strain IOH2T was enhanced by starch, glucose, maltodextrin and pyruvate as a carbon source, and elemental sulphur as an electron acceptor. Through genome analysis of strain IOH2T, arginine biosynthesis related genes were predicted, and growth of strain IOH2T without arginine was confirmed. The genome of strain IOH2T was assembled as a circular chromosome of 1 946 249 bp and predicted 2096 genes. The DNA G+C content was 39.44 mol%. Based on the results of physiological and phylogenetic analyses, Thermococcus argininiproducens sp. nov. is proposed with type strain IOH2T (=MCCC 4K00089T=KCTC 25190T).

Alkalibacter rhizosphaerae sp. nov., a CO-utilizing bacterium isolated from tidal flat sediment, and emended description of the genus Alkalibacter.

A novel anaerobic, rod-shaped, non-motile bacterium, designated strain ES005T, was isolated from tidal flat sediments near the rhizosphere of Phragmites australis at Eulsukdo Island, Republic of Korea. A polyphasic approach revealed that cells of the strain were Gram-stain-positive, catalase- and oxidase-negative, non-spore-forming rods. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain ES005T belonged to the family Eubacteriaceae, class Clostridia and showed the highest sequence similarity to Alkalibacter mobilis (97.52 %) and followed by Alkalibacter saccharofermentans Z-79820T (96.72%). The OrthoANI value between strain ES005T and A. mobilis was 69.67 %. Strain ES005T grew optimally at 33-37 °C, at pH 6.0-7.0 and in the presence of 1-2 % (w/v) NaCl. Growth in 12.5 % CO atmosphere was observed. Acetate and formate were end products of fructose fermentation and growth on CO. The major cellular fatty acids of strain ES005T were C14 : 0 (39.1 %) and C16 : 0 (26.6 %). The major polar lipids were diphoshatidylgycerol, phosphatidylglycerol and three unidentified phospholipids. The DNA G+C content of strain ES005T was 46.9 mol%. Based on the phenotypic, phylogenetic, genomic and chemotaxonomic features of the isloate, strain ES005T represents a novel species, for which the name Alkalibacter rhizosphaerae sp. nov. is proposed. The type strain is ES005T (=KCTC 25246T=JCM 34530T).

Establishment of Genome Based Criteria for Classification of the Family Desulfovibrionaceae and Proposal of Two Novel Genera, Alkalidesulfovibrio gen. nov. and Salidesulfovibrio gen. nov.

Bacteria in the Desulfovibrionaceae family, which contribute to S element turnover as sulfate-reducing bacteria (SRB) and disproportionation of partially oxidized sulfoxy anions, have been extensively investigated since the importance of the sulfur cycle emerged. Novel species belonging to this taxon are frequently reported, because they exist in various environments and are easy to culture using established methods. Due to the rapid expansion of the taxon, correction and reclassification have been conducted. The development of high-throughput sequencing facilitated rapid expansion of genome sequence database. Genome-based criteria, based on these databases, proved to be potential classification standard by overcoming the limitations of 16S rRNA-based phylogeny. Although standards methods for taxogenomics are being established, the addition of a novel genus requires extensive calculations with taxa, including many species, such as Desulfovibrionaceae. Thus, the genome-based criteria for classification of Desulfovibrionaceae were established and validated in this study. The average amino-acid identity (AAI) cut-off value, 63.43 ± 0.01, was calculated to be an appropriate criterion for genus delineation of the family Desulfovibrionaceae. By applying the AAI cut-off value, 88 genomes of the Desulfovibrionaceae were divided into 27 genera, which follows the core gene phylogeny results. In this process, two novel genera (Alkalidesulfovibrio and Salidesulfovibrio) and one former invalid genus ("Psychrodesulfovibrio") were officially proposed. Further, by applying the 95-96% average nucleotide identity (ANI) standard and the 70% digital DNA-DNA hybridization standard values for species delineation of strains that were classified as the same species, five strains have the potential to be newly classified. After verifying that the classification was appropriately performed through relative synonymous codon usage analysis, common characteristics were listed by group. In addition, by detecting metal resistance related genes via in silico analysis, it was confirmed that most strains display metal tolerance.

Bioconversion of CO to formate by artificially designed carbon monoxide:formate oxidoreductase in hyperthermophilic archaea.

Ferredoxin-dependent metabolic engineering of electron transfer circuits has been developed to enhance redox efficiency in the field of synthetic biology, e.g., for hydrogen production and for reduction of flavoproteins or NAD(P)+. Here, we present the bioconversion of carbon monoxide (CO) gas to formate via a synthetic CO:formate oxidoreductase (CFOR), designed as an enzyme complex for direct electron transfer between non-interacting CO dehydrogenase and formate dehydrogenase using an electron-transferring Fe-S fusion protein. The CFOR-introduced Thermococcus onnurineus mutant strains showed CO-dependent formate production in vivo and in vitro. The maximum formate production rate from purified CFOR complex and specific formate productivity from the bioreactor were 2.2 ± 0.2 µmol/mg/min and 73.1 ± 29.0 mmol/g-cells/h, respectively. The CO-dependent CO2 reduction/formate production activity of synthetic CFOR was confirmed, indicating that direct electron transfer between two unrelated dehydrogenases was feasible via mediation of the FeS-FeS fusion protein.

Proteiniclasticum aestuarii sp. nov., isolated from tidal flat sediment, and emended descriptions of the genus Proteiniclasticum and Proteiniclasticum ruminis.

A novel bacterium, designated SCR006T, was isolated from tidal flat sediment from Suncheon Bay, Republic of Korea. Cells of strain SCR006T were strictly anaerobic, motile cocci, Gram-reaction-negative, and catalase- and oxidase-negative. Growth was observed at 4-41 °C (optimum, 34-37 °C), at pH 6.5-10.0 (optimum, pH 7.0-7.5) and in presence of 0-8 % NaCl (optimum, 0-2 %). Fermentation products of peptone-yeast-glucose medium were acetate and ethanol. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that strain SCR006T had high sequence similarity to Proteiniclasticum ruminis D3RC-2T (97.9 %), followed by Youngiibacter multivorans DSM 6139T (95.9 %) and Youngiibacter fragilis 232.1T (95.0 %). The average nucleotide identity value between strain SCR006T and P. ruminis DSM 24773T was 72.7 %, which strongly supported that strain SCR006T reresents a novel species within the genus Proteiniclasticum. The major cellular fatty acids are iso-C15 : 0 (27.2 %) and anteiso-C15 : 0 (16.9 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unidentified phospholipids, an unidentified aminolipid and five unidentified lipids. The genomic size was 3.2 Mb with genomic DNA G+C content of 45.6 mol%. The results of 16S rRNA-based and genome-based phylogenetic tree analyses indicated that SCR006T should be assigned to the genus Proteiniclasticum. Strain SCR006T could be distinguished from P. ruminis D3RC-2T by its growth conditions, cell morphology and genomic characteristics. Based on the phenotypic, phylogenetic, genomic and chemotaxonomic features, strain SCR006T represents a novel species, for which the name Proteiniclasticum aestuarii sp. nov. is proposed, with the type strain SCR006T (=KCTC 25245T= JCM 34531T).

Blood KL-6 predicts prognosis in primary Sjögren's syndrome-associated interstitial lung disease.

Interstitial lung disease associated with primary Sjögren's syndrome (SJS-ILD) has a variable clinical course. We aimed to investigate the role of blood biomarkers in predicting prognosis for SJS-ILD. Clinical data of 46 SJS-ILD patients were retrospectively reviewed. Plasma biomarker levels, including Krebs von den Lungen-6 (KL-6), CC chemokine ligand 18 (CCL18), chitinase-3-like-1 (YKL-40), interleukin-4 receptor alpha (IL-4Ra), and matrix metalloproteinase-7 (MMP-7) were measured using the multiplex Luminex assays (R&D Systems, Minneapolis, USA). The median follow-up period was 69.0 months. The mean age of the patients was 59.4 years; 17.4% were men. The KL-6 level was significantly higher in non-survivors (n = 12; 119.6 vs. 59.5 pg/mL, P = 0.037) than survivors (n = 34), while the levels of the other biomarkers did not differ. Receiver operating characteristic analysis indicated that KL-6 shows the best performance for predicting survival (area under the curve = 0.705, P = 0.037; best cut-off value = 53.5 pg/mL). Multivariable Cox analysis that was adjusted by age and diffusing capacity for carbon monoxide suggested a high KL-6 level (> 53.5 pg/mL) as an independent prognostic factor for survival (hazard ratio = 5.939, 95% confidence interval 1.312-26.881, P = 0.021). Our results suggest that blood KL-6 might be a useful in predicting the prognosis for patients with SJS-ILD.

Multiplex Immunofluorescence Assay of Infiltrating Mononu-Clear Cell Subsets in Acute T-Cell-Mediated Rejection and BK Virus-Associated Nephropathy in the Allograft Kidney.

Renal allograft biopsy is the gold standard procedure for diagnosis of kidney rejection via specific pathological changes. To provide a better assessment of immunologic events in acute T-cell-mediated rejection (acute TCMR) and BK virus-associated nephropathy (BKVAN) cases, we used multiplex immunofluorescence staining to identify infiltrating mononuclear cell subsets in the cortex area of transplanted kidneys. Antibodies to CD4, CD8, CD20, CD68, Foxp3, and cytokeratin were used. In cortical interstitium, CD8+ cells were significantly more prevalent in acute TCMR than BKVAN cases (34% vs. 22.8%, p = 0.034). In medulla, CD20+ cells were significantly more prevalent in BKVAN than acute TCMR cases (51.9% vs. 11.3%, p = 0.028).

Immune profile by multiplexed immunohistochemistry associated with recurrence after chemoradiation in rectal cancer.

BACKGROUND AND AIM: Evidence has emerged that a pretreatment immune profile in rectal cancer is associated with response to chemoradiotherapy (CRT) and recurrence after CRT. However, few studies have evaluated the immune profile differences after CRT regarding recurrence and nonrecurrence. METHODS: We included patients with advanced rectal cancer treated with CRT and surgery with recurrence within 1 year in a recurrence group. After sex and age matching with the recurrence group, patients with no recurrence for 3 years after CRT were included in a nonrecurrence group. We extracted the immune profile, including CD3 and CD8, from the surgical specimen after CRT using multispectral fluorescence immunohistochemistry and compared the two groups. RESULTS: The immune profiles of 65 patients with rectal cancer were assessed; 30 were included in the recurrence group and 35 were included in the nonrecurrence group. CD3+ and CD8+ T lymphocyte densities were significantly higher in the nonrecurrence group than in the recurrence group (CD3+ ; P < 0.001, CD8+ ; P = 0.003) in the primary tumor. Consistent results were found in epithelial and stromal cells. Compared with the recurrence group, the distinct profiles of co-expressed immune markers in the nonrecurrence group were revealed (CD3+ CD8+ , P = 0.001; CD3+ CD8+ PD-L1- , P = 0.001; CD3+ CD8+ FOXP3- PD-L1- , P = 0.001). CONCLUSIONS: Vigorous CD3+ and CD8+ T cell priming post-CRT was prominent in the nonrecurrence group compared with that of the recurrence group. This finding suggests that differences in immune profiles may have clinical significance even after CRT.

Long-term clinical course and outcome in patients with primary Sjögren syndrome-associated interstitial lung disease.

Interstitial lung disease (ILD) is the most common lung manifestation in patients with Sjögren syndrome (SJS) and is associated with poor outcomes. This study aimed to investigate the long-term clinical course and prognostic factors in patients with SJS-ILD. Clinical data and high-resolution computed tomography (HRCT) images of 62 patients with primary SJS-ILD were retrospectively analyzed (biopsy-proven cases, n = 16). The mean patient age was 59.8 years; 83.9% of the patients were females, and 38.7% showed a usual interstitial pneumonia (UIP) pattern on HRCT. The median follow-up period was 61.5 months. During follow-up, 15 patients (24.2%) died, 7 (11.3%) experienced acute exacerbation (AE), and 27 (43.5%) progressed. The 1-, 3- and 5-year survival rates were 93.5%, 85.8%, and 81.1%, respectively. Age (hazard ratio [HR]: 1.158, P = 0.003), C-reactive protein (CRP) level (HR: 1.212, P = 0.045), FVC (HR: 0.902, P = 0.005), and a UIP pattern on HRCT (HR: 4.580, P = 0.029) were significant prognostic factors in multivariable Cox analysis. In conclusion, death, AE, and ILD progression occurred in 25%, 10%, and 50% of the patients with SJS-ILD, respectively. Older age, higher CRP level, lower FVC, and a UIP pattern on HRCT indicated poor prognosis.

Thermococcus indicus sp. nov., a Fe(III)-reducing hyperthermophilic archaeon isolated from the Onnuri Vent Field of the Central Indian Ocean ridge.

A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic archaeon, designated as strain IOH1T, was isolated from a new deep-sea hydrothermal vent (Onnuri Vent Field) area in the Central Indian Ocean ridge. Strain IOH1T showed > 99% 16S rRNA gene sequence similarity with Thermococcus celericrescens TS2T (99.4%) and T. siculi DSM 12349T (99.2%). Additional three species T. barossii SHCK-94T (99.0%), T. celer Vu13T (98.8%), and T. piezophilus (98.6%) showed > 98.6% of 16S rRNA gene sequence similarity, however, the maximum OrthoANI value is 89.8% for the genome of T. celericrescens TS2T. Strain IOH1T cells are coccoid, 1.2-1.8 µm in diameter, and motile by flagella. Growth was at 70-82°C (optimum 80°C), pH 5.4-8.0 (optimum pH 6.0) with 2-4% (optimum 3%) NaCl. Growth of strain IOH1T was enhanced by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon sources, and elemental sulfur as an electron acceptor; clearly different from those of related species T. celecrescens DSM 17994T and T. siculi DSM 12349T. Strain IOH1T, T. celercrescence DSM 17994T, and T. siculi DSM 12349T reduced soluble Fe(III)-citrate present in the medium, whereas the amount of total cellular proteins increased with the concomitant accumulation of Fe(II). We determined a circular chromosome of 2,234 kb with an extra-chromosomal archaeal plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA G + C content was 54.9 mol%. Based on physiological properties, phylogenetic, and genome analysis, we proposed that strain IOH1T (= KCTC 15844T = JCM 39077T) is assigned to a new species in the genus Thermococcus and named Thermococcus indicus sp. nov.

Paradesulfovibrio onnuriensis gen. nov., sp. nov., a chemolithoautotrophic sulfate-reducing bacterium isolated from the Onnuri vent field of the Indian Ocean and reclassification of Desulfovibrio senegalensis as Paradesulfovibrio senegalensis comb. nov.

An anaerobic, rod-shaped, mesophilic, chemolithoautotrophic, sulfate-reducing bacterial strain IOR2T was isolated from a newly found deep-sea hydrothermal vent (OVF, Onnuri Vent Field) area in the central Indian Ocean ridge (11°24'88″ S 66°25'42″ E, 2021 m water depth). The 16S rRNA gene sequence analysis revealed that the strain IOR2T was most closely related to Desulfovibrio senegalensis BLaC1T (96.7%). However, it showed low similarity with the members of the family Desulfovibrionaceae, such as Desulfovibrio tunisiensis RB22T (94.0%), D. brasiliensis LVform1T (93.9%), D. halophilus DSM 5663T (93.7%), and Pseudodesulfovibrio aespoeensis Aspo-2T (93.2%). The strain IOR2T could grow at 23-42°C (optimum 37°C), pH 5.0-8.0 (optimum pH 7.0) and with 0.5-6.5% (optimum 3.0%) NaCl. The strain could use lactate, pyruvate, H2, and glycerol as electron donors and sulfate, thiosulfate, and sulfite as electron acceptors. The major fatty acids of the strain IOR2T were iso-C15:0, iso-C17:0, ante-iso-C15:0, and summed feature 9 (C16:0 methyl/iso-C17:1ω9c). Both the strains IOR2T and BLaC1T could grow with CO2 and H2 as the sole sources of carbon and energy, respectively. Genomic evidence for the Wood-Ljungdahl pathway in both the strains reflects chemolithoautotrophic growth. The DNA G + C content of the strain IOR2T and BLaC1T was 58.1-60.5 mol%. Based on the results of the phylogenetic and physiologic studies, Paradesulfovibrio onnuriensis gen. nov., sp. nov. with the type strain IOR2T (= KCTC 15845T = MCCC 1K04559T) was proposed to be a member of the family Desulfovibrionaceae. We have also proposed the reclassification of D. senegalensis as Paradesulfovibrio senegalensis comb. nov.

CD56+CD57+ infiltrates as the most predominant subset of intragraft natural killer cells in renal transplant biopsies with antibody-mediated rejection.

Little is known about the characteristics and clinical implications of specific subsets of intragraft natural killer (NK) cells in kidney transplant recipients. We analyzed 39 for-cause renal transplant biopsies performed at our center from May 2015 to July 2017. According to histopathologic reports, 8 patients (20.5%) had no rejection (NR), 11 (28.2%) had T cell-mediated rejections (TCMR) only, and 20 (51.3%) had antibody-mediated rejection (ABMR). NK cells were defined as CD3-CD56+ lymphocytes that are positive for CD57, CD49b, NKG2A, or KIR. The density of NK cells was significantly higher in the ABMR group (2.57 ± 2.58/mm2) than in the NR (0.12 ± 0.22/mm2) or the TCMR (0.25 ± 0.34/mm2) group (P = 0.002). Notably, CD56+CD57+ infiltrates (2.16 ± 1.89) were the most frequently observed compared with CD56+CD49b+ (0.05 ± 0.13), CD56+NKG2A+ (0.21 ± 0.69), and CD56+KIR+ (0.15 ± 0.42) cells in the ABMR group (P < 0.001). Death-censored graft failure was significantly higher in patients with NK cell infiltration than those without (Log-rank test, P = 0.025). In conclusion, CD56+CD57+ infiltrates are a major subset of NK cells in kidney transplant recipients with ABMR and NK cell infiltration is significantly associated with graft failure post-transplant.

Domestication of the novel alcohologenic acetogen Clostridium sp. AWRP: from isolation to characterization for syngas fermentation.

BACKGROUND: Gas-fermenting acetogens have received a great deal of attention for their ability to grow on various syngas and waste gas containing carbon monoxide (CO), producing acetate as the primary metabolite. Among them, some Clostridium species, such as C. ljungdahlii and C. autoethanogenum, are of particular interest as they produce fuel alcohols as well. Despite recent efforts, alcohol production by these species is still unsatisfactory due to their low productivity and acetate accumulation, necessitating the isolation of strains with better phenotypes. RESULTS: In this study, a novel alcohol-producing acetogen (Clostridium sp. AWRP) was isolated, and its complete genome was sequenced. This bacterium belongs the same phylogenetic group as C. ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii based on 16S rRNA homology; however, the levels of genome-wide average nucleotide identity (gANI) for strain AWRP compared with these strains range between 95 and 96%, suggesting that this strain can be classified as a novel species. In addition, strain AWRP produced a substantial amount of ethanol (70-90 mM) from syngas in batch serum bottle cultures, which was comparable to or even exceeded the typical values obtained using its close relatives cultivated under similar conditions. In a batch bioreactor, strain AWRP produced 119 and 12 mM of ethanol and 2,3-butanediol, respectively, while yielding only 1.4 mM of residual acetate. Interestingly, the alcohologenesis of this strain was strongly affected by oxidoreduction potential (ORP), which has not been reported with other gas-fermenting clostridia. CONCLUSION: Considering its ethanol production under low oxidoreduction potential (ORP) conditions, Clostridium sp. AWRP will be an interesting host for biochemical studies to understand the physiology of alcohol-producing acetogens, which will contribute to metabolic engineering of those strains for the production of alcohols and other value-added compounds from syngas.

Mesenchymal stem cells prevent the progression of diabetic nephropathy by improving mitochondrial function in tubular epithelial cells.

The administration of mesenchymal stem cells (MSCs) was shown to attenuate overt as well as early diabetic nephropathy in rodents, but the underlying mechanism of this beneficial effect is largely unknown. Inflammation and mitochondrial dysfunction are major pathogenic factors in diabetic nephropathy. In this study, we found that the repeated administration of MSCs prevents albuminuria and injury to tubular epithelial cells (TECs), an important element in the progression of diabetic nephropathy, by improving mitochondrial function. The expression of M1 macrophage markers was significantly increased in diabetic kidneys compared with that in control kidneys. Interestingly, the expression of arginase-1 (Arg1), an important M2 macrophage marker, was reduced in diabetic kidneys and increased by MSC treatment. In cultured TECs, conditioned media from lipopolysaccharide-activated macrophages reduced peroxisomal proliferator-activated receptor gamma coactivator 1α (Pgc1a) expression and impaired mitochondrial function. The coculture of macrophages with MSCs increased and decreased the expression of Arg1 and M1 markers, respectively. Treatment with conditioned media from cocultured macrophages prevented activated macrophage-induced mitochondrial dysfunction in TECs. In the absence of MSC coculture, Arg1 overexpression in macrophages reversed Pgc1a suppression in TECs. These observations suggest that MSCs prevent the progression of diabetic nephropathy by reversing mitochondrial dysfunction in TECs via the induction of Arg1 in macrophages.

A rapid and sensitive enzymatic assay for 2,3-butanediol.

In this study, we developed a rapid and sensitive enzymatic assay for 2,3-butanediol (2,3-BDO) detection. The concentration of 2,3-BDO was determined by measuring the reduction of NADP+ using Clostridium ljungdahlii 2,3-butanediol dehydrogenase (CL-Bdh). The enzymatic assay could detect as low as 0.01 mM of 2,3-BDO, while the high-performance liquid chromatography (HPLC) method required a much higher concentration than 0.15 mM. Gratifyingly, the developed method was 15 times more sensitive than the HPLC method. When the enzymatic assay was applied to high-throughput screening, the enzymatic assay detected 14 positive samples out of 23 tested, as compared to 8 by the HPLC method. These results suggest that the enzymatic assay is an effective screening method for the detection of 2,3-BDO-producing microbes in a microtiter plate-based format.

A simple biosynthetic pathway for 2,3-butanediol production in Thermococcus onnurineus NA1.

The biosynthetic pathway of 2,3-butanediol (2,3-BDO) production from pyruvate under anaerobic conditions includes three enzymes: acetolactate synthase (ALS), acetolactate decarboxylase (ALDC), and acetoin reductase (AR). Recently, in anaerobic hyperthermophilic Pyrococcus furiosus, it has been reported that acetoin, a precursor of 2,3-BDO, is produced from pyruvate by ALS through a temperature-dependent metabolic switch. In this study, we first attempted to produce 2,3-BDO from Thermococcus onnurineus NA1 using a simple biosynthetic pathway by two enzymes (ALS and AR) at a high temperature. Two heterologous genes, acetolactate synthase (alsS) from Pyrococcus sp. NA2 and alcohol dehydrogenase (adh) from T. guaymacensis, were introduced and expressed in T. onnurineus NA1. The mutant strain produced approximately 3.3 mM 2,3-BDO at 80 °C. An acetyl-CoA synthetase IIIα (TON_1001) was further deleted to enhance 2,3-BDO production, and the mutant strain showed a 25% increase in the specific production of 2,3-BDO. Furthermore, when carbon monoxide (CO) gas was added as a reductant, specific production of 2,3-BDO increased by 45%. These results suggest a new biosynthetic pathway for 2,3-BDO and demonstrate the possibility of T. onnurineus NA1 as a platform strain for 2,3-BDO production at high temperatures.

MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers.

The loss of imprinting of MEST has been linked to certain types of cancer by promoter switching. However, MEST-mediated regulation of tumorigenicity and metastasis are yet to be understood. Herein, we reported that MEST is a key regulator of IL-6/JAK/STAT3/Twist-1 signal pathway-mediated tumor metastasis. Enhanced MEST expression is significantly associated with pathogenesis of breast cancer patients. Also, MEST induces metastatic potential of breast cancer through induction of the EMT-TFs-mediated EMT program. Moreover, MEST leads to Twist-1 induction by STAT3 activation and subsequently enables the induction of activation of the EMT program via the induction of STAT3 nuclear translocation. Furthermore, the c-terminal region of MEST was essential for STAT3 activation via the induction of JAK2/STAT3 complex formation. Finally, MEST is required for metastasis in an experimental metastasis model. These observations suggest that MEST is a promising target for intervention to prevent tumor metastasis.

Does the Apparent Diffusion Coefficient Value Predict Permanent Cerebral Ischemia/Reperfusion Injury in Rats?

RATIONALE AND OBJECTIVES: Variation in tissue damage after cerebral ischemia/reperfusion (I/R) can cause uncertainty in stroke-related studies, which can be reduced if the damage can be predicted early after ischemia by measuring the apparent diffusion coefficient (ADC). We investigated whether ADC measurement in the acute phase can predict permanent cerebral I/R injury. MATERIALS AND METHODS: The middle cerebral artery occlusion model was established using the intraluminal suture method to induce 60 minutes of ischemia followed by reperfusion in rats. T2-weighted images and diffusion-weighted images were obtained at 30 minutes and 24 hours after ischemia. Neuronal cell survival was assessed by neuronal nuclei (NeuN) immunofluorescence staining. The correlation between relative ADC (rADC) values at 30 minutes and I/R injury at 24 hours after ischemia was analyzed. Magnetic resonance imaging results were confirmed by histologic analysis. RESULTS: The correlation between rADC values at 30 minutes and 24 hours was strong in the ischemic core and peri-infarct region but moderate in the anterior choroidal and hypothalamic region. Histologic analysis revealed that the correlation between rADC values at 30 minutes and the number of NeuN-positive cells at 24 hours was strong in the ischemic core and peri-infarct region but moderate in the anterior choroidal and hypothalamic region. Furthermore, there was a strong positive correlation between the sum of rADC values of three regions at 30 minutes and the infarct volume at 24 hours. CONCLUSION: ADC measurement in the acute phase can predict permanent cerebral I/R injury and provide important information for the evaluation of ischemic stroke.

Spatiotemporal heterogeneity of tumor vasculature during tumor growth and antiangiogenic treatment: MRI assessment using permeability and blood volume parameters.

Tumor heterogeneity is an important concept when assessing intratumoral variety in vascular phenotypes and responses to antiangiogenic treatment. This study explored spatiotemporal heterogeneity of vascular alterations in C6 glioma mice during tumor growth and antiangiogenic treatment on serial MR examinations (days 0, 4, and 7 from initiation of vehicle or multireceptor tyrosine kinase inhibitor administration). Transvascular permeability (TP) was quantified on dynamic-contrast-enhanced MRI (DCE-MRI) using extravascular extracellular agent (Gd-DOTA); blood volume (BV) was estimated using intravascular T2 agent (SPION). With regard to region-dependent variability in vascular phenotypes, the control group demonstrated higher TP in the tumor center than in the periphery, and greater BV in the tumor periphery than in the center. This distribution pattern became more apparent with tumor growth. Antiangiogenic treatment effect was regionally heterogeneous: in the tumor center, treatment significantly suppressed the increase in TP and decrease in BV (ie, typical temporal change in the control group); in the tumor periphery, treatment-induced vascular alterations were insignificant and BV remained high. On histopathological examination, the control group showed greater CD31, VEGFR2, Ki67, and NG2 expression in the tumor periphery than in the center. After treatment, CD31 and Ki67 expression was significantly suppressed only in the tumor center, whereas VEGFR2 and α-caspase 3 expression was decreased and NG2 expression was increased in the entire tumor. These results demonstrate that MRI can reliably depict spatial heterogeneity in tumor vascular phenotypes and antiangiogenic treatment effects. Preserved angiogenic activity (high BV on MRI and high CD31) and proliferation (high Ki67) in the tumor periphery after treatment may provide insights into the mechanism of tumor resistance to antiangiogenic treatment.