Machine learning identifies cell-free DNA 5-hydroxymethylation biomarkers that detect occult colorectal cancer in PLCO Screening Trial subjects.

Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality, and CRC detection through screening improves survival rates. A promising avenue to improve patient screening compliance is the development of minimally-invasive liquid biopsy assays that target CRC biomarkers on circulating cell-free DNA (cfDNA) in peripheral plasma. In this report, we identify cfDNA biomarker candidate genes bearing the epigenetic mark 5-hydroxymethylcytosine (5hmC) that diagnose occult CRC up to 36 months prior to clinical diagnosis using the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial samples. Methods: Archived PLCO Trial plasma samples containing cfDNA were obtained from the National Cancer Institute (NCI) biorepositories. Study subjects included those who were diagnosed with CRC within 36 months of blood collection (i.e., case, n = 201) and those who were not diagnosed with any cancer during an average of 16.3 years of follow-up (i.e., controls, n = 402). Following the extraction of 3 - 8 ng cfDNA from less than 300 microliters plasma, we employed the sensitive 5hmC-Seal chemical labeling approach, followed by next-generation sequencing (NGS). We then conducted association studies and machine-learning modeling to analyze the genome-wide 5hmC profiles within training and validation groups that were randomly selected at a 2:1 ratio. Results: Despite the technical challenges associated with the PLCO samples (e.g., limited plasma volumes, low cfDNA amounts, and long archival times), robust genome-wide 5hmC profiles were successfully obtained from these samples. Association analyses using the Cox proportional hazards models suggested several epigenetic pathways relevant to CRC development distinguishing cases from controls. A weighted Cox model, comprised of 32-associated gene bodies, showed predictive detection value for CRC as early as 24-36 months prior to overt tumor presentation, and a trend for increased predictive power was observed for blood samples collected closer to CRC diagnosis. Notably, the 5hmC-based predictive model showed comparable performance regardless of sex and self-reported race/ethnicity, and significantly outperformed risk factors such as age and obesity according to BMI (body mass index). Additionally, further improvement of predictive performance was achieved by combining the 5hmC-based model and risk factors for CRC. Conclusions: An assay of 5hmC epigenetic signals on cfDNA revealed candidate biomarkers with the potential to predict CRC occurrence despite the absence of clinical symptoms or the availability of effective predictors. Developing a minimally-invasive clinical assay that detects 5hmC-modified biomarkers holds promise for improving early CRC detection and ultimately patient survival through higher compliance screening and earlier intervention. Future investigation to expand this strategy to prospectively collected samples is warranted.

5-Hydroxymethylcytosine signals in serum are a predictor of chemoresistance in high-grade serous ovarian cancer.

OBJECTIVE: The genome-wide profiling of 5-hydroxymethylcytosines (5hmC) on circulating cell-free DNA (cfDNA) has revealed promising biomarkers for various diseases. The purpose of this study was to investigate 5hmC signals in serum cfDNA and identify novel predictive biomarkers for the development of chemoresistance in high-grade serous ovarian cancer (HGSOC). We hypothesized that 5hmC profiles in cfDNA reflect the development of chemoresistance and elucidate pathways that may drive chemoresistance in HGSOC. Moreover, we sought to identify predictors that would better stratify outcomes for women with intermediate-sensitive HGSOC. METHODS: Women diagnosed with HGSOC and known platinum sensitivity status were selected for this study. Nano-hmC-Seal was performed on cfDNA isolated from archived serum samples, and differential 5hmC features were identified using DESeq2 to establish a model predictive of chemoresistance. RESULTS: A multivariate model consisting of three features (preoperative CA-125, largest residual implant after surgery, 5hmC level of OSGEPL), stratified samples from intermediate sensitive, chemo-naive women diagnosed with HGSOC into chemotherapy-resistant- and sensitive-like strata with a significant difference in overall survival (OS). Independent analysis of The Cancer Genome Atlas data further confirmed that high OSGEPL1 expression is a favorable prognostic factor for HGSOC. CONCLUSIONS: We have developed a novel multivariate model based on clinico-pathologic data and a cfDNA-derived 5hmC modified gene, OSGEPL1, that predicted response to platinum-based chemotherapy in intermediate-sensitive HGSOC. Our multivariate model applies to chemo-naïve samples regardless if the patint was treated with adjuvant or neoadjuvant chemotherapy. These results merit further investigation of the predictive capability of our model in larger cohorts.

Analysis of genome-wide 5-hydroxymethylation of blood samples stored in different anticoagulants: opportunities for the expansion of clinical resources for epigenetic research.

BACKGROUND: Elucidating epigenetic mechanisms could provide new biomarkers for disease diagnosis and prognosis. Technological advances allow genome-wide profiling of 5-hydroxymethylcytosines (5hmC) in liquid biopsies. 5hmC-Seal followed by NGS is a highly sensitive technique for 5hmC biomarker discovery in cfDNA. Currently, 5hmC Seal is optimized for EDTA blood collection. We asked whether heparin was compatible with 5hmC Seal as many clinical and biobanked samples are stored in heparin. METHODS: We obtained 60 samples in EDTA matched to 60 samples in heparin from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Samples were comprised of 30 controls and 30 individuals who were later diagnosed with colon cancer. We profiled genome-wide 5hmC in cfDNA using 5hmC-Seal assay followed by NGS. The 5hmC profiling data from samples collected in EDTA were systematically compared to those in heparin across various genomic features. RESULTS: cfDNA isolation and library construction appeared comparable in heparin vs. EDTA. Typical genomic distribution patterns of 5hmC, including gene bodies and enhancer markers, were comparable in heparin vs. EDTA. 5hmC analysis of cases and controls yielded highly correlated differential features suggesting that both anticoagulants were compatible with 5hmC Seal assay. CONCLUSIONS: While not currently recommended for the 5hmC-Seal protocol, blood samples stored in heparin were successfully used to generate analysable and biologically relevant genome-wide 5hmC profiling. Our findings are the first to support opportunities to expand the biospecimen resource to heparin samples for 5hmC Seal and perhaps other PCR-based technologies in epigenetic research.

A lncRNA from the FTO locus acts as a suppressor of the m6A writer complex and p53 tumor suppression signaling.

N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.

METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.

BACKGROUND: DNA N6-methyldeoxyadenosine (6mA) is rarely present in mammalian cells and its nuclear role remains elusive. RESULTS: Here we show that hypoxia induces nuclear 6mA modification through a DNA methyltransferase, METTL4, in hypoxia-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. Co-expression of METTL4 and 6mA represents a prognosis marker for upper tract urothelial cancer patients. By RNA sequencing and 6mA chromatin immunoprecipitation-exonuclease digestion followed by sequencing, we identify lncRNA RP11-390F4.3 and one novel HIF-1α co-activator, ZMIZ1, that are co-regulated by hypoxia and METTL4. Other genes involved in hypoxia-mediated phenotypes are also regulated by 6mA modification. Quantitative chromatin isolation by RNA purification assay shows the occupancy of lncRNA RP11-390F4.3 on the promoters of multiple EMT regulators, indicating lncRNA-chromatin interaction. Knockdown of lncRNA RP11-390F4.3 abolishes METTL4-mediated tumor metastasis. We demonstrate that ZMIZ1 is an essential co-activator of HIF-1α. CONCLUSIONS: We show that hypoxia results in enriched 6mA levels in mammalian tumor cells through METTL4. This METTL4-mediated nuclear 6mA deposition induces tumor metastasis through activating multiple metastasis-inducing genes. METTL4 is characterized as a potential therapeutic target in hypoxic tumors.

FTO mediates LINE1 m6A demethylation and chromatin regulation in mESCs and mouse development.

N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m6A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m6A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m6A demethylation by FTO in mammals.

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m6A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m6A modification, the methyltransferase responsible for the 18S rRNA m6A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m6A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m6A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m6A in noncoding RNAs.

A critical role of nuclear m6A reader YTHDC1 in leukemogenesis by regulating MCM complex-mediated DNA replication.

YTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in acute myeloid leukemia (AML) and that it is required for the proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We found that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem and progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides compelling evidence that shows an oncogenic role and a distinct mechanism of YTHDC1 in AML.

Control of Early B Cell Development by the RNA N6-Methyladenosine Methylation.

The RNA N6-methyladenosine (m6A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m6A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m6A reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA m6A methylation and its reader proteins in early B cell development.

Lampropedia aestuarii sp. nov., isolated from the estuary of a freshwater river.

A Gram-stain-negative, strictly aerobic, catalase-positive and oxidase-positive bacterium, designated strain YIM MLB12T, was isolated from estuary sediment sampled at Maliao River where it flows into a plateau lake (Dianchi) in Yunnan, south-west PR China. Cells were non-motile and rod-shaped. Growth was observed at 15-35 °C (optimum, 25-30 °C), pH 6.0-10.0 (optimum, pH 7.0-8.0) and in the presence of 0-7 % (w/v) NaCl (optimum, 0.5-2 %). Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM MLB12T formed a tight phylogenic lineage with members of the genus Lampropedia and was closely related to 'Lampropedia puyangensis' 2-bin with 98.3 % sequence similarity and had low similarities to the type strains of Lampropediahyalina ATCC 11041T (96 %) and Lampropedia cohaerens CT6T (95.5 %). Average nucleotide identity and in silico DNA-DNA hybridization values between strain YIM MLB12T and 'L. puyangensis' KCTC 32235 were 76.5 and 22.6 %, respectively. Strain YIM MLB12T contained ubiquinone-8 as the major quinone. The predominant cellular fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, C10 : 0 3-OH, summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), C12 : 0 3-OH and C14 : 0. The polar lipid profile of strain YIM MLB12T was composed predominantly of diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The major polyamine was spermidine. The genomic DNA G+C content of strain YIM MLB12T was 56.8 mol%. Based on its genotypic and chemotaxonomic features and results of phenotypic analyses, strain YIM MLB12T represents a novel species of the genus Lampropedia, for which the name Lampropediaaestuarii sp. nov. is proposed. The type strain is YIM MLB12T (=KCTC 42886T=CGMCC 1.17071T).

High expression of the ANKRD49 protein is associated with progression and poor prognosis of gastric cancer.

BACKGROUND: Gastric cancer is one of the most common malignant tumours. Identifying novel genes that govern the development of gastric cancer will help to elucidate its molecular mechanisms and find novel biomarkers. METHODS: Expression of the ANKRD49 protein was assessed by immunohistochemical analysis of tissue microarrays containing 92 sets of human gastric cancer specimens with adjacent non-cancerous tissue. Associations between ANKRD49 levels and clinicopathological characteristics of the patient were investigated. The correlation between ANKRD49 expression and patient survival was analysed by the Kaplan-Meier method. RESULTS: The results revealed that the expression level of the ANKRD49 protein in gastric cancer was significantly upregulated and correlated with the tumour size, tumour-node-metastasis (TNM) stage, histological grade, depth of invasion, vessel invasion, lymph node metastasis and distant metastasis. The mean survival time of patients with low expression levels of ANKRD49 was significantly longer than that of patients with high expression levels of ANKRD49. Multivariate Cox regression analysis demonstrated that the ANKRD49 protein expression level was an independent prognostic indicator for the survival rate of patients with gastric cancer. CONCLUSION: The results of the present study highlighted an important role of the ANKRD49 protein in the progression of gastric cancer. The ANKRD49 protein could act as a potential biomarker for prognosis evaluation of gastric cancer and may be used as a molecular target for gastric cancer treatment.

Mongoliibacter ruber gen. nov., sp. nov., a haloalkalitolerant bacterium of the family Cyclobacteriaceae isolated from a haloalkaline lake.

A novel haloalkalitolerant, rod-shaped bacterium, designated strain YIM 4-4T, was isolated from the surface water of the Dugerno lake, a haloalkaline lake in Inner Mongolia. The taxonomy of strain YIM 4-4T was investigated by a polyphasic approach. Strain YIM 4-4T was Gram-stain-negative, strictly aerobic, non-motile and formed red colonies. Optimal growth conditions were 28 °C, pH 8.0-11.0 and 0.5-2 % NaCl. The major respiratory quinone was menaquinone-7 (MK-7). The polar lipid profile was composed predominantly of phosphatidylethanolamine, six unidentified polar lipids, one phospholipid and one aminolipid. The predominant cellular fatty acids (>5 %) were iso-C15 : 0, iso-C17 : 1I/anteiso-C17 : 1B, iso-C16 : 1G, iso-C17 : 0 3-OH, C16 : 1ω7c/C16 : 1ω6c and iso-C16 : 1. The genomic DNA G+C content was 43.0 mol%. 16S rRNA gene sequence analysis indicated that the members of the genera Cecembia, Fontibacter, Aquiflexum and Indibacter of the family Cyclobacteriaceae (phylum Bacteroidetes) were the most closely related, with 16S rRNA gene sequence similarities ranging from 93.6 to 94.2 %. Other members of the family Cyclobacteriaceae showed sequence similarities < 93.0 %. On the basis of phenotypic, chemotaxonomic and phylogenetic properties, strain YIM 4-4T represents a novel species of a new genus, for which the name Mongoliibacter ruber gen. nov., sp. nov. is proposed. The type strain is YIM 4-4T ( = CCTCC AB 2012966T = DSM 27929T).

Griseusins F and G, spiro-naphthoquinones from a tin mine tailings-derived alkalophilic Nocardiopsis species.

Griseusins F (1) and G (2), two 2a-hydro-8a-(2-oxopropyl)-substituted spiro-naphthoquinones with a previously undescribed C23 polyketide skeleton, were isolated from a Yunnan tin mine tailings-derived alkalophilic actinomycete, Nocardiopsis sp. YIM DT266. Their complete structure assignments with the absolute stereochemistry were elucidated by spectroscopic data, X-ray crystal diffraction, calculation of optical rotation, and CD spectroscopic analysis. Compounds 1 and 2 exhibited strong cytotoxicity (IC50 0.37-0.82 µM) and antibacterial activity (MIC 0.80-1.65 µg/mL) against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) in vitro.

Fodinibius salinus gen. nov., sp. nov., a moderately halophilic bacterium isolated from a salt mine.

A novel, moderately halophilic, rod-shaped bacterium, designated strain YIM D17(T), was isolated from a sample of sediment from a salt mine in Yunnan, south-western China. The taxonomy of strain YIM D17(T) was investigated using a polyphasic approach. Strain YIM D17(T) was Gram-stain-negative, strictly aerobic and non-motile and formed pink colonies on marine agar. Optimal growth occurred at 37 °C, pH 7.5-8.0 and in the presence of 10-15 % (w/v) NaCl. The major menaquinone was MK-7. The polar lipid profile was composed predominantly of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, one phospholipid, one glycolipid and one aminolipid. Minor amounts of other lipids were also detectable. The predominant cellular fatty acids were iso-C(17 : 1)ω9c/10-methyl-C(16 : 0) (24.0 %), iso-C(15 : 0) (23.6 %) and C(16 : 1)ω7c/C(16 : 1)ω6c (13.8 %). The DNA G+C content was 43.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the isolate formed a distinct clade with the genera Gracilimonas and Balneola (both in the phylum Bacteroidetes) and was related to the species Gracilimonas tropica, Balneola vulgaris and Balneola alkaliphila, with sequence similarities of 85.6 %, 83.0 % and 82.8 % to the respective type strains. On the basis of its phenotypic, chemotaxonomic and phylogenetic features, strain YIM D17(T) represents a novel species of a new genus, for which the name Fodinibius salinus gen. nov., sp. nov. is proposed. The type strain is YIM D17(T) ( = ACCC 10716(T) = DSM 21935(T)).

Litoribacter ruber gen. nov., sp. nov., an alkaliphilic, halotolerant bacterium isolated from a soda lake sediment.

A novel alkaliphilic, halotolerant, rod-shaped bacterium, designated strain YIM CH208(T), was isolated from a soda lake in Yunnan, south-west China. The taxonomy of strain YIM CH208(T) was investigated by a polyphasic approach. Strain YIM CH208(T) was Gram-negative, strictly aerobic and non-motile and formed red colonies. Optimal growth conditions were 28°C, pH8.5 and 0.5-2.5 % NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the isolate formed a distinct line within a clade containing the genus Echinicola in the phylum Bacteroidetes and was related to the species Echinicola pacifica and Rhodonellum psychrophilum, with sequence similarity of 91.7 and 91.6 % to the respective type strains. The DNA G+C content was 45.1 mol%. The major respiratory quinone was menaquinone-7 (MK-7). The predominant cellular fatty acids were iso-C(17 : 1)ω9c (19.9 %), C(15 : 0) 3-OH (12.1 %), iso-C(17 : 0) 3-OH (11.3 %), summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c; 10.7 %) and C(17 : 1)ω6c (8.7 %). On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain YIM CH208(T) represents a novel species of a new genus, for which the name Litoribacter ruber gen. nov., sp. nov. is proposed. The type strain of Litoribacter ruber is YIM CH208(T) (=ACCC 05414(T) =KCTC 22899(T)).

Salinarimonas rosea gen. nov., sp. nov., a new member of the alpha-2 subgroup of the Proteobacteria.

A Gram-negative, rod-shaped, facultatively anaerobic, halotolerant bacterial strain, designated YIM YD3(T), was isolated from a salt mine in Yunnan, south-west China. The taxonomy of strain YIM YD3(T) was investigated by a polyphasic approach. Strain YIM YD3(T) was motile, formed pink colonies and was positive for catalase and oxidase activities. Q-10 was the predominant respiratory ubiquinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine and two unknown phospholipids. The major fatty acids (>10 % of total fatty acids) were C(18 : 1)omega7c, C(18 : 1)omega9c, C(16 : 0) and C(19 : 0) cyclo omega8c. The DNA G+C content was 71.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the isolate formed a distinct line within a clade containing the genera Balneimonas, Bosea, Chelatococcus and Microvirga in the order Rhizobiales, with highest levels of 16S RNA gene sequence similarity to the type strain of Balneimonas flocculans (93.5 %). On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain YIM YD3(T) represents a novel species in a new genus, for which the name Salinarimonas rosea gen. nov., sp. nov. is proposed, with strain YIM YD3(T) (=KCTC 22346(T)=CCTCC AA208038(T)) as the type strain.

Stackebrandtia albiflava sp. nov. and emended description of the genus Stackebrandtia.

A novel actinomycete, designated YIM 45751(T), was isolated from a soil sample collected from the Xishuang Banna tropical rainforest, Yunnan province, south-west China, and was subjected to a polyphasic taxonomic study. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. Whole-cell hydrolysates contained ribose, xylose and glucose. The predominant menaquinones were MK-10(H(4)), MK-10(H(6)), MK-11(H(4)) and MK-11(H(6)). The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and three unknown phospholipids. 16S rRNA gene sequence analysis showed that Stackebrandtia nassauensis DSM 44728(T) was the closest phylogenetic neighbour (95.8 % 16S rRNA gene sequence similarity). On the basis of phylogenetic and some common chemotaxonomic data, the isolate should belong to the genus Stackebrandtia. However, phylogenetic analysis and comparison of physiological and chemotaxonomic characteristics demonstrated that the isolate is distinct from S. nassauensis DSM 44728(T). Strain YIM 45751(T) represents a separate species of the genus Stackebrandtia, for which the name Stackebrandtia albiflava sp. nov. is proposed. The type strain is YIM 45751(T) (=DSM 45044(T)=CCTCC AA 206003(T)). Furthermore, the description of the genus Stackebrandtia is also required to be emended.

Virgibacillus kekensis sp. nov., a moderately halophilic bacterium isolated from a salt lake in China.

A Gram-positive, moderately halophilic, motile, strictly aerobic, endospore-forming, oxidase- and catalase-positive, rod-shaped bacterium, strain YIM kkny16(T), was isolated from a saline mud sample collected from the Keke salt lake in the Qaidam Basin, north-west China. This isolate grew in the presence of 0-25 % (w/v) NaCl and at pH 6.0-10.0 and 10-50 degrees C; optimum growth was observed with 10 % (w/v) NaCl and at pH 7.0 and 37 degrees C. Strain YIM kkny16(T) had meso-diaminopimelic acid as the diagnostic diamino acid, MK-7 as the predominant respiratory quinone, with a significant amount of MK-6, and anteiso-C(15 : 0), iso-C(14 : 0) and C(16 : 1)omega7c alcohol as major fatty acids. Major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C content was 41.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain YIM kkny16(T) was a member of the genus Virgibacillus, exhibiting sequence similarities of 94.9-97.3 % to the type strains of recognized Virgibacillus species. Strain YIM kkny16(T) could be differentiated from recognized Virgibacillus species based on phenotypic characteristics, chemotaxonomic differences, phylogenetic analysis and DNA-DNA hybridization data. On the basis of evidence from this polyphasic study, strain YIM kkny16(T) is considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus kekensis sp. nov. is proposed. The type strain is YIM kkny16(T) (=DSM 17056(T)=CGMCC 1.6298(T)).

Nocardiopsis quinghaiensis sp. nov., isolated from saline soil in China.

A previously unknown Gram-positive, obligately aerobic actinomycete, YIM 28A4(T), was isolated from a sample of saline soil collected from the Qaidam Basin in Qinghai Province, north-west China, and was investigated using a polyphasic taxonomic approach. The strain grew well on most of the media tested, producing white to pale-yellow substrate mycelium, white aerial mycelium and straight to flexuous hyphae. The substrate mycelium was well developed and fragmented with age; the aerial mycelium produced long, straight spore chains. The spore chains were composed of non-motile, smooth-surfaced, rod-shaped spores. No diffusible pigments were produced on any of the media tested. The strain grew in the presence of 0-10 % (w/v) NaCl and at pH 6.0-8.0, with optimum growth occurring at 3 % (w/v) NaCl and pH 7.0. It grew at 10-37 degrees C, the optimum growth temperature being 28 degrees C. Whole-cell hydrolysates of strain YIM 28A4(T) contained meso-diaminopimelic acid and no diagnostic sugars. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol. The predominant menaquinones were MK-10, MK-10(H(2)), MK-11 and MK-11(H(2)). The major cellular fatty acids were iso-C(16 : 0), anteiso-C(15 : 0) and anteiso-C(17 : 0). The DNA G+C content was 67.1 mol%. The morphological and chemotaxonomic characteristics of the isolate matched those described for Nocardiopsis species. A phylogenetic analysis based on 16S rRNA gene sequence comparisons confirmed that strain YIM 28A4(T) was a member of the genus Nocardiopsis and most closely related to the type strains Nocardiopsis aegyptia DSM 44442(T) and Nocardiopsis halotolerans DSM 44410(T), showing 98.1 and 97.8 % 16S rRNA gene sequence similarity, respectively. Strain YIM 28A4(T) can be differentiated from these type strains by using phenotypic, phylogenetic and DNA-DNA hybridization data. On the basis of the polyphasic evidence, strain YIM 28A4(T) represents a novel species of the genus Nocardiopsis, for which the name Nocardiopsis quinghaiensis sp. nov. is proposed. The type strain is YIM 28A4(T) (=DSM 44739(T) =CGMCC 4.3494(T)).

[Micirobial diversity and screening of antitumor activity of actinomycete strains from saline and alkaline environments in the Qinghai Province, P. R. China].

Soil and sediment samples were collected from saline and alkaline soils or lakes in the Qinghai Province, northwestern China. 145 actinomycete strains were isolated using Glucose-Peptone-Yeast extract agar (GPY) and ISP medium 2 agar supplemented with 1.0 - 3.0 mol/L NaCl at pH 7.5 - 10. The antitumor activities in vitro of the fermentation broth extracts from the 145 test strains were detected in 6 human tumor cell lines (gastric cancer GXF251L, lung cancer LXFL529L, mammary cancer MAXF401NL, melanoma cancer MEXF462NL, renal cancer RXF486L and uterus cancer UXF1138L). Out of 145 test strains, 26 strains were positive in antitumor activities (17.9%), among them 19 strains belong to the genus Nocardiopsis, 7 strains belong to the genus Streptomyces. Then 8 antitumor-positive strains were submitted for 16S rRNA gene amplification and phylogenetic analysis after a comparison of antitumor activities, morphological, physiological characteristics and whole cell amino acids analysis. The results suggested that strain YIM 80139 is a member of a known Streptomyces species S. griseus, while strain YIM 80038 may represent a potential new Streptomyce species, and that the other 6 strains may represent 4 potential new species of the genus Nocardiopsis. The results presented above showed that actinomycetes isolated from saline and alkaline samples are important resources for bioactive compounds, and the abundant microbial diversity in the saline and alkaline environments in the Qinghai Province, Northwestern China is attractive for further investigation.