Surgical removal of mandibular third molars results in some degree of post-operative pain, swelling and trismus. These can be controlled by proper administration of local anesthesia, careful bone removal, minimal trauma to adjacent soft tissues and administration of methylprednisolone and serratiopeptidase drugs. The aim of the present study was to compare the efficacy of methylprednisolone and serratiopeptidase in controlling post-operative pain, swelling and trismus after surgical removal of impacted mandibular third molars. The subjects were divided into two groups of 50 patients each undergoing surgical removal of mandibular third molars. Group A was given methylprednisolone 4mg orally every 8th hourly and Group B was given serratiopeptidase 10 mg every 12th hourly orally. Post-operatively pain, swelling and trismus were evaluated at the end of 1st, 3rd and 5thday. The results of this study showed that methylprednisolone is an effective analgesic, while serratiopeptidase has moderate analgesic activity. Serratiopeptidase is more effective than methylprednisolone in controlling post surgical swelling and trismus. Hence combination of these two drugs would be very effective than individual drug when widespread post-operative sequelae are expected after surgical removal of impacted lower third molars. We conclude that methylprednisolone affords better pain relief while serratiopeptidase exerts better anti-inflammatory and anti-swelling effects in the post-operative period. Synergistic combinations of these two drugs would however prove to be more effective when extensive post-operative sequelae are expected. Key words:Methylprednisolone, serratiopeptidase, pain, swelling, trismus, third molar. Surgical removal of teeth in oral surgery inevitably requires bone removal, which sometimes results in some degree of post-operative pain, swelling and trismus. Minimal trauma to adjacent soft tissues and proper wound closure minimizes pain, swelling and trismus. These can be controlled by proper administration of local anesthesia, good flap reflection, careful bone removal and minimal trauma to adjacent soft tissues with appropriate wound closure techniques . Pharmacet al. for establishing the anti-inflammatory effect of methylprednisolone, the author found that the methylprednisolone group had mean swelling volumes significantly lower than that of the control . However, statistical significance was observed on day 5. (Un paired t–test). Thus, serratiopeptidase has better efficacy in controlling post-operative edema than methylprednisolone , suggesting that pain control efficacy in group I was better than in group II (Chi-square test)  and 5 (n=0). Under the moderate pain category, group I had 9, 39 and 28 patients on day 1, 3 and 5 respectively, whereas group II had 1, 14 and 50 patients on day 1, 3 and 5 respectively. Under the severe pain category, group I had 36 patients on day 1 and 5 patients on day 3 but no pain was reported on day 5 (n=0), whereas group II had 48 and 36 patients on day 1 and 3 respectively. No pain was reported on day 5 (n=0). These observations indicated statistically significant differences between the two groups (re test) , Fig. 2.- Comparison of changes in trismus between two groups The mean difference in mouth opening post-operatively on day 1 in group I was 17.7 ± 5.5mm; this difference reduced to 9.4 ± 4.0 and 1.3 ± 2.8 on day 3 and 5 respectively. The mean difference in mouth opening post-operatively on day 1 in group II was 22.1 ± 4.2mm; this difference reduced to 13.8 ± 5.6 and 8.3 ± 4.6 on day 3 and 5 respectively.P<0.001) i.e. resolution of trismus was better in group II than in group I. (Un paired t–test) This attests to the probable fact that serratiopeptidase is better than methylprednisolone in resolving trismus post-operatively , from plant (papase and bromelain) and from animals (trypsin and chymotrypsin). Serratiopeptidase is a proteolytic enzyme produced by a microorganism Serratia species. This endopeptidase is prepared from the culture broth of genus Serratio SP E-15 (one of the enteric bacilli in silk worm). It has high enzyme activity, including a potent anti-inflammatory action, anti-swelling as well as bradykinin-decomposing activity and it also enhances the antibiotic action at the site of the lesion ,7.et al. suggested that intravenous administration of methylprednisolone combined with low oral doses in effective management of postoperative surgical sequale. In our study, methylprednisolone 4 mg was administered orally for 5 days, which showed apparently good improvement clinically (Troullos inically .Pederson concluded that steroid administration is effective in reducing post-operative complaints when pronounced post-operative reaction can be expected and that short-term administration is safe in the absence of contraindications for such administration. In the present study, methylprednisolone was found to be safe in the absence of contraindications .Panwar SK. concluded that a single preoperative dose of prednisolone reduces postoperative swelling. Statistical analysis and observations were done to determine the difference in swelling, trismus and pain among the control group and study group. A single pre-operative dose of oral prednisolone helped the patient to cope the postoperative sequela, without causing major side effects. In our study, methylprednisolone 4 mg administered orally for 5 days, showed good satisfactory results .et al. used serratiopeptidase for reduction of postoperative swelling in upper ankle joint surgery and concluded that significant reduction in swelling had been achieved post- operatively with the use of serratiopeptidase. In the present study similar effects were observed with serratiopeptidase proving it to be better in reducing swelling than methylprednisolone (Esch nisolone .Al-Khateeb and Nusair investigated the ability of serratiopeptidase to reduce post-operative pain, swelling and trismus after third molar surgery. There was a significant reduction in the pain intensity and extent of cheek swelling in the serratiopeptidase group at the 2nd, 3rd and 7th post-operative days. The present study also indicated similar results in which the serratiopeptidase group showed significantly reduced trismus and swelling on the day 1, 3 and 5 post-operatively. However, this enzyme preparation did not exert an appreciable analgesic effect post-operatively (Murugesan K. found that dexamethasone was more effective in reducing swelling and pain when compared with serratiopeptidase, however both had same effect on trismus. In our study, serratiopeptidase 10 mg orally produced an appreciable anti-edema effect and had no apparent effect on rate of wound healing .In the present study two different drugs methylprednisolone and serratiopeptidase were compared for clinical efficacy on post operative pain, swelling and trismus after surgical removal of mandibular third molars. Methylprednisolone affords better pain relief while serratiopeptidase exerts better anti-inflammatory effects in the post-operative period. Synergistic combinations of these two drugs would however prove to be more effective when extensive post-operative sequele are expected, but randomized case-control research trials need to be conducted in the future to attest to this probable fact.


Fusarium spp. 11β-Hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2), have been demonstrated to be the regulators of the local level of active glucocorticoid, which has a broad range of physiological actions. In the present study, the potency of zearalenone was tested for the inhibition of HSD11B1 and HSD11B2 in rat and human tissues. Zearalenone showed potent inhibition of HSD11B2 with the half-maximal inhibitory concentration (IC50) calculated at 49.63 and 32.22 μM for the rat and human, respectively. Results showed that zearalenone competitively inhibited HSD11B2 when a steroid substrate was used. However, it served as an uncompetitive inhibitory factor when the cofactor NAD+ was used. In contrast, the potency of zearalenone to inhibit both rat and human HSD11B1 was diminished, with the concentration of 100 μM causing almost no inhibitory effect on the isoform. In conclusion, we observed that zearalenone is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as kidney and placentas.Zearalenone is a mycotoxin produced by Fusarium spp. -cortisol (specific activity: 70–100 Ci/mmol) were purchased from DuPont-New England Nuclear . -11-Dehydrocorticosterone (3H-11DHC) and -cortisone were prepared from labeled 3H-corticosterone or 3H-cortisol as described earlier -11DHC or [3H]-cortisone, respectively, according to a previously described method [μM.HSD11B1 activity was measured in rat and human liver microsomes using [d method . The ass3H]-corticosterone or [3H]-cortisol, respectively, according to a previously described method [+ and 2 mM DTT. The microsomes were preincubated with zearalenone for 2 min and then added to the above tubes. The initial test concentration for zearalenone was 100 μM. When the inhibitory concentration was established, different concentrations of zearalenone were used.HSD11B2 activity was measured in rat kidney and human placental microsomes using [d method . The assOn each occasion, the reactions were stopped by adding 2 mL ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform and methanol , and the radioactivity was measured using a scanning radiometer . HSD11B1 activity was determined by the percentage conversion of 11DHC to corticosterone or cortisone to cortisol and HSD11B2 activity by the conversion of corticosterone to 11DHC or cortisol to cortisone by dividing the radioactive counts identified by the total counts associated with substrate and products.50) were calculated. Michaelis-Menten kinetics and Lineweaver-Burk plots were drawn for the analysis of the mode of inhibition of zearalenone.The dose-dependent inhibition of zearalenone on HSD11B was subjected to nonlinear analysis by GraphPad , and half-maximal inhibitory concentrations  of rat and human HSD11B2 activity at the 100 μM concentration . At first, we tested the mode of inhibition of zearalenone on this enzyme when cortisol was used for human enzyme. Michaelis-Menten kinetics  had reduced testosterone level . Adult m50 values of 32–49 μM, while it almost lacked effective inhibition of rat and human HSD11B1 even at 100 μM. Structurally, zearalenone is a phenol with a hydroxyl group, which is similar to 11-beta hydroxyl group in cortisol and cortisone. The action of zearalenone on HSD11B2 appears to be similar to that reported for another polyphenolic compound, that is, gossypol, which is also highly selective for HSD11B2 compared to HSD11B1 [+. When NAD+ was used, zearalenone showed mixed uncompetitive inhibition, indicating that zearalenone binds to NAD+-bounded enzyme complex.Overall, zearalenone has greater selectivity for HSD11B2 versus the HSD11B1 enzyme, because it inhibited rat and human HSD11B2 with IC HSD11B1 . HoweverWe tested the effects of zearalenone on HSD11B1 and HSD11B2 and found that zearalenone selectively inhibited both rat and human HSD11B2 enzyme. The mode of action of zearalenone on HSD11B2 is a competition when a glucocorticoid substrate is used.


Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe. Protein kinases (PK) are a large and diverse family of enzymes that catalyze the transfer of the γ-phosphate group from ATP to specific tyrosine, serine or threonine residues14The catalytic domain of PKs assumes a common fold consisting of two lobes, a smaller N-terminal lobe and a larger C-terminal one751112171011151617At the same time, H/D exchange experiments on PKA7811However, it has been long known that both inter-lobe hinge dynamics (clamshell-like)919201112172023The Src cytoplasmic non-receptor protein kinase, the first viral oncogenic protein discovered, is an ideal system for mechanistic explorations of PKs due to its high conformational flexibility, biomedical and historical importance3451491H,15N TROSY NMR spectrum of the triply isotopically labelled sample at 1 GHz is shown in SI The NMR assignment of Src catalytic domain  of the phosphorylated and unphosphorylated forms of Src , shows a1418As shown in To understand the role of the cofactor in achieving the fully active conformation, we recorded NMR spectra of Src and SrcP bound to ATP and ADP. Upon addition of ATP to unphosphorylated Src, we observe a fast trans-autophosphorylation of Src. Indeed, phosphorylation is achieved within minutes from the addition, as observed by NMR spectra and confirmed by MS , leading1H,15N TROSY spectrum are evident (see CSP in Addition of an excess of ATP produces the spectrum of phosphorylated Src bound to ATP (SrcP-ATP). When titrating SrcP with ATP, the changes in the Most importantly, the signals of SrcP complexed with ATP appear in very similar positions to the second form observed in both Src and SrcP . The molecular dynamics simulations were performed with the GROMACS simulation package33Samples were prepared in 20 mM phosphate at pH 6.5, 0.5 M NaCl, 1 mM MgClHow to cite this article: Pucheta-Martínez, E. et al. An Allosteric Cross-talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase. Sci. Rep. 6, 24235; doi: 10.1038/srep24235 (2016).


The use of new headless compression screws (HCSs) for scaphoid fixation is growing, but the nonunion rate has remained constant. The aim of this study was to compare the stability of fixation resulting from four modern HCSs using a simulated fracture model to determine the optimal screw design(s).We tested 40 fresh-frozen cadaver scaphoids treated with the Acumed Acutrak 2 mini (AA), the KLS Martin HBS2 midi (MH), the Stryker TwinFix (ST) and the Synthes HCS 3.0 with a long thread (SH). The bones with simulated fractures and implanted screws were loaded uniaxially into flexion for 2000 cycles with a constant bending moment of 800 Nmm. The angulation of the fracture fragments was measured continuously. Data were assessed statistically using the univariate ANOVA test and linear regression analysis, and the significance level was set at p < 0.05.2 = 0.827. This was followed by AA (R2 = 0.354), SH (R2 = 0.247) and ST (R2 = 0.019). Data assessed using an adapted ANOVA model showed no statistically significant difference (p = 0.291) between the screws.The median angulation of bone fragments φ allowed by each screw was 0.89° for AA, 1.12° for ST, 1.44° for SH and 2.36° for MH. With regards to linear regression, the most reliable curve was achieved by MH, with a coefficient of determination of RThe continuous development of HCSs has resulted in very comparable implants, and thus, at this time, other factors, such as surgeons’ experience, ease of handling and price, should be taken into consideration. Scaphoid fractures are common and problematic in young active males. The incFracture displacement is considered an absolute indication for fracture reduction and fixation. In particular, acute fractures of the proximal pol, fractures with preexisting cystic bone formation, transscaphoid perilunate dislocations, untreated fractures that are more than 4 weeks old and nonunion fractures require rigid stabilization. Internal fixation with a screw, via either the dorsal or the palmar approach, has become the gold standard,4–6. AddOver the last three decades, scaphoid fracture stabilization using a single screw has been developed. Although the first screw to use a pitch difference between the proximal and distal threads was biomechanically inferior to the conventional screw designs,10, the The mean time to union after internal screw fixation of acute, nondisplaced fractures of the scaphoid waist is approximately 6–8 weeks, which is consistently shorter than the time to union after nonsurgical treatment 12–15 weeks)–15 weeks. The maiThe purpose of this study was to compare the robustness of internal fixation with four leading HCSs of varying design under cyclic loading in a simulated scaphoid fracture. Each screw offers a different method of building compression. The primary outcome was the angulation allowed by each screw between the bone fragments after loading.We obtained 45 fresh-frozen scaphoids with written approval from the Ethics Committee of the Landesaerztekammer Rheinland-Pfalz, Mainz, Germany (Consent No. 837.088.07). Samples were procured from the donation center at The Institute of Anatomy of The Johannes Gutenberg University Mainz (JGU). The boneAfter performing a CT scan of all specimens , bones that were previously broken, were malformed by arthritis or were too small to fix in the loading device were excluded, as a typical patient with a scaphoid fracture is unlikely to have these features. Forty bones were included in the study. Further, 3D objects were built to calculate the mean voxel value (Hounsfield units). The air entrapments within bones were excluded prior to the calculation, as they do not provide support for the screw fixation. The mean voxel value in Hounsfield units was noted in previous studies to correlate with bone mineral density units, and thus, these values were accepted as sufficient for the purpose of our study. The meaBones of similar length were stratified into 10 groups of four bones each to minimize measurement error in the loading device. We compared differences in the bone density between the groups for AA, MH, ST and SH, but there was no statistical significance (p = 0.505). In every group of four bones, one screw was randomly assigned to each bone.  TablesWe chose four implants that are currently on the market from leading manufacturers. Each screw has a different mechanism for building compression . Three sAcumed Acutrak 2 Mini  has a conical screw design and a continuous, variable thread. The wider thread pitch at the tip of the screw penetrates the bone faster than the finer trailing thread, which gradually compresses the two fragments as the screw is advanced. Stryker TwinFix  has proximal and distal screw threads that work independently to allow for in situ dynamic adjustable interfragmentary compression. Edges at the screw body enable reaming at the core hole for the trailing screw thread. This screw requires a special screwdriver with two concentric hexagonal heads and a clutch. Synthes 3.0 HCS  has an additional instrument, a compression sleeve, which brings the fractured fragments together and allows for accurate placement of the screw under the cartilage surface. The screw is first screwed into the cannulated compression sleeve with a trailing thread. It is then inserted into the bone using a firmly connected compression sleeve. As the compression sleeve is wider than the head of the screw, once the tip of the compression sleeve lies on the bone, the fracture gap is closed and compressed by further turning the screw with the sleeve. Finally, the screw is countersunk into the bone with an additional screwdriver while the compression sleeve is held stationary. The pitch of the trailing thread is doubled, with the same lead between the leading and trailing thread. Therefore, with one rotation of 360°, both the leading and trailing threads move the same distance. As a result, no extra compression is generated during countersinking of the screw into the bone. We used screws with a long leading thread. KLS Martin HBS 2 Midi  offers a design with differential proximal and distal pitch to induce interfragmentary compression according to the idea of Herbert. It is sA new loading device was developed with the Physics Division of The RheinMain University of Applied Sciences, Wiesbaden, Germany. Each bone was first sawed at an angle perpendicular to the long axis in the waist, simulating the most common B2 fracture according to The Herbert Classification. AfterwaS, which resulted in the decomposition of vectors into FD and FB. R was the continuously measured distance between parallel axes. φ was the continuously measured angulation between the fragments during loading. lb was the distance from the middle of the distal axis to the osteotomy.The loading force coming from a screw gear driven by a stepping motor worked indirectly through a free-floating plate with the vector FThe cement cube was then interposed into the loading machine as shown in The actual loading of the scaphoid during daily motion remains unknown. In the pre-testing phase, we first checked the elasticity of the screws in sawbone models. In the next step, bone specimens with different loads were tested to identify the appropriate loading force at which no failure would occur before 2000 cycles and at which the results would be recognizable .The appropriate load (62.5–90 N) was computed for different bone lengths to assure the same bending moment of 800 Nmm for each specimen. Bones were then loaded over 2000 cycles. ´LabVIEW Professional 2011´  was used to control the device and collect the data.All statistical analyses presented in this report were produced using IBM SPSS Statistics version 22 . We performed a univariate analysis of variance (ANOVA). The effects of each screw on the φ were adjusted to the bone density of the specimens. P values <0.05 were considered statistically significant. Linear regression lines were drawn to analyze the individual properties of each screw.Thirty-six bones underwent the complete procedure . In one specimen, the head of ST lost grip with the screwdriver during the screw insertion due to a quick wearing of the screw head, and further closing of a fracture gap was not possible. This specimen had high bone density. Additional, there was one case of breakage of the blue ring holding a trailing thread in ST, but it did not significantly influence the residual stability of fixation. In the remaining three cases, there were problems building up compression in the osteoporotic bone specimens.2 = 0.327. After adjustment for bone density, no statistically significant difference (p = 0.291) between the screws could be shown. The factor that significantly influenced stability of the fixation was bone density (p = 0.003). In this case, further between-subject comparisons were redundant.The values of φ [grade] appeared skewed, so to adjust the statistical model, we performed a logarithmical conversion of the angle difference . Further2 = 0.827, followed by AM (R2 = 0.354), SH (R2 = 0.247) and ST (R2 = 0.019). The results of linear regression are shown in MH showed a coefficient of determination of RThe purpose of this study was to compare four different designs of leading HCSs in a simulated scaphoid fracture. The primary outcome was the degree of dislocation between bone fragments under cyclical loading.2 = 0.827). The results from the other screws showed a wider spread, and thus further conclusions are less reliable.No statistically significant difference between the screw designs could be shown. The median angle difference φ, as an indicator of loosening of the internal fixation, was lowest in AA (0.89°) and ST (1.12°), followed by SH (1.44°) and MH (2.36°). ST offered similar bone purchase for bones with low or high bone density. However, according to our findings, MH showed the most predictable results of dislocation of the fracture fragments expressed by the highest coefficient of determination  inclination. This faAfter the primary statistical analysis, two cases were found to be outliers. The rotating head of one ST screw was found to have broken free, although the screw provided high stability. In one SH, the interfragmentary compression could not be built due to a technical error during implantation. In this case, the SH was introduced too deep after leaving the compression sleeve. This issue is a known problem of the insertion technique that can cause almost complete lack of compression,20. DespThe question of whether different designs of modern HCSs have benefits for patients remains open. We seek better implants due to the increasing pressure from patients for greater treatment efficiency, quicker healing time and earlier return to work. However, an often unstated problem is that orthopedic surgeons often look for state-of-the-art devices solely to offer a novel device to fulfill patients’ desire for the latest trends in treatment. Based on our results, we advise surgeons to concentrate on problems such as appropriate surgical technique, screw length, placement and correct soft tissue management. These factors reduce the risk of poor outcomes after scaphoid fracture more than the choice of a novel implant.The continuous development of HCSs has resulted in comparable implants. According to our results, MH and AA offered slightly more stability in bones with higher density. The ST design offered consistent biomechanical results but may result in a malfunctioning implant, and the data we obtained are not reliable. SH is a cheaper alternative that is not statistically inferior to other screws. Finally, the choice of an implant should be made according to the surgeons’ experience with the device, its ease of use and the local price of the screw.S1 Fig(JPG)Click here for additional data file.S1 Table(SAV)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.S4 Text(PDF)Click here for additional data file.S5 Text(PDF)Click here for additional data file.


In each of these proteins the phosphorylation of a specific histidine residue regulates cellular signal transduction or channel activity. This article will therefore summarize our current knowledge on protein histidine phosphorylation and highlight its relevance for cardiovascular physiology and pathophysiology.Reversible phosphorylation of amino acid side chains in proteins is a frequently used mechanism in cellular signal transduction and alterations of such phosphorylation patterns are very common in cardiovascular diseases. They reflect changes in the activities of the protein kinases and phosphatases involving signaling pathways. Phosphorylation of serine, threonine, and tyrosine residues has been extensively investigated in vertebrates, whereas reversible histidine phosphorylation, a well-known regulatory signal in lower organisms, has been largely neglected as it has been generally assumed that histidine phosphorylation is of minor importance in vertebrates. More recently, it has become evident that the nucleoside diphosphate kinase isoform B (NDPK-B), an ubiquitously expressed enzyme involved in nucleotide metabolism, and a highly specific phosphohistidine phosphatase (PHP) form a regulatory histidine protein kinase/phosphatase system in mammals. At least three well defined substrates of NDPK-B are known: The β-subunit of heterotrimeric G-proteins (Gβ), the intermediate conductance potassium channel SK4 and the Ca Phosphorylation is a most ubiquitous post-translational modification that plays an essential role in the regulation of cellular function is. In higher eukaryotes the most common forms of phosphorylation of proteins involve serine/threonine or tyrosine protein kinases and counteracting phosphatases. Both types of enzymes can be regulated by cellular signaling events.In prokaryotes, but also in fungi and plants, phosphorylation of proteins on histidine residues is very common. The most widespread occurrence of histidine phosphorylation is in the two-component histidine kinase system. The histidine kinase is a membrane-bound sensor that on perceiving an extracellular signal undergoes autophosphorylation on a histidine residue. In the simplest two-component histidine kinase systems, this phosphoryl group is then transferred directly to an aspartate residue in a response regulator protein that catalyzes the reaction. The response regulator is often a transcription factor that is activated on phosphorylation to enhance expression of genes that provide the cellular response to the external stimulus .Phosphohistidine has a number of attributes that make it different from the phosphoester phosphoamino acids. The phosphoryl group is bonded to the imidazole ring of the amino acid via a phosphoramidate bond. Since the imidazole ring contains two nitrogens, there are three forms of phosphohistidine, 1- or 3-phosphohistidine or 1,3-diphosphohistidine , which was however not seen in cells overexpressing the histidine kinase deficient NDPK mutant NDPK-B-H118N (H118) or NDPK-A , in the NDPK-B-overexpressing H10 cells further increased Gβ phosphorylation and Gαormation . GβH266Lormation . Interesormation . Based oormation .s- and Gαi-subunits as well as the caveolae scaffold proteins caveolin-1 and -3 were reduced -induced cAMP synthesis and cardiac contractility, the requirement of NDPK-B for basal and βAR-stimulated cAMP synthesis was further analyzed by comparing wild-type NDPK-B and its catalytically inactive H118 mutant in several cellular models, including rat cardiomyocytes. Both, re-expression of human wild-type and H118 induced the re-appearance of Gs and caveolin-1 at the plasma membrane and thus enhanced the βAR-induced cAMP formation of NDPK-B—depleted cells to a similar extent. In contrast, the catalytically inactive H118 was less potent and less effective in rescuing basal cAMP production -VEGF receptor type 2 (VEGFR-2)-cascade in endothelial cells . Therefo1ARs and up-regulation of inhibitory G-protein-coupled receptor-kinases  induces complex remodeling processes in cardiomyocytes and changes in G-protein-signaling are a hallmark in this remodeling process. Chronic sympathetic stimulation results in a desensitization of βARs including reduced expression of βes GRKs; . Concomistage HF . This ishibition . Togethe disease .i protein signaling and thus the well-known reduction in cAMP formation in HF. A recent abstract , intermediate (SK4), and small (SK3) conductance Ca2+-activated K+-channels are expressed in the vasculature  indicated a constitutive activation of the SK4-channel in proliferating VSMCs of the wild-type but not of the NDPK-B-deficient mice. As this activation was completely inhibited by PHP, the phosphorylation of His358 in the de novo expressed SK4-channels by the constitutively expressed NDPK-B is pivotal to VSMC proliferation. A potential underlying mechanism is the driving of Ca2+-influx. In proliferating VSMC, TRPC channels and T-type Ca2+-channels are the main channels responsible for Ca2+-influx (SK4 due to the constitutive activation of the channel by NDPK-B will shift the membrane potential to more negative values. Such a hyperpolarization enhances the Ca2+-influx through TRPC and/or T-type Ca2+-channels. The resulting increase in the intracellular Ca2+-concentration likely triggers the activation of transcription factors and the induction of mitogenic immediate early genes (Recent evidence suggest that the activation of SK4 by NDPK-B in VSMC is required for neointima formation  Using a +-influx . In contly genes .–/–-mice with the selective SK4-channel blocker TRAM-34 significantly reduced not only proliferation and migration of VSMC, but also inhibited the infiltration of plaques by inflammatory cells (–/–-mice are protected from developing severe colitis in mouse models of inflammatory bowel disease (–/–- and SK4–/–-mice show similar profiles of cytokine production (Small molecules that inhibit the histidine kinase activity of NDPK-B , interfery cells , indicat disease  and CD4-oduction ,b, it isAlthough well known as an important regulatory event in many organisms, the role of protein histidine phosphorylation in mammals has long gone unrecognized. However, with the recent identification of NDPKs as protein histidine kinases, PHP as a specific counteracting phosphatase and the description of important substrates as Gβγ, SK4, and TRPV5, an understanding of the importance of NDPK-mediated protein histidine phosphorylation in the regulation of mammalian cell function is emerging, with the promise of much more to be discovered. As pointed out herein, two of these substrates Gβγ and SK4, both targeted by NDPK-B as protein histidine kinase, have important roles in the physiology and pathophysiology of the cardiovascular system. The apparent contribution of the NDPK-B/Gβγ- and the NDPK-B/SK4-interaction to HF and atherosclerosis, respectively, give raise to the assumption that interference with the histidine kinase activity of NDPK or even more specifically, the inhibition of the interaction of the NDPK-B with its target proteins by small molecule inhibitors might offer avenues for future treatments of cardiovascular diseases.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.


Dermatophagoides pteronissinus (Der p) in nasal lavage of patients with moderate to severe persistent allergic rhinitis, with or without intermittent asthma. Also, to correlate the levels of total IgE and specific IgE for Der p in the serum and in the nasal lavage.To evaluate the presence of total IgE and specific IgE for We selected 48 patients with moderate to severe rhinitis (12 of them had intermittent asthma) sensitized to Der p without prior treatment with allergen specific immunotherapy. The protocol was approved by the ethics committee. All participants signed informed consent. Determination of total IgE and specific IgE for Der p1 (D1) and Der p2 (D2) were performed in serum and nasal lavage, using ImmunoCAP method. Non parametric tests were used in the statistical analysis.The mean age was 35.1 years old . The mean total serum IgE was 367UL (234UL median and SD 433.9 UL) and in nasal lavage it was 32.2 UL (median 30.63UL and SD 13.2), ranging from 16 to 73UL. The mean serum specific IgE for D1 and D2 was 42.3 and 31.7 UL respectively. The mean specific IgE in nasal lavage for D1 and D2 was 1.7UL and 1.43 UL respectively and ranged from 0.5 to 14.9 UL for D1 and 0.4 to 12.75UL for D2. There was no statistical difference between the groups with moderate to severe persistent rhinitis alone or associated with asthma. We have estimated the value of total IgE in the nasal lavage by the linear regression model ; and specific IgE to D1 in the nasal lavage ; and the specific IgE in nasal lavage to D2 .It has been reported that 77% of allergic patients have nasal specific IgE. We have found nasal specific IgE in 100% of cases. In our case series we could estimate the value of total IgE and specific IgE in nasal lavage from the value found in the serum. However, there is no consensus in the literature about the values of total and specific IgE in nasal lavage in atopic patients.


Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl2 increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl In recent years, silver nanoparticles (NAg) have become an extremely popular nanotechnology product widely used in many applications. Their strong antibacterial potential is useful in medical products  is involved in neuronal cell death via excessive entry of calcium ions followed by an intracellular calcium imbalance. However, it has been shown that in addition to calcium, zinc (Zn) may have a negative effect on cell function in a process which leads to cell death  exposed to silver nanoparticles  cultured in the presence of nanosilver are studied under conditions of zinc deficiency and increased levels of zinc.45CaCl2 was produced by Polatom Sp. z o.o., Otwock—Swierk, Poland. Other chemicals, including nanosilver and cell culture materials were purchased from Sigma-Aldrich Chemical Co. . All reagents were of analytical grade.Fluo-3AM, FluoZin-1, FluoZin-3AM, 6-Carboxy-2′,7′-dichlorofluorescein diacetate (DCF), and rhodamine 123 (R123) were obtained from Molecular Probes . Commercially available nanosilver was used in the present study. Nanosilver is defined by the manufacturer (Sigma-Aldrich) as a mixture of polyvinylpyrrolidone-coated nanoparticles (0.2 % PVP-coated nanosilver) less than 100 nm in diameter. We previously characterized and investigated nanosilver produced by this manufacturer in toxicological studies  in order to avoid the release of Ag+ ions from the particle surface , containing Ag+ liberated from the nanoparticle surface, was used as a control to define the effects characteristic for ionic or “nano” form of silver. Absence of nanoparticles in supernatant was confirmed using TEM.The same volume of supernatant , and penicillin (50 U/mL). The CGCs were used in experiments after 7 days.Primary cultures of cerebellar granule cells (CGCs) were prepared from 7-day-old Wistar rats of both sexes as described previously  were added directly to BME growth medium to obtain final concentrations in the range of 2.5–10 or 75 µg/mL. In most experiments, nanosilver in concentration of 75 µg/mL (NAg 75) and supernatant obtained from this solution (super 75) were applied. In serum, nanoparticles sediment very slowly and this characteristic provides reproducibility during addition to the cultures. However, since serum contains trace amounts of its own glutamate, calcium, and zinc ions, it is also necessary to compare the results obtained for NAg with control culture supplemented with the volume of serum equal to the volume of NAg or supernatant added to the experimental system (control + serum 75). The final concentration of FCS in this experimental design was 10.75 %. Apart from this, untreated cells were also used as a negative control.On the 7th day of culture, serum solutions of nanosilver (NAg)/supernatant  ethylenediamine), 10–50 μM ZnCl2 or 0.5 µM of MK-801 (an antagonist of glutamatergic NMDA receptors), or 10–50 µM CNQX (an antagonist of AMPA/KA glutamatergic receptors). The cells were cultured for 24 h and then the viability of neurons was assessed by staining with 0.5 µg/ml propidium iodide (PI) or with the fluorescent dyes calcein and EtHD. PI is toxic for the neurons and stains only dead cells, so prior to staining with PI, the cells were fixed with 80 % methanol. Cells that were alive in the time of fixation differed morphologically from dead ones. Fluorescence microscopy was employed to count living and dead neurons  as described previously  was used. Images of fixed and PI-stained cells were recorded using 546 nm HeNe laser for excitation of fluorescence. The emission was measure at 2+ concentration were measured fluorometrically. The cells (1 × 106/well) were incubated at 37 °C for 60 min in growth medium with 3 μM FluoZin-3AM. Then, the cells were twice washed with Locke 5 medium and examined using confocal microscope  after addition of NAg/Ag+. The fluorescence of FluoZin-3 induced by argon laser at 488 nm was measured at 530 nm every 30 s during 15 min. The results are presented as percentage changes in the intensity of fluorescence in relation to its basal level (F/Fo). The data were obtained from three independent trials using separate CGC cultures and presented as means of 15 randomly selected objects.Changes in the intracellular Zn6/bottle) were transferred to Locke 25 medium and incubated 30 min with 75 µg/mL NAg or Ag+ in the presence of 0.75 % FCS. Then, to avoid interference with extracellular zinc from serum, the cells were three times washed with fresh Locke medium containing 154 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 2.3 mM CaCl2, 5 mM HEPES (pH 7.4), and 5 mM glucose. Since the probe does not penetrate the membrane, the cells from each bottle were lysed in 0.5 M NaOH to provide visualization of zinc in the intracellular space. Then 100 μL of solution was incubated with 1 μM FluoZin-1 and fluorescence was measured using a microplate reader  at 485 nm excitation and 538 nm emission wavelengths. Addition of NaOH alone had no effect on the readout of fluorescence (control with Locke solution with ZnCl2 versus the same concentration of ZnCl2 in NaOH).CGCs (15 × 106/well) were pre-incubated at 37 °C for 10 min in Locke 5 medium. Radioactive calcium (1 μCi/well) was added together with 75 μg/mL NAg/Ag+ or other substances and after 10 min incubation at 37 °C, the cells were washed 3× with ice-cold glucose and calcium-free medium containing 2 mM EGTA. After lysing the cells in 0.5 M NaOH, radioactive calcium uptake was measured using a Wallac 1409 liquid scintillation counter .The cells (4 × 106/well) were loaded with the fluorescent calcium-sensitive probe, 4 μM fluo-3 AM, at 37 °C for 30 min. The cells were washed with the Locke 5 buffer to terminate loading. Changes in fluorescence after addition of all tested compounds were recorded at 1-min intervals over a 30-min period, using a microplate reader  at 485-nm excitation and 538-nm emission wavelengths.CGCs (1 × 106/well) to a final concentration of 10 μM for 30 min at 37 °C. Decrease in mitochondrial membrane potential was monitored by increased intracellular fluorescence. After preincubation with R123, cells were washed with Locke 5 buffer and treated with 75 µg/mL NAg/Ag+ alone or mixed with other tested compounds. Changes of fluorescence were recorded every 3 min, over a 1-h period, using a microplate reader  at 485-nm excitation and 538-nm emission wavelengths.Rhodamine123 (R123) was added to the cultures (1 × 106/well) were washed with Locke 5 buffer and treated with 75 µg/mL nanosilver, supernatant alone, or in combination with other tested compounds. Changes in fluorescence were recorded at 1 min intervals, over a 30-min incubation period, using a microplate reader  at 485-nm excitation and 538-nm emission wavelengths.The fluorescent dye 2′,7′-dichlorofluorescin diacetate (DCF) was added to the cultures to a final concentration of 100 μM and used to evaluate free radical production. After a 30-min incubation period, cells  with Bonferroni correction for multiple comparisons. The significance level was set as Commercially available 0.2 % polyvinylpyrrolidone (PVP)-coated nanosilver particles <100 nm were used in the experiments. TEM studies revealed that the nanoparticles generally have a spherical shape and do not form aggregates in the stock solution either in the presence of serum or culture media (BME and Locke buffer). The size distribution estimation indicated that the vast majority (88 %) of nanosilver particles were between 5 and 25 nm in diameter and more than half (65 %) did not exceed 10 nm in diameter  over a longer period of time (after 30 min). FluoZin-1 is a cell-impermeable indicator designed to detect zinc concentrations in the 0–100 µM range with minimal interference with calcium sensitivity. Decreased fluorescence in cell lysates reflecting decrease in the intracellular zinc  was noted after exposure to both NAg and Ag+. However, destabilization of cellular zinc equilibrium was significantly more evident after exposure to nanosilver particles and reached about 20 % (*P < 0.05 versus control)  in the concentration range of 2.5–25 µg/mL exhibited no visible effect on the viability of neurons after 24 h of exposure  were generally comparable, but slightly greater in numbers of cells survived (55–70 %) relative to respective doses of NAg. Thus, in the current experiments, we used the concentration of silver nanoparticles in the presence of which viability of neurons decreased to about 50 % of control (75 µg/mL). Such marked dose–response effect allowed us to test modulatory effects of investigated substances on mechanisms leading to cell death.Results after addition of respective amounts of supernatant (Ag+) . Zinc chelation with 20 µM TPEN strongly aggravated cell death induced by NAg and Ag+. Addition of zinc to the cultured neurons in the concentration range of 10–50 µM increased the viability of the cells in a dose-dependent manner. In nanosilver-treated cells, the positive effect of zinc was less evident and did not exceed 20 %, whereas in Ag+-treated cells it was more pronounced in over 90 % of cells. Application of a noncompetitive antagonist of NMDA receptors (MK-801) significantly increased the number of living cells in nanosilver-treated cultures, exceeding the positive effect of zinc, by about 13 %. In Ag+-treated cultures, MK-801 gave neuroprotection at the same level as zinc. Positive effect of MK-801 was observed only in this type of experiments, under long-lasting exposure to Ag+ (24 h). In contrast to nanosilver-treated cells, further experiments performed on cells exposed for 30–60 min to Ag+ did not reveal positive effect of NMDAR antagonist.Cell viability was assessed by propidium iodide (PI) or with the fluorescent dyes calcein and EtHD 24 h after addition of nanosilver particles . However, that tendency was statistically insignificant  and TPEN to investigate the effect of zinc deficiency. As we stated before, applied concentrations of nanosilver particles did not result in cell death . TPEN added to neurons treated with 50 μg/mL nanosilver did not influence the calcium uptake. In the presence of the highest used concentration of nanosilver (75 μg/mL), TPEN stimulated calcium uptake to a level similar to that attained after addition of 100 μM glutamate i.e., 750 % over control value; *P < 0.05 and 66 % over NAg alone  increased relative to control untreated cells but was lower by about 310 % compared to CGCs treated with 75 μg/mL nanosilver TPEN exerted visible effect only when added together with the highest concentration of Ag+ (super 75), resulting in a slight increase in the rate of Ca uptake  Fig. a. TPEN aone Fig. a. Calciuake Fig. b.Fig. 4T2 resulted in strong and dose-dependent inhibition of calcium uptake in glutamate- or nanosilver-treated cultures , so as in control cells. In Ag+-exposed cultures, the effect was less pronounced (about 200 %)  by about 120 % relative to control cultures . Addition of MK-801 also reduces the level of fluorescence, although this effect was found to be unstable during the incubation time. The most pronounced and time-stable blockage of the increase in intracellular Ca2+/Zn2+ was observed after addition of 25 μM ZnCl2. The recorded signal decreased to the level of the control.These results confirm that in the presence of 75 μg/mL nanosilver, the intracellular calcium/zinc levels (as measured by fluorescence) increase significantly   Fig. a and mod %) Fig. b.Fig. 8T+ .ROS production increased significantly relative to the control after a 30-min incubation with NAg but not with Ag+ Fig. c. The prWe previously assumed that nanosilver exerts its toxicity in cultured neurons by increasing the rate of the calcium entry through the channels associated with NMDA receptors  was used to supplement cell cultures with zinc. In our experiments, we investigated cerebellar granule neurons which are glutamate- and aspartate-receptive glutamatergic cells  measured in cell lysates with FluoZin-1 after 30 min   was demonstrated in vitro  and TPEN induced characteristic apoptotic changes in the cell nuclei, although added alone TPEN did not exert such effect Fig. . It has 2+/Zn2+ fluorescence. The increased level of cytosolic zinc under nanosilver exposure is presumably connected to the overactivation of NMDAR as concluded from the reversing effect of MK-801 and ZnCl2. Thus, it is possible that nanosilver particles cause immediate disturbance of zinc homeostasis but to a low and variable extent. Possibly, a nanosilver-induced increase in cytosolic zinc measured in the experiment with fluo-3  but not short-time experiments (30–60 min) revealed positive effect of NMDAR antagonist. It is possible that NMDAR-mediated mechanisms are switched on during prolonged time of incubation with Ag+ as secondary mechanisms. Likewise, zinc homeostasis was altered in nanosilver-exposed cells. This represents the most pronounced effect of zinc supplementation and intensified TPEN toxicity.Effective protection provided by MK-801 is observed in most of the experiments in nanosilver- but not Ag+. Excessive increase in cytosolic level of zinc with its subsequent release from glutamatergic synapses are related to the contribution of zinc in processes mediated by overactivation of NMDARs, specifically under nanosilver exposure. However, the results indicate, in contrast to our expectations, that zinc homeostasis, although altered during exposure to nanosilver, does not contribute significantly to cell death. This conclusion is supported by the observation that the zinc chelator TPEN does not eliminate toxic changes evoked by nanosilver but instead tends to increase them leading to cell death. In contrast, zinc supplementation exerts a positive effect, which is likely due to inhibiting the NMDA-sensitive glutamate-gated calcium current.The results of this study stated the role of zinc in nanosilver-evoked CGC death. It is evident that exposure to increased concentrations of nanosilver leads to an imbalance between extra/intracellular zinc levels. The same does not hold true for exposure to Ag


Melosira arctica showed sign of different stages of degradation and physiological stoichiometries, with carbon to chlorophyll a ratios ranging from 110 to 66700, and carbon to nitrogen molar ratios of 8–35 and 9–40, respectively. Sub-ice algal aggregate densities ranged between 1 and 17 aggregates m−2, maintaining an estimated net primary production of 0.4–40 mg C m−2 d−1, and accounted for 3–80% of total phototrophic biomass and up to 94% of local net primary production. A potential factor controlling the buoyancy of the aggregates was light intensity, regulating photosynthetic oxygen production and the amount of gas bubbles trapped within the mucous matrix, even at low ambient nutrient concentrations. Our data-set was used to evaluate the distribution and importance of Arctic algal aggregates as carbon source for pelagic and benthic communities.Sea-ice diatoms are known to accumulate in large aggregates in and under sea ice and in melt ponds. There is recent evidence from the Arctic that such aggregates can contribute substantially to particle export when sinking from the ice. The role and regulation of microbial aggregation in the highly seasonal, nutrient- and light-limited Arctic sea-ice ecosystem is not well understood. To elucidate the mechanisms controlling the formation and export of algal aggregates from sea ice, we investigated samples taken in late summer 2011 and 2012, during two cruises to the Eurasian Basin of the Central Arctic Ocean. Spherical aggregates densely packed with pennate diatoms, as well as filamentous aggregates formed by  The endPlanktonic diatoms tend to collide and aggregate in nutrient depleted waters after a bloom due to mucus secretion during senescence Many questions remain about aggregate formation, decay, grazing and sinking, as well as their contribution to carbon fluxes in the Central Arctic. Due to the rapid warming of the Arctic leading to a decrease in sea-ice extent and thickness http://doi.pangaea.de/10.1594/PANGAEA.832345).Samples were collected during two cruises to international waters of the Central Arctic Ocean with RV Polarstern in August-September 2011 and 2012 (PS78/3 and PS80/3 respectively) between 81°55′–87°56′N and 31°7′–131°7′E . Six staMelosira were sampled at eight ice stations . Irradiance reaching the aggregates was calculated using the light attenuation coefficients of 1.5 m−1 for sea ice, 10 m−1 for snow −1 for Atlantic influenced Arctic seawater , and the daily average total incoming photosynthetically active radiation (PAR) measured with a pyranometer  mounted on the ship.Replicates of two different algal aggregate types dominated either by pennate diatoms or by stations . For eacMelosira arctica filaments 5 times (a and phaeopigments were measured using high-performance liquid chromatography (HPLC) as described in a measurements was generally below 2%. For transparent exopolymers (TEP), 1–20 ml of the algal slurry  were filtered in triplicate on to 0.4 µm pore size polycarbonate filters , stained with Alcian Blue and stored at −20°C. TEP concentration was measured with the colorimetric method according to Several aggregates (between 2 and 20) were sampled at each station together with some ambient water using a manually operated vacuum pump, when found in melt ponds , or a pl 5 times . AggregaThe qualitative algal aggregate composition was studied directly on board of the ship with a plankton chamber  and an inverted light microscope with phase contrast optics  and an integrated camera . The condition of the diatoms in each sample was defined according to the color and shape of the chloroplasts visible in the interior of the frustules. Green-brownish aggregates contained mostly healthy cells with bright green chloroplasts occupying the entire cytoplasm, while white-yellowish aggregates contained many dead cells with empty frustules or reduced yellowish chloroplasts. To determine bacterial abundance with Acridine Orange direct counts (AODC), sub-samples of the algal slurry were preserved with 2% formalin and sonicated (1×90 s). One milliliter was filtered on to a 0.2 µm pore size polycarbonate filter  previously stained with Irgalan black to eliminate autofluorescence −2.Although green-brownish sub-ice algal aggregates were observed at all ice stations a and POC as for the aggregate slurry. For the phytoplankton community in the water below the ice, samples were taken with Niskin bottles disposed in a rosette attached to the Conductivity-Temperature-Depth sensor (CTD) at discrete depths, the water was filtered, and Chl a and POC was analyzed as above. Water column phytoplankton biomass was integrated over the euphotic zone (1% incoming irradiance) while sea-ice algal biomass accounted for the ice thickness . Temperature was maintained stable at −1.3°C with a thermo bath . Three clear bottles with 10 mL each were incubated in the light and one in the dark. The estimated error of the method from the triplicates incubated in the light was 15% on average. Floating aggregate slurries collected at station PS80/3_224 and PS80/3_237 were incubated at 50 µmol photons m−2 s−1, which was a typical mid-day in situ value. Aggregate slurries from station PS80/3_349, as well as sea ice and water column of all stations were incubated under a range of irradiances  during 24 hours to calculate the in situ NPP from photosynthesis vs irradiance curves (PE curves). Using this incubation time NPP is assessed (gross PP minus respiration) 2>0.5 were retained. In situ NPP was calculated applying the irradiance measurements at the ice station to the PE curve equation.Depth-integrated NPP was estimated from Melosira filamentous biomass (Aggregate M5) was estimated from scaled images of the open melt pond  by the number of aggregates observed per m2 and by the carbon concentration of the aggregate per volume (mg C m−3). The aggregate NPP per m2 was calculated similarly, by multiplying the total aggregate volume per m2 by the measured carbon fixation rate. Chl a was estimated from the C:Chl a ratio measured in the algal slurry from the same aggregate type. A similar procedure was used for the C:N ratios. Since aggregates showed a very patchy distribution, these calculations reflect local estimates at the sampling spot and cannot be directly up scaled to the Arctic-wide ice-cover.To quantify the aggregated biomass and calculate NPP per area, aggregate abundance was quantified from scaled images. At stations PS80/3_224 (Aggregate P4) and PS80/3_237 (Aggregate P5) mean aggregate abundance under the ice was estimated from images taken by the upward looking camera of a Remotely Operated Vehicle (ROV) as described in elt pond . The aggin situ conditions: low light (8 µmol photons m−2 s−1) and cold temperature (0°C). The initial temperature of the melt pond water collected for the experiment was −1°C. After 12 h in the cold lab-container (0°C) under low light intensity (8 µmol photons m−2 s−1), the water reached 0°C and the piece of ice started melting. The vertical position of the aggregates was followed over time and as a function of the salinity gradient and the light intensity, to assess their buoyancy at different environmental conditions. In parallel, sub-samples of the algal slurry from the same type of aggregate were incubated under different conditions to assess oxygen, carbon and nitrogen fluxes.On 18 September 2012, station PS80/3_349 , a piece of ice from the frozen surface of an open melt pond was retrieved with algal aggregates attached . These w14C uptake rates, as described above. In addition, oxygen concentration was followed in the same vials using fiber optic oxygen sensors . Oxygen sensitive sensor spots were glued to the interior of the glass vials previous to the experiment. The sensor spots contain an oxygen quenchable fluorophore (oxygen sensitive dye) which changes its fluorescence properties according to the oxygen concentration 2>0.93) and a photosynthetic quotient of 1.25 was used to convert the oxygen exchange to carbon equivalents Five sub-samples were incubated under different light intensities to estimate NPP from −2 s−1 and nitrate was measured initially and after 1 and 4 days. In parallel, 18 subsamples of the algal slurry were incubated in 12 mL exetainers  to measure the denitrification and nitrification potential. Subsamples of the slurries were spiked either with 15NO3, with 15NH4+ or with a mixture of 14NO3 and 15NH4+ , using a modified version of the method described in −2 s−1, while the remaining sub-samples were placed in darkness. The gradual O2 consumption was followed in both set ups using the optode system described above. Anoxic conditions were reached after two days in the dark. The first time series was ended immediately by injecting 100 µL ZnCl2 into the exetainers, while the remaining samples were terminated as the oxygen level reached 67, 35, and 0%, after 0.8, 1.2 and 2 days respectively. The isotopic composition of the N2 was measured using a gas chromatograph coupled to an isotope ratio mass spectrometer through a ConFlo-III interphase x) were determined To measure the nitrate uptake, a 50 mL slurry sub-sample was incubated at 50 µmol photons mDuring August and September 2011 and 2012, FYI in a late melting stage was the dominant ice type at seven of the eight ice stations investigated . Sea-iceNitzschia sp., Navicula sp., Fragilariopsis sp., and Entomoneis sp.)  , and (2) arctica .−1 nitrate, 0.2 µmol L−1 phosphate, and 2 µmol L−1 silicate at the bottom of melt ponds or below the ice.Both the pennate and centric diatom aggregates were present in different degradation stages . Their ca mass ratios than green-brownish aggregates   of the POC. Greenish-brownish aggregates hosted one order of magnitude more bacteria than white-yellowish aggregates. The DOC concentrations of the algal aggregates ranged between 5 and 35 µmol DOC mg POC−1  to the particulate organic carbon pool of the integrated euphotic zone  were higher than the values for the phytoplankton (∼5) and the sea-ice algae (6–8). Green-brownish Melosira aggregates showed higher C:N ratios (9–28) than all pennate aggregates (8–11). In white-yellowish aggregates C:N ratios were generally higher (11–35 in pennate and 10–40 in Melosira aggregates).At the sampled sites, floating aggregates contributed with 0.5–26% (pennate), and 55–78%   and the sea ice in late September  . In conteptember . The daiMelosira arctica filaments found at station PS80/3_349, both carbon uptake and oxygen production were monitored in homogenized algal slurries at different light intensities. The fitted PE curve of the carbon uptake (R2 = 0.986), showed a photosynthetic maximum (Pmax) of 3.7 mg C L−1 d−1, an initial slope of 120 µg C L−1 d−1 (µmol photons m−2 s−1)−1 and no photoinhibition at irradiances lower than 90 µmol photons m−2 s−1 (−2 s−1). However, no net oxygen production took place at this irradiance, rather oxygen was consumed  anchored to newly formed ice from station PS80/3_349, composed of a mixture of chia sp. , Fig. 5Achia sp. . Once thchia sp. . It was chia sp.  bottom. chia sp. . After 2chia sp. . To testbuoyancy , indicatMelosira slurry showed a bulk oxygen consumption rate of 0.13±0.02 mmol O2 L−1 d−1 at low irradiance (8 µmol photons m−2 s−1). Non-invasive measurements with microsensors on one pennate-diatom also showed net oxygen consumption with almost anoxic conditions (10–120 µmol L−1 Oxygen) in the center of the aggregate (see The gate see .2 production to O2 respiration we transformed the oxygen consumption rate mentioned above into carbon respiration rate. Dividing the POC measured in each sample (Melosira slurry. The turnover of particulate carbon at low irradiance (8 µmol photons m−2 s−1) would be 8–11 days. Net oxygen consumption and carbon remineralization occurred at lower light intensities in the Melosira algal slurry than in the compact pennate-diatom aggregate. Floating green-brownish aggregates incubated for 4–5 days at low irradiances lost their buoyancy and became net heterotrophic. Oxygen production in both aggregate types was probably light regulated. However, this observation is based on single measurements, with the two respective types of aggregates. Further experiments are needed to fully conclude that light-regulated oxygen production is responsible for aggregate buoyancy.Assuming a 1∶1.25 ratio of COh sample  by the cMelosira algal slurry sample incubated under in situ representative light conditions (50 µmol photons m−2 s−1). During the first 24 h, the nitrate consumption rate was 0.94 µmol N L−1 and the ammonium consumption 2.47 µmol N L−1. Denitrification and nitrification potential was measured in slurry samples spiked with labeled nitrate and ammonium, respectively (15NO3 and 15NH4+). During light, nitrate was produced at a rate of 1.7±0.001 µmol L−1 d−1 and ammonium was consumed at a rate of 1.6±0.003 µmol L −1 d−1. In the dark, nitrate was produced at lower rates (0.4–0.5 µmol L−1 d−1) and high net ammonification rates were measured (1.5–5.8 µmol L−1 d−1). A potential for denitrification could be detected (2–5 nmol L−1 d−1) under anoxic conditions. Aggregates appeared to be hot spots for nitrogen cycling with potential for ammonification, nitrification and denitrification, and the rates seemed to be regulated by ambient light availability.Nitrate and ammonium concentrations were monitored in parallel in the Different types of aggregations of algae have been described in Arctic sea ice, mostly by observations made during summer, below pack ice Based on our measurements and a synthesis of previous studies, we propose a conceptual model for aggregate formation and degradation in the Arctic . This coMelosira arctica, grows forming chains and excretes high amounts of mucus that contribute to the formation of filament-shaped aggregates Melosira arctica, build up dense biomass accumulations despite nutrient constrains, that can be exported to the deep sea upon rapid melting of the sea ice Newly formed melt ponds are usually shallow, light blue, have low salinity, and contain little visible life a concentrations (2–6 µg Chl a m−2) when up scaling the contribution of the observed aggregates to the area of the entire ice floe. Here we assessed the standing stock and productivity at a local scale, for two different types of sea-ice algal aggregates. Our study took place at the end of the summer, when nutrient depletion limits pelagic productivity. During this period, we found that algal aggregates contributed significantly to total biomass at a local scale in and below the sea-ice.The spatial and temporal patchiness remains a challenge when estimating the importance of algal aggregates for ecosystem productivity and carbon flux a reported here for spherical aggregates formed by pennate diatoms . The overall relatively low mass ratios of C:Chl a of 110 and 212 for green-brownish pennate and centric diatom aggregates, respectively, indicate a healthy algal community. However, compared to the water column C:N ratio of 5–6, the green-brownish aggregates had an elevated C:N ratio . The white-yellowish aggregates containing many empty frustules even reached ratios of 40. This suggests an important contribution of exudate-carbon to the aggregates.Similar conclusions as to the relative contribution to biomass are reached using the POC values of the aggregates compared to the total sympagic or pelagic community. The contribution to total PON at a local scale was lower (0.3–18%) for pennate aggregates than for in situ integrated NPP at a local scale, and Melosira arctica filaments as much as 83–94% of the total NPP north of 80°N in mid-September, when very little production is taking place in the water column or in the sea-ice. Melosira filaments can be more productive than the integrated euphotic zone in and below the ice in the Central Arctic  than in the ambient water (105–107 cells mg POC−1) suggesting that aggregates are a rich substrate for bacteria. The higher concentrations of DOC in the aggregates compared to the average DOC concentrations of Arctic surface seawater Detrimental conditions to the aggregate community such as very low salinity from melt water, seawater warming, high grazing pressure, or nutrient depletion will eventually lead to buoyancy loss, degradation and sinking Melosira aggregate slurry, suggesting that nitrification might take place as well under photosynthetic conditions at high light intensities. This means that decaying algal aggregates could be sources of nitrate to the surrounding seawater in an otherwise nitrogen-limited system. Previously it has been speculated that internal nutrient regeneration by bacterial remineralization inside aggregates can support growth of at least a fraction of the algal population 2 production, may occur in the anoxic center of intact aggregates Melosira algal slurry were low (0.002–0.005 µmol L−1 d −1). This could indicate that the aggregates might remove considerable amounts of carbon and nitrogen from surface waters by export. Using the aggregate carbon export of 9 g C m−2 estimated previously for 2012 , and the median C:N ratio of 10 of the fresh aggregates (n = 4), approximately 45% of carbon fixed as new production since last winter and 36% of surface nitrogen consumed by sea-ice algae and surface water phytoplankton was exported to depths >4000 m.Net heterotrophic conditions were measured in floating sea-ice algal aggregates during summer indicating a rapid decomposition of the algal biomass in the aggregates . MoreoveMelosira arctica-based ice-algal aggregates contribute substantially to nutrient and carbon cycling and export in the Arctic. Preliminary experiments suggest that their buoyancy is regulated by photosynthetically-produced oxygen. With the current trend of warming of the Arctic causing sea ice retreat as well as thinning of the ice cover that increases drift speed, it is likely that sea-ice algae can grow faster and earlier in the season harvesting nutrients from a wider area of surface waters, but will more often meet unfavorable conditions during summer, e.g. nutrient limitation, freshening and melting of their habitat favoring aggregate formation and degradation. The advantages of the formation of aggregates compared to the single-cell or short-chain life style of pelagic sea ice algae is uncertain. Aggregation into large clumps and filaments may protect diatom species such as Melosira arctica from high irradiances. Trapping of gas bubbles in the aggregates enhancing floatation and eventually serving entrapment into the ice in late autumn might be another advantage. The intense nitrogen cycling observed in the aggregates indicates that they could play a role in nutrient cycling in the oligotrophic Arctic Ocean. It remains methodologically difficult, but important for a better understanding of carbon and nutrient budgets as well as for the ecology of the Arctic Ocean, to quantify temporal and regional variations in aggregate formation, distribution and fate on the ecosystem scale.In conclusion, we suggest that pennate diatom and Figure S1Oxygen profile inside degrading pennate diatom aggregate (P5). Oxygen microprofiles (n = 3) measured using an oxygen microoptode  in a 5 cm diameter spherical pennate-diatom aggregate incubated in a beaker in the lab for 3 days at 50 µmol photons m−2 s−1 and −1.3°C. Oxygen microprofiles across the water-aggregate interface were measured with steps of 0.5 mm and since the original spherical diatom aggregate started to flatten the diffusive oxygen uptake  was calculated using Fick’s first law of diffusion DOU = D0 (dC/dz), where D0 (cm−2 s−1) is the molecular diffusion coefficient in water, C (µmol L−1) is the solute concentration, and z (cm) is the depth within the aggregate. The total oxygen consumption rate of the aggregate was calculated integrating the diffusive flux over the entire aggregate surface area 2 consumption rate of a degrading pennate aggregate is 1.8±0.2 mmol O2 L−1 d−1 (n = 3).(TIFF)Click here for additional data file.Table S1Characteristics of pennate algal aggregates and sea ice stations investigated.(DOCX)Click here for additional data file.Table S2Characteristics of Melosira algal aggregates and sea ice stations investigated.(DOCX)Click here for additional data file.


The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP Student’s t-test with alpha level set to 0.05 or one-way ANOVA with Tukey's multiple comparisons test were performed using GraphPad Prism version 6.0d for Mac OS X, GraphPad Software, La Jolla California USA, 50 from 4.84 μM after 20 minutes to reach 0.87 μM after 60 minutes (50 for this parameter was found to be 0.79 μM (50 to be 3.37 μM (extracellular) presumably due to the leak of TPMP from mitochondria following the collapse of membrane potential  caused a rapid decrease of respiration on pyruvate/malate to 26.17% [23.21–29.12%] and of glutamate/malate respiration to 26.08% [24.66–27.50%] of basal respiration . When su50 of TPMP was estimated to be 3.93 [3.70–4.17] mM . We observed negligible inhibition of respiratory chain complexes and most enzymes of the Krebs cycle, however, there was a significant inhibition of the OGDHC . When tr4.17] mM .+ derivatives with respect to OGDHC inhibition in muscle homogenate enriched in mitochondrial fraction: 1 mM propyl-TPP+ reduced the activity to 60.70% [60.09–61.31%] of the untreated control, while pentyl-TPP+ caused even more inhibition to reach 15.87% [12.44–19.30%] of the control activity. Increasing the length of the alkyl side chain therefore appears to enhance the inhibitory effect on OGDHC activity , and significant correlation between Km and TPMP (R2 0.69). The pattern of decreasing Vmax and increasing Km fits in a mixed-inhibition model.In order to determine the type of inhibition of TPMP, we plotted the values of Vmax and Km  assumingWe also measured the effect of TPMP on the activity of the three components of OGDHC . The act+ derivatives, we were also interested whether similar detergents have similar effects. The cationic detergent benzyldodecyldimethylammonium (BDDA) exhibited a dose-dependent inhibitory action on OGDHC , which oxidises and decarboxylates 2-oxoglutarate and attaches coenzyme A to the product to form succinyl-CoA ,47. We eWe were also interested in the type of inhibition caused by TPMP. Our kinetic analysis suggests mixed inhibition with the inhibitor binding both to the enzyme and the enzyme-substrate complex. Since the OGDHC comprises three individual subunits we were interested if TPMP affects any or all of them. Our finding that individual subunits are affected much less than the intact complex raises a question about the possible molecular mechanism through which TPMP exerts its inhibitory effect.+ derivatives as lipophilic cations are similar in physicochemical properties to widely used cationic detergents . Our observation that the cationic detergent BDDA also causes an inhibition of OGDHC while the non-ionic detergent Triton X actually increases the activity of the enzyme complex suggests a possible similarity of action between the two cationic lipophilic species.Alkyl TPP+ on intact cell respiration. Why propylTPP+ is a more potent OGDHC inhibitor than TPMP but causes less inhibition in intact cells? One possible explanation could be that the concentration of the significantly more hydrophobic propylTPP+ in the mitochondrial matrix, where it is needed to interfere with OGDHC, would be lower than that of TPMP due to its preferential association with mitochondrial membranes.An intriguing question arises when one compares results presented here to our previous study , which s+ derivative TPMP and its more hydrophobic analogues are inhibitors of the Krebs cycle enzyme OGDHC most likely through the disruption of the enzyme complex. This finding should be taken into account when interpreting data from experiments using TPP+ salts. It also opens up new research opportunities into the possibility of manipulating Krebs cycle fluxes and intramitochondrial and intracellular availability of its intermediates such as 2-oxoglutarate.Our data show for the first time that the simplest TPPS1 Fig2O), followed by a sequential injection of 1 μM oligomycin, 1 μM FCCP, then 1 μM rotenone and antimycin A (R+A) mixture. ATP turnover-driven respiration is calculated by subtracting respiration after oligomycin from basal respiration . Maximum respiratory capacity is calculated by subtracting the residual respiration after rotenone and antimycin A treatment from the FCCP induced respiration . H+ leak is calculated as the difference between the respiration after oligomycin addition and the residual respiration after rotenone and antimycin A treatment . Data is expressed as the percentage of basal OCR (OCR%) and is presented as means ±95% CI, n = 4, measured in triplicate.The respiratory parameters concluded from measuring cellular respiration in intact C2C12 myoblasts after treatment with different concentrations of TPMP or vehicle (deionized H(TIF)Click here for additional data file.S2 Fig+ moieties in the assay mixture, except the OGDHC, which was significantly reduced. Data is presented as means ±95% CI, n = 3. Respiratory chain complexes were not altered by alkyl-TPP+ with chains ranging from C3 to C7 [The enzymatic activities of pyruvate dehydrogenase complex, glutamate dehydrogenase and other Krebs cycle enzymes were not affected by 1 mM concentration of the more lipophilic TPPC3 to C7 . PDHC, p(TIF)Click here for additional data file.


Streptomyces from soil.Tradeoffs among competing traits are believed to be crucial to the maintenance of diversity in complex communities. The production of antibiotics to inhibit competitors and resistance to antibiotic inhibition are two traits hypothesized to be critical to microbial fitness in natural habitats, yet data on costs or tradeoffs associated with these traits are limited. In this work we characterized tradeoffs between antibiotic inhibition or resistance capacities and growth efficiencies or niche widths for a broad collection of Streptomyces isolates tended to have either very little or very high inhibitory capacity. In contrast, Streptomyces isolates were most commonly resistant to antibiotic inhibition by an intermediate number of other isolates. Streptomyces with either very high antibiotic inhibitory or resistance capacities had less efficient growth and utilized a smaller number of resources for growth  than those with low inhibition or resistance capacities, suggesting tradeoffs between antibiotic inhibitory or resistance and resource use phenotypes.Streptomyces in natural communities.This work suggests that life-history tradeoffs may be crucial to the maintenance of the vast diversity of antibiotic inhibitory and resistance phenotypes found among The online version of this article (doi:10.1186/s12862-015-0470-6) contains supplementary material, which is available to authorized users. Soil bacteria produce an astounding array of antimicrobial compounds  AntibiotTradeoffs preclude an organism’s ability to optimize multiple traits , 10. It Tradeoffs associated with antibiotic production and resistance should be reflected in growth reductions for antibiotic-producing vs. non-producing bacteria in the absence of competition  or, morStreptomyces are filamentous, Gram-positive bacteria that are prolific producers of antibiotics and a significant reservoir of antibiotic resistance in soils [Streptomyces is often dependent on resource availability [Streptomyces vary extensively in the resources on which they can grow and in their relative growth efficiencies on different resources [Streptomyces that are both highly antagonistic and growth efficient on a wide array of resources. However, specific data on the relationships among Streptomyces resource use, antibiotic inhibition, and antibiotic resistance from natural settings are needed to understand the potential for fitness tradeoffs to constrain the accumulation of antibiotic inhibition or resistance capacities in soil microbes.in soils , 17. Antlability –24. Moreesources , suggestStreptomyces within natural soil populations. We examine patterns of antibiotic inhibition and resistance phenotypes among n = 263 Streptomyces isolates from natural soil settings in relation to their resource use on 95 sole carbon sources. Specifically, we 1) characterize antibiotic inhibition and resistance among Streptomyces and quantify inhibition and resistance phenotypes among isolates; 2) determine relationships between antibiotic inhibition, resistance, and resource use among Streptomyces isolates; and 3) explore evidence for tradeoffs among Streptomyces exhibiting distinct inhibition and resistance strategies. These data suggest that tradeoffs may be critical in structuring antibiotic production and resistance strategies among Streptomyces in soil.In this work we hypothesize that there are life-history tradeoffs associated with the antibiotic inhibitory and resistance capacities of www.cedarcreek.umn.edu), a NSF Long-Term Ecological Research site as described previously [2 sections of two plots in experiment E001 (plots 08-A and 10-D) for a total of six soil cores. Soil cores were transported to the lab on ice and processed immediately. Soils were dried overnight under a double-layer of sterile cheesecloth, serially diluted in phosphate buffer , and plated on oatmeal agar as described in Davelos et al. [Streptomyces densities were estimated based on characteristic colony morphology. Streptomyces colonies were randomly picked with a sterile toothpick, purified, and stored in 20 % glycerol at -80 C for further study. Detailed information on soil collection, processing, and isolation is published elsewhere [Soil samples were collected at the University of Minnesota Cedar Creek Ecosystem Science Reserve  were characterized for antibiotic inhibition profiles by evaluating their ability to inhibit each standard isolate using an agar-overlay method [8 spores/ml) were dotted onto 15 ml starch-casein agar (SCA). After incubation for 3 days at 28 C, isolates were killed by inverting each plate over a watch glass containing 4 ml chloroform. Plates were then left open in a laminar flow hood for 30 min to allow residual chloroform to evaporate, then overlaid with 15 ml of fresh 1 % water agar. After the agar solidified 100 ul of each test standard spore stock (~108 spores/ml) was spread on the plate. Plates were incubated for 3 days at 28 C and the size of inhibition zones around dotted isolates were measured. Each Streptomyces-standard interaction was replicated three times and only inhibition zones greater than 1 mm were considered to be inhibitory. The same approach was used to characterize the collection of Streptomyces isolates for resistance to antibiotic inhibition by each standard isolate. Spatial variation in the inhibition and resistance characteristics of a subset of the isolates used in this work have been published previously [s et al.  Standards et al. . Becausein 1 m2; ) and isoy method . BrieflyStreptomyces isolates on a diverse array of sole carbon sources. Fresh spore suspensions of each Streptomyces isolate were quantified to an absorbance of 0.22 at 590 nm, diluted according to the manufacturer’s instructions, and inoculated into Biolog SF-P2 plates. Plates were incubated at 28 C for 3 days and the absorbance of each well was measured (590 nm). The absorbance (au) from the water control well was subtracted from all 95 substrate-containing wells. After correction, all wells with an absorbance <0.005 were adjusted to 0 prior to analyses. We defined niche width as the number of substrates on which an isolate could grow (positive absorbance after adjustments). Growth efficiency was defined as the mean growth on used substrates . Variation in resource use of these isolates has been explored [Resource use was evaluated on Biolog SF-P2 microplates  using standard procedures as described in Schlatter et al. . Measureexplored , yet theStreptomyces inhibitory, resistance, and resource use phenotypes. Permutation tests  were used for Spearman correlations to estimate the significance of relationships using the ‘coin’ package in R [Streptomyces with antagonistic or resistant phenotypes and their non-antagonistic or resistant counterparts for each individual standard tested. Because antibiotic inhibitory phenotypes among Streptomyces fell into two distinct categories, those that did not inhibit any standards  and those that inhibited all 10 standards , Student’s t-tests were used to compare differences in resource use of Streptomyces among these categories. Similarly, although antibiotic resistance was normally distributed, resource use among Streptomyces with low  and high  resistance capacities was also compared using Student’s t-tests. Further, the ability of Streptomyces belonging to high- versus low-inhibition or resistance categories to utilize each of the n = 95 individual carbon sources was compared using Pearson’s Chi-squared tests, where p-values <0.05 after adjustment for multiple comparisons using an FDR correction were considered statistically significant.All statistical analyses were performed in the R statistical package  or Grapage in R . StudentStreptomyces isolates varied in their abilities to inhibit and resist standard strains, consistent with previous work [Streptomyces inhibited an average of 3.1 standards, though this average increased to 5.3 standards when considering only Streptomyces that could inhibit at least one standard. However, most isolates had either very little or very high inhibitory capacity, as reflected in the distinct bimodal distribution of isolate frequencies  of isolates was resistant to all ten standards. In contrast to the bimodal distribution of inhibitory phenotypes, frequencies of resistance were approximately normally distributed among isolates and fell around a distinct mode .Resistance phenotypes among age Fig. . ResistaStreptomyces capacities to inhibit and resist standards were weakly correlated. There was a marginally significant positive correlation between the number of standards inhibited and the number of standards resisted among individual Streptomyces , suggesting that Streptomyces with broad antibiotic inhibition capacities are slightly more likely to have broad resistance capacities.Streptomyces with the ability to inhibit specific standards versus those with no apparent inhibitory capacity against that standard sometimes differed in growth efficiency or niche width , though differences were only significant for four of the ten standards. These results suggest that having inhibitory capacity towards a standard sometimes comes at a cost with respect to growth efficiency or niche width, though costs may vary among antibiotics.th Table . InhibitStreptomyces that exhibited inhibitory capacities against particular standards, there were significant negative relationships between inhibition intensity (inhibition zone size) and growth efficiency for eight of ten standards , but not growth efficiency . Thus, isolates that inhibited a larger number of standards had smaller niche widths than those that inhibited fewer standards. However, correlations between total number of standards inhibited and niche width were weak, perhaps due to the variation in the costs of producing diverse antibiotics and inhibiting different standards. Notably, non-inhibitory isolates (inhibited none of the standards) and highly inhibitory ‘super-killers’  differed significantly in resource use. Super-killer Streptomyces had significantly smaller niche widths and grew less efficiently than non-inhibitors  and 6.8 % more efficient growth  than super-killers. Thus, super-killers with broad inhibitory capacities had more restricted niches and grew less efficiently than non-inhibitory Streptomyces. This suggests that there may be significant tradeoffs between cumulative inhibition capacity and both niche width and growth efficiency among Streptomyces. Niche width reductions in super-killers may result from evolutionary tradeoffs between genomic investments in primary versus secondary metabolism, specifically reductions in the diversity of metabolic capacities retained by highly inhibitory populations. In contrast, reduced growth efficiencies among super-killers may be an ecological consequence of energetic costs associated with antibiotic production that limit population growth rates, rather than the diversity of metabolic pathways.Considering cumulative inhibitory capacity, or the total number of standards that a Streptomyces with distinct inhibitory capacities (super-killers versus non-inhibitors) differed in their likelihood of using specific carbon sources for growth. Of the 95 sole carbon sources present on the Biolog SF-P2 plate, 21 carbon compounds were used significantly more or less frequently by super-killers than non-inhibitory Streptomyces , carboxylic acids (n = 3), amino acids (n = 2), polymers (n = 1), and miscellaneous compounds (n = 2). In general, super-killers used carbon sources less frequently than non-inhibitory Streptomyces , consistent with a niche width-inhibition capacity tradeoff. However, D-alanine and α-ketoglutaric acid were used at significantly higher frequencies among super-killers than among non-inhibitory strains .ces Fig. . These dStreptomyces that could resist inhibition by particular standards were sometimes less growth efficient or had smaller niche widths than isolates that were susceptible to that Streptomyces  or had smaller niche widths (7 of 10 standards) than Streptomyces susceptible to that standard.es Table . IsolateStreptomyces against environmental isolates) had no clear relationship with growth efficiency or niche width. This suggests that the costs of resistance may be largely associated with the presence of a resistant phenotype, rather than the degree of resistance conferred. However, the number of susceptible Streptomyces varied considerably for individual standards  and growth efficiency . Streptomyces with greater resistance capacity tended to have smaller niche widths and grow less efficiently than more susceptible isolates. Streptomyces with very high versus low antibiotic resistance capacities also differed significantly in resource use. Isolates with little resistance to inhibition by standards (those that could resist inhibition by ≤5 standards) had significantly larger niche widths and higher growth efficiency than highly resistant Streptomyces , and 28.0 % greater growth efficiency  than Streptomyces with a high number of resistances, suggesting substantial growth efficiency-resistance and niche width-resistance tradeoffs among Streptomyces isolates. Tradeoffs between niche width and resistance capacity may reflect distinct genomic investment in primary metabolism versus antibiotic resistance strategies, where highly resistant isolates are less capable of metabolizing diverse carbon sources. In contrast, growth efficiency-resistance tradeoffs may be a consequence of the direct physiological costs of bearing multiple resistance traits that limit population growth rates.The number of standards that Streptomyces with low versus high capacities to resist inhibition by standards differed significantly in the frequency with which they used specific carbon compounds for growth. Of the 95 sole carbon sources present on Biolog SF-P2 plates, nineteen carbon sources were used at different frequencies between Streptomyces with low versus high resistance capacities , polymers (n = 3), amino acids (n = 1), and miscellaneous compounds (n = 2). Similar to patterns observed for super-killers versus non-inhibitors, Streptomyces with low resistance capacities utilized most carbon compounds at significantly higher frequencies than those with high resistance capacities . Intriguingly, D-alanine was an exception and was used for growth by a greater proportion of highly resistant strains (82 % of strains) than those with low resistance capacities (40 % of strains). Together, these data indicate that reductions in niche width among Streptomyces with high versus low resistance capacities are often specific to individual carbon compounds.ies Fig. . DiffereStreptomyces were characterized by one of two distinct inhibitory strategies, tending to be either non-inhibitory, or highly inhibitory “super-killers”. Because antibiotic inhibitory interactions can be highly specific among Streptomyces isolates [Streptomyces to produce antibiotic compounds that act synergistically is thought to play a significant role in the evolution of Streptomyces secondary metabolism [Streptomyces strains may confer substantial and potentially synergistic fitness benefits to producers across time and space. As a result, optimal strategies for an antagonistic lifestyle may tend towards relatively high accumulation of antibiotic inhibitory phenotypes (super-killers), as observed here.Most isolates , 29, theisolates , 16, 30,isolates . Howeverisolates . Indeed,tabolism . Thus, ain vitro antibiotic production but rather to document life-history tradeoffs across a diverse collection of naturally-occurring Streptomyces isolates. Negative relationships between Streptomyces inhibitory phenotypes and niche width or growth efficiency and the substantially smaller growth efficiencies and niche widths for super-killer versus non-inhibitory Streptomyces suggest significant growth-inhibition and niche width-inhibition tradeoffs associated with these traits. These tradeoffs may represent physiological costs of constitutive antibiotic production, pleiotropic effects of genes involved in primary or secondary metabolism, or the costs of accumulating antibiotic biosynthetic pathways for multiple antibiotic compounds.Although antibiotic phenotypes are assumed to impose costs of production –13 our eStreptomyces, where genes encoding the production of many secondary metabolites have diverse and highly complex regulatory mechanisms which often require very specific growth conditions, nutrient limitation, and/or extra-cellular signals for expression [Streptomyces occurs when nitrogen, phosphorus, or carbon sources become limiting and the process of morphological differentiation is triggered [Streptomyces isolates. We observed growth efficiency and niche width tradeoffs associated with individual resistance phenotypes only for a small number of standards, suggesting that the cost of any specific resistance phenotype may be very small. This may be due in part to compensatory mutations, which are likely to minimize tradeoffs by rapidly alleviating the costs of antibiotic resistance [Streptomyces populations in soil.Negative relationships between inhibition intensity against standards and growth efficiency or niche width were common, yet significant differences in growth efficiency or niche width between inhibitors and non-inhibitors were observed for only a few standards. This suggests that the physiological costs of inhibition of a single target may be more strongly associated with inhibition intensity, while genomic costs were more evidenced in cumulative inhibitory capacities. Physiological tradeoffs associated with expressing a given biosynthetic pathway for antibiotic production may be offset by tightly-controlled regulation. This is frequently the case among pression –35. In griggered , 32. Morriggered . Becausesistance , 37 or asistance . Howeversistance , 40. ThuStreptomyces, and resistance to inhibition by an intermediate number of standards was the most common phenotype among isolates. In natural settings Streptomyces are commonly resistant to many antibiotics, even in environments with no history of antibiotic contamination from anthropogenic sources [Streptomyces isolates. These constraints combined with the substantial fitness cost of lacking resistance to antibiotic-producers may generate stabilizing selection that favors Streptomyces with moderate resistance capacities rather than those having very few or very many antibiotic resistances.In contrast to antibiotic inhibition, cumulative capacities to resist antibiotic inhibition (the number of standards that an isolate could resist) were approximately normally distributed among  sources , 30. ThiStreptomyces used many carbon sources at significantly lower frequencies than non-inhibitory or poorly resistant Streptomyces. This suggests that reductions in niche widths associated with these lifestyles are resource-specific  may confer resistance to multiple antibiotics, or may also impose different fitness costs [Streptomyces with similar inhibition or resistance capacities is not surprising. Further studies using population genomics tools to quantify the specific secondary metabolic pathways, resistance factors, and primary metabolic capacities of these populations will address these issues and offer critical insight into genomic and physiological tradeoffs among Streptomyces.Although we found compelling evidence for resource use tradeoffs with antibiotic inhibition and cumulative resistance capacity, there was substantial variability in niche width and growth efficiency among ss costs , 40. In Streptomyces with super-killer versus non-inhibitory life-history strategies than for those with highly resistant versus susceptible lifestyles. This suggests that it may be more costly to accumulate capacities to resist many antibiotics than to accumulate ability to inhibit (kill) many targets. This is a particularly novel finding considering that the fitness costs of antibiotic resistance in clinical settings are often found to be small [Intriguingly, reductions in growth efficiency and niche width were substantially smaller for be small  and thatStreptomyces. Moreover, greater apparent costs of accumulating multiple resistances versus inhibitory capacities are surprising, and appear to generate different distributions of these phenotypes among Streptomyces isolates. Further work exploring the roles tradeoffs play in the evolution of Streptomyces will be essential to elucidating the forces that generate and maintain the vast diversity of antibiotic inhibitory and resistance phenotypes in soil communities.Our findings suggest that tradeoffs are likely to constrain the evolution of extreme antibiotic inhibitory and resistance phenotypes among natural populations of soil-borne