IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

PURPOSE: Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. MATERIALS AND METHODS: To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. RESULTS: Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. CONCLUSIONS: These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

Invasiveness of Upper Tract Urothelial Carcinoma: Clinical Significance and Integrative Diagnostic Strategy.

Purpose: In this study, we aimed to determine the clinicopathologic, radiologic, and molecular significance of the tumor invasiveness to further stratify the patients with high-grade (HG) upper tract urothelial carcinoma (UTUC) who can be treated less aggressively. Materials and Methods: Clinicopathologic and radiologic characteristics of 166 surgically resected HG UTUC (48 non-invasive, and 118 invasive) cases were evaluated. Six non-invasive UTUC cases with intra-tumoral tumor grade heterogeneity were selected for whole exome sequencing (WES) to understand the underlying molecular pathophysiology. Barcode-tagging sequencing (BTSeq) was done for validation of the target genes from WES data. Results: Patients with non-invasive UTUC showed no cancer-specific death with better cancer-specific survival (p<0.001) and recurrence-free survival (p<0.001) compared to the patients with invasive UTUC. Compared to the invasive UTUC, non-invasive UTUC was correlated to a low grade on the preoperative abdominal computed tomography (CT) grading system (p<0.001), histologic intratumoral tumor grade heterogeneity (p=0.018), discrepancy in preoperative urine cytology diagnosis (p=0.018), and absence of urothelial carcinoma in situ (p<0.001). WES of the heterogeneous components showed mutually shared HRAS and FGFR3 mutations shared between the HG and LG components. HRAS mutation was associated with the lower grade on preoperative abdominal CT and intratumoral tumor grade heterogeneity (p=0.045 and p<0.001, respectively), whereas FGFR3 mutation was correlated to the absence of carcinoma in situ (p<0.001). Conclusion: According to our comprehensive analysis, HG non-invasive UTUC can be preoperatively suspected based on distinct preoperative radiologic, cytologic, histologic, and molecular features. Non-invasive HG UTUC shows excellent prognosis and thus should be treated less aggressively.

Glutathione dynamics is a potential predictive and therapeutic trait for neoadjuvant chemotherapy response in bladder cancer.

Radical cystectomy with preoperative cisplatin-based neoadjuvant chemotherapy (NAC) is the standard care for muscle-invasive bladder cancers (MIBCs). However, the complete response rate to this modality remains relatively low, and current clinicopathologic and molecular classifications are inadequate to predict NAC response in patients with MIBC. Here, we demonstrate that dysregulation of the glutathione (GSH) pathway is fundamental for MIBC NAC resistance. Comprehensive analysis of the multicohort transcriptomes reveals that GSH metabolism and immune-response genes are enriched in NAC-resistant and NAC-sensitive MIBCs, respectively. A machine-learning-based tumor/stroma classifier is applied for high-throughput digitalized immunohistochemistry analysis, finding that GSH dynamics proteins, including glutaminase-1, are associated with NAC resistance. GSH dynamics is activated in cisplatin-resistant MIBC cells, and combination treatment with a GSH dynamics modulator and cisplatin significantly suppresses tumor growth in an orthotopic xenograft animal model. Collectively, these findings demonstrate the predictive and therapeutic values of GSH dynamics in determining the NAC response in MIBCs.

PD-L1 Upregulation by the mTOR Pathway in VEGFR-TKI-Resistant Metastatic Clear Cell Renal Cell Carcinoma.

PURPOSE: Tyrosine kinase inhibitors (TKI) targeting vascular endothelial growth factor receptor (VEGFR) signaling pathways have been used for metastatic clear cell renal cell carcinoma (mCCRCC), but resistance to the drug develops in most patients. We aimed to explore the underlying mechanism of the TKI resistance with regard to programmed death-ligand 1 (PD-L1) and to investigate signaling pathway associated with the resistant mechanism. MATERIALS AND METHODS: To determine the mechanism of resistance, 10 mCCRCC patients from whom tumor tissues were harvested at both the pretreatment and the TKI-resistant post-treatment period were included as the discovery cohort, and their global gene expression profiles were compared. A TKI-resistant renal cancer cell line was established by long-term treatment with sunitinib. RESULTS: Among differentially expressed genes in the discovery cohort, increased PD-L1 expression in post-treatment tissues was noted in four patients. Pathway analysis showed that PD-L1 expression was positively correlated with the mammalian target of rapamycin (mTOR) signaling pathway. The TKI-resistant renal cancer cells showed increased expression of PD-L1 and mTOR signaling proteins and demonstrated aggressive tumoral behaviour. Treatment with mTOR inhibitors down-regulated PD-L1 expression and suppressed aggressive tumoral behaviour, which was reversed with stimulation of the mTOR pathway. CONCLUSION: These results showed that PD-L1 expression may be increased in a subset of VEGFR-TKI-resistant mCCRCC patients via the mTOR pathway.

Synaptophysin, CD117, and GATA3 as a Diagnostic Immunohistochemical Panel for Small Cell Neuroendocrine Carcinoma of the Urinary Tract.

Although SCNEC is based on its characteristic histology, immunohistochemistry (IHC) is commonly employed to confirm neuroendocrine differentiation (NED). The challenge here is that SCNEC may yield negative results for traditional neuroendocrine markers. To establish an IHC panel for NED, 17 neuronal, basal, and luminal markers were examined on a tissue microarray construct generated from 47 cases of 34 patients with SCNEC as a discovery cohort. A decision tree algorithm was employed to analyze the extent and intensity of immunoreactivity and to develop a diagnostic model. An external cohort of eight cases and transmission electron microscopy (TEM) were used to validate the model. Among the 17 markers, the decision tree diagnostic model selected 3 markers to classify NED with 98.4% accuracy in classification. The extent of synaptophysin (>5%) was selected as the initial parameter, the extent of CD117 (>20%) as the second, and then the intensity of GATA3 (≤1.5, negative or weak immunoreactivity) as the third for NED. The importance of each variable was 0.758, 0.213, and 0.029, respectively. The model was validated by the TEM and using the external cohort. The decision tree model using synaptophysin, CD117, and GATA3 may help confirm NED of traditional marker-negative SCNEC.

Genomic Landscape of Young-Onset Bladder Cancer and Its Prognostic Implications on Adult Bladder Cancer.

Due to the rare occurrence of young-onset bladder cancer (YBC), its genomic characteristics remain largely unknown. Twenty-nine biopsy-proven YBC cases were collected using a nation-wide search for bladder cancer diagnosed at 20 years or younger. Whole exome sequencing and RNA sequencing were carried out in 21 and 11 cases, respectively, and compared with those of adult bladder cancer (ABC) cases obtained from public databases. Almost all YBCs were low grade, non-invasive papillary tumors. YBC had a low mutation burden and less complex copy number alterations. All cases harbored putative driver mutations. Mutations were most commonly found in HRAS (10 cases), with a preference for exon 5. FGFR3 gene fusions were noted with various partner genes (7 cases). The alterations on HRAS and FGFR3 occurred in a mutually exclusive manner. Others included KRAS mutations (2 cases), chromosomes 4p and 10q arm-level deletions (1 case), and ERCC2 mutation (1 case). There were no point mutations in TP53 and FGFR3. The gene expression profiles of YBC were similar to those of the ABC group with good prognosis. None of the YBCs and ABCs with YBC-like mutations showed progression to muscle-invasive tumors. Our results suggest that bladder cancer with YBC-like mutations represents an indolent bladder tumor, regardless of age.

Involvement of the TNF-α Pathway in TKI Resistance and Suggestion of TNFR1 as a Predictive Biomarker for TKI Responsiveness in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Mechanism and predictive biomarkers for tyrosine kinase inhibitor (TKI) resistance of advanced clear cell renal cell carcinoma (ccRCC) have not been fully evaluated. METHODS: We performed gene expression profiling on samples from an acquired TKI resistance cohort that consisted of 10 cases of TKI-treated ccRCC patients with matched tumor tissues harvested at pre-treatment and TKI-resistant post-treatment periods. In addition, a public microarray dataset from patient-derived xenograft model for TKI-treated ccRCC (GSE76068) was retrieved. Commonly altered pathways between the datasets were investigated by Ingenuity Pathway Analysis using commonly regulated differently expressed genes (DEGs). The significance of candidate DEG on intrinsic TKI resistance was assessed through immunohistochemistry in a separate cohort of 101 TKI-treated ccRCC cases. RESULTS: TNFRSF1A gene expression and tumor necrosis factor (TNF)-α pathway were upregulated in ccRCCs with acquired TKI resistance in both microarray datasets. Also, high expression (> 10% of labeled tumor cells) of TNF receptor 1 (TNFR1), the protein product of TNFRSF1A gene, was correlated with sarcomatoid dedifferentiation and was an independent predictive factor of clinically unfavorable response and shorter survivals in separated TKI-treated ccRCC cohort. CONCLUSION: TNF-α signaling may play a role in TKI resistance, and TNFR1 expression may serve as a predictive biomarker for clinically unfavorable TKI responses in ccRCC.

Epithelial-mesenchymal transition as a mechanism of resistance to tyrosine kinase inhibitors in clear cell renal cell carcinoma.

Tyrosine kinase inhibitors (TKIs) are widely accepted as treatment for metastatic clear cell renal cell carcinoma (ccRCC). However, most patients eventually experience disease progression despite TKI treatment, even if the initial response is favorable. To define the underlying mechanism of TKI resistance, 10 TKI-treated metastatic ccRCC cases in which tumor samples were harvested before treatment and immediately after disease progression were examined. Gene expression profiles and copy number variations of matched pre- and post-treatment tumor samples were investigated. Altered biologic characteristics were confirmed in sunitinib-resistant ccRCC cell lines, which were generated by long-term treatment with sunitinib-containing media. Gene transcript levels related to the cell cycle and epithelial-mesenchymal transition (EMT) were significantly upregulated in the treated tumor samples compared with the pre-treatment samples. The mitotic count and sarcomatoid component were significantly increased in treated tumor samples. Alteration of EMT-related genes was also demonstrated in a sunitinib-resistant ccRCC cell line that showed enhanced migration and invasion compared to the parent cell line. siRNA-induced inhibition of EMT-related gene expression significantly suppressed the migration and invasion capacity of TKI-resistant cell lines. The present study shows that both ccRCC cases that progressed after TKI treatment and sunitinib-resistant ccRCC cell lines demonstrated alteration of EMT-related gene expression and enhancement of EMT-related behavior. These results suggest that EMT may explain the aggressive behavior of TKI-resistant ccRCC.

Development of Response Classifier for Vascular Endothelial Growth Factor Receptor (VEGFR)-Tyrosine Kinase Inhibitor (TKI) in Metastatic Renal Cell Carcinoma.

Vascular endothelial growth factor receptor (VEGFR)-targeted therapy improved the outcome of metastatic renal cell carcinoma (mRCC) patients. However, a prediction of the response to VEGFR-tyrosine kinase inhibitor (TKI) remains to be elucidated. We aimed to develop a classifier for VEGFR-TKI responsiveness in mRCC patients. Among 101 mRCC patients, ones with complete response, partial response, or ≥24 weeks stable disease in response to VEGFR-TKI treatment were defined as clinical benefit group, whereas patients with <24 weeks stable disease or progressive disease were classified as clinical non-benefit group. Clinicolaboratory-histopathological data, 41 gene mutations, 20 protein expression levels and 1733 miRNA expression levels were compared between clinical benefit and non-benefit groups. The classifier was built using support vector machine (SVM). Seventy-three patients were clinical benefit group, and 28 patients were clinical non-benefit group. Significantly different features between the groups were as follows: age, time from diagnosis to TKI initiation, thrombocytosis, tumor size, pT stage, ISUP grade, sarcomatoid change, necrosis, lymph node metastasis and expression of pAKT, PD-L1, PD-L2, FGFR2, pS6, PDGFRß, HIF-1α, IL-8, CA9 and miR-421 (all, P < 0.05). A classifier including necrosis, sarcomatoid component and HIF-1α was built with 0.87 accuracy using SVM. When the classifier was checked against all patients, the apparent accuracy was 0.875 (95% CI, 0.782-0.938). The classifier can be presented as a simple decision tree for clinical use. We developed a VEGFR-TKI response classifier based on comprehensive inclusion of clinicolaboratory-histopathological, immunohistochemical, mutation and miRNA features that may help to guide appropriate treatment in mRCC patients.

Prognostic Significance of Macroscopic Appearance in Clear Cell Renal Cell Carcinoma and Its Metastasis-Predicting Model.

Prognostic significance of macroscopic appearance of clear cell renal cell carcinoma (ccRCC) has not yet been studied. This study aimed to define the prognostic significance of macroscopic appearance and to propose a prognostic model for post-operative metastasis in ccRCC. A total of 1,025 patients with ccRCC were analyzed for the development dataset. A separate cohort of 399 such patients was used as an external validation dataset. Macroscopic appearances were initially divided into four groups, but were later divided into two groups: a simple nodular group (700 cases, 68.3%) and an irregular outline group (325 cases, 32.7%). During the 66.1-month mean follow-up period, 98 patients (9.6%) developed metastasis. Univariate analysis revealed that metastasis was associated with older age, radical nephrectomy, larger tumor size, higher tumor grade and stage, and the irregular outline group. On multivariate analysis, age, tumor size, and macroscopic appearance remained as independent prognostic factors. These factors were used to build a prognostic model, which divided into three risk groups. The probabilities of 5-year metastasis-free survival in the low-, intermediate-, and high-risk groups were 98%, 83%, and 53%, respectively. The results showed prognostic significance of macroscopic appearance in ccRCC and propose a prognostic model to guide post-operative management of patients with ccRCC.

Diverse Immunoprofile of Ductal Adenocarcinoma of the Prostate with an Emphasis on the Prognostic Factors.

BACKGROUND: Ductal adenocarcinoma (DAC) of the prostate is an uncommon histologic subtype whose prognostic factors and immunoprofile have not been fully defined. METHODS: To define its prognostic factors and immunoprofile, the clinicopathological features, including biochemical recurrence (BCR), of 61 cases of DAC were analyzed. Immunohistochemistry was performed on tissue microarray constructs to assess the expression of prostate cancer-related and mammalian target of rapamycin (mTOR) signaling-related proteins. RESULTS: During the median follow-up period of 19.3 months, BCR occurred in 26 cases (42.6%). DAC demonstrated a wide expression range of prostate cancer-related proteins, including nine cases (14.8%) that were totally negative for pan-cytokeratin (PanCK) immunostaining. The mTOR signaling-related proteins also showed diverse expression. On univariate analysis, BCR was associated with high preoperative serum levels of prostate-specific antigen (PSA), large tumor volume, predominant ductal component, high Gleason score (GS), comedo-necrosis, high tumor stage (pT), lymphovascular invasion, and positive surgical margin. High expressions of phospho-mTOR (p-mTOR) as well as low expressions of PSA, phospho-S6 ribosomal protein (pS6) and PanCK were associated with BCR. On multivariable analysis, GS, pT, and immunohistochemical expressions of PanCK and p-mTOR remained independent prognostic factors for BCR. CONCLUSIONS: These results suggest GS, pT, and immunohistochemical expressions of PanCK and p-mTOR as independent prognostic factors for BCR in DAC. Since DAC showed diverse expression of prostate cancer-related proteins, this should be recognized in interpreting the immunoprofile of DAC. The diverse expression of mTOR-related proteins implicates their potential utility as predictive markers for mTOR targeted therapy.

Ascidiaceihabitans donghaensis gen. nov., sp. nov., isolated from the golden sea squirt Halocynthia aurantium.

A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated RSS1-M3(T), was isolated from a golden sea squirt (Halocynthia aurantium) collected from the East Sea, South Korea. Strain RSS1-M3(T) grew optimally at 30 °C, at pH 7.0-8.0 and in presence of 2.0 % (w/v) NaCl. Strain RSS1-M3(T) exhibited the highest 16S rRNA gene sequence similarity (96.55 %) to the type strain of Pelagicola litoralis. Neighbour-joining and maximum-likelihood phylogenetic trees based on 16S rRNA gene sequences revealed that strain RSS1-M3(T) clustered with the type strains, or proposed type strains, of Planktotalea frisia, Pacificibacter maritimus, Roseovarius marinus and Halocynthiibacter namhaensis, showing sequence similarity of 94.88-96.32 %. Strain RSS1-M3(T) contained Q-10 as the predominant ubiquinone and C18 : 1ω7c and C16 : 0 as the major fatty acids. The polar lipid profile of strain RSS1-M3(T), containing phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified aminolipid and one unidentified lipid as major components, could be distinguished from those of the phylogenetically related genera. The DNA G+C content of strain RSS1-M3(T) was 55.8 mol%. On the basis of the phylogenetic, chemotaxonomic and phenotypic properties, strain RSS1-M3(T) is considered to represent a novel species of a new genus within the class Alphaproteobacteria, for which the name Ascidiaceihabitans donghaensis gen. nov., sp. nov. is proposed. The type strain is RSS1-M3(T) ( = KCTC 42118(T) = CECT 8599(T)).

Mesoflavibacter sabulilitoris sp. nov., isolated from seashore sand.

A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated, gliding and rod-shaped bacterial strain, designated GJMS-9(T), was isolated from seashore sand collected at Geoje island in the South Sea, South Korea. Strain GJMS-9(T) grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain GJMS-9(T) clustered with the type strain of Mesoflavibacter zeaxanthinifaciens, showing the highest sequence similarity of 99.1%. Strain GJMS-9(T) exhibited 16S rRNA gene sequence similarity of 96.5% to the type strain of Mesoflavibacter aestuarii and of less than 96.1% to the type strains of other recognized species. Strain GJMS-9(T) contained MK-6 as the only menaquinone and iso-C(15:1) G, iso-C(16:0) 3-OH, iso-C(15:0) and iso-C(17:0) 3-OH as the major fatty acids. The polar lipid profile of strain GJMS-9(T) containing phosphatidylethanolamine, one unidentified lipid and one unidentified glycolipid as major components was similar to that of the type strain of M. zeaxanthinifaciens. The DNA G+C content of strain GJMS-9(T) was 32.2 mol% and its DNA-DNA relatedness with M. zeaxanthinifaciens DSM 18436(T) was 38 ± 6.1%. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain GJMS-9(T) is separated from other species of the genus Mesoflavibacter. On the basis of the data presented, strain GJMS-9(T) is considered to represent a novel species of the genus Mesoflavibacter, for which the name Mesoflavibacter sabulilitoris sp. nov. is proposed. The type strain is GJMS-9(T) ( =KCTC 42117(T) =CECT 8597(T)).

Pseudohongiella acticola sp. nov., a novel gammaproteobacterium isolated from seawater, and emended description of the genus Pseudohongiella.

A Gram-negative, aerobic, non-spore-forming and rod-shaped or ovoid bacterial strain able to move by a single polar flagellum, designated GBSW-5(T), was isolated from seawater in the East Sea, South Korea. Strain GBSW-5(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain GBSW-5(T) clustered with the type strain of Pseudohongiella spirulinae. Strain GBSW-5(T) exhibited the highest 16S rRNA gene sequence similarity value (96.4%) to the type strain of P. spirulinae, and of less than 91.0% sequence similarities to the type strains of other recognized species. Strain GBSW-5(T) was found to contain Q-8 as the predominant ubiquinone and C(18:1)ω7c and summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c) as the major fatty acids. The major polar lipids of strain GBSW-5(T) were identified as phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The DNA G+C content of strain GBSW-5(T) was determined to be 59.1 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain GBSW-5(T) is separated from P. spirulinae. On the basis of the data presented, strain GBSW-5(T) is considered to represent a novel species of the genus Pseudohongiella, for which the name Pseudohongiella acticola sp. nov. is proposed. The type strain is GBSW-5(T) (= KCTC 42131(T) = CECT 8627(T)). An emended description of the genus Pseudohongiella is also given.

Granulosicoccus undariae sp. nov., a member of the family Granulosicoccaceae isolated from a brown algae reservoir and emended description of the genus Granulosicoccus.

A Gram-stain-negative, aerobic, non-flagellated and coccoid bacterial strain, W-BA3(T), which was isolated from a brown algae reservoir in Wando of South Korea, was characterized taxonomically. Strain W-BA3(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in presence of 2.0 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain W-BA3(T) clustered with the type strains of Granulosicoccus antarcticus and Granulosicoccus coccoides, with which it exhibited sequence similarity values of 98.4-99.3 %. Sequence similarity values of strain W-BA3(T) to the type strains of the other recognized species were less than 90.2 %. Strain W-BA3(T) was found to contain Q-8 as the predominant ubiquinone and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C18:1 ω7c and C16:0 as the major fatty acids. The major polar lipids of strain W-BA3(T), which were identified as phosphatidylethanolamine and phosphatidylglycerol, were similar to those of the type strains of G. antarcticus and G. coccoides. The DNA G+C content of strain W-BA3(T) was 56.0 mol % and its mean DNA-DNA relatedness values with the type strains of G. coccoides and G. antarcticus were 27 and 17 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, demonstrated that strain W-BA3(T) is separated from the two Granulosicoccus species. On the basis of the data presented, strain W-BA3(T) is considered to represent a novel species of the genus Granulosicoccus, for which the name Granulosicoccus undariae sp. nov. is proposed. The type strain is W-BA3(T) (=KCTC 42134(T) = NBRC 110411(T)). An emended description of the genus Granulosicoccus is also proposed.

Thalassotalea ponticola sp. nov., isolated from seawater, reclassification of Thalassomonas fusca as Thalassotalea fusca comb. nov. and emended description of the genus Thalassotalea.

A Gram-stain-negative, aerobic, non-flagellated and rod-shaped or ovoid bacterial strain, designated GJSW-36(T), was isolated from seawater at Geoje island in the South Sea, South Korea. Strain GJSW-36(T) grew optimally at pH 7.0-8.0, at 25 °C and in the presence of 2% (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain GJSW-36(T) fell within the clade comprising the type strains of species of the genus Thalassotalea and Thalassomonas fusca. Strain GJSW-36(T) exhibited 16S rRNA gene sequence similarity values of 94.2-96.0% to the type strains of species of the genus Thalassotalea and Thalassomonas fusca and of 93.8-94.5% to the type strains of the other species of the genus Thalassomonas. Strain GJSW-36(T) contained ubiquinone-8 (Q-8) as the predominant ubiquinone and summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c), C(1:1)ω8c and C(16:0) as the major fatty acids. The major polar lipids of strain GJSW-36(T) were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content of strain GJSW-36(T) was 45.1 mol%. Differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain GJSW-36(T) is separated from species of the genus Thalassotalea and Thalassomonas fusca. On the basis of the data presented, strain GJSW-36(T) is considered to represent a novel species of the genus Thalassotalea, for which the name Thalassotalea ponticola sp. nov. is proposed. The type strain is GJSW-36(T) ( =KCTC 42155(T) =CECT 8656(T)). From this study, it is also proposed that Thalassomonas fusca should be reclassified as a member of the genus Thalassotalea and the description of the genus Thalassotalea is emended.

Loktanella aestuariicola sp. nov., an alphaproteobacterium isolated from a tidal flat.

A Gram-stain negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, J-TF4(T), which was isolated from a tidal flat in the South Sea of South Korea, was characterized taxonomically. Strain J-TF4(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in presence of 2.0-3.0% (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain J-TF4(T) fell within the clade comprising the type strains of Loktanella species, clustering with the type strains of Loktanella cinnabarina, Loktanella hongkongensis, Loktanella soesokkakensis, Loktanella pyoseonensis and 'Loktanella variabilis' showing sequence similarity values of 97.2-98.4%. The strain exhibited 16S rRNA gene sequence similarity values of 94.1-95.4% to the type strains of the other Loktanella species. Strain J-TF4(T) was found to have Q-10 as the predominant ubiquinone and C(18:1) ω7c as the major fatty acid. The major polar lipids of strain J-TF4(T) were identified as phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid and an unidentified aminolipid. The DNA G+C content of strain J-TF4(T) was determined to be 67.9 mol% and its mean DNA-DNA relatedness values with the type strains of five phylogenetically related Loktanella species were 17.7-23.3%. Differential phenotypic properties, together with the phylogenetic and genetic data, demonstrate that strain J-TF4(T) is separated from other Loktanella species. On the basis of the data presented, strain J-TF4(T) is considered to represent a novel species of the genus Loktanella, for which the name Loktanella aestuariicola sp. nov. is proposed. The type strain is J-TF4(T) (=KCTC 42135(T)=NBRC 110408(T)).

Thalassobius aquaeponti sp. nov., an alphaproteobacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-motile and rod-shaped or ovoid bacterial strain, GJSW-22(T), which was isolated from seawater at Geoje island in South Korea, was characterized taxonomically. Strain GJSW-22(T) was observed to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain GJSW-22(T) grouped with the type strains of Thalassobius species, forming a stable cluster with the type strain of Thalassobius aestuarii (bootstrap value of 83.2 %). Strain GJSW-22(T) exhibited the highest 16S rRNA gene sequence similarity value (98.0 %) to the type strain of T. aestuarii. It exhibited 16S rRNA gene sequence similarity values of 95.6-96.1 % to the type strains of the other Thalassobius species. Strain GJSW-22(T) was found to contain Q-10 as the predominant ubiquinone and C18:1 ω7c and 11-methyl C18:1 ω7c as the major fatty acids. The major polar lipids of strain GJSW-22(T) were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G + C content of strain GJSW-22(T) is 60.3 mol % and its mean DNA-DNA relatedness value with the type strain of T. aestuarii was 23 %. Differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, confirmed that strain GJSW-22(T) is distinct from other Thalassobius species. On the basis of the data presented, strain GJSW-22(T) is considered to represent a novel species of the genus Thalassobius, for which the name Thalassobius aquaeponti sp. nov. is proposed. The type strain is GJSW-22(T) (=KCTC 42115(T) = NBRC 110378(T)).

Mariniflexile ostreae sp. nov., a member of the family Flavobacteriaceae isolated from an oyster.

A Gram-negative, aerobic, non-flagellated and rod-shaped bacterial strain able to move by gliding, designated TYO-10(T), was isolated from an oyster collected at Tongyoung on the South Sea, South Korea. Strain TYO-10(T) was found to grow optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain TYO-10(T) joined the cluster comprising the type strains of Mariniflexile species. Strain TYO-10(T) exhibited 16S rRNA gene sequence similarity values of 95.4-95.7 % to the type strains of Mariniflexile species and of less than 95.4 % to the type strains of other recognized species. Strain TYO-10(T) was found to contain MK-6 as the predominant menaquinone and iso-C15:0, iso-C15:1 G, anteiso-C15:0, iso-C17:0 3-OH and iso-C15:0 3-OH as the major fatty acids. The major polar lipids of strain TYO-10(T) were identified as phosphatidylethanolamine and an unidentified lipid, which is similar to those of the type strains of Mariniflexile species, but different from those of other phylogenetically related species. The DNA G+C content of strain TYO-10(T) was determined to be 35.9 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain TYO-10(T) is separated from other Mariniflexile species. On the basis of the data presented, strain TYO-10(T) is considered to represent a novel species of the genus Mariniflexile, for which the name Mariniflexile ostreae sp. nov. is proposed. The type strain is TYO-10(T) (= KCTC 42113(T) = CECT 8622(T)).

Loktanella variabilis sp. nov., isolated from a tidal flat.

Two Gram-negative, aerobic and rod-shaped or ovoid bacterial strains with different colony colours (greyish yellow and moderate orange), designated J-MR2-Y(T) and J-MR2-O, were isolated from a tidal flat in the South Sea of South Korea. The two novel strains grew optimally at 35-37 °C. Strains J-MR2-Y(T) and J-MR2-O showed no difference in their 16S rRNA gene sequences, and the mean DNA-DNA relatedness between them was 94%. Phylogenetic trees based on 16S rRNA gene sequences revealed that strains J-MR2-Y(T) and J-MR2-O clustered consistently with the type strains of Loktanella soesokkakensis, L. hongkongensis and L. cinnabarina, with which it exhibited 97.83-99.06% sequence similarity. Sequence similarities to the type strains of the other recognized species of the genus Loktanella were 94.01-96.26%. Both strains contained Q-10 as the predominant ubiquinone, C(18 : 1)ω7c as the major fatty acid and phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid and one unidentified aminolipid as the major polar lipids. The DNA G+C contents of strains J-MR2-Y(T) and J-MR2-O were 68.1 and 68.4 mol%, respectively, and DNA-DNA relatedness values with the type strains of L. soesokkakensis, L. hongkongensis and L. cinnabarina were 19-37%. Differential phenotypic properties, together with their phylogenetic and genetic distinctiveness, revealed that the two novel strains are separated from other species of the genus Loktanella. On the basis of the data presented, strains J-MR2-Y(T) and J-MR2-O are proposed to represent a novel species of the genus Loktanella, for which the name Loktanella variabilis sp. nov. is proposed. The type strain is J-MR2-Y(T) ( = KCTC 42074(T) = CECT 8572(T)).