High Mobility Group Box-1 (HMGB1) Participates in the Pathogenesis of Alcoholic Liver Disease (ALD)

Growing clinical and experimental evidence suggests that sterile inflammation contributes to alcoholic liver disease (ALD). High mobility group box-1 (HMGB1) is highly induced during liver injury; however, a link between this alarmin and ALD has not been established. Thus, the aim of this work was to determine whether HMGB1 contributes to the pathogenesis of ALD. Liver biopsies from patients with ALD showed a robust increase in HMGB1 expression and translocation, which correlated with disease stage, compared with healthy explants. Similar findings were observed in chronic ethanol-fed wild-type (WT) mice. Using primary cell culture, we validated the ability of hepatocytes from ethanol-fed mice to secrete a large amount of HMGB1. Secretion was time- and dose-dependent and responsive to prooxidants and antioxidants. Selective ablation of Hmgb1 in hepatocytes protected mice from alcohol-induced liver injury due to increased carnitine palmitoyltransferase-1, phosphorylated 5′AMP-activated protein kinase-α, and phosphorylated peroxisome proliferator-activated receptor-α expression along with elevated LDL plus VLDL export. Native and post-translationally modified HMGB1 were detected in humans and mice with ALD. In liver and serum from control mice and in serum from healthy volunteers, the lysine residues within the peptides containing nuclear localization signals (NLSs) 1 and 2 were non-acetylated, and all cysteine residues were reduced. However, in livers from ethanol-fed mice, in addition to all thiol/non-acetylated isoforms of HMGB1, we observed acetylated NLS1 and NLS2, a unique phosphorylation site in serine 35, and an increase in oxidation of HMGB1 to the disulfide isoform. In serum from ethanol-fed mice and from patients with ALD, there was disulfide-bonded hyperacetylated HMGB1, disulfide-bonded non-acetylated HMGB1, and HMGB1 phosphorylated in serine 35. Hepatocytes appeared to be a major source of these HMGB1 isoforms. Thus, hepatocyte HMGB1 participates in the pathogenesis of ALD and undergoes post-translational modifications (PTMs) that could condition its toxic effects.     

Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway: ROLE OF PALMITIC ACID*

Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt.     

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase–related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.     

Indoor-Air Microbiome in an Urban Subway Network: Diversity and Dynamics

Subway systems are indispensable for urban societies, but microbiological characteristics of subway aerosols are relatively unknown. Previous studies investigating microbial compositions in subways employed methodologies that underestimated the diversity of microbial exposure for commuters, with little focus on factors governing subway air microbiology, which may have public health implications. Here, a culture-independent approach unraveling the bacterial diversity within the urban subway network in Hong Kong is presented. Aerosol samples from multiple subway lines and outdoor locations were collected. Targeting the 16S rRNA gene V4 region, extensive taxonomic diversity was found, with the most common bacterial genera in the subway environment among those associated with skin. Overall, subway lines harbored different phylogenetic communities based on α- and β-diversity comparisons, and closer inspection suggests that each community within a line is dependent on architectural characteristics, nearby outdoor microbiomes, and connectedness with other lines. Microbial diversities and assemblages also varied depending on the day sampled, as well as the time of day, and changes in microbial communities between peak and nonpeak commuting hours were attributed largely to increases in skin-associated genera in peak samples. Microbial diversities within the subway were influenced by temperature and relative humidity, while carbon dioxide levels showed a positive correlation with abundances of commuter-associated genera. This Hong Kong data set and communities from previous studies conducted in the United States formed distinct community clusters, indicating that additional work is required to unravel the mechanisms that shape subway microbiomes around the globe.     

Obstructive sleep apnea and the prevalence and incidence of cancer

Background:

A link between obstructive sleep apnea and cancer development or progression has been suggested, possibly through chronic hypoxemia, but supporting evidence is limited. We examined the association between the severity of obstructive sleep apnea and prevalent and incident cancer, controlling for known risk factors for cancer development.

Methods:

We included all adults referred with possible obstructive sleep apnea who underwent a first diagnostic sleep study at a single large academic hospital between 1994 and 2010. We linked patient data with data from Ontario health administrative databases from 1991 to 2013. Cancer diagnosis was derived from the Ontario Cancer Registry. We assessed the cross-sectional association between obstructive sleep apnea and prevalent cancer at the time of the sleep study (baseline) using logistic regression analysis. Cox regression models were used to investigate the association between obstructive sleep apnea and incident cancer among patients free of cancer at baseline.

Results:

Of 10 149 patients who underwent a sleep study, 520 (5.1%) had a cancer diagnosis at baseline. Over a median follow-up of 7.8 years, 627 (6.5%) of the 9629 patients who were free of cancer at baseline had incident cancer. In multivariable regression models, the severity of sleep apnea was not significantly associated with either prevalent or incident cancer after adjustment for age, sex, body mass index and smoking status at baseline (apnea–hypopnea index > 30 v. < 5: adjusted odds ratio [OR] 0.96, 95% confidence interval [CI] 0.71–1.30, for prevalent cancer, and adjusted hazard ratio [HR] 1.02, 95% CI 0.80–1.31, for incident cancer; sleep time spent with oxygen saturation < 90%, per 10-minute increase: adjusted OR 1.01, 95% CI 1.00–1.03, for prevalent cancer, and adjusted HR 1.00, 95% CI 0.99–1.02, for incident cancer).

Interpretation:

In a large cohort, the severity of obstructive sleep apnea was not independently associated with either prevalent or incident cancer. Additional studies are needed to elucidate whether there is an independent association with specific types of cancer.Obstructive sleep apnea is a sleep-related breathing disorder characterized by repetitive episodes of upper-airway obstruction during sleep. Through sleep fragmentation, hypoxemia, hypercapnia, swings in intrathoracic pressure and increased sympathetic activity, these episodes lead to symptoms and health consequences.¹ In 2009, 23% of Canadian adults reported risk factors for obstructive sleep apnea, and 5% of the population 45 years and older reported being told by a health professional that they had the condition.²Obstructive sleep apnea has been postulated to cause cancer^3,4 or cancer progression,⁵ possibly through chronic intermittent hypoxemia,⁶ thus making it a potential modifiable risk factor for cancer development.⁷ However, the longitudinal evidence on this association is limited. Four cohort studies evaluated the longitudinal association between obstructive sleep apnea (expressed by the apnea–hypopnea index, oxygen desaturation or symptoms) and cancer development or cancer-related mortality (Appendix 1, available at www.cmaj.ca/lookup/suppl/doi:10.1503/cmaj.140238/-/DC1).^3–5,8 All had limitations. Of the 3 that reported a positive association,^3,5,8 2 studies included a small number of participants with severe obstructive sleep apnea, had a relatively small number of events and did not consider competing risk of death from other causes;^5,8 and 2 used less reliable sleep-testing devices to define obstructive sleep apnea,^3,8 which may have introduced measurement bias. In the only study that did not show an association between obstructive sleep apnea and cancer,⁴ the former was diagnosed on the basis of self-reported symptoms, which could have resulted in misclassification of exposure.There is a need for a sufficiently large cohort study with a long enough follow-up to allow for the potential development of cancer that adjusts for important potential confounders, examines common cancer subtypes and has a rigorous assessment of both obstructive sleep apnea and cancer.^7,9,10 Our study was designed to improve upon the methods of published studies. We examined the association between the severity of obstructive sleep apnea (expressed by the apnea–hypopnea index or oxygen desaturation) and prevalent or incident cancer after controlling for known cancer risk factors.     

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA^Leu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA^Leu (CAG)—a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.     

Substituting random forest for multiple linear regression improves binding affinity prediction of scoring functions: Cyscore as a case study

Background

State-of-the-art protein-ligand docking methods are generally limited by the traditionally low accuracy of their scoring functions, which are used to predict binding affinity and thus vital for discriminating between active and inactive compounds. Despite intensive research over the years, classical scoring functions have reached a plateau in their predictive performance. These assume a predetermined additive functional form for some sophisticated numerical features, and use standard multivariate linear regression (MLR) on experimental data to derive the coefficients.

Results

In this study we show that such a simple functional form is detrimental for the prediction performance of a scoring function, and replacing linear regression by machine learning techniques like random forest (RF) can improve prediction performance. We investigate the conditions of applying RF under various contexts and find that given sufficient training samples RF manages to comprehensively capture the non-linearity between structural features and measured binding affinities. Incorporating more structural features and training with more samples can both boost RF performance. In addition, we analyze the importance of structural features to binding affinity prediction using the RF variable importance tool. Lastly, we use Cyscore, a top performing empirical scoring function, as a baseline for comparison study.

Conclusions

Machine-learning scoring functions are fundamentally different from classical scoring functions because the former circumvents the fixed functional form relating structural features with binding affinities. RF, but not MLR, can effectively exploit more structural features and more training samples, leading to higher prediction performance. The future availability of more X-ray crystal structures will further widen the performance gap between RF-based and MLR-based scoring functions. This further stresses the importance of substituting RF for MLR in scoring function development.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-291) contains supplementary material, which is available to authorized users.     

Systematic transcriptome analysis of the zebrafish model of diamond-blackfan anemia induced by RPS24 deficiency

Background

Diamond–Blackfan anemia (DBA) is a class of human diseases linked to defective ribosome biogenesis that results in clinical phenotypes. Genetic mutations in ribosome protein (RP) genes lead to DBA phenotypes, including hematopoietic defects and physical deformities. However, little is known about the global regulatory network as well as key miRNAs and gene pathways in the zebrafish model of DBA.

Results

In this study, we establish the DBA model in zebrafish using an RPS24 morpholino and found that RPS24 is required for both primitive hematopoiesis and definitive hematopoiesis processes that are partially mediated by the p53 pathway. Several deregulated genes and miRNAs were found to be related to hematopoiesis, vascular development and apoptosis in RPS24-deficient zebrafish via RNA-seq and miRNA-seq data analysis, and a comprehensive regulatory network was first constructed to identify the mechanisms of key miRNAs and gene pathways in the model. Interestingly, we found that the central node genes in the network were almost all targeted by significantly deregulated miRNAs. Furthermore, the enforced expression of miR-142-3p, a uniquely expressed miRNA, causes a significant decrease in primitive erythrocyte progenitor cells and HSCs.

Conclusions

The present analyses demonstrate that the comprehensive regulatory network we constructed is useful for the functional prediction of new and important miRNAs in DBA and will provide insights into the pathogenesis of mutant rps24-mediated human DBA disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-759) contains supplementary material, which is available to authorized users.     

The Ancient Small GTPase Rab21 Functions in Intermediate Endocytic Steps in Trypanosomes

Endocytosis is an essential process in nearly all eukaryotic cells, including the African trypanosome Trypanosoma brucei. Endocytosis in these organisms is exclusively clathrin mediated, although several lineage-specific features indicate that precise mechanisms are distinct from those of higher eukaryotes. T. brucei Rab21 is a member of an ancient, pan-eukaryotic, endocytic Rab clade that is retained by trypanosomes. We show that T. brucei Rab21 (TbRab21) localizes to endosomes, partially colocalizing with TbRab5A, TbRab28, and TbVps23, the latter two being present at late endosomes. TbRab21 expression is essential for cellular proliferation, and its suppression results in a partial block in traffic to the lysosome. RNA interference (RNAi)-mediated knockdown of TbRab21 had no effect on TbRab5A expression or location but did result in decreased in trans expression of ESCRT (trypanosome endosomal sorting complex required for transport) components and TbRab28, while knockdown of ESCRT subunit TbVps23 resulted in decreased TbRab21 expression. These data suggest that TbRab21 acts downstream of TbRab5A and functions in intimate connection with the trypanosome ESCRT system.