Host genotype and exercise exhibit species-level selection for members of the gut bacterial communities in the mouse digestive system.

The mammalian gut microbiome can potentially impact host health and disease state. It is known that the mouse-genome, eating-behavior, and exercise-status promotes higher taxonomic rank-level alterations (e.g. family to phyla-level) of the gut microbiota. Here, host genotype or activity status was investigated to determine if selection of individual bacterial species or strains could be discerned within the murine digestive system. For this study, the fecal bacterial community of adenylyl cyclase 5 knock-out (AC5KO, n = 7) mice or their wild-type (WT, n = 10) littermates under exercise or sedentary conditions were profiled by sequencing rRNA operons. AC5KO mice were chosen since this genotype displays enhanced longevity/exercise capacity and protects against cardiovascular/metabolic disease. Profiling of rRNA operons using the Oxford MinION yielded 65,706 2-D sequences (after size selection of 3.7-5.7 kb) which were screened against an NCBI 16S rRNA gene database. These sequences were binned into 1,566 different best BLAST hits (BBHs) and counted for each mouse sample. Non-metric multidimensional scaling (NMDS) of the gut microbial community demonstrated clustering by physical activity (p = 0.001) but not by host genotype. Additionally, sequence similarity and phylogenetic analysis demonstrated that different bacterial species (closely related to Muribaculum intestinale and Parasutterella excrementihominis) inhabit AC5KO or WT mice depending on activity status. Other bacterial species of the gut microbiota did not follow such patterning (e.g. Turicibacter sanguinis and Turicimonas muris). Our results support the need of improved taxonomic resolution for better characterization of bacterial communities to deepen our understanding of the role of the gut microbiome on host health.

Toward Hyperpolarization of Oil Molecules via Single Nitrogen Vacancy Centers in Diamond.

Efficient polarization of organic molecules is of extraordinary relevance when performing nuclear magnetic resonance (NMR) and imaging. Commercially available routes to dynamical nuclear polarization (DNP) work at extremely low temperatures, relying on the solidification of organic samples and thus bringing the molecules out of their ambient thermal conditions. In this work, we investigate polarization transfer from optically pumped nitrogen vacancy centers in diamond to external molecules at room temperature. This polarization transfer is described by both an extensive analytical analysis and numerical simulations based on spin bath bosonization and is supported by experimental data in excellent agreement. These results set the route to hyperpolarization of diffusive molecules in different scenarios and consequently, due to an increased signal, to high-resolution NMR.

Nuclear magnetic resonance detection and spectroscopy of single proteins using quantum logic.

Nuclear magnetic resonance spectroscopy is a powerful tool for the structural analysis of organic compounds and biomolecules but typically requires macroscopic sample quantities. We use a sensor, which consists of two quantum bits corresponding to an electronic spin and an ancillary nuclear spin, to demonstrate room temperature magnetic resonance detection and spectroscopy of multiple nuclear species within individual ubiquitin proteins attached to the diamond surface. Using quantum logic to improve readout fidelity and a surface-treatment technique to extend the spin coherence time of shallow nitrogen-vacancy centers, we demonstrate magnetic field sensitivity sufficient to detect individual proton spins within 1 second of integration. This gain in sensitivity enables high-confidence detection of individual proteins and allows us to observe spectral features that reveal information about their chemical composition.

Spectroscopy of surface-induced noise using shallow spins in diamond.

We report on the noise spectrum experienced by few nanometer deep nitrogen-vacancy centers in diamond as a function of depth, surface coating, magnetic field and temperature. Analysis reveals a double-Lorentzian noise spectrum consistent with a surface electronic spin bath in the low frequency regime, along with a faster noise source attributed to surface-modified phononic coupling. These results shed new light on the mechanisms responsible for surface noise affecting shallow spins at semiconductor interfaces, and suggests possible directions for further studies. We demonstrate dynamical decoupling from the surface noise, paving the way to applications ranging from nanoscale NMR to quantum networks.

Nuclear magnetic resonance spectroscopy with single spin sensitivity.

Nuclear magnetic resonance spectroscopy and magnetic resonance imaging at the ultimate sensitivity limit of single molecules or single nuclear spins requires fundamentally new detection strategies. The strong coupling regime, when interaction between sensor and sample spins dominates all other interactions, is one such strategy. In this regime, classically forbidden detection of completely unpolarized nuclei is allowed, going beyond statistical fluctuations in magnetization. Here we realize strong coupling between an atomic (nitrogen-vacancy) sensor and sample nuclei to perform nuclear magnetic resonance on four (29)Si spins. We exploit the field gradient created by the diamond atomic sensor, in concert with compressed sensing, to realize imaging protocols, enabling individual nuclei to be located with Angstrom precision. The achieved signal-to-noise ratio under ambient conditions allows single nuclear spin sensitivity to be achieved within seconds.

Multiple intrinsically identical single-photon emitters in the solid state.

Emitters of indistinguishable single photons are crucial for the growing field of quantum technologies. To realize scalability and increase the complexity of quantum optics technologies, multiple independent yet identical single-photon emitters are required. However, typical solid-state single-photon sources are inherently dissimilar, necessitating the use of electrical feedback or optical cavities to improve spectral overlap between distinct emitters. Here we demonstrate bright silicon vacancy (SiV(-)) centres in low-strain bulk diamond, which show spectral overlap of up to 91% and nearly transform-limited excitation linewidths. This is the first time that distinct single-photon emitters in the solid state have shown intrinsically identical spectral properties. Our results have impact on the application of single-photon sources for quantum optics and cryptography.

Epithelioid haemangioma: a rare cause of painful erections and sleep deprivation.

Epithelioid haemangioma of the penis is a rare condition which usually presents a solid single nodule. We report a case in a 43-year-old man who presented with painful erections and sleep disturbance with two palpable penile nodules. Magnetic resonance imaging with an artificially induced erection revealed these as individual lesions, and local excision was successfully undertaken. Pathological diagnosis of epithelioid haemangioma was confirmed with positive staining for CD31. Although rare, penile epithelioid haemangioma should be considered as a differential in an atypical penile mass. Induction in of an artificial erection prior to MRI can aid diagnosis and treatment is typically with surgical excision.

Distribution of Hepatitis C virus genotypes among newly acquired HCV infections in British Columbia (2000-2013).

BACKGROUND: Characterization of newly acquired Hepatitis C virus (HCV) infections is important in order to understand the epidemiology and spread of HCV. OBJECTIVE: To describe the Hepatitis C virus (HCV) genotype distribution of newly acquired HCV infections in the province of British Columbia for the period 2000-2013. METHODS: A descriptive cross-sectional analysis of multi-year data on HCV genotypes. Time trends for the proportion of different HCV genotypes are presented only for newly acquired (incident) HCV infections. RESULTS: For acute cases, genotype 1a remains the dominant HCV type in circulation (50%), followed by genotype 3a (34%). HCV genotype 1b declined, while genotype 2 was relatively stable. Phylogenetically-related clusters of HCV strains were observed indicating a common source of infection. CONCLUSION: Enhanced hepatitis surveillance provides a mechanism for monitoring different HCV strains currently circulating in the community. While HCV genotype 1a continues to be the most prevalent, changes in the relative frequency of genotypes 1 and 3 have been observed. This may have important implications for the control and prevention of the infection.

Detecting and polarizing nuclear spins with double resonance on a single electron spin.

We report the detection and polarization of nuclear spins in diamond at room temperature by using a single nitrogen-vacancy (NV) center. We use Hartmann-Hahn double resonance to coherently enhance the signal from a single nuclear spin while decoupling from the noisy spin bath, which otherwise limits the detection sensitivity. As a proof of principle, we (i) observe coherent oscillations between the NV center and a weakly coupled nuclear spin and (ii) demonstrate nuclear-bath cooling, which prolongs the coherence time of the NV sensor by more than a factor of 5. Our results provide a route to nanometer scale magnetic resonance imaging and novel quantum information processing protocols.

Detection of a few metallo-protein molecules using color centers in nanodiamonds.

Nanometer-sized diamonds containing nitrogen-vacancy defect centers (NV) are promising nanosensors in biological environments due to their biocompatibility, bright fluorescence, and high magnetic sensitivity at ambient conditions. Here we report on the detection of ferritin molecules using magnetic noise induced by the inner paramagnetic iron as a contrast mechanism. We observe a significant reduction of both coherence and relaxation time due to the presence of ferritin on the surface of nanodiamonds. Our theoretical model is in excellent agreement with the experimental data and establishes this method as a novel sensing technology for proteins.

High spatial and temporal resolution wide-field imaging of neuron activity using quantum NV-diamond.

A quantitative understanding of the dynamics of biological neural networks is fundamental to gaining insight into information processing in the brain. While techniques exist to measure spatial or temporal properties of these networks, it remains a significant challenge to resolve the neural dynamics with subcellular spatial resolution. In this work we consider a fundamentally new form of wide-field imaging for neuronal networks based on the nanoscale magnetic field sensing properties of optically active spins in a diamond substrate. We analyse the sensitivity of the system to the magnetic field generated by an axon transmembrane potential and confirm these predictions experimentally using electronically-generated neuron signals. By numerical simulation of the time dependent transmembrane potential of a morphologically reconstructed hippocampal CA1 pyramidal neuron, we show that the imaging system is capable of imaging planar neuron activity non-invasively at millisecond temporal resolution and micron spatial resolution over wide-fields.

Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells.

Fluorescent particles are routinely used to probe biological processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biological environments. Here, we demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centres inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centres with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The experiments reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biological systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.

Microbial reduction of uranium under iron- and sulfate-reducing conditions: Effect of amended goethite on microbial community composition and dynamics.

There is a growing need for a better understanding of the biogeochemical dynamics involved in microbial U(VI) reduction due to an increasing interest in using biostimulation via electron donor addition as a means to remediate uranium contaminated sites. U(VI) reduction has been observed to be maximized during iron-reducing conditions and to decrease upon commencement of sulfate-reducing conditions. There are many unknowns regarding the impact of iron/sulfate biogeochemistry on U(VI) reduction. This includes Fe(III) availability as well as the microbial community changes, including the activity of iron-reducers during the uranium biostimulation period even after sulfate reduction becomes dominant. Column experiments were conducted with Old Rifle site sediments containing Fe-oxides, Fe-clays, and sulfate rich groundwater. Half of the columns had sediment that was augmented with small amounts of Fe(III) in the form of (57)Fe-goethite, allowing for a detailed tracking of minute changes of this added phase to study the effects of increased Fe(III) levels on the overall biostimulation dynamics. Mössbauer spectroscopy showed that the added (57)Fe-goethite was bioreduced only during the first thirty days of biostimultuion, after which it remained constant. Augmentation with Fe(III) had a significant effect on the total flux of electrons towards different electron acceptors; it suppressed the degree of sulfate reduction, had no significant impact on Geobacter-type bacterial numbers but decreased the bacterial numbers of sulfate reducers and affected the overall microbial community composition. The addition of Fe(III) had no noticeable effect on the total uranium reduction.

A single-cell pedigree analysis of alternative stochastic lymphocyte fates.

In contrast to most stimulated lymphocytes, B cells exposed to Toll-like receptor 9 ligands are nonself-adherent, allowing individual cells and families to be followed in vitro for up to 5 days. These B cells undergo phases typical of an adaptive response, dividing up to 6 times before losing the impetus for further growth and division and eventually dying by apoptosis. Using long-term microscopic imaging, accurate histories of individual lymphocyte fates were collected. Quantitative analysis of family relationships revealed that times to divide of siblings were strongly related but these correlations were progressively lost through consecutive divisions. A weaker, but significant, correlation was also found for death times among siblings. Division cessation is characterized by a loss of cell growth and the division in which this occurs is strongly inherited from the original founder cell and is related to the size this cell reaches before its first division. Thus, simple division-based dilution of factors synthesized during the first division may control the maximum division reached by stimulated cells. The stochastic distributions of times to divide, times to die, and divisions reached are also measured. Together, these results highlight the internal cellular mechanisms that control immune responses and provide a foundation for the development of new mathematical models that are correct at both single-cell and population levels.

Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing.

Stable isotope probing (SIP) was used to identify the active members in a benzene-degrading sulfidogenic consortium. SIP-terminal restriction fragment length polymorphism analysis indicated that a 270-bp peak incorporated the majority of the (13)C label and is a sequence closely related to that of clone SB-21 (GenBank accession no. AF029045). This target may be an important biomarker for anaerobic benzene degradation in the field.

Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP.

It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed.

13C-carrier DNA shortens the incubation time needed to detect benzoate-utilizing denitrifying bacteria by stable-isotope probing.

The active bacterial community able to utilize benzoate under denitrifying conditions was elucidated in two coastal sediments using stable-isotope probing (SIP) and nosZ gene amplification. The SIP method employed samples from Norfolk Harbor, Virginia, and a Long-Term Ecosystem Observatory (no. 15) off the coast of Tuckerton, New Jersey. The SIP method was modified by use of archaeal carrier DNA in the density gradient separation. The carrier DNA significantly reduced the incubation time necessary to detect the (13)C-labeled bacterial DNA from weeks to hours in the coastal enrichments. No denitrifier DNA was found to contaminate the archaeal (13)C-carrier when [(12)C]benzoate was used as a substrate in the sediment enrichments. Shifts in the activity of the benzoate-utilizing denitrifying population could be detected throughout a 21-day incubation. These results suggest that temporal analysis using SIP can be used to illustrate the initial biodegrader(s) in a bacterial population and to document the cross-feeding microbial community.

Hypothalamic STAT proteins: regulation of somatostatin neurones by growth hormone via STAT5b.

Signal transducers and activators of transcription (STATs) are a family of transcription factors linked to class I cytokine receptors. In the present study, we investigated whether their distribution in the hypothalamus reflects the feedback regulation by growth hormone and what role they might play in the functioning of target neurones. We demonstrate that each of the seven known STATs has a distinct distribution in the hypothalamus. Notably, the STAT5 proteins, that are important in growth hormone (GH) and prolactin signalling in peripheral tissues, were expressed in somatostatin neurones of the periventricular nucleus and dopamine neurones of the arcuate nucleus. Because somatostatin neurones are regulated by feedback from circulating GH, we investigated the importance of STAT5 in these neurones. We demonstrate that STAT5b protein expression, similar to somatostatin mRNA, is sexually dimorphic in the periventricular nucleus of rats and mice. Furthermore, chronic infusion of male dwarf rats with GH increased the expression of STAT5b, while a single injection of GH into similar rats induced the phosphorylation of STAT5 proteins. The cellular abundance of somatostatin mRNA in STAT5b-deficient mice was significantly reduced in the periventricular nucleus, effectively reducing the sexually dimorphic expression. These results are consistent with the hypothesis that STAT5 proteins are involved in the feedback regulation of somatostatin neurones by GH, and that these neurones may respond to patterned GH secretion to reinforce sexual dimorphism in the GH axis.

Examining the process of community development.

AIM OF THE STUDY: To present the results of evaluation research investigating a successful community development project. RATIONALE: Many governments, health care agencies, and organizations require community participation in health care. As a result, nurses and other health professionals are often required to practice using a philosophy of community development. Although the theoretical, philosophical, and practical components of community development are well articulated, there is little evidence that exemplifies the experience of initiating and participating in a community development project. This paper provides evidence of how one organization successfully engaged the broader community on a respite care project. METHOD: Using qualitative research methods and guided by the principles and practices of participatory action research, this evaluation engaged with the community in data collection, analysis, dissemination of finding, and in promoting effective change. RESULTS: Four themes emerged that provide insight into how one HIV/AIDS organization successfully undertook community development. These themes include: (a) identifying a community need; (b) addressing the various components identified in the community development process; (c) highlighting the strategies used to engage in successful community development; and (d) attending to factors that influence community development. CONCLUSION: The results of this research contribute to the body of knowledge related to engaging in the process of community development. By highlighting the experiences of one community group, it is hoped that nurses can learn and incorporate this knowledge into practice.

A secreted fluorescent reporter targeted to pituitary growth hormone cells in transgenic mice.

In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca2+ concentrations after loading with Ca2+ sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells.