Antibiotics, including polymyxins and neomycins, were orally administered in drinking water. Antibiotics attenuated hepatic stellate cell (HSC) activation and liver fibrosis via TGF-β and collagen in an experimental hepatic fibrosis model. The mechanism by which antibiotics attenuated LPS-TLR4 signaling and liver fibrosis was assessed. Notably, TLR4 mRNA level in the liver was elevated in the CDAA group and the CDAA-induced increase was significantly reduced by antibiotics. However, no significant differences were observed in the intestine among all groups. Elevated mRNA levels of LPS binding protein, which was correlated with serum endotoxin levels, were recognized in the CDAA group and the CDAA-induced increase was significantly reduced by antibiotics. The intestinal permeability of the CDAA group was increased compared with the choline-supplemented amino acid group. The tight junction protein (TJP) in the intestine, determined by immunohistochemical analysis was inversely associated with intestinal permeability. Antibiotics improved the intestinal permeability and enhanced TJP expression. Inhibition of LPS-TLR4 signaling with antibiotics attenuated liver fibrosis development associated with NASH via the inhibition of HSC activation. These results indicated that reduction of LPS and restoration of intestinal TJP may be a novel therapeutic strategy for treatment of liver fibrosis development in NASH.Previous clinical studies have demonstrated that endotoxin/toll-like receptor 4 (TLR4) signaling is critical in the inflammatory pathways associated with non-alcoholic steatohepatitis (NASH). In human and animal studies, NASH was associated with portal lipopolysaccharide (LPS) and the plasma LPS level was hypothesized to be associated with small intestinal bacterial overgrowth, change in composition of the microbiota and increased intestinal permeability. The aim of the present study was to investigate the roles of endogenous endotoxin and TLR4 in the pathogenesis of NASH. The effects of antibiotics were assessed  Non-alcoholic fatty liver disease (NAFLD) is the most common type of liver disease in the general population . NAFLD iPrevious clinical studies have reported that endotoxin/toll-like receptor 4 (TLR4) signaling is crucial in the activation of inflammatory pathways associated with NASH . TLR4 isIn human and animal studies, it has been reported that NASH is associated with portal LPS levels through mechanisms involving bacterial translocation ,10 and tEscherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and other members of the Enterobacteriaceae family due to their specificity for the LPS molecule, which exists within a number of Gram-negative outer membranes. They are produced by nonribosomal peptide synthetase systems in Gram-positive bacteria, including Paenibacillus polymyxa and disrupt the structure of the bacterial cell membrane by interacting with phospholipids. They are not absorbed through the gastrointestinal tract. In clinical settings, they are used for patients with Gram-negative bacterial infections and for endotoxin apheresis column treatment of endotoxemia  were housed in a room under controlled temperature and a 12/12 h light-dark cycle. The animals were divided into the following three experimental groups and fed for 8 weeks: i) Choline-deficient amino acid diet ; ii) choline-deficient amino acid diet plus antibiotics  and iii) choline-supplemented amino acid diet . All rats were sacrificed at the end of week 8. For selective intestinal decontamination, poorly absorbable antibiotics  and 3 g/l neomycin trisulfate salt hydrate  were administered to the rats in the CDAA+AB group by adding them to drinking water during the experimental period, excluding the first and fifth week. All animal procedures were performed according to standard protocols and in accordance with the standard recommendations for the proper care and use of laboratory animals. This study was approved by the ethics committee of Nara Medical University, Kashihara, Japan.Conventional histological examination was performed using hematoxylin and eosin and Sirius-red  staining of the excised liver sections, as described previously .® microscope  and images from ZO-1 and Claudin-4 staining were quantified using Axio software version 4® (Zeiss). For quantification, five images were randomly selected for quantification analysis from each sample and the software program quantified the staining intensity of the selected images based on a preselected threshold.For immunostaining of the α-smooth muscle actin (α-SMA), 5 μm-thick liver sections were stained using the indirect immunoperoxidase technique with mouse anti-human monoclonal anti-α-SMA antibody , as described previously . For the® , as described previously  in the liver and TLR4 in the intestine were measured by qPCR using the Applied Biosystems StepOnePlus™ Real-Time PCReviously . Primer ® . The TGF-β levels in the liver were measured using ELISA .The hepatic tissue was homogenized in lysis buffer  containing a mixture of protease and phosphatase inhibitors . The total collagen volume in the liver was measured using a Sircol collagen assay kitA total of 25 mg fluorescein isothiocyanate (FITC)-dextran  was orally administered per animal on the day of sacrifice. At 4 h after oral gavage of FITC-dextran, each rat was anesthetized and blood was drawn from its portal vein. The plasma was analyzed by fluorescence measurement at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.The results are presented as the mean ± standard deviation and were analyzed using Student’s t-test for unpaired data . P<0.05 was considered to indicate a statistically significant difference.The general findings of each experimental group at the time of sacrifice are shown in The present study initially examined the effects of poorly absorbable antibiotics on liver fibrosis, induced by CDAA intake. As shown in TLR4 enhances hepatic inflammation and fibrogenesis ,18. ThisThe elevated mRNA level of LBP suggested that the serum LPS level was increased in the CDAA group. The serum LPS level was hypothesized to be involved in gut permeability, therefore gut permeability was examined by analyzing the fluorescence levels of the portal vein following oral gavage loading with FITC-dextran. The fluorescence levels of the portal vein in the CDAA group were increased when compared with the CSAA group. The increase of intestinal permeability of the CDAA group was improved by the addition of poorly absorbable antibiotics . Since gIn the present study, the effect of the poorly absorbable antibiotics, polymyxin and neomycin, on the development of hepatic fibrosis and intestinal permeability was examined. It was demonstrated that the antibiotics not only reduced CDAA-induced hepatic fibrosis and HSC activation but also improved intestinal permeability.The liver is the main target of intestinally-derived bacterial products and the rate of bacterial translocation increases in various models of hepatic disease, rendering LPS a possible candidate mediator of TLR4-dependent profibrogenic effects. Accordingly, increased LBP mRNA expression was identified in the CDAA group, indicating that LPS was increased. In addition, LBP mRNA expression and fibrogenesis were reduced in rats treated with poorly absorbable antibiotics, suggesting that the intestinal flora is the main source of LPS and that intestinally-derived LPS drives fibrogenesis.et al (Translocated LPS derived from the gut microflora is considered to mediate TLR4 activation in the liver. However, this translocation may be independent of intestinal TLR4 . The mRNet al  reportedNAFLD is associated with increased intestinal permeability and small intestinal bacteria overgrowth ,23. Thesin vitro study, Caco-2 cells, which mimic intestinal epithelial cells, grown in zinc-deficient media have reduced TJP; therefore it was hypothesized that zinc deficiency may potentially be relevant to the increased intestinal permeability. In the NASH model used in the present study, CDAA-induced hepatic fibrosis, endogenous LPS and systemic increases in inflammatory cytokines may disrupt intestinal tight junction proteins. Considering these findings, the recruitment of TJPs using probiotics and zinc preparations, for example, offers a novel strategy for NASH treatment.Caco-2 cells grown in zinc-deficient media have reduced transepithelial electrical resistance and altered expression levels of ZO-1 and occludin, which are intestinal tight junction proteins, compared with Caco-2 cells grown in zinc-replete media . In clinin vivo model, dietary habits, which increase the percentage of intestinal endotoxin producers, including Gram-negative bacteria may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD (et al (The intestinal microflora is involved in liver fibrosis. In the present in NAFLD . Endo etD (et al  also demIn conclusion, the inhibition of LPS-TLR4 signaling with poorly absorbable antibiotics attenuated the liver fibrosis development in NASH via inhibition of HSC activation. These results indicated that reduction of LPS and restoration of the intestinal TJPs may be a new therapeutic strategy for the treatment of the development of liver fibrosis in NASH.


Today, in material science nanoscale structures are becoming more and more important. Not only for the further miniaturization of semiconductor devices like carbon nanotube based transistors, but also for newly developed efficient energy storage devices, gas sensors or catalytic systems nanoscale and functionalized materials have to be analysed. Therefore, analytical tools like near-edge X-ray absorption fine structure (NEXAFS) spectroscopy has to be applied on single nanostructures. Scanning transmission X-ray microscopes (STXM) as well as full-field transmission X-ray microscopes (TXM) allow the required spatial resolution to study individual nanostructures. In the soft X-ray energy range only STXM was used so far for NEXAFS studies. Due to its unique setup, the TXM operated by the Helmholtz-Zentrum Berlin (HZB) at the electron storage ring BESSY II is the first one in the soft X-ray range which can be used for NEXAFS spectroscopy studies which will be shown in this review. Here we will give an overview of the different microscopes used for NEXAFS studies and describe their advantages and disadvantages for different samples. In. In30]. In this review only methods for bulk sensitive NEXAFS spectroscopy using STXM and TXM in the soft X-ray range will be further discussed.The Stony Brook group at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory built the first STXM having a zone plate lens to produce the scanning spot –32. In oThe first TXM having a higher resolution than light microscopes was constructed by the group of Günter Schmahl, University of Göttingen at the electron storage ring ACO in Orsay, France –35. FiguE/ΔE) in the range of a few hundred hindering spectroscopic applications. The full field TXM at the undulator beamline U41-FSGM at the electron storage ring BESSY II operated by the Helmholtz-Zentrum Berlin (HZB) uses a different optical concept . It. It58]. After these first encouraging investigations further spectroscopic studies on nanostructures containing metal atoms like copper were performed. It was shown that chemical analysis of nanowires for solar cells can be performed by NEXAFS-TXM. As a result of this investigation it was found that neither Cd nor S diffuse into the CuO phase after the deposition of CdS on the CuO nanowires –61. The 3) could be demonstrated   . So far,xtracted . The anaBulk sensitive nanoscale NEXAFS spectroscopy was restricted in the past to STXMs as TXMs did not allow the necessary spectral resolution. With the recently developed setup of the HZB-TXM at the BESSY electron storage ring this restriction could be overcome. As shown in this review, several published NEXAFS-TXM studies of low dimensional nanostructures pave the way for understanding the electronic structure towards the atomic scale and will help in the design of tailored functional systems. The investigated carbon nanotubes, nanowires as well as nanoscale oxides have due to their high surface to volume ratio an enormous potential in nanoelectronics, catalysis, light harvesting and energy storage applications. The newly developed NEXAFS-TXM together with further complementary physicochemical methods and spectroscopic techniques will allow a more complete understanding of the function of low dimensional nanostructures.


In situ time-resolved XANES measurements of Au ions in an aqueous solution in the presence of support materials were performed under synchrotron X-ray irradiation. The synchrotron X-ray-induced reduction of Au ions leads to the formation of Au nanoparticles on the carbon particles, acrylic cell or polyimide window. The deposited Au metallic spots were affected by the wettability of carbon particles. in situ time-resolved XANES measurements of Au ions were performed under synchrotron X-ray irradiation. XANES spectra were obtained only when hydrophobic carbon particles were added to the precursor solution containing Au ions. Changes in the shape of the XANES spectra indicated a rapid reduction from ionic to metallic Au in the precursor solution owing to synchrotron X-ray irradiation. In addition, the effects of the wettability of the carbon particles on the deposited Au metallic spots were examined. The deposited Au metallic spots were different depending on the relationship of surface charges between metal precursors and support materials. Moreover, a Au film was obtained as a by-product only when hydrophilic carbon particles were added to the precursor solution containing the Au ions.Synchrotron X-ray-induced reduction of Au ions in an aqueous solution with or without support materials is reported. To clarify the process of radiation-induced reduction of metal ions in aqueous solutions in the presence of carbon particles as support materials,  Supported metal nanoparticles have been studied and developed for various applications in the fields of medicine, biology and catalysis. There are various reports on the radiolytic preparation of supported metal nanoparticles and their applications  device or soft X-rays using a synchrotron source while recording the XPS spectra; however, the metal salts were not in an aqueous solution during the measurements. In general, XPS measurement is performed under ultrahigh-vacuum conditions below 10−5 Pa and is therefore not suitable for solution samples. In contrast, the energy-dispersive X-ray absorption fine-structure (DXAFS) technique can be utilized under ordinary pressure and is therefore suitable for solid, solution and complex samples. In particular, the in situ time-resolved DXAFS technique has been recently applied to catalysts in solution for clarification of the reaction mechanism  was used as the metal precursor. Untreated carbon black powder  was used as the hydrophobic carbon particles. Hydrophilic carbon particles were prepared by immersing the hydrophobic carbon particles in concentrated nitric acid  for 45 min at 363 ± 5 K while stirring. Carboxylic acid groups (COOH) would be formed on the surface of the carbon particles  produced by a Direct-Q system (Millipore) was deaerated by freezing with liquid nitrogen and was then used as the solvent. Tetrachloroauric acid tetrahydrate (HAuCl2.2.in situ time-resolved XANES measurements of Au ions in the aqueous solution. The acrylic cell containing the precursor solution was placed in the synchrotron X-ray path in front of the polychromator. The precursor solution was irradiated with synchrotron X-rays at the NW2A beamline of Photon Factory Advanced Ring for Pulse X-rays (PF-AR) at High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. The precursor solution was continuously stirred during the irradiation. Chemical reductants such as hydrated electrons  were obtained using the DXAFS technique at the NW2A beamline while the precursor solution was irradiated with synchrotron X-rays, and consequently Au ions were reduced. To apply the DXAFS technique, the polychromator was controlled, and a one-dimensional X-ray detector was used. Each XANES spectrum was obtained for only 5 s. From the obtained XANES spectra, the ratio of ionic and metallic Au was calculated by linear-combination fitting.Changes in the chemical states of Au were investigated by comparing the time-resolved XANES spectra around the Au-Table 23.in situ time-resolved XANES spectra. In all the samples the Au ions are reduced via synchrotron X-ray irradiation, but the deposited Au metallic spots are different. Fig. 2via synchrotron X-ray irradiation. For sample 1, which has no carbon particles, metallic Au is observed on parts of the PI film  would be formed on the surface of the hydrophilic carbon particles, as previously reported efa)–2(f) indicate that (i) the synchrotron X-ray-induced reduction of the Au ions in an aqueous solution is not dependent on the wettability of support materials; (ii) metallic Au is easily deposited on hydrophobic surfaces; (iii) an increase in the solid–liquid interface area advances the reduction of Au ions.Table 3 Fig. 2a. Sample  Fig. 2e differs in situ time-resolved XANES spectra of these systems because the deposited metallic Au prevented the transmission of synchrotron X-rays, which resulted in inaccurate information regarding the changes in the chemical states of Au in the aqueous solution. In contrast, for sample 3, which had hydrophilic carbon particles, metallic Au was not deposited on the PI film; therefore, in situ time-resolved XANES spectra were successfully obtained during the reduction of Au ions via synchrotron X-ray irradiation.For samples 1, 2 and 4, metallic Au was deposited on the PI film; therefore, it was difficult to analyze in situ time-resolved XANES spectra obtained around the Au-LIII edge for sample 3 during the reduction of Au ions in the aqueous solution via synchrotron X-ray irradiation. As the irradiation time is increased, the shape of the XANES spectra of sample 3 approaches that of a Au foil. Fig. 44 and metallic Au, calculated using the XANES spectra in Fig. 3in situ time-resolved XANES measurements performed during synchrotron X-ray irradiation enable the tracking of radiation-induced reduction of Au ions in an aqueous solution.Fig. 34.In situ time-resolved XANES measurements of the precursor solution were simultaneously performed. The following are the two main results from the measurements. First, the deposited Au metallic spots were dependent on the wettability of the carbon particles added to the precursor solution, which suggests that the wettability of carbon particles enables the control of the deposited Au metallic spot. In particular, the addition of hydrophilic carbon particles to the precursor solution leads to the formation of a Au film. This phenomenon would be useful as a novel technique to form metal films via irradiation with, for example, X-rays, γ-rays and electron beams. Second, the addition of hydrophobic carbon particles to the precursor solution enabled in situ time-resolved XANES measurements for tracking the reduction of Au ions in an aqueous solution. This behaviour could also be applied to study other metal ions. The relationship of surface charges between metal precursor and supports materials must be carefully considered. In situ time-resolved XANES spectra could be obtained for tracking the reduction of metal ions in an aqueous solution in the presence of support materials only if the appropriate materials are selected. Further studies on the synchrotron X-ray-induced reduction of bimetallic ions in an aqueous solution and simultaneous monitoring by XANES spectroscopy are currently underway in our group and will be reported in a future publication (see Table 1In this study, synchrotron X-rays were irradiated to precursor solutions containing Au ions with or without carbon particles.


The Light-Oxygen-Voltage domain family of proteins is widespread in biology where they impart sensory responses to signal transduction domains. The small, light responsive LOV modules offer a novel platform for the construction of optogenetic tools. Currently, the design and implementation of these devices is partially hindered by a lack of understanding of how light drives allosteric changes in protein conformation to activate diverse signal transduction domains. Further, divergent photocycle properties amongst LOV family members complicate construction of highly sensitive devices with fast on/off kinetics. In the present review we discuss the history of LOV domain research with primary emphasis on tuning LOV domain chemistry and signal transduction to allow for improved optogenetic tools. Over the past 10 years, advancements in our understanding of photoactivated proteins have enabled genetic control of cellular events through light. These optogenetic approaches allow researchers to dictate biological signaling with exquisite spatial and temporal precision. The ability to remotely and non-invasively trigger signal transduction has led to unparalleled breakthroughs in neuroscience, cardiology and cell biology , a fungal circadian clock photoreceptor Vivid (VVD), a Bacillus subtilis stress response protein (YtvA), a FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 essential to plant flowering (FKF1), and a 222 amino acid LOV-transcription factor present in Erythrobacter litoralis (EL222)  contain an oxidized flavin cofactor that maximally absorbs blue-light at 450 nm  that is defined by a single broad absorption band centered at 390 nm. The photocycle is thermally reversible in the dark, decaying to the ground state on a timescale of seconds to days , intermediate cycling (1000 < τ < 10000 s), and slow cycling (10000 < τ s). Thus, existing optogenetic tools are either fast cycling , intermediate (YtvA~6000 s) or slow cycling , but retain peak sensitivity under low light conditions consistent with dusk/dawn (5–20 μmole/m2*s)  Factors that can stabilize increased electron density within the flavin system can stabilize the light state adduct. Thus, an increase in hydrogen bonding near the pyrimidine ring can stabilize the light-state species  adopts two possible configurations within the active site  to allow the development of an oxygen independent fluorescent reporter  or for possible implementation in super-resolution microscopy , involved fusing a peptide designed to interact with PDZ domains  . By creating a VVD pair with negatively charged resides at Ile52/Met55 and a positively charged version, Kawano et al. were able to design a robust system with ~40-fold activation upon light treatment . The resulting system imparted light-regulated control of kinase activity  following blue-light activation  following blue light treatment (Polstein and Gersbach, LOV proteins afford a unique platform for coupling blue-light activation to a wide range of signal transduction elements. Although significant detail is known for chemical and structural mechanisms in these systems, there still remains a limitation to the design and fidelity of LOV-based tools. Further research into light-state crystal structures of LOV proteins as well as LOV optogenetic tools is needed to enable improved, robust design of optogenetic devices. Several key areas are noted here for future development.Currently, structural studies of LOV proteins are, in most cases, limited to the isolated LOV domains. Few structures exist for full-length or multi-domain containing LOV proteins. These limit our understanding of allosteric mechanisms in LOV containing proteins and similarly optogenetic tools. Structural studies of LOV domains with extended N- and C-terminal regions indicate that these elements are essential to light-dark switching, even without the downstream effector domains. Subsequent studies have shown that targeting these elements for mutagenesis is a robust method for tuning optogenetic function. To further extend the utility of these tools it is essential for researchers to better understand the natural mechanisms coupling LOV dynamics to downstream effectors through the N- and C-terminal linkages.in vivo function or optogenetic tool utility. Going forward, it is recommended that studies of rate altering variants and corresponding light state structures be conducted in concert with their effect on in vivo function and optogenetic tool design. Such efforts may provide a global understanding of how LOV chemistry and structure regulate signal transduction and allostery.Extension of our understanding of allosteric mechanisms of signal transduction in LOV proteins and optogenetic tools should leverage existing efforts to tune LOV domain chemistry. Direct determination of light-state structures has been facilitated by rate-altering variants. These structures afford snapshots of the light and dark-adapted states that facilitate understanding of optogenetic tools. Unfortunately, few studies have examined the effects of these variants on The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acinetobacter baumannii (CP-AB) in Germany is still low an increasing number of outbreaks has been reported in recent years. In August 2014 we observed a cluster of 4 patients with CP-AB (OXA-23) on our surgical intensive care unit (SICU).Although the prevalence of carbapenemase-producing After excluding an environmental source we performed epidemiological analyses to gain insight into the spread of CP-AB in our university hospital.To characterize the CP-AB isolates molecular typing using amplified fragment length polymorphism was conducted. A genotypic comparison with strains collected in 2013 and the first half of 2014 was carried out. All new patient isolates of CP-AB were genotyped prospectively.Molecular typing revealed that 3 out of the 4 SICU-isolates were genotypically identical. Although temporally and locally coinciding, the 4th isolate of the patient cluster did not match.This 4th strain however was identical to a strain previously identified in a patient from the neurological department in July 2014. A physical link between these two patients could not be identified. Even more surprisingly, another isolate of CB-AB from a neurosurgical patient isolated in September 2014 was found to be identical with the three isolates of the SICU cluster, despite the absence of detectable temporal or local link to the SICU.Isolates of CP-AB from six patients fell into two clusters of two and four strains, respectively.In this case patient transfer within the hospital cannot explain these unexpected genotypical relationships between isolates from three departments. Transmission of CP-AB may have occurred in the context of consultants’ visits or when patients were temporarily moved to a diagnostic unit such as endoscopy or radiology.In the context of clusters of CP-AB isolated from several patients, genotyping provides the opportunity to follow the movement of these highly resistant bacteria within a hospital. Surprising genotyping results should engender efforts to analyse the pathways of transmission.None declared.


Synechocystis sp. PCC 6803 surprisingly produce both physico-chemically different polymers under conditions of high C to N ratios. For the first time, the function and interrelation of both carbon polymers in non-diazotrophic cyanobacteria are analyzed in a comparative physiological study of single- and double-knockout mutants , respectively. Most of the observed phenotypes are explicitly related to the knockout of glycogen synthesis, highlighting the metabolic, energetic, and structural impact of this process whenever cells switch from an active, photosynthetic ‘protein status’ to a dormant ‘glycogen status’. The carbon flux regulation into glycogen granules is apparently crucial for both phycobilisome degradation and thylakoid layer disassembly in the presence of light. In contrast, PHB synthesis is definitely not involved in this primary acclimation response. Moreover, the very weak interrelations between the two carbon-polymer syntheses indicate that the regulation and role of PHB synthesis in Synechocystis sp. PCC 6803 is different from glycogen synthesis.Classical microbial carbon polymers such as glycogen and polyhydroxybutyrate (PHB) have a crucial impact as both a sink and a reserve under macronutrient stress conditions. Most microbial species exclusively synthesize and degrade either glycogen or PHB. A few bacteria such as the phototrophic model organism  D-glucose residues that are interlinked via (1–6)-α-D-glucosidic linkages, forming a highly branched and rigid granule structure of about 107–108 Da in mass and 42 nm in size (R)-3-hydroxybutyrate units, forming 200–500 nm large inclusions , which act as cellular sinks as well as stable and yet readily accessible reservoirs for carbon and energy, to acclimate and to cope with starvation conditions, in particular nitrogen starvation leading to high C–to-N ratios of nutrients . Glycoge in size . The fir in size . The gluclusions . The firSynechocystis sp. PCC 6803 additionally synthesize PHB  with a very sensitive double-knockout mutant , followed by a conclusive discussion of the individual and mutual function of both glycogen and PHB in carbon-flux regulation and in acclimation to macronutrient stress, especially during nitrogen chlorosis.Most microorganisms synthesize either glycogen or PHB. The enzymatic settings for the metabolism of glycogen, the prevalent cyanobacterial carbon polymer, are conserved in all cyanobacteria . A few csize PHB . This dusize PHB . In recesize PHB , and tarsize PHB . A variesize PHB . In contphaC ORF (locus slr1830) were fused by an overlap-extension PCR  is described by The deletion–insertion mutagenesis of the glgC knockout, the glgC ORF (locus slr1176) was amplified in full length (including the promoter-region) by PCR  and 17.6 mM sodium nitrate as the nitrogen source (+N) or in BG110, which lacks sodium nitrate  and of the chlorophyll a content (determination according to the method as described by Growth characteristics of the strains (in liquid cultures) have been recorded by both daily measurement of the optical density at 750 nm (ODTo monitor the bleaching process (under nitrogen deprivation), whole-cell absorption spectra have been recorded in the wavelength range from 400 to 750 nm on a Specord200 plus spectrophotometer  equipped with a scattering position and were normalized at 750 nm  and with or without K2HPO4 (+Pi or –Pi), respectively. The plates were incubated at 28°C under alternating light/dark cycles or continuous illumination with a light intensity of 75 μmol photons m-2 s-1 for 7 days .For spot assay experiments, cells were grown in Erlenmeyer flasks to an ODFigure 4A).The intracellular glycogen levels were determined according to the protocol as described by Rhodobacter spheroides (Roche Diagnostics) in the presence of nicotinamide adenosine dinucleotide (NAD). The NADH synthesis was coupled to a phenazine methosulphate-p-iodonitrotetrazolium violet (PMS-INT) colorimetric assay (to prevent a backward reaction) as described by Figure 4B) was performed via the calibration of NADH series.Poly-β-hydroxybutyrate was extracted from dried cell pellets and simultaneously hydrolyzed into its monomers (3-hydroxybutyrate) by shaking in 0.5 M NaOH (300–400 μl) (1 h incubation at 85°C). After cooling, the solution was neutralized by the addition of 1 M HCl (volume ratio NaOH/HCl 4:1). Finally, the PHB content was spectrophotometrically quantified in an enzymatic assay using 3-hydroxybutyrate dehydrogenase from Figure 4C).Cyanophycin was extracted as described by Table 1).The extracellular pyruvate and 2-oxoglutarate levels were determined according to the protocol as described by For the determination of dry weight, cell pellets of 10–50 ml culture were washed twice with sterile water, dried completely at 80°C in a heating device, and finally weighed.Anabaena sp. PCC 7120 .Samples for Transmission electron microscopy (TEM) were prepared as described previously for PCC 7120 . FixatioglgC/ΔphaC) is not able to synthesize neither glycogen nor PHB (see below). The direct comparison of this mutant with the respective single-knockout mutant and the wild type  provide a deeper understanding of the individual and the mutual physiological roles as well as the metabolic interrelations of the two carbon-polymer syntheses.To completely abolish carbon-polymer biosynthesis, knockout mutants of either ADP-glucose pyrophosphorylase  or glycogen synthase (GlgA) for glycogen biosynthesis, or of either β-ketothiolase (PhaA), Acetoacetyl-CoA reductase (PhaB), or PHB synthase (PhaC/PhaE) for PHB synthesis were successfully created in different cyanobacterial strains previously . In thisglgC), of a PHB-deficient mutant (ΔphaC), and of a mutant deficient in the synthesis of both compounds (ΔglgC/ΔphaC) have been tested via spot assays as well as in liquid culture systems under different alternating light/dark regimes . Moreover, the negative effect on the viability and growth with increasing length of periodic darkness is more pronounced in the ΔglgC/ΔphaC mutant than in the ΔglgC single mutant. Cells deficient in both PHB and glycogen synthesis are not able to grow in conditions of periodic 12 h/12 h light/dark regimes . In conclusion, both carbon-polymer syntheses are presumably essential for the photoautotrophic lifestyle of Synechocystis sp. PCC 6803. However, the glycogen pool (and not the PHB pool) might be the main energy and carbon source for the maintenance metabolism in darkness and the PHB pool is dispensable.All cyanobacteria are able to perform a maintenance metabolism in the absence of light by predominantly using internal polymer reserves. Various studies showed the contribution of the carbon storage polymer glycogen to the maintenance metabolism in darkness. In contrast, the involvement of PHB metabolism has not been reported so far . The ΔglgC mutant is significantly impaired in both its viability  and its recovery capability  in prolonged starvation condition, as previously reported by Unbalanced growth conditions such as macronutrient starvation generally lead to an accumulation of reserve material as well as to a rapid degradation after replenishing conditions in all microorganisms including cyanobacteria . To undephaC behaves similar to the wild type. Upon nitrogen starvation both strains duplicate once more and enter chlorosis, being visualized by the yellow color due to the loss of pigments . The ΔphaC mutant recovers after starvation stress like the wild type, tested after 7 days of nitrogen depletion .The single, PHB-deficient mutant ΔglgC/ΔphaC mutant shows a similar phenotype as the ΔglgC mutant, indicating that the impaired growth, viability, and recovery capacity result rather from the block of glycogen synthesis than from the block of PHB synthesis in this double-knockout mutant . In conclusion, the glycogen synthesis (and not the PHB synthesis) appears to be the key player in Synechocystis’s short-term acclimation response toward macronutrient stress conditions, such as nitrogen starvation.In line with this finding, the ΔglgC- or ΔnglgA-mutant strains leads to three very characteristic, co-occurring phenotypes as short-term response to nitrogen starvation: (i) the incapability of carbon-excess redirection into glycogen-granule formation , (ii) impaired chlorosis . This phenotype is fully complemented by in-trans expression of the glgC gene under the control of its own promoter in the ΔglgC-mutant background  and is in agreement with results from phaC mutant responds to nitrogen-starvation conditions in a similar way as the wild type, which is in agreement with previous data obtained with a ΔphaA mutant (phaC mutant accumulate similar amounts of glycogen (approx. 25% of DW in 48 h) , degrade their phycobilisomes in a similar period . Under the nitrogen-replenishing conditions tested, the wild type and the ΔphaC mutant show a similar glycogen-degradation pattern (data not shown) and a similar cyanophycin accumulation .As previously shown, the block of glycogen biosynthesis as in the ΔA mutant . Both wiglgC and ΔglgC/ΔphaC are apparently not able to redirect the replenished nitrogen sources into cyanophycin biosynthesis, which is not detectable in cell extracts under the tested conditions . However, in response to nitrogen-starvation conditions, the single-knockout mutant ΔglgC significantly  redirect more of the carbon excess into PHB biosynthesis than the respective wild type, which is in agreement with data from In contrast, the glycogen-deficient mutants Δvice versa) as well as to a spilling of certain metabolites such as 2-oxoglutarate in response to macronutrient starvation.In conclusion, blocks in glycogen synthesis (but not blocks in PHB synthesis) lead to overflow reactions into other polymer syntheses : (i) a massive accumulation of electron-dense glycogen inclusions (approx. 40 nm in diameter) between the thylakoid layers , (ii) the degradation of the antenna complexes , and (iv) the formation of distinct electron-transparent PHB granules . Similar structural and morphological changes, except for the accumulation of PHB inclusions, occur in the PHB synthase-deficient mutant ΔphaC  as previously highlighted by Four major structural and morphological changes are observed in wild-type cells in response to nitrogen starvation  are not able neither to degrade their antenna complexes (phycobilisomes), nor to reduce the number and packing density of the thylakoid membrane-layer system, nor to accumulate glycogen granules in response to nitrogen depletion. Instead, several undefined vesicles (approx. 90 nm in diameter) appear inside the thylakoid stacks . Preparative artifacts could be excluded due to equal treatment of all samples . A similar effect of structural and morphological changes as in ΔglgC-mutant cells, except for the formation of definitive PHB granules, has been observed in the double-knockout mutant ΔglgC/ΔphaC .In contrast, AGPase-deficient mutant cells  has a substantial influence on the adaptation response to macronutrient starvation. The formation of glycogen granules appears as an important key player in the rearrangement and disassembling of the photosynthetic machinery such as phycobilisome degradation and thylakoid density and packing alterations. These changes are necessary to acclimate and trophically convert from an active photosynthetic cell to a dormant status.Figure 4). Only blocks of glycogen synthesis in Synechocystis sp. PCC 6803 lead to a prompt spilling of partially oxidized substrates such as 2-OG (to get rid of carbon excess) under unbalanced growth conditions, independent of the absence or presence of an active PHB synthesis (Table 1). However, this spilling effect is similar to PHB-synthesis knockouts in other bacteria naturally lacking glycogen metabolism  between the progression of both glycogen accumulation and phycobilisome degradation in wild-type cells  as a short-term response to nitrogen depletion. Cells deficient in glycogen synthesis are not able to perform the switch and remain in a metabolic arrest .The nitrogen-starvation response is characterized by a cascade of structural and metabolic events that finally switch the cellular mode from photosynthetically active to dormant status in non-diazotrophic cyanobacteria. These substantial changes are tightly related to active glycogen synthesis. The removal of external nitrate or ammonia sources stops the primary nitrogen assimilation processes mainly mediated by glutamine synthetase and glutamine/2-oxoglutarate aminotransferase. The reduction of cellular nitrogen is accompanied by the rapid rise of 2-oxoglutarate (2-OG) levels due to the absence of 2-OG dehydrogenase 2-OGDH; . Even intabolism . The dirtabolism  and the tabolism , are inenblA in Synechococcus elongatus PCC 7942 by glgC) of Synechococcus sp. PCC 7002 still maintained an efficient energy transfer from phycobilisomes to photosystem II reaction centers after prolonged nitrogen starvation  additionally supports the new role of glycogen synthesis/granules for the integrity of the thylakoid layer system. Such vesicles (around 90 nm in size) might represent spilling products (to remove destabilizing catabolites from the thylakoid membrane) or plastoglobuli-like structures, which have similar functions in the thylakoid biogenesis/degradation processes in plants (We hypothesize two mechanisms that do not necessarily exclude each other: a metabolic mechanism (i) and/or a structural mechanism (ii). (i) The concomitant directing of carbon excess (mainly from protein degradation) into glycogen synthesis allows the adjustment to an adequate cellular 2-OG concentration under conditions of nitrogen depletion, which in turn leads to an activation of the nitrogen signaling cascade, in particular of the PII signaling protein . An insuarvation . The incn plants . A potenn plants .Figure 4) and thus potentially do not interrelate under conditions of nitrogen starvation. Only a small proportion of primary carbon excess is redirected into PHB synthesis in cells blocked in glycogen synthesis . Most excessive carbon proportions are promptly spilled (Table 1). In this respect, it is not clarified whether parts of the carbon excess were directed into lipid biosynthesis, another abundant form of carbon storage in Synechocystis sp. PCC 6803  possibly results from the variety of negative pleiotropic  effects, summarized in an impaired acclimation response, than from the lack of carbon source. Solely PHB-deficient cells are neither affected under the conditions tested , which is in agreement with Synechocystis sp. PCC 6803 (and some purple bacteria) massively synthesize both types of the physically and chemically different carbon polymers, glycogen, and PHB, in response to macronutrient deficiency . Obviously, PHB synthesis does not act as a backup of glycogen synthesis and does not compensate knockouts in glycogen metabolism.The recovery incapability of the glycogen-deficient mutants (Synechocystis sp. PCC 6803 is given by the pronounced sensitivity and reduced growth rate of the double-knockout mutant ΔglgC/ΔphaC (certain loss of robustness) compared to the single knockouts even under standard growth conditions . In this respect, some already discussed functions of PHB are conceivable: (i) as a transient electron sink (Synechocystis sp. PCC 6803 and the non-compensatory effect in glycogen-deficient knockouts), and/or (iii) as a structural component during the equal separation of the multiple bacterial nucleoids and PHB granules in cell division processes (These observations are in line with the general metabolic principle that only one kind of carbon-polymer metabolism acts as both a sink and an accessible reserve. So far, the only indication for the specific role of PHB in ron sink  and/or (rocesses , that miRD, IM, and YZ made substantial contributions to the conception and the design of the study, the acquisition of data, and the analysis and interpretation of data; RD, IM, and YZ participated in drafting the article and revising it critically for important intellectual content; RD, IM, and YZ gave final approval of the version to be submitted. RD, IM, and YZ agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MicroRNAs regulate gene expression at the post-transcriptional level. Differential expression of miRNAs can potentially be used as biomarkers for early diagnosis and prediction for outcomes. Failure in validation of miRNA profiles is often caused by variations in experimental parameters. In this study, the performance of five extraction kits and three RT-qPCR systems were evaluated using BioMark high-throughput platform and the effects of different experimental parameters on circulating miRNA levels were determined. Differences in the performance of extraction kits as well as varying accuracy, sensitivity and reproducibility in qPCR systems were observed. Normalisation of RT-qPCR data to spike-in controls can reduce extraction bias. However, the extent of correlation for different qPCR systems varies in different assays. At different time points, there was no significant fold change in eight of the plasma miRNAs that we evaluated. Higher level of miRNAs was detected in plasma as compared to serum of the same cohort. In summary, we demonstrated that high-throughput RT-qPCR with pre-amplification step had increased sensitivity and can be achieved with accuracy and high reproducibility through stringent experimental controls. The information provided here is useful for planning biomarker validation studies involving circulating miRNAs. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level343678While miRNA profiling using microarray and next-generation sequencing (NGS) technologies are becoming common, reverse transcription - quantitative polymerase chain reaction (RT-qPCR) technique is still considered as the gold standard to validate differentially expressed miRNAs121314It is now known that handling bias can occur during the extraction step1617The aims of our study were to use a high-throughput platform to evaluate the performance of five extraction kits and three RT-qPCR systems as well as the effects of different experimental parameters on circulating miRNAs levels in a cohort of healthy individuals. Issues common in biomarker validation, including intra- and inter-platform variations (extraction kits and RT-qPCR systems) as well as intra-individuals variations (sampling time point and type) were investigated. MiRNAs selected for evaluation include common cancer-associated markers and miRNAs known to be abundant in blood.q values of the spike-in controls. Low Cq is an indication of high recovery while small CV would suggest less extraction bias across samples  as well as data from qPCR replicates without normalisation (R3). Our results indicated that RT replicates in the miScript system had the lowest reproducibility score and could be moderately improved by data normalisation to spike-in controls . OverallAccuracy of the two RT-qPCR systems was evaluated by data obtained from the standard curves . Accuracq values in plasma samples (S1) as well as in titration points (S2). Higher sensitivity scores were observed in the TaqMan system  were obtained in the TaqMan system  was observed in blood samples collected at different time points from the same set of individuals . There wSignificant high correlation  was also observed between paired plasma and serum samples . HoweverFor biomarker studies, it is now known that poor gene selection and validation strategies as well as technical biases including sampling errors, inconsistencies in sample processing and varying experimental parameters may lead to high prediction accuracy in the initial training set but low prediction accuracy in independent dataset2223Performance of extraction kit is usually indicated by the purity and yield of extracted RNA measured in spectrophotometer. However, in the context of cell-free miRNAs, quantification of small RNAs using spectrophotometer or capillary electrophoresis can be affected by contamination of phenol16q values and lower correlation observed in experiments carried out by the miScript system. Also, it is not surprising that lower detection sensitivity was observed in the miScript system, as the preamplification cycles we followed according to manufacturer's protocol were less for miScript as compared to TaqMan. It is likely that the miScript system will have improved detection sensitivity when protocol involving more pre-amplification cycles is optimised.In this study, we evaluated three qPCR systems for their adaptation to the high-throughput platform which required preamplification of RT products. This preamplification step increases the sensitivity to detect less abundant miRNAs in low amount of samples but caution has to be made during analysis to exclude non-specific and non-linear amplifications. We used titration curve from pooled cell lines to estimate the limit of linear amplification and no-template controls to rule out false positive. It was observed that using our current protocol, we could not adapt the commonly used RT-qPCR system, miRCURY to the BioMark high-throughput platform. This is evidenced by its result seen in melt-curve analysis (routine step to examine non-specific amplification) in the preamplified samples . DiscrepVarying circulating miRNA levels do not occur only in a diseased state, but sampling biases such as phlebotomy process16183017In summary, our study had evaluated the performance of five extraction kits and two qPCR systems adapted to the high-throughput RT-qPCR platform for circulating miRNAs. Our results suggested that at least three extraction kits were comparably good in terms of recovery and consistency. Two qPCR systems successfully adapted to the high-throughput RT-qPCR platform had varying performance in terms of reproducibility, accuracy and sensitivity. Normalisation of RT-qPCR data to spike-in controls will greatly reduce the technical bias from extraction but not technical bias from the use of different RT-qPCR system. Lastly, increased detection sensitivity and high reproducibility of miRNA validation study using high-throughput RT-qPCR platform can be achieved by following stringent experimental parameters demonstrated in this study.Ethics approval was obtained from the Medical Research & Ethics Committee (MREC), Ministry of Health Malaysia and methods were carried out according to the guidelines. Volunteer blood donors from the National Blood Blank and Institute for Medical Research were invited to donate blood samples for this study. Written informed consent was obtained from all 19 individuals prior to sample collection. All samples were collected from year 2012 to 2013. During each sampling, 5 ml of blood from healthy individual  was collected in EDTA (for plasma) or SST tube (for serum). Blood sample was centrifuged at 2500 rpm for 10 minutes at room temperature. Plasma or serum was then transferred to a new tube and centrifuged at 3000 rpm for 10 minutes at room temperature. Samples were aliquoted into microcentrifuge tubes and then kept at −80°C prior to RNA extraction. All blood samples were processed within two hours after sampling. For the comparison of plasma collected at different time points, repeated blood samples were collected from the same patient at a gap of approximately one year. Sample characteristics are shown in RNA from plasma samples were extracted using miRNeasy Serum/Plasma Kit , miRCURY™ RNA Isolation Kit - Biofluids , mirVana™ PARIS™ Kit , NucleoSpin® miRNA Plasma  and Plasma/Serum Circulating RNA Purification Kit (Slurry Format) . For optimal performance, all extractions were carried out according to manufacturer's protocol and both lysis buffer and isopropanol for the Plasma/Serum Circulating RNA Purification Kit (Slurry Format) were increased to 1 ml each. For paired plasma samples in time point experiment and the corresponding serum samples, automated RNA extraction was carried out using miRNeasy Serum/Plasma kit in QIAcube (Qiagen).At the beginning of each extraction procedure, exogeneous controls cel-miR-39 and cel-miR-54  were spiked into samples after the addition of lysis buffer. All eluted RNA samples were stored at −80°C until used. Details for comparison of extraction kits are indicated in High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system , miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen). The RT, preamplification and qPCR reactions were carried out according to previous publicationq) values were generated from Fluidigm Real-Time PCR Analysis. Wells with undetermined values were omitted from analysis. Average Cq was calculated from duplicate or triplicate wells using Microsoft Excel. Standard and titration curves were constructed from the Cq values of dilution points  undetermined Cq in more than 50% of the samples, (3) failure of melt curve analysis for the assays utilizing DNA-binding dye. Assays omitted from the analysis were listed in Raw quantification cycle  - measured (Cq standard point 1 - Cq standard point 2) |where ddCFold change over detection limit in q of highest detected value in linear amplification - normalised average Cq any assay.Fold change over detection limit = average CIn our experimental setting, ICC using two-way mixed model with absolute agreement was used as a measure for reproducibility when the same set of samples was processed and evaluated using the same experimental platform. In order to assess the correlation of data when different extraction kits, different RT-qPCR systems, different sampling time and type were used for the same set of samples, ICCs using two-way mixed model with consistency were calculated. For the computation of ICC in SPSS Statistics, only paired samples with determined values were included. Two-tailed paired T-test was used to evaluate the fold change of miRNA levels in plasma samples collected at different time points and in paired plasma and serum samples. All paired T-tests were calculated and graphs were plotted using GraphPad Prism.G.W.T. designed the study, performed the experiments, analysed and interpreted the data and drafted the manuscript. A.S.B.K. provided comments on the design of study and reviewed the manuscript. L.P.T. designed the study, interpreted the data, and revised the manuscript. All authors have approved of the final version of the manuscript.Supplementary Information


Aplysia californica. Sensorin (SRN) has a key role in learning and long-term memory storage in Aplysia. We have now identified NAT-SRN in the central nervous system (CNS) and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro-dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduction in levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.Despite the advances in our understanding of transcriptome, regulation and function of its non-coding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN), a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail  Recent high-throughput transcriptome studies have revealed widespread and extensive overlaps between genes and transcripts encoded on both strands of the genomic sequence. This overlapping gene organization, which produces sense-antisense transcript pairs, is capable of affecting regulatory cascades through established mechanisms. Natural antisense transcripts (NATs) are transcribed from the opposite strand to a protein coding or sense strand in the chromatin. Recent studies have provided ample evidence that more than 70% of the mammalian genome have antisense transcription potential.Antisense transcription has been recognized for roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level , a key transcript that plays an important role in the early stages of learning and memory formation  of wide spread antisense transcription in recent years, the functional significance of NATs and their physiological regulation remains poorly understood. However, the significant presence of NATs in the central nervous system suggests their potential role in brain function. The role of NATs in establishing memory formation has been suggested in Aplysia californica. For more than 50 years Aplysia has provided fundamental insights into the basic organization of neuronal functions. Aplysia's nervous system has large neurons, many of them can be uniquely identified and are associated with specific behaviors. These neurons can be isolated and cultured in vitro and they form circuits, which can be investigated at the molecular and cellular detail.To investigate physiological mechanisms that regulate expression and subcellular distribution of NATs, we have explored the expression of a NAT transcribed against the mRNA encoding the peptide neurotransmitter sensorin and its physiological regulation in the sensory neurons (SN) of the marine mollusk In vitro, SNs uniquely make synapses with their in vivo target motor neurons but not with their non-target motor neurons, providing an excellent model system to specifically study the effects of specific mRNAs in synapse formation and stabilization , is expressed exclusively in SNs and transported to distal neurites  analyses have confirmed the expression of sense (SRN) and antisense RNAs (NAT-SRN) of neuropeptide sensorin in SNs of Aplysia gill withdrawal reflex. We then examined whether expression of NAT-SRN transcripts is regulated in SNs during aging and in response to forskolin, an activator of CREB  has approved all of the experimental protocols (IBC Protocol 2010-019R1) described in this manuscript. Ethical approvals are not required for the research using invertebrate animals, such as the marine snail Aplysia californica maintained under standard conditions  at the National Aplysia Resource Facility  were used in the experiment. In our study, we used animals that correspond to two age groups (2 and 9 months old). Upon arrival in the laboratory, animals were kept in an aquarium at 16°C, under 12:12 light-dark conditions and was used for experiments within 2–3 days of arrival. Isolation of sensory neurons and culture were done as described earlier  (Roche Diagnostics). The molecular sizes of riboprobes were confirmed by gel electrophoresis. Electrophoresis and transfer of the RNA was performed using DIG Northern Starter Kit (Roche Applied Science) and followed manufacturer's protocols. Briefly, 5 and 10 μg of total RNA from Aplysia was run on 1% agarose gel, transferred to a positive nitrocellulose membrane following hybridization with strand-specific riboprobes at 68°C overnight with ExpressHyb.A 415-bp DNA fragment corresponding to exons 1 and 2 of sensorin mRNA was cloned into TOPO Vector with dual promoters T7 and SP6  and then Aplysia CNS using the standard RNA Trizol extraction method and dissolved in nuclease-free water. For the preparation of RNA from SNs, cell body and neurites were separated by manual micro-dissection followed by RNA extraction using Trizol. RNAs were amplified once using MessageAmp™ II aRNA Amplification Kit following manufacturer's instructions. For the CNS samples 1 μg of the total RNA, and for the cell body and neurites, 500 ng of RNAs were used for reverse transcription using qScript cDNA synthesis mix.Total RNA was extracted from the http://bioinfo.ut.ee/primer3-0.4.0/) based on cDNA and genome sequences listed in UCSC genome browser database (http://genome.ucsc.edu/)  forward and reverse primer. The reaction was carried out in a 7900HT Fast Real-Time PCR System  under the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. Five biological replicates and four technical replicates for each biological replicate were used in the qPCR. Quantification of the target transcripts was normalized to the Aplysia18S reference gene using the Pfaffl method  Figure . Briefly Pfaffl, . Data arin situ hybridization probes were prepared by in vitro transcription of cDNA templates by using SP6 or T7 RNA polymerase. 415 nt long coding region of sensorin was prepared by PCR using Aplysia abdominal ganglion cDNA as a template and sensorin specific PCR primers and ligated to pCRII-TOPO Vector with dual promoters T7 and SP6 . The Vector with the sensorin DNA was linearized with EcoR V (New England Biolab) for transcription using SP6 RNA polymerase to generate antisense probes and the corresponding sense probes were produced by linearizing with BamH I (New England Biolab) and transcribing with T7 RNA polymerase. A small aliquot (2 μ l) was run on 1.5% agarose gel to confirm the integrity of RNA probes.DIG labeled sense and antisense ribo probes for in situ hybridization analysis of sensory neurons were carried out as described in  − Mean background signal.Distributions of β-tubulin protein in both cell body and neuritis were measured to identify any non-specific changes in protein expression associated with aging or forskolin treatment.in situ hybridization analysis of expression of sensorin in sensory neurons using fluorescently labeled sense (S) and antisense (AS) riboprobes (unpublished data). Sensorin mRNA (SRN) is transcribed from psc1 gene on the reverse strand of scaffold 926 of Aplysia genome (available through UCSC genome browser), spanning 40 KB long region and contains four exons  by northern hybridization analysis. Figure Existence of sensorin antisense transcript (NAT-SRN) was previously suspected while performing s Figure . In ordeAplysia kinesin heavy chain (KHC1) and KLC2 , p = 0.027 (NAT-SRN)]. As a control we examined expression of Aplysia KHC1 transcripts in SN neurites. Specific primers for KHC1 were unable to detect expression of antisense or sense transcripts in SN neurites.Previous studies have shown that release of sensorin from the SN is required for both synapse formation and long-term facilitation (LTF) of SN to MN connections. Localization of sensorin is modulated by the formation of synapses between SN and its target motor neurons  indicating that SRN transcript level in the cell body is not affected by aging. However, we observed a significant increase in the distribution of NAT-SRN in the cell body of SNs from old animals . As an endogenous control we used the expression of β-tubulin and found that the β-tubulin protein levels in cell bodies of young and old neurons did not change significantly   Figure .N = 4, Students' t-test, p = 0.0864). Also, we did not observe any significant change in the level of NAT-SRN in the neurites of SNs from old animals when compared to young animals . The endogenous control β-tubulin protein levels in processes of young and old neurons did not change significantly   Figure . These rt-test; p = 0.0007). Interestingly, there was no change in the level of SRN in the neurites of SNs treated with forskolin within 30 min of treatment but the NAT-SRN level was increased by 50% after forskolin treatment in the neurites . β-tubulin protein levels in the cell body .We next studied whether NAT-SRN levels could be regulated by forskolin, an activator of cAMP-CREB signaling. SN cultures (4 DIV) were treated with 50 μM forskolin for 30 min  whether NAT-SRN is physiologically regulated and (b) whether NAT-SRN expression could be regulated by cellular activities that elicit specific signaling pathways leading to memory storage.Despite these elegant studies, we still do not know whether and how NATs are regulated in specific neural circuitries. To address this we used well-characterized neural circuitry in  Kandel,  and stud Kandel, . Sensori Kandel,  and tran Kandel, . To searAplysia. Our FISH analyses suggest that the amount of NAT-SRN increases in cell body of old neurons when compared to corresponding sense transcripts. However, the NAT-SRN level is decreased in neurites of sensory neurons cultured from old animals.To understand physiological regulation of NAT-SRN, we first determined whether its expression changes during aging and whether aging causes changes in subcellular distribution of NAT-SRN. Importantly, aging associated changes in specific NATs are poorly understood. We studied two age groups, young and old Lymnaea changes in the expression of NAT of nitric oxide synthase. Consistent with the idea that NATs could be physiologically regulated, we find that immediately after forskolin treatment, there is an increase in SRN transcripts in the cell body. However, forskolin did not cause an increase in the expression of NAT-SRN in the cell body. Interestingly we find that forskolin exposure resulted in a significant increase in NAT-SRN transcripts in the neurites.To determine whether specific cellular activities might regulate expression of NAT-SRN, we measured changes in expression of NAT-SRN in response to forskolin, an activator of cAMP-PKA-CREB pathway important for long-term memory storage Kandel, . RecentlThe differential subcellular localization of sense and NAT-SRN during aging and in response to forskolin treatment suggest that there might be specific mechanisms that mediate differential expression and transport of SRN and NAT-SRN transcripts. We consider three possible mechanisms: (a) regulation of transcription of NAT-SRN or (b) degradation of NAT-SRN in specific compartments, or (c) changes in axonal transport of NAT-SRN to neurites. It has been shown that expression of NATs could be regulated by epigenetic mechanisms (Conley and Jordan, In summary, we have identified expression of natural antisense RNA of sensorin, an important neuropeptide involved in memory storage. We find that its subcellular localization in sensory neurons is differentially regulated during aging and in response to activation of cAMP-PKA-CREB pathway. We now provide evidence that NATs could be differentially regulated in different sub-cellular compartments. Further studies are required to delineate mechanisms and understand physiological implications of such regulation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Further analysis of the exopolysaccharides from anaerobic granules, and reference polysaccharides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that exopolysaccharides from two of the anaerobic granular sludges studied exhibited spectra similar to that of alginate. The presence of sequences related to the synthesis of alginate was confirmed in the metagenomes of the granules. Collectively these results suggest that alginate-like exopolysaccharides are constituents of the EPS matrix in anaerobic granular sludge treating different industrial wastewater. This finding expands the engineered environments where alginate has been found as EPS constituent of microbial aggregates.Anaerobic granular sludge is composed of multispecies microbial aggregates embedded in a matrix of extracellular polymeric substances (EPS). Here we characterized the chemical fingerprint of the polysaccharide fraction of EPS in anaerobic granules obtained from full-scale reactors treating different types of wastewater. Nuclear magnetic resonance (NMR) signals of the polysaccharide region from the granules were very complex, likely as a result of the diverse microbial population in the granules. Using nonmetric multidimensional scaling (NMDS), the  The capacity of microorganisms to form aggregates  has been the key to the development of high-rate wastewater treatment reactors such as the up-flow and expanded anaerobic granular sludge bed (UASB and EGSB) reactors. Currently, various types of microbial granular communities are being used to treat diverse types of industrial and domestic wastewaters under anaerobic, aerobic or anoxic conditions1Granular sludge is composed of multispecies microbial consortia embedded in a matrix of inorganic and extracellular polymeric substances (EPS). Polysaccharides and proteins are the main constituents of EPS, with minor quantities of extracellular DNA, humic acids and lipids367Exopolysaccharides have been considered relevant in microbial aggregation since some can form viscous or gel-like matrices in which microorganisms can be embedded or attachedIn contrast to aerobic granules, the nature of the exopolysaccharides from anaerobic granules has not been investigated before using advanced techniques such as NMR and MALDI-TOF. Most studies in anaerobic granules have focused on bulk EPS characterizations describing EPS absorbance and or fluorescence properties131H NMR spectrum of polysaccharides can be divided into regions containing resonances corresponding to the sugar ring protons (3.2 to 4.4 ppm) and the anomeric protons (4.4 to 5.6 ppm)1H NMR spectra of the extracted exopolysaccharides from anaerobic granules displayed sugar ring resonances, at 3.2 to 4.4 ppm, that are typical of exopolysaccharides such as gellan, xanthan and alginate  obtained for the different granules were visually represented using NMDS analysis. Represented in two-dimensions, the exopolysaccharide fingerprints of all the granules were relatively close together , a large amount of sample is required to obtain 13C NMR signals necessary to characterize the chemical structure of complex molecules. The low EPS content of the granular sludge of this study (13C NMR-based characterization). Therefore, MALDI-TOF MS was tested as an alternative approach to further characterize the exopolysaccharides of the granular sludges.One drawback of NMR is that due to the low natural abundance of is study  hamperedLactobacillus, Sphingomonas and Xanthomonas, respectivelyMALDI-TOF MS can be considered as a rapid and sensitive approach for obtaining fingerprints of polysaccharidesDextran, gellan and xanthan, showed serial peaks with mass unit intervals of 162 m/z, which is consistent with hexose unit residues  and 4. HIn contrast, the spectra of alginate reproducibly exhibited a distinct bell-shape series of peaks  with a pThe polysaccharides from the AnG E granules (paper mill wastewater) yield some signals . Howeveret al.et al.et al.An alginate-like polysaccharide extracted from aerobic granules was characterized by Lin In the current study, the polysaccharides were not pre-treated  prior to their detection. Pre-treatment steps can be time consuming and laborious21In this study, 16S rRNA gene pyrosequencing was used to characterize the bacterial and archaeal community in the anaerobic granules see .et al.Candidatus Competibacter) whose abundance was ≥60% in aerobic granules from an acetate fed laboratory-scale reactor. However, Lin et al.vs municipal-abattoir water). However, a larger data set comprising microbial and exopolysaccharides fingerprints of full-scale granular sludge from diverse wastewater types would be necessary for recognition of patterns and data mining at higher resolution.Although the most abundant bacterial class differed among the three granular sludge samples, the exopolysaccharides NMR fingerprints of all the granules clustered together, and were closer to the fingerprint of the reference polysaccharide alginate than to the fingerprints of the other reference polysaccharides , suggestMethanobacteria and Methanomicrobia which comprise families of hydrogenotrophic and acetoclastic methanogens, respectively -mannose dehydrogenase (AlgD), which catalyzes the production of the key alginate precursor GDP-mannuronic acidalgD is exclusive for the synthesis of alginate and is not known to be required for any other cellular process. Additionally, genes encoding alginate modifying enzymes algI and algJ as well as regulatory genes, algB and algR, involved in initiating the transcription of the alginate operon were also found in the anaerobic granular sludges  alginate-like exopolysaccharides in anaerobic granules treating different industrial wastewaters in this study, (ii) alginate-like exopolysaccharides in aerobic granules and in flocculant activated sludge in a previous study−1 Therefore, it is relevant to investigate extracellular components  in microbial aggregates from full-scale systems because those components are the matrix constituents of granules under real conditions. Particularly identifying those matrix constituents and their amounts from physically- and microbially- well functioning granules in relation to operational characteristics and microbial community composition of the reactors can help to envisage strategies to promote and maintain granular sludge under real full-scale conditions.et al. (2005)There has not been a detailed study on characterization of exopolysaccharides in anaerobic granules. This is mainly due to the difficulty in characterizing polysaccharides despite current advances in analytical tools. Although full chemical characterization of all exopolysaccharides of the anaerobic granules was not possible with the current tools, our approach of integrating their obtained spectra with multivariate analysis provided a basis to realizing that a common exopolysaccharide type can be produced in various anaerobic granular sludges. The finding of alginate-like exopolysaccharides in anaerobic granules expands the engineered environments where alginate has been found as EPS constituent. Our previous knowledge regarding alginate-like exopolysaccharide was based on aerobic granular sludges that were cultivated under anaerobic-aerobic conditions1H NMR, and/or MALDI-TOF, combined with NMDS could potentially be used as a method for monitoring or comparing profiles of exopolysaccharides from various microbial aggregates such as granules and flocs as well as biofilms in biological wastewater treatment systems and as a function of operational conditions.Considering the relevance of exopolysaccharides in wastewater treatment systems and given that characterizing exopolysaccharides from mixed microbial systems is not straightforward, it is proposed that the use of All granular sludges were collected from full-scale reactors in The Netherlands. The anaerobic granules were from upflow anaerobic reactors treating paper mill (AnG E), brewery (AnG B) and sulfate-rich thermophilic (AnG W) wastewaters. Information regarding operational conditions of the papermill and the brewery reactors have been previously reported36et al.Two methods were used to extract (crude) EPS including the cation exchange resin (CER) method1H NMR spectra were recorded by collecting 1k scans with a recycle delay time of 3 sec. To suppress the water peak, each spectrum was induced with an excitation sculpting pulse sequence using the standard (zgespg) program from the Bruker pulse library. Chemical shifts were determined relative to 3-trimethylsilylpropane sulfonic acid  as internal chemical shift reference. The free induction decay (FID) data were collected with spectral width of 15243 Hz digitized into 64k data points. The FID signals were zero-filled and amplified by an exponential line-broadening factor of 0.3 Hz before Fourier transformation. The Topspin 2.1 software (Bruker) was used in all experiments to collect and analyze data.Samples of reference polysaccharides were prepared by dissolving separately 8 mg of alginate , 8 mg of gellan  or 3 mg of xanthan  in 1 mL of 0.1M NaOH in deuterium oxide The similarities between NMR spectra were visually represented using non-metric multidimensional scaling (NMDS). Compared to other ordination methods, NMDS does not assume linear relationships or normal distributions of data. The algorithm iteratively searches to place samples as points on k dimensions (axis) while attempting to maintain the relative distances of points as close as possible to the actual rank order of similarities between samples. Thus, samples having similar NMR spectra are plotted together in the ordination space.http://www.rstudio.org/) and the community ecology package vegan (http://CRAN.R-project.org/package=vegan) using the Bray-Curtis coefficient for calculating the dissimilarity matrix.The polysaccharide regions (3.2 to 5.6 ppm) of the NMR spectra were subdivided into sequential 0.2 ppm regions; each region was integrated (using the Topspin 2.1 software) and normalized  in proportion to the total area of the polysaccharide region. With this data set, NMDS analysis and hierarchical clustering with the complete linkage method was carried out using R Studio . A 2 μL volume of the sample-matrix mixture was spotted on a MTP Anchorchip 600–384  target plate. Similar to the samples, the reference polysaccharides dextran, gellan, xanthan and alginate (3 mg mL−1) were prepared for analysis. The DHB matrix has been previously used for the analysis of oligo and polysaccharidesSamples were mixed at a 1:1 ratio with 2,5-dihydroxybenzoic acid (DHB). The DHB matrix was prepared at 20 mg mLGenomic DNA was extracted from the granules using the Power Soil DNA isolation kit ; see SI. High-throughput pyrosequencing of 16S rRNA gene fragments and quantitative-PCR of bacteria and archaea was conducted as detailed in the Shotgun metagenome sequencing of the granules was conducted as described in the How to cite this article: Gonzalez-Gil, G. et al. NMR and MALDI-TOF MS based characterization of exopolysaccharides in anaerobic microbial aggregates from full-scale reactors. Sci. Rep.5, 14316; doi: 10.1038/srep14316 (2015).


Conversely, the wing regions have better quality with less stress as the dislocations propagated upwards when the GaN stripes are along [Uncoalesced a-plane GaN epitaxial lateral overgrowth (ELO) structures have been synthesized along two mask stripe orientations on a-plane GaN template by MOCVD. The morphology of two ELO GaN structures is performed by Scanning electronic microscopy. The anisotropy of crystalline quality and stress are investigated by micro-Raman spectroscopy. According to the Raman mapping spectra, the variations on the intensity, peak shift and the full width at half maximum (FWHM) of GaN E It is a very attractive material especially for application in electronic and optoelectronic devices. The majority of commercial GaN devices are grown with respect to the c-plane. The polarization field along c-axis could give rise to a significant band bending and thus cause the separation of holes and electrons which is beneficial to the High Electron Mobility Transistor (HEMT) devices, while this situation could also induce the reduction of optical efficiency of light-emitting diodes (LEDs), as well as an undesirable redshift in the emission spectra from the quantum wells what is called the quantum-confined Stark effect (QCSE)23458103) were used as source compounds. Hydrogen was used as the carrier gas. The growth temperature was kept at 1000 °C. A 100-nm-thick SiNx film was subsequently deposited onto the a-plane GaN template. Then, as shown in x film was patterned into 5-μm-wide mask stripes and 2-μm-wide window stripes oriented along the [0001] and  mask stripe direction, many TDs bend during the vertical growth. This phenomenon is explained by the dislocation following a path of minimum elastic energy per unit of growth length of materialsIn order to further investigate the spatial distributions of TDs in the two samples, SEM-CL studies have been carried out. Here, the region with higher density of TDs shows lower luminescence intensity which may be due to the TDs acting as nonradiative recombination centersFrom x mask stripes both parallel to [0001] and In summary, the spatial mapping of micro-Raman measurements have been performed on uncoalesced ELO GaN structures along SiNHow to cite this article: Jiang, T. et al. Spatially resolved and orientation dependent Raman mapping of epitaxial lateral overgrowth nonpolar a-plane GaN on r-plane sapphire. Sci. Rep.6, 19955; doi: 10.1038/srep19955 (2016).


Prosthetic heart valve (PHV) dysfunction remains difficult to recognise correctly by two-dimensional (2D) transthoracic and transoesophageal echocardiography (TTE/TEE). ECG-triggered multidetector-row computed tomography (MDCT), 18-fluorine-fluorodesoxyglucose positron emission tomography including low-dose CT (FDG-PET) and three-dimensional transoesophageal echocardiography (3D-TEE) may have additional value. This paper reviews the role of these novel imaging tools in the field of PHV obstruction and endocarditis.For acquired PHV obstruction, MDCT is of additional value in mechanical PHVs to differentiate pannus from thrombus as well as to dynamically study leaflet motion and opening/closing angles. For biological PHV obstruction, additional imaging is not beneficial as it does not change patient management. When performed on top of 2D-TTE/TEE, MDCT has additional value for the detection of both vegetations and pseudoaneurysms/abscesses in PHV endocarditis. FDG-PET has no complementary value for the detection of vegetations; however, it appears more sensitive in the early detection of pseudoaneurysms/abscesses. Furthermore, FDG-PET enables the detection of metastatic and primary extra-cardiac infections. Evidence for the additional value of 3D-TEE is scarce.As clinical implications are major, clinicians should have a low threshold to perform additional MDCT in acquired mechanical PHV obstruction. For suspected PHV endocarditis, both FDG-PET and MDCT have complementary value. Valvular heart disease often requires prosthetic heart valve (PHV) replacement in order to improve quality of life and survival. The number of PHV implantations is expected to rise due to ageing, the growth of the population and the development of catheter-based techniques for valve replacement in patients who were inoperable before these techniques emerged. The major drawback of both mechanical and biological PHVs is development of dysfunction that is accompanied by high morbidity and mortality . The repAs symptoms may be non-specific, echocardiography of PHVs plays a pivotal role and guides the therapeutic regimen. However, acoustic shadowing and reverberation of the metal of the PHVs hamper reliable imaging by transthoracic and transoesophageal echocardiography (TTE and TEE). For the detection of PHV leakages (not in the context of endocarditis) the combination of TTE and TEE has a high diagnostic accuracy for severity and location  and diagnostic dilemmas are mostly solved –6. PHV eStaphylococcus aureus is the causative bacterial agent [PHV endocarditis occurs with an incidence of 0.3–1.2 % per patient-year and is complicated by periannular extensions (pseudoaneurysms and abscesses) in at least 50 % of cases –9. This al agent –11. The al agent ; Table 1al agent , 10, 11.The diagnostic value of TTE and TEE has recently been systematically reviewed and meta-analysed . TEE wasMDCT has recently emerged as a promising novel imaging technique to evaluate PHVs. The technique uses electrocardiographic (ECG) gating to overcome artefacts related to the movement of the heart during the cardiac cycle. Reconstruction of the retrospectively ECG-gated acquired images provides 3D assessment of the PHV in any plane in each phase of the heart cycle. The acquisition is performed after the administration of contrast material. However, a non-contrast enhanced scan may have additional value to discriminate suture pledgets and calcifications of the PHV and can be performed using prospective ECG triggering in a single phase of the cardiac cycle. Contrast enhanced MDCT may detect PHV thrombi, pannus, vegetations, paravalvular leakages, mycotic aneurysms and even abscesses. In the field of PHV endocarditis three studies have been performed with MDCT –15. Thesthe desired imaging strategy in patients with high suspicion of PHV endocarditis but negative/inconclusive echocardiography  emits positrons. Unlike glucose, 18F-FDG is not metabolised but trapped in the human cell. Infected cells increase glucose consumption, resulting in accumulation of 18F-FDG . Howeverography ; Fig. 4)the desirography  is a 3D nuclear imaging technique. When using radioactively labelled leucocytes as the tracer it can be applied for the detection of infection. Images are obtained from multiple angles and are reconstructed in 3D. The study by Hyafil et al. investigated the additional value of detection of perivalvular infection in patients with a suspicion of PHV endocarditis and inconclusive TEE . DiagnosA major drawback of additional techniques such as MDCT and nuclear imaging is that they are expensive, expose patients to radiation and in case of MDCT iodinated contrast is administered. The use of contrast is relatively contraindicated when PHV endocarditis is complicated by renal failure. In most modern echo labs 3D-TEE is available for valve evaluation (anatomy) and guidance of treatment . This 3D technique may also be beneficial for detection of PHV endocarditis. 3D-TEE is performed with a multiplane probe including a 3D matrix-array. 3D full-volume datasets allow offline image editing using the multiple plane reconstruction mode and freehand cropping mode , 25. ThiCardiac MRI is safe beneath a value of 3 T for all modern PHVs  as no significant interruption of valve motion nor overheating have been observed , 27. Theand proven PHV endocarditis.PHV patients with a suspicion of endocarditis should undergo TTE and TEE by an experienced sonographer. If 3D-TEE is available when performing 2D-TEE, one should have a low threshold to acquire 3D images of the PHV, at least for confirmation of the 2D findings. Patients with a high suspicion of PHV endocarditis and negative or inconclusive echocardiography should undergo additional MDCT and FDG-PET. In line with the recently published ESC guideline on endocarditis , we suggMDCT may replace preoperative invasive coronary angiography, which is not desirable in patients with aortic PHV vegetations as mechanical manipulation by an invasive catheter may be complicated by distal embolisation. Furthermore, in case of PHV reoperation MDCT provides valuable information about the distance between the sternum and the right ventricle and also the relation of the sternum to bypass grafts if present. In contrast to non-PHV cases , non-invTTE is the first diagnostic screening tool with a good diagnostic accuracy for PHV obstruction. Transprosthetic valve gradients derived simultaneously by continuous wave Doppler and invasive catheter pressure measurements have a good correlation . BiologiPHV endocarditis and obstruction remain difficult to diagnose correctly by echocardiography alone. As clinical implications are major, this paper provides evidence that clinicians should have a low threshold to perform additional imaging by novel techniques in the field of PHV dysfunction, such as 3D-TEE, FDG-PET and/or MDCT.This work was supported by a grant from the Dutch Heart Foundation (NHS-2009B014)Undisclosed authors did not contribute to this manuscript.


Sacbrood virus (SBV) and Deformed wing virus (DWV) are evolutionarily related positive-strand RNA viruses, members of the Iflavirus group. They both infect the honeybee Apis mellifera but have strikingly different levels of virulence when transmitted orally. Honeybee larvae orally infected with SBV usually accumulate high levels of the virus, which halts larval development and causes insect death. In contrast, oral DWV infection at the larval stage usually causes asymptomatic infection with low levels of the virus, although high doses of ingested DWV could lead to DWV replicating to high levels. We investigated effects of DWV and SBV infection on the transcriptome of honeybee larvae and pupae using global RNA-Seq and real-time PCR analysis. This showed that high levels of SBV replication resulted in down-regulation of the genes involved in cuticle and muscle development, together with changes in expression of putative immune-related genes. In particular, honeybee larvae with high levels of SBV replication, with and without high levels of DWV replication, showed concerted up-regulated expression of antimicrobial peptides (AMPs), and down-regulated expression of the prophenoloxidase activating enzyme (PPAE) together with up-regulation of the expression of a putative serpin, which could lead to the suppression of the melanisation pathway. The effects of high SBV levels on expression of these immune genes were unlikely to be a consequence of SBV-induced developmental changes, because similar effects were observed in honeybee pupae infected by injection. In the orally infected larvae with high levels of DWV replication alone we observed no changes of AMPs or of gene expression in the melanisation pathway. In the injected pupae, high levels of DWV alone did not alter expression of the tested melanisation pathway genes, but resulted in up-regulation of the AMPs, which could be attributed to the effect of DWV on the regulation of AMP expression in response to wounding. We propose that the difference in expression of the honeybee immune genes induced by SBV and DWV may be an evolutionary adaptation to the different predominant transmission routes used by these viruses. Apis mellifera, is the most important managed insect pollinator worldwide. In recent decades a global decline in the number of honeybee colonies was reported, threatening security of the global food supply  a predominant viral pathogen in the Asian honeybee Apis cerana, accumulates to high levels and causes acute infections in orally inoculated honeybee larvae . We found that different sets of genes were differentially expressed (DE) in honeybee larvae with high levels of either DWV alone or SBV and DWV combined, and that high levels of SBV infection had a more significant impact on global gene expression in the honeybee compared to high levels of DWV, in particular on the expression of immune-related genes. We found, in both larval feeding and pupal injection experiments, that high levels of SBV were associated with up-regulation of the expression of antimicrobial peptide (AMP) genes and changes in expression of the genes involved in regulation of melanisation, which may suppress this function. These differential effects of DWV and SBV on expression of AMP and melanisation pathway genes may be an adaptation of these viruses to facilitate their vertical and horizontal routes of transmission respectively.Apis mellifera) with low managed levels of Varroa destructor infestation were maintained in Warwickshire, UK, and used as a source of larvae and pupae. DWV virus preparation was isolated from honeybee pupae sourced from a colony with high Varroa infestation levels. Virus preparations containing both SBV and DWV (SBV + DWV) were purified from larvae and pupae of the Varroa-infested colonies where some larvae showed typical SBV-induced symptoms. No other known honeybee viruses were detected in the prepared virus stocks. Virus isolation was carried out as described previously  were added to 50 µl of the honeybee rearing diet per bee, which was consumed within 12 h. No virus was added to the subsequent portions of the larval food. The controls in the feeding experiment included virus-free phosphate-buffered saline (PBS), and the UV-inactivated SBV + DWV virus preparation . The larvae were maintained for an additional 9 days up to the late fourth instar stage. Whole-body RNA samples were extracted from individual insects at 4 days post inoculation (dpi) or 9 dpi.Artificial rearing of the honeybee larvae was carried out essentially as described previously . For ora6 DWV virus particles in PBS), or with the mixture of SBV and DWV (106 SBV and 106 DWV virus particles in PBS). The pupae were reared at +33 °C as previously described  received injections into the haemolymph using a syringe with a 0.3 mm outer diameter needle  either wescribed . Whole-bPRJEB6511.Total RNA was extracted from individual pupae with Tri-reagent (Trizol)  according to the manufacturer’s instructions. The extracted column-purified total RNA from individual honeybees was used for high-throughput sequencing of the mRNA populations by RNA-Seq. The experiment and the reads were deposited into the European Nucleotide Archive under accession number Quantification of viral RNA and the honeybee transcripts were carried out by quantitative reverse transcription PCR (qRT-PCR) as described previously . In brieNC˙004830and AF092924respectively). The Next Generation Sequencing (NGS) gene expression profiles were used to identify differentially expressed (DE) genes using DESeq  below 0.05. Drosophila homologues of the honeybee genes were identified previously . Notably, this DWV dosage was 100 times higher than a dose used in oral infection of the adult bees which did not result in establishing high levels of DWV infection  and controls included UV-inactivated “SBV + DWV” virus preparation and PBS . The dosnfection . QuantifFor comprehensive characterization of honeybee gene expression in response to high levels of DWV and SBV, we used an RNA-Seq approach. The analysis was carried out using whole-body RNA extracted from individual honeybee pupae sampled at 9 dpi. Controls included pupae with low levels of DWV and SBV (samples 1 and 2) for comparison with the three virus-infected samples; one of these (sample 3) had high level of DWV and low level of SBV, and two samples had high levels of both SBV and DWV (samples 4 and 5) .NC˙004830and AF092924respectively) no other pathogens were detected. We observed a dramatic increase of DWV and SBV coverage, normalized to host actin mRNA coverage (GB44311), in infected honeybees compared to controls (Approximately 10 million 101 nt reads were produced for each library  and werecontrols . For exacontrols . SBV levcontrols . The obscontrols .We stratified the RNA-Seq samples according to the levels of DWV and SBV (high and low) into three groups, “Control” (samples 1 and 2), “DWV” (sample 3), and “SBV + DWV” (samples 4 and 5) and, by using both DESeq  and edgeThe highest numbers of DE genes were identified in the contrasts involving the “SBV + DWV” group. Of these, contrast 4 (high SBV + DWV vs control) had 1,638 DE genes, which included almost all  of those identified as DE in Contrast 2 . High commonality, 697 of 824 genes, was also observed between the DE genes in Contrast 5 and Contrast 4  was identified in Contrast 1 , and the low commonality between Contrasts 3 and 4 (n = 9) strongly suggested that transcriptional responses to high levels of DWV and SBV + DWV were different  was lower than those observed in Contrasts 2, 4 and 5, all of which involved the SBV + DWV group . A very ifferent . Indeed,ifferent  showed tifferent . Changesifferent , consistifferent . Surprisifferent . Howeverifferent . The oveifferent . In respifferent .GB47318) and hymenoptaecin (GB51223) are controlled by the Imd pathway (GB51741), persephone (GB55007), spatzle (GB52631) and one of the Toll receptors (GB50418), and down-regulation of two other Toll receptors (GB40699and GB43456) (GB50013), the only honeybee enzyme which proteolytically cleaves prophenoloxidase (GB48820and GB54611)  . We alsoloxidase  and up-rGB54611) . We propGB41428), or the Imd pathway , and the components of the melanisation pathway , we quantified gene expression levels in orally-infected larvae by qRT-PCR  and the expression level of honeybee immune genes of interest was quantified . Both AMin vitro  were significantly up-regulated in the DWV pupae and SBV + DWV-injected pupae compared to the PBS-injected control. There were no significant differences between the pupae groups with high levels of DWV alone and high levels of both SBV and DWV (GB48820) (GB48820) compared to orally infected larvae. At 2 dpi there was no significant difference between expression levels of defensin-1 and serpin  and 4G w and DWV  and 4H aA. mellifera with symptoms characteristic to most beekeepers. To better understand the influence of the route of virus transmission on disease development we investigated changes in gene expression resulting from orally administered or injected DWV and SBV. Transcriptome analysis of pupae showed strong up-regulation of the expression of AMPs (defensin-1 and hymenoptaecin) in orally infected larvae with high levels of SBV. Interestingly, high levels of DWV did not up-regulate these AMPs in orally infected larvae , it rem aegypti .Although the viral proteins encoded by SBV may directly and simultaneously activate the Toll and Imd pathways (which will require further studies) an alternative hypothesis is this results indirectly from SBV-induced pathogenesis. For example, the extensive disruption of the tracheal epithelial lining and pertrophic membranes caused by SBV infection  may alloVarroa mite vectoring, is transmitted vertically via queens and drone semen, and horizontally during trophylaxis (GB48820), may favour SBV transmission as melanisation contributes to virus resistance and could decrease infectivity of SBV particles in the larvae, thereby reducing horizontal transmission Fig. 5). Our results indicate that evolutionarily-related Iflaviruses SBV and DWV, evoke markedly different transcriptional responses in their honeybee host, including effects on the expression of immune-related genes. We also observed dominance of the SBV-induced transcriptome changes over the DWV-induced. Honeybee larvae with high levels of SBV replication, showed concerted up-regulated expression of antimicrobial peptides (AMPs) and down-regulated expression of the prophenoloxidase-activating enzyme (PPAE) together with up-regulation of the expression of a putative serpin, which could lead to the suppression of the melanisation pathway. The same effect was observed in the individuals with high levels of both SBV and DWV, but high levels of DWV alone did not affect expression of the AMPs and the genes involved in the regulation of melanisation. The effects of high SBV replication levels on expression of these immune genes were unlikely to be the consequences SBV-induced developmental changes, because some of them were observed in honeybees infected with SBV by injection at the pupal stage. It is possible that different impacts of SBV and DWV on the expression of immune-related genes may be an adaptation to horizontal and vertical transmission routes, the principal transmission routes of SBV and DWV respectively. These findings provide the basis for further studies of the contributions of AMPs and melanisation to virus-host interactions and the transmission of insect viruses, including economically important species such as honeybees.10.7717/peerj.1591/supp-1Table S1Click here for additional data file.10.7717/peerj.1591/supp-2Table S2Click here for additional data file.10.7717/peerj.1591/supp-3Table S3Click here for additional data file.10.7717/peerj.1591/supp-4Table S4Click here for additional data file.10.7717/peerj.1591/supp-5Table S5Click here for additional data file.10.7717/peerj.1591/supp-6Figure S1Click here for additional data file.