Pontederia (=Eichhornia) crassipes recorded the highest assimilation potential of heavy metals in water  and sediments , followed by a submerged native macrophyte, Stuckenia pectinatus, and three native emergent species, Typha capensis, Cyperus sexangularis, and Phragmites australis. Pollution indices clearly showed the promising assimilation by native and non-native macrophytes species; however, the Swartkops River was heavily influenced by multiple non-point sources along the system, compromising the assimilation effect. Furthermore, we emphasise that excessive anthropogenic inputs compromise the system’s ability to assimilate heavy metals inputs leading to water quality deterioration.There is continuous deterioration of freshwater systems globally due to excessive anthropogenic inputs, which severely affect important socio-economic and ecological services. We investigated the water and sediment quality at 10 sites along the severely modified Swartkops River system in the Eastern Cape Province of South Africa and then quantified the phytoremediation potential by native and non-native macrophyte species over a period of 6 months. We hypothesized that the presence of semi and permanent native and non-native macrophytes mats would reduce water and sediment contamination through assimilation downriver. Our results were variable and, thus, inconsistent with our hypotheses; there were no clear trends in water and sediment quality improvement along the Swartkops River. Although variable, the free-floating non-native macrophyte,  Aquatic ecosystems have been subjected to organic and inorganic pollution, which have worsened with poor waste water management . These iPrevious field and mesocosm trials have shown that reversing the impact of anthropogenic inputs in the environment is challenging, and that minimising the level of these inputs and waste management will help curb environmental contamination . EcologiHowever, Hanif et al.  showed tIt is clear that there is a need for innovative techniques with merits over traditional methods. Green technology, such as phytoremediation, which uses plants and associated microbes to assimilate and breakdown contaminants in natural environments, is one method that has been widely researched and applied ,14,15,16Typha capensis (Rohrb.) N.E.Br. (Typhaceae) (Bulrush), Phragmites australis (Cav.) Trin. ex Steud (Poaceae) (Common reed), and Cyperus sexagularis (L.) (Cyperaceae) (Swamp flat-sedge) . Th. Th47]. 2 wide catchment area where protected areas dominate the upper reaches of the catchment; the middle reaches is dominated by urban, formal settlements, and agricultural lands; and the lower reaches are surrounded by industries, formal and informal settlements before flowing into the ocean. The landscape activities contribute to the release of domestic effluents, industrial waste, untreated sewage, and other point and non-point source pollutants [Eucalyptus spp. (Gum trees) and Acacia spp. (Black Wattle and Port Jackson Willow) [The 155 km long Swartkops River drains the 42 kmllutants . The nat Willow) .P. crassipes and S. molesta (P. crassipes mat 1 (hereafter site 3), whereas site 4 was located ~0.6 km downstream from site 3 (P. crassipes mat 2 (hereafter site 6), and site 7 was about a kilometre downstream from site 6 (S. molesta mat 3 (hereafter site 9), and site 10 was located 0.6 km downstream from site 9  and non-native  macrophyte species were collected and analysed for heavy metal accumulation analysis  was collected ~20 cm below the water surface at each site using pre-rinsed clear polyethylene sample container for water chemistry analysis. Water samples were then stored on ice until they reached the laboratory, and, within 48 h after collection, water samples were sent to BEM-Labs, Cape Town, South Africa for water chemistry analysis, including Chemical Oxygen Demand (COD), Zinc (Zn), Iron (Fe), Cadmium (Cd), Arsenic (As), Chromium (Cr), Lead (Pb), Mercury (Hg), and Copper (Cu).Integrated water sample , the water samples were acidified to a pH of ±2 and digested to isolate all the metal ions in solution. Once cooled, the samples were filtered through a 0.45 µL syringe filter to remove any particulates. The resulting samples were then analysed using an Agilent ICP-OES 720 Axial instrument for total heavy metals. Since these were integrated water samples, it is possible that some properties, such as pH, or organic carbon, varied between the samples and may have influenced the speciation (and bioavailability) of pollutants.Using a gardening trowel, integrated soil sediment samples were collected at five areas per site at approximately 10 cm depth. Sediments samples were collected into plastic zip-lock bags and then stored on ice. Similar to the water chemistry samples, sediment samples were within 48 h after collection sent to BEM-Labs for sediment chemistry and heavy metal analysis, including Zn, Fe, Cd, As, Cr, Pb, Hg, and Cu.At BEM-Labs laboratory, a portion of the sediment sample was weighed into an Erlenmeyer flask. We added 20 mL nitric acid and 10 mL hydrogen peroxide to the flask, and the flask was then heated to allow the sample to digest. After digestion, the sample was transferred to a 100 mL volumetric flask, made up to volume, and then filtered. The resulting sample was then analysed on the Agilent ICP-OES 720 Axial instrument for heavy metals.T. capensis, C. sexangularis and P. australis; five matured floating plants i.e., P. crassipes and S. molesta, and about 200 g of submerged plant i.e., S. pectinatus, were collected by hand and rinsed with distilled water to remove any debris and periphyton biofilm.Native marginal and aquatic vegetation species together with non-native macrophytes were collected at each site for heavy metal analysis. Five stems of emergent plants i.e., Plant material were transferred into different zip lock bags (per plant species) and stored on ice until they reached the laboratory. In the laboratory, plant samples were immediately oven-dried at 60 °C for 72 h. During this procedure, all the cell processes  stopped, making sure that samples represents the nutrients composition per gram of leaf without the influence of water. Thereafter, dried leaves were homogenised into coarse material by grinding using a mortar and pestle.About 6.5 g of dried plants tissue was weight and packaged into aluminium foil envelopes and also sent to BEM-Labs, for heavy metal analysis, including Fe, Hg, Zn, Cd, As, Pb, and Cu. For each sample, 20 mL nitric acid and 5 mL hydrogen peroxide were added and the flask was heated to allow the sample to digest, until approximately 1 mL of the solution was left. The remaining sample was transferred into a 10 mL volumetric flask, made up to volume using distilled water and filtered. The filtered sample was analysed on the Agilent ICP-OES 720 Axial instrument for heavy metals.P. crassipes and S. molesta mat sites, the percentage reduction in water and sediment heavy metals concentrations were computed. Furthermore, to understand the current environmental condition at Swartkops River and the concentration of heavy metals, sediments and macrophyte indices were used to quantify heavy metal assimilation by both native and non-native macrophytes along the river system.To assess the reduction in water and sediment chemistry between upstream and downstream semi and permanent The geo-accumulation index (Igeo), which measures the degree of heavy metal contamination, was used to estimate heavy metal pollution in the Swartkops River during the study, and this was calculated following the equation defined by Muller :Igeo=logriations . The georiations .For further geo-accumulation interpretation, Muller  proposedCF) of all measured heavy metals on-site and was calculated following a formula adopted from Islam et al. [Secondly, the pollution load index (PLI), which is an important index in evaluating soil sediment quality was used to estimate heavy metal pollution in the sediments. The pollution load index is expressed as the product of the contamination factor  of each metal was computed separately per site using the metal concentration and the background value of the metal  , CF was  Meybeck . Accordi Meybeck , the PLIThe enrichment factor (EF) is a more comprehensive assessment of heavy metal contamination . The metBackground values used for the present study were given according to the world surface rock average by Martin and Meybeck . AccordiThen, to assess and estimate the native and non-native macrophyte species accumulation potential for heavy metal concentration in sediments, the bio-concentration factor (BCF) was calculated following Zayed et al. :BCF=[metexcluder .p < 0.05) nor were the variances homogenous . Thus, a non-parametric test, in this case, Kruskal–Wallis analysis of variance, with multiple comparison test was employed. All statistical analyses were conducted in R version 3.6.1 [To test the significant differences in sediment indices  between sites and the macrophyte assimilation factor (BCF) for each plant species, the Shapiro–Wilk test for normality and Levene test for homogenous variance were employed. The outcome of the tests revealed that none of the variables were normally distributed  with site 1 recording high Fe concentration (1.1 mg/L) and site 10 low concentration (0.09 mg/L) , the highest Zn concentration (0.12 mg/L) was recorded at site 10, and the lowest Zn concentration (0.02 mg/L) was recorded for all sites except sites 5 and 7  . There w 5 and 7 .H = 21.89, p = 0.001). The highest COD concentration was recorded at site 5 (57.4 mg/L) and the lowest at site 1 (14.64 mg/L) (The COD concentrations were significantly different between sites (64 mg/L) . The As 64 mg/L) .H = 24.32, p = 0.004), Zn , As , Cr , Pb , and Cu  concentrations were significantly different between sites , Cr (41.12 mg/kg), Cu (5.56 mg/kg), and P (2240.38) were high at site 5, and low at site 10 (Zn: 7.19 mg/kg and Cr: 8.15 mg/kg) and site 7  . The leaH = 17.16, p = 0.05), Cr , Cu , Fe , Pb , and Zn  at all sites  and Hg  were not significantly different at all sites, and showed negative (−) Igeo values, which are indicative of uncontaminated sediments (The geo-accumulation index (Igeo) was significantly different for heavy metals i.e., As  and Cr (11.06) whereas, site 1 recorded the highest Igeo value for Fe (15.11) . All sitH = 17.08, p = 0.05), Cr , Cu , Zn , and Pb , showed significant differences between sites (The enrichment factor (EF) revealed that five heavy metals, including As (en sites . Site 1 en sites .p > 0.05)  . All recPontederia crassipes and Salvinia molesta semi and permanent mats showed promising heavy metal assimilation, but this varied between sites. However, in some cases, the trend was clear showing heavy metal reduction between upstream and downstream P. crassipes and S. molesta mats, thus, indicating possible macrophyte assimilation potential , whereas Zn concentration showed a total reduction of 57%, 89%, and 65% between site 2 (62.55 mg/kg) and site 4 (26.92 mg/kg), site 5 (87.16 mg/kg) and site 7 (9.65 mg/kg), site 8 (22.4 mg/kg) and site 10 (7.19 mg/kg), respectively (The Fe concentration in the sediments showed a 46% reduction between the upstream site 2 (967.37 mg/kg) and downstream site 4 (520.02 mg/kg) of ectively . Arsenicectively .Chromium showed a 77% reduction between site 5 (41.12 mg/kg) and site 7 (9.50 mg/kg), and Pb showed a 68% reduction between site 5 (19.90 mg/kg) and site 7 (6.45 mg/kg), and 56% reduction between site 8 (8.42 mg/kg) and site 10 (3.67 mg/kg) . MercuryTypha capensis and Cyperus sexangularis BCF results were significant between sites for Cu  and Zn  , at all sites, except for site 5, which showed BCF values of less than 1 (Emergent native macrophytes species recorded the lowest bio-concentration factor (BCF) values when compared to both floating and submerged native macrophyte species . Typha c< 0.001) . Typha c, and 10 . Phrgamis than 1 .P. crassipes BCF results were significantly different between sites for As , Cr , Cu , Fe , Hg , and Zn   . Pontede, and 10 .S. pectinata BCF results were significantly different for four heavy metals, i.e., Cr , Fe , Hg , and Zn   . The hea < 0.01) . The siged to Cr .The present study reports that the Swartkops River system is heavily polluted by various heavy metals. Although our results show some degree of assimilation by native and non-native macrophyte stands, the continuous inputs, i.e., non-point sources, at different entry points along the river system surpass this potential. Research on macrophyte assimilation (or phytoremediation) has mainly been conducted in mesocosm settings, with limited in situ case studies or case studies in the natural environment ,66,67,68P. crassipes and S. molesta was tested in the present study and others , as well as site 8 and site 10 of S. molesta mat (site 9), we reported more than 45% reduction in heavy metal concentration   .P. crassipes and S. molesta mats acting as accumulators for heavy metals from upstream. The above findings corroborated with Mishra and Tripathi [P. crassipes in accumulation of Cr and Zn effluents, were P. crassipes efficiently assimilated more than 50% of the heavy metal concentration in only 11 days of exposure, further emphasizing the phytoremediation potential of these macrophytes.Reductions were attributed to the presence of Tripathi , whose sIt is possible that some of the pollutants were accumulated by sediments. This is because, as contaminants constantly wash off downriver, some slowly settles and gets assimilated in the sediments. Jernström et al.  indicateThese results were supported by Hadad et al.  and SchaThese findings were more evident at site 5, which recorded the highest heavy metal concentrations  in sediments, in addition, the EF and Igeo values were highest for Zn and Pb, revealing extreme sediment contamination by these heavy metals at site 5 . This emT. capensis, C. sexangularis, P. australis (native emergent macrophytes), S. pectinatus (native submerged macrophyte), and P. crassipes (invasive floating macrophyte) have promising assimilation potential. Various studies  index also showed that s , which were fully exposed to heavily polluted systems.In contrast, floating (non-native) and submerged (native) species revealed a greater uptake of heavy metals  with high BCF values compared to emergent macrophytes . This waP. crassipes was the most effective accumulator of heavy metals, followed S. pectinatus, P. australis, C. sexangularis, and T. capensis. The order of accumulation in heavy metals by macrophyte species  was similar to a study by Goulet et al. [Lemna minor (L.) (Araceae) (Common duckweed), submerged Potamogeton epihydrus (Raf.) (Potamogetonaceae) (Ribbon-leaf pondweed), Nuphar variegeta (Durand.) (Nymphaeaceae) (Yellow pond-lily), and emerged Typha latifolia (L.) (Typhaceae) (Common cattail) in the removal of heavy metals in a mesocosm study. The study revealed that, amongst all macrophytes, floating macrophytes were more effective in assimilating heavy metals, followed by submerged, and lastly emergent macrophytes, which was similar to the present study.The present study further revealed that t et al.  who testAlthough there was promising heavy metal assimilation, the Swartkops River did not show overall water and habitat quality improvement downriver. This indicates that heavy metal reductions (>45%) in concentration between native and non-native macrophyte stands did not improve the water and sediment quality contamination; however, this was not the same for some important sediments and macrophyte pollution indices, which were variable across sites.This could be due to constant influxes from multiple non-point and point sources  along the river system, meaning that the constant inputs have a significant effect on the system deterioration. Distance between sampled sites could have also influenced our findings, as some sites were located about one kilometre away from the non-native macrophyte stands, thus, allowing pollution inputs between sites, further suppressing the assimilation as seen in this study.In addition, field experiments are considered dynamic and difficult to work with because they are complex and are affected by multiple extraneous variables that are not easy to control and can affect the outcome of results. Since this study was the first of its kind in the highly impacted Swartkops River system, we show that the phytoremediation technique can be effective; however, the state and land-use pressure play a crucial role, and we recommend more field-based studies with limited alterations.The study showed the promising phytoremediation potential of native and non-native macrophytes to mitigate heavy metal contaminants from anthropogenic activities along the Swartkops River system. Water and sediment pollution indices were variable across sites showing no consistent trend in the reduction of water and sediment quality, and this was in contrast with our hypothesis. The lack of water and sediment quality improvement down river could have been due to constant pollution effluents from multiple non-point sources along the river system.It is also possible that the river system could have been severely polluted to the extent that ecosystem services provided by both native and non-native macrophytes  were supressed. This study showed that native and non-native macrophytes can be used to assimilate pollutants; however, this can be better achieved in more control settings, i.e., laboratory and mesocosm settings, compared to complex and dynamic field conditions.The screening of sediments and macrophytes (both native and non-native) provided an overview state of the Swartkops River system, and this may serve as an early warning or indication of changes in the system. Various authors ,30,31,40Thus, we propose that adjunctive studies should be conducted to assess phytoremediation using biological variables  to quantify phytoremediation success. The current study further emphasizes that physicochemical variables are not sensitive but variable and can only provide a snap-shot of habitat degradation. The sediment and macrophyte indices were reliable indicators of heavy metal contamination and macrophyte bio-accumulation potential; however, excessive anthropogenic input in the Swartkops River suppressed macrophyte ecosystem services. We therefore recommend more field studies to test various green technologies to mitigate the deterioration water and habitat quality using relevant biological indicators.


AGS is strongly associated with tick bites. An increased incidence of venom sensitization has been found in AGS patients. Here, we evaluated the frequency of wasp sensitization in Swedish AGS patients and the possible cross‐reactivity between wasp venom and tick proteins.Vespula spp.), the European tick Ixodes ricinus (Streptavidin ImmunoCAP), α‐Gal and total IgE by ImmunoCAP. The presence of α‐Gal on wasp venom proteins (Vespula vulgaris) was investigated by western blot (WB), and possible cross‐reactivity between wasp venom and tick proteins by enzyme‐linked immunosorbent assay and WB. Involvement of cross‐reactive carbohydrate domains (CCDs) was also assessed.Sera from 136 Swedish AGS patients and 29 wasp‐positive non‐AGS control sera were analyzed for IgE‐reactivity against wasp venom (in silico approach.Wasp sensitization was present in 54% of AGS patients, although the IgE levels were low. Wasp sensitized patients had higher IgE levels to α‐Gal and total IgE levels compared to non‐wasp sensitized AGS patients. α‐Gal was not detected in wasp venom, but cross‐reactivity between wasp and tick proteins was demonstrated which was not dependent on CCDs. The same cross‐reactivity was also observed in the control sera. Furthermore, 17 putative cross‐reactive peptides were identified using an For the first time, cross‐reactivity between wasp venom and tick proteins has been described. This may be a reason why the majority of Swedish AGS patients, who have all been tick bitten, are also sensitized against wasp. Interestingly, the disease onset is strongly associated with tick bites.The observed relation between tick bites and AGS has raised the question whether other biting or stinging arthropods may be associated with AGS as well. Until now, available data is limited. In the US, larvae of the Trombiculidae mites, or “chiggers,” were suspected to have caused α‐Gal sensitization in a few cases.Another class of insects that have been linked to AGS are the hymenoptera, including bees and wasps. Venom allergy has been found in 24% (12/50) of meat allergic patients from a Turkish case study, and honey bee venom allergy was the most frequent.Vespula spp.) and possible cross‐reactivity with tick proteins (Ixodes ricinus), in order to examine if properties of wasp venom proteins could be involved in the increased wasp sensitization in AGS patients. Finally, we elucidated if the same results could be observed in a wasp‐positive, non‐AGS control group.In this study, we explored wasp sensitization in AGS patients on a molecular level. We analyzed wasp‐specific IgE levels in a Swedish AGS cohort. Furthermore, we investigated the presence of α‐Gal on wasp venom proteins , tick (I. ricinus), and total IgE were determined for all sera by ImmunoCAP (Phadia AB/Thermo Fisher Scientific). IgE antibodies against tick were measured by coupling biotinylated tick protein extract to Streptavidin ImmunoCAP,  as previously described.A/L. A selection of sera, both from AGS patients and controls, were either used individually or as serum pools in experiments described below  was purchased from Citeq Biologics. The lyophilized product was reconstituted in Phosphate‐buffered saline (PBS), and the protein concentration was determined using the Bradford method.I. ricinus adults (IS Insect Service GmbH) were prepared as previously described.Wasp venom (2.3Wasp venom and tick protein extract (each 30 μg per lane) were separated under reducing conditions on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) at TGX™ gradient precast gels  using a Mini Protean Cell II system (Bio‐Rad Laboratories). Protein bands were stained with Coomassie Brilliant Blue. For western blot (WB), proteins were transferred to polyvinylidene difluoride membranes (0.2 μm pore size) using a Bio‐Rad Turbo system. Membranes were blocked with 1% human serum albumin in phosphate buffered saline containing 0.05% Tween for 2 h at room temperature (RT).To detect α‐Gal carrying proteins, the membrane was incubated with 1:7500 dilution of chicken anti‐α‐Gal single‐chain antibody variable‐region fragment 2O2 substrate  on a ChemiDoc instrument (Bio‐Rad Laboratories).IgE binding to wasp venom and tick proteins was tested by incubating the membrane with serum pools from AGS patients  was used to investigate the inhibitory capacity of wasp venom and tick protein extract on the IgE binding to tick and wasp venom protein, respectively. Microtiter plates were coated with 10 μg/ml wasp venom or tick protein extract in coating buffer (50 mM sodium bicarbonate buffer pH 9.6) overnight at +4°C. Serum pools .In order to identify cross‐reactive epitopes, sequence similarity between tick hemelipoglycoprotein (A0A0D3RJ94) and wasp vitellogenin (G8IIT0) was assessed by calculating the property distance (PD) value using the peptide similarity tool of the Structural database of allergenic proteins . These wasp‐sensitized AGS patients showed significantly higher levels of α‐Gal‐specific IgE and total IgE compared to non‐wasp sensitized AGS patients .Of the 136 included AGS patients, 73 (53.7%) had IgE reactivity against wasp Figure , with a s Figure . A poor n = 60) had IgE reactivity to tick. Of these double‐positive patients, 41 had higher tick‐specific than wasp‐specific IgE levels and 19 higher IgE levels to wasp than to tick. Furthermore, tick‐specific IgE levels were also significantly higher in wasp‐sensitized AGS patients compared to non‐wasp sensitized AGS patients .More than 80% of the 73 wasp‐positive AGS patients . Eight sera were found to be tick‐positive . Four of these sera were both α‐Gal‐ and tick‐positive.We compared the results with 29 wasp‐positive non‐AGS sera . A total of 17 peptide combinations was considered to be possible cross‐reactive peptides due to their PD value of <7  as the cross‐reacting protein in tick extract, which has previously been detected in Vitellogenin is an egg yolk precursor which is present in almost all female oviparous animals. It has been recognized as a pan‐allergen in different mite species,IgE reactivity to CCDs is notorious for causing cross‐reactivity in plants and insects.We noted that the majority of our random population of wasp‐positive sera was solely wasp‐IgE positive. These sera showed only binding to Ves v 5 and Ves v 1, which were not inhibited by tick protein extract Figure . Howeverin silico analysis confirms our experimental data by showing that cross‐reactivity is likely based on the primary structure of the proteins, and provides candidate peptides for further research focusing on the identification of the allergen epitopes in vitellogenin.Although vitellogenin has been described as an allergen in multiple species, the exact allergen epitopes have not been identified yet, and it is not known which part of the protein is cross‐reactive with vitellogenin and vitellogenin‐like proteins from other species. Sequence similarity assessment including the whole tick hemelipoglycoprotein and wasp vitellogenin (G8IIT0) revealed a list of 17 possible cross‐reactive peptides. This In conclusion, for the first time cross‐reactivity between wasp venom and tick has been described. This may be one reason why more than half of Swedish AGS patients, who have all been tick bitten, are also sensitized against wasp, in addition to other factors, for example, environmental factors. Furthermore, the clinical relevance of the observed cross‐reactivity between tick and wasp proteins needs further research.Dr. van Hage reports personal fees from Thermo Fisher Scientific, outside the submitted work. Dr. Starkhammar reports fees from Mylan, ALK, AstraZeneca and Chies. The rest of the authors declare no conflict of interest.Supporting Information S1Click here for additional data file.


Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p38 and ERK, mitogen-activated protein kinases (MAPKs) that have established roles in activating cytosolic PLA2α (cPLA2α) and 5-LOX. The reduction of p38 and ERK phosphorylation is associated with a decrease in cPLA2α phosphorylation and attenuated leukotriene production. Furthermore, KIRA6 inhibits p38 activity, and molecular modelling indicates that it can directly interact with the ATP-binding pocket of p38. This potent and unexpected, non-canonical effect of KIRA6 on p38 and ERK MAPKs and leukotriene biosynthesis may account for some of the immune-modulating properties of this widely used IRE1α inhibitor.The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B Together with the accessory proteins in the pathway, these enzymes form the leukotriene biosynthetic complex and control cellular production of leukotrienes (Leukotrienes (LTs) are a class of lipid mediators derived from arachidonic acid (AA), predominantly produced in myeloid cells . There are two types of leukotrienes-leukotriene B (SRS-A) . These m (SRS-A) . The bions (PGs) . In a seotrienes .2α and 5-LOX are regulated by cytosolic calcium and protein phosphorylation , which also modulates the activity of the two enzymes , inositol-requiring enzyme 1α (IRE1α) plays an important role in sensing misfolded proteins and maintaining homeostasis of the endoplasmic reticulum (ER) . Exposur2-mediated pain with a comparable efficacy as celecoxib  s have been designed to allosterically inhibit the RNase activity of IRE1α by stabilizing the ATP-binding kinase domain and preventing oligomerization . As an oelecoxib , whereas2α (p-cPLA2α) (Ser-505), p-p38 (Thr180/Tyr182), p-ERK (Thr202/Tyr204), and p-5-LOX (Ser271 and Ser663) were from Cell Signaling Technology . LPS, Thapsigargin, fMLP 4μ8C, RPMI-1640 cell culture medium, HEPES solution, 1,25-dihydroxy vitamin D3, horseradish peroxidase (HRP)-linked mAb against β-actin were from Sigma-Aldrich . TGF-β1 and M-CSF were from Thermofisher Scientific . SB203580 was from Tocris Bioscience .KIRA6 was from Cayman Chemicals . Monoclonal antibodies (mAb) against phosphorylated cPLAHuman monocyte-derived macrophages (hMDMs) and neutrophils were isolated as previously described . BrieflyMM6 cells were transfected with lentiviral shRNA against IRE1α (NM_001433) and non-target shRNA to generate IRE1α knockdown (KD) and control MM6 cells. The knockdown efficiency was evaluated by qPCR analysis of the mRNA expression of IRE1α.4, cys-LTs, and PGE2 in cell supernatants were measured with ELISA kits  according to the manufacturer’s instructions. The absorbance at 405 nm was recorded by a TECAN Infinite M200 plate reader.LTBCells were lysed with RIPA buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails . SDS-PAGE and western blot transfer were carried out with NuPAGE Novex 4–12% Bis-Tris gels and iBlot2 blotting system (Thermofisher). Afterwards, the membranes were incubated with 3% non-fat milk for 1 h and further incubated with HRP-linked β-actin antibody (Sigma-Aldrich) for 1 h, or desired primary antibodies overnight at 4°C followed by HRP-linked anti-rabbit antibody . The enhanced chemiluminescence substrate was then added to the membranes to visualize the immunoreactive protein bands.50 values were determined with the GraphPad Prism program.The p38 and ERK kinase assays were performed according to the manufacturer’s protocol (Promega) with optimized conditions. The p38 assay was carried out with 10 ng of the p38 enzyme, 150 µM ATP and 0.2 µg/µL p38 peptide as substrates, and 0–10 µM of KIRA6, 4 µ8C or SB203580 as the positive control. The ERK kinase assay was performed with 3 ng of ERK1 or ERK2, 50 µM ATP and 0.5 µg MAP kinase substrate (MBP), together with 0–100 µM of KIRA6 or K252a as the positive control. Produced ADP was converted to ATP by ADP-Glo Assay (Promega) and the luminescence recorded using the TECAN Infinite M200 plate reader. Fluorescence values were normalized to vehicle group and the dose response curve was created using non-linear regression from which IChttps://www.globalphasing.com). Prior to the docking, SB203580 was removed from the X-ray structure. Docking simulations with KIRA6 was carried out with the AutoDock Vina tool  was used as a template to perform docking simulations with KIRA6. KIRA6 as the ligand was prepared using the Grade Web Server , for instance MKK3/6 and MKK1/2, respectively, which would in turn reduce p38 and ERK phosphorylation and thereby leukotriene biosynthesis. Moreover, the important decreases of p38 and ERK phosphorylation also predict a reduction of 5-LOX phosphorylation at Ser271 and Ser663, which could contribute to the decreased leukotriene production. However, we were not able to detect endogenous 5-LOX phosphorylation with commercially available antibodies (data not shown), suggesting that 5-LOX phosphorylation, or its inhibition, is not involved in this model.Inasmuch as KIRA6 blocked leukotriene biosynthesis with equal, or even better potency compared to its action against IRE1α, we initially hypothesized that this arm of the UPR was closely linked to the leukotriene cascade, in analogy with what has been reported for prostaglandin synthesis . However and ERK , establi ERK (2α , and 1 µ ERK (2α . This su50 = 1 μM, 50 = 0.6 µM, (2α (Besides reducing p38 and ERK phosphorylation, KIRA6 was also found to directly interact with p38 and inhibit its kinase activity. Through molecular docking, KIRA6 was assigned binding affinity to p38 and a potential to enter its ATP-binding domain. Moreover, the inhibitory potency of KIRA6 against p38 kinase activity (IC 0.6 µM, ]. It is  µM, (2α . This pr µM, (2α .in vivo (2α axis of leukotriene synthesis may account for some of these drug effects. Moreover, p38 and ERK participate in a variety of cellular processes by integrating different stress, metabolic, and inflammatory signals suggesting that KIRA6 may interfere with other fundamental cellular processes, yet to be characterized.As a potent allosteric inhibitor of IRE1α, KIRA6 has been developed to inhibit IRE1α-mediated cell death, and has been demonstrated to promote cell survival and prevent ER stress-induced cell degeneration in vivo . Of notein vivo . In lighIn summary, we have uncovered a novel potent action of KIRA6, in which this small molecule interferes with p38 and ERK signaling pathways, leading to inhibition of leukotriene production. While KIRA6 is a promising lead for pharmacological intervention in the IRE1α-XBP1 pathway, further work is required to detail the full pharmacological repertoire of this drug.


Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identified by our methods, as well as those previously reported in the literature. The “KANPHOS”  database and its web portal were built based on a next-generation XooNIps neuroinformatics tool. To explore the functionality of the KANPHOS database, we obtained phosphoproteomics data for adenosine-A2A-receptor signaling and its downstream MAPK-mediated signaling in the striatum/nucleus accumbens, registered them in KANPHOS, and analyzed the related pathways. Protein phosphorylation and dephosphorylation are critical post-translational modifications in the regulation of cell signaling networks, especially in the nervous system, where these mechanisms contribute to brain development, functions and disorders ,2,3,4,5.With the advancement of mass spectrometry (MS)-based proteomics approaches, a variety of databases have been developed to document experimentally verified in vivo and/or in vitro phosphorylation sites from numerous biological samples. Several phosphoproteomics databases have been developed, including UniProtKB , PhosphoKinase-Associated Neural PHOspho-Signaling), (available online: https://kanphos.neuroinf.jp/ (accessed on 2 December 2021)) which provides phosphorylation signaling information, including data on phosphoproteins, phosphorylation sites and responsible kinases, in the brain. Moreover, we performed KIOSS to obtain novel phosphoproteomics data for adenosine-A2a-receptor signaling, including downstream MAPK-mediated signaling, in the striatum/nucleus accumbens and registered them in KANPHOS.In this study, we developed and presented an online database named KANPHOS (https://xoops.org/ (accessed on 1 December 2021)), which is an OS-independent content management system (CMS) web application written in PHP ). In KANPHOS, XooNIps runs on CentOS (available online: https://www.centos.org/ (accessed on 1 December 2021)) using Apache ) as the web server and MySQL (available online: https://www.mysql.com/ (accessed on 24 November 2021)) as the database management system.The KANPHOS database is designed as a web-based database that is accessible from a web browser to allow simple and widespread use by researchers. It was built based on the neuroinformatics platform system named XooNIps (XOOPS module for Neuroinformatics) version 4.0  . XooNIpsThe stable operation and updating of web-based databases requires substantial familiarity with web and network systems. To solve these problems, CMSs for the construction and management of web-based databases have become popular. The database we sought to develop needed to be able to handle a very large and continuously increasing amount of experimental data on protein kinases and phosphorylation sites. XooNIps is an easy-to-use software that can facilitate the construction of web-based databases for organizing vast amounts of information with metadata in various electronic formats and sharing it with users around the world on the internet.As of October 2021, approximately 10,000 records have been registered in KANPHOS. Each record is composed of a pair, a protein phosphorylation site and its modifier . Of these, 6228 records were identified based on our experimental results, while 3736 records were based on publications from other groups. To facilitate analysis, KANPHOS supports three search modes: (1) search for substrates phosphorylated by a specific kinase; (2) search for kinases phosphorylating a specific protein; and (3) search for kinases and their target substrates in a specific signaling pathway. All proteins registered in KANPHOS are associated with a UniProt ID to facilitate linkage with 12 external databases . In part3− buffer . Coronal brain slices (350 μm) were prepared using a VT1200S vibratome . After dissecting of the whole striatum/NAc, coronal slices were incubated at 30 °C in Krebs-HCO3− buffer with 10 μg/mL adenosine deaminase  for 30 min under constant oxygenation with 95% O2/5% CO2. The buffer was replaced with fresh Krebs-HCO3− buffer and preincubated for 30 min. Coronal slices were pretreated with the indicated inhibitors  and then stimulated with the indicated activators . After stimulation, slices were immediately frozen in liquid nitrogen and stored at −80 °C. The slices were lysed in lysis buffer , protease inhibitor cocktail (Roche), pH 7.0) and sonicated for 20 s. The protein concentration of lysates was determined by BCA assay (FUJIFILM Wako). The A2AR-specific agonist CGS21680 , D2R-specific agonist quinpirole , phosphatase inhibitor okadaic acid (OA) (primarily a PP2A inhibitor) , and MEK1/2 inhibitor U0126  were used for slice culture treatment.Coronal slices were prepared from male C57BL/6 mice as described previously . In our ζ (500 pmol) immobilized on glutathione sepharose 4B beads  for 60 min at 4 °C. The beads were washed with lysis buffer three times, followed by wash with wash buffer  three times. Using guanidine solution (7 M guanidine and 50 mM Tris), bound proteins were removed from the beads, followed by reduction with 5 mM dithiothreitol for 30 min, and alkylation by10 mM iodoacetamide for 1 h at dark. The proteins were digested with Trypsin/Lys-C (Promega) and incubated overnight at 37 °C. Demineralization was performed using SPE c-tips  and phosphopeptide was enriched by TiO2 column  according to the manufacturer’s instructions. Finally, all the peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) using an Orbitrap Fusion mass spectrometer , which was coupled with an UltiMate3000 RSLCnano LC system  using a nano High-performance liquid chromatography (HPLC) capillary column, C18, 3 µm, 150 mm × 75 µm  via a nanoelectrospray ion source. Reversed-phase chromatography was performed with solvent A  and solvent B  with a linear gradient  at a flow rate of 300 nL/min. Prior to MS/MS analysis, a precursor ion scan was carried out using a 400–1600 mass to charge ratio (m/z) and then MS/MS analysis was performed by isolation at 0.8 Th with the quadrupole, HCD fragmentation with a normalized collision energy of 30%, and rapid scan MS analysis in the ion trap. Precursor ions with charge states of 2–6 were sampled for MS2 only. The instrument was run in a top speed mode with 3 s per cycle. Phosphoprotein identification was performed by MaxQuant software (version 1.4.1.2)  against the reference Mus musculus proteome database obtained from UniProtKB (release in November 2018) (accessed on 4 June 2021) [https://david.ncifcrf.gov/ (accessed on 7 November 2021)) and Reactome analysis .Mass spectrometry (MS) was performed as previously described  with somne 2021) . We set The proteins were separated via SDS-PAGE using 8% polyacrylamide gels  and transferred to polyvinylidene difluoride membranes . The membranes were blocked for 30 min at room temperature with Blocking-One  and then incubated with primary antibody for overnight at 4 °C. Following primary antibodies were used: rabbit monoclonal anti-phospho-GluR1 (S845) (RRID: AB_10860773) from CST; mouse monoclonal anti-GluR1 (RRID: AB_11212678) from EMD Millipore; rabbit monoclonal anti-phospho-p44/42 MAPK (ERK1/2) (T202/Y204) (RRID: AB_2315112), mouse monoclonal anti-p44/42 MAPK (ERK1/2) (RRID: AB_10695739) from CST; anti-phospho-Rap1gap (S563) , rabbit To construct the KANPHOS database of protein phosphorylation modification in the brain, we integrated data mainly from our own developed phosphoproteomic method (45%), from the literature (37%) and from Protoarray (18%) A. To obtζ protein, tryptophan–tryptophan (WW) domain, and forkhead-associated (FHA) domain as the phospho-Ser/Thr-binding modules because these modules contain proteins that broadly regulate the function of their target proteins in a phosphorylation state-dependent manner and contribute to cellular phospho-signaling networks [In the KIOSS method, cell or tissue cultures were treated with an agonist, kinase inhibitor, and/or phosphatase inhibitor, and phosphoproteins were enriched from the lysate with phospho-Ser/Thr-binding proteins, followed by LC-MS/MS analyses to identify phosphoproteins and their phosphorylation sites B. We chonetworks . We perfnetworks , PKA andnetworks . The rat® microarray  [Published phosphoproteomics datasets were collected from the literature by searching PubMed using keywords including ‘phosphoproteomic’, ‘phosphoproteomics’ and ‘phosphoproteome’. The retrieved literature was manually reviewed and curated by our professional staff; experimentally identified candidate substrates and phosphorylation site information were collected and cross-checked with other databases, such as UniProt, PhosphositePlus, and PhosphoELM. In addition to manual curation from the literature, data were also collected from Protoarray, which provides data generated by substrate screening for proteins phosphorylated by recombinant kinases. Each kinase was assayed at 5 nM and 50 nM in the presence of γ33P ATP to explore the phosphorylation of more than 9000 human proteins printed on the Protoarrayitrogen) ,39. HoweThe distribution of phosphoproteins and their phosphorylation sites, different residue types, and species are summarized and represented in To facilitate access to the KANPHOS database in a user-friendly manner, a web interface was developed with browsing and search options to facilitate the query of protein phosphorylation events. An overview of the KANPHOS workflow is shown in To examine how the KANPHOS database works, we performed KIOSS to obtain phosphoproteomic data downstream of the adenosine signaling pathway in the striatum/NAc. We used this study as a model to demonstrate the ability of the KANPHOS database to identify novel phosphorylation-dependent signaling dynamics in the brain.olf protein, whereas D1R-MSNs express A1R, which couples to the Gi protein [Adenosine is an important neuromodulator in the striatum/NAc that acts through adenosine A1 receptor (A1R) or adenosine A2A receptor (A2AR) . Approxi protein ,44,45. T protein ,47. Trea2 column to enrich for phosphopeptides, followed by LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites ) revealed several potassium and Hyperpolarization-activated cyclic nucleotide gated (HCN) channels that may potentially be involved in neuronal excitability  is an abundant protein in the brain that regulates axonal growth and neuronal polarity, is phosphorylated by CDK5 at S522 and GSK3β at T509, T514, and S518, and is involved in the pathogenesis of Alzheimer’s disease [Protein phosphorylation orchestrates neurotransmitter release, neuronal polarity, and neuronal signaling pathways and is involved in the pathogenesis of many neurological diseases; thus, pathway analysis using the KANPHOS database could provide a means to understand how phosphorylation events can regulate neuronal signaling events. In the previous section, we identified MAPK candidate substrates. Here, we propose a workflow for analyzing MAPK phosphoproteomics data using KANPHOS pathway analysis as a model study to identify novel phosphoproteins downstream of the MEK-MAPK pathway in A2AR . First, p-value C. Here, p-value D. After p-value E. Previop-value . In the  disease ,54. Thes disease . Thus, uProtein phosphorylation is one of several general mechanisms that regulates the function of proteins and signal transduction in the cell, which regulates many biological processes, including cell proliferation, growth, differentiation, and metabolism. To understand the biological role of phosphorylation, it is necessary to characterize upstream signal transduction, including the kinases responsible for phosphorylation and the functional changes in downstream molecules. Phosphorylation signaling cascades vary among tissues, and completely different cellular reactions can result from the same protein phosphorylation event in different tissues due to slight differences in the cascade components. Our KANPHOS database is designed to refer not only to phosphorylation and the responsible kinases but also to related diseases, genetic polymorphisms, and knockout mice. By examining these associated data and pathway analysis of the protein phosphorylation selected in the KANPHOS search results, the signaling cascades participating in the phosphorylation of the target protein can be predicted. Compared to the other conventional databases, brain-specific, signal transduction and kinase-oriented phosphorylation data collection is unique to KANPHOS, and would provide high quality data with good usability for neuroscientists.Our current phosphorylation data are derived mainly from the mouse striatum and from cell lines such as HeLa cells, but other tissues such as the mouse forebrain, hippocampus, liver, muscle, and cancer cell lines could be further targets of our KISS/KIOSS phosphoproteomics research. With recent improvements to the CRISPR system, kinases can be easily knocked out in various cell lines. Our in vivo protein phosphorylation screening method, KIOSS, can be used to monitor phosphorylation-dependent signaling cascades that completely lack the target kinases. However, there remain several limitations for improving in KANPHOS. The tissues and cells deposited in the database are mainly brain and neuronal cells. We need to obtain and process phosphoproteomic data from other repositories, along with empirically examining other tissues and cell lines to produce additional tissues/cell-specific phosphoproteomic profiles, to add to our database. The number of target kinases in KIOSS is limited because we need to establish effective agonist/antagonist treatment conditions for each kinase, but knockdown of each kinase by improved gene targeting methods will help us to systematically perform KIOSS analysis on kinases of interest. As a future perspective, we will continuously update KANPHOS by incorporating phosphoproteomics data based on our experimental results and links with other databases. We are preparing the data of phosphorylation-dependent signaling by several neurotransmitters in the specific brain regions, and mounting search options to narrow down the result by brain regions as the next step.Protein kinases such as Pak3, Limk1, ERK, and RSK2 have been implicated in intellectual disability ,57. Howe


Prosthetic rehabilitation of a partial or total maxillectomy with an obturator is the most acceptable treatment option. The hollowing of the obturator prosthesis is beneficial as it reduces the stresses over the underlying and surrounding tissues. A simple technique of fabricating a hollow bulb obturator has been discussed in this article. At the step of the packing of a denture, the hollow wax pattern of the defect area is formed with modeling wax. This hollow wax pattern is filled with water and is allowed to freeze to form an ice block. This ice block is removed from the wax pattern and is interposed between two layers for heat-cured acrylic resin and is then cured. After processing the denture, the water is retrieved by making a small hole in denture base, which is packed after hollowing with a cold cure acrylic resin. A lightweight prosthesis with a uniform thickness was achieved with a readily available and easily retrievable material, i.e., ice. Maxillectomy is a term used for the removal of the partial or total maxilla in patients with benign or malignant tumors. Rehabilitating such maxillary defects with free flap surgery has shown the best results functionally as well as esthetically. Prosthetic rehabilitation is the preferred modality only when surgical intervention is not possible . PatientThe hard and/or soft palate defect leading to oro-nasal or oro-antral communication often leads to physical and psychological trauma to the patient. Patients planned for maxillectomy often ask about the quality of life (QoL) that they should expect after surgery. There are many studies documented in the literature supporting a good quality of life provided by well-designed obturators prosthesis following maxillectomy –8.Obturator prosthesis is a removable appliance used to close the congenital or acquired tissue opening primarily of the hard palate and/ or soft palate. This bulb of the obturator engages the defect to gain retention for the prosthesis and form a barrier for oral and nasopharyngeal communication. The bulb of the obturator prosthesis increases the weight of the prosthesis. An increase in weight hampers the retention of the prosthesis that makes the prosthesis unacceptable by the patient .et al. [et al. [To avoid this, the obturator prosthesis should be made hollow. Various methods for hollowing the prosthesis with the use of different materials have been well documented in studies –15. Howeet al.  and Holt [et al. , respectThis article describes the technique for the fabrication of hollow obturator prosthesis using a freely accessible material, i.e., ice. A hollow wax pattern was formed by adapting modeling wax on the borders of the defect cavity. Water was filled in a hollow wax pattern to form an ice block. This ice block was placed at the time of curing as a hollowing material. After processing the denture, the ice water was retrieved by making a small hole in the denture base.The ice block used in this method as a material for hollowing could withstand the pressure of the compression molding technique and did not cause discoloration of the denture, as seen with many materials used for hollowing. It also provided ease of retrievability. Moreover, this technique maintained the even thickness of the hollow portion and also aided in the complete curing of the bulb portion.All clinical and laboratory procedures for fabricating the obturator prosthesis from preliminary impression until de-waxing of the trial denture were done conventionally  and 2. AThe wax pattern was removed carefully from the flask and was checked for the seal . Water wAfter setting the ice, the wax pattern was removed to get the ice block. . The heaOnce the curing was complete, the flask was allowed to bench cool for an hour. Then, it was opened, and the prosthesis was retrieved. A small hole was drilled in the prosthesis for the retrieval of water to make it hollow. Water effortlessly came out of the prosthesis, and then the hole was closed with the self-cure acrylic resin (DPI).The obturator prosthesis was weighted before and after hollowing  and 10. While restoring a large defect, reducing the weight of the prosthesis by making it hollow is beneficial, especially for obturator prosthesis, wherein the heavyweight of the acrylic resin threatens the retention, stability, and support of the prosthesis, making the patient feel uncomfortable. Moreover, the heavyweight of the prosthesis increases the stress over the residual alveolar ridge and supporting bone that leads to further resorption of the denture-bearing foundation .Several techniques and materials for the hollowing of the obturator prosthesis have been described in the literature to fabricate a lightweight prosthesis –16. Mateet al. also used ice to hollow the obturator prosthesis. The authors advocated for the use of crushed ice in the defect area for hollowing. However, crushed ice can not completely fill the defect space and will result in uneven thickness of the hollow bulb, resulting in a further increase in the weight of the prosthesis [In 1978, Schneider osthesis . To overThe prosthesis weighed 32.49 grams before and 19.75 grams t after retrieval of water. A reduction of 12.74 grams of weight after retrieval of water is remarkable, which is achieved by the hollowing of the prosthesis. The uniform thickness of the prosthesis, of around 1.3 mm, was measured with a vernier caliper. Hollowing the prosthesis affects the flexural strength. If the clinician is more concerned about reducing the weight of the prosthesis, then the strength will be compromised, affecting the mechanical properties of the material .When the boiling process is held in the pressure vessel, the water starts to boil at a higher temperature than 100°C. At a certain point, the energy absorbed by the water molecules makes them vibrate fast, and it attempts to escape and evaporate. However, the atmosphere around them exerts pressure on and thus limits the number of water molecules that can escape. Therefore, a minimal volume of water will expand, but the closed flask will limit it and not affect the accuracy of the obturator prosthesis.Ease of retrievability;Maintains even thickness of the hollow portion;Ice does not adhere to acrylic resin;The ice block can withstand the pressure of compression molding technique;Ice does not create porosities, so it does not compromise the strength of the prosthesis;Ice leaves no lines of demarcation to discolor the denture;Ice aids in the complete curing of the bulb portion;Total removal of ice is possible so complete hollowing of a prosthesis can be achieved;The procedure is not technique sensitive;Ease of availability;Inexpensive.The flexural strength of the hollowed structure was not determined.Other prostheses such as finger prosthesis, vaginal stent, complete dentures for severely atrophic ridges, and prosthesis for mandibular resection can also make use of ice to make them hollow. Future studies are suggested to evaluate the changes in the properties of the heat cure acrylic resin after processing with this technique. An experimental test program is recommended using numerical modeling as a tool to predict the behavior of the hollowed structure in terms of its mechanical properties . Also, tAn alternative, simple, economical, time-saving, and predictable approach for the hollowing of the prosthesis has been described in this article. This technique overcomes the disadvantages of the older techniques. The ice block has the advantage of retrievability ease, and it does not adhere to acrylic resin. It also provides an even thickness of acrylic resin during the packing stage and resists deformation during the closure of the mold.


Canine eumycetoma is a rare granulomatous disease caused by dematiaceous fungi.Madurella pseudomycetomatis was molecularly characterized.A 2-year-old Great Dane dog had a subcutaneous mass in the right thigh that was surgically removed. Grossly, numerous black-grains were visible. Histologically subcutaneous pyogranulomas were centered on myriads of pigmented fungal elements.  Curvularia spp., Cladophialophora bantiana, Pseudollechiaria bodydii and Madurella mycetomatis are the most common fungi that cause canine eumycetoma, with either subcutaneous, osseous, intrabdominal or disseminated lesions . Thi. ThiMaduMadurella pseudomycetomatis so far only reported in human literature.In addition, the molecular characterization performed in this study allowed identification of the new species Madurella pseudomycetomatis should also be considered as possible etiology of canine eumycetoma in dogs.With this report, the presence of There are none.


In addition, our work reveals the specific modulation of the phosphorylation of eIF4G1 at Ser1147 in the vulnerable region, with increased levels and colocalization with eIF4E in response to IR. These findings contribute to elucidate the molecular mechanism of protein translation regulation that underlies in the balance of cell survival/death during pathophysiological stress, such as cerebral ischemia.Ischemic strokes are caused by a reduction in cerebral blood flow and both the ischemic period and subsequent reperfusion induce brain injury, with different tissue damage depending on the severity of the ischemic insult, its duration, and the particular areas of the brain affected. In those areas vulnerable to cerebral ischemia, the inhibition of protein translation is an essential process of the cellular response leading to delayed neuronal death. In particular, translation initiation is rate-limiting for protein synthesis and the eukaryotic initiation factor (eIF) 4F complex is indispensable for cap-dependent protein translation. In the eIF4F complex, eIF4G is a scaffolding protein that provides docking sites for the assembly of eIF4A and eIF4E, binding to the cap structure of the mRNA and stabilizing all proteins of the complex. The eIF4F complex constituents, eIF4A, eIF4E, and eIF4G, participate in translation regulation by their phosphorylation at specific sites under cellular stress conditions, modulating the activity of the cap-binding complex and protein translation. This work investigates the phosphorylation of eIF4G1 involved in the eIF4E/eIF4G1 association complex, and their regulation in ischemia-reperfusion (IR) as a stress-inducing condition. IR was induced in an animal model of transient cerebral ischemia and the results were studied in the resistant cortical region and in the vulnerable hippocampal CA1 region. The presented data demonstrate the phosphorylation of eIF4G1 at Ser Worldwide, a stroke is the second most common cause of death and a major origin of long term disability in adults ,2,3. Brirestored ,8,9,10. restored ,12.Cellular protein synthesis is generally determined by a rate-limiting stage, the initiation of translation. The initiation step is a highly regulated mechanism with an important checkpoint in the 40S ribosomal subunit recruitment to the 5′ end of a specific mRNA ,14. In s1108, Ser1148, and Ser1192; equivalent positions to Ser1147, Ser1187, and Ser1231, respectively, in the human canonical sequence of eIF4G1 , sequence referred from here on. In addition, some reports have confirmed that an intense inhibition of mTOR activity occurs as a consequence of ischemic brain damage [1231 site is a multitarget residue that can be also phosphorylated by the extracellular signal-regulated kinase 1 and 2 (ERK1/2) [1185, regulated by protein kinase C alpha (PKCα) [The scaffolding protein eIF4G consists of three protein family members, eIF4G1 (referred as eIF4GI)—the main form with the highest expression—eIF4G3 —a minor form with the lowest expression—and eIF4G2 , which share homology with eIF4G1 and eIF4G3 in the middle and C-terminal regions ,20. eIF4n damage ,26,27,28(ERK1/2) —whose ex(ERK1/2) —and addi(ERK1/2) . Finallya (PKCα) . Despite1147, Ser1185, and Ser1231 phosphorylation sites as three different targets of the mTOR, PKCa, and ERK1/2 signaling pathways, respectively, related with ischemic stress. The aim of this study was to investigate the changes in these phosphorylations in response to IR stress, by analyzing their consequences in the eIF4E/eIF4G1 association found in resistant and vulnerable brain regions following IR damage. The presented data demonstrate the phosphorylation of eIF4G1 at Ser1147, Ser1185, and Ser1231 in brain regions both in control and ischemic conditions, being the phosphorylation of eIF4G1 at Ser1147 the only one found in the eIF4E/eIF4G association complex from the cap-containing matrix (m7GTP-Sepharose). In addition, our work reveals the specific modulation of the phosphorylation of eIF4G1 at Ser1147 in the hippocampal CA1 region, with increased levels and colocalization with eIF4E in this vulnerable brain region in response to ischemia-reperfusion.With all these phosphorylation regulation sites reported, we decided to study Ser1147, Ser1185, and Ser1231 in the human eIF4G1 sequence [1140, Ser1178, and Ser1222 in the rat eIF4G1 sequence, respectively, and we could consider them as potential phospho-regulatory sites of interest to study under IR stress in our experimental model. Moreover, these eIF4G1 phospho-sites were localized in the interdomain linker (IDL), a dynamic region described in between HEAT  domains 1 and 2 of eIF4G1, enriched in phosphorylation sites that respond to cell stimulation signaling [To explore the post-translational modification of eIF4G1 by protein phosphorylation, we analyzed the main specific phosphorylation region of eIF4G1 described in the human amino acid sequence, and then we compared the sequence of eIF4G1 in humans and rats with bioinformatics tools. Our sequence-based alignment revealed that this region is highly conserved and that rat eIF4G1 is a counterpart to human eIF4G1 A. Thus, sequence ,31, corrignaling ,31  and eIF4G1 immunoprecipitation assays. To assess the above results, we analyzed the colocalization of eIF4G1 phosphorylated at Ser animals A. The imn in R3d A. The co animals B, confirThere is a strong correlation between translation inhibition in vulnerable regions to ischemia, and ischemic-induced neuronal death . The tra1147, Ser1185, and Ser1231, belonging to the IDL domain, that could be phosphorylated by three different signaling pathways, mTOR, PKCα, and ERK1/2, respectively, that are related to ischemic stress. These phospho-sites have a precise sequence alignment when comparing human and rat eIF4G1, and are specifically recognized by the same anti-phospho antibodies. It is worthwhile to note that the selected phospho-sites can differ by one amino acid in the sequence of published reports [Eukaryotic cells have two homolog isoforms of eIF4G, eIF4GI (eIF4G1), and eIF4GII (eIF4G3) that contain a similar C-terminal domain, eIF4G1 being the main form in mammalian cells . The C-t reports ,31,32, t1147, Ser1185, and Ser1231 is present in brain tissue, without changes between the different regions studied nor between the control or the ischemic condition; (ii) among the three phospho-sites studied, the phosphorylation of eIF4G1 at Ser1147 was the only one found in the eIF4E/eIF4G association complex from the cap-containing matrix (m7GTP-Sepharose); (iii) phosphorylation of eIF4G1 at Ser1147 is increased in the vulnerable CA1 region in response to IR stress, as demonstrated eIF4G1 immunoprecipitation assays; and (iv) phosphorylation of eIF4G1 at Ser1147 is colocalized with eIF4E in brain sections, with higher levels in the CA1 region after IR.In this report, we explore the hypothesis that phosphorylation of eIF4G1 can be a differential control mechanism between brain regions that are resistant and vulnerable to ischemia. For this purpose, we studied whether the phosphorylation of eIF4G1 could operate in the eIF4E/eIF4G association complex as a regulatory mechanism in the translation inhibition induced by ischemia-reperfusion, analyzing their phosphorylation status in SHC3d control and R3d ischemic samples of the cortical resistant and CA1 vulnerable regions. This study has been conducted in ischemic young adult animals, although aging and co-morbidities are important determinants of outcome after an ischemic stroke. Since this study investigates a molecular mechanism in brain regions vulnerable to ischemia, the inclusion of a co-morbidity in the study would mean that the molecular mechanism could be associated with processes related to co-morbidity, rather than with a precise knowledge of the ischemic pathophysiology. In addition, the results obtained may be specific to the co-morbidity studied and exhibit new effects in a different co-morbidity. Therefore, co-morbidity has been avoided in this study. From the results achieved in this report, several novel findings can be concluded: (i) phosphorylation of eIF4G1 at Ser1147, Ser1187, and Ser1231 in response to serum-stimulation, resulting in a fully active eIF4G1 [1185 and Ser1231, respectively, in a mitogenic signal transduction, inducing a rearrangement of the complex with eIF4A/eIF4B/eIF3 and increasing the mitogen-activated protein kinase-interacting kinase Mnk1 association [1147, Ser1185, and Ser1231 was detected in both hippocampal and cortical brain regions, in control and ischemic conditions, although changes in these phospho-sites were not found under IR stress. Interestingly, while the role of phosphorylation of eIF4G1 is not well established [7GTP-Sepharose), eIF4G1 immunoprecipitation and eIF4E/phospho-eIF4G1 colocalization, only phosphorylation of eIF4G1 at Ser1147 was detected. Since the eIF4G1 present in the cap-containing matrix is bound to eIF4E, it means that it is “active” eIF4G1 for protein translation—i.e., eIF4G1 associated with eIF4E forming active eIF4F complex—and because it is only phosphorylated at Ser1147 (not on Ser1185 nor Ser1231), it can be concluded that this phosphorylation characterizes to “active” eIF4G1, even though the precise mechanism by which this phosphorylation may affect the activity of eIF4G1 is unknown. Consistent with different intracellular pools of eIF4G1, changes in intracellular compartmentalization of eIF4G and eIF4G1 phosphorylated at Ser1147 have been reported in different brain regions [1147 with protein aggregates associated with translation inhibition, our results identify this phosphorylation in the eIF4E/eIF4G1 complex, active for protein translation, in agreement with other reports that associate this phosphorylation with increased translation [Several studies have shown different signaling pathways involved in phosphorylation of eIF4G1 ,29,31,32 regions . Howevernslation . 1147 in the CA1 region under IR stress, phosphorylation that has been related to an active eIF4E/eIF4G1 complex assembly and with increased protein translation rates [1147 may serve to enhance the recruitment of eIF4E in response to a stress situation—IR stress—and make feasible a minimal protein synthesis required, e.g., for programmed cell death mechanisms. In this sense, the results that show higher levels of eIF4G1 phosphorylated at Ser1147 after IR stress  and translation inhibition where neuronal death occurs several days after ischemia [1147 to compete with 4E-BPs) bound to eIF4E and maintain a limited cap-dependent translation to progress in the apoptotic mechanism. To confirm the physiological role of eIF4G1 phosphorylation at Ser1147 in the initiation step of protein translation and elucidate whether this phosphorylation could be a target to recover brain regions vulnerable to ischemia, further studies would be needed to assess the implications of this phosphorylation in the survival/death balance in brain tissue under IR stress. Overall, our results study three phosphorylation sites, Serischemia ,51,52. OAll general products were purchased from Sigma-Aldrich  except those indicated in the text. Chemicals used in the gel electrophoresis were obtained from Bio-Rad  and Cytiva . Rabbit polyclonal anti-phospho-eIF4G1 (Ser1108)—Ser1147 in the human canonical sequence Q04637-1 from UniProt, see above—antibody (#2441) was from Cell Signalling Technology . Rabbit polyclonal anti-phospho-eIF4G1 (Ser1185) antibody (bs-4003R) was from Bioss . Rabbit polyclonal anti-phospho-eIF4G1 (Ser1231) antibody (GTX79087) was from GeneTex . Mouse monoclonal anti-eIF4E antibody (610269) was from BD Transduction Laboratories . Mouse monoclonal anti-eIF4G1  and goat polyclonal anti-eIF4G1  antibodies were from Santa Cruz Biotechnology . Mouse monoclonal anti-β-tubulin antibody (T5201) was from Sigma-Aldrich . Secondary antibodies used for immunodetection were anti-mouse (NA931), -rabbit (NA934) , or -goat (sc-2020) IgG peroxidase-conjugated antibodies . Secondary antibodies used for immunohistochemistry were donkey anti-rabbit IgG (H+L) Alexa Fluor 488-conjugated antibody (A-21441) from Thermo Fisher Scientific  and goat anti-mouse IgG (H+L) Alexa Fluor 568-conjugated antibody (A-11031) from Thermo Fisher Scientific .https://www.uniprot.org/, accessed on 16 December 2021). Sequence alignment analysis of eIF4Gs was performed with pairwise bioinformatics tools (EMBOSS Needle) of EMBL-EBI , prior to sequence similarities analysis performed by ESPript3.0 program  [http://smart.embl-heidelberg.de/, accessed on 10 August 2021). The sequences of human and rat eIF4G1, Q04637 (IF4G1_HUMAN) and D4AD15 (D4AD15_RAT), respectively, were obtained from the UniProt database (er 2021) . The ana2/20% O2) during the dissection of common carotid arteries, then ischemia was induced by carotid occlusion with atraumatic clips for 15 min. Next, the clips were removed to allow reperfusion   by the standard four-vessel occlusion (4VO) model, as described previously ,54. In berfusion . The boderfusion . g for 15 min to collect post-mitochondrial supernatant (PMS). All procedures were performed at 4 °C. The PMS fraction was kept at −80 °C until use, and protein concentrations in each sample were determined by Bradford assay . For brain section preparations, SHC3d and R3d animals were sacrificed by intracardiac perfusion under deep anesthesia with 200 mL of saline with heparin through the left ventricle, followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for brain fixation. After that, brains were removed and subsequently post-fixed by immersion in the same fixative solution overnight at 4 °C. The brains were then washed sequentially with 10%, 20%, and 30% sucrose/PBS (w/v), and included in Tissue-Tek O.C.T. , before freezing at −80 °C.After euthanasia, the cerebral cortex and hippocampal CA1 region from both control and R3d ischemic animals were rapidly dissected and instantly frozen in dry ice for future procedures. The samples of brain regions were homogenized 1:5 (w/v) in buffer A , as previously described ,55. The 7GTP)-Sepharose  was used, as described previously [7GTP-Sepharose and incubated for 30 min at 4 °C in modified buffer A containing 100 mM potassium chloride and 100 μM GTP. The beads were centrifuged at 2500× g for 5 min and washed three times (5 min each), with the same buffer. Bound proteins were eluted from m7GTP-Sepharose with loading buffer  and analyzed by Western blotting after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) (see below). To study eIF4G binding to eIF4E, a cap-containing matrix-7-methyl-GTP  for SDS-PAGE and Western blotting (see below). Control experiments were performed in parallel using samples without anti-eIF4G antibody incubation.PMS samples (100 μg) were incubated with goat polyclonal anti-eIF4G antibody (1 μg) overnight, and then further incubated with Protein G-Agarose 4  for 1 h at 4 °C on a rotary shaker . The imm7GTP-Sepharose-bound proteins, or eIF4G immunoprecipitates of each experimental condition were resolved by SDS-PAGE  . Proteins were transferred onto PVDF membranes  and blocked  in 0.1 M PBS pH 7.4 for 1.5 h at room temperature. The membranes were then incubated for 1 h at room temperature or overnight at 4 °C with a primary antibody against the specific protein to be detected, washed three times (10 min each), and incubated for 1 h with peroxidase-conjugated anti-mouse, -rabbit or -goat IgG secondary antibody. Finally, blots were developed with the Clarity Western ECL Substrate reagent  and ECL-Prime, or ECL-Select reagent . Detection of phosphoproteins was performed sequentially with phospho-specific primary antibodies and re-probed after stripping with a total-protein-specific primary antibody. The blots were quantified using Quantity One software  with β-tubulin as internal standard. Protein levels were calculated in arbitrary units (A.U.) or expressed relative to total reference protein levels (ratios). Protein markers (range: 12–225 kDa)  were used to calculate the apparent molecular mass.PMS samples (35 μg), mCoronal brain sections, with the hippocampus region included, were prepared by cryostat sections at the interaural level +5.7 ± 0.2 mm on Real Capillary Gap microscope slides , and were subsequently post-fixed with 4% PFA/PBS for 5 min at room temperature. After washing three times in PBS (5 min each), the sections were permeabilized with 10 mM sodium citrate pH 6.0 for 3 min at 95 °C, followed by cooling for 20 min at room temperature and washed in PBS for three times. Brain sections were then incubated with blocking solution  for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. After PBS washing three times, the sections were incubated with fluorochrome-conjugated secondary antibodies for 1 h at room temperature and washed three times again in PBS. Sections were mounted with coverslips in anti-fade solution containing p-phenylenediamine in glycerol-buffer and 30 μM bisbenzimide (Hoechst 33342) for nuclear staining. The fields of the cerebral cortex and hippocampal CA1 region of a given section were examined using a confocal laser-scanning microscope MRC-1024  at a wavelength excitation of 488 and 568 nm controlled by LaserSharp software . The acquisition settings were kept constant for each image. Data acquisition was carried out sequentially to avoid interference with emission spectra between fluorochromes. Experiments with fluorochrome-conjugated secondary antibodies that omitted the primary antibody were used to check the background staining. Image acquisitions of more than 6 different fields and sections per sample were made with a 40x objective (1.8 zoom). LaserSharp software provides quantitative analysis of the degree of colocalization by evaluating the percentage of green objects that colocalize with red objects in the area of interest.t-test for comparisons between the cerebral cortex and hippocampal CA1 region. All statistical analyses were performed with Prism software  and the level of significance was established at α = 0.05.Data are expressed as mean ± SE for the indicated number of experiments. Statistical significance between experimental groups was determined using the one-way ANOVA test and, when significant, it was followed by the Newman–Keuls post-test for multiple group comparisons, or the Student’s


Km for ADP twofold. The half maximal inhibitory concentration (IC50) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the Km for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose‐dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31P‐magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP‐stimulated state 3 respiration and increased the apparent  AACADP/ATP carrierANTadenine nucleotide translocaseGPMglutamate, pyruvate, malateIFMinterfibrillar mitochondriaLCACoAlong chain acyl Co enzyme APCoApalmitoyl CoAPCarnpalmitoyl carnitineSSMsubsarcolemmal mitochondriaT2Dtype 2 diabetes1In type 2 diabetes (T2D), cardiac fatty acid (FA) uptake, oxidation, and storage are all increased, resulting in increased dependence on FA for energy generation and increased accumulation of lipid intermediates within the cardiomyocyte.Mitochondrial respiration accounts for 95% of ATP generation in the heart, and regeneration of ATP from ADP and phosphate (Pi) requires transport of these molecules across the highly impermeable mitochondrial inner membrane. Under normal physiological settings, ADP is imported into and ATP exported from the matrix by the ADP/ATP carrier (AAC), also known as the adenine nucleotide translocase (ANT). Whereas the phosphate carrier (PiC) has relatively weak control of the rate of oxidative phosphorylation, as shown by 60% depletion of PiC having no effect on mitochondrial respiration,Research over many decades has shown that LCACoAs can reversibly inhibit the activity of the AAC.This study set out to investigate whether oxidative phosphorylation is acutely regulated by LCACoA in the heart, and to understand how this regulation changes in the lipotoxic environment of diabetes. Here we demonstrate that PCoA inhibits respiration in control mitochondria via regulation of the phosphorylation apparatus, but this effect is blunted in mitochondria from diabetic hearts. This has consequences during ischemia, as in the healthy heart PCoA serves to protect mitochondria by inhibiting the AAC, and preventing ATP hydrolysis via reverse ATP synthase activity. This protection of energy metabolism during ischemia is lost in diabetes, with diabetic hearts losing membrane potential and ATP more rapidly during ischemia.22.1All animal experiments conformed to Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 and were approved by a local ethics committee. T2D was induced as previously described,2.2Subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated from the heart according to our previously published protocol.Km and Vmax values, mitochondrial respiration in the presence of PCoA or PCoA + Carn were carried out as described above (following a 3‐minutes incubation), with the exception that the concentration of ADP was varied between 40 and 1000 µM, prior to the fitting of data to Michaelis‐Menton kinetics via nonlinear regression. For the measurement of inhibitory concentration (IC50) values, experiments were performed as described above with 200 µM ADP to induce state 3 respiration, but with the concentration of PCoA varied between 0 and 52 µM. All derived quantities were calculated using nonlinear regression analysis in GraphPad Prism 8.For the generation of 2.3−ΔΔCT method.RNA was extracted from frozen cardiac tissue using the Qiagen RNeasy Mini Kit, and converted into cDNA using a high‐capacity cDNA reverse transcriptase kit (Applied Biosystems). Quantitative polymerase chain reaction was performed using the SYBR detection method, and a StepOnePlus Real‐Time PCR system (Applied Biosystems). Primers for AAC1 , AAC2  and GAPDH  were used. Analysis was carried out using the 22.4Lysates prepared from frozen cardiac tissue and isolated mitochondria were used for western blotting as previously described.2.5Citrate synthase activity was measured according to the protocol of Srere.Acetylation of AAC in SSM and IFM was assessed using mass spectrometry, following immunoprecipitation using an anti‐acetyl lysine antibody  to isolate acetylated proteins. The immobilised proteins were digested by adding SMART trypsin (Thermo Fisher), and the eluted peptides were desalted using SepPak reverse phase columns and evaporated to dryness. Mass Spectrometry data was acquired by an Orbitrap Fusion Lumos after chromatographic separation with a Dionex Ultimate 3000 (Thermo Fisher) according to our previously published protocol.Long chain acyl CoAs were extracted from cardiac tissue according to the method of Hoppel et al.Using the values obtained, the mitochondrial LCACoA was calculated using the equation below:With the values taken from the literature for the specific gravity of cardiac tissue,2.6Respiration media was supplemented with 5‐µM safranine O in 96‐well black plates, and membrane potential was measured using a FLUOstar Omega plate reader using an excitation wavelength of 495 nm and emission wavelength of 586 nm. Absolute values were calculated using a potassium clamp as described by Akerman and Wikstrom,Substrates glutamate (20 mM), malate (5 mM) and pyruvate (10 mM), ADP (200 µM), ATP (10 mM), and inhibitors myxothiazol (8 µM), carboxyatractyloside (CATR 2.5 mM), oligomycin (10 µM), and BMS199264 (3 µM) were added, and fluorescence measured after stabilisation.2.731P‐MRS spectra . This was followed by the acquisition of time‐resolved spectra  throughout 15 minutes of full flow perfusion, followed by 32 minutes of low‐flow ischemia at 0.5 mL/minutes/gww. After the end of this acquisition, a further 50 µL of PPA was injected into the left ventricular balloon prior to the acquisition of another fully‐relaxed spectrum, with this process repeated to allow for absolute quantification of metabolite levels.Rat hearts were excised and arrested in ice‐cold Krebs Henseleit (KH) buffer, the aorta was cannulated, and the hearts were perfused in Langendorff‐mode with warmed KH buffer according to our previous publication.After acquisition, spectra were quantified using a MATLAB implementation of the AMARES algorithm using previously published prior knowledge that takes into account the known spectral locations of ATP and its J‐coupling, PCr, PPA, and Pi, which permits the absolute quantification of intracellular energy metabolite concentrations.2.8P value of less than .05, using GraphPad Prism 8. Data were analyzed using one‐way ANOVA with Dunnett's test for multiple comparison  as substrates. Incubation of the mitochondria for 3 minutes with this GPM mix supplemented with PCoA, resulted in decreased state 3 respiration in SSM and IFM from control and diabetic hearts Figure . For PCo3.2Km measurements .In order to investigate this change in PCoA kinetics further, ADP‐stimulated respiration rates were measured after incubation with a range of PCoA concentrations, to determine the ICn Figure . IC50 va3.3To try to understand the changes in sensitivity to PCoA inhibition in diabetes, we looked at expression of the phosphorylation apparatus. Given the high control coefficient of the AACThe different AAC isoforms have different kinetics for ADP transport, which may also result in different sensitivities to LCACoA inhibition. The mRNA expression of AAC1 was significantly increased in diabetic hearts compared with control hearts, whereas the mRNA expression of AAC2 was not significantly different between groups Figure . To see Given that no difference in isoform protein expression could be identified, specific post‐translational modifications, acetylation and palmitoylation, were measured, which have been mechanistically implicated in high fat exposure and nutrient excess. There were no significant differences in AAC1 or AAC2 acetylation between control and diabetic SSM or IFM Figure . In addi50 values. Liepinsh et al showed in response to ischemia cardiac LCACoA concentrations increased to 11 nmol/gww,50 values. Thus, the inhibitory effects of LCACoA inhibition on the AAC in diabetes may not be physiologically relevant under baseline conditions, but instead become significant in response to pathological ischemia.The concentration of the long chain acyl CoA's was measured within the myocardium, and was found to be increased by 74% in diabetic hearts Figure . Due to 3.4To test our hypothesis that PCoA inhibition of the AAC may serve a role in ischemia, a model of mitochondrial ischemia was developed. Mitochondrial membrane potential was measured in the presence of substrate and was found to be between −167 and −174 mV across the different mitochondrial populations Figure , in agreIn both SSM and IFM populations, PCoA was also able to prevent this membrane repolarisation by ATP, in a concentration‐dependent manner, with significance present from 12 µM onwards in control mitochondria, but only at 15 µM in diabetic mitochondria Figure . When a 3.531P‐spectroscopy was used to measure high energy phosphates ATP and PCr, within the myocardium (Figure Intact contracting control and diabetic hearts were perfused within an 11.7 T magnet, and m Figure . Under bm Figure . Hearts m Figure . The ATPm Figure . This ocm Figure . Whilst 4Lipid intermediates accumulate within the heart in diabetes, and understanding their role in regulating cellular processes will help us understand why cardiomyocyte function is compromised in diabetes. Here we demonstrate that PCoA can acutely inhibit mitochondrial respiration in the heart, via inhibition of the phosphorylation apparatus. We demonstrate that this inhibition is blunted in the diabetic heart, an environment with elevated concentrations of LCACoA's. While this regulation may not be sufficient to inhibit respiration under normal physiological concentrations, we demonstrate a beneficial role for PCoA‐mediated AAC inhibition during ischemia in control hearts. Thus, lipid intermediates can play a role in protecting the heart in ischemia, but this is blunted and protection is decreased in diabetes during myocardial ischemia.4.150 and no significant increase in the Km for ADP when PCoA is present. This insensitivity to the effects of PCoA may represent an adaptation to the high fat environment of the T2D heart, given that diabetic mitochondria will be chronically bathed in higher concentrations of lipid intermediates.Control mitochondria acutely decrease mitochondrial respiration when exposed to PCoA, an effect that is exclusive to the CoA ester form of palmitate, and is absent if the PCoA can be transported into the mitochondria for oxidation. The site of PCoA‐mediated respiratory inhibition can be localised to the phosphorylation apparatus, and both experimental and computational‐based modular kinetic analysis have confirmed the predominant site of PCoA control is the AAC.Suppression of mitochondrial respiration via competitive inhibition of the AAC by LCACoA's has been reported previously in liver and skeletal muscle. Here we show this is a preserved mechanism also occurring in the heart, and is an interesting observation given that the heart is a primary consumer of FAs and has higher ATP demand per gram of tissue than any other organ. At first glance, it would appear undesirable for the heart to have its energy generation compromised by an intermediate of its predominant energy generating fuel, which led us to consider situations when it may be beneficial for this regulation to occur.4.250 values and LCACoA concentrations, we would suggest any control under normal conditions would be minimal. However, our data provides an alternative role for this regulation in the pathological setting of ischemia, when the concentrations of LCACoA have been demonstrated to increase by up to 300%.It has been debated for many years as to whether there is a physiological role for regulation by PCoA of the AAC. Based on our measured ICIn diabetes, we have shown that the ability of PCoA to preserve the membrane potential is suppressed. This would result in diabetic hearts losing ATP at a greater rate during ischemia, and becoming energy depleted more rapidly. To extrapolate our findings from isolated mitochondria to the intact beating heart, we demonstrate that as the diabetic heart enters ischemia the rate of ATP loss is greater than that seen in control hearts, under the same ischaemic flow rate. The diabetic heart suffers a “double whammy” under these ischaemic conditions, as not only is ATP hydrolysis by the mitochondria accelerated but the upregulation of anaerobic glycolysis and cytosolic ATP synthesis is suppressed.4.3Genetic studies on AAC have generated confusing findings, with both overexpression and deletion resulting in improved insulin sensitivity.Understanding the effects of lipid intermediates on mitochondrial and cellular functions is important in lipid‐overloaded settings such as diabetes and ischemia, to understand the pathophysiological mechanisms of these diseases. Previous research has shown that long chain acyl carnitines, such as PCarn can exert deleterious effects on calcium handling, membrane permeability and respiratory coupling in cardiac mitochondria.In conclusion, LCACoA can inhibit the phosphorylation apparatus and decrease respiration via interaction with AAC, and while this is unlikely to play a significant role in regulating energetics under normal physiological conditions, in ischemia this prevents wasteful ATP hydrolysis to regenerate the membrane potential. In T2D, this regulation of the AAC is blunted and mitochondria become resistant to the suppressive effects of PCoA, resulting in more rapid ATP depletion during ischemia.The authors have stated explicitly that there are no conflicts of interest in connection with this article.M. Kerr, W. Fuller, K. Clarke and L.C. Heather designed research. M. Kerr, K.M.J.H. Dennis, C.A. Carr, G. Berridge, S. Rohling, C.L. Aitken, and C. Lopez performed research. M. Kerr, D.J. Tyler, J.J. Miller and L.C. Heather analyzed data. M. Kerr and L.C. Heather wrote the manuscript.Fig S1Click here for additional data file.