Coastal acid sulfate soils (CASS) constitute a serious and global environmental problem. Oxidation of iron sulfide minerals exposed to air generates sulfuric acid with consequently negative impacts on coastal and estuarine ecosystems. Tidal inundation represents one current treatment strategy for CASS, with the aim of neutralizing acidity by triggering microbial iron- and sulfate-reduction and inducing the precipitation of iron-sulfides. Although well-known functional guilds of bacteria drive these processes, their distributions within CASS environments, as well as their relationships to tidal cycling and the availability of nutrients and electron acceptors, are poorly understood. These factors will determine the long-term efficacy of “passive” CASS remediation strategies. Here we studied microbial community structure and functional guild distribution in sediment cores obtained from 10 depths ranging from 0 to 20 cm in three sites located in the supra-, inter- and sub-tidal segments, respectively, of a CASS-affected salt marsh . Whole community 16S rRNA gene diversity within each site was assessed by 454 pyrotag sequencing and bioinformatic analyses in the context of local hydrological, geochemical, and lithological factors. The results illustrate spatial overlap, or close association, of iron-, and sulfate-reducing bacteria (SRB) in an environment rich in organic matter and controlled by parameters such as acidity, redox potential, degree of water saturation, and mineralization. The observed spatial distribution implies the need for empirical understanding of the timing, relative to tidal cycling, of various terminal electron-accepting processes that control acid generation and biogeochemical iron and sulfur cycling. Coastal acid sulfate soils (CASS) constitute a major global environmental problem , both of which can promote the immobilization of arsenic, a common toxic metalloid in CASS environments , northeast Australia  and two control sites located (i) outside of the tidal bund-wall  and (ii) at an CASS site not treated with tidal inundation (“Acidic Site”). The samples were freeze-dried, homogenized and Soxhlet-extracted (48 h) in a mixture of dichloromethane (90%) and methanol (10%). Elemental sulfur was removed with activated copper pellets. The extracts were separated into three fractions  by silica gel column chromatography using eluents of increasing polarity  at ~3 scans/s and a source temperature of 230°C. Peak assignments were based on correlation of GC retention time and mass spectral data with reference compounds, library spectra or other published data.Gas chromatography-mass spectrometry (GC-MS) analyses of aromatic and derivatised polar fractions were performed on an Agilent 6890/5973 GC-MS equipped with an Agilent 6890 autosampler and a 60 m × 0.25 mm i.d. × 0.25 μm film thickness DB5-MS column (J&W Scientific). Aromatic fractions were injected in splitless mode. Polar fractions were injected in splitless cool on column mode into an Alltech pre-column (2 m x 0.53 mm i.d.) fitted to the DB5-MS column. Helium was used as a carrier gas at a constant flow of 1.1 and 1.2 mL/min for aromatic and polar fractions, respectively. The GC oven was heated from 40 to 325°C at 3°C/min with initial and final hold times of 1 and 30 min for aromatic fractions and from 50 to 320°C at 6°C/min with initial and final hold times of 1 and 20 min for polar fractions. The MS was operated at 70 E. coli position 2305–2322, Brosius et al., E. coli position 2908–2923, Brosius et al., 2 at a final concentration of 2 mM and BSA at a final concentration of 0.3 mg/mL. Thermal cycling had an annealing temperature of 55°C with 30 cycles. ACE has done extensive testing with this protocol and biases in amplication have been minimized. Then 2 μL of the first PCR product was used for a second PCR with no clean up to add barcodes , using the same reagents and conditions as for the first PCR but for 10 cycles. The PCR products were then quantified on the TapeStation and pooled at equal concentrations. The pooled DNA was gel extracted and amplified by emulsion PCR for sequencing. The Roche 454 sequencing (GS FLX Titanium chemistry) was performed with Roche 454 protocols.Each DNA extraction used ~0.3 g of sediments (wet weight) with the PowerSoil DNA Isolation Kit  according to the manufacturer's protocol. DNA samples were sent for 454 pyrosequencing at the Australian Centre for Ecogenomics . A first round of PCR was conducted with SSU803F  for each sample. The evenness values were used to evaluate the distribution evenness of relative OTU abundances. The beta diversity, which represents differentiation among each sample, was calculated and represented in two ways: a dendrogram which was calculated using the Jaccard index, then clustered using the UPGMA algorithm; and a principle coordinate analysis (PCoA) calculated using the Yue and Clayton index  represent less kinetic or thermodynamic limitations, or that the reaction is far from equilibrium and the forward direction overwhelms the reverse direction. If the reaction is close to equilibrium, the factors decrease toward to 0, which means little energy is available for microorganisms. This rate law is built on Monod and Michaelis-Menten kinetics  and absolute temperature (298 K was used in this study), χ is average stoichiometric number with a suggested value of 6 used in this study :i is the activity coefficient, [i] is the concentration of reactant or product i, and the ΔG0T is the standard Gibbs free energy at absolute temperature, T°K. The activity coefficient γi used in this study was derived using the Geochemist's Workbench software  for acetotrophic sulfate reduction  for the upper portions (0–10 cm) of each core, with values from 3.29 to 6.13 at site A1, 4.38 to 5.92 at site A2, and 6.08 to 6.43 at site A3. In contrast, pH values were similar for all three sites for the lower portions (10–20 cm), ranging from 5.97 to 6.79. The Eh values decreased with depth within each core , but generally increased along the transect from the sea toward the upland site at each depth.The polar fractions from surface sediments of each transect site, as well as from the acidic control site, were dominated by a suite of plant-derived pentacyclic 3-oxy triterpenoids, including oleanolic, betulinic and ursolic acids, which showed a similar distribution in all samples. However, these compounds were absent in the polar fractions obtained from the mangrove control site, which was dominated by the triterpenoid taraxerol. In the aromatic fractions, pentacyclic triterpenoid derivatives were highly abundant Figure . HoweverP < 0.001, ANOVA). Chao richness estimates, which estimate the number of phylogenetically different OTUs (3% or more different in sequence composition), displayed a significant decrease from site A3 to A1 , with the highest values in the upper part of site A3 (3668 reads), followed by sites A2 (1707 reads) and A1 (1128 reads), respectively. Both Shannon and Simpson diversity indices showed a significant difference among the three sites , which is shown on the pH and Eh diagram with the inverse Simpson index . The gamma-, beta-, and alpha-proteobacteria exhibited higher sequence abundances at Zone 1 . The Chloroflexi, Bacteroidetes, Firmicutes, Euryarcheota, and Crenarchaeota showed greater abundances in Zone 2 (22%) and Zone 3 (39%), compared to Zone 1 (8%).Bacteria comprised ~89% and archaea ~11% of the prokaryotic community structure, taken across all sites within the East Trinity wetlands. In total 30 bacterial and three archaeal phyla  organic carbon in the organic horizon at East Trinity wetlands  became the predominant plant species in the drained lands, but at locations with re-introduced tidal flows, such as our study site, they died off and the original mangrove vegetation  returned  values Figure . Site A1in situ soil layering, water saturation, and pH values, the boundaries of these three zones were consistent with major variations in these environmental parameters , microbes may not need to compete for electrons and carbon  to those representing known iron-reducing bacteria or close relatives for Zone 3 , and there are not enough samples in Zone 2 to demonstrate correlation convincingly. Site A3, depths 4–6 cm, differed from all other samples and can be explained by the observation that the sulfuric horizon is much closer to the tidal zone in site A3 than in site A2 . When the energy available in the environment is in excess of energy conserved by microbial metabolism, the thermodynamic factor moves toward a greater value, which means the reaction is far from equilibrium and the reaction has a greater tendency to move in the forward direction. Taking acetotrophic sulfate reduction as an example, the concentrations of sulfate and organic carbon (reactants) are higher than the concentrations of sulfide and bicarbonate (products) in the study site, and higher thermodynamic factors 0.75–0.89) were observed (Figure 9 were obK2−4SO and K3COO−CH by at least one order of magnitude, and therefore the kinetic factor moves toward unity (Figure In this case, the concentrations of sulfate and acetate were greater than the half-reaction constant y Figure  for the k and microbial biomass concentrations. Biomass in the thermodynamic-kinetic model and other kinetic models does not account for dormant microbial cells (Jin et al., Microbial metabolic rates depend on a the rate constant Microbial distributions in the East Trinity wetlands support the paradigm of community selection by from a homogenous population (De Wit and Bouvier, Tidal re-inundation is being tested as an effective means for natural remediation of CASS systems. However, tidal fluctuations can make CASS systems highly dynamic environments with respect to redox states and the flux of nutrients and electron donors or acceptors. Correspondingly, microbial communities living in tidal zones experience both static and fluctuating environmental conditions that, in turn, are modulated by lithological compositions and hydrological connectivity. For researchers attempting to construct complete biogeochemical process models, these factors must also be considered.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.


Immunoglobulin (Ig) E is well-known to play a critical role in allergic diseases. We investigated the association between longitudinal change in total IgE level and the asthma control in patients with adult asthma.For this retrospective study, 154 patients with asthma aged 21–82 years were recruited from the allergy and pulmonary units of the Showa University Hospital. Data on longitudinal changes in IgE over the preceding 10 years were collected and logarithmically transformed. Associations between longitudinal change in IgE and clinical characteristics including asthma control test (ACT) score, asthma control, pulmonary function test, and antigen specific IgE, were assessed.1 and FEV1% were lower in patients with increased IgE than in those with decreased or unchanged IgE. Moreover, the prevalence of Aspergillus-specific IgE was higher in patients with increased IgE than in those with decreased or unchanged IgE.Patients with increased IgE tended to have significantly higher mean age, more episodes of acute exacerbation within a year, lower ACT scores, and used oral corticosteroids more frequently than those with decreased or unchanged IgE. The prevalence of uncontrolled asthma was higher in patients with increased IgE than in those with decreased or unchanged IgE. Mean %FEVAspergillus-specific IgE in patients with adult asthma.These data suggest that a longitudinal increase in total IgE is associated with both poor asthma control and  Immnoglobulin E (IgE), the fifth and last immunoglobulin to be identified, was first reported in 1966 . EpidemiTotal IgE level is higher in children with severe asthma than in those with mild to moderate asthma . In The In general, serum total IgE concentration seems to peak in early adolescence and decrease with age -19. HoweThe aim of the present study was to investigate the association between longitudinal change in total IgE and asthma control to elucidate the role of longitudinal change in IgE in patients with asthma.This was a retrospective study conducted in the allergy and pulmonary units of the Showa University Hospital, Tokyo, Japan, between January 2011 and December 2012. The study protocol was approved by the Showa University ethics committee and written informed consent was obtained.1) of 12% or 200 mL after the inhalation of salbutamol.Out of 854 asthmatic patients, we recruited 154 patients, whose longitudinal changes in total IgE in the preceding 10 (±2) years could be obtained. All patients had been regularly followed at Showa University Hospital. Individuals with chronic obstructive pulmonary disease (COPD) or other lung disease, smoking history greater than 20 pack-years, being treated with omalizumab, vocal cord dysfunction, or neurological disease were excluded. No participants, even those who had high IgE level, had parasitic infections. Asthma was diagnosed based on cough, breathlessness or dyspnea, and demonstration of airflow variability. The latter was determined according to a reversible airflow limitation that represented an increase in forced expiratory volume in 1 s (FEVDermatophagoides pteronissinus (house dust mite: HDM), Cryptomeria japonica (cedar), Ambrosia artemisiifolia (ragweed), Candida albicans (candida), Aspergillus fumigatus (aspergillus), Alternaria alternata , dog dander, cat dander, Bombyx mori (moth), and Blatta orientalis (cockroach)] were collected at enrollment. The definition of atopic type was being positive to crude house dust-specific IgE.Baseline data including medical history, Asthma Control Test (ACT) score, spirometry, fractional exhaled nitric oxide (FeNO), percent peripheral eosinophils, total and specific IgE [Asthma control was assessed using the validated Japanese version of the ACT. Patients were subjectively evaluated for the degree of impairment caused by their asthma during the preceding 4 weeks by responding to five questions using a five-point scale.1, forced vital capacity (FVC), and FEV1/FVC (FEV1%). The highest value from three technically satisfactory attempts was recorded. FEV1 and FVC values were expressed as a percentage of the predicted value.Spirometry was performed using an AS-302 spirometer  in accordance with American Thoracic Society/European Respiratory Society guidelines ,24 to deFeNO was measured by a portable device  at an expiratory flow rate of 50 mL/s for 10 s.Serum total IgE levels, measured with a fluorescent enzyme immunoassay  were transformed logarithmically to normalize their distribution. A zero was replaced with a value half of the lowest value observed before log transformation. Antigen-specific IgE was also measured with ImmunoCAP-FEIA, and levels higher than class 2 were considered as positive. The longitudinal change in total IgE (ΔIgE) was calculated as log total IgE at present – log total IgE 10 years ago. An increase or decrease in total IgE was arbitrarily designated as a variation of >0.15 log10 kU/L.t-test. Categorical variables were analyzed with χ2 tests. A value of P <0.05 was considered significant for all statistical assessments.Results were expressed as mean ± SD for continuous variables. FeNO values were transformed logarithmically to normalize their distribution. All analyses were performed using JMP version 10 . Differences in the continuous variables between groups were tested by analysis of variance (ANOVA); if the differences between groups were significant, comparisons were made by unpaired 1 were 2.5 ± 0.9 L (%FVC: 88.2 ± 19.3%), and 1.8 ± 0.8 L (%FEV1: 75.0 ± 23.0%), respectively.One hundred and fifty-four patients with asthma with a median age of 62.5 years (range 21–82) were included. The mean BMI was 23.1 ± 3.6, and 62 patients (40.3%) were men. Thirteen patients (8.4%) were current smokers, and 31 patients (20.1%) were ex-smokers at the time of enrollment. The mean ACT score and FeNO were 20.7 ± 3.7 and 51.3 ± 42.9 ppb, respectively. In the pulmonary function test, the mean FVC and FEV10 kU/L 10 years ago and 2.37 ± 0.66 log10 kU/L at the time of the study. An increase or decrease in IgE was arbitrarily designated as a variation of >0.15 log10kU/L. Figure Mean total IgE was 2.43 ± 0.65 logDemographic details for the patient population are shown in Table To identify patients with poor asthma control, several tools can be used globally, one of which is ACT. This test comprises five questions to assess activity limitation, shortness of breath, nighttime symptoms, use of rescue medication, and the patient’s overall rating of asthma control over the previous 4 weeks. Mean ACT score was significantly lower for patients with increased IgE  guidelines, the most widely used asthma guidelines in the world, emphasize the importance of evaluating asthma control rather than asthma severity. The GINA guidelines provide a categorical scale for assessing asthma control, consisting of well-controlled, partly controlled, or uncontrolled asthma . Asthma 1 and FEV1%. The patients with increased IgE had lower %FEV1 than did the patients with no change in IgE (Figure 1% than patients with decreased or unchanged IgE (Figure Lung function, as an assessment of variable airway obstruction, is an objective of asthma control. There was no significant difference in %FVC between the groups Figure A. In conThe prevalence of IgE specific to inhaled antigens is shown in Table In the present study, we found that patients with a longitudinal increase in total IgE used on-demand OCS more frequently, were more likely to have been treated at levels above Step 4, had more episode of acute exacerbation within a year, had lower ACT scores, and were more likely to have uncontrolled asthma than patients with a longitudinal decrease in IgE or no change in IgE. In addition, more airway obstructions were observed among patients with a longitudinal increase in IgE. These results suggest that a longitudinal increase in IgE is associated with poor asthma control. To our knowledge, this is the first study to investigate the clinical role of longitudinal change in total IgE in patients with adult asthma and to demonstrate the possible association between a longitudinal increase in IgE and poor control of adult asthma. Recent global trials showed no association between absolute IgE levels and asthma severity in patients with adult asthma ,14. ConsLongitudinal changes in IgE vary considerably among individual in patients with adult asthma, as shown in Figure Previous studies have shown that males and current smokers have higher IgE levels than females and non-current smokers, respectively -29. In tAspergillus fumigatus contributed to an increased risk of adult onset of asthma [The total IgE level reflects both specific and unspecific IgE. Matricardi et al. followed total and allergen-specific IgE levels in children from birth to 13 years of age . This stf asthma . To the e the association between longitudinal change in aspergillus-specific IgE and longitudinal change in total IgE and control levels.There are several limitations to our study. First, although we showed that patients with increased IgE had a higher prevalence of aspergillus-specific IgE than patients with decreased or unchanged IgE, a longitudinal change in aspergillus-specific IgE could not be shown. Thus, to demonstrate directly the contribution of aspergillus-specific IgE to the increase in total IgE and poor control of asthma, we should assessSecond, the treatments of patients were not taken into consideration in this study. The treatments of all patients enrolled in this study have changed, usually a number of times, during the past 10 years. Therefore, we could not account for the treatments in our analysis. It remains unclear how treatments affect longitudinal changes in total IgE and antigen-specific IgE.In conclusion, this is the first study to investigate the clinical roles of longitudinal change in serum total IgE in patients with asthma. We demonstrated that longitudinal increase in total IgE is associated with poor asthma control and a higher prevalence of aspergillus-specific IgE in patients with adult asthma, suggesting a significant contribution of IgE in the pathophysiology of severe asthma. Further prospective analysis is needed to validate our data.


In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11–7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11–7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11–7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach “Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target” (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity. In three consecutive enzymatic reactions, G6PDH (reaction 1), followed by 6-phosphogluconolactonase  and 6PGDH (reaction 3), glucose-6-phosphate (G6P) is catabolised supplying cells with ribulose-5-phosphate maintaining the antioxidative power by generating 2 NADPH molecules. NADPH is an absolute requirement for reductive metabolism and maintenance of cellular redox homeostasis (Glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the oxidative (irreversible) branch of the pentose phosphate pathway (oxPPP), has multiple functions in both pro- and eukaryotic cells. Another NADPeostasis .Long-term inhibition of G6PDH activity and the associated impairment of the NADPH-generating system and glutathione (GSH)-replenishment system significantly increase the vulnerability of the affected cells to apoptosis. Thus, proliferating tumour cells as well as erythrocytes infected with malaria parasites with their high demand for NADPH and GSH can be effectively eliminated by inhibition of G6PDH. Disruption of G6PDH activity has been shown to repress proliferation and simultaneously promote apoptosis in growing tumour cellsin vitro and/or in vivo such as the naturally occurring adrenal steroid dehydroepiandrosterone (DHEA)in vitro growth inhibitory effect on parasites’ culture of Trypanosoma brucei (own unpublished data). This growth inhibitory effect might also be attributed to G6PDH inhibition.Numerous compounds have been used to inhibit the activity of endogenous mammalian G6PDH +), respectively (for review seeMany of the hitherto applied inhibitors of G6PDH contain sugar phosphates or various nucleotides competing with the substrate (G6P) or cofactor  in affected subjects with cardiovascular diseasesUpregulation of pro-oxidative enzymes NADPH oxidase (NOX) and nitric oxide synthase (NOS), fuelled by G6PDH-derived NADPH, leads to the production of high levels of superoxide anion  as a result of GSH depletion after treatment of human erythrocytes with DMFHowever, the role of the metabolic enzymes responsible for GSH regeneration and their involvement in eryptosis under inhibitor treatment conditions has not been addressed yet. In order to confirm eryptosis induction by the inhibitors and to analyse their death-inducing capacity, erythrocyte populations from the same donors were treated with increasing concentrations of Bay 11–7082, parthenolide or DMF, under otherwise identical conditions. Eryptosis was measured by Annexin V binding to PS translocated to the outer leaflet of the erythrocyte membrane (scrambling). The strongest effect could be observed with Bay 11–7082, followed by parthenolide and DMF  and 2A. It is known that hemolysis is induced as a result of unspecific drug interaction with erythrocytesThe anti-inflammatory substances Bay 11–7082, parthenolide or DMF are capable to directly and irreversibly bind to GSH in a 1:1 ratio thereby completely curbing the intracellular GSH levels1819The physiological concentration of GSH is millimolar, whereas that of GSSG is micromolar. Therefore, a painstaking attention to technical details was given for GSH and GSSG analysis, as oxidation of even 1% of a given GSH concentration results in an increase of GSSG of one order of magnitude.et al.50 values of the compounds varied considerably with 7.6 μM for Bay 11–7082, 10 μM for parthenolide and 20 μM for DMF. The affinity of the individual drugs to GSH may differ and thus may result in differing IC50 values.In order to directly measure GSH levels in erythrocytes, we applied the newly developed HPLC-based method of Giustarini +-dependent redox-relevant enzymes like G6PDH and 6PGDH as well as NADPH-dependent GR function as upstream enzymes of GSH recycling and replenishment, we further investigated the effects of Bay 11–7082, parthenolide and DMF on these enzymes.The decrease in GSH and thus the cellular resistance against oxidative stresses observed in inhibitor-treated RBCs was not accompanied by a concomitant increase in GSSG concentration. On the contrary, we could observe a slight decrease also of GSSG, most evident at the highest concentrations of the tested drugs . Figure 2O2) decrease the existing intracellular high GSH/GSSG ratio according to the principle of an inverse correlation in favour of a high GSSG/GSH ratio. This dynamic reciprocal proportionality in mature human erythrocytes between intracellular GSH and GSSG concentrations was not observed, when cells were treated with the anti-inflammatory compounds Bay 11–7082, parthenolide, or DMF. In our study, GSH depletion was not accompanied by an increase of the intracellular GSSG concentration , pointing to a specific inhibitory effect on the activity of the first NADPH generating enzyme, i.e. the G6PDH in erythrocytes. The similarities between DHEA as an uncompetitive inhibitor of mammalian and lower eukaryotes G6PDH and the compounds Bay 11–7082, parthenolide and DMF used in our study are intriguing. DHEA exclusively inhibits G6PDH, but not 6PGDHThe idea that Bay 11–7082, parthenolide or DMF might exert hitherto unrecognized regulatory/inhibitory effects on other redox-relevant enzymes, prompted us to investigate a possible inhibitory effect of these compounds on GR activity. Among other functions, G6PDH serves to fuel GR with NADPH. GR, in turn, as the central enzyme of glutathione redox metabolism triggers via its co-factor NADPH the conversion of its physiologic substrate GSSG into GSH.Each monomer of the dimeric mammalian flavoprotein GR is composed by four structural domains: the flavin adenine dinucleotide (FAD) as prosthetic group, NADPH, central and interface domains. The highly reactive site thiols Cys58 and Cys63 constitute a reversible redox system and act as part of the active centre. Thus, these thiols provide GR with a profound reductive capacity to convert its physiological substrate GSSG into its reduced form GSHIn our experimental set up, however, we observed an interesting phenomenon: Whereas control samples (lysates of the DMSO-treated erythrocytes) and all other inhibitor-treated samples showed an increased GR activity (at least in tendency) after addition of FAD , the samin vitro, only 8 compounds were capable to inhibit more than 50% of the G6PDH activity at concentrations <100 μMTargeting G6PDH is a pharmacological meaningful approach to treat a variety of pathologic conditions including cancer and parasitic infections. Recently, approximately three million commercially available substances have been scrutinized by various established approaches regarding their putative inhibitory effects on the G6PDH activity. Out of the selected 250 potential candidates studied in vivo moieties. Due to high doses that are given orally (120 to 240 mg DMF per tablet) high local concentrations can be assumed after release in the gut lumen. Due to high lipophilicity DMF can penetrate into the mucosa and may affect immune cells and red blood cells in the local vasculature. Unfortunately, there is no published literature about local DMF concentration in the small intestine neither in animals nor in man. Parthenolide, a naturally occurring sesquiterpene lactone exhibits broad-spectrum anti-cancer activities and has already been tested in cancer clinical trials  salts) is registered for treatment of moderate-to-severe psoriasis since 1984 in Germany, while in recent years, Tecfidera was approved in EU and US for treatment of multiple sclerosis. In both inflammatory diseases, the exact mechanisms of action of DMF have not been conclusively deciphered yet, even though the roles of GSH depletion and the switch of different immune cells have been demonstrated2●−)2●− 2●− generating enzymes such as NADPH-dependent oxidases (NOXs) which are major sources of O2●− production in human myocardium2●− concentrations causes apoptosis of myocardial cells (for review see2●− is metabolised to other reactive oxygen species (i.e. hydrogen peroxide) by superoxide dismutase .The inhibitory effects of these compounds are not restricted to the targets described above, but they may exhibit multifunctional properties, among them the inhibition of the NFκB signalling pathway involved in several inflammatory and survival processes2942At this moment, we are not able to explain the underlying mechanisms how Bay 11–7082, parthenolide or DMF inhibit the activity of the lipogenic enzyme G6PDH. One explanation might be that these compounds influence the upstream regulators of G6PDH phosphorylation and/or dephosphorylation. Numerous studies have already shown the stimulatory or inhibitory effects of phosphorylation on G6PDH activity44Human erythrocytes possess a highly active GSH synthesis machinery. Apart from liverin vivoi to S-nitrosoglutathione (GSNO), thus depleting [GSH]i Mature erythrocytes possess functional eNOS that modulates vascular nitric oxide (NO) signalling Bay 11–7082-, parthenolide- or DMF-mediated G6PDH inhibition  may causfalciparum-infected erythrocytes (it is 78 times higher than that in uninfected erythrocytes) can be tremendously diminished thereby leading to early death of the parasites , whose progression strongly depends on the bioavailability of NADPH and GSH .In summary, our study, which to our knowledge is the first of its kind, provides evidence that the susceptibility of erythrocytes to eryptosis mediated by Bay 11–7082, parthenolide or dimethyl fumarate (DMF), is partially based on the inhibition of G6PDH and GR activities.Highly purified erythrocyte suspensions from healthy volunteers with white blood cell (WBC) or thrombocyte contaminations below 0.1%4, 32 N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid (HEPES)/NaOH (pH 7.4), 5 glucose, and 1 CaCl2. Annexin-binding buffer contained (in mM): 125 NaCl, 10 HEPES/NaOH (pH 7.4), and 5 CaCl2. Where indicated, Bay 11–7082 (1–20 μM), parthenolide (1–50 μM) and dimethyl fumarate  were added. Dimethyl sulfoxide (DMSO)-treated erythrocytes served as solvent controls (0.2% (v/v) DMSO). Bay 11–7082, parthenolide, DMF, N-ethylmaleimide (NEM), glutathione reductase (GR) and glutathione disulfide (GSSG) were purchased from Sigma . Annexin V-FLUOS was purchased from Roche Diagnostics . 10 mg Bay 11–7082 and 5 mg parthenolide were dissolved in 965 μl and 400 μl DMSO, respectively to achieve a 50 mM stock solution. These stocks were aliquoted and stored at −20 °C for up to 3 months. 70 mM DMF stock solution (100 mg dissolved in 9.9 ml DMSO) was freshly prepared. All required solutions were freshly prepared immediately before use.Experiments analysing the suicidal death of erythrocytes (0.4% Hct), determinations of glutathione content, MCV and MCHC values, as well as measurements of enzyme activities (0.6% Hct), were carried out in Ringer solution in a total volume of 2, 30, 30, or 13 ml, respectively. Ringer solution was composed of (in mM): 125 NaCl, 5 KCl, 1.2 MgSO6 RBCs were washed in 500 μl Annexin-binding buffer. RBC pellets were then vortexed gently to get a homogenous cell suspension. To detect phosphatidylserine (PS) on the outer leaflet of the plasma membrane, these cells were subsequently stained with 32 μl Annexin V-FLUOS at a 1:33 dilution and mixed gently on a vortex mixer. After 20 min incubation in the dark at room temperature, 200 μl of Annexin-binding buffer was added to each sample, thoroughly vortexed to achieve single cell suspensions, and analysed by flow cytometry on a FACS-Calibur . Cell volume was determined by forward scatter (FSC), and Annexin V-FLUOS binding was measured in the FL1-channel.Erythrocyte concentrates suspended in 2 ml Ringer solution (0.4% Hct) were treated in the absence or presence of Bay 11–7082, parthenolide or DMF for 24 h or 48 h. After incubation, 2·107 RBCs were centrifuged for 4 min at 420 g, 4 °C, and the supernatants were harvested. To quantify hemolysis, the hemoglobin concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of RBCs lysed in distilled water was defined as 100% hemolysis.RBC concentrates (0.4% Hct) were treated with varying concentrations of Bay 11–7082, parthenolide or DMF for 24 h or 48 h, then hemolysis was determined. Briefly, after incubation, 600 μl suspension containing 1.2·10Erythrocyte concentrates (0.6% Hct), suspended in 30 ml Ringer solution were treated with Bay 11–7082 (20 μM), parthenolide (50 μM) and DMF (140 μM) for 24 h, respectively. DMSO (0.2% (v/v))-treated erythrocytes served as negative controls. After incubation, samples were centrifuged at 210 g for 10 min at room temperature in order to obtain RBC pellets. Ionomycin (0.25 μM)-treated erythrocytes served as positive controls, and were first incubated in Ringer solution in the absence of ionomycin for 21.5 h. Subsequently, the cells were treated with ionomycin for 2.5 h, and the MCV and MCHC values were determined. The MCV and MCHC analysis was carried out with an ADVIA 120 Hematology System Instrument .2O. NEM functioned as the best alkylating agent for GSH conjugation to prevent sample manipulation-induced oxidation of GSH. After a 2-min incubation at room temperature, samples were centrifuged at 210 g for 10 min in order to obtain RBC pellets. These were immediately stored at −20 °C until GSH and GSSG analyses were performed. All measurements were carried out within one week after the treatment. GSH and GSSG were measured in the clear supernatant obtained by treatment of 0.1 ml RBCs with 0.12 ml 15% (w/v) trichloroacetic acid. GSH analysis was carried out by high-performance liquid chromatography (HPLC), as recently reported2OErythrocyte concentrates (0.6% hematocrit), suspended in 30 ml Ringer solution were treated with varying concentrations of Bay 11–7082, parthenolide or DMF for 24 h. DMSO-treated erythrocytes served as negative controls. After incubation, erythrocytes were treated with 0.6 mM  NEM prepared as a 310 mM stock in H+ to NADPH in the presence of glucose-6-phosphate (600 μM). The activity of 6PGDH alone was then measured by the conversion of NADP+ to NADPH in the presence of 6-phosphogluconate (600 μM). Finally, G6PDH activity was calculated by subtracting 6PGDH activity from total dehydrogenase activity.RBC concentrates (0.6% hematocrit), suspended in 13 ml Ringer solution were treated with varying concentrations of Bay 11–7082, parthenolide or DMF for 24 h. DMSO-treated erythrocytes served as negative controls. Determinations of the enzymatic activities of G6PDH, 6PGDH and GR (+/− FAD) were performed in leukocytes- and platelets-free erythrocyte lysates according to standard biochemical proceduresData are presented as the mean values ± SEM of at least 3 independent experiments with different blood samples. A total of 18 different blood samples were used in this study. Unless otherwise stated, one-way ANOVA with Dunnet’s post test was used for statistical comparisons of treated samples with controls. Differences of the means were considered to be statistically significant when the calculated p value was less than 0.05 .How to cite this article: Ghashghaeinia, M. et al. Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11–7082, parthenolide and dimethyl fumarate. Sci. Rep.6, 28754; doi: 10.1038/srep28754 (2016).


Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2’,4’-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2’,4’-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 μM, the 2’,4’-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2’,4’-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2’,4’-bridged thymidine. Molecular dynamics (MD) simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD) values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor) as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2’,4’-bridged thymidine suggests the potential of bridged nucleosides as drug candidates.Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2’,4’-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of  DNA replication is an essential process for the proliferation of all forms of life. The importance of the inhibition of DNA replication is hardly understated in modern therapeutic strategy against numerous diseases including viral infections and cancer. One potential venue to inhibit DNA replication is in the active site of DNA polymerase, which can be classified into at least five different families based on amino acid sequence comparison and crystal structure analyses ,2. RegarHerpes simplex virus, binds to the active site of the viral DNA polymerase  is the concentration of substrate, [I] denotes the concentration of inhibitor, Km represents the Michaelis-Menten constant, Ki is the inhibition constant. All experiments were independently conducted at least twice.Data obtained by initial velocities were fit to the rate equation describing a competitive inhibition model (Eq ).v=VmaxTaq DNA polymerase and nucleosides was performed using AutoDock Vina [Taq DNA polymerase structure, a 2.40 Å resolution X-ray structure (PDB code 1TAQ) was used [Molecular docking between ock Vina . For thewas used . For theTaq DNA polymerase was set for configuration in Autodock Vina, which uses Autodock Vina force field (based on Amber force field). The catalytic sites were assigned as flexible residues. The exhaustiveness configuration was set to 8. The results produced by the Autodock Vina showed the binding energies and hydrogen bonding interactions between the protein and nucleosides. The structure with lowest energy is chosen for computing intermolecular binding energies. In the case of T7 DNA polymerase (PDB code 1SKR), a grid box of 30 Å × 30 Å × 30 Å with 1.0 Å grid spacing centered on x-y-z coordinates  near the potential binding pocket was set for configuration in Autodock Vina; in the case of HIV-1 RT (human immunodeficiency virus-1 reverse transcriptase), a grid box of 30 Å × 30 Å × 30 Å with 1.0 Å grid spacing centered on x-y-z coordinates  near the potential binding pocket was set. All experiments were performed in triplicate.A grid box of 25 Å × 25 Å × 25 Å with 1.0 Å grid spacing centered on x-y-z coordinates  near the potential binding pocket of + counterions by replacing water molecules and were added with 0.05 M KCl. The energy of the structures using the steepest descent approach was minimized with a tolerance of 1000 kJ/mol to avoid high energy interactions and steric clashes. The energy minimized system was treated for 100 ps in an equilibration run, and 1000 ps MD simulations were carried out with a time-step of 2fs at the NPT canonical ensemble and the periodic boundary conditions were used in all three dimensions. The MD simulations were run in a temperature bath at 300 K with a constant pressure of 1 bar using the V-rescale method for temperature and the Parrinello-Rahman method for pressure [Molecular dynamics (MD) simulations were carried out using the GROMACS 5.0.4 package with AMBER99SB force field ,30,31. Tpressure ,36. The pressure ,38 and ppressure . Root meTaq DNA polymerase and 100 μM of nucleosides showed that only two nucleosides, 2',4'-bridged 5-methylcytidine and 2',4'-bridged thymidine, suppressed the amplification of PCR products as shown in Taq DNA polymerase, the concentrations of the two nucleosides were varied from 100 μM to 0.1 μM. In the case of 2',4'-bridged 5-methylcytidine, the 100 μM sample exhibited, as was seen in the initial testing, effective inhibition (no amplification was detected), but the 10 μM sample elicited a marginal inhibition  obtained by the TaqMan method  of the atomic positions was analyzed. The analysis of the binding mode of the ligand obtained after MD simulation showed an approximate 40° rotation counter clockwise of the ligand with an approximate 45° rotation clockwise of the sugar moiety with respect to the initial state . Before Taq DNA polymerase focused on modified nucleosides containing a bridge on the sugar moiety. As a result, the compound 2’,4’-bridged thymidine was found to effectively inhibit the polymerase in a competitive fashion. The observation could possibly spark a great deal of interest in such bridged nucleosides as lead compounds. In addition, a novel assay method was used to determine kinetic parameters using qPCR. Particularly, for the initial inhibition tests, the use of the SYBR Green dye to probe the inhibition possibility and the subsequent use of TaqMan to gain the information about kinetic parameters combine to facilitate a facile method of study for any Taq DNA polymerase inhibition experimentation. Docking analysis has provided the insight into the initial docking by the ligand 2’,4’-bridged thymidine to the active site of the enzyme. The fact that the Asp610 residue was a unique binding site for 2’,4’-bridged thymidine compared to other nucleosides docking sites provides an idea that the residue appears to be a critical binding site. The MD simulations supported the idea that the ligand was still stabilized inside the active pocket. The hydrogen bond between the residue Arg587 of the enzyme and the ligand appeared to be the most important anchoring and pivotal interaction, allowing the ligand to twist into its most stable state. From these observations, we can conclude that the in silico analyses not only soundly supported the experimental data but also provided indispensible knowledge of how the inhibitor binds to the enzyme.Effort to find compounds that would inhibit S1 FigA primer extension assay was carried out at the 2’4’-bridged thymidine concentrations of 0.01 μM (A), 10 μM (B), 50 μM (C), and 500 μM (D).(TIFF)Click here for additional data file.S2 FigThe line colored in yellow represents 500 μM 2',4'-bridged thymidine; the line colored in pink denotes 10 μM 2',4'-bridged thymidine; the lines colored in purple are 1 μM (bottom) and 0.01 μM (top) 2',4'-bridged thymidine compounds.(TIFF)Click here for additional data file.S3 FigA primer extension assay was carried out with ddCTP concentrations of 0.01 μM (A), 250 μM (B), 500 μM (C), and 2.5 mM (D).(TIFF)Click here for additional data file.S4 FigThe line colored in purple represents 2500 μM ddCTP; the line colored in red denotes 500 μM ddCTP; the lines colored in green are 1 μM (bottom) and 0.01 μM (top) ddCTP.(TIFF)Click here for additional data file.


Using these data, together with other δ13C and δ18O records from the Atlantic, we assess the impact of the early Pliocene CAS shoaling phase on deep-water circulation. We find that NADW formation was vigorous prior to 4.7 Ma and showed limited subsequent change. Hence, the overall structure of the deep Atlantic was largely unaffected by the early Pliocene CAS shoaling, corroborating other evidence that indicates larger changes in NADW resulted from earlier and deeper shoaling phases. This finding implies that the early Pliocene shoaling of the CAS had no profound impact on the evolution of climate.The early Pliocene shoaling of the Central American Seaway (CAS), ~4.7–4.2 million years ago (mega annum-Ma), is thought to have strengthened Atlantic Meridional Overturning Circulation (AMOC). The associated increase in northward flux of heat and moisture may have significantly influenced the evolution of Pliocene climate. While some evidence for the predicted increase in North Atlantic Deep Water (NADW) formation exists in the Caribbean and Western Atlantic, similar evidence is missing in the wider Atlantic. Here, we present stable carbon (δ Changes in major ocean-gateways during the late Neogene are considered to have had far-reaching impacts on the evolution of climate, mediated through large-scale changes in ocean circulation. Notably, it has been proposed that the progressive closure of the CAS, which occurred during the Pliocene223569Elucidating the timing and nature of the CAS closure is crucial for evaluating its impact on AMOC and climate. However, geological records reveal it as a complex process, with many details remaining unclear and contested1112131415171918O-gradient between the Pacific and Caribbean Sea  Sites 10883437et al.40In order to better assess early Pliocene changes in AMOC, we present benthic stable oxygen and carbon isotope data from ODP Site 1264 , recently published by Bell 18O and δ13C data, together with published data from a number of other sites in the wider Atlantic  Atlantice.g.4613C signature than deep-waters originating from the south18O values are less straightforward to predict: it is possible that an increase in AMOC will be accompanied by an increase in δ18O, reflecting higher salinities and a low-latitude δ18Oseawater signature18O during the equilibrium precipitation of calcite and/or local meltwater enriched in 16O from the north.Benthic foraminiferal δ13C and δ18O data from all Atlantic sites are presented in 18O stack of Lisiecki and RaymoTimeseries of benthic δ13C records generally record high values, averaging between 0.5–1.0‰, directly comparable to values in the modern ocean . On the other hand, Sites 704 and 999 record low average values (~0–0.5‰) in early Pliocene, with Site 999 displaying a long-term increase from values averaging ~0.2‰ at the start of the Pliocene, rising to an average of ~0.65‰ by 3.7–3.6 Ma.Early Pliocene Atlantic δrn ocean , and sho18O records show a spread in average values similar in range to δ13C records. Sites 1264 and 704 have consistently higher δ18O values than the LR04 stack, while Sites 929 and 982 have consistently lower values. Long-term trends are generally small, with the notable exception of Site 925, which shows a large increase in δ18O at ~4.7 Ma.Atlantic δ18O-δ13C anomalies for each site calculated by subtracting the mean ocean δ18O-δ13C changes, as recorded by ODP Site 849 in the deep Pacificp = < 0.05 for student t-tests) in δ13C is only seen at Site 999 (Δδ13C = +0.18‰). δ13C changes in the remaining sites are small and not significant. Meanwhile, a significant and prominent shift in δ18O of +0.28‰ is observed for Site 925.13C increase at Site 999, together with a concomitant increase in carbonate preservationThe δ13C-gradients gradients are expected to be sensitive to latitudinal movements in the extent of NADW, and hence reflect the strength of NADW formation. Throughout the interval studied, and with no apparent change in response to CAS shoaling, Site 1264–704 δ13C-gradients varied in the range of ~0.2–0.6‰, although sparse data from Site 704 indicate short periods of lower gradients. This is similar to the modern δ13C-gradient for this region, which is spatially variable and in the range of ~0.2–0.4‰13C–gradients between Site 1264 and sites in the North Atlantic are also similar to the modern situation, with the exception of Site 982–1264 gradients, which are even lower. Therefore, these observations provide a convincing basis for inferring that NADW formation during the early Pliocene was generally strong, with periods possibly stronger than today, and was seemingly unaffected by the CAS shoaling phase.Data from Site 1264 provide a reference for Site 704, forming a latitudinal transect (~28–47°S) of the Southeast Atlantic at ~2500 m depth. Since this region and depth is situated along a major export pathway for NADW13C data from Site 704 in order to reconstruct the relative strength of AMOC for intervals that encompass the early Pliocene355213C changes, these studies speculated that NADW formation was already strong during the late Miocene, from ~6–7 Ma onwards, and that the early Pliocene progressive shoaling of the CAS (~4.7–4.2 Ma) had little impact. Similar conclusions were made with data from Site 108813C-gradient within the South Atlantic; a finding which is independent of remaining uncertainties in Southern Ocean ventilation e.g.Previous studies have utilized δ13C-signal penetrating into the South Atlantic, we are able to quantitatively estimate the relative contribution of NADW versus SSW to waters bathing Site 1264. For a given site (x), this may be calculated using the equation of Oppo and FairbanksThus, given the confirmation of a northern δ13CSSW is the end-member carbon isotopic value for SSW, δ13CNADW is the end-member carbon isotopic value for NADW, and δ13Cx is the carbon isotopic value at site x in the Atlantic. For Site 1264, the calculation yields values of ~70%, both before and after the CAS shoaling phase (4.7–4.2 Ma), using NADW and SSW end-member values of Sites 607 and 704, respectively. By comparison, based on phosphate measurementsThe δ18O-gradient between Sites 925 and 607  , an abrupt increase in % sand fraction variability is seen after ~4.6 Ma3  was restricted to a strengthening of upper NADW formation and an altered flow regime of the DWBC, sedimentary evidence of increased carbonate preservation suggests a further increase in the prevalence of low-corrosive NADW. An increase in the sedimentary carbonate fraction from ~0% to ~40% is seen during the early Pliocene in the eastern North Atlantic Site 665, 4755 m.~4.6 Ma3 , likely ~4.6 Ma3 , it is coS, suggesting that NADW formation was active despite the open gateway. Low δ13C-gradients between various sites across the Atlantic indicate that deep-water circulation was rigorous, comparable to and frequently stronger than today. In particular, our new data confirm the dominance of NADW in the Southeast Atlantic. However, upper-NADW formation in the Labrador Sea was reduced and, taking changes in δ18O data at face value, deep-waters within the DWBC were distinct from those filling the wider Atlantic, implying limited mixing with deep-waters in the basin interior, possibly due to low flow speeds.The early Pliocene progressive CAS shoaling phase had a limited effect on the overall geometry of Atlantic deep-waters . This ise.g.3435465052We conclude that the lack of change in large parts of the Atlantic Ocean demonstrates that the early Pliocene shoaling phase of the CAS had little impact on AMOC and, therefore, climate. Our data corroborate data and modeling studies that propose significant NADW flow existed with an open CAS3 samples were taken from Site 1264 cores at 1.5 cm intervals, using the shipboard splice to create a meters composite depth (mcd) scale. For the large majority of Site 1264 samples (30.01–57.45 mcd = 3.715–5.300 Ma), processing and measurements were carried out at the University of Florida. Benthic species, Cibicidoides wuellerstorfi, were picked from the >150 μm size fraction. The foraminifera were then cleaned in an ultrasonic bath to remove fine-grained particles and soaked in 15% H2O2 to remove surface organic contaminants prior to analysis. The number of specimens of C. wuellerstorfi varied from 1 to 4 and foraminiferal tests were crushed before analysis. The foraminiferal calcite was reacted in 70 oC orthophosphoric acid using a Kiel III carbonate preparation device. Evolved CO2 gas was measured online with a Finnigan-MAT 252 mass spectrometer.10 cmC. wuellerstorfi, selected from the >200 μm size fraction, using a Finnigan 251 Gas Source mass spectrometer equipped with Kiel type automated carbonate extraction line.For samples between 27.80–30.01 mcd (=3.600–3.715 Ma), samples were processed and measured at Vrije Universiteit, Amsterdam. Measurements were performed on 1–3 18O and δ13C is better than ±0.1‰. For all sites assessed, δ18O values are adjusted by +0.64‰ to account for disequilibrium with surrounding seawaterAll stable isotope results are reported relative to Vienna Pee Dee Belemnite and calibrated using in house standards that are correlated to the international standard, NBS19. Analytical precision for both δ18O records, generated by pooling data from different labs. Indeed, our experience from a small number of duplicate measurements taken between different laboratories indicates potential offsets of up to 0.1–0.2‰, in line with previous findings of up to 0.3‰C. wuellerstorfi specimens, such offsets are difficult to correct for. Hence, we are aware of potential offsets of <0.3‰.Systematic inter-laboratory offsets are a possible artifact in δ18O time series to the global benthic δ18O stack of Lisiecki and Raymoet al.All age models used in this study were derived from the alignment of δHow to cite this article: Bell, D. B. et al. Atlantic Deep-water Response to the Early Pliocene Shoaling of the Central American Seaway. Sci. Rep. 5, 12252; doi: 10.1038/srep12252 (2015).


Chronic myeloid leukemia (CML) is driven by the expression of the BCR-ABL oncoprotein. STAT5 is a BCR-ABL substrate and persistently activated by tyrosine phosphorylation in CML cells. Activated STAT5 (pSTAT5) drives proliferation and survival of leukemic cells and contributes to initial transformation and maintenance of the disease. In cytokine-induced STAT5 signaling, phosphorylation of STAT5A on Y694 leads to nuclear accumulation of the transcription factor, followed by DNA-binding and gene induction. However, Src-family kinases (SFK) mediate cytoplasmic retention of pSTAT5A leading to attenuated target gene expression and colony formation in CML cells.Y694 phosphorylation by SFK, but at the same time promotes the persistent cytoplasmic localization of the transcription factor as found in BCR-ABL+ leukemia. As a consequence of the complex formation between tyrosine-phosphorylated SFK and the SH2 domain of STAT5A, the dimerization of STAT5A is impaired. We further demonstrate that constitutively active STAT5AS710F escapes from SFK-mediated cytoplasmic retention by enhancing STAT5A dimer stability.In this study we show that autophosphorylation of Y416 in the highly conserved activation loop of SFK generates a potent recruitment site for the SH2 domain of STAT5A. Binding of the SH2 domain to the activation loop is required for STAT5AOur results reveal important structural aspects of cytoplasmic pSTAT5A found in myeloid leukemias and will contribute to the understanding of STAT5A mediated cytoplasmic signaling. Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that transmit signals from membrane-bound surface receptors to the nucleus. STAT proteins regulate proliferation, differentiation and apoptosis, involved in diverse biological processes such as hematopoiesis, tissue remodeling, immune responses and inflammation .The STAT family is composed of seven members  that share a structurally related and conserved domain architecture: The N-terminal multimerization domain, a coiled-coil domain, followed by the DNA-binding domain (DB), a linker domain (L), a single Src homology domain (SH2) and the transactivation domain (TA). In canonical Janus kinase (JAK)/STAT signaling, phosphorylated tyrosine residues in the cytoplasmic part of activated receptors serve as docking sites for SH2 domain containing proteins including STATs. Activation of STAT family members is mediated by the phosphorylation of a single conserved tyrosine residue, which results in dimerization via reciprocal phosphotyrosine-SH2 domain interactions, subsequent nuclear accumulation, DNA-binding and target gene expression [c) receptors [STAT5 is of central importance for hematopoiesis in health and disease. Lymphocyte development is regulated by a variety of cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, which activate the JAK3/STAT5 signaling pathway through common γ chain , essential thrombocythemia , systemic mastocytosis , acute myeloid leukemia  and chronic myeloid leukemia  [In contrast to physiological transient signaling, persistent STAT5 activation is a hallmark of many hematological malignancies including myeloproliferative neoplasms (MPNs) such as polycythemia vera  . DespiteBCR-ABL) . ActivatBCR-ABL) ,12.S710F) was found in a complex with Gab2, which promotes the cytoplasmic localization of the persistently phosphorylated transcription factor in myeloid leukemias [Mounting evidence suggests that STAT5A-mediated prosurvival signaling emerges from the cytoplasmic compartment, based on a cooperative crosstalk with the PI3-K/Akt signaling pathway. While mechanistic details of this crosstalk remain elusive, constitutively active STAT5A  resistance in CML cells -16. MoreIn this study, we provide evidence that the STAT5A SH2 domain tightly binds to the phosphorylated Y416 in the activation loop of SFK, resulting in the cytoplasmic localization and an impaired dimerization of the transcription factor. These processes are dominant over the BCR-ABL induced nuclear accumulation of STAT5A and therefore give a rational explanation for the cytoplasmic localization of STAT5A in a number of myeloid leukemias.In order to analyze STAT5A nucleocytoplasmic transport dynamics, we evaluated the subcellular distribution of fluorescently labeled STAT5A Figure A in an eR618Q-eYFP, which harbors an inactivating mutation in the SH2 domain, in the presence of vSrc-dsRed in HeLa T-REx FRT cells   experiments in HeLa T-REx FRT cells. Co-expression of vSrc-dsRed and STAT5A-eYFP resulted in a robust interaction of both proteins and subsequent phosphorylation of STAT5Ad Figure E which iY694 phosphorylation, we analyzed Src kinase tyrosine to phenylalanine mutants to identify the phosphorylation site that facilitates STAT5A recruitment  and SH2 (R175L) domains of vSrc-dsRed had no impact on STAT5AY694 phosphorylation levels independent of the Src kinase mutant used in this experiment  compared to non-transfected cells   or have been described to be phosphorylated .To support this idea, we studied the interaction between STAT5A-eYFP and two kinase activity impairing vSrc-dsRed mutants. These mutants are characterized by either the absence (Y416F) or a drastically reduced K295N) phosphorylation of Y416 in the activation loop , Yes and Fyn  nor vSrcY416F-dsRed (lower panel) was capable of preventing the nuclear accumulation of STAT5A-eYFP in these cells. Consistently, the co-expression of BCR-ABL and kinase dead vSrcK295N-dsRed did not alter the phosphorylation status of Y416, demonstrating that the activation loop of Src kinases is not phosphorylated by BCR-ABL  and Src kinases, we expressed STAT5A-FLAG in doxycycline treated HeLa T-REx HA-EpoR and HeLa T-REx vSrc-dsRed cells, which were stably transfected with STAT5A-eYFP. EpoR expressing cells were either treated with Epo to induce STAT5A activation or were left untreated  to mimic a constitutive serine phosphorylation or to alanine (STAT5AS710A) to prevent serine phosphorylation. The subcellular distribution of these mutants was analyzed in the presence of vSrc-dsRed in HeLa T-REx FRT cells by confocal microscopy. In contrast to STAT5AS710F-eYFP, STAT5AS710D-eYFP and STAT5AS710A-eYFP predominantly resided in the cytoplasm of vSrc-dsRed expressing cells, suggesting that the hydrophobic properties of phenylalanine at this position are of importance for the dimer stabilization of STAT5AS710F.These results are substantiated by the fact that STAT5AConclusively, our results indicate that binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK leads to a redistribution of the activated transcription factor from the nucleus to the cytoplasm, which is accompanied by impaired STAT5A dimer formation  [STAT5 signaling is implicated in proliferation, survival and myeloid differentiation of hematopoietic cells. Aberrant STAT5 activity is linked to the expression of dysregulated upstream tyrosine kinases such as BCR-ABL, JAK2s (MPNs) .D816V positive neoplastic mast cells [In CML, STAT5 represents a key signaling node since pSTAT5 is essential for the proliferation and survival of leukemic cells and contributes to both the initial transformation and maintenance of the disease . In cytost cells ,19,30. Gst cells ,31-35.in-vitro kinase assays, providing strong evidence for a direct interaction, which is further substantiated by the co-localization of pSTAT5 with constitutively active Hck in podosomes [Most recently, we identified Src family kinases (SFK) to be involved in mediating the cytoplasmic retention of pSTAT5A in BCR-ABL expressing cells, a process that involves the SH2 domain of STAT5A. As a consequence, STAT5A target gene expression was shown to be attenuated, which could be rescued through the specific inhibition of SFK . Furtherodosomes ,36.R618Q-eYFP with the SFK members Hck-dsRed and vSrc-dsRed. We confirmed the observation that the SH2 domain of STAT5A is involved in the formation of a stable complex with both SFK, which contributes to the cytoplasmic localization of pSTAT5A. Moreover, phosphorylation of STAT5AY694 by SFK requires an intact STAT5A SH2 domain, which supports the idea of an exceptional interaction between the kinase and its substrate. Interestingly, the inactivating mutation R618Q in the SH2 domain of STAT5A did not result in a complete loss in binding to SFK, which indicates that multiple domains contribute to the interaction.However, the role of the STAT5A SH2 domain in this context is still unresolved. In order to clarify the mechanism underlying the Src kinase mediated cytoplasmic retention of STAT5A, we co-expressed STAT5A-eYFP or STAT5AIn line with this concept, the SFK mediated activation of the STAT family members STAT3 and STAT5B was shown to be largely independent of a functional SH2 domain (data not shown) . ConsistFurthermore, the specific knockdown of STAT5B, but not STAT5A, was shown to be associated with a loss of CML cell proliferation. In the context of BCR-ABL signaling, stress protection through the regulation of reactive oxygen species could be attributed to STAT5A functions independent of its transcriptional activity, suggesting a cytoplasmic role of pSTAT5A in this context . In contY416F-dsRed, which lacks the phosphorylation site in the activation loop, resulted in a decreased STAT5AY694 phosphorylation. This observation is not surprising, since the Y416F mutation negatively affects kinase activity. However, subsequent interaction studies revealed that binding of STAT5A to vSrcY416F-dsRed and vSrcK295N-dsRed is significantly reduced compared to vSrc-dsRed, which correlates with a substantial loss of Y416 phosphorylation and a decreased capacity to induce the cytoplasmic localization of STAT5A. In addition, STAT5A could be successfully precipitated with a phosphorylated peptide corresponding to the sequence of the activation loop of SFK in a SH2 domain dependent fashion. However, our experiments also demonstrate that the binding of STAT5A to SFK is not limited to phosphotyrosine-SH2 domain interactions, which has also been shown for STAT5/Hck complexes in BCR-ABL transformed haematopoietic cells and TEL-ABL expressing Ba/F3 cells [In order to further characterize the SFK/STAT5A protein interaction and its contribution to the cytoplasmic localization of pSTAT5A, tyrosine to phenylalanine mutations were introduced into vSrc-dsRed. Out of seven Y to F mutations only the expression of vSrcF3 cells ,44.K295N and absent in vSrcY416F, is required for the SFK induced cytoplasmic localization of STAT5A in the presence of BCR-ABL. Taken into account that the SFK members Hck and Lyn are typically expressed in myeloid cells and are constitutively activated by the p210 isoform of BCR-ABL, it is tempting to speculate that activated SFK contribute to the persistent cytoplasmic localization of pSTAT5A observed in primary CD34+ CML cells [Accordingly, our findings suggest that phosphorylation of the activation loop, which is drastically reduced in kinase dead vSrcML cells ,17,19,45Having shown that SFK/STAT5A protein complexes are stabilized by a phosphotyrosine-SH2 domain mediated interaction, which is accompanied by a lack of nuclear accumulation, we wondered whether this interaction affects STAT5A dimerization. Native gels revealed that the dimer formation of STAT5A in response to activation through SFK is significantly reduced compared to Epo stimulation, despite equal phosphorylation levels. Interestingly, our unpublished data suggest that in response to SFK, STAT5A is also phosphorylated at residues different from Y694, which has also been reported for STAT5B . Taken iS710F but also STAT5AS710A to rescue STAT5A−/− T cell proliferation, only STAT5AS710F was shown to result in the induction of leukemia upon expression in the bone marrow of wild-type and STAT5−/− mice, demonstrating that the hydrophobic properties of phenylalanine are required for the oncogenic potential of STAT5AS710F. However, a detailed understanding of the mechanism rendering STAT5AS710F constitutively active is lacking.The role of constitutive STAT5 activation and its contribution to the development of blood malignancies has been intensively investigated by the use of STAT5 activating mutations. A single point mutation in the transactivation domain (S710F) renders STAT5A constitutively active and promotes factor independent growth of Ba/F3 cells . DespiteS710F-eYFP, which is in line with the observation that STAT5AS710F-eYFP escapes from the SFK mediated cytoplasmic retention and predominantly localizes to the nuclear compartment. Phosphorylation of S710 is most likely not involved in the regulation of the subcellular distribution of STAT5A, since mutants designed to impair (STAT5AS710A) or to mimic (STAT5AS710D) serine phosphorylation could not rescue the SFK mediated block in nuclear accumulation of STAT5A. Conceivably, our data suggest that the oncogenic properties of STAT5AS710F are a consequence of increased dimer stability that either interferes with the deacetylation/sumoylation cycle, which is involved in STAT5 inactivation, or impairs the recruitment of STAT5 phosphatases such as SHP-2 and PTP1B [SFK failed to interfere with the dimerization of STAT5And PTP1B -49.+ cells, which is accompanied by an impaired dimerization. The oncogenic STAT5AS710F mutant escapes from cytoplasmic retention through SFK as a consequence of increased dimer stability. These results reveal important structural aspects of the cytoplasmic localization of pSTAT5A in BCR-ABL expressing cells and will contribute to our understanding of STAT5A mediated cytoplasmic signaling.Our findings identify Y416 in the conserved activation loop of SFK as a recruitment site for the SH2 domain of STAT5A. The phosphotyrosine-SH2 domain interaction is required for the activation of STAT5A. At the same time, this interaction promotes the cytoplasmic localization of the transcription factor in BCR-ABL694/699-STAT5A/B (#9351), anti-pY416-Src (#2101), anti-pY412-Abl (#2865), anti-Hsp70 , anti-STAT5A , Vienna, Austria), anti-GFP , anti-FLAG (F3165), anti-α-tubulin , anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc , anti-vSrc  and anti-HA  antibodies were used for immunoblotting. The anti-pY416-Src antibody was used to detect phosphorylated Y390 of Hck. Anti-rabbit, anti-mouse and anti-goat antibodies conjugated to horseradish peroxidase (HRP) were ordered from DAKO .Erythropoietin  was used at a concentration of 1 or 5 U/ml. Anti-pYThe cDNA of murine STAT5A was either cloned into the pcDNA3.1, pcDNA5/FRT/TO  or pLentiLox  expression vectors and fused to the cDNA of the enhanced yellow fluorescent protein (eYFP) or the FLAG-tag (N’-DYKDDDDK-C’). The cDNA of vSrc and human Hck  was fused to monomeric dsRed and cloned into pcDNA3.1 or pcDNA5/FRT/TO expression vectors. The cDNA of BCR-ABL  was cloned into the pcDNA5/FRT/TO expression vector. The cDNA of the human erythropoietin receptor (EpoR) was cloned into the pcDNA5/FRT/TO expression vector and fused to the cDNA of the hemagglutinin tag (HA). Fluorescent proteins and the FLAG-tag were fused to the C-terminus of target proteins, whereas the HA-tag was fused to the N-terminus of the human EpoR. Point mutants of STAT5A, Hck and vSrc were generated with the PCR based QuikChange XL Site-Directed Mutagenesis Kit according to manufacturer’s instructions .TransIT®-LT1  according to manufacturer’s instructions. Stable transfection of HeLa T-REx cells for the inducible expression of BCR-ABL, HA-EpoR, vSrc-dsRed and Hck-dsRed were generated with the Flp-In system  using 250 μg/ml hygromycin B  and 15 μg/ml blasticidin  for selection. Non-inducible HeLa T-REx FRT cells were maintained in DMEM medium supplemented with 200 μg/ml Zeocin  and 15 μg/ml blasticidin. The expression of the gene of interest was induced with 5 ng/ml doxycycline  for 24 h or as indicated. All media were supplemented with 10% FCS  and 25 U/ml penicillin/streptomycin . Cells were incubated at 37°C in a water-saturated atmosphere with 5% CO2.HeLa T-REx  cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/GlutaMAX . Transient transfection of HeLa T-REx cells was performed using S710F-eYFP were cloned into the lentiviral pLentiLox expression vector downstream of the CMV promoter . HEK293-T packaging cells were seeded on 6-cm dishes on the first day to be 80-90% confluent at the time of transfection. On the second day, HEK293-T cells were transiently co-transfected with the lentiviral vector, psPAX2 (packaging plasmid) and pMD2.G (envelope plasmid). Eighteen hours after transfection the culture medium was replaced by high serum growth medium containing 30% FCS. Viruses were harvested 24 and 48 hours after medium exchange. For the infection of target cells, virus-containing supernatant was supplemented with 8 μg/ml polybrene  prior to incubation. For sorting by fluorescence-activated cell sorter analysis (FACS), cells were grown for 10 days and sorted by eYFP gating.To achieve stable and constitutive expression of STAT5A constructs in inducible HeLa T-REx cells, self-inactivating lentiviral constructs were used according to the guidelines of the RNAi consortium. Briefly, the cDNA of STAT5A-eYFP and STAT5A2HPO4, 1.5 mM KH2PO4, adjusted to pH 7.4) once and lysed with RIPA lysis buffer  supplemented with phosphatase/protease inhibitors , 5 μg/ml aprotinin, 2.5 μg/ml leupeptin) according to standard procedures. The proteins were separated by SDS-PAGE and transferred to a PVDF membrane with subsequent immunodectection using specific antibodies. Primary and HRP-conjugated secondary antibodies were diluted in TBS-N buffer . Membrane-bound antibody complexes were detected by chemiluminescence .HeLa T-REx cells were washed with PBS  supplemented with 2% digitonin . The lysates were cleared by centrifugation and incubated with Coomassie brilliant blue G-250 and separated over night at 4°C using the NativePAGE™ Bis-Tris gel system with a gradient polyacrylamide gel (4-16%) according to manufacturer’s instructions. Dimerization of STAT5A-eYFP was analyzed by the detection of the eYFP fluorescence with a typhoon fluorescence scanner  by excitation with a 488 nm laser line. The emission was detected using a 515–555 nm bandpass filter.R618Q-eYFP were lysed with storage buffer  supplemented with phosphatase/protease inhibitors. Lysates were cleared by ultracentrifugation . Biotinylated peptides corresponding to the activation loop sequence of Src family kinases (N’-RLIEDNE(p)YTARQGAK-C’) were synthesized with >95% purity . 25 nmol of the peptides were coupled to NeutrAvidin Agarose Resin  for 2 h at 4°C, washed twice with storage buffer and incubated with 1 mg of the lysate over night at 4°C. Peptide/protein complexes were washed 3 times with storage buffer for 10 minutes and the precipitates were analyzed by SDS-PAGE and immunoblotting.HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5AHeLa T-REx cells were lysed with RIPA lysis buffer  supplemented with phosphatase/protease inhibitors and the lysates were cleared by ultracentrifugation . Protein G Sepharose  was coupled to 1 μg anti-GFP antibody for 16 h at 4°C, washed twice with RIPA buffer and subsequently incubated with 1 mg of lysates over night at 4°C. Antibody/protein complexes were washed 3 times with RIPA buffer for 10 minutes and precipitates were analyzed by SDS-PAGE and immunoblotting.HeLa T-REx cells expressing fluorescently labeled fusion proteins were grown on glass cover slips. Methanol fixation was performed at room temperature as has been described previously . Primaryt-test.HeLa T-REx cells expressing STAT3-eYFP and STAT5A-eYFP fusion constructs were fixed as described previously. Tilescan images  of adherent cells were generated with a Zeiss LSM 710 confocal microscope , using the Zeiss LD C-apochromat 40x/1.1 water objective. Tilescan images represent confocal slices of approximately 1 μm. Mean fluorescent intensities (MFI) of the YFP fluorescence in the cytoplasm and nucleus were calculated for double transfected cells using the profile function of the ZEN 2012 software . Relative ratios of nuclear versus cytoplasmic fusion proteins were plotted using Graph Pad Prism 4 software and statistically evaluated by Student’s


ROCK inhibition has been largely applied as a strategy to treat neurodegenerative diseases (NDDs) and promising results have been obtained in the recent years. However, the underlying molecular and cellular mechanisms are not fully understood and different models have been proposed for neurodegenerative disorders. Here, we aim to review the current knowledge obtained for NDDs identifying common mechanisms as well as disease-specific models. In addition to the role of ROCK in different cell types such as neurons and microglia, we focus on the molecular signaling-pathways which mediate the beneficial effects of ROCK. Besides canonical ROCK signaling, modulation of neighboring pathways by non-canonical ROCK-crosstalk is a recurrent pattern in many NDD-model systems and has been suggested to mediate beneficial effects of ROCK-inhibition. Signaling events downstream of ROCK are responsible for key neuronal processes in axonogenesis, growth cone dynamics, and stability of synapses. ROCK has a number of downstream targets including profilins and cofilin (via LIM kinase) as actin-binding proteins as well as myosin light chain phosphatase (MLCP) Figure . Upstreain vitro and in vivo NDD models. Thus, ROCK inhibition might at least in part exert its beneficial effects on NDDs by rescuing pathomechanistic changes. Thereby, two dimensions of elevated ROCK-activity have to be taken into account: (i) The pathway dimension, and (ii) the cell-type dimension. (i) Canonical ROCK signaling has been extensively studied and associated with the control of actomyosin contractility via phosphorylation of downstream actin-binding proteins such as cofilin or profilin as well as myosin-binding proteins such as myosin light chain phosphatase  In microglia, ROCK-activity is critically involved in neurodegeneration. Despite microglia, other inflammatory cells are likely involved in neurodegeneration. However, in the context of ROCK-inhibition in NDDs microglia have been most extensively studied. Microglia, also termed “macrophages of the nervous system”, have ambiguous roles in NDDs dependent on their activation state  or Fibroblast Growth Factor 1 and 2   has been extensively employed as an MS-animal model. Auto-immunization with myelin-sheet protein components such as myelin, oligodendrocyte glycoprotein (MOG), or Proteolipid Protein (PLP) induces pathological features similar to MS including neuroinflammation, demyelination, and neuronal damage -gene -gene  morphology, and muscle atrophy . PTEN mainly antagonizes the phosphatidylinositol 3 kinase (PI3K) pathway by hydrolyzing the second messenger phosphatidylinositol -triphosphate (PIP3) thereby blocking the recruitment and activation of Akt (reviewed in Song et al., G93A (Synofzik et al., ALS is a motoneuron disease not only affecting lower motoneurons in spinal cord but also leading to upper motoneuron and brainstem degeneration. Clinical symptoms include muscle weakness and paralysis leading to death 3–5 years after onset, which typically develops in advanced adulthood (reviewed in Chen et al., G93A-mice exhibited an enhanced ROCK-activity, elevated PTEN-activity while the Akt-pathway was down-regulated. Moreover, ROCK-inhibition by Fasudil-treatment completely rescued PTEN/Akt activity (Takata et al., G93A-mice (Takata et al., G93A-mice. Moreover, motoneuron degeneration and nerve fibers were partially rescued (Tonges et al., in vitro data (Zhang et al., T109M represents a phospho-site mutation (Ingre et al., Interestingly, SOD1Taken together, ROCK-inhibition ameliorates clinically relevant outcomes in rodent models of several NDDs making it a promising future treatment-strategy. However, with regard to chronic diseases detrimental long-term effects have to be considered highlighting alternative approaches such as non-systemic application routes as well as ROCK-isoform specific inhibition. With regard to the underlying mechanisms, neurons as well as neuroinflammatory cells such as microglia seem to be involved in mediating the beneficial effects of ROCK-inhibition. Genetic models with cell specific ROCK-depletion would greatly improve our understanding of the exact contribution by different cell-types. Moreover, little is known about the ROCK-dependent activation-mechanisms of neuroinflammatory cells on the molecular level. While ROCK-crosstalk with neurotrophic pathways is a relevant mechanism in neurons, such mechanisms have not been demonstrated in microglia yet. At this point, it has to been considered that elucidiation and analyses of single pathways and crosstalking molecules may limit our view on regulatory mechanisms. Instead, future work has to determine important nodes and edges as basic components of a molecular network with ROCK at its center. For example, network analyses could reveal new feedback loops and other network motifs relevant to determine direct and indirect effects of inhibitory drugs. Such a network should include multi-dimensional data on expression as well as on activation states to truly understand the flow of information in physiological and diseased conditions.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.


C. acetobutylicum ATCC 824 gene CA_C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA_C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105.The crystal structure of the protein product of the  Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR. The core structure of GH105 hydrolases are closely related to those of GH88, although they differ in substrate specificity and sequence identity  as a URH  was used for routine growth and cells were propagated in an anaerobic environment as described previously  were harvested as described previously  and 359XhoR (AAGTGTTTCGTATTCGTAAGATGCAAGTAAGCTCGAGGATC) to introduce restriction-enzyme sites for NdeI and XhoI at the putative transcriptional start site and the 3′ end of the gene, also removing the stop codon, respectively. The PCR product was cloned into pTXB1 using standard protocols  cells (New England Biolabs) were transformed with p359-intein using the manufacturer’s protocol. Cultures of transformed cells were grown to an OD600 of 0.6 in 1 l LB Lennox broth supplemented with 50 µg ml−1 ampicillin at 37°C in a shaking incubator and were then transferred to 21°C for 30 min in a shaking incubator. Expression and purification of CA_C0359 protein was performed using previously established protocols  for 24 h at 4°C. The dialyzed protein sample was further purified on a Superdex 75 size-exclusion column  in S-75 buffer. The fractions containing the CA_C0359 protein, as determined by SDS–PAGE, were concentrated via an Amicon Ultra centrifugal device .Partially purified protein was concentrated to 5 ml using an Amicon Ultra centrifugal device  and dialyzed in a MWCO 9K dialysis cassette (Pierce) into 4 l S-75 buffer (150 m2.5.−1) with 2 µl reservoir solution consisting of 0.1 M Tris pH 7.75, 16%(w/v) polyethylene glycol 4000 (Hampton Research) at 21°C using the hanging-drop vapor-diffusion method. Crystals were cryoprotected by soaking in 20% glycerol in reservoir solution, which was followed by liquid-nitrogen flash-cooling. Diffraction data were collected on the NSLS X29a beamline at Brookhaven National Laboratory using an ADSC Quantum 315 CCD detector at a wavelength of 1.075 Å.Native crystals were obtained by mixing 2 µl protein sample . Docking was performed using the SwissDock web service . Growth on galacturonic acid monomers did not induce CA_C0359, indicating that a degradation product of pectin or PGA is required for gene induction. Additionally, a lack of induction during growth on galacturonic acid, arabinose or lactose indicates that CA_0359 is not subject to catabolite repression.Examination of the CAZy database identified eight genes with possible pectin-backbone degradation activity in et al., 2011et al., 2010et al., 1999C. acetobutylicum is capable of subsisting on galacturonic acid monomers and therefore may not have a strict requirement to process polysaccharides intracellularly . When UGL was removed, several solvent-accessible residues in the active pockets were found to be conserved among the five proteins . The conserved residues represented by YteR are Tyr41, Asp88, His132, Trp141, Asp143, Met147, His189, Trp211 and Trp217. These have all been structurally and experimentally shown to be involved in catalytic activity . The protein adopts a six-(α/α)-hairpin barrel fold with a small two-stranded β-sheet and helix overlaying the end of the barrel near the active pocket. The CA_C0359 structure closely adopts the fold of GH105 proteins based on a comparison made by Collén et al. . Conserved residues included the highlighted residues from the active-site pocket of YteR . Of these, Asp130, His176, Asp75 and Tyr25 are also considered to be functionally important for UGL . Analysis of sugar binding within the B. subtilis UGL structure stressed the important role of Trp42 in providing a stabilizing stacking interaction essential in binding the ΔGlcA of ΔGlcA-GalA  comprising residues 320–330 in CA_C0359.Itoh and coworkers postulated the effect of steric hindrance from a protruding loop (gate loop), also seen in CA_C0359, YesR and op Fig. 2a compri3.3.et al., 2006ac). The character of the CA_C0359 active-site pocket is somewhere between that of YteR and UGL. The pocket surrounding the active site of CA_C0359 is lined with aromatic residues in a similar fashion to the UGL active site  may provide some gating action, it is unlikely to completely block access to the active-site pocket. Some docking results demonstrated that some mobile loop residues (including Asp326) may furnish additional positive interactions with the sugar to facilitate attracting it to the active-site pocket. Other solvent-accessible residues with possible function during saccharide binding in the CA_C0359 structure include Lys120, Lys325 and Lys346.ΔGlcA-GalNAc, ΔGalA-Rha and ΔGalA-GalA were docked into the active site of CA_C0359 using l. 2006b. Both thet al., 2006abl-threo-5-hexosulose-uronate. The CA_C0359 protein will hereafter be referred to as Cac_GH105 owing to the substrate source, sequence identity, structural analysis and conserved active-site configuration.Using the enzymatic reaction model of YteR with ΔGalA-Rha substrate of Itoh and coworkers, we hypothesize, based on conservation between active sites, that Asp130 of CA_C0359 will form a hydrogen bond to a water molecule that is stabilized by His176 . For example, the loops surrounding the active site of Nu_GH105 have a much more open configuration than in YteR, YesR and Cac_GH105 , which the docking study indicates may interact with bound oligosaccharide but does not affect the ability of the disaccharides listed above to fit into the active site. It is not clear whether this loop affects the size of the sugar or the length of the oligosaccharide that is able to bind to the active site. Taken with the electrostatic comparison and growth on pectin and PGA, Cac_GH105 may cleave a wider range of substrates than or a different range of substrates to YteR, including ΔGalA-Rha and ΔGalA-GalA, which may be decorated with side chains or be longer-chained oligosaccharides.Substrate specificity in the GH105 family correlates with the loops surrounding the active site (Collén op Fig. 2b, whichC. acetobutylicum ATCC 824 glycoside hydrolase, 4wu0PDB reference: 10.1107/S2053230X15012121/tb5084sup1.pdfSupporting Information.. DOI:


Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily focuses on novel chemical and biotechnological strategies for the site-directed attachment of drugs that are currently validated for 2nd generation ADCs to promote conjugate homogeneity and overall stability.Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals Chemotherapeutic strategies have long been used as the primary treatment against a broad range of cancers. However, tumor-cell specificity is only addressed with regard to rapid cell division rates present in most cancers, a feature that is true for a lot of non-malignant cell types as well, leading to systemic side-effects. Thus, targeted cancer treatments with therapeutic antibody biologics have gained major interest in the pharmaceutical and biopharmaceutical industry. In recent years however, huge efforts have been made to merge the positive features of chemical and biological cancer treatments with the development of antibody drug conjugates (ADCs) that deliver the highly cytotoxic drugs directly at the tumor site. As such, ADCs widen the therapeutic window in comparison to chemotherapeutics: The tumor-targeted, antibody-mediated drug delivery approach decreases the minimum effective dose and at the same time elevates the maximum tolerated dose Fig. .Fig. 1NoWith such promising properties and the recent FDA approval of the first two ADCs, Kadcyla® and Adcetris®, the ADC field has gained momentum in all relevant directions with increasing knowledge about the major challenges, drawbacks and screws to turn for improving the quality and efficacy of modern cancer treatments covered by recent technology reviews –3 and cuMany choices have to be made for the successful generation and application of ADCs. The antibody defines the cellular target. To date various different molecular targets in both solid and haematological cancers are being exploited for ADC development . While tMylotarg® (Gemtuzumab ozogamicin) targeting CD33 was the first ADC to be approved by the FDA for the treatment of myeloid leukemia in 2000 and statistically conjugated via surface exposed lysine residues of the antibody molecule. Ten years later, Pfizer voluntarily withdrew it from the US and European market due to low efficacy and possible toxicity observed in a second phase III study . This maThe selective reduction of the four intermolecular disulfide bonds within an IgG molecule followed by maleimide-thiol conjugation to cytotoxic drugs was one of the first attempts to increase the control of the conjugation sites and to reduce the heterogeneity observed using lysine-based conjugation methods . AdcetriA different approach to reduce heterogeneity of cysteine-based conjugated ADCs is the engineering of additional cysteine residues to the antibody leaving the interchain disulfides untouched , 24. IniCysteine mediated production of ADCs and the ideas arising thereof laid the foundation for site-specific conjugations of drugs to antibodies. Ever since it was shown that site-specific conjugation positively influences the characteristics of ADCs, the field has rapidly adapted and a significant number of novel techniques has emerged for drug loading at defined location within the antibody.p-acetylphenylalanine at distinct sites within the antibodies and aminooxy-auristatin F and auristatin D derivatives were conjugated by oxime ligation. Although the final conjugation step took up to 4 days at slightly acidic pH, the auristatin F conjugates had a similar pharmacokinetic profile in rats to that of unconjugated Trastuzumab. The auristatin D ADCs showed superior efficacy (in vitro and in rats) and in vivo pharmacokinetics compared to cysteine conjugated counterparts [E. coli based cell-free system for the production of heterogenous ADCs. However, the antibody titer that they were able to achieve by this was even lower (250 mg/L) and all antibodies generated by cell-free expression systems are not glycosylated [Amber suppression pioneered by Schultz is the most frequently used method for the incorporation of unnatural amino acids to proteins and antibodies , 29. Theterparts , 32. Oneterparts , 32. Morosylated . Even thosylated , the impWithin the last years, several enzymatic and chemoenzymatic methods for the site-specific functionalization of antibodies have been developed, among them the use of engineered glycotransferases, transglutaminases, the bacterial derived formyl glycine generating enzyme (FGE) and the transpeptidase Sortase A. In nature glycotransferases promote the transfer of an activated glycosyl donor to a lipid or glycoprotein and thereby play important roles in the posttranslational modification of proteins and in signaling pathways. Qasba et al. were able to show, that by the mutation of a few amino acids within the binding pocket of the beta-1,4-galactosyltransferase I, unnatural monosaccharides that carry a unique reactive group  are accepted as substrates and can thereby be incorporated to glycan modified proteins . Ever siStreptoverticillium mobaraense resulting in DAR of 1.2–2 [In 2010, the Schibli group was showed site-specific functionalization of IgGs (rituximab and the anti-L1-CAM chCE7) using an amide bond forming transglutaminase. After removal of the antibodies N-glycan using PNGase, various amine-containing substrates were coupled to Q295 by isopeptide bond formation . Using aof 1.2–2 . These cof 1.2–2 . The Groof 1.2–2 . HoweverStaphylococcus aureus) has intensively been studied for the site-specific modification of proteins and antibodies [The transpeptidase Sortase A  oxidizes the cysteine side chain of the peptide sequence CXPXR to a formylglycine was pioneered and developed in the labs of Diercks and Bertozzi , 54. OxiTub-tag labeling is a novel approach for the site-specific modification of antibodies that combines the above mentioned use of UAA incorporation with a highly efficient chemoenzymatic system . The tecThe still increasing number of ADCs in clinical trials and the constantly growing number of ADC-related publications do underline the significance of the ‘magic bullet’ oncology approach that has first been postulated in 1908 by Paul Ehrlich. Nonetheless, the field has witnessed a number of recent pitfalls that exposed the major technological vulnerabilities where improvement is needed. Most of these weak spots revolve around ADC efficacy, toxicity, clearance and stability and are currently tackled from various directions. This includes the identification of novel drugs, antibody engineering to identify regions that are well suited for drug attachment and versatile chemical linker strategies to modulate the drug load, release and overall ADC stability.Moreover, novel site-specific coupling strategies to allow for homogeneous ADC species with defined and controllable therapeutic properties are under investigation. All existing site-specific conjugation strategies have advantages and disadvantages and negotiate scientific parameters such as the extent of antibody manipulation and in consequence its functional integrity, conjugation efficiencies, conjugate stability, chemical versatility as well as applied ‘real world’ parameters such as technical handling for cost-effective, industrial ADC production.With an ever increasing portfolio of ADC enabling technologies at hand that together allow for better, versatile and tailor made conjugates, probably one of the most promising and pressing tasks within the ADC field lies in the search for novel antibody conjugate indications beyond classical oncology targets. This particularly includes pathogens that have previously been hard or impossible to tackle, as has recently been exemplified for novel Antibody-antibiotic conjugates targeting well-hidden, intracellular populations of pathogenic bacteria .