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Objective: To develop evidence-informed recommendations to support the delivery of best practice therapeutic exercise for people with knee and/or hip osteoarthritis (OA). Design: A multi-stage, evidence-informed, international multi-disciplinary consensus process that included: 1) a narrative literature review to synthesise existing evidence; 2) generation of evidence-informed proposition statements about delivery of exercise for people with knee and/or hip OA by an international multi-disciplinary expert panel, with statements refined and analysed thematically; 3) an e-Delphi survey with the expert panel to gain consensus on the most important statements; 4) a final round of statement refinement and thematic analysis to group remaining statements into domains. Results: The expert panel included 318 members (academics, health care professionals and exercise providers, patient representatives) from 43 countries. Final recommendations comprised 54 specific proposition statements across 11 broad domains: 1) use an evidence-based approach; 2) consider exercise in the context of living with OA and pain; 3) undertake a comprehensive baseline assessment with follow-up; 4) set goals; 5) consider the type of exercise; 6) consider the dose of exercise; 7) modify and progress exercise; 8) individualise exercise; 9) optimise the delivery of exercise; 10) focus on exercise adherence; and 11) provide education about OA and the role of exercise. Conclusion: The breadth of issues identified as important by the international diverse expert panel highlights that delivering therapeutic exercise for OA is multi-dimensional and complex.

BACKGROUND: An association between increasing anaesthetic depth and decreased postoperative survival has been shown in observational studies; however, evidence from randomised controlled trials is lacking. Our aim was to compare all-cause 1-year mortality in older patients having major surgery and randomly assigned to light or deep general anaesthesia.METHODS: In an international trial, we recruited patients from 73 centres in seven countries who were aged 60 years and older, with significant comorbidity, having surgery with expected duration of more than 2 h, and an anticipated hospital stay of at least 2 days. We randomly assigned patients who had increased risk of complications after major surgery to receive light general anaesthesia (bispectral index [BIS] target 50) or deep general anaesthesia (BIS target 35). Anaesthetists also nominated an appropriate range for mean arterial pressure for each patient during surgery. Patients were randomly assigned in permuted blocks by region immediately before surgery, with the patient and assessors masked to group allocation. The primary outcome was 1-year all-cause mortality. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12612000632897, and is closed to accrual.FINDINGS: Patients were enrolled between Dec 19, 2012, and Dec 12, 2017. Of the 18 026 patients screened as eligible, 6644 were enrolled, randomly assigned to treatment or control, and formed the intention-to-treat population (3316 in the BIS 50 group and 3328 in the BIS 35 group). The median BIS was 47·2 (IQR 43·7 to 50·5) in the BIS 50 group and 38·8 (36·3 to 42·4) in the BIS 35 group. Mean arterial pressure was 3·5 mm Hg (4%) higher (median 84·5 [IQR 78·0 to 91·3] and 81·0 [75·4 to 87·6], respectively) and volatile anaesthetic use was 0·26 minimum alveolar concentration (30%) lower (0·62 [0·52 to 0·73] and 0·88 [0·74 to 1·04], respectively) in the BIS 50 than the BIS 35 group. 1-year mortality was 6·5% (212 patients) in the BIS 50 group and 7·2% (238 patients) in the BIS 35 group (hazard ratio 0·88, 95% CI 0·73 to 1·07, absolute risk reduction 0·8%, 95% CI -0·5 to 2·0). Grade 3 adverse events occurred in 954 (29%) patients in the BIS 50 group and 909 (27%) patients in the BIS 35 group; and grade 4 adverse events in 265 (8%) and 259 (8%) patients, respectively. The most commonly reported adverse events were infections, vascular disorders, cardiac disorders, and neoplasms.INTERPRETATION: Among patients at increased risk of complications after major surgery, light general anaesthesia was not associated with lower 1-year mortality than deep general anaesthesia. Our trial defines a broad range of anaesthetic depth over which anaesthesia may be safely delivered when titrating volatile anaesthetic concentrations using a processed electroencephalographic monitor.FUNDING: Health Research Council of New Zealand; National Health and Medical Research Council, Australia; Research Grant Council of Hong Kong; National Institute for Health and Research, UK; and National Institutes of Health, USA.

The holographic principle has become an extraordinary tool in theoretical physics, most notably in the form of the Anti-deSitter Conformal Field Theory (AdS/CFT ) correspondence, in which classical gravitational degrees of freedom in N -dimensions are related quantum field theory degrees of freedom in N 1 dimensions in the limit of a large number of fields. Here we present an account of the AdS/CFT correspondence, also known as the gauge/gravity duality, from its origins in the large N ’tHooft expansion, up to Maldacena’s proposal that type IIB string theory in the presence of D-branes at low energy is dual to an N = 4, d = 4, U(N) super Yang-Mills on AdS5 ⇥ S 5 . We begin with an extensive review of (super)string theory including D-branes. We then present the general formulation of the AdS/CFT in the supergravity background of AdS5 ⇥ S 5 , along with several examples of how it is used in terms of the identification of bulk fields with operators on the boundary of a CFT. We move on to discuss two applications of the gauge/gravity duality. The first is the application of the holographic gauge/gravity correspondence to the QCD k-string. The second applies the AdS/CFT formalism to a Kerr black hole solution embedded in 10-dimensional heterotic sting theory. These two applications of the holographic gauge/gravity duality comprise the original work presented here. We follow with summaries and discussions of the background material, the original work, and future investigations. vi TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii CHAPTER

Planning is a massive field, and the types and variations of models used are enormous. This chapter (and the related collection), therefore, makes no pretence to cover all the various forms of models that are used across the entire gambit of planning applications. It focuses very much on the types of mathematical models that have been developed by social scientists in their efforts to explain, in particular, spatial location patterns. We feel that it is preferable to systematically treat an area of modelling in a particular context rather than pursue a shot-gun approach embracing a a number of planning applications which would mean the pellets spreading across a large area without any real penetration at any point.

Objectives: (a) To identify whether differences exist in the pattern of recovery with respect to functional outcomes for acutely ruptured anterior cruciate ligament deficient (ACLD) copers, adapters, and non-copers. (b) To identify clinically relevant outcomes that could distinguish between three functional subgroups. Methods: A longitudinal study was used to measure gait variables and distance hop at regular intervals after injury using a digital camcorder and computer for quantitative analysis. A sample of 63 ACLD subjects entered the study; 42 subjects were measured at least three times. At 12–36 months after injury, subjects were classified as functional copers, adapters, or non-copers on the basis of which of their preinjury activities they had resumed. To determine the pattern of recovery, repeated measurements were analysed using a least squares fit of the data. Results: 17% of ACLD subjects were classified as functional copers, 45% as adapters, and 38% as non-copers. Only 5% of those who participated in high demand activities before injury returned to them. ACLD copers had recovered above the control mean for all gait variables by 40 days after the injury. Hopping distance did not recover to the control mean. Non-copers struggled to recover to control limits and remained borderline for all the gait variables. Conclusions: Distinctive patterns of functional recovery for three subgroups of ACLD subjects have been identified. Gait variables and activity level before injury were the most useful variables for distinguishing between the subgroups. If potential for recovery is identified early after injury, then appropriate treatment can be given.

DRASTIC––Database Resource for the Analysis of Signal Transduction In Cells (http://www.drastic.org.uk/) has been created as a first step towards a data-based approach for constructing signal transduction pathways. DRASTIC is a relational database of plant expressed sequence tags and genes up- or down-regulated in response to various pathogens, chemical exposure or other treatments such as drought, salt and low temperature. More than 17700 records have been obtained from 306 treatments affecting 73 plant species from 512 peer-reviewed publications with most emphasis being placed on data from Arabidopsis thaliana. DRASTIC has been developed by the Scottish Crop Research Institute and the University of Abertay Dundee and allows rapid identification of plant genes that are up- or down-regulated by multiple treatments and those that are regulated by a very limited (or perhaps a single) treatment. The INSIGHTS (INference of cell SIGnaling HypoTheseS) suite of web-based tools allows intelligent data mining and extraction of information from the DRASTIC database. Potential response pathways can be visualized and comparisons made between gene expression patterns in response to various treatments. The knowledge gained informs plant signalling pathways and systems biology investigations.

Oligobacteria are bacteria adapted to growth in organic nutrient conditions of the marine and other aquatic environments. Their main food source is dissolved photosynthetically produced organic material. The major products are carbon dioxide, minerals, and cellular biochemicals. Oligobacteria evolved to sustain at nutrient concentrations that are small. The properties of oligobacteria are based largely on copeotrophs such as Escherichia coli because oligobacteria have been unavailable in sufficient mass for investigation. Phytoplankton collect nutrients and reduce them to concentrations that are small as well. Some, such as Synechococcus and Prochlorococcus, are very small giving a large surface- to-volume ratio for mineral collection. This ability is smaller than for oligobacteria because phytoplankton must synthesize their own organics as well. The problem of oligobacterial growth in natural water systems is one of nutrient acquisition. Rates measured are generally near upper limits for oligobacteria because the focus is often on photic zone processes in active regions. However, the cosmopolitan distribution of oligobacteria among the various phyla suggest that metabolic patterns could have since evolved and that metabolic strategies may have converged. The major direct control of oligobacterial activity is the concentration of utilizable nutrients at the surface of the cell. The ability to concentrate many nutrients in this way appears to be cosmopolitan so that successful members of the oligobacteria have coevolved across many genetic lineages.

Expanded specific affinity theory specifies the advantages of small dry mass and dilute cytoarchitecture in impoverished systems. For marine samples, bacterioplankton mean dry mass, according to flow cytometry, was near 24.5 fg cell -1 . Conversion to volume with buoyant density gave a cell volume of 0.124 μm 3 . Total DNA was 2.9 fg cell -1 . This compared with the size of a single genome of a small extinction-culture isolate, Sphingomonas sp. RB2256, of 3.96 fg or 3.6 Mb. The genome size of such isolates and other cultures decreased with metabolic simplicity. It was found that bacterioplankton could be exposed to radiolabeled amino acids and then sorted for size and that the specific affinities of the fraction of small organisms were as great as the fraction of large cells. Size, DNA, and metabolic-complexity distributions were concordant with the concepts that cell volume approaches a minimum set by sufficient space for the smallest genome that can provide sufficient information for competitive dissolved-nutrient acquisition, and that space requirements are further alleviated by the expression of few cytoplasmic-enzyme molecules in each of the various pathways and a dilute cytoplasm. Bacterioplankton approached a minimum genome size of 1.7 Mb with a minimum cell volume of about 0.06 μm 3 and a DNA content of 16% dry weight. The property of small dry mass with a low DNA content was common in in situ bacteria but absent from cultivated representatives, which led to speculation that failure to grow in the laboratory is related to missing regulatory information.

Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to Escherichia coli . The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible; it was named Cycloclasticus oligotrophus . A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant K A for toluene was low at 1.3 μg/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme; K A increased with increasing specific affinities. Specific affinities, a° s , were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high a° s and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present.

We show that bacterial viability can be calculated from the number of species arising in cultures comprised of seawater diluted to near extinction of the propagating organisms. Natural conditions are simulated by partitioning the culture against illuminated raw seawater to maintain a source of nutrients and a sink for waste products, and predators are eliminated by dilution to a statistical absence. Mean values for viability can be corrected for contaminants that may enter the culture by computing their probable entrance frequency as well. Viability determinations were found to be mildly sensitive to the number of species estimated to be present in the original sample. This novel procedure provides a significant improvement in the cultivation success of typical marine bacteria, and the formulations presented here lay the foundation for the quantitative analysis of the population structure.

When a heterotrophic (Rhodotorula rubra) and a phototrophic (Selenastrum capricornutum) plankton were grown together in dilute phosphate (Pi) continuous cultures, coexistence occurred only when the heterotroph was growthrate limited by organic carbon (C). Because of its higher affinity for Pi, and because C starvation does not affect the heterotrophic yeast's ability to transport Pi, the concentration of organic carbon indirectly controlled the biomass of the phototroph. The results support a threshold model of microbial growth.

We investigate a holographic description of k-strings in higher representations via D5 branes with worldvolume fluxes. The D-brane configurations are embedded in supergravity backgrounds dual to confining field theories in 3 and 4 dimensions. We compute the tensions and find qualitative agreement for the totally symmetric and totally anti-symmetric representations with the results of other methods such as lattice as well as the Hamiltonian approach of Karabali and Nair. A one-loop computation on the D-brane configurations yields the Luescher term and allows us to confirm a previously proposed universal expression following from holography., 32 pages, 2 figures, 1 table. v2 references added; comparison with other methods enhanced

In Lake Zurich, a deep subalpine mesotrophic lake, phosphate was low or limiting at 0.2 to 1 μM relative to combined nitrogen at 50 μM. Heterotrophic bacteria were responsible for 53% of the observed microbial wet biomass in our depth profile while phytoplankton, largely Planktothrix (Oscillatoria) rubescens , contributed most of the remainder. Most cell carbon was contributed by this carbon-sufficient cyanobacterium. A material balance indicated that most of the phosphate was sequestered by the bacteria due to a higher phosphate content and specific affinity for this nutrient. Size distributions of the heterotrophic bacteria were narrow; 90% of organisms were from 0.06 to 0.06 μ 3 in volume. Several subpopulations of bacteria were resolved by flow cytometry, and bivariate fluorescence (DAPI-DNA) and light scatter (cell-size) histogram profiles varied with depth. One or two of these subpopulations appeared to be bacteria with sufficient cytoplasmic constituents to produce a normal light-scatter signal but retained only a small amount of DNA; an apparent content of 200 kbp or 5% of a usual oligobacterial genome. These helped increase the oligobacterial population to 6 × 10 6 ml −1 . Application of published specific affinities and measured nutrient concentrations to formulations of system kinetics led to the conclusion that growth rates of the heterotrophic bacterial fraction were carbon limited with cell size, and thus populations were controlled by grazing. The depth profile indicated that phototrophs affected concentrations in a significant way. Considerations of nutrient uptake kinetics suggested that much potential capacity remained in the dissolved phosphate pool to support additional phytoplanktonic biomass. Computations led to the conclusion that, if phosphate is generally limiting in lakes, then additional mechanisms exist which limit populations of phytoplankton to sufficiently small values to allow phosphate accumulation to observed levels. Bacterial biomass then depends on the organic carbon from these phosphate-controlled organisms.

Forward scatter signals (0–20°) from a flow cytometer can be converted into cellular volumes or cellular wet or dry biomasses by the calculations presented here. The results are applicable to bacteria, and including very small bacteria (0.02–0.6 μm3), rods as well as cocci. The physical limitations and the mathematical basis of the calculations are discussed to justify the application of the proposed formula for practical use in microbial ecology and physiology of extremely small aquatic bacteria as well as the more well-studied forms. Although the method was developed for the small bacteria present in natural populations that are difficult to size by alternative methods, it will also be useful for laboratory cultures of bacteria when present in very low concentrations as well as for the characterization of subpopulations and different growth phases. Bacteria, in general, are small enough so that inclusion of the phase shifts of the light passing through the bacteria (the Mie theory) is superfluous. That theory is, however, useful for instrumental calibration with latex microspheres and for some blue-green algae. Even the interference between rays scattered from different parts of the cell (the Rayleigh-Gans theory), although significant, is minor, particularly from elongated particles which are oriented by the fluidics. The computer programs, provided here, correct for intraparticle interference and generate cell volumes (or wet or dry masses) from the forward scatter intensity. The result of the calculations of the light scattered into the collection system of the flow cytometer is presented. From analysis of these values a rule is developed to relate the observed intensities, when scaled by an empirical constant and raised to a power between 0.5 and 0.75, to the cell biomass.

We compute the asymptotic symmetry group of the four dimensional near-extremal Kerr-Sen black hole within an AdS2/CFT1 correspondence. We do this by performing a Robinson-Wilczek two dimensional reduction and construct a quantum theory of the remaining field content. The resulting energy momentum tensor generates an asymptotic Virasoro algebra, to s-wave, with a calculable central extension. This center in conjunction with the proper regularized lowest Virasoro eigen-mode yields the near-extremal Kerr-Sen entropy via the statistical Cardy formula. Finally we analyze quantum holomorphic fluxes of the dual CFT giving rise to a finite Hawking temperature weighted by the central charge of the near-extremal Kerr-Sen metric., Comment: 16 pages, references added, revised for clarity. To appear in JHEP

Specific affinity theory is compared with traditional ways of understanding the nutrient concentration dependency of microbial growth. It is demonstrated that the Michaelis constant increases with the ratio of metabolic enzyme to membrane permease content of bacteria so that small values can reflect specialization for nutrient collection. When compared to the specific affinity, Kt gives a measure of oligotrophic capacity. Specific affinity, on the other hand, reflects nutrient collection ability directly, and increases with the number of permeases. It can be estimated, along with the other kinetic constant, Vmax, by use of isotopes in natural samples. Because of systematic errors in estimating Vmax, specific affinity is the preferred measure of substrate accumulation ability. The advantage of simultaneous collection of multiple substrates in dilute solution is demonstrated. The structural basis of this advantage is computed from collision frequency and recollision probability, computations that further show that multisubstrate usage is essential for bacterial growth under low-nutrient conditions. Computed growth rates from specific affinities require that several substrates be used simultaneously for growth at measured concentrations. Formulations anticipate that the surface of oligobacteria should be occupied by a diversity of transporter types, that each type of transporter should occupy only a small portion of the cell surface, and the number of cytoplasmic enzymes can be small, allowing small cell size to give a large surface-to-volume ratio for high specific affinity. The large number of substrate types that may be accumulated by a single oligobacterial species is consistent with extensive species diversity.

We show that the near horizon regime of a Kerr-Newman-$AdS$ (KN$AdS$) black hole, given by its two dimensional analogue $a la$ Robinson and Wilczek (2005 Phys. Rev. Lett. 95 011303), is asymptotically $AdS_2$ and dual to a one dimensional quantum conformal field theory (CFT). The s-wave contribution of the resulting CFT's energy-momentum-tensor together with the asymptotic symmetries, generate a centrally extended Virasoro algebra, whose central charge reproduces the Bekenstein-Hawking entropy via Cardy's Formula. Our derived central charge also agrees with the near extremal Kerr/CFT Correspondence (2009 Phys. Rev. D 80, 124008) in the appropriate limits. We also compute the Hawking temperature of the KN$AdS$ black hole by coupling its Robinson and Wilczek two dimensional analogue (RW2DA) to conformal matter., 15 pages. Major edits and revisions. Final version accepted for publication in IJMPA

Turnover times for toluene in Resurrection Bay after the Exxon Valdez grounding were determined to be decades, longer than expected considering that dissolved hydrocarbons were anticipated to drift with the current and stimulate development of additional hydrocarbon-utilizing capacity among the microflora in that downcurrent location. These turnover times were based on the recovery of 14CO2 from added [14C]toluene that was oxidized. The concentrations of toluene there, 0.1 to 0.2 microgram/liter, were similar to prespill values. Oxidation rates appeared to be enhanced upstream near islands in the wake of the wind-blown slick, and even more within the slick itself. Specific affinities of the water column bacteria for toluene were computed with the help of biomass data, as measured by high-resolution flow cytometry. They were a very low 0.3 to 3 liters/g of cells.h-1, indicating limited capacity to utilize this hydrocarbon. Since current-driven mixing rates exceeded those of oxidation, dissolved spill components such as toluene should enter the world-ocean pool of hydrocarbons rather than biooxidize in place. Some of the floating oil slick washed ashore and permeated a coarse gravel beach. A bacterial biomass of 2 to 14 mg/kg appeared in apparent response to the new carbon and energy source. This biomass was computed from that of the organisms and associated naphthalene oxidation activity washed from the gravel compared with the original suspension. These sediment organisms were very small at approximately 0.06 microns 3 in volume, low in DNA at approximately 5.5 g per cell, and unlike the aquatic bacteria obtained by enrichment culture but quite similar to the oligobacteria in the water column.(ABSTRACT TRUNCATED AT 250 WORDS)

Formulations are presented that describe the concentration dependency of nutrient-limited transport and growth in molecular terms. They relate the rate of transport at steady state through a two-sequence process, transport and metabolism, to ambient concentrations according to the amounts and kinetic characteristics of the two rate-limiting proteins in these sequences. Sequences are separated by a metabolic pool. A novel feature of these formulations is the translation coefficient, which relates the transport rate attained at given ambient nutrient concentrations and membrane transporter characteristics to the nutrient concentrations sustained in the metabolic pools. The formulations, termed janusian kinetics, show that hyperbolic kinetics are retained during independent changes in transporter and enzyme contents or characteristics. Specific affinity ( a ° A ) depends strongly on the amount and kinetic characteristics of the transporters; it is also mildly affected by the amount and characteristics of the rate-limiting enzyme. This kinetic constant best describes the ability to accumulate substrate from limiting concentrations. Maximal velocity ( V max ) describes uptake from concentrated solutions and can depend strongly on either limiting enzyme content or the associated content of transporters. The whole-cell Michaelis constant ( K T ), which depends on the ratio of rate-limiting enzyme to transporter, can be relatively independent of change in a ° A and is best used to describe the concentration at which saturation begins to occur. Theory specifies that good oligotrophs have a large a ° A for nutrient collection and a small V max for economy of enzyme, giving a small K T . The product of the two constants is universally rather constant so that oligotrophy is scaled on a plot of a ° A versus K T , with better oligotrophs toward one end. This idea is borne out by experimental data, and therefore typical small difficult-to-culture aquatic bacteria may be classified as oligobacteria.

The array of chemiresistors made of individual pristine SnO2, surface doped (Ni)-SnO2 nanowires, and TiO2 and In2O3 mesoscopic whiskers was fabricated on a Si/SiO2 wafer. Their conductance was measured under pulses of H2 and CO reducing gases in oxygen as background gas. The nanostructures were shown to be n-type semiconductors possessing high sensitivity to the target gases. Following the “electronic nose” concept, correlation analysis of response of three-chemiresistor array is shown to be sufficient to discriminate between H2 and CO signals.

A theory for solute uptake by whole cells was derived with a focus on the ability of oligobacteria to sequester nutrients. It provided a general relationship that was used to obtain the kinetic constants for in situ marine populations in the presence of naturally occurring substrates. In situ affinities of 0.9 to 400 liters g of cells −1 h −1 found were up to 10 3 times smaller than those from a “ Marinobacter arcticus ” isolate, but springtime values were greatly increased by warming. Affinities of the isolate for usual polar substrates but not for hydrocarbons were diminished by ionophores. A kinetic curve or Monod plot was constructed from the best available data for cytoarchitectural components of the isolate by using the theory together with concepts and calculations from first principles. The order of effect of these components on specific affinity was membrane potential > cytoplasmic enzyme concentration > cytoplasmic enzyme affinity > permease concentration > area of the permease site > translation coefficient > porin concentration. Component balance was influential as well; a small increase in cytoplasmic enzyme concentration gave a large increase in the effect of permease concentration. The effect of permease concentration on specific affinity was large, while the effect on K m was small. These results are in contrast to the Michaelis-Menten theory as applied by Monod that has uptake kinetics dependent on the quality of the permease molecules, with K m as an independent measure of affinity. Calculations demonstrated that most oligobacteria in the environment must use multiple substrates simultaneously to attain sufficient energy and material for growth, a requirement consistent with communities largely comprising few species.

Basic Concepts Kinetic Formulations from Collision Frequency Some Problems that May be Solved by Using Microbial Kinetics Keywords: growth kinetics; kinetics, microbial

The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell −1 was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell −1 .

Publisher Summary The chapter discusses the methods for isolation of oligobacteria. Oligobacteria are defined as bacteria that can accumulate dissolved organics from low concentrations. Oligobacteria are smaller than copeotrophs giving rise to the term ultramicrobacteria. They are low in solids content and DNA probably as a way of maximizing use of their energy reserves for growth and increasing their surface to volume ratio to maximize their specific affinities for nutrients. There are two methods of isolation discussed: (1) spread plate isolation and (2) extinction culture isolation. The procedures outlined are seen as a significant improvement over conventional techniques for isolating and thus understanding pelagic aquatic bacteria. The frequency of isolation success has been increased by use of extinction cultures where viabilities can approach 10% even in oligotrophic low-productivity waters. The extinction culture technique is based on the observation that when bacteria are filtered from seawater and then re-inoculated into filtered seawater, the population will regenerate. The resulting populations were first called “seawater cultures.” When the inoculum is large—that is, 10% unfiltered seawater—the rate of increase can be used to determine the rate of growth. When it is small, just a few organisms, it can be used in combination with the number of organisms inoculated to determine the culturability or viability of those originally present.

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus , for which dry weight was determined by CHN analysis and 14 C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli . The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10 3 cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.

The far-ranging distribution of genes for aromatic hydrocarbon catabolism, predominantly studied in soil pseudomonads, is extended to a marine oligobacterium by finding five homologous sequences in a 5.7-kb chromosomal DNA from a new isolate, Cycloclasticus oligotrophus RB1. RB1 is capable of growth in unamended seawater or mineral salts media supplemented with a variety of aromatic compounds, including toluene, o-, m-, or p-xylenes, as sole carbon sources. The five open reading frames, designated xylM, K, G, C1, and C2, are 57% A+T-rich. XylM is predicted to be an integral membrane protein; XylK and XylG possess glutathione S-transferase (GST) and 2-hydroxy-5methyl-6-oxohexa2,4-dienoate dehydrogenase activities, respectively; XylC1C2 are homologs of the large and small subunits of the iron sulfur protein component of the biphenyl dioxygenase (e.g., BphA1A2).

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 × 10 9 to 1.07 × 10 9 cells per liter. The mean cell volume varied between 0.042 and 0.074 μm 3 , and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 × 10 5 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 μm 3 and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids ( a° A , 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy ( a° A , ± 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.

Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10 4 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10 4 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.

Traditional concepts of nutrient uptake and growth kinetics as linked by cell yield are presented. Phenomena affecting the kinetics are examined along with a discussion of those which lead to ambiguity. Concepts of flux control are presented to help understand the distribution of material along metabolic pathways. Specific affinity is described to relate nutrient accumulation rates to transporter density. It is shown to be a primary kinetic constant and the best available index of nutrient collection ability. As an aid to understanding, specific affinity is reexpressed in terms of membrane permeability. Formulations of nutrient transport rate as a function of cellular composition, particularly transporter and enzyme content and known as janusian kinetics, are described as an improvement to specific affinity theory. Procedures for quantified unidirectional fluxes are reviewed to identify the difference between gross and net transport rates of substrate. Collision frequency theory is used to show that in addition to total biomass, cell size and transporter density should also be included in rate equations describing microbial growth. Theory diversity suggests that one reason for microbial metabolic is that the likelihood of additional collisions of substrate molecules with a cell surface, after an initial collision, requires only a sparse distribution of transporter sites for maximal rate, leaving room for additional transporters able to collect other substrate types.

The transport properties and gas-sensing performance of chemiresistors based on quasi-1D, single-crystal, SnO2 nanostructures with deliberately synthesized (encoded) segmented morphology are explored. Such nanostructures were obtained using programmable modulation of the gas supersaturation ratio during their growth. Using hydrogen and oxygen as model reducing and oxidizing gases, we show that the responsiveness of these structurally modulated nanowires to gases is improved over that of straight nanowires of the same average diameter. This is due to the presence of small-diameter segments, which dominate the nanostructure's transport properties and to the better tolerance of such nanostructures towards contact effects. The narrow segments approximate the excellent responsiveness of 'necks' between particles in traditional thin film gas sensors but with the significant advantage of greater morphological integrity and stability.

The steady-state growth rate of a marine isolate was related to the concentrations of several carbon and energy source substrates when these substrates limited growth simultaneously in continuous culture. Glucose limitation was characterized by a threshold of 0.21 mg/liter for growth, a half-maximal growth rate at 0.48 mg/liter, U-shaped curves in extractable pool concentration-versus-growth velocity plots, and slow maximal growth rates. Arginine addition reduced the glucose threshold to 0.008 mg/liter, more than doubled the maximal growth rate, and stabilized pool concentrations at low growth rates. Addition of a third substrate, glutamate, caused further reduction of the glucose concentration a steady state. Maximal reduction of the glucose concentration was effected by adding a mixture of 20 amino acids. Steady-state limiting nutrient concentration was dependent on the specific identity of the auxiliary nutrients and on the concentration ratio at which they were supplied. When glucose was supplemented with an equal quantity of an amino acid mixture, the external steady-state glucose remained below 10 mug/liter. When 1 mug of glucose was added to a 2.5-mg/liter amino acid mixture, at least 70% of it was consumed at steady state in spite of the threshold observed. Lack of crossover between metabolic pathways, suggested by the absence of glucose carbon in pool glutamate of arginine-glucose-grown cells, may have been partly responsible for the mixed carbon source stimulation of nutrient accumulation observed. The affinity observed is sufficient to account for normal growth at a total organic substrate concentration of only 0.11 mg/liter when supplied from a suitable mixture.

Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters, collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 micron 3 as calibrated by Coulter impedance. Plastic spheres down to 0.014 micron 3, 0.3 micron in diameter, were resolved. Aquatic bacteria 0.05 micron 3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence. These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.