The molecular characteristics could supplement the staging system of pT2/T3N0M0 esophageal squamous cell carcinoma: a translational study based on a cohort with over 20 years of follow-up.

OBJECTIVE: This study aimed to construct a model based on 23 enrolled molecules to evaluate prognoses of pT2/3N0M0 esophageal squamous cell carcinoma (ESCC) patients with up to 20 years of follow-up. METHODS: The lasso-Cox model was used to identify the candidate molecule. A nomogram was conducted to develop the survival model (molecular score, MS) based on the molecular features. Cox regression and Kaplan-Meier analysis were used in this study. The concordance index (C-index) was measured to compare the predicted ability between different models. The primary endpoint was overall survival (OS). RESULTS: A total of 226 patients and 23 proteins were enrolled in this study. Patients were classified into high-risk (MS-H) and low-risk (MS-L) groups based on the MS score of 227. The survival curves showed that the MS-L cohort had better 5-year and 10-year survival rates than the MS-H group (5-year OS: 51.0% vs. 8.0%; 10-year OS: 45.0% vs. 5.0%, all p < 0.001). Furthermore, multivariable analysis confirmed MS as an independent prognostic factor after eliminating the confounding factors (Hazard ratio 3.220, p < 0.001). The pT classification was confirmed to differentiate ESCC patients' prognosis (Log-rank: p = 0.029). However, the combination of pT and MS could classify survival curves evidently (overall p < 0.001), which showed that the prognostic prediction efficiency was improved significantly by the combination of the pT and MS than by the classical pT classification (C-index: 0.656 vs. 0.539, p < 0.001). CONCLUSIONS: Our study suggested an MS for significant clinical stratification of T2/3N0M0 ESCC patients to screen out subgroups with poor prognoses. Besides, the combination of pT staging and MS could predict survival more accurately for this cohort than the pT staging system alone.

Terrirubrum flagellatum gen. nov., sp. nov. of Terrirubraceae fam. nov. and Lichenibacterium dinghuense sp. nov. from forest soil and proposal of Rhodoblastaceae fam. nov.

Two Gram-negative, aerobic, rod-shaped bacterial strains, 7MK25T and 6Y81T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Based on the results of 16S rRNA gene sequence analysis, strain 7MK25T showed the highest similarity (93.6 %) to Methyloferula stellata AR4T, followed by Bosea thiooxidans DSM 9653T (93.3 %). Strain 6Y81T had the highest similarity of 97.9 % to Lichenibacterium minor RmlP026T, followed by Lichenibacterium ramalinae RmlP001T (97.2 %). Phylogenomic analysis using the UBCG and PhyloPhlAn methods consistently showed that strain 7MK25T formed a sister clade to Boseaceae, while strain 6Y81T formed an independent clade within the genus Lichenibacterium, both in the order Hyphomicrobiales. The digital DNA-DNA hybridization and average nucleotide identity values between strains 7MK25T, 6Y81T and their close relatives were in the ranges of 19.1-29.9 % and 72.5-85.5 %, respectively. The major fatty acids of 7MK25T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C19 : 0 cyclo ω8c, C16 : 0 and C17 : 0 cyclo, while those of 6Y81T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C16 : 0 and C16 : 0 3-OH. Strains 7MK25T and 6Y81T took diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine as their dominant polar lipids, and Q-10 as their major respiratory quinone. On the basis of phenotypic and phylogenetic data, strain 7MK25T is proposed to represent a novel species of a novel genus with name Terrirubrum flagellatum gen. nov., sp. nov., within a novel family Terrirubraceae fam. nov., with 7MK25T (=KCTC 62738T=GDMCC 1.1452T) as its type strain. Strain 6Y81T represents a novel species in the genus Lichenibacterium, for which the name Lichenibacterium dinghuense sp. nov. (type strain 6Y81T=KACC 21 727T=GDMCC 1.2176T) is proposed. Rhodoblastaceae fam. nov. with Rhodoblastus as the type genus is also proposed to solve the non-monophylectic problem of the family Roseiarcaceae.

Reconsidering N component of cancer staging for T1-2N0-2M0 small-cell lung cancer: a retrospective study based on multicenter cohort.

BACKGROUND: The current nodal (pN) classification still has limitations in stratifying the prognosis of small cell lung cancer (SCLC) patients with pathological classifications T1-2N0-2M0. Thus. This study aimed to develop and validate a modified nodal classification based on a multicenter cohort. MATERIALS AND METHODS: We collected 1156 SCLC patients with pathological classifications T1-2N0-2M0 from the Surveillance, Epidemiology, and End Results database and a multicenter database in China. The X-tile software was conducted to determine the optimal cutoff points of the number of examined lymph nodes (ELNs) and lymph node ratio (LNR). The Kaplan-Meier method, the Log-rank test, and the Cox regression method were used in this study. We classified patients into three pathological N modification categories, new pN#1 (pN0-#ELNs > 3), new pN#2 (pN0-#ELNs ≤ 3 or pN1-2-#LNR ≤ 0.14), and new pN#3 (N1-2-#LNR > 0.14). The Akaike information criterion (AIC), Bayesian Information Criterion, and Concordance index (C-index) were used to compare the prognostic, predictive ability between the current pN classification and the new pN component. RESULTS: The new pN classification had a satisfactory effect on survival curves (Log-rank P < 0.001). After adjusting for other confounders, the new pN classification could be an independent prognostic indicator. Besides, the new pN component had a much more accurate predictive ability in the prognostic assessment for SCLC patients of pathological classifications T1-2N0-2M0 compared with the current pN classification in the SEER database (AIC: 4705.544 vs. 4731.775; C-index: 0.654 vs. 0.617, P < 0.001). Those results were validated in the MCDB from China. CONCLUSIONS: The multicenter cohort developed and validated a modified nodal classification for SCLC patients with pathological category T1-2N0-2M0 after surgery. Besides, we propose that an adequate lymph node dissection is essential; surgeons should perform and consider the situation of ELNs and LNR when they evaluate postoperative prognoses of SCLC patients.

Trinickia mobilis sp. nov. and Trinickia acidisoli sp. nov., isolated from soil.

Two novel Gram-stain-negative, aerobic and rod-shaped bacterial strains, designated DHG64T and 4D114T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. DHG64T grew at 12-37 °C (optimum 33 °C), pH 4.5-10.0 (optimum 6.5-7.5) and in the presence of 0-2.0 % NaCl (w/v); while 4D114T grew at 12-37 °C (optimum 20-33 °C), pH 4.0-7.0 (optimum 4.5-6.0) and in the presence of 0-1.0 % NaCl (w/v). DHG64T and 4D114T showed 97.1-98.0 and 97.5-98.4 % 16S rRNA gene sequence similarities with seven species of the genus Trinickia with validly published names, respectively. In the phylogenetic trees based on 16S rRNA gene and genome sequences, both strains formed a clade with the members of genus Trinickia but well separated from each other. The average nucleotide identity and digital DNA-DNA hybridisation values for the novel strains to all species of the genus Trinickia with validly published names were in the ranges of 80.6-85.0 and 22.4-28.0 %, respectively. DHG64T contained C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c, while 4D114T had C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c and summed feature 2 (iso-C16 : 1 I and/or C14 : 0 3-OH) as the major cellular fatty acids. The major polar lipids for strains DHG64T and 4D114T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C contents of DHG64T and 4D114T were 63.0 and 62.8 mol%, respectively. Genomic analyses indicated that DHG64T and 4D114T may have potential for various applications, such as developing drugs against certain health problems and restoring environments polluted with metal ions and/or benzoate. On the basis of the results of morphological, physiological, biochemical and phylogenetic analyses, strains DHG64T and 4D114T were classified as representing two novel species of the genus Trinickia, for which the names Trinickia mobilis sp. nov. (type strain DHG64T = KACC 21223T = GDMCC 1.1282T) and Trinickia acidisoli sp. nov. (type strain 4D114T = KCTC 82876T = GDMCC 1.2131T) are proposed.

Trinickia violacea sp. nov. and Trinickia terrae sp. nov., isolated from forest soil.

Two Gram-stain negative, aerobic and rod-shaped bacterial strains, DHOD12T and 7GSK02T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Strain DHOD12T grew at 4-42 °C (optimum, 28-33 °C), pH 4.0-8.5 (optimum, pH 5.5-6.5) and in the presence of 0-1.5 % (w/v; optimum, 0-0.5 %)NaCl; while strain 7GSK02T grew at 12-42 °C (optimum, 28-33 °C), pH 4.0-8.5 (optimum, pH 5.0-6.0) and in the presence of 0-0.5 % (w/v; optimum, 0 %) NaCl. Strains DHOD12T and 7GSK02T had the highest 16S rRNA sequence similarities of 98.0 and 98.3 % with the same species Trinickia mobilis DHG64T, respectively, and 98.4 % between themselves. In the 16S rRNA phylogeny, they formed a clade that was sister to a major cluster consisting of all described Trinickia species. Phylogenomic analyses with the UBCG and PhyloPhlAn methods consistently showed that strains DHOD12T and 7GSK02T formed a clade with T. mobilis DHG64T that was a sister of a cluster containing the remainder of the Trinickia species. The DNA G+C contents of strains DHOD12T and 7GSK02T were 63.1 and 64.6 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity values of strains DHOD12T, 7GSK02T and their closely related strains were in the ranges of 21.6-31.4 % and 77.1-86.9 %, respectively. These two strains had the same major respiratory quinone, ubiquinone-8, and both had C16 : 0, C17 : 0 cyclo and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) as their major fatty acids. Their major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Genomic analysis indicated that the two strains could have the potential to degrade aromatic compounds like other Trinickia species. On the basis of phenotypic and phylogenetic results, strains DHOD12T and 7GSK02T represent two novel species of the genus Trinickia, for which the names Trinickia violacea sp. nov. (type strain DHOD12T=LMG 30258T=CGMCC 1.15436T) and Trinickia terrae sp. nov. (type strain 7GSK02T=CGMCC 1.15432T=KCTC 62468T) are proposed.

Paraburkholderia flagellata sp. nov. and Paraburkholderia adhaesiva sp. nov., two novel species isolated from forest soil in Dinghushan Biosphere Reserve in Guangdong, China.

Two Gram-stain-negative, aerobic, motile and short rod strains, designated 4D117T and ZD32-2T, were isolated from the forest soils. Strains 4D117T and ZD32-2T grew optimally at pH 4.0-6.5, 20-33 °C and pH 4.5-7.0, 33 °C, respectively, and both at 0.5% (w/v) NaCl concentration. Strains 4D117T and ZD32-2T shared the highest 16S rRNA gene sequence similarity with P. acidiphila 7Q-K02T (99.1%) and P. ferrariae NBRC 106233T (98.7%), respectively. The genome size and G + C contents of strains 4D117T and ZD32-2T were 9,002,095 bp, 62.9% and 6,974,420 bp, 61.7%, respectively. The dDDH and ANI values between strains 4D117T, ZD32-2T and closely related Paraburkholderia species were in the ranges of 21.9-51.6% and 82.9-94.4%, and 81.7% and 25.4% between themself, respectively. Functional genomic analysis showed both strains were capable of degrading contaminants, such as benzoate, anthranilic acid and catechol for 4D117T, and benzene and catechol for ZD32-2T, indicating that they may have potentials for soil pollutant treatment. The main polar lipids of strains 4D117T and ZD32-2T were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Strain 4D117T contained C16:0, C19:0 cyclo ω8c and C18:1 ω7c and/or C18:1 ω6c, while strain ZD32-2T had C16:0 and C17:0 cyclo as their major cellular fatty acids (> 10%). Based on the phenotypic characters and genomic data, strains 4D117T and ZD32-2T represent two novel species of genus Paraburkholderia, for which the names Paraburkholderia flagellata sp. nov. (type strain 4D117T = GDMCC 1.2617T = NBRC 115278T) and Paraburkholderia adhaesiva sp. nov. (type strain ZD32-2T = GDMCC 1.2622T = NBRC 115282T) are proposed.

Postoperative survival effect of the number of examined lymph nodes on esophageal squamous cell carcinoma with pathological stage T1-3N0M0.

BACKGROUND: The postoperative survival effect of the number of examined lymph nodes on patients of R0-resected esophageal squamous cell carcinoma with pathological stage T1-3N0M0 is still unclear. METHODS: Patients diagnosed with pathological stage T1-3N0M0 esophageal squamous cell carcinoma from two cancer databases-our cancer center (N = 707), and Surveillance Epidemiology and End Results (N = 151). The primary clinical endpoint was overall survival. The X-tile software was used to determine the optimal cutoff value of the number of examined lymph nodes, and propensity score matching was conducted to reduce selection bias according to the results of X-tile software. The cohort of 151 patients from another database was used for validation. RESULTS: X-tile software provided an optimal cutoff value of 15 examined lymph nodes based on 707 patients, and 231 pairs of matched patients were included. In the unmatched cohort, Cox proportional hazard regression analysis revealed better overall survival in patients with more than 15 examined lymph nodes (adjusted hazard ratio, 0.566, 95% confidence interval, 0.445-0.720; p < 0.001) compared with patients with 15 or fewer examined lymph nodes. In the validation cohort, patients with more than 15 examined lymph nodes also had better overall survival (adjusted hazard ratio 0.665, p = 0.047). CONCLUSIONS: The number of examined lymph nodes is a significant prognostic factor in esophageal squamous cell carcinoma patients with pathological stage T1-3N0M0, and more than 15 examined lymph nodes are associated with better overall survival. Although the difference is not significant, the survival curve of patients with examined lymph nodes > 30 is better than those with examined lymph nodes 15-30. We believe that the number of examined lymph nodes can provide prognostic guidance for those patients, and the more examined lymph nodes cause lesser occult lymph nodes metastasis and lead to a better prognosis. Therefore, surgeons and pathologists should try to examine as many lymph nodes as possible to evaluate the pathological stage precisely. However, we need more validation from other studies.

Paracidobacterium acidisoli gen. nov., sp. nov. and Alloacidobacterium dinghuense gen. nov., sp. nov., two acidobacteria isolated from forest soil, and reclassification of Acidobacterium ailaaui and Acidipila dinghuensis as Pseudacidobacterium ailaaui gen. nov., comb. nov. and Silvibacterium dinghuense comb. nov.

Two aerobic and obligately acidophilic bacteria, designated 4G-K13T and 4Y35T, were isolated from the forest soil sampled at Dinghushan Biosphere Reserve, Guangdong Province, PR China. These two strains were Gram-stain-negative, non-motile and short rods that multiplied by binary division. Strains 4G-K13T and 4Y35T had the highest 16S rRNA gene sequence similarity of 97.0 and 97.2 % to Silvibacterium bohemicum DSM 103733T and Acidisarcina polymorpha SBC82T, respectively. Phylogenetic trees based on the 16S rRNA gene and whole genome sequences showed consistently that these two strains formed a major clade with members of the genera Acidipila, Acidisarcina, Silvibacterium and Acidobacterium in the family Acidobacteriaceae, but each occupied an unique position. In both the UBCG and the PhyloPhlAn phylogenomic trees, strains 4G-K13T and 4Y35T congruently formed a highly supported subclade with Acidobacterium capsulatum DSM 11244T and Acidobacterium ailaaui DSM 27394T, respectively. The major fatty acids (>5 %) of strain 4G-K13T were iso-C15 : 0, iso-C17 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), while that of strain 4Y35T were C16 : 0, C18 : 1 ω9c, iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl). Strain 4G-K13T contained phosphatidylethanolamine, four unidentified phospholipids, four glycolipids, two unidentified aminolipids and two unknown lipids, while strain 4Y35T had phosphatidylethanolamine, three unidentified phospholipids, two glycolipids, five unidentified aminolipids and one unknown polar lipid. The DNA G+C contents of 4G-K13T and 4Y35T were 60.5 and 55.8 mol%, respectively. Based on all these phylogenetic, physiological and chemotaxonomic data, we suggest that strains 4G-K13T and 4Y35T represent two novel species of two novel genera in the family Acidobacteriaceae, for which the names Paracidobacterium acidisoli gen. nov., sp. nov. (type strain: 4G-K13T=GDMCC 1.1195T=NBRC 113249T) and Alloacidobacterium dinghuense gen. nov., sp. nov. (type strain: 4Y35T=KACC 21728T=NBRC 114261T) are proposed. We also propose to reclassify Acidobacterium ailaaui and Acidipila dinghuensis as Pseudacidobacterium ailaaui gen. nov., comb. nov. and Silvibacterium dinghuense comb. nov., respectively, based mainly on the results of phylogenomic analysis.

Incidence and survival analyses for occult lung cancer between 2004 and 2015: a population-based study.

BACKGROUND: This study aimed to investigate the incidence and long-term survival outcomes of occult lung cancer between 2004 and 2015. METHODS: A total of 2958 patients were diagnosed with occult lung cancer in the 305,054 patients with lung cancer. The entire cohort was used to calculate the crude incidence rate. Eligible 52,472 patients (T1-xN0M0, including 2353 occult lung cancers) were selected from the entire cohort to perform survival analyses after translating T classification according to the 8th TNM staging system. Cancer-specific survival curves for different T classifications were presented. RESULTS: The crude incidence rate of occult lung cancer was 1.00 per 100 patients, and it was reduced between 2004 and 2015 [1.4 per 100 persons in 2004; 0.6 per 100 persons in 2015; adjusted risk ratio = 0.437, 95% confidence interval (CI) 0.363-0.527]. In the survival analysis, there were 2206 death events in the 2353 occult lung cancers. The results of the multivariable analysis revealed that the prognoses with occult lung cancer were similar to patients with stage T3N0M0 (adjusted hazard ratio = 1.054, 95% CI 0.986-1.127, p = 0.121). Adjusted survival curves presented the same results. In addition, adjusted for other confounders, female, age ≤ 72 years, surgical treatment, radiotherapy, adenocarcinoma, and non-squamous and non-adenocarcinoma non-small cell carcinoma were independent protective prognostic factors (all p < 0.05). CONCLUSIONS: Occult lung cancer was uncommon. However, the cancer-specific survival of occult lung cancer was poor, therefore, we should put the assessment of its prognoses on the agenda. Timely surgical treatment and radiotherapy could improve survival outcomes for those patients. Besides, we still need more research to confirm those findings.

Dyella telluris sp. nov. and Dyella acidiphila sp. nov., isolated from forest soil of Dinghushan Biosphere Reserve, China.

Cells of bacterial strains G9T and 7MK23T, isolated from forest soil samples collected from the Dinghushan Biosphere Reserve, Guangdong Province, PR China, were Gram-stain-negative, aerobic and rod-shaped. Strain G9T was motile with single polar flagellum and grew at 12-37 °C (optimum, 28 °C), pH 4.5-8.0 (optimum, pH 6.0-7.5) and in the presence of 0-3.5 % NaCl (optimum, 1.5%, w/v); while strain 7MK23T was non-motile and grew at 12-42 °C (optimum, 28-33 °C), pH 2.5-8.5 (optimum, pH 4.5-6.5) and NaCl levels of 0-1.0 % (optimum, 0-0.5 %, w/v). Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates fell within the cluster of the genus Dyella. The closely related species (with a 16S rRNA gene sequence similarity >98.65%) of strain G9T were Dyella terrae JS14-6T (99.0 %), D. kyungheensis THG-B117T (98.8 %) and D. amyloliquefaciens DHC06T (98.7 %) while that of strain 7MK23T were D. mobilis DHON07T (99.2 %) and D. flava DHOC52T (99.1 %), but the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strains G9T, 7MK23T and the closely related Dyella species listed above were in the ranges of 77.5-83.8 % and 22.0-27.0 %, much lower than the species demarcation lines of 95.5 and 70 %, respectively. Phylogenomic analyses using UBCG and Phylophlan also supported that these two strains represent two novel species of Dyella. The major fatty acids of strain G9T were iso-C15 : 0, iso-C17 : 1 ω9c and iso-C17 : 0 while that of strain 7MK23T were iso-C15 : 0 and anteiso-C15 : 0. Ubiquinone-8 was the only respiratory quinone detected in both strains. The polar lipids of strain G9T consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and several unknown phospholipids, aminophospholipids, aminolipids and lipid while strain 7MK23T contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and several unknown phospholipids and aminophospholipids. The DNA G+C contents of strains G9T and 7MK23T were 64.7 and 63.4 mol%, respectively. On the basis of 16S rRNA gene sequence phylogenetic and phylogenomic analyses as well as phenotypic data obtained, we propose that strains G9T and 7MK23T represent two novel species of the genus Dyella, for which the names Dyella telluris sp. nov. (type strain G9T=KACC 21725T=GDMCC 1.2132T) and Dyella acidiphila sp. nov. (type strain 7MK23T=KCTC 62739T=GDMCC 1.1446T) are proposed.

A prognostic model for stratification of stage IB/IIA esophageal squamous cell carcinoma: a retrospective study.

BACKGROUND: To explore the postoperative prognosis of esophageal squamous cell carcinoma (ESCC) patients with stage IB/IIA, using a prognostic score (PS). METHODS: Stage IB/IIA ESCC patients who underwent esophagectomy from 1999 to 2010 were included. We retrospectively recruited 153 patients and extracted their medical records. Moreover, we analyzed the programmed death ligand-1 (PD-L1) expression of their paraffin tissue. The cohort were randomly divided into a training group (N = 123) and a validation group (N = 30). We selected overall survival (OS) as observed endpoint. Prognostic factors with a multivariable two-sided P < 0.05 met standard of covariate inclusion. RESULTS: Univariable and multivariable analyses identified pTNM stage, the number of lymph nodes (NLNs) and PD-L1 expression as independent OS predictors. Primary prognostic score which comprised above three covariates adversely related with OS in two cohorts. PS discrimination of OS was comparable between the training and internal validation cohorts (C-index = 0.774 and 0.801, respectively). In addition, the PS system had an advantage over pTNM stage in the identification of high-risk patients (C-index = 0.774 vs. C-index = 0.570, P < 0.001). Based on PS cutoff, training and validation datasets generated low-risk and high-risk groups with different OS. Our three-factor PS predicted OS (low-risk subgroup vs. high-risk subgroup 60-month OS, 74% vs. 23% for training cohort and 83% vs. 45% for validation cohort). CONCLUSION: Our study suggested a PS for significant clinical stratification of IB/IIA ESCC to screen out subgroups with poor prognosis.

Histone demethylase Jmjd3 modulates osteoblast apoptosis induced by tumor necrosis factor-alpha through directly targeting RASSF5.

Purpose: Regulation of gene expression is fine-tuned by a dynamic equilibrium between repressive modifications and transcriptional activation of histone tails. Jumonji domain-containing 3 (Jmjd3), also known as KDM6B, is a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3) that specifically removes the methylation of H3K27me3 and promotes gene expression. Our previous study showed that Jmjd3 inhibits serum deprivation-induced osteoblast apoptosis. In this study, we clarified the role of Jmjd3 in tumor necrosis factor-alpha (TNF-α)-induced osteoblast apoptosis. Materials and Methods: Jmjd3 activity was inhibited by GSK-J4. Transfection of osteoblastic murine MC3T3-E1 cells with short hairpin RNA (shRNA) was used to establish stable Jmjd3 knockdown cells. Osteoblast apoptosis was detected using Annexin V-APC/PI staining, cysteinyl aspartate specific protease-3 (caspase-3) activity assays, and Western blot. Real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assays were performed to clarify the mechanism responsible for Jmjd3-regulated osteoblast apoptosis induced by TNF-α. Results: Based on Annexin V-APC/PI staining, caspase-3 activation, and poly ADP-ribose polymerase (PARP) cleavage, pretreatment with GSK-J4 and knockdown of Jmjd3 by shRNA transfection each inhibited osteoblast apoptosis. Furthermore, knockdown of Jmjd3 decreased the expression of Ras association domain family 5 (RASSF5), which is a pro-apoptotic gene of the Ras associated domain family. H3K27me3 levels in the promoter region of RASSF5 were up-regulated in the Jmjd3 knockdown cells. Conclusions: Jmjd3 regulated TNF-α-induced osteoblast apoptosis by targeting RASSF5.

Pararobbsia silviterrae gen. nov., sp. nov., isolated from forest soil and reclassification of Burkholderia alpina as Pararobbsia alpina comb. nov.

A novel Gram-stain-negative, aerobic, non-spore-forming, non-motile and rod-shaped bacterial strain, DHC34T, was isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, China (112° 31' E 23° 10' N). It grew optimally on R2A medium at 28 °C, at pH 6.0-7.0 and in the presence of 0-1 % (w/v) NaCl. Strain DHC34T was closely related to Burkholderia alpina LMG 28138T (98.5 % 16S rRNA gene sequence similarity). 16S rRNA gene sequence analysis showed that strain DHC34T formed a clade with B. alpina LMG 28138T, which is next to but branched deeply with Robbsia andropogonis ICMP 2807T. The phylogenetic relationships among these three strains were also supported with the phylogram based on concatenated partial gyrB, recA and trpB gene sequences. The phylogenomic tree generated with the UBCG tool showed that strains DHC34T and R. andropogonis ICMP 2807T were in a different clade. The DNA-DNA relatedness values between strain DHC34T and B. alpina LMG 28138T and R. andropogonis ICMP 2807T were much lower than 70 %. Strain DHC34T contained ubiquinone 8 as the major respiratory quinone. Its major fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The DNA G+C content of strain DHC34T was 64.2 mol%. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, three unidentified aminophospholipids, four unidentified phospholipids, one unidentified aminolipid and a polar lipid. The phenotypic, phylogenetic, genotypic and chemotaxonomic data showed that strain DHC34T represents a novel species of a new genus in the family Burkholderiaceae, for which the name Pararobbsia silviterrae gen. nov., sp. nov. is proposed. The type strain of Pararobbsia silviterrae is DHC34T (=KCTC 42628T=LMG 28845T). On the basis of the current data, Burkholderia alpina is renamed as Pararobbsia alpina comb. nov.

Dyella amyloliquefaciens sp. nov., isolated from forest soil.

A Gram-stain-negative, aerobic, yellow-pigmented, rod-shaped and motile with single polar flagellum bacterial strain, designated DHC06T, was isolated from forest soil sampled at Dinghushan Biosphere Reserve, Guangdong Province, PR China. The strain grew at 4-37 °C (optimum, 28 °C), pH 4.5-8.0 (pH 6.0-7.5) and in the presence of 0-4.0 % (2.0 %, w/v) NaCl. In the 16S rRNA gene sequence phylogram, strain DHC06T formed a clade with Dyella solisilvae DHG54T and Dyella terrae KACC 12748T within the genus of Dyella. Strain DHC06T had 16S rRNA gene sequence similarities of 98.6, 98.3, 98.3 and 98.2 % to Dyella japonica DSM 16301T, Dyella terrae JS14-6T, Dyella soli KACC 12747T and Dyella solisilvae DHG54T, respectively. The distinctiveness of strain DHC06Tfrom all described Dyellaspecies was also supported by the results of phylogenomic analysis based on 92 single-copy gene sequences. The DDH values among strain DHC06T and closely related Dyella species were all lower than 70 %. Strain DHC06T contained Q-8 as the only respiratory quinone. Its main fatty acids were iso-C15 : 0, iso-C17 : 1 ω9c and summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c). The DNA G+C content of strain DHC06T was 64.6 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of phenotypic, 16S rRNA gene sequence and genomic analyses and chemotaxonomic data, strain DHC06T represents a novel species of the genus Dyella, for which the name Dyella amyloliquefaciens sp. nov. (type strain DHC06T=GDMCC 1.1186T=LMG 30090T) is proposed.

Paraburkholderia pallida sp. nov. and Paraburkholderia silviterrae sp. nov., isolated from forest soil.

Two Gram-stain-negative, aerobic, motile, white-pigmented and rod-shaped bacterial strains, 7MH5T and 4 M-K11T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Strain 7MH5T grew at 4-37 °C (optimum, 28-33 °C), pH 3.5-9.0 (pH 4.0-5.5) and in the presence of 0-3 % (w/v) NaCl (0-1.5 w/v); while strain 4 M-K11T grew at 4-42 °C (20-33 °C), pH 3.5-8.5 (pH 4.5-6.0) and in the presence of 0-2.5 % (w/v) NaCl (0-1.5 w/v). Strains 7MH5T and 4 M-K11T have the highest 16S rRNA gene sequence similarities of 98.6 and 98.7 % to Paraburkholderia peleae PP52-1T, and 98.4 % between themselves. In the 16S rRNA gene sequence phylogram, strains 4 M-K11T and Paraburkholderia ferrariae NBRC 106233T formed a clade while 7MH5T were relatively distinct from other Paraburkholderia species. Based on the UBCG phylogenomic analysis, strains 7MH5T and 4 M-K11T formed a clade with Paraburkholderia oxyphila NBRC 105797T and Paraburkholderia sacchari LMG 19450T in the genus of Paraburkholderia. The DNA G+C contents of strains 7MH5T and 4 M-K11T were 64.2 and 64.3 %, respectively. Digital DNA-DNA hybridization and the average nucleotide identity values of strains 7MH5T, 4 M-K11T and closely related strains were in the ranges of 25.2-63.6 % and 81.0-95.5 %, respectively. The two strains had the same major respiratory quinone: ubiquinone-8. Strain 7MH5T had C16 : 0, C17 : 0cyclo, C19 : 0cyclo ω8c and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) as its major fatty acids, while strain 4 M-K11T had major fatty acids of C16 : 0, C17 : 0cyclo and summed feature 2 (iso-C16 : 1 I/C14 : 0-3OH). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of phenotypic and phylogenetic analyses based on both 16S rRNA gene and whole genome sequences, as well as chemotaxonomic data, strains 7MH5T and 4 M-K11T represent two novel species of the genus Paraburkholderia, for which the names Paraburkholderia pallida sp. nov. (type strain 7MH5T=GDMCC 1.1450T=KACC 19962T) and Paraburkholderia silviterrae sp. nov. (type strain 4 M-K11T=GDMCC 1.1284T=CGMCC 1.15450T=KACC 19961T=LMG 29217T) are proposed.

Dyella dinghuensis sp. nov. and Dyella choica sp. nov., isolated from forest soil.

Two aerobic, Gram-stain-negative, non-motile, rod-shaped bacterial strains, designated as DHOA06T and 4 M-K27T, were isolated from soil samples collected from the forest of Dinghushan Biosphere Reserve, Guangdong Province, PR China (112° 31' E 23° 10' N). Strains DHOA06T and 4 M-K27T grew at pH 4.5-7.0 (optimum, pH 5.0-6.0) and pH 4.5-6.5 (pH 6.0), respectively. Both strains grew at 12-37 °C (optimum, 28 °C) and NaCl levels up to 1.0 % (optimum 0 %, w/v). Phylogenetic analysis based on both 16S rRNA gene sequences and the concatenated partial atpD, gyrB andlepA gene sequences showed that strains DHOA06T and 4 M-K27T formed two isolated clades with members of the genus Dyella, but they each occupied a distinctive position within the genus. Strains DHOA06T and 4 M-K27T showed the highest 16S rRNA gene sequence similarities to Dyellacaseinilytica DHOB09T (98.7 %) and Dyellaacidisoli 4M-Z03T (98.8 %), respectively. DNA-DNA hybridization values of strains DHOA06T/DHOB09T and 4 M-K27T/4M-Z03T were 27.4±2.4 % and 38.8±1.0 %, respectively. Ubiquinone-8 was the only respiratory quinone detected in both strains. Their major fatty acids consisted of iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1ω9c, and strain DHOA06T had iso-C17 : 0 in addition. Their polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid, one unidentified phospholipid and two unidentified aminolipids, and strain DHOA06T had phosphatidylmethylethanolamine and one unidentified lipid in addition. The DNA G+C contents of strains DHOA06T and 4 M-K27T were 59.1 and 61.7 mol%, respectively. Based on the above results, we propose that strains DHOA06T and 4 M-K27T represent two novel species of the genus Dyella, namely Dyelladinghuensis sp. nov. (type strain DHOA06T = KCTC 52129T=NBRC 111978T) and Dyellachoica sp. nov. (type strain 4 M-K27T=GDMCC 1.1189T=LMG 30267T).

Paraburkholderia phosphatilytica sp. nov., a phosphate-solubilizing bacterium isolated from forest soil.

A novel Gram-stain-negative, aerobic, non-spore-forming, non-motile and rod-shaped bacterial strain, 7QSK02T, was isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, China. It grew at 12-37 °C, at pH 4.0-7.5 and in the presence of 0-1.0 % (w/v) NaCl on R2A agar medium, with optimum growth at 28 °C, pH 5.5 and 0 % NaCl. Strain 7QSK02T was closely related to members of the genus Paraburkholderia: P. acidipaludis NBRC 101816T (98.1 % 16S rRNA gene sequence similarity), P. piptadeniae STM 7183T (97.6 %), P. kururiensis JCM 10599T (97.3 %), P. caballeronis TNe-841T (97.3 %) and P. diazotrophica JPY461T (97.1 %). 16S rRNA gene sequence analysis showed that strain 7QSK02T and two closely strains, P. kururiensis JCM 10599T and P. caballeronis TNe-841T, formed a clade within the genus Paraburkholderia, but was clearly separated from the established species. The genomic G+C content of strain 7QSK02T was 64.9 mol% based on total genome calculations. The average nucleotide identity and digital DNA-DNA hybridization value for the complete genomes were 79.2-81.5 and 23.2-24.9 % between strain 7QSK02T and its closely related species listed above. Strain 7QSK02T contained ubiquinone 8 as the major respiratory quinone. Major fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylmethylethanolamine, one unidentified aminophospholipid, aminolipid and polar lipid. The phenotypic, chemotaxonomic and phylogenetic properties, and genome analysis suggest that strain 7QSK02T represents a novel species of the genus Paraburkholderia, for which the name Paraburkholderia phosphatilytica sp. nov. is proposed. The type strain is 7QSK02T (=GDMCC 1.1283T=CGMCC 1.15470T=KCTC 62473T).

Paraburkholderia telluris sp. nov., isolated from subtropical forest soil.

Strain DHOC27T is a Gram-stain-negative, aerobic, non-motile, light yellow-pigmented and rod-shaped bacterium isolated from the forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. It grew at 4-37 °C (optimal 28-33 °C), pH 4.0-8.5 (optimal 4.5-6.0) and 0-1.5 (optimal 0-0.5) % (w/v) NaCl. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain formed a clade with Paraburkholderia phenazinium LMG 2247T, Paraburkholderia. sartisoli LMG 24000T and Paraburkholderia. pallidirosea DHOK13T, with a sequence similarity of 98.5, 97.5 and 98.1 % to the above strains, respectively. The DNA G+C content of DHOC27T was 62.3 mol%. The digital DNA-DNA relatedness values and the average nucleotide identities between strain DHOC27T and P. phenazinium LMG 2247T and P. sartisoli LMG 24000T were 26.9 and 24.3 % and 82.3 and 79.9 %, respectively. C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c were the major fatty acids, and ubiquinone-8 was the major respiratory quinone detected, all of which supported the affiliation of DHOC27T to the genus Paraburkholderia. On the basis of the data presented above, strain DHOC27T represents a novel species of the genus Paraburkholderia and the name Paraburkholderia telluris sp. nov. is proposed. The type strain is DHOC27T (=LMG 30263T=GDMCC 1.1281T).

Dyella monticola sp. nov. and Dyella psychrodurans sp. nov., isolated from monsoon evergreen broad-leaved forest soil of Dinghu Mountain, China.

Cells of bacterial strains 4 G-K06T and 4MSK11T, isolated from soil samples collected from monsoon evergreen broad-leaved forest of the Dinghushan Mountain (112° 31' E 23° 10' N), Guangdong Province, PR China, were Gram-stain-negative, aerobic, non-spore-forming, non-motile and rod-shaped. Strain 4 G-K06T grew at 10-37 °C, pH 3.5-7.5 and 0-3.5 % (w/v) NaCl; while 4MSK11T grew at 4-42 °C, pH 3.5-7.5 and 0-2.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed strain 4 G-K06T formed a clade with Dyellaflagellata 4 M-K16T, Dyella acidisoli 4M-Z03T, Dyellahumi DHG40T and Dyellanitratireducens DHG59T, while strain 4MSK11T formed a clade with Dyellacaseinilytica DHOB09T and Dyellamobilis DHON07T, both within the genus Dyella. The result of the partial atpD, gyrB and lepA gene sequence analysis supported the conclusion based on 16S rRNA gene sequence analysis, which showed that these two strains represent two novel species of Dyella. The average nucleotide identity and digital DNA-DNA hybridization value for the whole genomes were 75.0-79.0 and 20.3-22.6 % between strains 4 G-K06T, 4MSK11T and those described Dyella species with genome sequences; while the DNA-DNA hybridization rates between strains 4 G-K06T, 4MSK11T and closely related Dyella species (without genome sequence) were 29.5-41.8 %. The major cellular fatty acids of these two strains were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1ω9c, while the major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several unidentified phospholipids and aminophospholipids. The only ubiquinone of these two strains was ubiquinone-8. The DNA G+C contents of 4 G-K06T and 4MSK11T were 60.4 and 61.3 mol%, respectively. On the basis of the evidence presented here, strains 4 G-K06T and 4MSK11T represent two novel species of the genus Dyella, for which the names Dyella monticola sp. nov. (type strain 4 G-K06T=LMG 30268T=GDMCC 1.1188T) and Dyella psychrodurans sp. nov. (type strain 4MSK11T=KCTC 62280T=GDMCC 1.1185T) are proposed.

Dyella solisilvae sp. nov., isolated from mixed pine and broad-leaved forest soil.

A Gram-stain-negative, aerobic, motile, yellow-pigmented, rod-shaped with a single polar flagellum bacterial strain, designated strain DHG54T, was isolated from a forest soil sample of Dinghushan Biosphere Reserve, Guangdong Province, China. Strain DHG54T grew at 12-37 °C (optimum, 28 °C), pH 4.5-8.0 (optimum, pH 6.0-7.0) and in the presence of 0-3.0 % (w/v) NaCl (optimum, 0-1.5 %, w/v). Based on 16S rRNA gene sequence analysis, strain DHG54T formed a clade with the members of the genus Dyella and showed highest sequence similarities of 98.2 % to Dyella japonica DSM 16301T and Dyella terrae KACC 12748T. This was also supported by phylogenetic analysis based on the concatenated partial gyrB, lepA and recA housekeeping gene sequences. DNA-DNA hybridization results between strain DHG54T and closely related Dyella species were all lower than 70 %. Ubiquinone-8 was the only respiratory quinone, and iso-C15 : 0, iso-C17 : 0 and iso-C17 : 1 ω9c were major fatty acids. The DNA G+C content of strain DHG54T was 65.4 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of the polyphasic characterization results presented here, strain DHG54T represents a novel species of the genus Dyella, for which the name Dyellasolisilvae sp. nov. (type strain DHG54T=GDMCC 1.1187T = LMG 30091T) is proposed.