We here analyze the effect of S47 modification in fibroblast cell lines stably expressing S47E phosphomimetic Cytc, unphosphorylated WT, or S47A Cytc. Our results show that S47E Cytc results in partial inhibition of mitochondrial respiration corresponding with lower mitochondrial membrane potentials (ΔΨm) and reduced reactive oxygen species (ROS) production. When exposed to an oxygen-glucose deprivation/reoxygenation (OGD/R) model simulating ischemia/reperfusion injury, the Cytc S47E phosphomimetic cell line showed minimal ROS generation compared to the unphosphorylated WT Cytc cell line that generated high levels of ROS upon reoxygenation. Consequently, the S47E Cytc cell line also resulted in significantly lower cell death upon exposure to OGD/R, confirming the cytoprotective role of S47 phosphorylation of Cytc. S47E Cytc also resulted in lower cell death upon H2O2 treatment. Finally, we propose that pro-survival kinase Akt (protein kinase B) is a likely mediator of the S47 phosphorylation of Cytc in the brain. Akt inhibitor wortmannin abolished S47 phosphorylation of Cytc, while the Akt activator SC79 maintained S47 phosphorylation of Cytc. Overall, our results suggest that loss of S47 phosphorylation of Cytc during brain ischemia drives reperfusion injury through maximal electron transport chain flux, ΔΨm hyperpolarization, and ROS-triggered cell death.We previously reported that serine-47 (S47) phosphorylation of cytochrome  It functions at the intersection between cellular respiration and intrinsic apoptosis, linking the two pathways. Cytc plays a role in cellular respiration by functioning as the electron carrier between complex III and complex IV  in the mitochondrial electron transport chain (ETC) [c from the mitochondria into the cytosol is considered the committing step for intrinsic apoptosis. Cytc acts as a trigger of apoptosis by interacting with apoptosis protease activating factor-1 (Apaf-1) to form the apoptosome, which activates caspase-9 to initiate the caspase cascade, the executioner pathway of intrinsic apoptosis [c serves as a key metabolic regulator and orchestrator of cellular life and death decisions. In addition to respiration and apoptosis, Cytc has multiple other cellular functions, such as reactive oxygen species (ROS) scavenging, cardiolipin peroxidase activity, redox-coupled protein import, and ROS formation via the p66Shc protein [c to oxygen is the proposed rate-limiting step of the ETC [c are tightly regulated by all major regulatory mechanisms, such as allosteric regulation by ATP, expression of tissue-specific isoforms, and reversible post-translational modifications (PTMs), of which phosphorylations are physiologically most relevant [Cytochrome in (ETC) . In contpoptosis ,4. Cytc  protein . The COX the ETC ,6,7. Therelevant .c—Y97 [c pool, making it a biologically significant modification. Furthermore, this phosphorylation was found to be completely lost under ischemia, implying a critical role for this modification in the brain under healthy basal conditions. Our initial study used in vivo phosphorylated and S47E phosphomimetic Cytc for the in vitro functional characterization with consistent results between the two systems, demonstrating that our phosphomimetic replacement serves as a good model. We reported that brain COX activity was reduced by 54.3%, caspase-3 activity was reduced by 64.5%, and cardiolipin peroxidase activity was reduced by about 50% in the presence of the S47 phosphomimetic S47E compared to unphosphorylated WT Cytc [So far, five tissue-specific phosphorylations have been mapped on mammalian Cytc—Y97 ,9, Y48 [c—Y97 , T28 [11c—Y97 , and T58c—Y97 . These mc—Y97 ,14. We r WT Cytc . These e WT Cytc .c, which are lost during ischemia, play a central role in brain ischemia/reperfusion injury through the following mechanism: Under ischemic stress calcium builds up in the mitochondria, leading to dephosphorylation of Cytc and other ETC proteins. This causes hyperactivation of the mitochondrial ETC during reperfusion when oxygen becomes available again, resulting in pathologically high mitochondrial membrane potentials (ΔΨm). In turn, this triggers an exponential increase in the generation of ROS, which causes further tissue damage and initiates cell death cascades [c-centered mechanism mediated by loss of its phosphorylations. Furthermore, we propose that S47 phosphorylation is likely mediated by the pro-survival kinase Akt under normal conditions.Globally, strokes are the second leading cause of death and the third leading cause of disability . Most stcascades . Our datc cloned into pBABE-puro expression plasmid  was used to generate S47A and S47E Cytc variants using site-directed mutagenesis , as previously described [c expression constructs were stably transfected into Cytc double-knockout mouse lung fibroblasts  using Transfast transfection reagent  in a 1:1 transfection reagent to DNA ratio, as described in the manufacturer’s protocol. The transfected cells were cultured in DMEM supplemented with 10% FBS , 100 µg/mL primocin , 1 mM sodium pyruvate, and 50 mg/mL uridine in selection media with 3 µg/mL puromycin at 37 °C in 5% CO2.WT Cytescribed , with thc expression in stably-transfected Cytc double-knockout mouse lung fibroblasts was confirmed with a mouse Cytc antibody  used at a 1:1000 dilution in 5% dry milk. Briefly, cells were lysed in RIPA lysis buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich). The cellular protein lysates were quantified using the DC protein assay  based on the Lowry method. Thirty micrograms of total cell lysate of each cell line were run on a 10% tris-tricine SDS-PAGE gel in the presence of anode  and cathode buffers . The gel was transferred onto a PVDF membrane (Bio-Rad) using a semi-dry apparatus (Bio-Rad) and incubated with the primary antibody overnight at 4 °C. The same lysates were also probed for subunits of the ETC complexes with the following antibodies at a dilution of 1:1000: NDUFB6 (NADH:Ubiquinone Oxidoreductase Subunit B6) for complex I , SDHA (Succinate Dehydrogenase Complex Flavoprotein Subunit A) for complex II , UQCRC1 (Ubiquinol-Cytochrome c Reductase Core Protein 1) for complex III , COX4 (Cytochrome c Oxidase Subunit 4) for complex IV , and ATP5A (ATP Synthase Subunit Alpha) for complex V . Cell lysates were also probed with a 4-hydroxynonenal monoclonal antibody (clone 12F7) . The blots were probed for tubulin  as a loading control. HRP-conjugated mouse and rabbit secondary antibodies  were used at a 1:5000 dilution. The blots were visualized using the HyGLO chemiluminescent HRP detection reagent .Cyt2-free incubator for 1 h, and the basal oxygen consumption rate (OCR) was measured, followed by mitochondrial stress test with sequential injections of oligomycin (1 μM), trifluoromethoxy carbonylcyanide phenylhydrazone , and rotenone/antimycin A (1 μM) in an XFe24 Seahorse bioanalyzer , according to the manufacturer’s protocol. Extracellular acidification rate (ECAR) was determined as a measure of the rate of glycolysis in Seahorse medium supplemented with 10 mM sodium pyruvate and 10 mM glucose.Cells were seeded at a density of 25,000/well in a gelatin-coated XF24 plate . After overnight incubation, the growth medium was replaced with 675 µL of Seahorse media (#D5030 DMEM) supplemented with 10 mM galactose without FBS or phenol red. In independent experiments, the growth medium was also replaced with 675 μL of seahorse media supplemented with 10 mM sodium pyruvate and 10 mM glucose. The cells were incubated in a COm) was measured using the ratiometric JC-1  probe . Cells were seeded  in a black 96-well plate . The growth medium was changed into FBS-free, phenol red-free high glucose (4.5 g/L) DMEM cell culture medium supplemented with 0.5 µM JC-1 and incubated for 30 min. Cells were washed with 1× PBS twice. Green fluorescence (excitation 485 nm/emission 527 nm) and red fluorescence (excitation 485 nm/emission 590 nm) of the cells were measured using a Fluorskan Ascent FL plate reader (Thermoscientific). ΔΨm was represented as a ratio of red/green fluorescence. ΔΨm uncoupler FCCP (1 µM) was used as a negative control.Mitochondrial membrane potential . Cells were seeded  on a 24-well plate and incubated in FBS-free, phenol red-free high glucose (4.5 g/L) DMEM cell culture medium supplemented with 5 µM MitoSOX for 30 min at 37 °C. Cells were washed with 1× PBS. Fluorescence (excitation 510 nm/emission 580 nm) was measured using a Synergy H1 plate reader .Cultured fibroblasts were collected by scraping. The pellets were immediately frozen in less than 90 s and stored at −80 °C until analysis. The samples were boiled in the presence of 300 µL of boiling buffer  and sonicated on ice. The sample lysates were diluted 300-fold. The ATP concentration of 40 µL diluted lysate was measured using the ATP bioluminescence assay kit HS II , according to the manufacturer’s protocol, using an Optocomp 1 luminometer . The results were normalized to the protein concentration determined using the DC protein assay kit (Bio-Rad).c variants were exposed to transient oxygen-glucose deprivation (OGD). Cells were seeded and cultured according to our standard protocol described above. The media was exchanged into glucose-free, FBS-free, phenol red-free DMEM  bubbled with 95% N2 and 5% CO2 (ischemia-mimetic media). Cells were incubated in ischemia-mimetic media with a 1% O2 and 5% CO2 atmosphere in a hypoxic chamber under the control of ProOx 110 oxygen and ProCO2 carbon dioxide controllers  for 90 min at 37 °C unless otherwise stated. After ischemia, cells were reoxygenated for 30 min with glucose-containing, FBS-free, phenol red-free DMEM culture medium  supplemented with the respective probe.To model ischemia/reperfusion conditions, stable cell lines expressing Cyt2O2 (400 µM for 16 h). Cells were harvested, washed with cold 1× PBS twice, and resuspended in 1× annexin V binding buffer . A total of 1 × 106 cells were stained with annexin V and propidium iodide (PI), as described in the annexin V/PI staining apoptosis kit manual . The cells were resuspended in 1 mL of annexin V binding buffer, and the data were collected using a flow cytometer . The results were analyzed using FCS Express 7 software .Cells were exposed to OGD/R (12 h ischemia/1 h reperfusion), as previously described , or trea2, 1 mM EGTA, 200 µM ATP, 0.01% Brij-35 buffer with 2 µM Cytc at 30 °C. The serine phosphorylation state of Cytc was determined by Western blotting at times 0, 15, and 30 min. The blots were probed with a 1:5000 dilution of phosphoserine antibody  in 5% BSA-TBST buffer, followed by a 1:10,000 dilution of HRP-conjugated rabbit secondary antibody .Recombinant Akt 1 (#A16-10G), Akt 2 (#A17-10G), and Akt 3 (#A18-10G) isoforms were purchased from SignalChem . The in vitro kinase reaction was performed in 50 mM HEPES (pH 7.5), 10 mM MgClc was purified, as previously described [Freshly harvested pig brain tissue was thoroughly washed with ice-cold 1× PBS, cut into smaller pieces, and minced using a commercial meat grinder. All procedures involving animal tissues were approved by the Wayne State University Institutional Animal Care and Use Committee. The tissue was suspended in 250 mM sucrose, 20 mM Tris-Cl buffer supplemented with 10 mM potassium fluoride (KF), 2 mM ethylene glycol tetraacetic acid (EGTA), and 1 mM Phenylmethylsulfonyl fluoride (PMSF). The tissue homogenate was divided into 3 equal volumes. The fractions were treated with 13.7 µM SC79, an Akt activator , 10 µM wortmannin , an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway, or DMSO as solvent control. Tissues were incubated at 37 °C for 30 min with stirring to allow adequate tissue respiration and oxygenation. The pH was adjusted to 7.5. Following treatment, brain Cytescribed .g for 10 min to remove the tissue debris. The supernatant was filtered through a cheesecloth and spun at 14,000× g for 20 min to collect the mitochondria. The pellet was homogenized with a glass Dounce homogenizer and centrifuged at 300× g for 8 min. The supernatant was again spun at 14,000× g for 20 min to collect the mitochondria. The pellet was resuspended in 10 mM HEPES buffer (pH 7.5) containing 210 mM mannitol, 70 mM sucrose, and 1 mM EDTA. Mitochondrial protein content was quantified using the DC protein assay (Bio-Rad). Two sucrose solutions (1.2 M and 1.6 M) of varying density were prepared, and 16 mL of 1.6 M sucrose solution was added to a Beckmann ultra-clear tube (#344058), followed by 19 mL of 1.2 M sucrose solution. Each sucrose gradient was loaded with 5 mg of mitochondrial protein, and the tubes were spun at 27,000 rpm in a Beckmann SW28 swing rotor for 2 h at 4 °C. The mitochondrial fraction was extracted from the tube using a 5 cc syringe with an 18 gauge needle. The collected fraction was centrifuged at 50,000 rpm in a Beckmann type 75 Ti rotor. The pellet was resuspended in 250 mM sucrose, 10 mM Tris-Cl, and 20 mM EDTA buffer (pH 7.6) and spun at 22,000× g for 15 min. The pellet was then resuspended in a hypertonic solution  and allowed to swell for 20 min on ice, as previously described [2 were added and further incubated for 5 min. The suspension was centrifuged at 15,000× g for 10 min at 4 °C. The supernatant was collected as the mitochondrial IMS (intermembrane space) fraction, and the pellet was collected as a mixture of mitoplasts, OMM , IMM , and residual IMS. The fractions were immunoblotted with the following antibodies: pAkt , total-Akt , cytochrome c , COX4 , heat shock protein 60 (HSP60) , and tubulin .Freshly harvested pig brain tissue was homogenized in 250 mM sucrose, 20 mM Tris-Cl buffer supplemented with 10 mM KF, 2 mM EGTA, and 1 mM PMSF. All procedures involving animal tissues were approved by the Wayne State University Institutional Animal Care and Use Committee. The homogenate was spun at 650× escribed . After 2c were fully reduced with sodium dithionite and desalted on a NAP5 column . The initial rate of Cytc oxidation with 100 µM H2O2 within the first 10 s was calculated, as previously described [c oxidation is represented as % of the WT.WT, S47A, and S47E variants were overexpressed in C41(DE3) competent bacterial cells  and purified using ion-exchange chromatography, as previously described . WT, S47escribed . The ratp < 0.05.Statistical analyses of the data were performed with MSTAT version 6.1  using the Wilcoxon rank-sum test. Data are reported as means ± SEM with three or more replicates and were considered statistically significant (*) with c in an intact cell culture system, we created cell lines stably expressing S47E Cytc, WT Cytc, and S47A Cytc as an additional control that cannot be phosphorylated. These Cytc variants were transfected into a Cytc double-knockout mouse lung fibroblast cell line where both the rodent testes and somatic isoforms of Cytc were knocked out [c, along with a Cytc-null empty vector (EV) control, were used for the functional studies  in the S47E phosphomimetic Cyted media  and 10 med media , respect chamber . The S47 chamber . The ratm) in our cell lines was assessed using the ratiometric JC-1 probe. The JC-1 probe diffuses into the mitochondria and accumulates and aggregates when ΔΨm is high, resulting in red fluorescence. At low ΔΨm, it exists as a monomer that emits green fluorescence. Therefore, the ratio of red/green fluorescence provides a relative measure for ΔΨm. FCCP, a potent uncoupler of ΔΨm, was used as a control. The phosphomimetic S47E Cytc cell line resulted in a statistically significant 21% decrease in fluorescence compared to the WT Cytc expressing cell line, indicating that the phosphomimetic replacement led to a reduction in ΔΨm  model. This system was used to model ischemia/reperfusion injury in cultured cells [c expressing cells doubled upon reperfusion. In striking contrast, MitoSOX fluorescence in the S47E cell line and S47A cell line showed a non-significant increase of 8.8% and 9.2%, respectively  compared to the WT and S47A cell lines that resulted in 32.3% and 30% cell death, respectively , whereas cells expressing S47E Cytc and S47A Cytc were better protected with only 11.2% and 13% cell death, respectively  staining, followed by flow cytometry analysis. Phosphatidylserine (PS) is a membrane lipid that is normally present in the inner leaflet of the plasma membrane. However, during apoptosis, PS flips to the outer leaflet of the plasma membrane. Annexin V, a calcium-dependent protein that binds to PS when fluorescently labeled, can be used to detect PS that is exposed on the outside of apoptotic cells. Apoptotic cells can be distinguished from necrotic cells by co-staining with PI . PI is nectively A,B. We fe stress . Annexinectively A,B. The ant Cytc .c for phosphorylation. We performed an in silico analysis using NetPhos 3.1 software [c. We assessed the likelihood of S47 phosphorylation by performing an in vitro kinase assay with all three isoforms of Akt . S47 is the only serine residue present in rodent Cytc. After probing the Akt kinase reactions with a phosphoserine antibody, we observed that all three isoforms of Akt could phosphorylate Cytc in a time-dependent manner in vitro  domain of Akt and changes the conformation that makes it favorable for phosphorylation by upstream kinases [c was purified from these tissue samples and assessed for its serine phosphorylation state. In the control sample, there was a basal level of serine phosphorylation. Upon treatment with the Akt inhibitor wortmannin, complete dephosphorylation of Cytc was observed, suggesting that this phosphorylation might be mediated by Akt. Upon treatment with the Akt activator SC79, serine phosphorylation of Cytc was observed  and functional studies using in vivo S47 phosphorylated and S47E Cytc side by side [c in Cytc double-knockout lung fibroblasts [c are knocked out from these cells because, in the absence of the somatic isoform, the testes isoform may be induced [c encode Cytc isoforms consisting of 104 amino acids, with 86% identical residues between the two [c in human and mouse genomes [c that shows features of both the rodent somatic and testes isoforms. Once Cytc is stably reintroduced into the Cytc double-knockout cells, they start expressing higher levels of complexes I, III, and IV, which are known to form supercomplexes and are downregulated in the absence of Cytc [c in the assembly and stability of respiratory supercomplexes [Our lab previously showed that S47 phosphorylation of Cytan brain . In this by side . We overroblasts . It is i induced . Both so the two . There a genomes . However of Cytc ,42. Thisomplexes .c was similar to the decrease in COX activity reported in vitro with a Clark-type oxygen electrode [c further supported our model that the reaction between Cytc and COX can serve as the rate-limiting step of the ETC controlling overall ETC flux [c may also interact with the bc1 complex [bc1 complex, lowering overall ETC activity. The phosphomimetic S47E cell line showed higher glycolytic flux compared to the unphosphorylated WT cell line, likely compensating for the lower mitochondrial oxygen consumption. The ATP level for S47E was lower than that of WT and S47A; however, the difference was not statistically significant. It should also be noted that in vivo phosphorylation of the Cytc pool in the brain will never reach 100%, whereas all Cytc in the cells expressing S47E Cytc possess the phosphomimetic modification. Therefore, the effect of Cytc phosphorylation on ATP levels may be negligible in the mammalian brain.The effect on intact cell respiration observed in the presence of S47E Cytlectrode   avoids excessive ROS generation but provides the full capability to produce ATP because maximal rates of ATP synthesis by ATP synthase take place at an intermediate ΔΨm of 100–120 mV [m > 140 mV, ROS production at ETC complexes I and III increases exponentially, whereas mitochondria of resting cells with an optimal, intermediate ΔΨm do not produce significant amounts of ROS [m and ROS is evident from our results and further tested in the context of ischemia/reperfusion injury by using an OGD/R cell culture model. The unphosphorylated WT Cytc expressing cells showed a significant increase in ROS following OGD/R. The phosphomimetic cell line attenuated ROS production, further establishing a protective role for S47 phosphorylation in the context of ischemia/reperfusion injury. Our model proposes that cell signaling pathways target the ETC. However, under conditions of stress, such as ischemia/reperfusion, there is an influx of calcium into the mitochondria [c, mediated by calcium-dependent phosphatases [m levels and a burst of ROS that triggers cell death as a result of ischemia/reperfusion injury (reviewed in [c was evident from significantly lower cell death reported upon exposure to OGD/R. In addition, the phosphomimetic cell line showed greater cell viability upon treatment with hydrogen peroxide. Other contributing factors explaining reduced cell death could be lowered caspase-3 activity and cardiolipin peroxidase activity as was previously reported for S47 phosphomimetic Cytc [c resulted in 38% and 35% caspase-3 activity, respectively, compared to WT. Both the mutants also resulted in significantly lower cardiolipin peroxidase activity compared to WT at varying ratios of cardiolipin to Cytc (c is modified.The partial inhibition of respiration observed in the S47E Cytal cells . Mainten0–120 mV . At ΔΨm s of ROS ,47. Thischondria ,49,50. Ichondria . This hyphatases . The inctic Cytc . S47A an to Cytc . These fc, making it a unique regulatory site in mammals. In vitro kinase assays with all three Akt isoforms resulted in a time-dependent increase in serine phosphorylation. We also used pharmacological Akt activator SC79 and inhibitor wortmannin in pig brain tissue lysates to modulate the signaling pathway. The analysis of Cytc phosphorylation state post-treatment resulted in complete dephosphorylation of Cytc in the presence of wortmannin, suggesting a potential role for Akt in mediating this pathway. Interestingly, several studies have shown that Akt activator SC79 is an effective treatment for cerebral ischemia/reperfusion injury in animal models [c resides [c is targeted for S47 phosphorylation by Akt, which, in turn, results in optimal, intermediate ΔΨm levels, lower ROS production, and significant protection from cell death upon OGD/R and other stresses. This experimental model is further supported by our recently discovered specific wavelengths of infrared light that partially inhibits the reaction between Cytc and COX, mimicking a protective effect similar to that observed with S47 phosphorylation of Cytc [Finally, we presented data suggesting that pro-survival kinase Akt could target S47 for phosphorylation. S47 is the only serine residue present in rodent Cytl models ,55,56. W resides ,35,36. T of Cytc ,58. In c


Protanthomyzagrimaldiisp. nov. , is described from Baltic amber  based on two  inclusions. It is the ninth species of the †genus Protanthomyza Hennig, 1965 and †subfamily Protanthomyzinae Roháček, 1998. Adult morphology of P.grimaldiisp. nov. revealed that the rich chaetotaxy of the thoracic pleuron, two anal veins and presence of the anteroventral process of the epandrium are plausibly shared by all species of Protanthomyza. Relationships of the new species, which belongs to a group lacking the ctenidial spine on the fore femur, are discussed.A new fossil species,  Anthomyzidae (Diptera) were reviewed by Anthomyzidae were described from Baltic amber (48–34 Ma) and only one, viz. Grimalanthavulnerata Roháček, 1998, originates from Dominican amber . Only four of the ancient species belong to the “modern” subfamily Anthomyzinae Czerny, 1903, viz. the above Grimalanthavulnerata from the Miocene  and Lacrimyzalacrimosa Roháček, 2013, L.christelae Roháček, 2013 and Reliquanthaeocena Roháček, 2014 from the Eocene. The latter species belongs to a genus originally described by R.variipes Roháček, 2013 from Great Britain. All remaining ancient Anthomyzidae are members of the exclusively fossil subfamily Protanthomyzinae Roháček, 1998 and belong to its only genus Protanthomyza Hennig, 1965. Eight named species of this genus are currently recognized, viz. P.collarti Hennig, 1965, P.hennigi Roháček, 2013, P.hoffeinsorum Roháček, 2013, P.krylovi Roháček, 2013, P.loewi Roháček, 2013, P.meunieri Roháček, 2013, P.presli Roháček, 2013 and P.tschirnhausi Roháček, 2013, all of which were treated in detail by Protanthomyza which was left unnamed due to insufficient material . This unnamed species was considered distinctly different from all other known members of the genus and, therefore, it was also counted among them for the estimation of species diversity of Anthomyzidae in the Eocene Baltic amber forest ecosystem  in which both sexes are known.Fossil taxa of cosystem : 470. ReAMNH – American Museum of Natural History, New York, USA; CCHH – collection of C. and H. W. Hoffeins, Hamburg, Germany; CMTB – collection of M. von Tschirnhaus, Bielefeld, Germany; SDEI – Senckenberg Deutsches Entomologisches Institut  in Müncheberg, Germany.Two amber pieces with 2 anthomyzid inclusions were examined. Abbreviations of amber sources and depositories: CCHH.The methods of preparation of amber stones with fly inclusions were described in detail by 3Cs : 4Cs , index dm-cur-m\ : dm-cu (= ratio of length of section between r-m and dm-cu on cell dm : length of dm-cu) and index dm-cur-m\ : 1CuA .The amber inclusions were examined, drawn and measured using two types of binocular stereoscopic microscopes . Legs were drawn on squared paper using a Reichert binocular microscope with an ocular screen. The specimens were either photographed by a Canon EOS 60D digital camera with Canon MP-E 65 mm 1–5× macro lens or by a Canon EOS 5D Mark III digital camera with a Nikon CFI Plan 10×/0.25NA 10.5mm WD objective attached to a Canon EF 70–200 mm f/4L USM zoom lens. The specimen photographed by means of the latter equipment was repositioned upwards between each exposure using a Cognisys StackShot Macro Rail and the final photograph was compiled from multiple layers (35) using Helicon Focus Pro 7.0.2. The final images were edited in Adobe Photoshop CS6. Some illustrations were drawn from these and some other macrophotographs and details were inked based on direct observation at higher magnification using a binocular microscope. Measurements: Six characteristics were measured – body length , wing length (from wing base to wing tip), wing width (maximum width), index Morphological terminology follows that used in Diptera 2A second anal vein ac acrostichal (seta) (acr)ar aristaavp anteroventral process of epandrium;C costace cercus1CuA cubitus dc dorsocentral (seta)dm discal medial celldm-cu discal medial-cubital  cross-veinep epandrium1f, 2f, 3f fore, mid, hind femurha halterehu humeral  (seta)lbl labellumM media mspl mesopleural  (seta)npl notopleural (seta)oc ocellar (seta)ors orbital (seta) (orb)pa postalar (seta) pk preapical kinkplp maxillary palpuspoc postocular (setulae)ppl propleural  (seta)prs presutural  (seta)prsc prescutellar acrostichal (seta)pvt postvertical  (seta)1R, 2+3R, 4+5R 1st, 2nd, 3rd branches of radiusr-m radial-medial cross-veinS1–S8 abdominal sternasa supraalar (seta) sc scutellar (seta) (sctl)Sc subcostastpl sternopleural  (seta)T1–T8 abdominal terga1t, 2t, 3t fore, mid, hind tibiavi vibrissa (vb)vte outer vertical (seta) (o vt)vti inner vertical (seta) (i vt)Diptera Linnaeus, 1758Order Opomyzoidea Fallén, 1820Superfamily Anthomyzidae Czerny, 1903Family Protanthomyzinae Roháček, 1998Subfamily Taxon classificationAnimaliaDipteraAnthomyzidaeGenusHennig, 19652E50143B-0BCB-5B3D-AA7A-9B23ACCACF1BProtanthomyzacollarti Hennig, 1965; Baltic amber (Eocene).Taxon classificationAnimaliaDipteraAnthomyzidaeE5AF0B3A-36C3-549A-AAC7-9C1FE665BEE0http://zoobank.org/D95A90D3-4F55-46E2-814A-4D8117555C47Protanthomyza sp. nov.: The species is dedicated to Prof. Dr. David Grimaldi , the distinguished American dipterist and palaeontologist, in recognition of his valuable contribution to the knowledge of amber fossil flies, including acalyptrates (largely from Dominican amber).Holotype ♂ labelled “Faszination Bernstein, Christel Hoffeins, Hans Werner Hoffeins” (framed on obverse), ”1040-5a Diptera: Acalyptratae, Anthomyzidae ♂” , “Baltic amber, Russia: Kaliningrad region, Yantarny”, “obtained in May 2010 from Dr. Andrey Krylov, Kaliningrad, Russia”, and “Holotypus ♂, Protanthomyzagrimaldii sp.n., J. Roháček det. 2020” (red label) , deposited in SDEI (inventory number Dip-00821). The original amber stone (in form of an icicle = Zapfenschlaube in German), size about 45 × 20 × 11 mm, with multiple layers and aggregation of inclusions, was cut in two pieces; that with the inclusion of P.grimaldii (No. 1040-5a) was separated, manually prepared and embedded in polyester resin by H. W. Hoffeins in August 2015. Syninclusions in 1040-5a: 1 stellate hair, pollen grains. Syninclusions in the remaining part : Diptera: Empididae: 1 Rhamphomyia sp. female; 2 Mycetophilidae males + fragment; 1 Simuliidae; 2 Chironomidae female and male; Trichoptera, not identifiable; Coleoptera fragment; Araneae fragment; stellate hairs. Paratype ♀ labelled “47b-1, , 15.1 × 10.7 × 3.3 [mm]” (handwritten), “Protanthomyza sp.n. ♀, J. Roháček det. 2011” (yellow label) and “Paratypus ♀, Protanthomyzagrimaldii sp.n., J. Roháček det. 2020” (yellow label) , temporarily held in CMTB; it will be deposited in AMNH. Syninclusions: only stellate hairs.Russia: Kaliningrad region, Yantarny mine. Middle to Late Eocene, 48–34 Ma cf. .st antennal flagellomere normal, not enlarged; arista bare; 3 or 4 dc macrosetae; 1f without a ctenidial spine; male epandrium elongate and posteriorly tapered, with a robust, hook-like, curved anteroventral process; female T7 short; female S6 and S7 broadly transverse.Ocellar triangle delimited by a groove; frontal triangle not delimited; 1Male  relatively short, convergent but not crossed; vti longest of cephalic setae, slightly inclinate; vte strongly exclinate and only slightly shorter than vti; 3 distinct ors, all slightly reclinate, posterior ors longest (about as long as vte), others becoming slightly shorter anteriorly; oc relatively thin (not longer than middle ors), proclinate , arising inside ocellar triangle; anterior half of frons with about 5 or 6 pairs of microsetae, mostly medially in front of ocellar triangle but a few (1 or 2) also between anterior and middle ors; vi distinct  and only 2 or 3 setulae on humeral callus ; 2 long npl, anterior slightly longer; 1 distinct prs (about as long as posterior npl); 1 long sa; 2 pa, external very long (longer than sa), internal shorter  becoming shorter anteriorly, the hindmost longest ; ac microsetae dense, in 9 or 10 rows on suture, those in medial rows reaching up to posterior dc; prescutellar ac macrosetae long and strong, as long as sa; 2 sc, laterobasal shorter (about as long as prs), apical very long; no additional setulae on scutellum; 2 upcurved ppl, anterior distinct (as long as hu), posterior small. Mesopleuron (anepisternum) and sternopleuron (katepisternum) setose  distinct widely-spaced setae in posterodorsal row; setae in posteroventral row more numerous but short and weak. 2f with 2 or 3 anterior setae near middle  and 2 or 3 small setae adjacent to the latter; 1t, 3t and all tarsi simply setulose but basitarsi of all legs with ventrobasal setulae somewhat longer than others.cf. Fig. , otherwiWing , finely short-pilose more distally (on 3Cs and 4Cs); Sc distinct, separate almost along its entire length, only apically fused with 1R to form preapical kink ; 1R short, dilated distally due to fusion with Sc; 2+3R long, very slightly sinuate to almost straight and only its extreme apex slightly upcurved to C; 4+5R slightly recurved in distal half, divergent from 2+3R and apically slightly convergent with M; M almost straight. Cell dm of moderate length, narrow proximally and much widened distally, with angle of anterior outer corner obtuse while that of posterior outer corner distinctly acute  as in other Protanthomyza species  distinctly different from those in other Protanthomyza species where known  visible ventrally, subapically and apically. Mesonotum with 3 strong dc in left row and with 4 dc  in right row; prescutellar ac macrosetae longer than sa; about 8 rows of ac microsetae on suture. Legs more slender but with same chaetotaxy as in male, except t2  obviously darker brown than sterna, relatively narrow, hence pleural part of preabdominal segments large; setae on T3–T5  longer than those on adjacent sterna. Preabdominal sterna pale brown to ochreous. S1–S5 becoming distinctly wider posteriorly, S5 widest and largest.men Figs , 13 onlyPostabdomen.T6 distinctly shorter than T5, transverse; T7 yet shorter and also narrower than T6. S6 strikingly broad and transverse, wider but shorter than S5 and apparently larger than adjacent T6 and laterally almost reaching the latter; S7 only half length of S6, strongly transverse and laterally meeting with sides of T7  sclerite  and P.tschirnhausi , see Protanthomyzagrimaldii belongs to a group of species lacking a ctenidial spine on the fore femur and both sexes are correctly keyed by P. sp. nov.). With its bare arista and generally similar chaetotaxies of the head and thorax, it most closely resembles P.loewi , which could be its nearest relative. However, P.grimaldii can be easily distinguished from P.loewi by the distinctly smaller 1st flagellomere of the antenna . Only in P.tschirnhausi is the epandrial process similarly robust and flat but it is simply triangular (not hooked) and the epandrium is short, almost globose (not elongate)  Protanthomyza species extends the morphological diversity evident in the male and female terminalia of this genus. This adds to the previously documented wide morphological diversity among species seen in the head structures, chaetotaxies, and formation of the sclerites of the abdomen besides the male and female terminalia.(2) The description of a new 1A, 2A) can continue to be treated as a subfamily/generic character for Protanthomyzinae and Protanthomyza, respectively.(3) Two anal veins (mspl macrosetae + numerous setulae more anteriorly) and sternopleuron (with 1 posterior stpl macroseta + setulae) seems to be rather uniform within the genus, although in a few species some setulae on the mesopleuron can be enlarged and/or one  mspl macroseta is reduced (cf. Roháček 1913a).(4) The chaetotaxy of the mesopleuron  The anteroventral process of the epandrium is a synapomorphic character of


MMP-2 activity in heart homogenates was also higher in the I/R group (p = 0.04). ALC1 gene expression and protein synthesis were significantly increased in I/R ventricles . VLC1 content in coronary effluents was increased in the I/R group (p = 0.02), confirming the increased degradation of VLC1 by MMP-2 and probably an adaptive production of ALC1 during I/R. This mechanism of adaptation to oxidative stress led to improved heart mechanical function.Ischemia/reperfusion (I/R) injury induces post-translational modifications of myosin light chains (MLCs), increasing their susceptibility to degradation by matrix metalloproteinase 2 (MMP-2). This results in the degradation of ventricular light chains (VLC1) in heart ventricles. The aim of the study was to investigate changes in MLCs content in the mechanism of adaptation to oxidative stress during I/R. Rat hearts, perfused using the Langendorff method, were subjected to I/R. The control group was maintained in oxygen conditions. Lactate dehydrogenase (LDH) activity and reactive oxygen/nitrogen species (ROS/RNS) content were measured in coronary effluents. Atrial light chains (ALC1) and ventricular light chains (VLC1) gene expression were examined using RQ-PCR. ALC1 and VLC1 protein content were measured using ELISA tests. MMP-2 activity was assessed by zymography. LDH activity as well as ROS/RNS content in coronary effluents was higher in the I/R group ( The restoration of blood flow to pre-ischemic myocardium leads to ischemia–reperfusion injury (IRI), which subsequently results in deterioration of cardiac function ,3,4,5,6.The main aim of this study was to investigate whether ALC1 expression was increased in rat heart ventricles as an adaptive mechanism to ischemia–reperfusion injury. We explored changes in gene expression and concentration of ALC1 and VLC1 proteins in isolated rat heart ventricles. We also measured reactive oxygen/nitrogen species (ROS/RNS) and MMP-2 content, as well as MMP-2 activity in hearts as indicators of oxidative stress. We analyzed the hemodynamic parameters of perfused hearts and examined the contractility of isolated myocytes to assess the impact of VLC1 degradation on heart function.Heart rate (HR), coronary flow (CF), and recovery of heart mechanical function (RPP) (rate pressure product at 77 min versus 25 min of perfusion—the product of HR and left ventricular developed pressure (LVDP)—systolic minus diastolic ventricular pressure) were significantly decreased in hearts subjected to global no-flow ischemia and reperfusion in comparison to aerobic control .p < 0.001, n = 13) after ischemia and reperfusion , was also significantly decreased  (Lactate dehydrogenase (LDH) activity measured in coronary effluents was significantly increased in the I/R group in comparison to the Aero group (n = 5–7) , confirmp = 0.04, n = 6–8)  was observed (Reactive oxygen/nitrogen species (ROS/RNS) content in coronary effluents was increased after ischemia/reperfusion (I/R) in relation to aerobic control (n = 6–8) a. A posiobserved b, suggesp = 0.03, pro-MMP-2: p = 0.04, MMP-2 active: p = 0.03, Total-MMP-2: p = 0.04, respectively, n = 7)  a–c.p = 0.03, r = 0.64; p = 0.03, r = 0.66, respectively)  a,b.MYL4 gene (coding ALC1 protein) was significantly higher in the I/R group in relation to the Aero group , while the MYL3 gene (coding the VLC1 protein) expression level result was not significant  (Expression level of = 12–14) a,b.p = 0.04, n = 6–7)  , confirming that VLC1 was released into extracellular space  a, and non = 7–9) b. Howevear space c.p < 0.001, r = 0.87), ROS/RNS content in coronary effluents , and Total-MMP-2 activity in heart homogenates , while the negative correlation between VLC1 content in coronary effluents and HR  and recovery of rate pressure product (RPP)  was observed  and myosin heavy chain β (MHC-β), and two pairs of light chains: regulatory (RLC/MLC2) and essential ELC/MLC1), divided into atrial (ALC1) and ventricular (VLC1) ,34,35,36, dividedIn many previous studies, MMP-2 had been shown to be involved in the degradation of contractile proteins ,38,39. IOur results show that I/R of heart tissue led to a decreased content of VLC1 and an increased tissue expression of ALC1 in ventricular cardiomyocytes in comparison to the Aero group. We found no correlation of ALC1 content in heart homogenates with MMP-2 activity in heart homogenates and LDH activity in coronary effluents. ALC1 content in coronary effluents showed no differences between the aerobic control group and I/R group. Those results may mean that ALC1 did not constitute a target for degradation by MMP-2 and did not contribute to heart damage. It rather might be induced by oxidative stress in order to prevent any further deterioration of cardiac function. The increased expression of ALC1 in heart ventricles was considered as an adaptive mechanism to I/R conditions in cardiac hypertrophy in the previous studies ,29. ALC1In this research, we showed an increased tissue content of ALC1 and decreased content of VLC1 in an ischemia–reperfusion injury model. We revealed an increased amount of ALC1 already at the level of gene expression in rat heart cardiomyocytes after I/R. This proportion was also maintained at the stage of protein synthesis. Arrell et al. 2001) and Morano et al. (1996) showed that a small amount of ALC1 appeared in the ventricles of adult people with chronic cardiovascular disease and hypertrophic, dilated cardiomyopathy, similarly to our I/R mechanism, which increased the maximal shortening velocity, rate of tension redevelopment, and isometric force generation ,20. Khal01 and MoHowever, there are also reports showing that the mechanism of compensation based on the replacement of degraded VLC1 by ALC1 occurs only in human heart, while rodents present a compensatory mechanism based on change in the expression of MHC ,29,41,42This investigation conforms to the Guide to the Care and Use of Experimental Animals published by the Polish Ministry of Science and Higher Education and was approved by the local Ethics Committee for Experiments on Animals at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland (Resolution 14/2016 of 20 April 2016).2PO4, 1.2 mmol/L MgSO4, 3.0 mmol/L CaCl2, 25 mmol/L NaHCO3, 11 mmol/L glucose, and 0.5 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4. Next, hearts were suspended on a blunt end needle of the Langendorff system  with aorta and maintained at 37 °C. Hearts were perfused at a constant pressure of 60 mm Hg with Krebs–Henseleit Buffer at pH 7.4, at 37 °C and gassed continuously with 5% carbogen. After aerobic stabilization (25 min), hearts were subjected to global, no-flow ischemia  and aerobic reperfusion (30 min). Control hearts were perfused aerobically for 77 min. CF, HR, and LVDP were defined as hemodynamic end points of cardioprotection. The cardiac mechanical function was expressed as the recovery of RPP. Coronary effluents for biochemical tests were collected at the beginning of reperfusion (47 min) to achieve the constant volume (15 mL)  were used in these experiments as a surrogate model for analysis of heart protection. Rats were treated with buprenorfin , anaesthetized with sodium pentobarbital , and hearts were rapidly excised. Immediately after removal, spontaneously beating hearts were rinsed by immersion in ice-cold Krebs–Henseleit Buffer containing 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH (15 mL) . Next, a2, 1 mmol/L Na2HPO4, 20 mmol/L NaHCO3, 5 mmol/L glucose, 9 mmol/L taurine, and 10 mmol/L CaCl2, pH 7.4. Next, hearts were suspended on a blunt end needle of a Langendorff system with the aorta and maintained at 37 °C. Hearts were perfused in a water-jacketed chamber of the Langendorff mode at a constant flow of 10 mL/min with MIB buffer containing 1 mmol/L CaCl2, pH 7.4, at 37 °C, and gassed continuously with 5% carbogen for 5 min.The hearts were rapidly excised from rats anesthetized with sodium pentobarbital  as described above. Immediately after removal, spontaneously beating hearts were rinsed by immersion in ice-cold Myocyte Isolation Buffer (MIB) containing 120 nmol/L NaCl, 5 mmol/L KCl, 2 mmol/L NaAc, 2 mmol/L MgCl2, the buffer was replaced with MIB buffer containing 5 µmol/L CaCl2. Then, hearts were perfused for another 5 min. Low CaCl2 level induced a loss of cardiomyocytes contractility. Mild myocardial swelling was obtained using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer  containing 40 µmol/L CaCl2, 25 mg of collagenase, and 2 mg of protease at pH 7.4. Then, the right ventricle was excised from the heart, rinsed with HEPES buffer containing 100 µmol/L CaCl2, 150 mg bovine serum albumin (BSA), and minced thoroughly in the digestion solution . The minced tissue was digested up to six times for 10 min in a water bath [37 °C]), and the third–sixth fraction were used for further experiments [After 5 min of heart perfusion in a Langendorff system with MIB buffer containing 1 mmol/L CaCl2 and 150 mg BSA, chemical ischemia was induced by resuspending cells pellets in HEPES buffer containing 4 mmol/L 2-deoxyglucose and 40 mmol/L sodium cyanide. The optimal duration of ischemia—3 min—was established in earlier studies [g); next, the cells pellet was suspended in the fresh portion of HEPES buffer containing 100 µmol/L CaCl2 and 150 mg BSA. After reperfusion (20 min), cells were centrifuged (5 min 1500× g), and the cells pellet was suspended in HEPES buffer (100 µmol/L CaCl2 and 150 mg BSA) and used for contractility measurement. The aerobic control group was kept for 38 min in atmospheric air (After 15 min of treatment with HEPES buffer containing 100 µmol/L CaCl studies . Three meric air .2 (4 mL/min) at 37 °C. Cardiomiocytes were continuously paced with 1 Hz and 5 V . The contractility, which was expressed as percentage of peak shortening in comparison to the length of the diastolic cell, was measured on an average of five cells per aliquot. At least five aliquots per heart were examined A total of 13 hearts were tested in each group [A 100 µl aliquot of cell suspension was placed in the rapid change stimulation chamber of the IonOptix Contractility System  for 3 min of aerobic stabilization, followed by perfusion using oxygenated HEPES buffer containing 2 mmol/L CaClch group .g at 4 °C for 15 min. Supernates were collected and stored at −80 °C.Hearts previously frozen at −80 °C were crushed with use of a mortar and pestle in liquid nitrogen. Then, hearts underwent three cycles of freezing (in liquid nitrogen) and thawing (at 37 °C) in the homogenization buffer containing 50 mmol/L Tris-HCl (pH 7.4), 3.1 mmol/L sucrose, 1 mmol/L dithiothreitol, 10 mg/mL leupeptin, 10 mg/mL soybean trypsin inhibitor, 2 mg/mL aprotinin, and 0.1% Triton X-100. Homogenates were centrifuged at 10,000× Protein concentration in cardiac tissue homogenates was determined using Bradford Protein Assay  and BSA  which served as a protein standard.A lactate dehydrogenase activity assay kit  was used to determine LDH activity in coronary effluents collected during reperfusion, following the manufacturer’s instruction. Briefly, lactate dehydrogenase catalyzes the interconversion of pyruviate and lactate with the reduction of NAD to NADH, which was specifically detected with a colorimetric assay at 450 nm. Lactate dehydrogenase served as a marker of tissue damage. It is naturally located in the cytoplasm and immediately released into extracellular space during cell membrane damage/increased permeability.The total ROS/RNS in the rat heart tissue was assessed using an OxiSelect™ In Vitro ROS/RNS Assay Kit . The assay measures total ROS and RNS, including hydrogen peroxide, nitric oxide, peroxyl radical, and peroxynitrite anion, using a proprietary fluorogenic probe, dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ). The probe is primed with a dequenching reagent to the highly reactive DCFH form. In the presence of ROS and RNS, the DCFH is rapidly oxidized to the highly fluorescent DCF. Fluorescence intensity is proportional to the total ROS/RNS level in the sample. The total ROS/RNS level was assessed in coronary effluents and normalized to CF .MMP-2 content in heart homogenates was measured using quantitative Quantikine ELISA Assay for Total MMP-2  according to the manufacturer’s instructions. Total MMP-2 Quantikine ELISA Assay recognized recombinant MMP-2, natural human, mouse, rat, porcine and canine active, pro- and TIMP complexed MMP-2. MMP-2 immobilized with monoclonal antibody specific to this protein was detected with use of anti-Total-MMP-2 polyclonal antibody conjugated to Streptavidin-Horse Radish Peroxidase (HRP). Next, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added to develop the reaction. The minimum detectable concentration was 0.033 ng/mL. MMP-2 concentration in heart homogenates was expressed as ng per µg of total protein.ν:ν). Samples containing 120 µg of protein were applied to 8% polyacrylamide gel copolymerized with gelatin (2 mg/mL) and 0.1% SDS . After electrophoresis, gels were rinsed three times for 20 min in 2.5% Triton X-100 to remove SDS. Then, gels were placed in incubation buffer  and incubated overnight at 37 °C. After gelatin was digested, gels were stained in staining solution  for 2 h and destained in destaining solution  until bands were clearly visible. MMP-2 activity was visualized as bright bands on a dark background. Zymograms were scanned using a GS-800 Calibrated Densitometer (model PowerLook 2100 XL-USB) and analyzed using Quantity One v. 4.6.9 software . The relative activity of MMP-2 was established and expressed in arbitrary units (AU) as an activity per µg of total protein.MMP-2 activity in heart homogenates was analyzed using the gelatin zymography method with Heussen and Dowdle protocol modified by us ,45. InitMYL4 (coding ALC1) and MYL3 (coding VLC1) genes in relation to the GAPDH gene was analyzed by relative RQ-PCR using CFX96 Touch . iTaq Universal Sybr Green Supermix , forward and reverse primers , Ultra Pure DEPC-Treated water and cDNA (100 ng) were used in a final volume of 30 µL. The 5′-3′ sequences of primers were as follows: MYL4 F: AGGTGGAGCAGCTGTTGACT, MYL4 R: GAGACTCCACACTGGGCTTC, MYL3 F: GCTGAGCCTCTCAGGAAGC, MYL3 R: GACAGAAAGGGTACCACGGG, GAPDH F: AGTGCCAGCCTCGTCTCATA, GAPDH R: GATGGTGATGGGTTTCCCGT. The amount of mRNA in relation to GAPDH was calculated as 2−ΔCt. The relative expression of respective genes was compared in hearts exposed to aerobic conditions and hearts subjected to global ischemia.Total RNA was isolated from heart tissue using TRIZOL Reagent  in accordance with the manufacturer’s instruction. Reverse transcription of 2 µg of pure RNA to cDNA using iScript cDNA Synthesis Kit  was performed. The expression of −3 ng/mL; Bioassay Technology Laboratory, Shanghai, China). Briefly, capture antibodies bound ALC1/VLC1 from heart homogenates/coronary effluents. Next, antigens were detected by biotinylated antibodies. HRP conjugated with biotinylated ALC1/VLC1 antibody. TMB substrate solution was added to enable complex visualization. Concentrations of ALC1/VLC1 in heart homogenates were expressed as ng per µg of total protein and as ng/mL normalized to coronary flow in coronary effluents.ALC1 and VLC1 content in heart homogenates and coronary effluents were determined using a Rat Myosin Light Chain 4 (MYL4) ELISA Kit  and Rat Myosin Light Chain 3 ELISA Kit (sensitivity: 6.99 × 10t test, unpaired t test, and Mann–Whitney U-test. Correlations were estimated using Pearson’s or Spearman’s tests. Results were expressed as mean ± SEM. p < 0.05 was accepted as a statistically significant difference.GraphPad Prism v. 6 was used for statistical analysis of the results. A Shapiro–Wilk normality test or Kolmogorov–Smirnov test was used to assess the normality of variances changes. Next, results were analyzed by appropriate tests: paired In conclusion, oxidative stress caused the increase in MMP-2 activity, which led to cell damage visible in substantially decreased hemodynamic parameters and VLC1 expression changes. Oxidative stress also induced ALC1 gene expression and synthesis in hearts, which may constitute an adaptive mechanism to I/R conditions. This study might also be a good foundation for further research into actin–myosin interactions in I/R conditions.


HL, PLT, and PRN) contained four antibiotics: HCN, PHL, PLT, and PRN, while OTU RZ (PRN and PHZ) contained three: HCN, PRN, and PHZ. OTU H1, H2, H3, H4, H5, H6, and H7 (PHL1-7) contained two antibiotics: HCN and PHL, while OTU H8 (PHL8) contained one: PHL. Isolates belonging to OTU HLR and RZ suppressed damping-off disease in cabbage seedlings caused by Rhizoctonia solani. Effective strains belonging to OTU HLR and RZ were related to Pseudomonas protegens and Pseudomonas chlororaphis, respectively. Antibiotic biosynthesis gene-possessing fluorescent pseudomonads are distributed among different geographical sites in Japan and plant species.More than 3,000 isolates of fluorescent pseudomonads have been collected from plant roots in Japan and screened for the presence of antibiotic-synthesizing genes. In total, 927 hydrogen cyanide (HCN)-, 47 2,4-diacetylphloroglucinol (PHL)-, 6 pyoluteorin (PLT)-, 14 pyrrolnitrin (PRN)-, and 8 phenazine (PHZ)-producing isolates have been detected. A cluster analysis (≥99% identity) identified 10 operational taxonomic units (OTUs) in antibiotic biosynthesis gene-possessing pseudomonads. OTU HLR (P Pseudomonas is one of the most ubiquitous bacteria in multiple environments, including soil and water, and is frequently found in animals and phytospheres , 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), pyrrolnitrin (PRN), and phenazines (PHZ) (The genus diseases . On the tilizers . Fluoreses (PHZ) . Each ofPseudomonas fluorescens or P. putida, the diversity of fluorescent pseudomonads found in Japan has not yet been investigated in detail. On the other hand, some plant-protecting isolates of P. fluorescens were recently re-identified as a novel species .The surfaces of individual 1-g root samples collected from various fields in Japan were washed with 9 mL of sterile 15 mM phosphate buffer (pH 7.0) and then sonicated with an ultrasonic washer  at 42 kHz for 1 min to release microbial cells from the roots. Serial dilutions were cultivated on King’s B (KB) medium agar containing 50 μg mLhcnAB and hcnBC for HCN; phlD for PHL; pltB and pltC for PLT; prnC and prnD for PRN; and phzCD and phzF for PHZ) were described previously  liquid medium (Sigma-Aldrich Japan) and incubated at 25°C for 24 h on a reciprocal shaker (140 rpm). Bacterial cells were collected by centrifugation, and total bacterial genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions. Specific primer sets for the amplification of antibiotic-synthesizing genes (AGAGTTTGATCMTGGCTCAG-3′) and 1525R (5′-AAGGAGGTGWTCCARCC-3′). The thermal cycling program began with an initial denaturation at 94°C for 3 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 10 min. Sequencing was conducted on 16S rRNA genes using the 27F, 1525R, f1L (5′-GTATTACCGCGGCTGCTGG-3′), f2L (5′-CCAGCAGCCGCGGTAATAG-3′), and 926f (5′-AAACTCAAAGGAATTGACGG-3′) primers from the Takara Dragon Genomic Center (Takara Bio). Sequences were placed in a taxonomic hierarchy using the Classifier in Ribosomal Database Project (RDP) II  from genomic DNA obtained from bacterial isolates. The primers used were as follows: 27F (5′-d method  with typd method . The tred method .N-acyl homoserine lactone (AHL), a quorum-sensing signal, may be detected by the AHL reporter strains, Chromobacterium violaceum CV026 and VIR24  of R. solani MAFF726551 was cut from colonies grown on potato sucrose agar (PSA) and placed on a water agar (WA) plate (55 mm in diameter). Plates were incubated at 28°C for 7 d in the dark. Bacterial strains were incubated on KB medium agar at 28°C in the dark for 3 d. Cabbage (Brassica oleracea var. capitate L. ‘Shosyu’) was used as the test plant in the present study, and seeds were surface-sterilized in 70% ethanol for 30 s and in 2.5% sodium hypochlorite for 1 min, then rinsed three times with sterile distilled water. Seeds were then inoculated with bacterial cells and placed on WA plates. Bacterial cells were detected at approximately 5×108 to 1×109 colony-forming units (CFU) seed–1, and plates were incubated at 28°C for 24 h in the dark. In comparisons, the anti-microbial agent fluazinam (Ishihara Sangyo) was used as a chemical control.To assess the effectiveness of isolates against phytopathogens, a cabbage-R. solani and overlaid with sterilized vermiculite (Takamura). Three plates were tested with each treatment. Plates were transferred into a sterilized plant box (Asahi Glass), and plant boxes were incubated at 28°C for 14 d in a multi thermo incubator . Disease severity was evaluated according to the following damage index: 0=no symptoms, 1=discoloration of the hypocotyl, 2=damping-off, and 3=collapse of seedlings. Disease severity was calculated by the following formula: ([0×n0+1×n1+2×n2+3×n3]/24). Each experiment was replicated three times. The disease severity of the control treatment was calculated and set to 100% in order to represent the disease incidence for the control. The disease incidence of the bacterial treatment was compared with the control treatment using Dunnett’s test. Statistical analyses were performed using BellCurve software .Eight bacterized seeds were inoculated onto WA plates containing The nucleotide sequences of the 16S rRNA genes have been deposited in the DDBJ/ENA/GenBank database under accession numbers LC420166–LC420220.Pseudomonas.Approximately 500 root samples from 88 distinct plant species, including crops and weeds, were collected from 135 fields located in 19 different prefectures  in Japan. Five colonies that showed fluorescence on KB agar under UV light were isolated from each sample. In total, 3,115 fluorescent pseudomonads were isolated. The results of 16S rRNA gene sequencing revealed that all isolates belonged to the genus hcn, and two isolates only possessed the PHL synthesis gene, phl. Thirty-eight isolates possessed both hcn and phl, eight possessed, hcn, prn, and phz, and only five possessed all four genes, .The presence of antibiotic biosynthesis genes in the DNA of isolates was assessed via PCR with specific primers. Antibiotic biosynthesis genes were not detected in 2,186 isolates. Nine hundred and twenty-seven isolates only possessed the HCN synthesis gene, phl, plt, prn, and phz , was related to P. baetica  revealed the presence of 10 OTUs among isolates possessing the antibiotic biosynthesis genes  and phz . Strainsectively . 16S rRNrotegens  and S1. roraphis  and S2. ectively  and S3.  baetica  and S4.  agarici  and S4, aligenes  and S4.Previous studies showed that the biosynthesis of PHZ and PRN was regulated by the quorum sensing system via AHL .R. solani pathosystem. Isolates belonging to OTU HLR and RZ exhibited strong antifungal and biocontrol activities  Diversity of Antibiotic Biosynthesis Gene-possessing Rhizospheric Fluorescent Pseudomonads in Japan and Their Biocontrol Efficacy. https://doi.org/10.1264/jsme2.ME19155Supplementary Material


In parallel experiments, the hearts isolated from untreated animals were perfused with Ticagrelor (3.70 μM) before ischemia and subjected to IR. The hearts of animals pretreated with Ticagrelor showed a significantly reduced infarct size  when compared to control IR group (69 ± 2% of AAR). Similarly, ex vivo administration of INF before the IR injury resulted in significant IS reduction (38 ± 3% of AAR). Myocardial IR induced the NLRP3 inflammasome complex formation, which was attenuated by either INF pretreatment ex vivo, or by repeated oral treatment with Ticagrelor. The beneficial effects induced by either treatment were associated with the protective Reperfusion Injury Salvage Kinase (RISK) pathway activation and redox defence upregulation. In contrast, no protective effects nor NLRP3/RISK modulation were recorded when Ticagrelor was administered before ischemia in isolated heart, indicating that Ticagrelor direct target is not in the myocardium. Our results confirm that Ticagrelor conditioning effects are likely mediated through platelets, but are not additives to the ones achieved by directly inhibiting NLRP3.Inhibition of either P2Y12 receptor or the nucleotide-binding oligomerization domain- (NOD-) like receptor pyrin domain containing 3 (NLRP3) inflammasome provides cardioprotective effects. Here, we investigate whether direct NLRP3 inflammasome inhibition exerts additive effects on myocardial protection induced by the P2Y12 receptor antagonist Ticagrelor. Ticagrelor (150 mg/kg) was orally administered to rats for three consecutive days. Then, isolated hearts underwent an ischemia/reperfusion  protocol. The selective NLRP3 inflammasome inhibitor INF (50  Ischemic heart disease remains the leading cause of morbidity in the Western world, and the number of deaths from acute myocardial infarction (AMI) is also rapidly rising in the developing world. Although restoration of early blood flow to the ischemic myocardium with thrombolysis is presently the most effective therapy to limit infarct size, reperfusion alone is inadequate to salvage the damaged myocardium and may result in myocardial ischemia/reperfusion (IR) injury, which is characterized by excessive oxidative stress and inflammatory response –3. In favia inhibition of the equilibrative nucleoside transporter 1 (ENT1) by protecting the extracellular adenosine from intracellular metabolism [In clinical practice, P2Y12 adenosine disphosphate (ADP) receptor antagonists are standard of care in AMI patients undergoing primary percutaneous intervention. Several preclinical studies have convincingly shown that these drugs significantly protect against IR injury, suggesting that these pleiotropic effects could be even more important than their antiaggregant properties in this specific clinical setting –8. Clinitabolism –12. Thistabolism –15, despAlthough different cell types  expressIn the present study, we evaluate (1) the ability of Ticagrelor and INF, alone and in combination, to reduce infarct size following IR injury, (2) the potential mechanisms of cross-talk between the two drug treatments underlying their myocardial protection, and (3) the relevance of the presence of blood in mediating cardioprotective effects and the platelet mediators released after Ticagrelor exposure.3 25, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.25, and Glucose 11. The buffer was gassed with 95% O2 : 5% CO2. The hearts were perfused in constant flow mode to achieve a perfusion pressure of about 80 mmHg. To assess the conditions of experimental preparation, coronary perfusion pressure was monitored during all experiments [Male Wistar rats  5–6 months old, reaching a body weight of 450–550 g, were anesthetized with sodium pentothal (50 mg/kg) by intraperitoneal injections and heparinized  before being culled by cervical dislocation. The hearts were then rapidly excised, placed in an ice-cold buffer solution, and weighed. The excised hearts were rapidly perfused by the Langendorff technique with Krebs-Henseleit bicarbonate buffer containing (mM) NaCl 118, NaHCOeriments , and floThe protocol was approved by the Institutional Animal Care and Use Committee of the University of Turin and conformed to the European Directive 2010/63/EU on the protection of animals used for scientific purposes.n = 6 − 8 per group) received water or Ticagrelor  by oral gavage for 3 days (oTIC). Then, the isolated hearts were submitted to ischemia/reperfusion as described above (IR and oTIC groups). A subgroup of isolated hearts from oTIC rats were exposed to the selective NLRP3 inflammasome inhibitor INF (50 μM) in the perfusate for 20 min before ischemia (oTIC+exINF). In a subsequent series of experiments, the isolated hearts from control rats were pretreated with 3.70 μM Ticagrelor or 50 μM exINF or both in the perfusate for 20 min before ischemia . After stabilization, sham hearts underwent 90 min perfusion only and served as control group . As previously documented [−1 s−1, measured as second-order rate constant (k2) for the reaction with cysteamine. Ticagrelor was dissolved at 3.70 μM concentration in Krebs solution. The in vivo dose of Ticagrelor and the in vitro concentrations of both Ticagrelor and INF were chosen according to previous studies demonstrating their efficacy against myocardial IR injury [A stock solution of 200 mM INF in DMSO was prepared and was then diluted at a final concentration of 50 o et al.  and selecumented , 30, INFR injury , 28, 31.Infarct areas were assessed at the end of the 60 min reperfusion with the nitro-blue-tetrazolium (NBT) technique. The basal part of the left ventricle was dissected by transverse sections into two/three slices. Following 20 min of incubation at 37°C in 0.1% solution NBT  in phosphate buffer, unstained necrotic tissue was carefully separated from stained viable tissue by an independent observer, who was unaware of the protocols. Since the ischemia was global and we analyzed only the basal part of the ventricle, the necrotic mass was expressed as a percentage of the analyzed ischemic tissue .w/v) in a Potter-Elvehjem homogenizer  using a homogenization buffer , 0.5 mM phenylmethyl sulphonyl fluoride (PMSF), 0.5% Nonidet P-40, phosphatase, and protease inhibitors) and centrifuged at 1300 × g for 5 min at 4°C. To obtain the cytosolic fraction, supernatants were removed and centrifuged at 16000 × g at 4°C for 40 minutes. The pelleted nuclei were resuspended in extraction buffer containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 20% glycerol, 1 mM EGTA, phosphatase, and protease inhibitors and incubated in ice for 30 minutes followed by centrifugation at 16000 × g for 20 min at 4°C. The resulting supernatants containing nuclear proteins were carefully removed, and protein content was determined on both nuclear and cytosolic extracts using the bicinchoninic acid (BCA) protein assay following the manufacturer's directions . Protein extracts were stored at −80°C until use.As previously described , the heaβ in tissue homogenates, according to the manufacturer's instructions.Commercially available ELISA kit  was used to measure concentrations of IL-1473Akt , rabbit anti-total Akt , rabbit anti-Ser9 GSK-3β , anti-total GSK-3β , anti-Ser660 PKC and total PKC , SOD2 , and NRF2  followed by incubation with appropriate HRP-conjugated secondary antibodies (BioRad). Proteins were detected with Clarity Western ECL substrate  and quantified by densitometry using analytic software . Results were normalized with respect to densitometric value of mouse anti-tubulin , and autoradiograms showing statistically significant differences in terms of gel-loading homogeneity were excluded from the following biomarkers analyses.Equal amounts of total protein extracts were separated by SDS-PAGE and electrotransferred to nitrocellulose membrane . Membranes were probed with rabbit anti-NLRP3 , rabbit anti-caspase-1 , mouse anti-Sern. 155/2017, and informed consent was obtained in accordance with the 1964 Declaration of Helsinki and its later amendments. The platelet-rich plasma, obtained by centrifugation at 100 × g for 20 min, underwent further centrifugation at 2000 × g for 10 min, and pellet was washed 2 times at 37°C in HEPES-Na buffer (mmol\L): 10 HEPES Na, 140 NaCl, 2.1 MgSO4, 10 D-glucose, and pH 7.4. Platelets were counted by automatic blood cells counter  and resuspended to a final concentration of 2 × 1011 cells/L in phosphate-buffered saline containing 1% BSA. The contamination of white blood cells was less than 1/104 platelets. Platelet samples were subjected to stirring (1200 rpm speed at 37°C) in both the absence and presence of Ticagrelor  or thrombin receptor-activating peptide (TRAP-6)  , then centrifuged at 4000 rpm for 10 min. Supernatants were stored at -20°C until sphingosine, S1P, and adenosine measurements.Fasting venous blood sample from four male healthy volunteers (mean age: 38 ± 2 years) was withdrawn without stasis and anticoagulated with citrate-dextrose solution . The human platelet study was authorized by “Comitato Etico Interaziendale San Luigi Gonzaga,” authorization μL of platelet samples and heart homogenates were added with 2 mL of 0.1% trifluoroacetic acid in chloroform/methanol 1/1 and with internal standards (adenosine and S1P d7) at 300 μg/L as final concentration. After vortex in g for 30 seconds, 0.5 mL of chloroform and 0.5 mL of water were added. After centrifugation, organic phase was recovered and extracted twice with 1 mL of chloroform. The solution was dried overnight under vacuum  and reconstituted with 100 μL of eluents A/B 7/3.100 The analyses of sphingolipids and adenosine were performed using a Nexera  UHPLC coupled through an ESI source to a Qtrap5500 triple quadrupole analyzer .μm, 100 × 2.1 mm, 100 Å, Phenomenex, Bologna, Italy) with 0.1% formic acid in water/acetonitrile 8/2 (eluent A) and 0.1% formic acid in isopropanol/acetonitrile 8/2 (eluent B). The separation gradient was from 5 to 100% of B in 7 minutes, followed by column reconditioning. Flow rate was set at 400 μL min−1, and injection volume was 3 μL. The LC column effluent was delivered to the ESI ion source, using air as both 1 and 2 gasses , and the ion voltage was 5.0 kV. Curtain gas (nitrogen) was 30 arbitrary units.The chromatographic separation was achieved with a Kinetex column (1.7 m/z) 300@282 CE 13 V; C18-S1P (m/z) 380@264 CE 21 V; and adenosine (m/z) 268@136 CE 21 V. For the internal standard, the MRM transition was C18-S1P d7 (m/z) 387@271 CE 19 V. The lower limit of detection (LLOQ) was 0.50 μg/L for all analytes.The multiple reaction monitoring (MRM) transitions and parameters were C18-Sph .t-test. A P value < 0.05 was considered statistically significant.All values are expressed as means ± SEM and were analyzed by ANOVA test followed by Bonferroni's posttest and Student's P < 0.01 vs. IR group). No protective effects were recorded when Ticagrelor was added ex vivo to the perfusate of excised hearts from untreated animals , thus suggesting that Ticagrelor protection was only triggered in the intact organism . Infarct size was significantly reduced by rat pretreatment for 3 days with Ticagrelor , we observed a significant reduction in infarct size , comparable to the one achieved by oTIC alone . Similarly, no priming effects on exINF protection were recorded when Ticagrelor was coadministered ex vivo in the perfusate only .Interestingly, exINF did not exert additive effects on protection against infarct size evoked by oral Ticagrelor pretreatment, as the reduction in infarct size evoked by the combination was almost identical to that obtained herein with either Ticagrelor or exINF alone . No additive effects were recorded when the two treatments were combined. Interestingly, ex vivo Ticagrelor exposure 20 min prior to ischemia only did not significantly modify the activation of the RISK pathway evoked by IR and/or INF.Since RISK pathway is activated by both pre- and postconditioning treatments , we quanP < 0.05 vs. sham). All treatments blunted IR-induced SOD upregulation. However, in oTIC, oTIC+exINF, and exTIC+exINF groups, the levels of SOD2 were significantly lower than that of IR group  did not further increase S1P or adenosine accumulation in the heart in comparison to oTIC alone.As shown in P < 0.05). Adenosine concentration increased significantly compared to control in the supernatant of Ticagrelor-treated platelets. The positive control was obtained by incubating human platelet with TRAP-6 . The values of the release of S1P and adenosine in the supernatant in TRAP-6 group were significantly higher than control and Ticagrelor-treated platelets (data not shown).Sphingosine, S1P, and adenosine were evaluated in the supernatant of human platelet samples subjected to stirring (1200 rpm speed at 37°C) with or without Ticagrelor  . Ticagrein vivo, as adding the P2Y12 antagonist ex vivo to the perfusate in excised hearts does not counteract the IR injury. Here, we confirm our previous data [ex vivo does not enhance the heart response to exINF, showing no interactions between the two treatments.The present study further extends previous findings on Ticagrelor cardioprotective effects, confirming that the protection was dependent upon its administration ous data  that theIndeed, Ticagrelor has been shown to modulate the expression on blood cells of toll-like receptors, key receptors involved in NLRP3 regulation . Here, win vivo, evokes significant decrease of protein levels of NLRP3, resulting in lower activation of caspase-1, thus counteracting the IR-induced accumulation of active IL-1beta proteins in the heart.To the best of our knowledge, so far, only another study has suggested that cardioprotection of Ticagrelor tested in models of acute myocardial injury can be partially attributable to inhibition of mRNA levels of NLRP3 and IL-1beta in the heart of diabetic rats . Here, win vivo studies did not definitively rule out a contribution from the antiplatelet effect in limiting IR injury. Our study, in which the P2Y12 antagonist was administered in vivo and myocardial infarct subsequently induced ex vivo, in the absence of platelets, definitively confirms that the protective effect is mainly due to a preconditioning effect in vivo that lasts throughout the IR procedure ex vivo. The lack of cardioprotection by Ticagrelor when administered in the ex vivo model further support the idea of a blood cell-mediated preconditioning effect [in vivo only.A pharmacological approach with cardioprotective inhibitors has suggested that cardioprotection induced by P2Y12 antagonists is due to a conditioning phenomenon rather than to their antiplatelet effect . In the g effect , 8, 31. g effect , 17, it g effect , 16, 17.g effect . Plateleg effect , and plag effect , 39. Ticg effect , 14, 16.g effect , 41. Thig effect –15. Howeg effect , 43. Herin vivo vs. ex vivo treatments has been reported in the literature. Thus, our study adds a further interesting piece of evidence on the ability of Ticagrelor to cause blood cells to release substances, which may contribute to cardioprotection. In fact, either S1P or adenosine has been already demonstrated to evoke protective effects throughout activation of the protective survival RISK pathway in the heart [The contribution of blood-derived S1P and adenosine in mediating the myocardial protective effects of Ticagrelor was confirmed by showing that both S1P and adenosine reached the highest concentrations in the heart of rats orally exposed to Ticagrelor. While the increase in myocardial adenosine levels has been already documented in the IR heart of rats orally pretreated with Ticagrelor , so far,he heart , 45. As he heart , 46. Pothe heart , 47–49. he heart –52, but ex vivo.The role of oxidative stress in contributing to IR injury is not clear , 54. OveOur results confirm Ticagrelor conditioning effects, which are not additives to the cardioprotection achieved by directly inhibiting NLRP3. It is likely that this lack of additive effect is due to the activation of RISK pathway by both treatments. Other studies demonstrated additive effect when P2Y12 and a downstream NLRP3 factor, namely, caspase-1, were inhibited , 7, thusIn conclusion, we confirm that Ticagrelor requires the presence of blood to act as conditioning agent. Importantly, we demonstrate that the cardioprotective effects of Ticagrelor are not due to a direct action on the myocardial tissue nor to its antiaggregating effect, whereas the NLRP3 inhibitor, INF, is able to act directly on the heart. Nevertheless, these two drugs given before ischemia activate a similar protective pathway, involving RISK pathway and redox modulation, without additive cardioprotective effects.


Salvia sclarea L.) to 100 μM cadmium (Cd) in hydroponics was sufficient to increase Cd concentrations significantly in roots and aboveground parts and affect negatively whole plant levels of calcium (Ca) and magnesium (Mg), since Cd competes for Ca channels, while reduced Mg concentrations are associated with increased Cd tolerance. Total zinc (Zn), copper (Cu), and iron (Fe) uptake increased but their translocation to the aboveground parts decreased. Despite the substantial levels of Cd in leaves, without any observed defects on chloroplast ultrastructure, an enhanced photosystem II (PSII) efficiency was observed, with a higher fraction of absorbed light energy to be directed to photochemistry (ΦPSΙΙ). The concomitant increase in the photoprotective mechanism of non-photochemical quenching of photosynthesis (NPQ) resulted in an important decrease in the dissipated non-regulated energy (ΦNO), modifying the homeostasis of reactive oxygen species (ROS), through a decreased singlet oxygen (1O2) formation. A basal ROS level was detected in control plant leaves for optimal growth, while a low increased level of ROS under 5 days Cd exposure seemed to be beneficial for triggering defense responses, and a high level of ROS out of the boundaries (8 days Cd exposure), was harmful to plants. Thus, when clary sage was exposed to Cd for a short period, tolerance mechanisms were triggered. However, exposure to a combination of Cd and high light or to Cd alone (8 days) resulted in an inhibition of PSII functionality, indicating Cd toxicity. Thus, the rapid activation of PSII functionality at short time exposure and the inhibition at longer duration suggests a hormetic response and describes these effects in terms of “adaptive response” and “toxicity”, respectively.Five-day exposure of clary sage ( Cadmium (Cd) is occurring in soils at low concentrations but can arise to high concentrations as a result of numerous human activities, while being not biodegradable in the soil, it is considered as one of the most toxic elements, and also a non-essential element for plants ,3,4,5,6.−1) is considered extraordinary and a limit value for Cd hyperaccumulation ,91S. sclcontrols d. In othchanisms ,93. Highchanisms . This dineration . Control at PSII a, show ar 2 days c. In conexposure c, H2O2 p control a, being  control , while a control a and was control . Thus, sn plants ,27,83,89S. sclarea can be described as a hormetic response  ,25, withresponse , represeSedum alfredii exposed to 5 μM Cd. Similar results with stimulation of growth when Noccea caerulescens was exposed to 100 μM Cd were reported by Lombi et al. [Arabidopsis halleri was also described recently [Although excess Cd accumulation is detrimental to plants, different strategies of Cd tolerance and accumulation are adopted by plants . Tang eti et al. . A stimurecently . Małkowsrecently  reportedrecently ,105,106.recently ,107 has recently ,26,27.S. sclarea leaves exposed to Cd for 2 and 5 days (−1) remained higher than the adequate range limit (2000 µg g−1) [A negative impact of Cd on photosynthesis has been assigned to decreases in chlorophylls; ascribed to Cd-induced damage in chloroplasts’ ultrastructure ,109. Howd 5 days a cannot d 5 days f. Howeve µg g−1) .2 fixation [S. sclarea Cd-treated plants, an increase in deposited electron-dense material was observed, as reported also by Mizushima et al. [Cadmium has been reported to alter chloroplast ultrastructure, reduce photosynthesis ,106,111,fixation . Ultrastfixation ,114,115 fixation . In Cd tfixation ,102,117,fixation ,118, a pa et al. , but no a et al. ,120,121,a et al. ,34,52.S. sclarea plants to a combination of Cd and high light (900 μmol photons m−2 s–1) resulted in an inhibition of PSII functionality . After germination on soil in a growth room for about a month, the seedlings were transferred to pots containing continuously aerated modified Hoagland nutrient solution (described in detail before) [−2 s−1.Seeds of  before) . The nutS. sclarea plants in the hydroponic culture experiments were subjected to 0 or 100 μM Cd (as 3CdSO4 8H2O) for a period up to five days. The pots containing only Hoagland nutrient solution served as the control, while all solutions were renewed every two days.Two-month-old Salvia plants were harvested, separated in roots and aboveground (shoots-leaves) tissues, washed three times in deionized water, and then dried at 65 °C to constant biomass, milled and finally sieved. Dried sieved samples of 0.3 g were transferred in 10 mL quartz vessels with 65% (v/v) nitric acid  and 30% (v/v) hydrogen peroxide  in 3:1 ratio. Digestion was carried out in the microwave assisted digestion system Ethos One . The process run out in 3 stages: ramp time—20 min to reach 200 °C and 1500 W; hold time—30 min at 200 °C and 1500 W; cooling—30 min. The next step was the quantitative transfer of digested samples into polypropylene tubes and dilution with demineralized water . All prepared samples were diluted immediately prior to inductively coupled plasma mass spectrometer (ICP-MS) analysis. Samples were analyzed in an ICP-MS model ELAN DRC II  [After 5 days treatment with 0 (control) or 100 μM Cd,  Canada) . ICP-MS a (Chla) and chlorophyll b (Chlb) content was determined according to Lichtenthaler [v/v) acetone and centrifuged at 5000× g for 5 min at 4 °C. The absorbance of the supernatant was measured at 646.8 and 663.2 nm  and Chla and Chlb content was estimated from the equations: Chla = 12.25 A663.2 − 2.79 A646.8; Chlb = 21.50 A646.8 − 5.10 A663.2 [−1 FW.Chlorophyll enthaler . Leaf ti0 A663.2 . The meaS. sclarea plants, treated for 2 and 5 days with 0 (control) or 100 μM Cd, using an Imaging PAM M-Series system  as described in detail previously [S. sclarea leaves, a LL, similar to the growth light (220 μmol photons m−2 s−1), and a HL (900 μmol photons m−2 s−1). In each leaf, representative areas of interest (AOIs) were selected so as to have measurements of the whole leaf area. The definitions of the five main chlorophyll fluorescence parameters  measured by the Imaging PAM M-Series system are presented in v/Fm after dark adaptation and of ΦPSΙΙ and ΦNO, after 5 min illumination with 220 μmol photons m–2 s–1 (LL) or/and 900 μmol photons m–2 s–1 (HL).Chlorophyll fluorescence measurements were conducted on dark adapted (20 min) leaves of eviously . Two lig2O2) and malondialdehyde (MDA) content. The level of lipid peroxidation in S. sclarea leaves of control, and 5- and 8-days Cd-treated plants was measured as malondialdehyde (MDA) content determined by the reaction with 2-thiobarbituric acid (TBA), according to the method of Hodges et al. [2O2) was extracted by homogenization with 50 mM K-phosphate buffer pH (6.5) and determined as described by Hossain et al. [4 in 20% H2SO4.Leaf samples were frozen in liquid nitrogen and stored at −80 °C for analysis of hydrogen peroxide . Statistically significant differences among the means were determined using one-way analysis of variance or two-way ANOVA. Means (±SD) were considered statistically different at a level of Although surplus Cd accumulation is detrimental to most plants, different strategies of Cd tolerance and accumulation are adopted by different plant species . When cl


RAD54B, NBS1, RAD51B, and PRKDC were significantly amplified and over-expressed in ESCC. Amplification and high expression of DSB repair pathway genes were associated with poorer overall survival. Gene set variation analysis further showed that DSB repair pathways were up-regulated in ESCC. Besides, we firstly demonstrated that combination of mirin and NU7441, two inhibitors for HR and NHEJ respectively, with ionizing radiation treatment significantly enhanced DSBs, reduced clonogenic cell survival, inhibited cell proliferation, and promoted cell apoptosis in ESCC cells with DSB pathway gene amplification. These findings suggest that DSB repair pathways were significantly altered in ESCC and inhibiting DSB repair pathways might enhance the radio-sensitivity of ESCC with DSB repair up-regulation.Esophageal squamous cell cancer (ESCC) is a common malignancy with a poor 5-year overall survival in China. Altered DNA damage repair (DDR) pathways are associated with a predisposition to cancer and contribute to therapeutic response and resistance in cancers. However, alterations of DDR pathway genes in ESCC are still largely unknown. In this study, we employed genome sequencing data of 192 samples, comparative genomic hybridization data of 123 cases, and gene expression microarray data of 119 patients to firstly perform a comprehensive analysis of the gene alterations of 7 DDR pathways in ESCC. Gene mutations and copy number variations (CNVs) were observed in all 7 DDR pathways, and especially, CNVs were the dominant alteration types. Compared with other pathways, two DNA double-strand break (DSB) repair pathways homologous recombination (HR) and non-homologous end joining (NHEJ), carried significant gene mutations and CNVs especially gene amplifications. Most genes including  Esophageal cancer, principally comprising of two pathological types: esophageal squamous cell cancer (ESCC) and esophageal adenocarcinoma, is a global problem and the sixth leading cause of cancer mortality annually worldwide. The overall 5-year survival of patients with esophageal cancer ranges from 15 to 25%. ESCC accounts for 70% of cases of esophageal cancer globally and is the dominant type of esophageal cancer in China (DNA damage repair (DDR) genes have crucial roles in maintaining genomic stability of human cells. According to biochemical and mechanistic criteria, DDR genes can be grouped into seven main functional pathways. Base excision repair (BER) and nucleotide excision repair (NER) are involved in DNA base damage repair, while mismatch repair (MMR) mainly corrects base mis-pairs. Homologous recombination (HR) and non-homologous end joining (NHEJ) are two pathways which contribute to DNA double-strand break (DSB) repair. In addition, the Fanconi anemia (FA) pathway is associated with the repair of DNA inter-strand crosslinks in the genome, and specialized DNA polymerases in trans-lesion synthesis (TLS) pathway synthesize DNA to bypass unrepaired DNA lesions , 4. DysrXRCC6 and XRCC5, two genes in NHEJ pathway, were related to higher risk of ESCC  was conducted to analyze the pathway activity changes of HR and NHEJ, and DSB repair pathways were observed to be up-regulated in ESCC. We finally investigated the effect of combination of mirin and NU7441 with ionizing radiation (IR) treatment on ESCC cell phenotypes, and found that mirin and NU7441 could enhance the radio-sensitivity of ESCC cells with DSB pathway gene amplification. These findings suggest that alterations of DSB repair pathways might be involved in ESCC radio-resistance, and mirin and NU7441 might have potential application in ESCC treatment.A workflow was designed to identify the gene alterations in DDR pathways , 19, andhttps://www.ncbi.nlm.nih.gov/geo/) database. To extract gene expression information of DDR genes in GSE53624, we re-annotated probes from Agilent-038314 CBC Homo sapiens lncRNA + mRNA microarray V2.0 platform (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL18109). Human protein-coding transcript sequences (release 29) were downloaded from GENCODE (https://www.gencodegenes.org/) database. All probes in Agilent-038314 platform were then re-annotated as follow: 1, All probes sequences were aligned to human protein-coding transcript sequences with BLASTN. 2, The probes that were matched to one transcript or multiple transcripts from same genes were reserved. 3, The max expression value of multiple probes that were mapped to the same gene was calculated to represent the expression level of the gene. The clinical characteristics of 119 ESCC patients in GSE53624 were summarized in Additionally, in order to analyze the mRNA expression of DSB repair pathway genes in ESCC, we downloaded GSE53624 dataset, the mRNA expression profile of paired cancer, and adjacent normal tissues from 119 ESCC patients , from GE2. The source of YES2 and KYSE30 cell lines have been recently authenticated and tested for mycoplasma contamination, and no contamination was found.The human ESCC cell lines YES2 and KYSE30 were obtained from Y. Shimada’s lab in Kyoto University. YES2 and KYSE30 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10% fetal bovine serum (FBS), and were grown at 37°C in humidified air with 5% COvia R “GSVA” package with the following parameters: method = “gsva,” mx.diff = “TRUE,” and kcdf = “Gaussian.”GSVA, using a nonparametric approach to transform a gene-by-sample matrix into a gene set-by-sample matrix, facilitates to determine the variation of pre-defined gene set activities over the samples based on gene expression data . Express4 YES2 and KYSE30 cells were seeded into confocal dishes for 24 h prior to treatment with mirin  (50 µM) and NU7441  (5 µM). After treatment with inhibitors for 1 h, cells were exposed to 6 Gy of IR. Then, cells were cultured with inhibitors for 24 h. Subsequently, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature, then were permeabilized with PBST (0.5% Triton X-100 in PBS) for 10 min on ice. Nonspecific binding was blocked with 1% bovine serum albumin (BSA) in PBST for 30 min. Then, the cells were incubated in the diluted antibody against γ-H2AX  in 1% BSA (1:200) in a humidified chamber for overnight at 4°C and followed by incubation with Alexa Fluor 488-conjugated secondary antibody  in PBS for 1 h at room temperature in the dark. Immunofluorescence images were taken by using laser-scanning confocal microscope .A total of 1×10To assess how combination of inhibitors with IR treatment affects clonogenic cell survival, YES2, and KYSE30 cells were seeded into six-well plates at a density of 1,500 and 1,000 cells per well, respectively. The cells were incubated for 10 days. Then, cells were treated with mirin (50 µM) and NU7441 (5 µM) for 1 h and irradiated afterward once with 6 Gy. After incubation with inhibitors for an additional 3 days, the cells were fixed with methanol for 5 min, and stained with 0.05% crystal violet  for 5 min. Colonies were counted by using ImageJ 1.52V software.To examine the effect of combination of inhibitors with IR treatment on cell proliferation, YES2 and KYSE30 cells were seeded into 96-well plates at a density of 5,000 cells per well for 24 h. Then, cells were incubated with mirin (50 µM) and NU7441 (5 µM) for 1 h, followed by being exposed to 6 Gy of IR. Subsequently, the optical density (OD) value at 490 nm was detected after 0, 24, 48, 72, and 96 h with a microplate reader  after treatment with  (MTS) (Promega) solution (10% MTS in RPMI 1640 medium) for 1 h. The experiment was repeated three times, and the ratio of OD value (hours 24–96) to the average value of 0 h was calculated and plotted as MTS curves.5 YES2 and KYSE30 cells were seeded into 6 cm dishes and cultured for 24 h. Subsequently, cells were treated with mirin (50 µM) and NU7441 (5 µM) for 1 h, and were then exposed to 6 Gy of IR. After being cultured with inhibitors for 24 h, cells were collected and stained with annexin V and propidium iodide (PI) according to the manufacturer’s instruction provided in Annexin V-FITC/PI apoptosis assay kit . Flow cytometry (BD LSR) was used to determine the percentage of apoptotic cells.A total of 2×10t-test was used to compare the GSVA scores in ESCC and normal samples, and to analyze the correlations between the GSVA scores and clinical characteristics of ESCC patients. ESCC patients were divided into two groups (high and low groups) according to the median value of gene expression or GSVA scores, and survival analysis was conducted by Kaplan-Meier method. Besides, the correlations between gene expression and clinical characteristics of ESCC patients were analyzed with Fisher’s exact test. The other statistical analyses were performed with Student’s t-test. Each P was two-sided, and P < 0.05 was considered statistically significant.All statistical tests and graphing were performed by R 3.6.0 and GraphPad Prism 7.0. All of the experiments in this study were independently performed in triplicate, and the data was presented as mean ± standard deviation (S.D.). Fisher’s exact test was applied to gene mutation enrichment analysis. Survival curves were performed by Kaplan-Meier method, and the differences between the curves were estimated by log-rank test. Welch’s unequal variances In order to investigate the landscape of genetic alterations in DDR pathways, we defined a “core DDR” gene set of 79 DNA repair pathway-specific genes (genes annotated to more than one specific DDR pathway were not included), encompassing 7 major DDR pathways: BER, NER, MMR, HR, NHEJ, FA, and TLS , 22.POLB, LIG1, and LIG3) mutated in BER pathway, each of which only carried one mutation. In NER pathway, both XPC and ERCC6 had two mutations and ERCC2 had one mutation. Similarly, three genes  in MMR pathway each had one mutation event and MLH1 was mutated in two samples. In HR pathway, BRCA1/2 showed four mutation events . We also observed that MRE11-RAD50-NBS1 (MRN) complex genes, which play important roles in the sensing, processing and repair of DSBs , carried the most frequent mutations . Furthermore, both TP53BP1 and LIG4 had two mutations. FANCM was the highest frequently mutated gene of FA pathway , and REV3L was the most frequently  mutated gene in TLS pathway. Interestingly, we observed that most ESCC samples only had one DDR gene mutation, indicating a mutually exclusive tendency.Firstly, we investigated the non-silent somatic mutation profile of DDR pathway genes in ESCC. As shown in  of DSBs , were muP = 0.022 and P = 0.004, respectively). Similarly, HR and NHEJ pathways tended to be enriched in ESCC . We combined HR and NHEJ as the “DSB repair pathway” and conducted enrichment analysis. The result showed that the “DSB repair pathway” mutation was significantly enriched in ESCC  of cases and the incidence of amplification of APEX1/2 was 14.3% (22/154). Besides, PARP1 revealed 5.8% (9/154) of amplification frequency and 0.6% (1/154) of deletion frequency. On the contrary, some genes in NER pathway had more deletions instead of amplifications. CUL5 had the highest deletion frequency , and ERCC1 and ERCC2 had the same CNV profile (3.9% of amplification and 6.5% of deletion) due to proximal genomic location. Gene polymorphisms of XPA and XPC were reported to be associated with increasing risk of ESCC (XPA was amplified in 8 ESCC patients (5.2%) and deleted in 2 ESCC cases (1.3%), whereas the incidences of amplification and deletion of XPC were 1.3% (2/154) and 5.2% (8/154), respectively. MLH1, one of the important genes in MMR process, was observed to be deleted in 5.2% (8/154) of ESCC cases. Other genes in MMR pathway mainly had more amplifications than deletions.Next, we identified the CNV profile of DDR pathway genes in ESCC. As the complex heatmap in 10.4% 16/4 of caseRAD54B  which is related to multiple cancers  of ESCC patients. Cheng and colleagues reported that XRCC3 was up-regulated in ESCC and was a potential target to improve the radiotherapy effect of ESCC (XRCC3 was amplified in 11.7% (18/154) of ESCC cases, indicating that up-regulation of XRCC3 might be due to gene amplification. Other genes such as SHFM1, RAD52, RAD51B, and MUS81 were also remarkably amplified in ESCC with the frequency 16.2% (25/154), 12.3% (19/154), 11% (17/154), and 10.4% (16/154), respectively. In NHEJ pathway, PRKDC was one of significantly amplified DDR pathway genes with the amplification frequency 21.4% (33/154). Another notably amplified gene was POLM , which has not been studied in cancers yet. Besides, XRCC6 had more amplification events  than deletions   of ESCC cases. Conversely, FANCL, FANCM, FACC, UBE2T, and FANCI obviously had more amplification events. In TLS pathway, although POLQ did not have significant mutation events, this gene was obviously amplified in ESCC . Similarly, the main CNV type of POLH and REV1 was deletion. REV3L, the most mutated gene in TLS pathway, carried 8 (5.2%) amplification events .Given the fact that HR and NHEJ, the DSB repair pathways, had significant gene amplifications, we were interested to investigate the correlation between amplification of DSB repair pathways and clinical characteristics of ESCC patients. Survival analysis showed that ESCC samples with amplification of HR or NHEJ pathway had shorter overall survival . Besides, XRCC5 expression was related to lymph node metastasis (P = 0.027).We also investigated the correlations between expression of DSB repair pathway genes and clinical traits of ESCC patients. Survival analysis showed that high expression of five genes including P < 0.0001, P < 0.0001, and P < 0.0001, respectively; P = 0.0186, P = 0.0187, and P = 0.0180, respectively; P = 0.0004; P = 0.0334; P = 0.0468; In order to further compare the activities of DSB repair pathways between ESCC and normal tissues, we conducted GSVA based on expression of DSB repair pathway genes. We observed that pathway activities of HR, NHEJ and the combined “DSB repair pathway” were all significantly up-regulated in ESCC based on GSVA scores . Therefore, we used YES2 and KYSE30 cells to investigate the effects of mirin and NU7441. We firstly tested if mirin and NU7441 could induce DSBs. As phosphorylation of H2AX (γ-H2AX) is a hall marker of DSBs , we condNext, to investigate the effect of mirin and NU7441 on clonogenic cell survival of ESCC cells, we conducted clonogenic assay. IR treatment alone did not have a significant influence on clonogenic survival of both YE2 and KYSE30 cells , NCBI Sequence Read Archive  and Gene Expression Omnibus . Additionally, gene mutation and clinical information within this study can be found in the supplementary materials of the previously published studies (https://www.nature.com/articles/nature13176) (https://www.cell.com/ajhg/fulltext/S0002-9297(15)00100-7).Publicly available datasets were analyzed in this study. This data can be found here: European Genome-phenome Archive , Beijing Municipal Commission of Health and Family Planning Project (PXM2018_026279_000005).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.