A novel targeted/untargeted GC-Orbitrap metabolomics methodology applied to Candida albicans and Staphylococcus aureus biofilms

Introduction: Combined infections from Candida albicans and Staphylococcus aureus are a leading cause of death in the developed world. Evidence suggests that Candida enhances the virulence of Staphylococcus—hyphae penetrate through tissue barriers, while S. aureus tightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state, C. albicans enhances the antimicrobial resistance characteristics of S. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence. Objectives: To explore the interaction, we used a novel GC-Orbitrap-based mass spectrometer, the Q Exactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards. Methods: Optimised C. albicans and S. aureus mono- and co-cultured biofilms were analysed using the new instrument in addition to the fresh and spent bacterial growth media. Results: The targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. Untargeted analysis resulted in the detection of 465 features from fresh and spent medium and 405 from biofilm samples. Three significantly changing compounds that matched to high scoring library fragment patterns were chosen for validation. Conclusion: Evaluation of the results demonstrates that the Q Exactive GC is suitable for metabolomics analysis using a targeted/untargeted methodology. Many of the results were as expected: e.g. rapid consumption of glucose and fructose from the medium regardless of the cell type. Modulation of sugar-phosphate levels also suggest that the pentose phosphate pathway could be enhanced in the cells from co-cultured biofilms. Untargeted metabolomics results suggested significant production of cell-wall biosynthesis components and the consumption of non-proteinaceous amino-acids

Evidence for Cloud Disruption in the L/T Dwarf Transition

Clouds of metal-bearing condensates play a critical role in shaping the emergent spectral energy distributions of the coolest classes of low-mass stars and brown dwarfs, L and T dwarfs. Because condensate clouds in planetary atmospheres show distinct horizontal structure, we have explored a model for partly cloudy atmospheres in brown dwarfs. Our model successfully reproduces the colors and magnitudes of both L and T dwarfs for the first time, including the unexpected brightning of the early- and mid-type T dwarfs at J-band, provided that clouds are rapidly removed from the photosphere at T_eff ~ 1200 K. The clearing of cloud layers also explains the surprising persistence and strengthening of gaseous FeH bands in early- and mid-type T dwarfs. The breakup of cloud layers is likely driven by convection in the troposphere, analogous to phenomena observed on Jupiter. Our results demonstrate that planetary-like atmospheric dynamics must be considered when examining the evolution of free-floating brown dwarfs.Comment: 12 pages, 3 figures, accepted to ApJ Letters for June 200

Physiology and evolution of nitrate acquisition in Prochlorococcus

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is ~17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.Gordon and Betty Moore Foundation (Grant GBMF495)National Science Foundation (U.S.) (Grant OCE-1153588)National Science Foundation (U.S.) (Grant DBI-0424599

The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors

The Brown Dwarf Kinematics Project (BDKP) I. Proper Motions and Tangential Velocities for a Large Sample of Late-type M, L and T Dwarfs

We report proper motion measurements for 427 late-type M, L and T dwarfs, 332 of which have been measured for the first time. Combining these new proper motions with previously published measurements yields a sample of 841 M7-T8 dwarfs. We combined parallax measurements or calculated spectrophotometric distances and computed tangential velocities for the entire sample. We find that kinematics for the full and volume-limited 20 pc samples are consistent with those expected for the Galactic thin disk, with no significant differences between late-type M, L, and T dwarfs. Applying an age-velocity relation we conclude that the average kinematic age of the 20 pc sample of ultracool dwarfs is older than recent kinematic estimates and more consistent with age results calculated with population synthesis models. There is a statistically distinct population of high tangential velocity sources whose kinematics suggest an even older population of ultracool dwarfs belonging to either the Galactic thick disk or halo. We isolate subsets of the entire sample, including low surface-gravity dwarfs, unusually blue L dwarfs, and photometric outliers in J-Ks color and investigate their kinematics. We find that the spectroscopically distinct class of unusually blue L dwarfs has kinematics clearly consistent with old age, implying that high surface-gravity and/or low metallicity may be relevant to their spectral properties. The low surface-gravity dwarfs are kinematically younger than the overall population, and the kinematics of the red and blue ultracool dwarfs suggest ages that are younger and older than the full sample, respectively. We also present a reduced proper motion diagram at 2MASS Ks for the entire population and find that a limit of H_Ks > 18 excludes M dwarfs from the L and T dwarf population regardless of near-infrared color.Comment: Accepted for publication in the Astronomical Journal, 21 pages text, 12 tables, 12 figure

Carcinoma and multiple lymphomas in one patient: establishing the diagnoses and analyzing risk factors

Multiple malignancies may occur in the same patient, and a few reports describe cases with multiple hematologic and non-hematologic neoplasms. We report the case of a patient who showed the sequential occurrence of four different lymphoid neoplasms together with a squamous cell carcinoma of the lung. A 62-year-old man with adenopathy was admitted to the hospital, and lymph node biopsy was positive for low-grade follicular lymphoma. He achieved a partial remission with chemotherapy. Two years later, a PET-CT scan showed a left hilar mass in the lung; biopsy showed a squamous cell carcinoma. Simultaneously, he was diagnosed with diffuse large B cell lymphoma in a neck lymph node; after chemo- and radiotherapy, he achieved a complete response. A restaging PET-CT scan 2 years later revealed a retroperitoneal nodule, and biopsy again showed a low-grade follicular lymphoma, while a biopsy of a cutaneous scalp lesion showed a CD30-positive peripheral T cell lymphoma. After some months, a liver biopsy and a right cervical lymph node biopsy showed a CD30-positive peripheral T cell lymphoma consistent with anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. Flow cytometry and cytogenetic and molecular genetic analysis performed at diagnosis and during the patient’s follow-up confirmed the presence of two clonally distinct B cell lymphomas, while the two T cell neoplasms were confirmed to be clonally related. We discuss the relationship between multiple neoplasms occurring in the same patient and the various possible risk factors involved in their development

A Combined Subaru/VLT/MMT 1--5 Micron Study of Planets Orbiting HR 8799: Implications for Atmospheric Properties, Masses, and Formation

We present new 1--1.25 micron (z and J band) Subaru/IRCS and 2 micron (K band) VLT/NaCo data for HR 8799 and a rereduction of the 3--5 micron MMT/Clio data first presented by Hinz et al. (2010). Our VLT/NaCo data yields a detection of a fourth planet at a projected separation of ~ 15 AU -- "HR 8799e". We also report new, albeit weak detections of HR 8799b at 1.03 microns and 3.3 microns. Empirical comparisons to field brown dwarfs show that at least HR 8799b and HR8799c, and possibly HR 8799d, have near-to-mid IR colors/magnitudes significantly discrepant from the L/T dwarf sequence. Standard cloud deck atmosphere models appropriate for brown dwarfs provide only (marginally) statistically meaningful fits to HR 8799b and c for unphysically small radii. Models with thicker cloud layers not present in brown dwarfs reproduce the planets' SEDs far more accurately and without the need for rescaling the planets' radii. Our preliminary modeling suggests that HR 8799b has log(g) = 4--4.5, Teff = 900K, while HR 8799c, d, and (by inference) e have log(g) = 4--4.5, Teff = 1000--1200K. Combining results from planet evolution models and new dynamical stability limits implies that the masses of HR 8799b, c, d, and e are 6--7 Mj, 7--10 Mj, 7--10 Mj and 7--10 Mj. 'Patchy" cloud prescriptions may provide even better fits to the data and may lower the estimated surface gravities and masses. Finally, contrary to some recent claims, forming the HR 8799 planets by core accretion is still plausible, although such systems are likely rare.Comment: 27 pages, 15 figures, Accepted for publication in The Astrophysical Journa

Myoferlin Depletion in Breast Cancer Cells Promotes Mesenchymal to Epithelial Shape Change and Stalls Invasion

Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET). These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin) and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNAMYOF transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF