Circulating MiR-21 expression is upregulated after 30 days of head-down tilt bed rest.

Relative expression of miR-21-5p in serum was upregulated in response to 30 days of bed rest, and miRNA fold changes were positively associated with serum calcium changes. INTRODUCTION: Circulating miRNAs (c-miRNAs) have potential as biomarkers of cellular activity, and they may play a role in cell-to-cell communication. The purpose of this study was to examine c-miRNA and bone marker responses to a 30-day six-degree head-down bed rest protocol at an ambient 0.5% CO2. METHODS: Eleven participants (6 males/5 females, 25-50 years) had fasting blood draws taken 3 days before and immediately after completing the 30-day bed rest protocol at the Institute for Aerospace Medicine in Germany. Serum relative expression of miRNAs associated with bone function (miR-21-5p, -100-5p, -125b-5p, -126-3p) were analyzed using qPCR, and serum bone markers were quantitated using ELISA. RESULTS: Serum bone markers, sclerostin, and calcium significantly increased (p ≤ 0.036), and total hip aBMD significantly decreased (p = 0.003) post bed rest. Serum miR-21-5p relative expression was significantly upregulated (p = 0.018) post bed rest. Fold changes in miR-126-3p (r = 0.82, p = 0.002) and miR-21-5p (r = 0.62, p = 0.042) were positively correlated with absolute change in serum calcium. There were no sex differences in miRNA responses; women had greater percent increases in TRAP5b (37.3% vs. 16.9% p = 0.021) and greater percent decreases in total hip aBMD (- 2.15% vs. - 0.69%, p = 0.034) than men. CONCLUSION: c-miR-21-5p has potential as a biomarker of bone resorption and bone loss in an unloading condition. The upregulation of miR-21-5p may reflect an increase in osteoclast activity after bed rest, which is corroborated by the increase in TRAP5b.

Geospatial epidemiology of Staphylococcus aureus in a tropical setting: an enabling digital surveillance platform.

Delivery of information to clinicians on evolving antimicrobial susceptibility needs to be accurate for the local needs, up-to-date and readily available at point of care. In northern Australia, bacterial infection rates are high but resistance to first- and second-line antibiotics is poorly described and currently-available datasets exclude primary healthcare data. We aimed to develop an online geospatial and interactive platform for aggregating, analysing and disseminating data on regional bacterial pathogen susceptibility. We report the epidemiology of Staphylococcus aureus as an example of the power of digital platforms to tackle the growing spread of antimicrobial resistance in a high-burden, geographically-sparse region and beyond. We developed an online geospatial platform called HOTspots that visualises antimicrobial susceptibility patterns and temporal trends. Data on clinically-important bacteria and their antibiotic susceptibility profiles were sought from retrospectively identified clinical specimens submitted to three participating pathology providers (96 unique tertiary and primary healthcare centres, n = 1,006,238 tests) between January 2008 and December 2017. Here we present data on S. aureus only. Data were available on specimen type, date and location of collection. Regions from the Australian Bureau of Statistics were used to provide spatial localisation. The online platform provides an engaging visual representation of spatial heterogeneity, demonstrating striking geographical variation in S. aureus susceptibility across northern Australia. Methicillin resistance rates vary from 46% in the west to 26% in the east. Plots generated by the platform show temporal trends in proportions of S. aureus resistant to methicillin and other antimicrobials across the three jurisdictions of northern Australia. A quarter of all, and up to 35% of methicillin-resistant S. aureus (MRSA) blood isolates in parts of the northern Australia were resistant to inducible-clindamycin. Clindamycin resistance rates in MRSA are worryingly high in regions of northern Australia and are a local impediment to empirical use of this agent for community MRSA. Visualising routinely collected laboratory data with digital platforms, allows clinicians, public health physicians and guideline developers to monitor and respond to antimicrobial resistance in a timely manner. Deployment of this platform into clinical practice supports national and global efforts to innovate traditional disease surveillance systems with the use of digital technology and to provide practical solutions to reducing the threat of antimicrobial resistance.

A caffeine containing weight loss supplement augments hemodynamic responses after exercise.

BACKGROUND: Since the effects of supplements can be potentially harmful and/or ineffective to obtain desired positive benefits, there is a need to investigate supplementation to understand the responses of physiological systems, to educate consumers, and to provide feedback for businesses creating these supplements. The purpose of the current study was to test hemodynamic responses of a weight loss supplement and determine its effects on hemodynamic variables. METHODS: 31 participants underwent a randomized, double-blind, crossover study design and received a placebo or supplement on two separate days. Baseline measures of all variables were assessed prior to exercise. During exercise, each participant performed treadmill running at 80% VO2PEAK until volitional fatigue. Immediately post-exercise, hemodynamic measures were recorded at multiple time points. RESULTS: There was a significant condition∗time interaction with the supplement having a higher PWV for the carotid to femoral segment (p=0.004). There were also significant condition∗time interactions for heart rate (p=0.001). Large arterial elasticity was significantly lower for the supplement (p=0.005). Systolic blood pressure was conditionally higher (p=0.001), as was diastolic blood pressure (p=0.003) and mean arterial pressure (p=0.003). Vascular resistance was conditionally higher for the supplement (p=0.044). CONCLUSIONS: Ingredients in the supplement caused multiple negative effects within hemodynamics and were ineffective at increasing running time.

RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Socioeconomic deprivation and NHS orthodontic treatment delivery in Scotland.

OBJECTIVE: The purpose of this observational study was to investigate the relationship between deprivation and the delivery of primary care NHS orthodontic services across Scotland. METHOD: Deprivation was measured using the Scottish Index of Multiple Deprivation (SIMD). The Information Services Division, NHS National Services Scotland, supplied data on all claims for orthodontic treatments in Scotland for the years 2008 and 2009. Each claim was assigned to a SIMD quintile (SIMD 1 being the most deprived, and SIMD 5 the least deprived), and odds ratios were calculated. RESULTS: Uptake of orthodontic services is highest in the least deprived areas. Patients from the least deprived areas are nearly twice as likely to receive orthodontic treatment as those from the most deprived areas (odds ratio of 1.90 with a 95% confidence interval (CI) 1.86 to 1.94). CONCLUSION: Patients from more the most deprived backgrounds are less likely to receive orthodontic treatment than those from more affluent backgrounds, which does not necessarily reflect need.

Plasmon-dispersion corrections and constraints for surface selection rules of single molecule SERS spectra.

The problem of extracting information from relative intensities of Raman peaks in surface-enhanced-Raman-scattering (SERS) is intimately related to several important topics in the technique. Among them: (i) the possibility (or sometimes impossibility) of observing surface selection rules in different situations, (ii) the role of analyte resonance conditions, (iii) the crucial inclusion of plasmon-resonance dispersion corrections in the analysis of relative Raman intensities among peaks, and (iv) the connection of these phenomena with (broader) issues like surface-enhanced fluorescence (SEF). This paper deals with the underlying connections among these (apparently disconnected at first sight) topics. The technique is now at a mature stage to review the aforementioned phenomena from a unified point of view; thus pinpointing the most important issues, clarifying concepts that have been historically confusing (or treated in isolation), and paving the road for future developments.

Mapping water table depth using geophysical and environmental variables.

Despite its importance, accurate representation of the spatial distribution of water table depth remains one of the greatest deficiencies in many hydrological investigations. Historically, both inverse distance weighting (IDW) and ordinary kriging (OK) have been used to interpolate depths. These methods, however, have major limitations: namely they require large numbers of measurements to represent the spatial variability of water table depth and they do not represent the variation between measurement points. We address this issue by assessing the benefits of using stepwise multiple linear regression (MLR) with three different ancillary data sets to predict the water table depth at 100-m intervals. The ancillary data sets used are Electromagnetic (EM34 and EM38), gamma radiometric: potassium (K), uranium (eU), thorium (eTh), total count (TC), and morphometric data. Results show that MLR offers significant precision and accuracy benefits over OK and IDW. Inclusion of the morphometric data set yielded the greatest (16%) improvement in prediction accuracy compared with IDW, followed by the electromagnetic data set (5%). Use of the gamma radiometric data set showed no improvement. The greatest improvement, however, resulted when all data sets were combined (37% increase in prediction accuracy over IDW). Significantly, however, the use of MLR also allows for prediction in variations in water table depth between measurement points, which is crucial for land management.

Functional neuroimaging with MEG: normative language profiles.

The reliability of language-specific brain activation profiles was assessed using Magnetoencephalography (MEG) in five experiments involving ninety-seven normal volunteers of both genders ranging in age from seven to eighty-four years. MEG data were analyzed with a fully automated method to eliminate subjective judgments in the process of deriving the activation profiles. Across all experiments, profiles were characterized by significant bilateral activity centered in the superior temporal gyrus, and in activity lateralized to the left middle temporal gyrus. These features were invariant across age, gender, variation in task characteristics, and mode of stimulus presentation. The absolute amount of activation, however, did decline with age in the auditory tasks. Moreover, contrary to the commonly held belief that left hemisphere dominance for language is greater in men than in women, our data revealed an opposite albeit a not consistently significant trend.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

Disrupted in Schizophrenia 1 (DISC1) is a multicompartmentalized protein that predominantly localizes to mitochondria.

DISC1 is disrupted by a chromosomal translocation cosegregating with schizophrenia and recurrent major depression in a large Scottish family and has also been reported as a potential susceptibility locus in independent populations. We reveal a widespread and complex pattern of DISC1 expression, with at least five forms of Disrupted in Schizophrenia 1 DISC1 detectable. Mitochondria are the predominant site of DISC1 expression with additional nuclear, cytoplasmic, and actin-associated locations evident. Although the subcellular targeting of DISC1 is clearly complex, the association with mitochondria is of interest as many mitochondrial deficits have been reported in schizophrenia and other neuropsychiatric illnesses. Moreover, of the many cellular functions performed by mitochondria, their role in oxidative phosphorylation, calcium homeostasis, and apoptosis may hold particular relevance for the neuronal disturbances believed to be involved in the pathogenesis of schizophrenia.

The plug domain of a neisserial TonB-dependent transporter retains structural integrity in the absence of its transmembrane beta-barrel.

Transferrin binding protein A (TbpA) is a TonB-dependent outer membrane protein expressed by pathogenic bacteria for iron acquisition from human transferrin. The N-terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of Escherichia coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB-dependent transporters. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. These experiments suggest that the plug domain may fold independently of the beta-barrel, but extracellular loops of the beta-barrel are required for ligand binding.

The promise of structural genomics in the discovery of new antimicrobial agents.

Structural Genomics stands out among the emerging fields of proteomics since it influences the drug discovery process at so many points. Recent developments in protein expression technologies, x-ray crystallography and NMR spectroscopy provide the essential elements for high-throughput structure determination platforms. Bioinformatics methods to interrogate the resulting data will provide comprehensive, genome-wide databases of protein structure. Genomic sequencing and methods for high-throughput expression and protein purification are furthest advanced for microbial genes and so these have been the early targets for structural genomics initiatives. The information will be invaluable in understanding gene function, designing broad-spectrum small molecule inhibitors and in better understanding drug-host interactions.

Comparative changes in plasma concentrations of progesterone, oestradiol and LH during the ovulatory cycle in a multiple ovulating male line and a single ovulating traditional line of turkeys.

The aim of this study was to compare the profile of circulating concentrations of LH, progesterone and oestradiol in a multiple ovulating male line with that of a single ovulating line of traditional turkeys. Plasma samples from seven traditional and 12 male-line turkeys were obtained every 3 h for 36 h. Male-line and traditional turkeys had single peaks of LH and progesterone that were of similar duration in both lines. The mean height of the plasma peaks of LH and progesterone were similar in the two lines and there was no detectable peak plasma oestrogen concentration. Mean plasma concentrations of LH and oestrogen were higher in single compared with multiple ovulating turkeys, whereas there were no differences in mean plasma progesterone concentrations. The results indicate that the multiple ovulation state in genetically selected high-growth lines of turkey may be the result of a correlated response in the steroidogenic capacity of ovarian tissue associated with low plasma concentrations of oestrogen rather than of a disturbance in the hormone profile of the ovulatory cycle.

Sequestration and phosphorylation of the prostaglandin E2 EP4 receptor: dependence on the C-terminal tail.

The prostaglandin E2 (PGE2) EP4 subtype is one of four prostanoid receptors that use PGE2 as the preferred ligand. We have investigated the agonist-mediated regulation of EP4 using a multifaceted approach. Short-term (30 min) agonist challenge of recombinant EP4 expressed in human embryonic kidney 293 cells (EP4-HEK293 cells) with PGE2 (1 microM) resulted in the desensitization of intracellular cyclic AMP (cAMP) accumulation and a reduction in cell surface [3H]PGE2 specific binding sites. These events correlated with sequestration of EP4, as visualized by immunofluorescence confocal microscopy and phosphorylation, as shown by [32P]orthophosphate labeling of the receptor. Stimulation of protein kinase A activity in EP4-HEK293 cells (10 microM forskolin or 1 mM 8-bromo-cAMP) did not induce EP4 desensitization, sequestration, or phosphorylation. In contrast, stimulation of protein kinase C activity (100 nM phorbol 12-myristate 13-acetate) attenuated PGE2-induced adenylyl cyclase activity and increased EP4 phosphorylation, but did not induce sequestration or a reduction in [3H]PGE2 specific binding sites. EP4 receptors containing a third intracellular loop deletion [EP4 (del. 215-263)] or a carboxyl-terminal tail truncation [EP4 (del. 355)] of EP4 were used to demonstrate that the C-terminal tail governs sequestration as well as phosphorylation of the receptor.

A prospective randomised concurrent comparison of the COBE Spectra Version 4.7, COBE Spectra Version 6 (auto PBSC) and Haemonetics MCS+ cell separators for leucapheresis in patients with haematological and non-haematological malignancies.

A prospective study of the CD34+ cell collection efficiency of three cell separators was undertaken comparing the mononuclear cell, CD34+ cell and CFU-GM yield. Twenty patients were entered in the study, all had received mobilising chemotherapy and daily G-CSF (5 microg/kg subcutaneously). The first leucapheresis was performed when the peripheral blood absolute CD34+ cell count was > or = 20 cells/microl. All patients underwent two leucaphereses on consecutive days. The patients were randomised to undergo either the first or second leucapheresis using the COBE Spectra Version 4.7 and then randomised to either the COBE Spectra Version 6 or Haemonetics MCS+ for the other leucapheresis. The target durations of the procedure on the COBE Spectra Version 4.7 and Version 6 were 180 min or two total blood volumes (TBV), and for the Haemonetics MCS+ was 20 cycles with four recirculations. All machines were operated on the 1997 software supplied by the respective manufacturers. The time taken for the procedure was significantly longer with both the Haemonetics MCS+ and the COBE Spectra Version 6 than the COBE Spectra Version 4.7. Both COBE Spectra versions processed significantly larger volumes of blood than the Haemonetics MCS+. The absolute yield of mononuclear cells, CFU-GM and CD34+ cells were all significantly lower with the Haemonetics MCS+ compared with both COBE Spectra Versions, as were the yields per unit volume of blood processed. There was no significant difference in the reduction in the platelet count following leucapheresis with any of the machines.

A structural genomics approach to the study of quorum sensing: crystal structures of three LuxS orthologs.

BACKGROUND: Quorum sensing is the mechanism by which bacteria control gene expression in response to cell density. Two major quorum-sensing systems have been identified, system 1 and system 2, each with a characteristic signaling molecule (autoinducer-1, or AI-1, in the case of system 1, and AI-2 in system 2). The luxS gene is required for the AI-2 system of quorum sensing. LuxS and AI-2 have been described in both Gram-negative and Gram-positive bacterial species and have been shown to be involved in the expression of virulence genes in several pathogens. RESULTS: The structure of the LuxS protein from three different bacterial species with resolutions ranging from 1.8 A to 2.4 A has been solved using an X-ray crystallographic structural genomics approach. The structure of LuxS reported here is seen to have a new alpha-beta fold. In all structures, an equivalent homodimer is observed. A metal ion identified as zinc was seen bound to a Cys-His-His triad. Methionine was found bound to the protein near the metal and at the dimer interface. CONCLUSIONS: These structures provide support for a hypothesis that explains the in vivo action of LuxS. Specifically, acting as a homodimer, the protein binds a methionine analog, S-ribosylhomocysteine (SRH). The zinc atom is in position to cleave the ribose ring in a step along the synthesis pathway of AI-2.

Ovarian steroid hormone production in a multiple ovulating male line and a single ovulating traditional line of turkeys.

Ovarian production of oestradiol and progesterone was investigated in a single ovulating traditional line and a multiple ovulating male line of turkeys. The oestradiol output from small follicles in response to LH stimulation and the aromatase activity of the residual ovary were also compared. The multiple hierarchy of follicles in the male line was shown to consist of a series of follicles of a similar size and stage of physiological maturation. The follicles of the traditional line produced significantly greater quantities of oestradiol than those of the male line. Impaired oestradiol production may have resulted in the lower plasma oestradiol concentration observed in the male line. There was no difference in follicular progesterone output between the traditional-line and male-line turkeys. The oestradiol output of whole small white follicles in response to LH stimulation was similar in the two lines and the depressed steroidogenesis of the male line was not changed. The aromatase activity of the small follicles was also lower in the male line, and there was no evidence that the male line ovary produced oestradiol in quantities proportional to its size. The results demonstrated that the hierarchy of follicles in the male line consisted of groups of follicles of similar mass and hormone output and indicated that selection for increased meat yield may have resulted in reduced ovarian steroidogenesis in male-line turkeys, in comparison with traditional, unselected turkeys.

Type I secretion and multidrug efflux: transport through the TolC channel-tunnel.

The crystal structure of TolC from Escherichia coli was recently determined to 2.1-A resolution and shows a unique type of channel architecture: a 12-stranded beta-barrel spans the outer membrane and is attached to a long alpha-helical channel that penetrates far into the periplasm. The structure suggests a mechanism for its role in secretion of proteins and in efflux of toxic small molecules. The TolC export pathway is compared with several import pathways of gram-negative bacteria where the outer membrane protein structures are also known.