Linking carbon metabolism to carotenoid production in mycobacteria using Raman spectroscopy

Bacteria can utilize multiple sources of carbon for growth, and for pathogenic bacteria like Mycobacterium tuberculosis, this ability is crucial for survival within the host. In addition, phenotypic changes are seen in mycobacteria grown under different carbon sources. In this study, we use Raman spectroscopy to analyze the biochemical components present in M. smegmatis cells when grown in three differently metabolized carbon sources. Our results show that carotenoid biosynthesis is enhanced when M. smegmatis is grown in glucose compared to glycerol and acetate. We demonstrate that this difference is most likely due to transcriptional upregulation of the carotenoid biosynthesis operon (crt) mediated by higher levels of the stress-responsive sigma factor SigF. Moreover, we find that increased SigF and carotenoid levels correlate with greater resistance of glucose-grown cells to oxidative stress. Thus, we demonstrate the use of Raman spectroscopy in unraveling unknown aspects of mycobacterial physiology and describe a novel effect of carbon source variation on mycobacteria.

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M-tuberculosis

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38?350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional Delta APSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.

Dual role of arginine metabolism in establishing pathogenesis

Arginine is an integral part of host defense when invading pathogens are encountered. The arginine metabolite nitric oxide (NO) confers antimicrobial properties, whereas the metabolite ornithine is utilized for polyamine synthesis. Polyamines are crucial to tissue repair and anti-inflammatory responses. iNOS/arginase balance can determine Th1/Th2 response. Furthermore, the host arginine pool and its metabolites are utilized as energy sources by various pathogens. Apart from its role as an immune modulator, recent studies have also highlighted the therapeutic effects of arginine. This article sheds light upon the roles of arginine metabolism during pathological conditions and its therapeutic potential.

Integration of holographic sensors into microfluidics for the real-time pH sensing of L. casei metabolism

We describe developments in the integration of analyte specific holographic sensors into PDMS-based microfluidic devices for the purpose of continuous, low-impact monitoring of extra-cellular change in micro-bioreactors. Holographic sensors respond to analyte concentration via volume change, which makes their reduction in size and integration into spatially confined fluidics difficult. Through design and process modification many of these constraints have been addressed, and a microfluidics-based device capable of real-time monitoring of the pH change caused by Lactobacillus casei fermentation is presented as a general proof-of-concept for a wide array of possible devices.

Influence of nutrient intake on antioxidant capacity, muscle damage and white blood cell count in female soccer players

11 p.

Nutrient Limitation of Periphyton in a Spring-Fed, Coastal Stream in Florida, USA.

There is strong evidence to suggest that ground-water nitrate concentrations have increased in recent years and further increases are expected along portions of the central Gulf coast of Florida. Much of the nitrate enriched groundwater is discharged into surface waters through numerous freshwater springs that are characteristic of the area and the potential for eutrophication of their receiving waters is a legitimate concern. To test the potential effects of elevated nutrient concentrations on the periphyton community an in situ nutrient addition experiment was conducted in the spring-fed Chassahowitzka River, FL, USA, during the summer of 1999. Plastic tubes housing arrays of glass microscope slides were suspended in the stream. Periphyton colonizing the microscope slides was subjected to artificial increases in nitrogen, phosphorus or a combination of both. Slides from each tube were collected at 3- to 4- day intervals and the periphyton communities were measured for chlorophyll concentration. The addition of approximately 10 μg/L of phosphate above ambient concentrations significantly increased the amount of periphyton on artificial substrates relative to controls; the addition of approximately 100 μg/L of nitrate above ambient concentrations did not. The findings from this experiment implicated phosphorus, rather than nitrogen, as the nutrient that potentially limits periphyton growth in this system.(PDF contains 4 pages.)

Regulation of Energy Metabolism by the Extracytoplasmic Function (ECF) σ Factors of Arcobacter butzleri

11 p.

MBARI procedures for automated nutrient analyses using a modified Alpkem Series 300 Rapid Flow Analyzer

This report documents the methods used at the Monterey Bay Aquarium Research Institute (MBARI) for analyzing seawater nutrient samples with an Alpkem Series 300 Rapid Flow Analyzer (RFA) system. The methods have been optimized for the particular requirements of this laboratory. The RFA system has been used to analyze approximately 20,000 samples during the past two years. The methods have been optimized to run nutrient analyses in a routine manner with a detection limit of better than -±1% and a within run precision of -±1% of the full scale concentration range. The normal concentration ranges are 0-200 ~M silicate, 0-5 ~M phosphate, 0-50 ~M nitrate, 0-3 ~M nitrite, and 0-10 ~M ammonium. The memorandum is designed to be used in a loose-leaf binder format. Each page is dated and as revisions are made, they should be inserted into the binder. The revisions should be added into the binder. Retain the old versions in order to maintain a historical record of the procedures. (88 pages)

A Comparison of Two Methods for Enhancing the Recovery of Seagrasses into Propellor Scars: Mechanical Injection of a Nutrient and Growth Hormone Solution vs. Defecation by Roosting Seabirds: Final Report.

Based on the recovery rates for Thalassia testudinum measured in this study for scars of these excavation depths and assuming a linear recovery horizon, we estimate that it would take ~ 6.9 years (95% CI. = 5.4 to 9.6 years) for T. testudinum to return to the same density as recorded for the adjacent undisturbed population. The application of water soluble fertilizers and plant growth hormones by mechanical injection into the sediments adjacent to ten propellor scars at Lignumvitae State Botanical Site did not significantly increase the recovery rate of Thalassia testudinum or Halodule wrightii. An alternative method of fertilization and restoration of propellor scars was also tested by a using a method of “compressed succession” where Halodule wrightii is substituted for T. testudinum in the initial stages of restoration. Bird roosting stakes were placed among H.wrightii bare root plantings in prop scars to facilitate the defecation of nitrogen and phosphorus enriched feces. In contrast to the fertilizer injection method, the bird stakes produced extremely high recovery rates of transplanted H. wrightii. We conclude that use of a fertilizer/hormone injection machine in the manner described here is not a feasible means of enhancing T. testudinum recovery in propellor scars on soft bottom carbonate sediments. Existing techniques such as the bird stake approach provide a reliable, and inexpensive alternative method that should be considered for application to restoration of seagrasses in these environments. Document contains 40 pages)

Some aspects of the temperature, oxygen and nutrient distributions in Monterey Bay, California. Annual Report, Part 1, 1973

Seasonal variations in temperature, dissolved oxygen, and nutrients in the nearshore areas and in the canyon area of Monterey Bay, California during 1971-1972 were similar~ During upwelling periods, however, water in the nearshore areas was higher in temperature and oxygen and lower in nutrients than water in the canyon area~ This was caused by upwelled water moving north and south of the canyon into counterclockwise and clockwise flow in the northern and southern ends of the bay respectively. The water was heated by insolation and depleted of its nutrients by photosynthesis during this movement. The residence time of water in the nearshore northern and southern bay during upwelling is estimated to be 3 to 8 days, and this fits well into the above circulation pattern and average measured current velocities of 10 to 15 cm/sec~ There is sorne evidence that this circulation pattern and the estimated residence time may be also valid for on-upwelling periods. Upwelling apparently occurred in Monterey Submarine Canyon at rates of 0.4 to 2.9 m/day and was stronger in 1971 than 1972. (PDF contains 107 pages)

Nutrient distributions for Georges Bank and adjacent waters in 1979

In this report we describe the temporal and spatial distributions of inorganic nutrients over Georges Bank and in adjacent waters and discuss major features with respect to tbe nutrient environments of pbytoplankton. Nitrate and orthophosphorus were rapidly depleted from the surface layer of much of the study area in spring, but major differences were found between the shallow areas on Georges Bank and the surrounding stratified waters. In the "well-mixed" area of Georges Bank, the depletion encompassed the entire water column and ammonium became the dominant form of inorganic nitrogen throughout. Dissolved silicon was depleted slowly over central Georges Bank, reaching a minimum concentration in September while orthophosphorus gradually increased during the summer. The nutrient environment of phytoplankton over central Georges Bank may be described as vertically uniform but temporally changing in the relative availability of the various nutrients. In areas that undergo stratification (e.g., the central Gulf of Maine), a quasi-steady state was established as the surface water layer formed, consisting of declining nutrient gradients from below the euphotic layer to the top of the water column. These intergrading nutrient environments are relatively stable through time. Destratification reintroduced nutrients to depleted areas beginning in October; however, dissolved silicon was again depleted over shallow Georges Bank in late autumn though nitrate remained abundant. Slope water has been found to enter the bottom layer of the Gulf of Maine via the Northeast Channel. High nutrient concentrations observed in the bottom water of the Northeast Channel are consistent with this mechanism being the nutrient source for the Gulf of Maine. (PDF file contains 40 pages.)

Nutrient dynamics of seasonal tanks in the dry zone of Sri Lanka in relation to their hydrological regimes

Village tanks are put to a wide range of uses by the rural communities that depend on them for their survival. As the primacy of irrigation has decreased under these tanks due to a variety of climatic and economic reasons there is a need to reevaluate their use for other productive functions. The research presented in this paper is part of a programme investigating the potential to improve the management of living aquatic resources in order to bring benefits to the most marginal groups identified in upper watershed areas. Based on an improved typology of seasonal tanks, the seasonal changes and dynamics of various water quality parameters indicative of nutrient status and fisheries carrying capacity are compared over a period of one year. Indicators of Net (Primary) Productivity (NP): Rates of Dissolved Oxygen (DO) change, Total Suspended Solids (TSS): Total Suspended Volatile solids (TVSS) ratios are the parameters of principle interest. Based on these results a comparative analysis is made on two classes of ‘seasonal’ and ‘semi-seasonal’ tanks. Results indicate a broad correlation in each of these parameters with seasonal trends in tank hydrology. Highest productivity levels are associated with periods of declining water storage, whilst the lowest levels are associated with the periods of maximum water storage shortly after the NW monsoon. This variation is primarily attributed to dilution effects associated with depth and storage area. During the yala period, encroachment of the surface layer by several species of aquatic macrophyte also has progressively negative impacts on productivity. The most seasonal tanks show wider extremes in seasonal nutrient dynamics, overall, with less favourable conditions than the ‘semi-seasonal’ tanks. Never the less all the tanks can be considered as being highly productive with NP levels comparable to fertilised pond systems for much of the year. This indicates that nutrient status is not likely to be amongst the most important constraints to enhancing fish production. Other potential management improvements based on these results are discussed. [PDF contains 19 pages]

Global pressures, specific responses: effects of nutrient enrichment in streams from different biomes

13 p. + 2 p. (Erratum)

Changes in white adipose tissue metabolism induced by resveratrol in rats

Background: A remarkable range of biological functions have been ascribed to resveratrol. Recently, this polyphenol has been shown to have body fat lowering effects. The aim of the present study was to assess some of the potential underlying mechanisms of action which take place in adipose tissue. Methods: Sixteen male Sprague-Dawley rats were randomly divided into two groups: control and treated with 30 mg resveratrol/kg body weight/d. All rats were fed an obesogenic diet and after six weeks of treatment white adipose tissues were dissected. Lipoprotein lipase activity was assessed by fluorimetry, acetyl-CoA carboxylase by radiometry, and malic enzyme, glucose-6P-dehydrogenase and fatty acid synthase by spectrophotometry. Gene expression levels of acetyl-CoA carboxylase, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase, adipose triglyceride lipase, PPAR-gamma, SREBP-1c and perilipin were assessed by Real time RT-PCR. The amount of resveratrol metabolites in adipose tissue was measured by chromatography. Results: There was no difference in the final body weight of the rats; however, adipose tissues were significantly decreased in the resveratrol-treated group. Resveratrol reduced the activity of lipogenic enzymes, as well as that of heparin-releasable lipoprotein lipase. Moreover, a significant reduction was induced by this polyphenol in hormone-sensitive lipase mRNA levels. No significant changes were observed in other genes. Total amount of resveratrol metabolites in adipose tissue was 2.66 +/- 0.55 nmol/g tissue. Conclusions: It can be proposed that the body fat-lowering effect of resveratrol is mediated, at least in part, by a reduction in fatty acid uptake from circulating triacylglycerols and also in de novo lipogenesis.

Recent Developments in In Situ Nutrient Sensors: applications and future directions, Savannah, Georgia, December 11-13, 2006: workshop proceedings

The Alliance for Coastal Technologies (ACT) convened a Workshop on "Recent Developments in In Situ Nutrient Sensors: Applications and Future Directions" from 11-13 December, 2006. The workshop was held at the Georgia Coastal Center in Savannah, Georgia, with local coordination provided by the ACT partner at the Skidaway Institute of Oceanography (University System of Georgia). Since its formation in 2000, ACT partners have been conducting workshops on various sensor technologies and supporting infrastructure for sensor systems. This was the first workshop to revisit a topic area addressed previously by ACT. An earlier workshop on the "State of Technology in the Development and Application of Nutrient Sensors" was held in Savannah, Georgia from 10-12 March, 2003. Participants in the first workshop included representatives from management, industry, and research sectors. Among the topics addressed at the first workshop were characteristics of "ideal" in situ nutrient sensors, particularly with regard to applications in coastal marine waters. In contrast, the present workshop focused on the existing commercial solutions. The in situ nutrient sensor technologies that appear likely to remain the dominant commercial options for the next decade are reagent-based in situ auto-analyzers (or fluidics systems) and an optical approach (spectrophotometric measurement of nitrate). The number of available commercial systems has expanded since 2003, and community support for expanded application and further development of these technologies appears warranted. Application in coastal observing systems, including freshwater as well as estuarine and marine environments, was a focus of the present workshop. This included discussion of possible refinements for sustained deployments as part of integrated instrument packages and means to better promote broader use of nutrient sensors in observing system and management applications. The present workshop also made a number of specific recommendations concerning plans for a demonstration of in situ nutrient sensor technologies that ACT will be conducting in coordination with sensor manufacturers.[PDF contains 40 pages]