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Microorganisms play an essential role in sulfide removal. Alkaline absorption solution facilitates the sulfide’s dissolution and oxidative degradation, so haloalkaliphile is a prospective source for environmental-friendly and cost-effective biodesulfurization. In this research, 484 sulfide oxidation genes were identified from the metagenomes of the soda-saline lakes and a haloalkaliphilic heterotrophic bacterium Halomonas salifodinae IM328 (=CGMCC 22183) was isolated from the same habitat as the host for expression of a representative sequence. The genetic manipulation was successfully achieved through the conjugation transformation method, and sulfide: quinone oxidoreductase gene (sqr) was expressed via pBBR1MCS derivative plasmid. Furthermore, a whole-cell catalyst system was developed by using the engineered strain that exhibited a higher rate of sulfide oxidation under the optimal alkaline pH of 9.0. The whole-cell catalyst could be recycled six times to maintain the sulfide oxidation rates from 41.451 to 80.216 µmol·min−1·g−1 dry cell mass. To summarize, a whole-cell catalyst system based on the engineered haloalkaliphilic bacterium is potentiated to be applied in the sulfide treatment at a reduced cost.

Soda-saline lakes are polyextreme environments inhabited by many haloalkaliphiles, including one of the most abundant Spiribacter species. However, its mechanisms of adaptation are not ecophysiologically characterized. Based on a large-scale cultivation strategy, we obtained a representative isolate of this Spiribacter species whose relative abundance was the highest (up to 15.63%) in a wide range of salinities in the soda-saline lakes in Inner Mongolia, China. This species is a chemoorganoheterotrophic haloalkaliphile. It has a small and streamlined genome and utilizes a wide variety of compatible solutes to resist osmotic pressure and multiple monovalent cation/proton antiporters for pH homeostasis. In addition to growth enhancement by light under microaerobic conditions, cell growth, organic substrate consumption and polyhydroxybutyrate biosynthesis were also improved by inorganic sulfide. Both quantitative RT-PCR and enzymatic assays verified that sulfide:quinone oxidoreductase was upregulated during this process. Metatranscriptomic analysis indicated that all genes related to environmental adaptation were transcribed in natural environments. Overall, this study has identified a novel abundant haloalkaliphile with multiple and highly integrated adaptive strategies and found that inorganic sulfide was able to improve the adaptation of a heterotroph to polyextreme environments.

The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer cells to regulate anabolic metabolism. Although PKM2 was recently reported to regulate lipid homeostasis, the molecular mechanism remains unclear. Herein, we discovered an ER transmembrane protein 33 (TMEM33) as a downstream effector of PKM2 that regulates activation of SREBPs and lipid metabolism. Loss of PKM2 leads to up-regulation of TMEM33, which recruits RNF5, an E3 ligase, to promote SREBP-cleavage activating protein (SCAP) degradation. TMEM33 is transcriptionally regulated by nuclear factor erythroid 2-like 1 (NRF1), whose cleavage and activation are controlled by PKM2 levels. Total plasma cholesterol levels are elevated by either treatment with PKM2 tetramer-promoting agent TEPP-46 or by global PKM2 knockout in mice, highlighting the essential function of PKM2 in lipid metabolism. Although depletion of PKM2 decreases cancer cell growth, global PKM2 knockout accelerates allografted tumor growth. Together, our findings reveal the cell-autonomous and systemic effects of PKM2 in lipid homeostasis and carcinogenesis, as well as TMEM33 as a bona fide regulator of lipid metabolism.

Cold seeps are globally widespread seafloor ecosystems that feature abundant methane production and flourishing chemotrophic benthic communities. Chemical evidence indicates that cold seep methane is largely biogenic; however, the primary methane-producing organisms and associated pathways involved in methanogenesis remain elusive. This work detected methane production when glycine betaine (GBT) or trimethylamine (TMA) was added to the sediment microcosms of the Formosa cold seep, South China Sea. The methane production was suppressed by antibiotic inhibition of bacteria, while GBT was accumulated. This suggests that the widely used osmoprotectant GBT could be converted to cold seep biogenic methane via the synergistic activity of bacteria and methanogenic archaea because archaea are not sensitive to antibiotics and no bacteria are known to produce ample methane (mM). 16S rRNA gene diversity analyses revealed that the predominant bacterial and archaeal genera in the GBT-amended methanogenic microcosms included Oceanirhabdus and Methanococcoides. Moreover, metagenomic analyses detected the presence of grdH and mtgB genes that are involved in GBT reduction and demethylation, respectively. Two novel species were obtained, including bacterium Oceanirhabdus seepicola, which reduces GBT to TMA, and a methanogenic archaeon, Methanococcoides seepicolus, which produces methane from TMA and GBT. The two strains reconstituted coculture efficiently converted GBT to methane at 18°C; however, at 4°C addition of dimethylglycine (DMG), the GBT demethylation product, was necessary. Therefore, this work demonstrated that GBT is the precursor not only of the biogenic methane but also of the cryoprotectant DMG to the microorganisms at the Formosa cold seep. IMPORTANCE Numerous cold seeps have been found in global continental margins where methane is enriched in pore waters that are forced upward from sediments. Therefore, high concerns have been focused on the methane-producing organisms and the metabolic pathways in these environments because methane is a potent greenhouse gas. In this study, GBT was identified as the main precursor for methane in the Formosa cold seep of the South China Sea. Further, synergism of bacteria and methanogenic archaea was identified in GBT conversion to methane via the GBT reduction pathway, while methanogen-mediated GBT demethylation to methane was also observed. In addition, GBT-demethylated product dimethyl glycine acted as a cryoprotectant that promoted the cold seep microorganisms at cold temperatures. GBT is an osmoprotectant that is widely used by marine organisms, and therefore, the GBT-derived methanogenic pathway reported here could be widely distributed among global cold seep environments.

BackgroundArchaea were originally discovered in extreme environments, and thrive in many extreme habitats including soda lakes with high pH and salinity. Characteristic and diverse archaeal community played a significant role in biogeochemical cycles; however, the archaeal community and their functions are still less-studied in the intricate sediment of soda lakes. ResultsIn this article, the archaeal community of the deep sediment (40-50 cm depth) of five artificially-separated ponds with a salinity range from 7.0% to 33.0% in a soda saline lake was systematically surveyed using culture-independent metagenomics combined with the next-generation sequencing of the archaeal 16S rRNA amplicons. Nine archaeal phyla were detected, which accounted for 2.2% to 35.73% of microbial community in the five deep sediments. Besides the well-known class Halobacteria, one novel archaeal order (Candidatus Natranaeroarchaeales) of the class Thermoplasmata was even more abundant than Halobacteria in some deep sediment samples. Of 69 dereplicated archaeal metagenome-assembled genomes (MAGs), 30 MAGs belonged to Ca. Natranaeroarchaeales. Different genera of the Ca. Natranaeroarchaeales preferred to inhabit in the different salinities, and the divergent halophilic adaptation strategies (salt-out or salt-in) suggested the fast evolution adaptation within this lineage. Most high-quality MAGs had the genes of Wood-Ljungdahl pathway, organic acid fermentation and sulfur respiration, suggesting the putative functions in carbon fixation and sulfur reduction. Interestingly, heterodisulfide reductase and F420-non-reducing hydrogenase complex HdrABC-MvhADG were widely distributed in Ca. Natranaeroarchaeales and may play the core roles in energy metabolism from hydrogen. The regeneration of CoM-S-S-CoB was coupled to succinate or 2-oxoglutarate production in Ca. Natranaeroarchaeales instead of methanogenesis in the close related Methanomassiliicoccales. It suggested that methyl-coenzyme M reductase in Methanomassiliicoccales may be laterally transferred from other methanogens. ConclusionA novel archaeal order Ca. Natranaeroarchaeales of Thermoplasmata was found by culture-independent approaches. This order was the most abundant archaeal lineage in the deep sediment of soda lakes, with the characteristic environmental adaptation and biogeochemical potentials in carbon fixation and sulfur reduction. The difference in fermentation products coupled to energy metabolism between methanogens and Ca. Natranaeroarchaeales provided additional insights into the origination of methanogenesis in Thermoplasmata from the energy metabolism perspective.

A haloalkaliphilic strain XQ-INN 246T was isolated from the sediment of a salt pond in Inner Mongolia Autonomous Region, China. Cells of the strain were rods, motile and strictly aerobic. The strain was able to grow in the presence of 2.6–5.3 M NaCl (optimum concentration is 4.4 M) at 30–50 °C (optimum temperature is 42 °C) and pH 7.0–10.0 (optimum pH is 8.0–8.5). The whole genome sequencing of strain XQ-INN 246T revealed a genome size of 4.52 Mbp and a DNA G+C content of 62.06 mol%. Phylogenetic tree based on 16S rRNA gene sequences and concatenated amino acid sequences of 122 single-copy conserved proteins revealed a robust lineage of the strain XQ-INN 246T with members of related genera of the family Natrialbaceae . The strain possessed the polar lipids of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Based on phylogenetic analysis, phenotypic characteristics, chemotaxonomic properties and genome relatedness, the isolate was proposed as the type strain of a novel species of a new genus within the family Natrialbaceae, for which the name Salinadaptatus halalkaliphilus gen. nov., sp. nov. is proposed. The type strain is XQ-INN 246T (=CGMCC 1.16692T=JCM 33751T).

Small RNAs guard CRISPR-Cas The microbial adaptive immunity system CRISPR-Cas benefits microbes by warding off genetic invaders, but it also inflicts a fitness cost because of occasional autoimmune reactions, rendering CRISPR loci evolutionarily unstable. Li et al. identified previously unnoticed toxin-antitoxin RNA pairs embedded within diverse CRISPR-Cas loci. The antitoxin RNA mimics a CRISPR RNA and repurposes the CRISPR immunity effector to transcriptionally repress a toxin RNA that would otherwise arrest cell growth by sequestering a rare transfer RNA. These small RNAs thus form a symbiosis with CRISPR, rendering CRISPR addictive to the host despite its fitness cost. These findings reveal how CRISPR-Cas can operate as a selfish genetic element. Science , this issue p. eabe5601

Background: The chloride-carbonate-sulfate lakes (also called Soda-Saline lakes) are double-extreme ecological environments with high pH and high salinity values but can also exhibit high biodiversity and high productivity. The diversity of metabolic process that functioned well in such environments remains to be systematically investigated. Deep sequencing and species-level characterization of the microbiome in elemental cycling of carbon and sulfur will provide novel insights into the microbial adaptation in such saline-alkaline lakes. Results: In this study, we characterized environmental factors and performed deep metagenomic sequencing of the brine and sediment samples from nine ponds with different salinities of two chloride-carbonate-sulfate lakes in Inner Mongolia of China. The results showed that the concentration of chloride most significantly influenced the microbial community in both of the brine and sediment environments. Of 385 high-quality metagenome-assembled genomes (MAGs) belonging to archaea (56 Euryarchaeota, 12 Candidatus Nanohaloarchaeota, and 12 Candidatus Woesearchaeota) and bacteria (119 Proteobacteria and 186 other diverse bacterial phyla, including 18 from Candidate Phyla Radiation, CPR), 38 MAGs were observed to be abundant species at least in one of the eighteen niches. These abundant taxa represented most branches of a phylogenomic tree at phylum level. Interestingly, almost half of the abundant MAGs had the potential to drive dissimilatory sulfur cycling, including four autotrophic Ectothiorhodospiraceae MAGs, one Cyanobacteria MAG and nine heterotrophic MAGs with potential to oxidize sulfur, as well as four abundant MAGs containing genes for elemental sulfur respiration, which may increase the ability of environmental adaptation in such saline-alkaline environments. In addition, some subgroups in Ca. Nanohaloarchaeota and Ca. Woesearchaeota of the DPANN superphylum were also observed abundant. We found 1,4-alpha-glucans phosphorylation and complete glycolysis pathway in the abundant Nanohaloarchaeota MAG NHA-1, and this efficient energy regeneration pathway (compared with hydrolysis) may be a key factor for Ca. Nanohaloarchaeota in adaptation to hypersaline environments. Conclusions: The abundant taxa including carbon fixing microbes, versatile heterotrophs and the nanosized DPANN superphyla, and the superiority of energy production and thermodynamics of these abundant species were systematically investigated. This has provided novel insights into the microbial process in chloride-carbonate-sulfate lakes, and further understanding of the metabolic mechanism of adaptation to such extremely alkaline and saline conditions.

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication., Author summary Replication of positive-strand RNA viruses, which infect plants and animals, depends on many cellular resources. These viruses subvert cellular membranes and co-opt host proteins to build replication compartments that produce the viral progeny. The viral replication process also requires cellular metabolites and energy in the form of ATP. Using plant host as well as the model host yeast, the authors discovered that tomato bushy stunt tombusvirus (TBSV) co-opts the fermentation pathway to facilitate the local generation of ATP within the replication compartments. TBSV replication induces high expression of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes. With the help of the viral replication protein, TBSV recruits Pdc1 and Adh1 into the viral replication compartment. The authors propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses by facilitating rapid and efficient replication in the infected hosts. The authors also showed that the replication of five other plant viruses depends on the fermentation pathway in plants.

Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei. The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYK(Hm) [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPS(Hm) [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pyk(Hm) gene and pps(Hm) was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYK(Hm) catalyzed the conversion from PEP to pyruvate and that PPS(Hm) catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials. IMPORTANCE Archaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea. However, this process is still uncharacterized in the haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.

Growing evidence suggests that hepatic macrophages play an important role in tissue repair after liver injury by coordinating the induction and resolution of inflammation, removing apoptotic cells, and promoting hepatocyte proliferation. Understanding the role of macrophages in the pathogenesis of liver injury will help pave the way to future therapeutics. Here, we investigated whether macrophage p38α plays a regulatory role in the tissue repair following d-galactosamine (GalN)/tumor necrosis factor-α (TNF-α)-induced acute liver injury. We found that macrophage p38α-deficient mice displayed decreased mortality and relieved liver injury as evident from less apoptosis, accelerated regeneration, decreased granulocytes recruitment, monocytes infiltration, and cytokine production after GalN/TNF-α treatment. Mechanistically, we found that p38 signaling was activated by lipopolysaccharide/interferon-γ treatment but not by inteleukin-4 stimulation, while pharmaceutical inhibition of p38α induced a shift in polarization from M1 macrophages to M2 macrophages. Together, our results indicated that macrophage p38α signaling is involved in the pathogenesis of liver injury induced by GalN/TNF-α, and inhibition of p38α signaling in macrophage could ameliorate liver injury and accelerate regeneration, probably by promoting the polarization of macrophages from the M1 phenotype to the M2 phenotype.

Transcriptional dysregulation induced by disease-defining genetic alterations of proteins in transcriptional machinery is a key feature of cancers. Mediator complex subunit 12 (MED12) is the central architectural subunit in the kinase module of Mediator, a large transcriptional regulatory complex that controls essential steps of transcription. Emerging evidence links deregulated MED12 to human cancers. MED12 is frequently mutated in benign tumors and cancers. Although the missense mutations of MED12 in benign tumors disrupt the kinase activity of Mediator, MED12 mutations in cancers could eliminate the interaction between Mediator complex and RNA polymerase II, leading to severe transcriptional misregulation. Aberrant expression of MED12 is associated with the prognosis of various types of human cancers. Loss of MED12 function has been associated with the development of resistance to chemotherapeutics. Moreover, MED12 is modified by posttranscriptional regulations. Arginine methylation of MED12 has been shown to regulate MED12-mediated transcriptional regulation and response to chemotherapeutics in human cancer cell lines. In this mini-review, the authors provide an overview of the roles of MED12 in the development of benign and malignant tumors as well as its roles in chemoresistance. The studies of MED12 exemplify that aberrant transcriptional programming is a therapeutic vulnerability for certain types of cancer.

The metabolic regulation of cell death is sophisticated. A growing body of evidence suggests the existence of multiple metabolic checkpoints that dictate cell fate in response to metabolic fluctuations. However, whether microRNAs (miRNAs) are able to respond to metabolic stress, reset the threshold of cell death, and attempt to reestablish homeostasis is largely unknown. Here, we show that miR-378/378* KO mice cannot maintain normal muscle weight and have poor running performance, which is accompanied by impaired autophagy, accumulation of abnormal mitochondria, and excessive apoptosis in skeletal muscle, whereas miR-378 overexpression is able to enhance autophagy and repress apoptosis in skeletal muscle of mice. Our in vitro data show that metabolic stress-responsive miR-378 promotes autophagy and inhibits apoptosis in a cell-autonomous manner. Mechanistically, miR-378 promotes autophagy initiation through the mammalian target of rapamycin (mTOR)/unc-51-like autophagy activating kinase 1 (ULK1) pathway and sustains autophagy via Forkhead box class O (FoxO)-mediated transcriptional reinforcement by targeting phosphoinositide-dependent protein kinase 1 (PDK1). Meanwhile, miR-378 suppresses intrinsic apoptosis initiation directly through targeting an initiator caspase—Caspase 9. Thus, we propose that miR-378 is a critical component of metabolic checkpoints, which integrates metabolic information into an adaptive response to reduce the propensity of myocytes to undergo apoptosis by enhancing the autophagic process and blocking apoptotic initiation. Lastly, our data suggest that inflammation-induced down-regulation of miR-378 might contribute to the pathogenesis of muscle dystrophy.

// Xiuchao Wang 1, * , He Ren 1, * , Tiansuo Zhao 1 , Weidong Ma 1 , Jie Dong 1 , Shengjie Zhang 1 , Wen Xin 1 , Shengyu Yang 2 , Li Jia 1, 3 , Jihui Hao 1 1 Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Cancer Prevention and Therapy, National Clinical Research Center for Cancer, Tianjin 300060, China 2 Department of Tumor Biology and Comprehensive Melanoma Research Center, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA 3 Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, United Kingdom * These authors have contributed equally to this work Correspondence to: Jihui Hao, e-mail: haojihui@tjmuch.com Keywords: hypoxia-inducible factor-1 alpha (HIF-1α), microRNA-199a (miR-199a), pancreatic ductal adenocarcinoma (PDAC), single nucleotide polymorphism (SNP) Received: December 30, 2015 Accepted: January 29, 2016 Published: February 8, 2016 ABSTRACT Hypoxia-inducible factor-1 alpha (HIF-1α) is over-expressed in many cancers including pancreatic ductal adenocarcinoma (PDAC) and correlated with poor prognosis. We aim to determine the effect of germline genetic variants on the regulation of the homeostasis of the miRNA-gene regulatory loop in HIF1A gene and PDAC risk. HIF1A rs2057482 single nucleotide polymorphism (SNP) was genotyped in 410 PDAC cases and 490 healthy controls. The CC genotype SNP HIF1A is significantly correlated with PDAC risk (OR = 1.719, 95% CI: 1.293–2.286) and shorter overall survival (OS, P

Nipped-B-like protein plays a pivotal role as a cohesin loading factor in the segregation of chromosomes when cells divide. Accumulating evidence indicates that alterations of this protein are involved in human carcinogenesis, especially in the regulation of chemotherapeutic drug response. However, the role of Nipped-B-like protein in esophageal squamous cell carcinoma remains unknown. In this study, we investigated the relevance of Nipped-B-like protein in the regulation of cisplatin sensitivity in esophageal squamous cell carcinoma. Ectopic expression of Nipped-B-like protein inhibited the growth of COLO-680N cells with low endogenous expression levels of Nipped-B-like protein, and increased sensitivity to cisplatin, a commonly used chemotherapy drug for patients with esophageal squamous cell carcinoma. In contrast, loss of Nipped-B-like protein stimulated the growth of EC9706 and Eca-109 cells with high levels of the protein, and resulted in resistance to cisplatin. P53-upregulated modulator of apoptosis, which is essential in the modulation of cisplatin sensitivity in a variety of cancers, acts as a downstream effector of Nipped-B-like protein. Restoration of this pro-apoptotic protein in Nipped-B-like protein-overexpressing esophageal squamous cell carcinoma cells effectively increased cisplatin sensitivity. Conversely, the silencing of P53-upregulated modulator of apoptosis in Nipped-B-like protein-depleted esophageal squamous cell carcinoma rendered cells resistant to cisplatin. Moreover, Nipped-B-like protein could bind directly to the promoter region of P53-upregulated modulator of apoptosis. In summary, our study addresses the involvement of Nipped-B-like protein in the development of esophageal squamous cell carcinoma, and the modulation of cisplatin sensitivity via regulation of P53-upregulated modulator of apoptosis.

p38 has long been known as a central mediator of protein kinase A (PKA) signaling in brown adipocytes, which positively regulate the transcription of uncoupling protein 1 (UCP-1). However, the physiological role of p38 in adipose tissues, especially the white adipose tissue (WAT), is largely unknown. Here, we show that mice lacking p38α in adipose tissues display a lean phenotype, improved metabolism, and resistance to diet-induced obesity. Surprisingly, ablation of p38α causes minimal effects on brown adipose tissue (BAT) in adult mice, as evident from undetectable changes in UCP-1 expression, mitochondrial function, body temperature (BT), and energy expenditure. In contrast, genetic ablation of p38α in adipose tissues not only markedly facilitates the browning in WAT upon cold stress but also prevents diet-induced obesity. Consistently, pharmaceutical inhibition of p38α remarkably enhances the browning of WAT and has metabolic benefits. Furthermore, our data suggest that p38α deficiency promotes white-to-beige adipocyte reprogramming in a cell-autonomous manner. Mechanistically, inhibition of p38α stimulates the UCP-1 transcription through PKA and its downstream cAMP-response element binding protein (CREB), which form a positive feedback loop that functions to reinforce the white-to-beige phenotypic switch during cold exposure. Together, our study reveals that inhibition of p38α is able to promote WAT browning and confer metabolic benefits. Our study also indicates that p38α in WAT represents an exciting pharmacological target to combat obesity and metabolic diseases., Author summary The functional brown adipose tissue (BAT) identified in human adults consists of not only classic brown adipocytes but also brown-like adipocytes (beige adipocytes), both of which are important for energy homeostasis. Due to the same ability to convert fat into heat as brown adipocytes, beige adipocytes have been considered as a novel pharmacological target to combat obesity. Growing evidence suggests that promoting the development and formation of beige adipocytes in white adipose tissue (WAT), also called the browning of WAT, is able to prevent diet-induced obesity and improve metabolism in rodents. Thus, understanding the molecular basis for the regulation of browning in WAT may help us to develop new strategies to counteract obesity and metabolic diseases. In this study, adipocyte-specific p38α knockout (Fp38αKO) mice are generated that display a lean phenotype, improved metabolism, and resistance to diet-induced obesity. Interestingly, we found that adipocyte p38α deficiency facilitates the browning in WAT. Then, we show that pharmaceutical inhibition of p38α enhances the browning in WAT and has metabolic benefits. We propose that inhibiting p38α in WAT, possibly combined with cold exposure, could constitute an exciting pharmacological target to combat obesity and metabolic diseases.

FK228 is a selective inhibitor of histone deacetylases that exhibits marked antitumor activity in cancer cells and xenograft models. However, the effect of FK228 on the global profile of histone lysine acetylation and the proteome of EC109 cells remains poorly understood. The present study aimed at analyzing histone lysine acetylation and identifying the proteomic changes in EC109 cells following treatment with FK228, using the stable isotope labelling by amino acids in cell culture technique and a high-sensitivity mass spectrometer. In total, 87 acetylation sites and 3,515 proteins revealed changes in response to FK228 treatment. Of the 87 acetylation sites, 25 were quantifiable and 19 were quantified with ratio of >1.3. Notably, no downregulated lysine acetylation (Kac) sites were quantified in the present study and the 62 unquantified Kac sites were only identified in the FK228-treated cells. Bioinformatic analysis revealed that these quantifiable proteins were primarily involved in multiple biological functions and metabolic pathways as well as in protein complexes. The results of the present study revealed the extensive lysine acetylome and proteome in EC109 cells and expanded upon the current understanding of the anticancer mechanism of FK228 in EC109 cells.

Symptoms of cardiovascular diseases are frequently found in patients with hypothyroidism and hyperthyroidism. However, it is unknown whether arachidonic acid metabolites, the potent mediators in cardiovascular system, are involved in cardiovascular disorders caused by hyperthyroidism and hypothyroidism. To answer this question, serum levels of arachidonic acid metabolites in human subjects with hypothyroidism, hyperthyroidism and mice with hypothyroidism or thyroid hormone treatment were determined by a mass spectrometry-based method. Over ten arachidonic acid metabolites belonging to three catalytic pathways: cyclooxygenases, lipoxygenases, and cytochrome P450, were quantified simultaneously and displayed characteristic profiles under different thyroid hormone status. The level of 20-hydroxyeicosatetraenoic acid, a cytochrome P450 metabolite, was positively correlated with thyroid hormone level and possibly contributed to the elevated blood pressured in hyperthyroidism. The increased prostanoid (PG) I2 and decreased PGE2 levels in hypothyroid patients might serve to alleviate atherosclerosis associated with dyslipidemia. The elevated level of thromboxane (TX) A2, as indicated by TXB2, in hyperthyroid patients and mice treated with thyroid hormone might bring about pulmonary hypertension frequently found in hyperthyroid patients. In conclusion, our prospective study revealed that arachidonic acid metabolites were differentially affected by thyroid hormone status. Certain metabolites may be involved in cardiovascular disorders associated with thyroid diseases.

Skeletal muscle has a major role in locomotion and muscle disorders are associated with poor regenerative efficiency. Therefore, a deeper understanding of muscle regeneration is needed to provide a new insight for new therapies. CaMKK2 plays a role in the calcium/calmodulin-dependent kinase cascade; however, its role in skeletal muscle remains unknown. Here, we found that CaMKK2 expression levels were altered under physiological and pathological conditions including postnatal myogensis, freeze or cardiotoxin-induced muscle regeneration, and Duchenne muscular dystrophy. Overexpression of CaMKK2 suppressed C2C12 myoblast proliferation and differentiation, while inhibition of CaMKK2 had opposite effect. We also found that CaMKK2 is able to activate AMPK in C2C12 myocytes. Inhibition of AMPK could attenuate the effect of CaMKK2 overexpression, while AMPK agonist could abrogate the effect of CaMKK2 knockdown on C2C12 cell differentiation and proliferation. These results suggest that CaMKK2 functions as an AMPK kinase in muscle cells and AMPK mediates the effect of CaMKK2 on myoblast proliferation and differentiation. Our data also indicate that CaMKK2 might inhibit myoblast proliferation through AMPK-mediated cell cycle arrest by inducing cdc2-Tyr15 phosphorylation and repress differentiation through affecting PGC1α transcription. Lastly, we show that overexpressing CaMKK2 in the muscle of mice via electroporation impaired the muscle regeneration during freeze-induced injury, indicating that CaMKK2 could serve as a potential target to treat patients with muscle injury or myopathies. Together, our study reveals a new role for CaMKK2 as a negative regulator of myoblast differentiation and proliferation and sheds new light on the molecular regulation of muscle regeneration.

Ginsenoside Rb1, an active ingredient in Panax ginseng, was widely used for its various biological activities. To clarify the role of the gut microbiota in pharmacokinetics and metabolism of Rb1, a comprehensive and comparative study of colonic deglycosylation metabolism and systemic exposure of ginsenoside Rb1 in normal rats, antimicrobials (ATMs) treated rats, and restraint stressed rats was conducted. ATMs treated rats received oral administration of non-absorbable antimicrobial mixtures for 7 consecutive days. Restraint stressed rats were subjected to repeated restraint stress for a period of 2 h once daily for 7 days. Plasma concentration dynamics, urine and fecal excretion of Rb1 and its deglycosylation metabolites (Rd, F2, and C-K) were studied. Moreover, the in vitro metabolism of Rb1 in fecal suspension and the fecal β-d-glucosidase activity were profiled. Systemic exposure of the deglycosylation metabolites of ginsenoside Rb1 (F2, C-K) were significantly higher in restraint stressed rats, but ATMs treated rats exhibited a decreased plasma levels of F2 and C-K, compared with normal rats. Further studies illustrated that altered systemic Rb1 and its deglycosylation metabolites exposure in restraint-stressed rats and ATMs treated rats may be partially attributed to alternations in cumulative fecal excretion. The distinguishing fecal β-d-glucosidase, in vitro elimination of Rb1, and formation of these deglycosylation metabolites afforded further evidence for the in vivo data. In conclusion, the dys-regulated fecal β-d-glucosidase activity and deglycosylation metabolism may contribute to the altered pharmacokinetic of ginsenoside Rb1 and its hydrolysis metabolites after ATMs treatment or restraint stress exposure. Our results may offer valuable insights into the pharmacological changes of bioactive ginsenosides in dys-regulated gut microbiota statue.

SummaryUnderstanding the fiber-type specification and metabolic switch in skeletal muscle provides insights into energy metabolism in physiology and diseases. Here, we show that miR-182 is highly expressed in fast-twitch muscle and negatively correlates with blood glucose level. miR-182 knockout mice display muscle loss, fast-to-slow fiber-type switching, and impaired glucose metabolism. Mechanistic studies reveal that miR-182 modulates glucose utilization in muscle by targeting FoxO1 and PDK4, which control fuel selection via the pyruvate dehydrogenase complex (PDHC). Short-term high-fat diet (HFD) feeding reduces muscle miR-182 levels by tumor necrosis factor α (TNFα), which contributes to the upregulation of FoxO1/PDK4. Restoration of miR-182 expression in HFD-fed mice induces a faster muscle phenotype, decreases muscle FoxO1/PDK4 levels, and improves glucose metabolism. Together, our work establishes miR-182 as a critical regulator that confers robust and precise controls on fuel usage and glucose homeostasis. Our study suggests that a metabolic shift toward a faster and more glycolytic phenotype is beneficial for glucose control.

Activation of hypoxia-inducible factor 1α (HIF1α) controls the transcription of genes governing angiogenesis under hypoxic condition during tumorigenesis. Here we show that hypoxia-responsive miR-182 is regulated by HIF1α at transcriptional level. Prolyl hydroxylase domain enzymes (PHD) and factor inhibiting HIF-1 (FIH1), negative regulators of HIF1 signaling, are direct targets of miR-182. Overexpression of miR-182 in prostate cancer cells led to a reduction of PHD2 and FIH1 expression and an increase in HIF1α level either under normoxic or hypoxic condition. Consistently, inhibition of miR-182 could increase PHD2 and FIH1 levels, thereby reducing the hypoxia-induced HIF1α expression. Matrigel plug assay showed that angiogenesis was increased by miR-182 overexpression and vice versa. miR-182 overexpression in PC-3 prostate cancer xenografts decreased PHD2 and FIH1 expression, elevated HIF1α protein levels and increased tumor size. Lastly, we revealed that the levels of both miR-182 and HIF1α were elevated, while the expression PHD2 and FIH1 was downregulated in a mouse model of prostate cancer. Together, our results suggest that the interplay between miR-182 and HIF1α could result in a sustained activation of HIF1α pathway, which might facilitate tumor cell adaption to hypoxic stress during prostate tumor progression.

Background NIPBL, the sister chromatid cohesion 2 (SCC2) human homolog, is a cohesin loading factor which is essential for deposition of cohesin onto the sister chromatid. Recent studies have shown that NIPBL contribute to sister chromatid cohesion and plays a critical role in development, DNA repair, and gene regulation. In this study, we measured the expression of NIPBL in clinical non-small cell lung cancer specimens, and determined its effects on cellular processes and chemosensitivity in vitro. Methods NIPBL immunohistochemistry was performed on 123 lung adenocarcinoma samples. Through knockdown of NIPBL protein expression, non-small cell lung cancer cell lines were used to test the potential involvement of NIPBL silencing on cell proliferation, migration, invasion, and apoptosis. Chemosensitivity was assessed with clonogenic assays, and chromatin immunoprecipitation assays were performed to analyze the relationship between NIPBL and signal transducers and activators of transcription 3 (STAT3). Results Immunohistochemical analysis showed that high expression of NIPBL was strongly correlated with poor prognosis, tumor differentiation, and lymph node metastasis. Survival analysis further indicated that NIPBL expression was a potential prognostic factor for non-small cell lung cancer. Knockdown of NIPBL in non-small cell lung cancer cell lines significantly reduced cellular proliferation, migration, and invasion, and enhanced cellular apoptosis and sensitivity to cisplatin, paclitaxel, and gemcitabine hydrochloride. NIPBL bound to the promoter region of the STAT3 gene, directly regulating the expression of STAT3. Conclusions These data suggested that NIPBL played a significant role in lung carcinogenesis. NIPBL expression conferred poor prognosis and resistance to chemotherapy in non-small cell lung cancer, suggesting that NIPBL may be a novel therapeutic target.

This study aimed to investigate the interaction and regulatory mechanism of microRNA185 (miR185) and hypoxia-inducible factor-1 (HIF-1) in pancreatic cancer. The significance of miR185 on the clinicopathologic characteristics and prognosis was further explored. qRT-PCR and immunohistochemistry examined miR185 and HIF-1 expression of tumor tissues. Western blot analyzed HIF-1 protein expression. We regulated HIF-1 via transfection to observe the impact of HIF-1 on miR185 expression. ChIP sequencing and dual luciferase identified binding sites of HIF-1 and miR185. MiR185 expression was significantly higher in pancreatic tumors. MiR185 closely associated with tumor size, pTNM stage, lymph node, and perneural invasion. After hypoxic culture, both HIF-1 and miR185 expression of MiaPaCa2 and AsPc1 cells increased significantly. Up- or down-regulating HIF-1 expression via transfection leads to synchronous alteration of miR185. In ChIP sequencing, only the HRE2 (−938 bp) was significantly brighter under hypoxia among four hypoxia response elements’ (HREs) sequence of miR185 promoter. After pGL3-miR185 and HIF-1 over-expressing plasmids co-transfect the MiaPaCa2 cells, its relative expression of bioluminescence increased. MiR185 expression was significantly higher in tumor tissues and closely associated with the clinical features of pancreatic cancer. Expression of HIF-1 in pancreatic cancer cells increased in hypoxia. HIF-1 may bind to HRE2 of miR185 and initiate its transcription.

Potential target genes of microRNA (miR)-494 have been reported in many types of cancers. However, the role of miR-494 in esophageal squamous cell carcinoma (ESCC) remains unknown. This study focused on the expression and biological function of miR-494 in ESCC. Using bioinformatics analyses, we found that cleft lip and palate transmembrane 1-like (CLPTM1L) was a potential target of miR-494. We performed quantitative real-time (qRT) PCR assays in 37 ESCC tumor tissues to determine the expression of miR-494 and CLPTM1L mRNA, and we analyzed the correlation between both of these factors and clinical characteristics. The cell counting kit-8 and colony formation assays were used to evaluate the effects of miR-494 expression on the proliferation of ESCC cells. The transwell migration assay and flow cytometric apoptosis assay were performed to study the influence of miR-494 on the invasion and apoptosis of ESCC cells. Western blotting, luciferase assays, and CLPTM1L knockdown experiments were used to determine whether CLPTM1L was a target of miR-494. The qRT-PCR assays showed significant downregulation of miR-494 (P

Background & Aims It is proposed that p38 is involved in gluconeogenesis, however, the genetic evidence is lacking and precise mechanisms remain poorly understood. We sought to delineate the role of hepatic p38α in gluconeogenesis during fasting by applying a loss-of-function genetic approach. Methods We examined fasting glucose levels, performed pyruvate tolerance test, imaged G6Pase promoter activity, as well as determined the expression of gluconeogenic genes in mice with a targeted deletion of p38α in liver. Results were confirmed both in vivo and in vitro by using an adenoviral dominant-negative form of p38α (p38α-AF) and the constitutively active mitogen-activated protein kinase 6, respectively. Adenoviral dominant-negative form of AMP-activated protein kinase α (DN-AMPKα) was employed to test our proposed model. Results Mice lacking hepatic p38α exhibited reduced fasting glucose level and impaired gluconeogenesis. Interestingly, hepatic deficiency of p38α did not result in an alteration in CREB phosphorylation, but led to an increase in AMPKα phosphorylation. Adenoviral DN-AMPKα could abolish the effect of p38α-AF on gluconeogenesis. Knockdown of up-steam transforming growth factor β-activated kinase 1 decreased the AMPKα phosphorylation induced by p38α-AF, suggesting a negative feedback loop. Consistently, inverse correlations between p38 and AMPKα phosphorylation were observed during fasting and in diabetic mouse models. Importantly, adenoviral p38α-AF treatment ameliorated hyperglycemia in diabetic mice. Conclusions Our study provides evidence that hepatic p38α functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, dysregulation of this regulatory network contributes to unrestrained gluconeogenesis in diabetes, and hepatic p38α could be a drug target for hyperglycemia.

This study aimed to investigate the interaction and regulatory mechanism of microRNA185 (miR185) and hypoxia-inducible factor-1 (HIF-1) in pancreatic cancer. The significance of miR185 on the clinicopathologic characteristics and prognosis was further explored. qRT-PCR and immunohistochemistry examined miR185 and HIF-1 expression of tumor tissues. Western blot analyzed HIF-1 protein expression. We regulated HIF-1 via transfection to observe the impact of HIF-1 on miR185 expression. ChIP sequencing and dual luciferase identified binding sites of HIF-1 and miR185. MiR185 expression was significantly higher in pancreatic tumors. MiR185 closely associated with tumor size, pTNM stage, lymph node, and perneural invasion. After hypoxic culture, both HIF-1 and miR185 expression of MiaPaCa2 and AsPc1 cells increased significantly. Up- or down-regulating HIF-1 expression via transfection leads to synchronous alteration of miR185. In ChIP sequencing, only the HRE2 (-938 bp) was significantly brighter under hypoxia among four hypoxia response elements' (HREs) sequence of miR185 promoter. After pGL3-miR185 and HIF-1 over-expressing plasmids co-transfect the MiaPaCa2 cells, its relative expression of bioluminescence increased. MiR185 expression was significantly higher in tumor tissues and closely associated with the clinical features of pancreatic cancer. Expression of HIF-1 in pancreatic cancer cells increased in hypoxia. HIF-1 may bind to HRE2 of miR185 and initiate its transcription.

Semaphorin 4D (Sema4D/CD100) is a 150-kDa transmembrane glycoprotein expressed by platelets and T-cells. When these cells are activated, Sema4D is cleaved proteolytically, generating a biologically active 120-kDa fragment (soluble Sema4D) capable of targeting receptors on platelets, B-cells, endothelial cells and tumor cells. However, its plasma levels and significance in heart failure (HF) have not been reported. In this study, we established an ELISA and detected soluble Sema4D in human plasma. In healthy controls, plasma Sema4D levels were higher in men than women (5.15±3.30 ng/mL, n = 63, vs. 4.19±2.39 ng/mL, n = 63, P

During cancer development, tumor suppressor genes were silenced by promoter methylation or histone deacetylation. Histone deacetylases (HDACs) are important to maintain histone deacetylation. HDAC inhibitors (HDACis) were thus proposed as a new therapeutic approach to cancer. The current study aims to understand the effect and molecular mechanisms of HDACis on gastric cancer cells. Trichostatin A (TSA) significantly inhibited the growth of gastric cancer cells by inducing apoptosis. Gene profiling results showed PUMA (p53 upregulated modulator of apoptosis) as one of 122 genes upregulated in TSA-treated gastric cancer cells. PUMA was downregulated in gastric cancer cell lines and primary gastric carcinoma tissues. Patients with low PUMA expression had significant decreases in overall survival (HR, 2.04; p = 0.047). Ectopic PUMA expression inhibited the growth of gastric cancer cells while PUMA depletion promoted cellular growth. The knockdown of HDAC3 but not other HDACs upregulated PUMA expression. HDAC3 could bind to PUMA promoter, which was abrogated after TSA treatment. In contrast to TSA and SB, HDAC3 siRNA failed to upregulate p53 expression but promoted the interaction of p53 with PUMA promoter. In summary, proapoptotic PUMA was downregulated in gastric cancer and its mRNA expression level is a valuable prognosis factor for gastric cancer. HDAC3 is important to downregulate PUMA expression in gastric cancer and HDACis, like TSA, promoted PUMA expression through stabilizing p53 in addition to HDAC3 inhibition. In combination with chemotherapy, targeting HDAC3 might be a promising strategy to induce apoptosis of gastric cancer cells.

As a member of the GLI-Kruppel family of transcriptional factors, Yin Yang-1 (YY1) functions as an oncogene in various types of cancers. However, the role of YY1 in hepatocellular carcinogenesis remains unknown. In this report, we investigated the relevance of YY1 to hepatocellular carcinoma (HCC) development. We found that YY1 was upregulated in HCC cell lines. Ectopic YY1 expression promoted the growth of non-tumor liver cells that expressed low level of YY1. In contrast, YY1 depletion inhibited the growth of HCC cells which was accompanied with distinct morphological changes. Moreover, the phenotypic changes induced by YY1 depletion were attributed to cellular differentiation rather than cellular senescence. CCAAT/enhancer-binding protein alpha (CEBPA) which was important to regulate differentiation of hepatocytes was found as the direct target downregulated by YY1. Restoration of CEBPA in YY1-expressing HCC cells induced cellular differentiation and growth inhibition while knockdown of CEBPA expression in non-tumor liver cells promoted cell growth. In summary, our study demonstrated that YY1 could promote hepatocellular carcinogenesis and inhibit cellular differentiation through the downregulation of CEBPA expression.

ObjectivesWe aimed to investigate the prognostic value of RRM1 in GC patients.MethodsA total of assessable 389 GC patients with clinicopathological and survival information were enrolled from City of Hope (COH, n = 67) and Zhejiang University (ZJU, n = 322). RRM1 protein expression was determined by immunohistochemistry on FFPE tissue samples. Kaplan-Meier and Cox analyses were used to measure survival. Ras/Raf activity and invasion assays were used to evaluate the role of RRM1 in GC cell lines.ResultsIn vitro experiments demonstrated RRM1 activated Ras/Raf/MAPK signal transduction and promoted GC cell proliferation. Meanwhile, RRM1 expression was significantly associated with lymph node involvement, tumor size, Ki67 expression, histological subtype and histological grade in the GC tissue samples (pConclusionsRRM1 overexpression predicts poor survival in GC patients with advanced TNM stage. RRM1 could potentially serve as prognostic biomarker and therapeutic target for GCs.