Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis

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First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73–82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype–phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.Mammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia (OMIM 261600) (1). PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2, 3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5, 6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric conformer (3).There are >500 disease-associated missense variants of human PAH; the amino acid substitutions are distributed throughout the 452-residue protein and among all its domains (Fig. 1A) (7–9). Of those disease-associated variants that have been studied in vitro (e.g., ref. 10), some confound the allosteric response, and some are interpreted as structurally unstable. We also suggest that the activities of some disease-associated variants may be dysregulated by an altered equilibrium among conformers having different intrinsic levels of activity, arguing by analogy to the enzyme porphobilinogen synthase (PBGS) and its porphyria-associated variants (11). Consistent with this notion, we have recently established that PAH can assemble into architecturally distinct tetrameric conformers (12), and propose that these conformers differ in activity due to differences in active-site access. This idea has important implications for drug discovery, as it implies that small molecules could potentially modulate the conformational equilibrium of PAH, as has already been demonstrated for PBGS (e.g., ref. 13). Deciphering the relationship among PAH structure, dynamics, and function is a necessary first step in testing this hypothesis.Open in a separate windowFig. 1.The structure of PAH. (A) The annotated domain structure of mammalian PAH. (B) The 2.9 Å PAH crystal structure in orthogonal views, colored as in part A, subunit A is shown in ribbons; subunit B is as a Cα trace; subunit C is in sticks; and subunit D is in transparent spheres. In cyan, the subunits are labeled near the catalytic domain (Top); in red, they are labeled near the regulatory domain (Bottom). The dotted black circle illustrates the autoregulatory domain partially occluding the enzyme active site (iron, in orange sphere). (C) Comparison of the subunit structures of full-length PAH and those of the composite homology model; the subunit overlay aligns residues 144–410. The four subunits of the full-length PAH structure (the diagonal pairs of subunits are illustrated using either black or white) are aligned with the two subunits of 2PAH (cyan) and the one subunit of 1PHZ (orange). The catalytic domain is in spheres, the regulatory domain is in ribbons, and the multimerization domain is as a Cα trace. The arrow denotes where the ACT domain and one helix of 2PAH conflict.Numerous crystal structures are known for one- and two-domain constructs of mammalian PAH (14).

Table S1.

Mammalian PAH structures available in the PDB (August 2015)

Cingulo-opercular control network and disused motor circuits joined in standby mode

Whole-brain resting-state functional MRI (rs-fMRI) during 2 wk of upper-limb casting revealed that disused motor regions became more strongly connected to the cingulo-opercular network (CON), an executive control network that includes regions of the dorsal anterior cingulate cortex (dACC) and insula. Disuse-driven increases in functional connectivity (FC) were specific to the CON and somatomotor networks and did not involve any other networks, such as the salience, frontoparietal, or default mode networks. Censoring and modeling analyses showed that FC increases during casting were mediated by large, spontaneous activity pulses that appeared in the disused motor regions and CON control regions. During limb constraint, disused motor circuits appear to enter a standby mode characterized by spontaneous activity pulses and strengthened connectivity to CON executive control regions.

Disuse is a powerful paradigm for inducing plasticity that has uncovered key organizing principles of the human brain (1–4). Monocular deprivation—prolonged covering of one eye—revealed that multiple afferent inputs can compete for representational territory in the primary visual cortex (1). Similar competition between afferents also shapes the somatomotor system. Manipulations such as peripheral nerve deafferentation, whisker trimming, and limb constraint all drive plasticity in the primary somatosensory and motor cortex (2–4). Most plasticity studies to date have used focal techniques, such as microelectrode recordings, to study local changes in brain function. As a result, little is known about how behavior and experience shape the brain-wide functional networks that support complex cognitive operations (5).The brain is composed of networks of regions that cooperate to perform specific cognitive functions (5–8). These functional networks show synchronized spontaneous activity while the brain is at rest, a phenomenon known as resting-state functional connectivity (FC) (9–11). FC can be measured noninvasively in humans using resting-state functional MRI (rs-fMRI) and has been used to parse the brain into canonical functional networks (12, 13), including visual, auditory, and somatomotor networks (14, 15); ventral and dorsal attention networks (8, 16); a default mode network with roles in internally directed cognition and episodic memory (7, 11); a salience network thought to assess the homeostatic relevance of external stimuli (17); a frontoparietal control network supporting error processing and moment-to-moment adjustments in behavior (18–20); and a cingulo-opercular control network (CON), which maintains executive control during goal-directed behavior (18, 19, 21). Each functional network likely carries out a variety of additional functions.A more recent advance in human neuroscience has been the recognition of individual variability in network organization (22–25). Most early rs-fMRI studies examined central tendencies in network organization using group-averaged FC measurements (10, 12, 13). Recent work has demonstrated that functional networks can be identified in an individual-specific manner if sufficient rs-fMRI data are acquired, an approach termed precision functional mapping (PFM) (22, 23, 26–30). PFM respects the unique functional anatomy of each person and avoids averaging together functionally distinct brain regions across individuals.We recently demonstrated that PFM can be used to follow the time course of disuse-driven plasticity in the human brain (31). Three adult participants (Nico, Ashley, and Omar) were scanned at the same time of day for 42 to 64 consecutive days (30 min of rs-fMRI per day) before, during, and after 2 wk of dominant upper-extremity casting (Fig. 1 A and B). Casting caused persistent disuse of the dominant upper extremity during daily behaviors and led to a marked loss of strength and fine motor skill in all participants. During casting, the upper-extremity regions of the left primary somatomotor cortex (L-SM1_ue) and right cerebellum (R-Cblm_ue) functionally disconnected from the remainder of the somatomotor network. Disused motor circuits also exhibited large, spontaneous pulses of activity (Fig. 1C). Disuse pulses did not occur prior to casting, started to occur frequently within 1 to 2 d of casting, and quickly waned after cast removal.Open in a separate windowFig. 1.Experimental design and spontaneous activity pulses. (A) Three participants (Nico, Ashley, and Omar) wore casts covering the entire dominant upper extremity for 2 wk. (B) Participants were scanned every day for 42 to 64 consecutive days before, during, and after casting. All scans included 30 min of resting-state functional MRI. (C) During the Cast period, disused somatomotor circuits exhibited large pulses of spontaneous activity. (C, Left) Whole-brain ANOVA showing which brain regions contained disuse-driven pulses. (C, Right) Time courses of all pulses recorded from the disused primary somatomotor cortex.Somatomotor circuits do not function in isolation. Action selection and motor control are thought to be governed by complex interactions between the somatomotor network and control networks, including the CON (18). Prior studies of disuse-driven plasticity, including our own, have focused solely on somatomotor circuits. Here, we leveraged the whole-brain coverage of rs-fMRI and the statistical power of PFM to examine disuse-driven plasticity throughout the human brain.     

In Vitro Activity of Daptomycin in Combination with β-Lactams,Gentamicin, Rifampin,and Tigecycline against Daptomycin-Nonsusceptible Enterococci

Enterococci that are nonsusceptible (NS; MIC > 4 μg/ml) to daptomycin are an emerging clinical concern. The synergistic combination of daptomycin plus beta-lactams has been shown to be effective against vancomycin-resistant Enterococcus (VRE) species in vitro. This study systematically evaluated by in vitro time-kill studies the effect of daptomycin in combination with ampicillin, cefazolin, ceftriaxone, ceftaroline, ertapenem, gentamicin, tigecycline, and rifampin, for a collection of 9 daptomycin-NS enterococci that exhibited a broad range of MICs and different resistance-conferring mutations. We found that ampicillin plus daptomycin yielded the most consistent synergy but did so only for isolates with mutations to the liaFSR system. Daptomycin binding was found to be enhanced by ampicillin in a representative isolate with such mutations but not for an isolate with mutation to the yycFGHIJ system. In contrast, ampicillin enhanced the killing of the LL-37 human antimicrobial peptide against daptomycin-NS E. faecium with either the liaFSR or yycFGHIJ mutation. Antagonism was noted only for rifampin and tigecycline and only for 2 or 3 isolates. These data add support to the growing body of evidence indicating that therapy combining daptomycin and ampicillin may be helpful in eradicating refractory VRE infections.     

Non‐muscle invasive bladder cancer and bacillus Calmette‐Guerin treatment: a review of the literature

Bladder cancer is the second most common urological cancer in the UK, with over 10 000 cases diagnosed annually. With 80% of urothelial bladder cancers being non‐muscle invasive, it is important to understand the treatments available. This review aims to identify and review the literature regarding bacillus Calmette‐Guerin (BCG) treatment. An integrative‐based review was conducted to generate a broad overview of the existing knowledge for BCG treatment. An open search of online databases was conducted to identify articles published in English from the earliest date available to September 2013, using key terms related to BCG. A significant number of articles were identified. To narrow the results and identify the most relevant articles, the search terms were cross‐referenced. The resulting articles were then reviewed using the critical appraisal skills programme framework. The tools provided by CASP give a systematic, transparent and rigorous approach to the quality assessment of research studies. The research articles were then categorized under the following headings: side effects, including local, systemic and age; quality of life; and attrition. The major conclusion from this literature review is that BCG treatment, when given through an induction and maintenance regime, significantly reduces the risk of progression and recurrence. However, there are potential side effects which the patient and the nurse need to be aware. This review also highlighted that there is a lack of research from the UK and that there is a paucity of research showing why patients withdraw from BCG treatment     

Alternative Gnas gene products have opposite effects on glucose and lipid metabolism

Gnas is an imprinted gene with multiple gene products resulting from alternative splicing of different first exons onto a common exon 2. These products include stimulatory G protein alpha-subunit (G(s)alpha), the G protein required for receptor-stimulated cAMP production; extralarge G(s)alpha (XLalphas), a paternally expressed G(s)alpha isoform; and neuroendocrine-specific protein (NESP55), a maternally expressed chromogranin-like protein. G(s)alpha undergoes tissue-specific imprinting, being expressed primarily from the maternal allele in certain tissues. Heterozygous mutation of exon 2 on the maternal (E2m-/+) or paternal (E2+/p-) allele results in opposite effects on energy metabolism. E2m-/+ mice are obese and hypometabolic, whereas E2+/p- mice are lean and hypermetabolic. We now studied the effects of G(s)alpha deficiency without disrupting other Gnas gene products by deleting G(s)alpha exon 1 (E1). E1+/p- mice lacked the E2+/p- phenotype and developed obesity and insulin resistance. The lean, hypermetabolic, and insulin-sensitive E2+/p- phenotype appears to result from XLalphas deficiency, whereas loss of paternal-specific G(s)alpha expression in E1+/p- mice leads to an opposite metabolic phenotype. Thus, alternative Gnas gene products have opposing effects on glucose and lipid metabolism. Like E2m-/+ mice, E1m-/+ mice had s.c. edema at birth, presumably due to loss of maternal G(s)alpha expression. However, E1m-/+ mice differed from E2m-/+ mice in other respects, raising the possibility for the presence of other maternal-specific gene products. E1m-/+ mice had more severe obesity and insulin resistance and lower metabolic rate relative to E1+/p- mice. Differences between E1m-/+ and E1+/p- mice presumably result from differential effects on G(s)alpha expression in tissues where G(s)alpha is normally imprinted.     

Intracellular trafficking during liver regeneration. Alterations in late endocytic and transcytotic pathways

BACKGROUND/AIMS: Liver growth, induced by partial hepatectomy of the organ is a precisely regulated process during which a radical reorganisation of metabolism occurs as the hepatocytes become committed to enter the cell cycle. Recent studies have shown the importance of the endocytic compartment in the control of lipid and protein intracellular trafficking but also in the control of the signal transduction events, which eventually will trigger the initiation of DNA synthesis and the subsequent cell division. METHODS: We isolated endosomes at different times after partial hepatectomy in male rats and compared with endosomes isolated from sham-operated animals. Also, bile was collected and analysed by 2D-gel electrophoresis. RESULTS: The amount of late endosomes isolated from regenerating livers decreased, concomitant with decreased cathepsin D specific enzyme activity. Furthermore, secretion of horseradish peroxidase, pIgA and transferrin increased in the pre-replicative phase of liver regeneration. CONCLUSIONS: At the early stages of liver regeneration, the hepatocellular transport pathway towards degradation (late endosomes and lysosomal pathway) decreases, but the transcytosis and the bile secretion of several major proteins increases.     

Is There a Weekly Pattern for Health Searches on Wikipedia and Is the Pattern Unique to Health Topics?

Background

Online health information–seeking behaviors have been reported to be more common at the beginning of the workweek. This behavior pattern has been interpreted as a kind of “healthy new start” or “fresh start” due to regrets or attempts to compensate for unhealthy behavior or poor choices made during the weekend. However, the observations regarding the most common health information–seeking day were based only on the analyses of users’ behaviors with websites on health or on online health-related searches. We wanted to confirm if this pattern could be found in searches of Wikipedia on health-related topics and also if this search pattern was unique to health-related topics or if it could represent a more general pattern of online information searching—which could be of relevance even beyond the health sector.

Objective

The aim was to examine the degree to which the search pattern described previously was specific to health-related information seeking or whether similar patterns could be found in other types of information-seeking behavior.

Methods

We extracted the number of searches performed on Wikipedia in the Norwegian language for 911 days for the most common sexually transmitted diseases (chlamydia, gonorrhea, herpes, human immunodeficiency virus [HIV], and acquired immune deficiency syndrome [AIDS]), other health-related topics (influenza, diabetes, and menopause), and 2 nonhealth-related topics (footballer Lionel Messi and pop singer Justin Bieber). The search dates were classified according to the day of the week and ANOVA tests were used to compare the average number of hits per day of the week.

Results

The ANOVA tests showed that the sexually transmitted disease queries had their highest peaks on Tuesdays (P<.001) and the fewest searches on Saturdays. The other health topics also showed a weekly pattern, with the highest peaks early in the week and lower numbers on Saturdays (P<.001). Footballer Lionel Messi had the highest mean number of hits on Tuesdays and Wednesdays, whereas pop singer Justin Bieber had the most hits on Tuesdays. Both these tracked search queries also showed significantly lower numbers on Saturdays (P<.001).

Conclusions

Our study supports prior studies finding an increase in health information searching at the beginning of the workweek. However, we also found a similar pattern for 2 randomly chosen nonhealth-related terms, which may suggest that the search pattern is not unique to health-related searches. The results are potentially relevant beyond the field of health and our preliminary findings need to be further explored in future studies involving a broader range of nonhealth-related searches.     

What Online Communities Can Tell Us About Electronic Cigarettes and Hookah Use: A Study Using Text Mining and Visualization Techniques

Background

The rise in popularity of electronic cigarettes (e-cigarettes) and hookah over recent years has been accompanied by some confusion and uncertainty regarding the development of an appropriate regulatory response towards these emerging products. Mining online discussion content can lead to insights into people’s experiences, which can in turn further our knowledge of how to address potential health implications. In this work, we take a novel approach to understanding the use and appeal of these emerging products by applying text mining techniques to compare consumer experiences across discussion forums.

Objective

This study examined content from the websites Vapor Talk, Hookah Forum, and Reddit to understand people’s experiences with different tobacco products. Our investigation involves three parts. First, we identified contextual factors that inform our understanding of tobacco use behaviors, such as setting, time, social relationships, and sensory experience, and compared the forums to identify the ones where content on these factors is most common. Second, we compared how the tobacco use experience differs with combustible cigarettes and e-cigarettes. Third, we investigated differences between e-cigarette and hookah use.

Methods

In the first part of our study, we employed a lexicon-based extraction approach to estimate prevalence of contextual factors, and then we generated a heat map based on these estimates to compare the forums. In the second and third parts of the study, we employed a text mining technique called topic modeling to identify important topics and then developed a visualization, Topic Bars, to compare topic coverage across forums.

Results

In the first part of the study, we identified two forums, Vapor Talk Health & Safety and the Stopsmoking subreddit, where discussion concerning contextual factors was particularly common. The second part showed that the discussion in Vapor Talk Health & Safety focused on symptoms and comparisons of combustible cigarettes and e-cigarettes, and the Stopsmoking subreddit focused on psychological aspects of quitting. Last, we examined the discussion content on Vapor Talk and Hookah Forum. Prominent topics included equipment, technique, experiential elements of use, and the buying and selling of equipment.

Conclusions

This study has three main contributions. Discussion forums differ in the extent to which their content may help us understand behaviors with potential health implications. Identifying dimensions of interest and using a heat map visualization to compare across forums can be helpful for identifying forums with the greatest density of health information. Additionally, our work has shown that the quitting experience can potentially be very different depending on whether or not e-cigarettes are used. Finally, e-cigarette and hookah forums are similar in that members represent a “hobbyist culture” that actively engages in information exchange. These differences have important implications for both tobacco regulation and smoking cessation intervention design.