CTCF binding site sequence differences are associated with unique regulatory and functional trends during embryonic stem cell differentiation

CTCF (CCCTC-binding factor) is a highly conserved multifunctional DNA-binding protein with thousands of binding sites genome-wide. Our previous work suggested that differences in CTCF’s binding site sequence may affect the regulation of CTCF recruitment and its function. To investigate this possibility, we characterized changes in genome-wide CTCF binding and gene expression during differentiation of mouse embryonic stem cells. After separating CTCF sites into three classes (LowOc, MedOc and HighOc) based on similarity to the consensus motif, we found that developmentally regulated CTCF binding occurs preferentially at LowOc sites, which have lower similarity to the consensus. By measuring the affinity of CTCF for selected sites, we show that sites lost during differentiation are enriched in motifs associated with weaker CTCF binding in vitro. Specifically, enrichment for T at the 18^th position of the CTCF binding site is associated with regulated binding in the LowOc class and can predictably reduce CTCF affinity for binding sites. Finally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions. Our results suggest that the regulatory control of CTCF is dependent in part on specific motifs within its binding site.     

Calling dynamics and call synchronization in a local group of unison bout callers

In many species of chorusing frogs, callers can rapidly adjust their call timing with reference to neighboring callers so as to maintain call rate while minimizing acoustic interference. The rules governing the interactions, in particular, who is listening to whom are largely unknown, presumably influenced by distance between callers, caller density, and intensities of interfering calls. We report vocal interactions in a unison bout caller, the green tree frog (Hyla cinerea). Using a microphone array, we monitored bouts from a local group of six callers embedded in a larger chorus. Data were analyzed in a 21-min segment at the peak of the chorus. Callers within this group were localized and their voices were separated for analysis of spatio-temporal interactions. We show that callers in this group: (1) synchronize with one another, (2) prefer to time their calls antiphonally, almost exactly at one-third and two-thirds of the call intervals of their neighbors, (3) tolerate call collision when antiphonal calling is not possible, and (4) perform discrete phase-hopping between three preferred phases when tracking other callers. Further, call collision increases and phase-locking decreases, with increasing inter-caller spacing. We conclude that the precise phase-positioning, phase-tracking, and phase-hopping minimizes acoustic jamming while maintaining chorus synchrony.     

Glutamine in the Pathogenesis of Hepatic Encephalopathy: The Trojan Horse Hypothesis Revisited

Hepatic encephalopathy (HE) is major neuropsychiatric disorder occurring in patients with severe liver disease and ammonia is generally considered to represent the major toxin responsible for this condition. Ammonia in brain is chiefly metabolized (“detoxified”) to glutamine in astrocytes due to predominant localization of glutamine synthetase in these cells. While glutamine has long been considered innocuous, a deleterious role more recently has been attributed to this amino acid. This article reviews the mechanisms by which glutamine contributes to the pathogenesis of HE, how glutamine is transported into mitochondria and subsequently hydrolyzed leading to high levels of ammonia, the latter triggering oxidative and nitrative stress, the mitochondrial permeability transition and mitochondrial injury, a sequence of events we have collectively termed as the Trojan horse hypothesis of hepatic encephalopathy.     

Sialylation of Outer Membrane Porin Protein D: A Mechanistic Basis of Antibiotic Uptake in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is an environmentally ubiquitous, extracellular, opportunistic pathogen, associated with severe infections of immune-compromised host. We demonstrated earlier the presence of both α2,3- and α2,6-linked sialic acids (Sias) on PA (PA^+Sias) and normal human serum is their source of Sias. PA^+Sias showed decreased complement deposition and exhibited enhanced association with immune-cells through sialic acid binding immunoglobulin like lectins (Siglecs). Such Sias-siglec-9 interaction between PA^+Sias and neutrophils helped to subvert host immunity. Additionally, PA^+Sias showed more resistant to β-lactam antibiotics as reflected in their minimum inhibitory concentration required to inhibit the growth of 50% than PA^−Sias. Accordingly, we have affinity purified sialoglycoproteins of PA^+Sias. They were electrophoresed and identified by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry analysis. Sequence study indicated the presence of a few α2,6-linked, α2,3-linked, and both α2,3- and α2,6-linked sialylated proteins in PA. The outer membrane porin protein D (OprD), a specialized channel-forming protein, responsible for uptake of β-lactam antibiotics, is one such identified sialoglycoprotein. Accordingly, sialylated (OprD^+Sias) and non-sialylated (OprD^−Sias) porin proteins were separately purified by using anion exchange chromatography. Sialylation of purified OprD^+Sias was confirmed by several analytical and biochemical procedures. Profiling of glycan structures revealed three sialylated N-glycans and two sialylated O-glycans in OprD^+Sias. In contrast, OprD^−Sias exhibit only one sialylated N-glycans. OprD^−Sias interacts with β-lactam antibiotics more than OprD^+Sias as demonstrated by surface plasmon resonance study. Lyposome-swelling assay further exhibited that antibiotics have more capability to penetrate through OprD^−Sias purified from four clinical isolates of PA. Taken together, it may be envisaged that sialic acids on OprD protein play important role toward the uptake of commonly used antibiotics in PA^+Sias. This might be one of the new mechanisms of PA for β-lactam antibiotic uptake.Sialic acids (Sias)¹ are nine carbon atom containing acidic residues characteristically found in the terminal position of glycoproteins and glycolipids (1–4). Structural diversity of sialic acids is because of the modification of one or more hydroxyl groups in various positions of the core structure by different groups like acetyl-, methyl-, sulfate-, lactyl-, or phosphate (1, 5–7). More than fifty derivatives of Sias has been reported both in vertebrate and invertebrate systems. It functions as ligand for various cellular communications and also act as masking element for glycoconjugates (8–12).Sialic acid binding immunoglobulins (Ig)-like lectins (siglecs) selectively expressed on the hematopoetic cells and interact with an array of linkage-specific Sias on a glycan structure express on the same cells or other cells (13). Siglecs can also recognize terminal sialylated glycoconjugates on several pathogens (14–16). After recognizing, they carry out various functions like internalization, attenuation of inflammation, restraining cellular activation along with inhibition of natural killer cell activation (17).Pseudomonas aeruginosa (PA) is a Gram-negative, rod-shaped bacterium. This human pathogen has remarkable capacity to cause diseases in immune compromised hosts. This colonizing microbial pathogen is responsible for infection in chronic cystic fibrosis, nosocomial infections; severe burn, transplantation, cancer, and AIDS and other immuno-supressed patients (18).We have reported earlier the presence of linkage-specific Sias on PA. Normal human serum (NHS) is possibly one of the sources of these Sias (19). PA utilizes these Sias to interact through siglecs present on the surface of different immune cells. PA^+Sias showed enhanced association with neutrophils through α2,3-linked Sias-siglec-9 interaction which facilitated their survival by subverting innate immune function of host (20).The treatment of PA-infected patient depends upon the extent of the disease and the concerned organs. Conventional β-lactam, cephalosporins, and aminoglycosides group of antibiotics are most common for such treatment (21). β-lactam antibiotics inhibit cell wall synthesis by disrupting the synthesis of the peptidoglycan layer of bacterial cell walls (22). When PA showed resistant to β-lactam antibiotics, new generation of β-lactam with increased doses or other broad spectrum antibiotics like tetracyclines or fluoroquinolones are prescribed (23). PA isolates from intensive care unit (ICU) patients in general showed higher rates of β-lactam resistance among other hospitalized patients (24). The increasing frequency of resistance to ceftazidime, piperacillin, imipenem, fluoroquinolone, and aminoglycoside were 36.6%, 22.3%, 22.8%, 23.8%, and 17.8% respectively in PA (25).The outer membrane of Gram-negative bacteria is, in general, semipermeable through which hydrophilic molecules including antibiotics of below exclusion limit size (0.6 kDa) can pass through the channel-forming proteins generally called porins e.g. OprD, OprF, OprG etc. (26, 27). PA shows lower outer membrane permeability with respect to many other Gram-negative bacteria like Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia cepacia, hence the diffusion rate of β-lactam antibiotics is decreased (27).Additionally, PA uses MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM as efflux pumps along with important regulatory factors MexR/NalB, NfxB, NfxC/MexT, and MexZ respectively on their membrane to pump out undesirable chemicals, detergent and antibiotics (28–32). Other Gram-negative bacteria also uses similar types of efflux pumps for such purposes. Moreover, PA produces antibiotic-resistance genes by some mutation (33). Furthermore, β-lactamase and aminoglycoside-modifying enzymes produced by PA are capable of breaking down the antibiotics (34). Alternatively, these enzymes can directly modify the drug. Hence these antibiotics become functionally ineffective (27).The presence of lipopolysaccharides (LPS) containing O-specific polysaccharides with tri-saccharide repeats of 2-acetamido-2,6-dideoxy-d-glucose, 2-acetamido-2,6-dideoxy-d-galactose, and 5-acetamido-3,5,7,9-tetyradeoxy-7-[(R)-3-hydroxybutyramidol]-3-l-glycerol-l-manno-nonulosonic acid are known for PA serogroup O11 (35). The genes for key enzymes required for complex protein glycosylation are found in the genome of PA14 (36). Moreover, glycosylation in PA1244 has been reported in the form of an O-linked glycan in pilin (37). A cluster of seven genes known as the pel genes, encode proteins with similarity to components involved in polysaccharide biogenesis. Among these genes, PelF is a putative glycosyltransferase (GT) of the type IV glycosyltransferase (GT4) family (36). PA secreted sialidase in culture medium (38). Genome search reveals that PA14 has the sialidase gene, which may be responsible for cleaving sialic acids (39). PA1 also has sialic acid transporter gene, which possibly transport sialic acids inside the cells (Gene ID: 17688338, Source: http://www.ncbi.nlm.nih.gov/gene/17688338). Additionally, CMP-sialic acid transferase, which is responsible for converting sialic acids to CMP-sialic acid, was purified from PAO12 (40). This enzyme shows close similarity with the enzyme found in E. coli.However, PA being such a notorious organism, it might have many other different mechanisms to fight against antibiotics for their survival. Therefore, it is worthwhile to explore newer mechanism to understand how antibiotics penetrate inside this bacterium. Here we addressed the following questions. Does sialylation of glycoproteins demonstrated on PA play any role in the entry of antibiotics that might facilitate their survival within host?Accordingly, we have affinity purified a few sialoglycoproteins from PA. Sequence analysis identified twenty six α2,3- and α2,6-linked sialoglycoproteins. One such identified sialoglycoprotein is OprD porin protein. The presence of Sias on OprD was conclusively confirmed. We have demonstrated that Sias on OprD protein isolated four different clinical isolates hampered its interaction with β-lactam antibiotics. This might be one of the new mechanisms for β-lactam antibiotic resistance of PA and thereby facilitates their survival in host.     

Predetermined Flake Production at the Lower/Middle Paleolithic Boundary: Yabrudian Scraper-Blank Technology

While predetermined débitage technologies are recognized beginning with the middle Acheulian, the Middle Paleolithic is usually associated with a sharp increase in their use. A study of scraper-blank technology from three Yabrudian assemblages retrieved from the early part of the Acheulo-Yabrudian complex of Tabun Cave (ca. 415–320 kyr) demonstrates a calculated and preplanned production, even if it does not show the same complexity and elaboration as in the Levallois technology. These scraper dominated assemblages show an organization of production based on an intensive use of predetermination blank technology already in place at the end of the Lower Paleolithic of the Levant. These results provide a novel perspective on the differences and similarities between the Lower and Middle Paleolithic industries. We suggest that there was a change in the paradigm in the way hominins exploited stone tools: in many Middle Paleolithic assemblages the potential of the stone tools for hafting was a central feature, in the Lower Paleolithic ergonometric considerations of manual prehension were central to the design of blanks and tools.     

No Correlation between TIMP2 -418 G>C Polymorphism and Increased Risk of Cancer: Evidence from a Meta-Analysis

Aim

Tissue inhibitor of metalloproteinase (TIMP2) is involved in the regulation of matrix metalloproteinase 2 (MMP2) and shown to implicate in cancer development and progression. The results from the published studies based on the association between TIMP2 -418 G>C polymorphism and cancer risk are inconsistent. In this meta-analysis, we aimed to evaluate the potential association between TIMP2 -418 G>C polymorphism and cancer risk.

Methodology

We searched PubMed (Medline) and EMBASE web databases to cover all studies based on relationship of TIMP2 -418 G>C polymorphism and risk of cancer until October 2013. The meta-analysis was performed for selected case-control studies and pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for all genetic models.

Results

A total of 2225 cancer cases and 2532 controls were included from ten eligible case-control studies. Results from overall pooled analysis suggested no evidence of significant risk between TIMP2 -418 G>C polymorphism and cancer risk in any of the genetic models, such as, allele (C vs. G: OR = 1.293, 95% CI = 0.882 to 1.894, p = 0.188), homozygous (CC vs. GG: OR = 0.940, 95% CI = 0.434 to 2.039, p = 0.876), heterozygous (GC vs. GG: OR = 1.397, 95% CI = 0.888 to 2.198, p = 0.148), dominant (CC+GC vs. GG: OR = 1.387, 95% CI = 0.880 to 2.187, p = 0.159) and recessive (CC vs. GG+GC: OR = 0.901, 95% CI = 0.442 to 1.838, p = 0.774) models. No evidence of publication bias was detected during the analysis.

Conclusions

The present meta-analysis suggests that the TIMP2 -418 G>C polymorphism may not be involved in predisposing risk factor for cancer in overall population. However, future larger studies with group of populations are needed to analyze the possible correlation.     

Down-regulated Peroxisome Proliferator-activated Receptor γ (PPARγ) in Lung Epithelial Cells Promotes a PPARγ Agonist-reversible Proinflammatory Phenotype in Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition and a leading cause of death, with no available cure. We assessed the actions in pulmonary epithelial cells of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor with anti-inflammatory effects, whose role in COPD is largely unknown. We found that PPARγ was down-regulated in lung tissue and epithelial cells of COPD patients, via both reduced expression and phosphorylation-mediated inhibition, whereas pro-inflammatory nuclear factor-κB (NF-κB) activity was increased. Cigarette smoking is the main risk factor for COPD, and exposing airway epithelial cells to cigarette smoke extract (CSE) likewise down-regulated PPARγ and activated NF-κB. CSE also down-regulated and post-translationally inhibited the glucocorticoid receptor (GR-α) and histone deacetylase 2 (HDAC2), a corepressor important for glucocorticoid action and whose down-regulation is thought to cause glucocorticoid insensitivity in COPD. Treating epithelial cells with synthetic (rosiglitazone) or endogenous (10-nitro-oleic acid) PPARγ agonists strongly up-regulated PPARγ expression and activity, suppressed CSE-induced production and secretion of inflammatory cytokines, and reversed its activation of NF-κB by inhibiting the IκB kinase pathway and by promoting direct inhibitory binding of PPARγ to NF-κB. In contrast, PPARγ knockdown via siRNA augmented CSE-induced chemokine release and decreases in HDAC activity, suggesting a potential anti-inflammatory role of endogenous PPARγ. The results imply that down-regulation of pulmonary epithelial PPARγ by cigarette smoke promotes inflammatory pathways and diminishes glucocorticoid responsiveness, thereby contributing to COPD pathogenesis, and further suggest that PPARγ agonists may be useful for COPD treatment.     

Decreased astrocytic thrombospondin‐1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin‐1 (TSP‐1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5–2.5 mM) for 1–10 days, and TSP‐1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra‐ and extracellular TSP‐1 levels. Exposure of cultured neurons to conditioned media from ammonia‐treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP‐1 over‐expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP‐1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP‐1 synthesis in other cell types, also reversed the ammonia‐induced TSP‐1 reduction. Likewise, we found a significant decline in TSP‐1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP‐1 may represent an important therapeutic target for CHE.