The Paf1 complex is required for RNA polymerase II removal in response to DNA damage.

Rpb1, the largest subunit of RNA polymerase II (RNAPII), is rapidly polyubiquitinated and degraded in response to DNA damage; this process is considered to be a "mechanism of last resort'' employed by cells. The underlying mechanism of this process remains elusive. Here, we uncovered a previously uncharacterized multistep pathway in which the polymerase-associated factor 1 (Paf1) complex (PAF1C, composed of the subunits Ctr9, Paf1, Leo1, Cdc73, and Rtf1) is involved in regulating the RNAPII pool by stimulating Elongin-Cullin E3 ligase complex-mediated Rpb1 polyubiquitination and subsequent degradation by the proteasome following DNA damage. Mechanistically, Spt5 is dephosphorylated following DNA damage, thereby weakening the interaction between the Rtf1 subunit and Spt5, which might be a key step in initiating Rpb1 degradation. Next, Rad26 is loaded onto stalled RNAPII to replace the Spt4/Spt5 complex in an RNAPII-dependent manner and, in turn, recruits more PAF1C to DNA lesions via the binding of Rad26 to the Leo1 subunit. Importantly, the PAF1C, assembled in a Ctr9-mediated manner, coordinates with Rad26 to localize the Elongin-Cullin complex on stalled RNAPII, thereby inducing RNAPII removal, in which the heterodimer Paf1/Leo1 and the subunit Cdc73 play important roles. Together, our results clearly revealed a new role of the intact PAF1C in regulating the RNAPII pool in response to DNA damage.

Atrachinenins D-S, novel meroterpenoids with geranyl hydroquinone moiety from Atractylodes chinensis by the LC/MS-based molecular decoy and targeted isolation.

To mine fascinating molecules from the rhizomes of Atractylodes chinensis, the known molecular formula of atrachinenin A was used as a bait to search LC-HRMS data in different subfractions. Sixteen new meroterpenoids, atrachinenins D-S (1-16) including three unprecedented carbon skeletons (1-5) and eleven new oxygen-bridged hybrids (6-16) were obtained by the targeted isolation. Their structures and absolute configurations were elucidated by the spectroscopic data and electronic circular dichroism (ECD) calculations. The isolated compounds were evaluated for their inhibitory activity of NO production and compounds 1, 4, 8, and 13 showed moderate anti-inflammatory activity. The proposed biosynthetic pathways of 1-5 were also discussed.

Biochemical insights into Paf1 complex-induced stimulation of Rad6/Bre1-mediated H2B monoubiquitination.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.

[Development of Sample Size Formulas for Least Square Regression-Based Consistency Evaluation of Quantitative Indicators].

Objective To develop and verify the sample size formulas for quantitative data consistency evaluation based on the least square regression method. Methods According to the principle of least square regression-based quantitative consistency evaluation,statistical inference,and formula derivation,we developed the formulas for calculating sample size based on regression constant and regression coefficient.Furthermore,the accuracy of the formulas was verified by the data of three examples,and the results were compared with those of the sample size formula established based on the Bland-Altman(BA)method. Results The sample size formulas for regression-based quantitative consistency evaluation were deduced,and the accuracy of the formulas was verified by three examples.In addition,the results obtained with this formula had differences compared with those of the sample size formula established based on the BA method.Furthermore,consistent conclusions could be obtained by regression analysis and BA analysis with the sample size calculated with the regression method.However,with the sample size calculated based on the BA method,the consistency conclusion of regression analysis and BA analysis was sometimes not valid. Conclusion A sample size formula for quantitative consistency evaluation based on the regression method was proposed for the first time,which provided methodological support for the research in this field.

Association between calf circumference and incontinence in Chinese elderly.

BACKGROUND: The objective of this study was to analyze the association between calf circumference and incontinence in Chinese elderly, and to find out the maximal cut-off point by gender for the use of calf circumference in screening for incontinence. METHODS: In this study, participants were from the 2018 Chinese Longitudinal Healthy Longevity Survey (CLHLS). The maximal calf circumference cut-off point and other incontinence-related risk factors were explored using receiver operating characteristic (ROC) curves and logistic regression analysis. RESULTS: The study included 14,989 elderly people (6,516 males and 8,473 females) over 60. The prevalence of incontinence in elderly males was 5.23% (341/6,516), significantly lower than females, which was 8.31% (704/8,473) (p < 0.001). There was no correlation between calf circumference < 34 cm in males and < 33 cm in females and incontinence after adjusting the confounders. We further stratified by gender to predict incontinence in elderly based on the Youden index of ROC curves. We found the association between calf circumference and incontinence was the strongest when the cut-off points were < 28.5 cm for males and < 26.5 cm for females, with an odds rate (OR) value of 1.620 (male, 95%CI: 1.197-2.288) and 1.292 (female, 95%CI: 1.044-1.600) after adjusting the covariates, respectively. CONCLUSIONS: Our study suggests that calf circumference < 28.5 cm in males and < 26.5 cm in females is a risk factor for incontinence in the Chinese elderly population. Calf circumference should be measured in routine physical examination, and timely interventions should be made to reduce the risk of incontinence in subjects with calf circumference less than the threshold.

Hly176B, a low-salt tolerant halolysin from the haloarchaeon Haloarchaeobius sp. FL176.

Extracellular proteases of haloarchaea can adapt to high concentrations of NaCl and can find useful applications in industrial or biotechnology processes where hypersaline conditions are desired. The diversity of extracellular proteases produced by haloarchaea is largely unknown though the genomes of many species have been sequenced and are publicly available. In this study, a gene encoding the extracellular protease Hly176B from the haloarchaeon Haloarchaeobius sp. FL176 was cloned and expressed in Escherichia coli. A related gene homolog to hly176B, hly176A, from the same strain was also expressed in E.coli, but did not show any proteinase activity after the same renaturation process. Therefore, we focus on the enzymatic properties of the Hly176B. The catalytic triad Asp-His-Ser was confirmed via site-directed mutagenesis, indicating that Hly176B belongs to the class of serine proteases (halolysin). Unlike previously reported extracellular proteases from haloarchaea, the Hly176B remained active for a relatively long time in an almost salt-free solution. In addition, the Hly176B displayed prominent tolerance to some metal ions, surfactants and organic solvents, and exerts its highest enzyme activity at 40 °C, pH 8.0 and 0.5 M NaCl. Therefore, this study enriches our knowledge of extracellular proteases and expands their applications for various industrial uses.

[Excretion of three alkaloids from Simiao Pills in urine, feces, and bile between normal and type 2 diabetic rats].

This study investigated the differences in excretion kinetics of three alkaloids and their four metabolites from Simiao Pills in normal and type 2 diabetic rats. The diabetes model was established in rats by injection of streptozotocin, and the alkaloids in urine, feces, and bile of normal and diabetic rats were detected by LC-MS/MS to explore the effect of diabetes on alkaloid excretion of Simiao Pills. The results showed that 72 h after intragastric administration of the extract of Simiao Pills, feces were the main excretion route of alkaloids from Simiao Pills. The total excretion rates of magnoflorine and berberine in normal rats were 4.87% and 56.54%, which decreased to 2.35% and 35.53% in diabetic rats, which had statistical significance(P<0.05). The total excretion rates of phellodendrine, magnoflorine, and berberine in the urine of diabetic rats decreased significantly, which were 53.57%, 60.84%, and 52.78% of those in normal rats, respectively. After 12 h of intragastric administration, the excretion rate of berberine in the bile of diabetic rats increased significantly, which was 253.33% of that of normal rats. In the condition of diabetes, the excretion rate of berberine metabolite, thalifendine significantly decreased in urine and feces, but significantly increased in bile. The total excretion rates of jateorrhizine and palmatine in the urine increased significantly, and t_(1/2) and K_e changed significantly. The results showed that diabetes affected the in vivo process of alkaloids from Simiao Pills, reducing their excretion in the form of prototype drug, affecting the biotransformation of berberine, and ultimately increasing the exposure of alkaloids in vivo, which would be conducive to the hypoglycemic effect of alkaloids. This study provides references for the clinical application and drug development of Simiao Pills in diabetes.

[Recent research on machine learning in the diagnosis and treatment of necrotizing enterocolitis in neonates]. / æœºå™¨å­¦ä¹ åœ¨æ–°ç”Ÿå„¿åæ­»æ€§å°è‚ ç»“è‚ ç‚Žè¯Šç–—ä¸­çš„ç ”ç©¶è¿›å±•.

Necrotizing enterocolitis (NEC), with the main manifestations of bloody stool, abdominal distension, and vomiting, is one of the leading causes of death in neonates, and early identification and diagnosis are crucial for the prognosis of NEC. The emergence and development of machine learning has provided the potential for early, rapid, and accurate identification of this disease. This article summarizes the algorithms of machine learning recently used in NEC, analyzes the high-risk predictive factors revealed by these algorithms, evaluates the ability and characteristics of machine learning in the etiology, definition, and diagnosis of NEC, and discusses the challenges and prospects for the future application of machine learning in NEC.

Halorhabdus amylolytica sp. nov. and Halorhabdus salina sp. nov., isolated from hypersaline environments.

Two novel extremely halophilic archaeal strains, designated H27T and FL145T, were isolated from a salt mine and a kelp salt sample, respectively. Cells of both strains were Gram-stain-negative, motile and pleomorphic. The 16S rRNA and rpoB' gene sequence similarities between strains H27T and FL145T were 96.60 and 88.77%. Strains H27T and FL145T were both closely related to Halorhabdus rudnickae WSM-64T, Halorhabdus tiamatea SARL4BT and Halorhabdus utahensis AX-2T, with a 16S rRNA gene sequence similarities of 98.14, 96.34 and 96.27% for strain H27T and 96.42, 95.82 and 96.17% for strain FL145T. The genome-based average nucleotide identity (ANI) values between strains H27T and FL145T, and these three species were 83.93, 79.79 and 79.09% (for strain H27T), and 78.32, 77.95 and 77.05% (for strain FL145T), respectively. The ANI value between strains H27T and FL145T was 78.65 %. The digital DNA-DNA hybridization values between strains H27T and FL145T, and these three species were less than 27.40%, which were below the recommended threshold for membership of the same species. The major polar lipids of both strains were found to consist of sulfated diglycosyl diether, triglycosyl diether, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol. The DNA G+C content was determined from genome to be 62.10 mol% for strain H27T and 61.51 mol% for strain FL145T. Based on phylogenetic, phenotypic, chemotaxonomic and genomic analyses, these two new isolates should be classified as representing two novel species in the genus Halorhabdus, with strain H27T (=CGMCC 1.16342T=NBRC 113589T) as the type strain of a new species for which we propose the name Halorhabdus amylolytica sp. nov., and strain FL145T (=CGMCC 1.13888T=NBRC 114260T) as the type strain of another new species for which we propose the name Halorhabdus salina sp. nov.

Comparative pharmacokinetics, intestinal absorption and urinary excretion of six alkaloids from herb pair Phellodendri Chinensis cortex-Atractylodis Rhizoma.

Phellodendri Chinensis Cortex (PCC) and Atractylodis Rhizoma (AR) are frequently used as herb pair to treat eczema and gout owing to their synergistic effects. Alkaloids are the major ingredients from PCC and the effect of their combination on the in vivo processing of alkaloids remains unclear. In this study, a simple and reliable UPLC-MS/MS method for simultaneous determination of six alkaloids in rat plasma was developed. This method was applied to a comparative pharmacokinetic study between PCC and PCC-AR in rats. Effect of AR on absorption of alkaloids was investigated by a single-pass intestinal perfusion study. The effect of AR on urinary excretion of alkaloids was studied. Pharmacokinetic studies showed that the values of rea under the concentration-time curve of phellodendrine, magnoflorine and palmatine were greater in the PCC-AR group than in the PCC group. The intestinal absorptive parameters absorption rate constant and effective permeability of phellodendrine and jatrorrhizine in PCC-AR groups were higher than those in the PCC group. Urinary excretion studies revealed that the excreted amount of alkaloids in the PCC-AR group was lower than that in the PCC group. The results revealed that the combination of PCC and AR improves intestinal absorption of alkaloids and reduces their urinary excretion, which enhances their systemic exposure. This study may explain the synergetic effects of PCC and AR in clinical applications.

Different Inhibition of Nrf2 by Two Keap1 Isoforms α and ß to Shape Malignant Behaviour of Human Hepatocellular Carcinoma.

Nrf2 (nuclear factor E2-related factor 2, encoded by Nfe2l2) acts as a master transcriptional regulator in mediating antioxidant, detoxification, and cytoprotective responses against oxidative, electrophilic, and metabolic stress, but also plays a crucial role in cancer metabolism and multiple oncogenic pathways, whereas the redox sensor Keap1 functions as a predominant inhibitor of Nrf2 and, hence, changes in its expression abundance directly affect the Nrf2 stability and transcriptional activity. However, nuanced functional isoforms of Keap1 α and ß have rarely been identified to date. Herein, we have established four distinct cell models stably expressing Keap1-/-, Keap1ß(Keap1Δ1-31), Keap1-Restored, and Keap1α-Restored aiming to gain a better understanding of similarities and differences of two Keap1 isoforms between their distinct regulatory profiles. Our experimental evidence revealed that although Keap1 and its isoforms are still localized in the cytoplasmic compartments, they elicited differential inhibitory effects on Nrf2 and its target HO-1. Furthermore, transcriptome sequencing unraveled that they possess similar but different functions. Such functions were further determined by multiple experiments in vivo (i.e., subcutaneous tumour formation in nude mice) and in vitro (e.g., cell cloning, infection, migration, wound healing, cell cycle, apoptosis, CAT enzymatic activity, and intracellular GSH levels). Of note, the results obtained from tumourigenesis experiments in xenograft model mice were verified based on the prominent changes in the PTEN signaling to the PI3K-AKT-mTOR pathways, in addition to substantially aberrant expression patterns of those typical genes involved in the EMT (epithelial-mesenchymal transition), cell cycle, and apoptosis.

VOLN27B: A New Head-Tailed Halovirus Isolated from an Underground Salt Crystal and Infecting Halorubrum.

A novel halovirus, VOLN27B, was isolated from a drill core sample taken at a depth of approximately 430 m, from a layer formed during the Cretaceous period (Anhui, China). VOLN27B infects the halophilic archaeon Halorubrum sp. LN27 and has a head-tailed morphotype with a contractile tail, typical of myoviruses. The average head diameter is 64 ± 2.0 nm, and uncontracted tails are 15 ± 1.0 × 65 ± 2.0 nm. The latent period is about 10 h. The maturing time of VOLN27B in cells of Halorubrum sp. LN27 was nearly 8 h. The adsorption time of VOLN27B on cells of Halorubrum sp. LN27 was less than 1 min. Virus particles are unstable at pH values less than 5 or when the NaCl concentration is below 12% (w/v). VOLN27B and Halorubrum sp. LN27 were recovered from the same hypersaline environment and provide a new virus-host system in haloarchaea.

Monocytic MDSCs skew Th17 cells toward a pro-osteoclastogenic phenotype and potentiate bone erosion in rheumatoid arthritis.

OBJECTIVES: While myeloid-derived suppressor cells (MDSCs) were previously shown to promote a proinflammatory T helper (Th) 17 response in autoimmune conditions, a potential impact of the MDSC-Th17 immune axis on abnormal bone destruction in RA remains largely unknown. METHODS: We investigated the correlation between the frequency of MDSCs or its subsets and joint destruction in RA patients. The reciprocal actions of patient-derived MDSCs and Th17 cells were studied using osteoclast (OC) differentiation and bone resorption assays in vitro, which were further validated using mouse models of RA. Contribution of MDSCs to osteoclastogenesis and bone erosion in vivo was determined by depletion or transfer of MDSCs. RESULTS: Human MDSCs, particularly monocytic MDSCs (M-MDSCs), exhibit inherent OC-differentiating capacity and positively correlate with clinical bone erosion in RA patients. Strikingly, patient-derived M-MDSCs can program Th17 cells towards a pro-osteoclastogenic phenotype, which in return potentiates OC differentiation via the receptor activator of nuclear factor κΒ ligand (RANK-L)-RANK signalling. This enhanced osteolysis driven by the reciprocal actions of M-MDSCs and Th17 cells is further confirmed using mouse models of RA. Selective depletion of M-MDSCs significantly ameliorates osteoclastogenesis and disease severity in arthritic mice, whereas transfer of M-MDSCs aggravates bone erosion associated with increased OCs in recipient mice. CONCLUSION: Our findings highlight the functional plasticity of MDSCs and identify a novel pro-osteoclastogenic pathway governed by interplay between myeloid cells and T lymphocytes in autoimmune RA.

The I510V mutation in KLHL10 in a patient with oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia is a human infertility syndrome caused by defects in spermatogenesis, spermiogenesis, and sperm maturation, and its etiology remains unclear. Kelch-like 10 (KLHL10) is a component of ubiquitin ligase E3 10 (KLHL10) and plays an important role in male fertility. Deletion or mutation of the Klhl10 gene in Drosophila or mice results in defects in spermatogenesis or sperm maturation. However, the molecular mechanisms by which KLHL10 functions remain elusive. In this study, we identified a missense mutation (c.1528A→G, p.I510V) in exon 5 of KLHL10, which is associated with oligoasthenoteratozoospermia in humans. To investigate the effects of this mutation on KLHL10 function and spermatogenesis and/or spermiogenesis, we generated mutant mice duplicating the amino acid conversion using the clustered regularly interspaced palindromic repeat/caspase 9 (CRISPR/Cas9) system and designated them Klhl10I510V mice. However, the Klhl10I510V mice did not exhibit any defects in testis development, spermatogenesis, or sperm motility at ten-weeks-of-age, suggesting that this mutation does not disrupt the KLHL10 function, and may not be the cause of male infertility in the affected individual with oligoasthenoteratozoospermia.

Snf5 and Swi3 subcomplex formation is required for SWI/SNF complex function in yeast.

The SWI/SNF chromatin remodeling complex, which alters nucleosome positions by either evicting histones or sliding nucleosomes on DNA, is highly conserved from yeast to humans, and 20% of all human cancers have mutations in various subunits of the SWI/SNF complex. Here, we reported the crystal structure of the yeast Snf5-Swi3 subcomplex at a resolution of 2.65 Å. Our results showed that the Snf5-Swi3 subcomplex assembles into a heterotrimer with one Snf5 molecule bound to two distinct Swi3 molecules. In addition, we demonstrated that Snf5-Swi3 subcomplex formation is required for SWI/SNF function in yeast. These findings shed light on the important role of the Snf5-Swi3 subcomplex in the assembly and functional integrity of the SWI/SNF complex.

Loss of Fbxw7 in Sertoli cells impairs testis development and causes infertility in mice†.

F-box and WD-40 domain protein 7 (Fbxw7) is a component of the Skp1-Cdc53/Cullin-F-box-protein complex (SCF/ß-TrCP), which is an E3 ubiquitin ligase that mediates protein degradation. This complex has recently been shown to negatively regulate spermatogonial stem cell self-renewal; however, its roles in Sertoli cell (SC) proliferation, differentiation, and function remain to be established. In this study, we generated conditional mutant mice with SC-specific deletion of Fbxw7 via the Cre-loxP system. Fbxw7 deficiency in SCs impaired testis development, which is characterized by age-dependent tubular atrophy, excessive germ cell loss, and spermatogenic arrest, and the mutant males were infertile at 7 months old. Fbxw7 ablation also compromised cytoskeletal organization and cell polarity of SCs, as well as integrity of the blood-testis barrier. In addition, the transcript levels of cell markers for germ cells, Leydig cells, and SCs were significantly decreased in Fbxw7 mutant mice. Importantly, protein levels of GATA-4, a transcription factor that plays a crucial role in SC maturation and testis development, were progressively decreased in control SCs after postnatal day 14, whereas levels were aberrantly elevated in Fbxw7-deleted SCs. Interestingly, the Gata-4 messenger RNA levels remained stable following Fbxw7 deletion. Fbxw7 silencing in SCs also induced progressive Leydig cell inefficiency and testosterone insufficiency. Collectively, these results demonstrate that Fbxw7 expression is required for SC maturation and function, potentially through degradation of GATA-4, to support pubertal testis development and spermatogenesis.

Adenylate kinase 1 deficiency disrupts mouse sperm motility under conditions of energy stress†.

Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in nonobstructive azoospermia patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis, or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis, or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

Halomicroarcula amylolytica sp. nov., a novel halophilic archaeon isolated from a salt mine.

A halophilic archaeon named strain LR21T was isolated from a salt mine in Yunnan Province, PR China. Cells were spherical, Gram-stain-negative and motile. Strain LR21T grew at 20-50 °C (optimum, 42 °C), with 8-30 % (w/v) NaCl (optimum, 23 %) and at pH 5.5-9.5 (optimum, pH 7.5-8.5). Mg2+ was not required for growth. The major polar lipid profile comprised phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Strain LR21T had two dissimilar 16S rRNA genes (rrnA and rrnB) and they were closely related to Halomicroarcula limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T with sequence similarities of 95.3-99.0, 93.0-96.2 and 93.2-95.9 %, respectively, and much lower values to other members. The rpoB' gene sequence similarities between strain LR21T and Hma. limicola YGHS32T, Hma. pellucida BNERC31T and Hma. salina YGHS18T were 95.2, 91.2 and 91.2 % respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain LR21T and Hma. limicola YGHS32T, were 89.0 and 90.1 %, respectively. DNA relatedness between strains LR21T and Hma. limicola YGHS32T determined by in silico DNA-DNA hybridization was 36.8 %. Values of ANI and AAI between strain LR21T and other members in the genus Halomicroarcula were far below 95 % and the DNA-DNA relatedness values between strain LR21T and its close relatives were much lower than 70 %, which is far below the boundary for delineation of a new species prokaryote. The DNA G+C content of strain LR21T was 62.0 mol% (genome). The results suggested that strain LR21T represents a novel species of the genus Halomicroarcula, for which the name Halomicroarcula amylolytica sp. nov. is proposed. The type strain is LR21T (=CGMCC 1.13611T=NBRC 113588T).

Halomicrococcus hydrotolerans gen. nov., sp. nov., an extremely halophilic archaeon isolated from a subterranean salt deposit.

A halophilic archaeon, strain H22T, was isolated from a subterranean salt deposit sampled at Yunnan salt mine, PR China. Colonies of strain H22T were light pink-pigmented. Cells were coccus, non-motile, Gram-stain-negative, and did not lyse in distilled water. The strain was aerobic and grew at 20-55 °C (optimum, 37 °C), in the presence of 10-30 % (w/v) NaCl (20 %) and at pH 6.5-9.0 (pH 7.0). Mg2+ was required for growth (optimum, 0.005 M). Major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and sulfated mannosyl-glucosyl-glycerol diether-1. Sequence similarity search based on the multiple 16S rRNA genes (rrnA, rrnB and rrnC) of strain H22T revealed that it was most closely related to species of the genera Haloarchaeobius, Haladaptatus, Halorussus and Halorubellus with relative low sequence similarities (91.9-93.7 %). The strain, however, shared highest rpoB' gene sequence identities with Halorussus rarus TBN4T (90.8 % rpoB' gene sequence similarity). Phylogenetic trees based on 16S rRNA and rpoB' gene sequences revealed a robust lineage of the strain H22T with members of related genera of the family Halobacteriaceae. The DNA G+C content of strain H22T was 62.9 mol%. Genome-based analysis of average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) between strains H22T and its closest relative were equal or lower than 77.7 and 22.4 %, respectively, which were far below the threshold for delineation of a new species. Based on ANI values, in silico DDH, and distinct morphological and physiological differences from the previously described taxa, we suggest that strain H22T represents a novel species of a new genus within the family Halobacteriaceae, for which the name Halomicrococcus hydrotolerans gen. nov., sp. nov. is proposed. The type strain is H22T (=CGMCC 1.16291T=NBRC 113231T).

Halobellus captivus sp. nov., an extremely halophilic archaeon isolated from a subterranean salt mine.

An extremely halophilic archaeon, strain ZY21T, was isolated from a subterranean rock salt sample in Yunnan, China. Colonies of strain ZY21T on nutrient-rich agar plates are orange, wet and transparent. Cells are pleomorphic, motile, Gram-stain negative and lyse in distilled water. Cells can grow at 20-55 °C (optimum 42 °C), in the presence of 15-30% (w/v) NaCl (optimum 18-20%) and at pH 6.0-9.5 (optimum 7.5). Mg2+ is required for growth (optimum 0.3 M). The major polar lipids of strain ZY21T are phosphatidylglycerol, phosphatidylglycerol sulfate and phosphatidylglycerol phosphate methyl ester, sulfated mannosyl-glucosyl-glycerol diether-1 and seven unidentified glycolipids. Sequence similarity searches with the 16S rRNA gene and rpoB' gene showed that strain ZY21T is closely related to Halobellus rufus CBA1103T (sequence similarities: 97.5% for 16S rRNA gene and 93.3% for rpoB' gene). The DNA G+C content of strain ZY21T was determined to be 63.0 mol% based on the draft genome sequence. Genome-based sequence similarity analysis showed that the values of the ANI, AAI, and DDH were far below the boundary for delineation of new species. Phenotypic, chemotaxonomic characteristics and phylogenetic properties suggest that strain ZY21T represents a novel species in the genus Halobellus, for which the name Halobellus captivus sp. nov. is proposed. The type strain is ZY21T (= CGMCC 1.16343T = NBRC 113439T).