Germline Signals Deploy NHR-49 to Modulate Fatty-Acid β-Oxidation and Desaturation in Somatic Tissues of C. elegans

In C. elegans, removal of the germline extends lifespan significantly. We demonstrate that the nuclear hormone receptor, NHR-49, enables the response to this physiological change by increasing the expression of genes involved in mitochondrial β-oxidation and fatty-acid desaturation. The coordinated augmentation of these processes is critical for germline-less animals to maintain their lipid stores and to sustain de novo fat synthesis during adulthood. Following germline ablation, NHR-49 is up-regulated in somatic cells by the conserved longevity determinants DAF-16/FOXO and TCER-1/TCERG1. Accordingly, NHR-49 overexpression in fertile animals extends their lifespan modestly. In fertile adults, nhr-49 expression is DAF-16/FOXO and TCER-1/TCERG1 independent although its depletion causes age-related lipid abnormalities. Our data provide molecular insights into how reproductive stimuli are integrated into global metabolic changes to alter the lifespan of the animal. They suggest that NHR-49 may facilitate the adaptation to loss of reproductive potential through synchronized enhancement of fatty-acid oxidation and desaturation, thus breaking down some fats ordained for reproduction and orchestrating a lipid profile conducive for somatic maintenance and longevity.     

Clonal Expansion of Early to Mid-Life Mitochondrial DNA Point Mutations Drives Mitochondrial Dysfunction during Human Ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17–78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

Pathophysiological characterization of asthma transitions across adolescence

Background

Adolescence is a period of change, which coincides with disease remission in a significant proportion of subjects with childhood asthma. There is incomplete understanding of the changing characteristics underlying different adolescent asthma transitions. We undertook pathophysiological characterization of transitional adolescent asthma phenotypes in a longitudinal birth cohort.

Methods

The Isle of Wight Birth Cohort (N = 1456) was reviewed at 1, 2, 4, 10 and 18-years. Characterization included questionnaires, skin tests, spirometry, exhaled nitric oxide, bronchial challenge and (in a subset of 100 at 18-years) induced sputum. Asthma groups were “never asthma” (no asthma since birth), “persistent asthma” (asthma at age 10 and 18), “remission asthma” (asthma at age 10 but not at 18) and “adolescent-onset asthma” (asthma at age 18 but not at age 10).

Results

Participants whose asthma remitted during adolescence had lower bronchial reactivity (odds ratio (OR) 0.30; CI 0.10 -0.90; p = 0.03) at age 10 plus greater improvement in lung function (forced expiratory flow 25-75% gain: 1.7 L; 1.0-2.9; p = 0.04) compared to persistent asthma by age 18. Male sex (0.3; 0.1-0.7; p < 0.01) and lower acetaminophen use (0.4; 0.2-0.8; p < 0.01) independently favoured asthma remission, when compared to persistent asthma. Asthma remission had a lower total sputum cell count compared to never asthma (31.5 [25–75 centiles] 12.9-40.4) vs. 47.0 (19.5-181.3); p = 0.03). Sputum examination in adolescent-onset asthma showed eosinophilic airway inflammation (3.0%, 0.7-6.6), not seen in persistent asthma (1.0%, 0–3.9), while remission group had the lowest sputum eosinophil count (0.3%, 0–1.4) and lowest eosinophils/neutrophils ratio of 0.0 (Interquartile range: 0.1).

Conclusion

Asthma remission during adolescence is associated with lower initial BHR and greater gain in small airways function, while adolescent-onset asthma is primarily eosinophilic.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0153-7) contains supplementary material, which is available to authorized users.     

NF2/Merlin Is a Novel Negative Regulator of mTOR Complex 1, and Activation of mTORC1 Is Associated with Meningioma and Schwannoma Growth

Inactivating mutations of the neurofibromatosis 2 (NF2) gene, NF2, result predominantly in benign neurological tumors, schwannomas and meningiomas, in humans; however, mutations in murine Nf2 lead to a broad spectrum of cancerous tumors. The tumor-suppressive function of the NF2 protein, merlin, a membrane-cytoskeleton linker, remains unclear. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a novel mediator of merlin''s tumor suppressor activity. Merlin-deficient human meningioma cells and merlin knockdown arachnoidal cells, the nonneoplastic cell counterparts of meningiomas, exhibit rapamycin-sensitive constitutive mTORC1 activation and increased growth. NF2 patient tumors and Nf2-deficient mouse embryonic fibroblasts demonstrate elevated mTORC1 signaling. Conversely, the exogenous expression of wild-type merlin isoforms, but not a patient-derived L64P mutant, suppresses mTORC1 signaling. Merlin does not regulate mTORC1 via the established mechanism of phosphoinositide 3-kinase-Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase-mediated TSC2 inactivation and may instead regulate TSC/mTOR signaling in a novel fashion. In conclusion, the deregulation of mTORC1 activation underlies the aberrant growth and proliferation of NF2-associated tumors and may restrain the growth of these lesions through negative feedback mechanisms, suggesting that rapamycin in combination with phosphoinositide 3-kinase inhibitors may be therapeutic for NF2.Meningiomas are mesenchymal tumors that arise from the arachnoid layer covering the brain and spinal cord and account for approximately 30% of all primary intracranial neoplasms (30). Most sporadic meningiomas (60%) display somatic inactivation of the NF2 gene. Germ line mutations of NF2 are associated with neurofibromatosis 2 (NF2), a dominantly inherited disorder characterized by multiple nervous system tumors, including schwannomas and meningiomas (33). Although most meningiomas are benign (WHO grade I), they often cause significant morbidity due to compression of the adjacent brain or spinal cord. Benign meningiomas also have recurrence rates of up to 20% over 10 years. Ten percent of meningiomas are classified as atypical (WHO grade II) or anaplastic (WHO grade III) and display more aggressive clinical behavior, with rapid growth and increased recurrence rates (6, 21). The current standard of care is maximal surgical resection, with adjuvant radiation reserved for progressive tumors or those with aggressive features (e.g., WHO grade II or III). The treatment strategy for meningiomas that progress despite surgery and radiation remains limited, and currently there is no effective chemotherapy.The development of effective therapies has been hampered, in part, by our incomplete understanding of the signals influencing meningioma cell growth. Enhanced expression of certain peptide and steroid growth factors and receptors in meningioma tissue suggests that specific autocrine growth-stimulatory loops may be functionally important in meningioma cell proliferation (20, 38). The scarcity of established meningioma models that would allow for the assessment of growth-regulatory mechanisms has also hampered progress. Recently, we have developed reliable meningioma models that overcome the challenges of the low growth rates and senescence of primary benign meningioma cells (19).Biallelic inactivation of the NF2 gene is detected in the majority of sporadic meningiomas and nearly all schwannomas (11). The tumor suppressor gene NF2 encodes merlin (also called schwannomin), a member of the ezrin-radixin-moesin (ERM) protein family that functions to link membrane proteins to the cortical actin cytoskeleton (31, 41). Like the ERM proteins, merlin has been implicated in the regulation of membrane organization and cytoskeleton-based cellular processes such as adhesion, migration, cell-cell contact, spreading, proliferation, and signal transduction (27). The loss of contact-dependent inhibition of proliferation is seen in several types of NF2-deficient cells (23, 29). Merlin controls cell proliferation in response to cell contact via CD44 (28) and functions together with the related tumor suppressor Expanded via the Hippo/Mst pathway in both Drosophila and some types of mammalian cells (14, 49). Although merlin is implicated in a wide range of cellular activities, the precise mechanism by which merlin mediates growth-inhibitory functions in human arachnoidal and Schwann cells and the way in which its loss results in tumor formation in NF2 remain poorly understood.We recently reported that primary human merlin-deficient meningioma cells exhibit a striking, enlarged-cell phenotype compared to nonneoplastic arachnoidal cell counterparts derived from the same patient (19). Interestingly, the tuberous sclerosis complex (TSC) tumor suppressor syndrome is characterized by widespread benign tumors that possess abnormally large cells (22). Mutations in the tumor suppressor genes TSC1 and TSC2 result in TSC syndrome, and the corresponding protein products, hamartin and tuberin (referred to as TSC1 and TSC2), function together as a complex that potently inhibits mammalian target of rapamycin complex 1 (mTORC1) (17). mTOR is an evolutionarily conserved Ser/Thr kinase that exists in one of two distinct functional complexes, TORC1 and TORC2. TORC1, which regulates autophagy, protein translation, and ribosome biogenesis, is potently and specifically inhibited by rapamycin (10, 46). TORC2, which is less sensitive to rapamycin, is important for cytoskeletal regulation and Akt/protein kinase B activation (16, 18, 36).The TSC1-TSC2 complex inhibits mTORC1 by acting as a GTPase-activating protein for the small GTPase Rheb (Ras homolog enriched in brain). Inactivation of the TSC1-TSC2 complex results in the accumulation of GTP-bound Rheb, which activates mTORC1 (10). In addition to naturally occurring mutations in the TSC1 and TSC2 genes, growth factor stimulation of the phosphoinositide 3-kinase (PI3K)-Akt pathway, as well as Ras/mitogen-activated protein kinase (MAPK) pathways, leads to the phosphorylation and inactivation of the TSC1-TSC2 complex and consequent activation of mTORC1 (17). The activation of mTORC1 results in the phosphorylation of two well-characterized effectors, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1), leading to an increase in ribosomal biogenesis and the selective translation of specific mRNA populations. As a critical regulator of cell growth and proliferation, the mTORC1 pathway is dysregulated in several hamartoma syndromes, as well as in many cancers (10).In this report, we identify the NF2 tumor suppressor protein, merlin, as a novel negative regulator of the mTORC1 pathway to control cell growth (cell size). We show that mTORC1 is constitutively activated in merlin-deficient human meningioma cells, leading to increased cell size. Furthermore, we suggest that the slow growth of merlin-deficient meningioma cells is due to a rapamycin-sensitive, mTORC1-S6K-dependent negative feedback loop that diminishes PI3K-Akt signaling in response to growth factor stimulation. The findings of these studies provide insight into the mechanism of merlin tumor suppressor activity and, moreover, indicate that rapamycin or rapamycin analogs in combination with PI3K inhibitors may provide promise as new therapeutics in the treatment of meningiomas and schwannomas.     

Antithetical effects of corticosterone and dibutyryl cAMP on adenosine deaminase in the gastrointestinal tract of chicken during postnatal development

Liver fatty acid binding protein (L-FABP) is highly expressed in both enterocytes and hepatocytes and binds multiple ligands, including saturated (SFA), unsaturated fatty acids (PUFA), and cholesterol. L-fabp ^?/? mice were protected against obesity and hepatic steatosis on a high saturated fat (SF), high cholesterol “Western” diet and manifested a similar phenotype when fed with a high SF, low cholesterol diet. There were no significant differences in fecal fat content or food consumption between the genotypes, and fatty acid (FA) oxidation was reduced, rather than increased, in SF-fed L-fabp ^?/? mice as evidenced by decreased heat production and serum ketones. In contrast to mice fed with a SF diet, L-fabp ^?/? mice fed with a high PUFA diet were not protected against obesity and hepatic steatosis. These observations together suggest that L-fabp ^?/? mice exhibit a specific defect in the metabolism of SFA, possibly reflecting altered kinetics of FA utilization. In support of this possibility, microarray analysis of muscle from Western diet-fed mice revealed alterations in genes regulating glucose uptake and FA synthesis. In addition, intestinal cholesterol absorption was decreased in L-fabp ^?/? mice. On the other hand, and in striking contrast to other reports, female L-fabp ^?/? mice fed with low fat, high cholesterol diets gained slightly less weight than control mice, with minor reductions in hepatic triglyceride content. Together these data indicate a role for L-FABP in intestinal trafficking of both SFA and cholesterol.     

Plant Growth-Promoting and Rhizosphere-Competent <Emphasis Type="Italic">Acinetobacter rhizosphaerae</Emphasis> Strain BIHB 723 from the Cold Deserts of the Himalayas

A phosphate-solubilizing bacterial strain BIHB 723 isolated from the rhizosphere of Hippophae rhamnoides was identified as Acinetobacter rhizosphaerae on the basis of phenotypic characteristics, carbon source utilization pattern, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The strain exhibited the plant growth-promoting attributes of inorganic and organic phosphate solubilization, auxin production, 1-aminocyclopropane-1-carboxylate deaminase activity, ammonia generation, and siderophore production. A significant increase in the growth of pea, chickpea, maize, and barley was recorded for inoculations under controlled conditions. Field testing with the pea also showed a significant increment in plant growth and yield. The rifampicin mutant of the bacterial strain effectively colonized the pea rhizosphere without adversely affecting the resident microbial populations.