Hemin and iron increase synthesis and trigger export of xanthine oxidoreductase from hepatocytes to the circulation.

We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 µM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 µU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control µU/mg). Interestingly, free iron (Fe2+ and Fe3+) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 µM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.

NUDT7 regulates total hepatic CoA levels and the composition of the intestinal bile acid pool in male mice fed a Western diet.

Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the ß-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.

Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM): a method for detection of antimicrobial resistance in environmental samples.

Effective monitoring of antibiotic resistance genes and their dissemination in environmental ecosystems has been hindered by the cost and efficiency of methods available for the task. We developed the Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM), a method implementing TruSeq high-throughput sequencing to simultaneously sequence thousands of antibiotic resistant gene targets representing a full-spectrum of antibiotic resistance classes common to environmental systems. In this study, we demonstrated DARTE-QM by screening 662 antibiotic resistance genes within complex environmental samples originated from manure, soil, and livestock feces, in addition to a mock-community reference to assess sensitivity and specificity. DARTE-QM offers a new approach to studying antibiotic resistance in environmental microbiomes, showing advantages in efficiency and the ability to scale for many samples. This method provides a means of data acquisition that will alleviate some of the obstacles that many researchers in this area currently face.

Measurement of Fatty Acid ß-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes.

Fatty acid ß-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid ß-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid ß-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid ß-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid ß-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid ß-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Drug resistance occurred in a newly characterized preclinical model of lung cancer brain metastasis.

BACKGROUND: Cancer metastasis and drug resistance have traditionally been studied separately, though these two lethal pathological phenomena almost always occur concurrently. Brain metastasis occurs in a large proportion of lung cancer patients (~ 30%). Once diagnosed, patients have a poor prognosis surviving typically less than 1 year due to lack of treatment efficacy. METHODS: Human metastatic lung cancer cells (PC-9-Br) were injected into the left cardiac ventricle of female athymic nude mice. Brain lesions were allowed to grow for 21 days, animals were then randomized into treatment groups and treated until presentation of neurological symptoms or when moribund. Prior to tissue collection mice were injected with Oregon Green and 14C-Aminoisobutyric acid followed by an indocyanine green vascular washout. Tracer accumulation was determined by quantitative fluorescent microscopy and quantitative autoradiography. Survival was tracked and tumor burden was monitored via bioluminescent imaging. Extent of mutation differences and acquired resistance was measured in-vitro through half-maximal inhibitory assays and qRT-PCR analysis. RESULTS: A PC-9 brain seeking line (PC-9-Br) was established. Mice inoculated with PC-9-Br resulted in a decreased survival time compared with mice inoculated with parental PC-9. Non-targeted chemotherapy with cisplatin and etoposide (51.5 days) significantly prolonged survival of PC-9-Br brain metastases in mice compared to vehicle control (42 days) or cisplatin and pemetrexed (45 days). Further in-vivo imaging showed greater tumor vasculature in mice treated with cisplatin and etoposide compared to non-tumor regions, which was not observed in mice treated with vehicle or cisplatin and pemetrexed. More importantly, PC-9-Br showed significant resistance to gefitinib by in-vitro MTT assays (IC50 > 2.5 µM at 48 h and 0.1 µM at 72 h) compared with parental PC-9 (IC50: 0.75 µM at 48 h and 0.027 µM at 72 h). Further studies on the molecular mechanisms of gefitinib resistance revealed that EGFR and phospho-EGFR were significantly decreased in PC-9-Br compared with PC-9. Expression of E-cadherin and vimentin did not show EMT in PC-9-Br compared with parental PC-9, and PC-9-Br had neither a T790M mutation nor amplifications of MET and HER2 compared with parental PC-9. CONCLUSION: Our study demonstrated that brain metastases of lung cancer cells may independently prompt drug resistance without drug treatment.

Regulation of coenzyme A levels by degradation: the 'Ins and Outs'.

Coenzyme A (CoA) is the predominant acyl carrier in mammalian cells and a cofactor that plays a key role in energy and lipid metabolism. CoA and its thioesters (acyl-CoAs) regulate a multitude of metabolic processes at different levels: as substrates, allosteric modulators, and via post-translational modification of histones and other non-histone proteins. Evidence is emerging that synthesis and degradation of CoA are regulated in a manner that enables metabolic flexibility in different subcellular compartments. Degradation of CoA occurs through distinct intra- and extracellular pathways that rely on the activity of specific hydrolases. The pantetheinase enzymes specifically hydrolyze pantetheine to cysteamine and pantothenate, the last step in the extracellular degradation pathway for CoA. This reaction releases pantothenate in the bloodstream, making this CoA precursor available for cellular uptake and de novo CoA synthesis. Intracellular degradation of CoA depends on specific mitochondrial and peroxisomal Nudix hydrolases. These enzymes are also active against a subset of acyl-CoAs and play a key role in the regulation of subcellular (acyl-)CoA pools and CoA-dependent metabolic reactions. The evidence currently available indicates that the extracellular and intracellular (acyl-)CoA degradation pathways are regulated in a coordinated and opposite manner by the nutritional state and maximize the changes in the total intracellular CoA levels that support the metabolic switch between fed and fasted states in organs like the liver. The objective of this review is to update the contribution of these pathways to the regulation of metabolism, physiology and pathology and to highlight the many questions that remain open.

Investigating the dispersal of antibiotic resistance associated genes from manure application to soil and drainage waters in simulated agricultural farmland systems.

Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment.

Investigational chemotherapy and novel pharmacokinetic mechanisms for the treatment of breast cancer brain metastases.

In women, breast cancer is the most common cancer diagnosis and second most common cause of cancer death. More than half of breast cancer patients will develop metastases to the bone, liver, lung, or brain. Breast cancer brain metastases (BCBM) confers a poor prognosis, as current therapeutic options of surgery, radiation, and chemotherapy rarely significantly extend life and are considered palliative. Within the realm of chemotherapy, the last decade has seen an explosion of novel chemotherapeutics involving targeting agents and unique dosage forms. We provide a historical overview of BCBM chemotherapy, review the mechanisms of new agents such as poly-ADP ribose polymerase inhibitors, cyclin-dependent kinase 4/6 inhibitors, phosphatidyl inositol 3-kinaseinhibitors, estrogen pathway antagonists for hormone-receptor positive BCBM; tyrosine kinase inhibitors, antibodies, and conjugates for HER2+ BCBM; repurposed cytotoxic chemotherapy for triple negative BCBM; and the utilization of these new agents and formulations in ongoing clinical trials. The mechanisms of novel dosage formulations such as nanoparticles, liposomes, pegylation, the concepts of enhanced permeation and retention, and drugs utilizing these concepts involved in clinical trials are also discussed. These new treatments provide a promising outlook in the treatment of BCBM.

Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance.

Use of antibiotics in human and animal medicine has applied selective pressure for the global dissemination of antibiotic-resistant bacteria. Therefore, it is of interest to develop strategies to mitigate the continued amplification and transmission of resistance genes in environmental reservoirs such as farms, hospitals and watersheds. However, the efficacy of mitigation strategies is difficult to evaluate because it is unclear which resistance genes are important to monitor, and which primers to use to detect those genes. Here, we evaluated the diversity of one type of macrolide antibiotic resistance gene (erm) in one type of environment (manure) to determine which primers would be most informative to use in a mitigation study of that environment. We analyzed all known erm genes and assessed the ability of previously published erm primers to detect the diversity. The results showed that all known erm resistance genes group into 66 clusters, and 25 of these clusters (40%) can be targeted with primers found in the literature. These primers can target 74%-85% of the erm gene diversity in the manures analyzed.

The direct identification of core-collapse supernova progenitors.

To place core-collapse supernovae (SNe) in context with the evolution of massive stars, it is necessary to determine their stellar origins. I describe the direct identification of SN progenitors in existing pre-explosion images, particularly those obtained through serendipitous imaging of nearby galaxies by the Hubble Space Telescope I comment on specific cases representing the various core-collapse SN types. Establishing the astrometric coincidence of a SN with its putative progenitor is relatively straightforward. One merely needs a comparably high-resolution image of the SN itself and its stellar environment to perform this matching. The interpretation of these results, though, is far more complicated and fraught with larger uncertainties, including assumptions of the distance to and the extinction of the SN, as well as the metallicity of the SN environment. Furthermore, existing theoretical stellar evolutionary tracks exhibit significant variations one from the next. Nonetheless, it appears fairly certain that Type II-P (plateau) SNe arise from massive stars in the red supergiant phase. Many of the known cases are associated with subluminous Type II-P events. The progenitors of Type II-L (linear) SNe are less established. Among the stripped-envelope SNe, there are now a number of examples of cool, but not red, supergiants (presumably in binaries) as Type IIb progenitors. We appear now finally to have an identified progenitor of a Type Ib SN, but no known example yet for a Type Ic. The connection has been made between some Type IIn SNe and progenitor stars in a luminous blue variable phase, but that link is still thin, based on direct identifications. Finally, I also describe the need to revisit the SN site, long after the SN has faded, to confirm the progenitor identification through the star's disappearance and potentially to detect a putative binary companion that may have survived the explosion.This article is part of the themed issue 'Bridging the gap: from massive stars to supernovae'.

Depression comes in many disguises to the providers of primary care: recognition and management.

Depression is encountered frequently in the primary care setting. Its appearance is dominated by the physical symptoms of the syndrome. This factor, when combined with the residual stigma that mitigates against acceptance of the diagnosis, probably accounts for how often the diagnosis is missed. The depressive illnesses are serious, disrupting occupational and social functioning to a significant degree. They are life-threatening for some in the short-term, but for many more over a lifetime, as depression is more often recurrent or chronic than a one time experience. With a psychiatric nomenclature now available that is user-friendly, screening tests that are available and easy to administer, and treatments that are successful, it is important that the physician learn to recognize and manage this common set of problems. Depression is typically co-morbid with serious medical illness, and often co-morbid with complicating emotional disorders. It may appear in a form that takes a bipolar course, including episodes of mania and hypomania. The physician in practice must decide which patients with depression he or she will treat, and who to refer for specialty care. The SSRI anti-depressants are usually the frontline treatment of choice. Bipolar, treatment-resistant, and difficult patients with co-morbid psychiatric illnesses should be referred to psychiatrists. It is valuable for the physician to have psychiatrists he or she knows to facilitate consultation, communication and coordination. The value of brief psychotherapy in the treatment of a depressive episode underlines the need for a psychiatrist with whom the physician can work collaboratively. The depressed patient presents the physician with a situation in which he or she can make a positive difference in the life of a person and his or her family. The need to model and teach the treatment of depression in primary care is evident, with the likelihood that this will be the arena in which these patients will continue to receive care.

Primary care and psychiatry: anticipating an interfaith marriage.

The emergence of a managed-care--driven health delivery system in the United States has had a major impact on both primary care physicians and psychiatrists. Since a significant portion of psychiatric disorders present in primary care settings and a significant number of patients with emotional disorders receive their care from primary care physicians, the convergence of the two fields of medicine seems likely. Surveys document that both the diagnosis and the treatment of emotional disorders are inadequate in primary care. The authors review possible approaches to rectifying this situation and show that the attachment of the psychiatrist to the primary care team offers many advantages to the provision of clinical services, the education of a diverse group of trainees, and the collaboration of two categories of physician not typically seen as working closely together. The authors also describe an outpatient county medical clinic in which the concept of "attachment-liaison" of the psychiatrist to the primary care setting is tested on a daily basis. The authors (a psychiatrist and an internist), who met in this setting, detail their initial expectations and the observed outcomes of having a psychiatrist join the clinic team. The various tasks of the psychiatrist in this primary care setting are enumerated, along with several typical case presentations.

The effect of cyproheptadine on hunger, calorie intake and body weight in man.

The appetite stimulating action and the weight gaining potential of one month's treatment with the antihistamine and antiserotonergic compound cyproheptadine (Periactin) 4 mg three times daily was compared to placebo in a double-blind crossover trial in sixteen thin but otherwise normal volunteers who wanted to gain weight. Subjects gained significantly more weight on cyproheptadine than on placebo. There was also a corresponding relative increase in subjective hunger ratings and food intake during the period on active drug. Drowsiness was the most frequent side effect observed. These findings are discussed in relation to a possible serotonergic feeding mechanism.