Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasma

Background

The human microbiome plays a significant role in maintaining normal physiology. Changes in its composition have been associated with bowel disease, metabolic disorders and atherosclerosis. Sequences of microbial origin have been observed within small RNA sequencing data obtained from blood samples. The aim of this study was to characterise the microbiome from which these sequences are derived.

Abundant non-human small RNA sequences were identified in plasma and plasma exosomal samples. Assembly of these short sequences into longer contigs was the pivotal novel step in ascertaining their origin by BLAST searches. Most reads mapped to rRNA sequences. The taxonomic profiles of the microbes detected were very consistent between individuals but distinct from microbiomes reported at other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Many contigs were from plants, presumably of dietary origin.  In addition, extremely abundant small RNAs derived from human Y RNAs were detected.

A characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status. The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37 degreesC using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs+-rich pipette solutions to block K+ currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V-1/2 of -45 +/- 5 mV. In contrast, the T current inactivated with a V-1/2 of -80 +/- 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 +/- 54 nm) and Ni2+ (ED50 = 65 +/- 16 muM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by TTX, reduction in external Na+, application of nifedipine at concentrations below 300 nm or substitution of external Ca2+ with Ba2+, but was reduced by Ni2+ with an ED50 of 6 +/- 1 mum. When cells were stepped from -100 to -30 mV in Ca2+-free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg2+ with an ED50 of 32 +/- 16 mum. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents.

'A Breath of Fresh Air': The experiences of undergraduate nursing students' participating in a creative learning project:This paper presents the experiences of undergraduate nursing students who participated in a creative learning project to explore the cells, tissues and organs of the human body through felt making.

Aim of the study
This paper presents the experiences of undergraduate nursing students who participated in a creative learning project to explore the cells, tissues and organs of the human body through felt making.

Context and Background
This project was funded by a Teaching Innovation Award from the School of Nursing and Midwifery, Queen’s University Belfast to explore creative ways of engaging year one undergraduate nursing students in learning anatomy and physiology. The project was facilitated through collaboration between University Teaching staff and Arts Care, a unique arts and health charity in Northern Ireland.

Methodology
Twelve year one students participated in four workshops designed to explore the cells, tissues and organs of the human body through the medium of felt. Facilitated by an Arts Care artist, students translated their learning into striking felt images. The project culminated in the exhibition of this unique collection of work which has been viewed by fellow students, teaching staff, nurses from practice, and artists from Arts Care, friends, family and members of the public.

Key Findings and conclusions
The opportunity to learn in a more diverse way within a safe and non-judgmental environment was valued, with students’ reporting a greater confidence in life science knowledge. Self- reflection and group discussion revealed that the project was a unique creative learning experience for all involved – students, teaching staff and artist – resulting in individual and collective benefits far beyond knowledge acquisition. As individuals we each felt respected and recognised for our unique contribution to the project. Working in partnership with Arts Care enabled us to experience the benefits of creativity to well-being and reflect upon how engagement in creative activities can help healthcare professionals to focus on the individual patient’s needs and how this is fundamental to enhancing patient-centred care

TRP channel expression in human gingival fibroblasts

Background: The transient receptor potential (TRP) super family of ion channels is believed to play a critical role in sensory physiology, acting as transducers for thermal, mechanical and chemical stimuli. Our understanding of the role of TRP channel expression in gingival fibroblasts is currently limited. The role of non-neuronal TRP channel expression is an area of much research interest particularly since TRP channel activation has recently been hypothesised to be associated with inflammation. Objectives: The present study was designed to determine the expression of TRPV1, TRPV2, TRPV3 and TRPV4 on human gingival fibroblasts. Methods: Human gingival fibroblasts were derived by explant culture from surgical tissue following ethical approval. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum (FCS) in 5% CO2. Cell lysates of gingival fibroblasts were electrophoresed and blotted on to nitrocellulose before probing with specific anti-TRP antibodies. Immunoreactive bands were detected using anti-species antibodies and chemiluminescent detection. Results: Gingival fibroblasts were shown to express proteins corresponding to the TRPV1, TRPV2, TRPV3 and TRPV4 channels as determined by western blotting. Conclusion: This study reports for the first time the expression of TRPV1, TRPV2, TRPV3 and TRPV4 by gingival fibroblasts. Knowledge of the expression of TRP channels by human gingival fibroblasts will guide future research on the roles of TRP channels in sensing the external environment in the oral cavity.

'Heat-sensing' TRP channel expression in human odontoblasts

Background: Thermal changes in the oral cavity are a common trigger of dental pain. Several members of the transient receptor potential (TRP) super family of ion channels are believed to play a critical role in sensory physiology, where they act as transducers for thermal, mechanical and chemical stimuli. Objectives: The present study was designed to determine the expression and functionality of the TRPV1 channel in human odontoblasts. Methods: Cultured human odontoblasts were derived from dental pulp cells induced with 2 mM beta-glycerophosphate. Molecular and protein expression of TRPV1 was confirmed by PCR, western blotting and immunohistochemistry. Functional expression of the ‘heat-sensing' TRPV1 channel was investigated using a Ca2+ microfluorimetry assay in the presence of agonists/antagonists or with appropriate adjustment of the recording chamber temperature. Results: The odontoblastic phenotype of the cells was confirmed by the expression of the odontoblast markers dentin sialophosphoprotein (DSPP) and nestin. Expression of TRPV1 in human odontoblastic cells was confirmed by PCR, western blotting and immunohistochemistry. Odontoblasts were shown to respond to pharmacological agonists and to increasing temperature by an increase in intracellular Ca2+. Both the pharmacological and temperature responses could be blocked by specific antagonists. These results indicate that odontoblasts may sense heat via TRPV1. Conclusion: This study reports that TRPV1 is expressed by human odontoblasts and is activated by specific pharmacological agonists and by heat.
This work was supported by Research Grants from the Royal College of Surgeons of Edinburgh and the British Endodontic Society

Human odontoblasts express functional TRPV2 channels

Background: The transient receptor potential (TRP) ion channels play a critical role in sensory physiology, where they act as transducers of thermal, mechanical and chemical stimuli. We have previously shown the functional expression of several TRP channels by human odontoblast-like cells and proposed their significance in odontoblast sensory perception. Functional expression of the mechano-sensitiveTRPV2 channel by human odontoblasts would further support a role for TRP channels in odontoblast physiology. Objective: The objective of the current study was to determine the functional expression of TRPV2 by human odontoblasts. Methods: Human dental pulp cells were cultured in the presence of 2 mM β-glycerophoshate to induce an odontoblast phenotype. TRPV2 gene expression was determined by qPCR employing custom designed FAM TRPV2 specific primers and probes (Roche, UK) and the Light Cycler 480 Probes Master (Roche). TRPV2 protein expression was determined following SDS-PAGE and Western blotting of cell lysate preparations. Functional expression of TRPV2 was investigated by Ca2+ microfluorimetry. Results: qPCR data indicated robust expression of TRPV2 in odontoblast-like cells. Western blotting revealed a discrete immunoreactive protein band indicating expression of TRPV2 in cell lysates. In functional assays, the chemical agonist of TRPV2, cannabidiol, was shown to elicit [Ca2+]i transients, that were reduced to baseline in the presence of the TRPV2 antagonist Tranilast, suggesting channel functionality in odontoblast-like cells. Conclusion: These results provide the first evidence for the functional expression of TRPV2 in human odontoblast-like cells, providing further support for the role of TRP channels in odontoblast physiology.

Functional expression of TRPV4 in human periodontal ligament cells

Background: Periodontal ligament (PDL) cells are exposed to physical forces in vivo in response to mastication, parafunction, speech and orthodontic tooth movement. Although it has been shown that PDL cells perceive and respond directly to mechanical stimulation, the nature of the ion channels that mediate this mechanotransduction remain to be fully elucidated. The transient receptor potential (TRP) superfamily of ion channels is believed to play a critical role in sensory physiology, where they act as transducers for thermal, chemical and mechanical stimuli. Recent studies have shown that members of the vanilloid (TRPV) and ankyrin (TRPA) subfamilies encode mechanosensitive TRPs. The vanilloid family member TRPV4 is one such non selective calcium permeable cationic channel which has been shown to be activated by chemical ligands, hypotonicity, and mechanical stimuli. Objectives: The objective of the current study was to investigate functional expression of TRPV4 in cultured human PDL cells. Methods: Human PDL cells were grown in Dulbecco's Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100UI/ml penicillin and 100μg/ml streptomycin. Cells in passage 4-6 were used in all experiments. TRPV4 functional expression was determined using ratiometric calcium imaging. Cultured cells were loaded with intracellular Ca2+ probe fura-2 and cells were then stimulated with the TRPV4 agonists, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), GSK1016790A or hypotonic solution. The TRPV4 antagonist RN 1734 was used to block the corresponding agonist responses. Results: PDL fibroblasts responded to application of TRPV4 agonists and hypotonic stimuli by an increase in intracellular calcium which was attenuated in the presence of the TRPV4 antagonist. Conclusions: We have shown for the first time the functional expression of the mechanosensitive TRPV4 channel in human PDL cells. The molecular identity and mechanisms of activation of mechanosensitive TRP channels in PDL cells merit further investigation.

Diversity in levels of intracellular total creatine and triglycerides in human skeletal muscles observed by 1H-MRS

We used1H-magnetic resonance spectroscopy to noninvasively determine total creatine (TCr), choline-containing compounds (Cho), and intracellular (IT) and extracellular (between-muscle fibers) triglycerides (ET) in three human skeletal muscles. Subjects' (n = 15 men) TCr concentrations in soleus [Sol; 100.2 ± 8.3 (SE) mmol/kg dry wt] were lower (P < 0.05) than those in gastrocnemius (Gast; 125.3 ± 9.2 mmol/kg dry wt) and tibialis anterior (TA; 123.7 ± 8.8 mmol/kg dry wt). The Cho levels in Sol (35.8 ± 3.6 mmol/kg dry wt) and Gast (28.5 ± 3.5 mmol/kg dry wt) were higher (P < 0.001 andP < 0.01, respectively) compared with TA (13.6 ± 2.4 mmol/kg dry wt). The IT values were found to be 44.8 ± 4.6 and 36.5 ± 4.2 mmol/kg dry wt in Sol and Gast, respectively. The IT values of TA (24.5 ± 4.5 mmol/kg dry wt) were lower than those of Sol (P < 0.01) and Gast (P < 0.05). There were no differences in ET [116.0 ± 11.2 (Sol), 119.1 ± 18.5 (Gast), and 91.4 ± 19.2 mmol/kg dry wt (TA)]. It is proposed that the differences in metabolite levels may be due to the differences in fiber-type composition and deposition of metabolites due to the adaptation of different muscles during locomotion.

Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model

BACKGROUND: Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status. OBJECTIVES: To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance. METHODS: Healthy swine were randomized to receive by intracoronary route either 30x106 MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups. RESULTS: CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted. CONCLUSION: Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy.

Metabolic and respiratory effects of infused sodium acetate in healthy human subjects.

The metabolic and respiratory effects of intravenous 0.5 M sodium acetate (at a rate of 2.5 mmol/min during 120 min) were studied in nine normal human subjects. O2 consumption (VO2) and CO2 production (VCO2) were measured continuously by open-circuit indirect calorimetry. VO2 increased from 251 +/- 9 to 281 +/- 9 ml/min (P < 0.001), energy expenditure increased from 4.95 +/- 0.17 kJ/min baseline to 5.58 +/- 0.16 kJ/min (P < 0.001), and VCO2 decreased nonsignificantly (211 +/- 7 ml/min vs. 202 +/- 7 ml/min, NS). The extrapulmonary CO2 loss (i.e., bicarbonate generation and excretion) was estimated at 48 +/- 5 ml/min. This observation is consistent with 1 mol of bicarbonate generated from 1 mol of acetate metabolized. Alveolar ventilation decreased from 3.5 +/- 0.2 l/min basal to 3.1 +/- 0.2 l/min (P < 0.001). The minute ventilation (VE) to VO2 ratio decreased from 22.9 +/- 1.3 to 17.6 +/- 0.9 l/l (P < 0.005), arterial PO2 decreased from 93.2 +/- 1.9 to 78.7 +/- 1.6 mmHg (P < 0.0001), arterial PCO2 increased from 39.2 +/- 0.7 to 42.1 +/- 1.1 mmHg (P < 0.0001), pH from 7.40 +/- 0.005 to 7.50 +/- 0.007 (P < 0.005), and arterial bicarbonate concentration from 24.2 +/- 0.7 to 32.9 +/- 1.1 (P < 0.0001). These observations indicate that sodium acetate infusion results in substantial extrapulmonary CO2 loss, which leads to a relative decrease of total and alveolar ventilation.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

Effects of infused amino acids on glucose production and utilization in healthy human subjects.

Amino acids have been reported to increase endogenous glucose production in normal human subjects during hyperinsulinemia: however, controversy exists as to whether insulin-mediated glucose disposal is inhibited under these conditions. The effect of an amino acid infusion on glucose oxidation rate has so far not been determined. Substrate oxidation rates, endogenous glucose production, and [13C]glucose synthesis from [13C]bicarbonate were measured in six normal human subjects during sequential infusions of exogenous glucose and exogenous glucose with (n = 5) or without (n = 5) exogenous amino acids. Amino acids increased endogenous glucose production by 84% and [13C]glucose synthesis by 235%. Glucose oxidation estimated from indirect calorimetry decreased slightly after amino acids, but glucose oxidation estimated from [13C]glucose-13CO2 data was increased by 14%. It is concluded that gluconeogenesis is the major pathway of amino acid degradation. During amino acid administration, indirect calorimetry underestimates the true rate of glucose oxidation, whereas glucose oxidation calculated from the 13C enrichment of expired CO2 during [U-13C]glucose infusion does not. A slight stimulation of glucose oxidation during amino acid infusion, concomitant with an increased plasma insulin concentration, indicates that amino acids do not inhibit glucose oxidation.

Determination of displacement, stress- and strain-distribution in the human heart: a FE-model on the basis of MR imaging.

The determination of characteristic cardiac parameters, such as displacement, stress and strain distribution are essential for an understanding of the mechanics of the heart. The calculation of these parameters has been limited until recently by the use of idealised mathematical representations of biventricular geometries and by applying simple material laws. On the basis of 20 short axis heart slices and in consideration of linear and nonlinear material behaviour we have developed a FE model with about 100,000 degrees of freedom. Marching Cubes and Phong's incremental shading technique were used to visualise the three dimensional geometry. In a quasistatic FE analysis continuous distribution of regional stress and strain corresponding to the endsystolic state were calculated. Substantial regional variation of the Von Mises stress and the total strain energy were observed at all levels of the heart model. The results of both the linear elastic model and the model with a nonlinear material description (Mooney-Rivlin) were compared. While the stress distribution and peak stress values were found to be comparable, the displacement vectors obtained with the nonlinear model were generally higher in comparison with the linear elastic case indicating the need to include nonlinear effects.

Functional and structural basis for a bacteriophage homolog of human RAD52.

In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis[4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.

Early processing in the human lateral occipital complex is highly responsive to illusory contours but not to salient regions.

Human electrophysiological studies support a model whereby sensitivity to so-called illusory contour stimuli is first seen within the lateral occipital complex. A challenge to this model posits that the lateral occipital complex is a general site for crude region-based segmentation, based on findings of equivalent hemodynamic activations in the lateral occipital complex to illusory contour and so-called salient region stimuli, a stimulus class that lacks the classic bounding contours of illusory contours. Using high-density electrical mapping of visual evoked potentials, we show that early lateral occipital cortex activity is substantially stronger to illusory contour than to salient region stimuli, whereas later lateral occipital complex activity is stronger to salient region than to illusory contour stimuli. Our results suggest that equivalent hemodynamic activity to illusory contour and salient region stimuli probably reflects temporally integrated responses, a result of the poor temporal resolution of hemodynamic imaging. The temporal precision of visual evoked potentials is critical for establishing viable models of completion processes and visual scene analysis. We propose that crude spatial segmentation analyses, which are insensitive to illusory contours, occur first within dorsal visual regions, not the lateral occipital complex, and that initial illusory contour sensitivity is a function of the lateral occipital complex.