Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies. Neoplastic diseases represent one of the leading causes of death in humans and small animals ,2,3. DesAn interesting approach, coming into focus over the past years, is represented by viral oncolysis. The first incidental and anecdotal descriptions of tumor regressions after a spontaneous virus infection or vaccination date several decades back . Since tInitially, tumor regression induced by oncolytic viruses was thought to occur through direct lysis of tumor cells . HoweverCanine distemper virus (CDV), a member of the family Paramyxoviridae, genus Morbillivirus, represents an interesting candidate for viral oncolysis . SimilarResults from a previously established murine xenotransplantation model of canine histiocytic sarcomas clearly indicated that expression of MMP-2, MMP-9, MMP-14 and TIMP-1 by tumor cells and tumor-associated macrophages and the positive correlation between MMP expression and microvessel density contributed to tumor progression and invasion . Moreove3 (~1.5 cm in diameter). Therefore, the last planned time point (77 dpt) was excluded from further analyses. The clinically determined tumor size is shown in Subcutaneously xenotransplanted DH82 cells formed tumors with a diameter of approximately 0.5 cm 35 days post-transplantation (dpt), displaying no significant differences between the groups. At this time point, treatment by intratumoral infection with CDV was initiated and repeated 10 times every second day and compared to controls. The latter comprised groups receiving either an intratumoral application of UV-inactivated CDV or cell culture medium or were left untreated. Animals receiving intratumoral injections with UV-inactivated CDV or medium were preliminary sacrificed at 70 dpt because tumors exceeded a volume of 1.6875 cmp values ranging from 0.0002 to 0.0303) in tumor volume between 35 dpt and the day of death.Over time, all groups displayed a significant increase (p = 0.0058). At 41 dpt non-treated neoplasms (p = 0.0006) and tumors of both intratumorally treated control groups exhibited a significantly larger size (p = 0.0040 and p = 0.0006) than neoplasms infected with CDV. Similar findings were present over time with few exceptions at 44 dpt and 54 dpt. In the former time point (44 dpt), no significant difference was observed between the CDV-infected group and the group treated with UV-inactivated CDV or left untreated. At the latter time point (54 dpt), only untreated control tumors were significantly larger than neoplasms infected with CDV (p = 0.0306). Unfortunately, the size of both intratumorally injected control groups (DH82-UV-CDVai and DH82-medium) was not determined at this time point. Starting at 65 dpt, no significant differences were found between CDV-infected and medium-injected neoplasms and untreated controls. Tumors injected with UV-inactivated CDV were significantly larger (p = 0.0225) than neoplasms infected with CDV until 70 dpt at which animals treated with UV-inactivated virus were sacrificed. Taken together, a tenfold intratumoral injection with CDV-Ond led to a significant reduction in tumor growth from day 35 to day 70 compared to controls.Interestingly, the volume of tumors infected with CDV was already significantly smaller at 37 dpt when compared to neoplasms injected with medium  and distinct cell boundaries. In addition, the cells showed homogenous to finely granulated or vacuolated, eosinophilic cytoplasm and oval to polygonal, central to eccentric nuclei with coarse chromatin and prominent eosinophilic nucleoli. A moderate to high anisocytosis and anisokaryosis was present. The mitotic index varied from 8–11 mitotic figures per high-power field. Tumors from all groups showed a mild multifocal infiltration with mononuclear cells. In addition to HE-stained sections, tumor cells were highlighted using immunohistochemical staining of neoplastic cells with a CD44 antibody. Tumors of all groups displayed a strong CD44 expression within neoplastic cells.Areas of necrosis were detected in all groups, which appeared as hypereosinophilic areas with loss of cellular details and indistinct cell boundaries in addition to nuclear pyknosis, karyorrhexis and karyolysis.p values between 0.0051 and 0.0306). At 54 dpt DH82-CDVai xenografts displayed significantly larger areas of necrosis than non-treated DH82 neoplasms (p = 0.0075) or DH82-UV-CDVai tumors (p = 0.0225). Areas of necrosis were significantly larger in DH82-CDVai xenografts than non-treated DH82 transplants (p = 0.0081) at 63 dpt. At 44 and 54 dpt, no significant differences were observed between the control groups. At 63 dpt, DH82-medium xenografts displayed significantly larger necrosis areas than non-treated DH82 neoplasms (p = 0.0082), whereas no significant differences were observed between the other control groups.DH82-CDVai xenografts showed a higher median area of necrosis compared to non-infected controls at all time points . StatistIn none of the groups, statistically significant changes over time were observed. All data are summarized in p values between 0.0081 and 0.0200; CDV nucleoprotein was detected in DH82-CDVai and DH82-UV-CDVai xenografts using immunohistochemistry. Immunoreactivity was characterized by a localized intracytoplasmic or membranous staining within tumor cells. Morphometric analysis of DH82-CDVai xenografts exhibited a median positive area of 0.126% at 44 dpt , 0.227% at 54 dpt , and 0.121% at 63 dpt (10 days after last treatment). DH82-UV-CDVai xenografts exhibited a median positive area of 0.014% at 44 dpt , 0.003% at 54 dpt , and 0.002% at 63 dpt . Although the replication of the virus was limited to a small percentage of area in both investigated groups, CDV immunoreactivity was present in larger areas in DH82-CDVai compared to DH82-UV-CDVai at all time points investigated , whereas no significant differences were observed between the different control groups. At 63 dpt, no significant differences were found between the different groups. The cleaved caspase-3 expressing area decreased significantly in DH82-CDVai over time . In non-infected DH82 transplants, the cleaved caspase-3 immunopositive area was significantly smaller (p = 0.0306) at 54 dpt compared to 44 dpt. Furthermore, there was an increase in the cleaved caspase-3 immunopositive area at 63 dpt in DH82-UV-CDVai xenografts compared to tumors at 54 dpt in this group (p = 0.0453). DH82-medium xenotransplants displayed no changes in the cleaved caspase-3 immunopositive area over time. All data are summarized in Apoptotic tumor cells were detected by measuring the area exhibiting cleaved caspase-3 immunoreactivity . In genep values ranging from 0.0051 to 0.0453; p = 0.0306). At 63 dpt, no significant differences were observed between the different groups.Tumor-associated macrophages were investigated by determining the Mac3 (CD107b/LAMP2) immunoreactive area . A mild p = 0.0453). Furthermore, a significant increase between 44 dpt and 54 dpt (p = 0.0306) or 63 dpt (p = 0.0306), respectively, was found in DH82-medium transplants. In all other groups, no significant changes were present over time. All data are summarized in In non-infected DH82 xenotransplants, a significant increase in the Mac3-positive area was present from 44 dpt to 63 dpt . The only exceptions were present at 63 dpt, where MMP-2 in DH82-CDVai and MMP-14 in DH82-CDVai, and DH82-UV-CDVai lacked significant differences between the different localizations. Considering that the periphery of the tumor is supposed to be the most important area with the highest biological relevance representing the invasive front, further analyses concentrated on this localization [The immunopositive area for MMP-2 , MMP-9 , MMP-14 lization ,36. The p < 0.0001) and DH82-UV-CDVai tumors (p = 0.0005). In addition, a significantly larger peripheral area expressing MMP-2 was noted at 63 dpt in DH82-UV-CDVai neoplasms compared to untreated controls (p = 0.0017) and DH82-medium xenografts (p = 0.0038). All other comparisons between different groups at the same time point lacked significant differences. A comparison of the MMP-2 expression over time revealed a significantly smaller peripheral immunopositive area in non-treated controls at 54 dpt (p < 0.0001) and 63 dpt (p < 0.0001) compared to 44 dpt. Similarly, DH82-medium xenografts comprised a smaller-sized MMP-2-positive area at 54 dpt (p = 0.0036) and 63 dpt (p < 0.0001) within the tumor periphery compared to 44 dpt. An opposite development was noted in DH82-UV-CDVai xenografts, which exhibited a larger peripheral MMP-2 immunoreactive area at 63 dpt compared to 54 dpt (p = 0.0059). Besides the aforementioned, no significant differences were present over time within the different groups (The MMP-2-positive area within the tumor periphery was at 54 dpt significantly larger in DH82-CDVai xenografts compared to untreated controls (t groups A.p < 0.0001). A similar result was obtained at 63 dpt. At this time point DH82-CDVai neoplasms exhibited a larger peripheral MMP-9 immunopositive area than DH82-UV-CDVai tumors (p = 0.0033) or DH82-medium xenografts (p < 0.0001). Furthermore, a significantly larger peripheral MMP-9 area was observed in DH82-medium tumors at 63 dpt compared to untreated control neoplasms (p = 0.0002). All other comparisons lacked statistically significant differences. Interestingly, the peripheral MMP-9 expression in DH82-CDVai xenografts decreased significantly over time . With the exception of DH82-medium xenografts, which exhibited an increase in the peripheral MMP-9 immunopositive area from 44 dpt to 63 dpt (p = 0.0428), all other groups lacked statistically significant changes over time . Additionally, untreated control neoplasms revealed a smaller MMP-14 immunopositive area at 44 dpt compared to DH82-UV-CDVai tumors (p < 0.0001). No other significant changes were observed between the other groups independent of the time point. Interestingly, DH82-CDVai xenografts revealed a decrease in the peripheral MMP-14 immunopositive area from 44 dpt to 63 dpt (p = 0.0001). In contrast, DH82-UV-CDVai and DH82-medium tumors developed a reduction of the peripheral MMP-14 immunopositive area from 44 dpt to 54 dpt (p = 0.0004 and p < 0.0001). This decrease was followed by significant enlargement of the peripheral MMP-14 immunopositive area from 54 dpt to 63 dpt in both groups (p < 0.0001). All other groups lacked statistically significant changes over time . A contrary result was obtained at 54 dpt. At this time point, DH82-UV-CDVai neoplasms exhibited significantly larger peripheral TIMP-1 immunopositive areas than xenografts of all other groups (p < 0.0001). At the last time point investigated (63 dpt), DH82-CDVai tumors revealed a significantly smaller peripheral TIMP-1 immunopositive area than DH82-medium xenografts (p < 0.0001). Furthermore, DH82-medium neoplasms displayed a smaller peripheral TIMP-1 immunopositive area than DH82-UV-CDVai (p < 0.0001) xenografts or untreated controls (p < 0.0001). Interestingly, DH82-CDVai, DH82-medium tumors and untreated controls showed a significant smaller peripheral TIMP-1 immunopositive area at 54 dpt (p < 0.0001) and 63 dpt (p < 0.0001) compared to 44 dpt. In DH82-UV-CDVai xenografts, an increase in the peripheral TIMP-1 immunopositive area was observed from 44 dpt to 54 dpt (p = 0.0124), followed by a decrease from 54 dpt to 63 dpt (p < 0.0001). This resulted in a significantly smaller peripheral TIMP-1 immunopositive area at 63 dpt compared to 44 dpt  and untreated controls (p = 0.0453). At 63 dpt, a significantly lower microvessel density was present in DH82-CDVai xenografts compared to DH82-UV-CDVai (p = 0.0137) and untreated controls (p = 0.0080). All other groups lacked significant differences in the microvessel density at the respective time point. The microvessel density significantly increased over time in all control groups , with the exception of DH82-medium xenografts lacking a further increase in microvessel density from 54 dpt to 63 dpt. A contrary finding was present in DH82-CDVai tumors, which displayed a stable number of vessels per area as reflected by lacking statistically significant differences over time . Interestingly, this was associated with large areas of necrosis within CDV-infected tumors, which were only detectable in limited amounts in control groups, while apoptosis seems to play a minor role in all groups. In contrast to the present findings, cell death via apoptosis was suspected in CCT cells, another canine histiocytic sarcoma cell line, after infection with CDV . This isThe concept of reduced angiogenesis as a viral oncolysis mechanism has been described before for various virus families, including Poxviridae and Rhabdoviridae ,43,44. SAn important factor interfering with virus spread and angiogenesis within neoplasms is represented by the tumor microenvironment , with meMMPs and TIMP-1 are often secreted directly by tumor cells ,57. HoweSummarized, the present study demonstrates that an intratumoral CDV-infection of canine histiocytic sarcoma xenografts led to reduced tumor growth. In this context, inhibition of angiogenesis in combination with an enhancement of the innate immune response seems to play an important role in modulating the expression of matrix modifying enzymes as one factor. However, the exact interplay between angiogenesis and innate immune response as represented by tumor-associated macrophages needs to be detailed in further studies.DH82 cells, a permanent canine histiocytic sarcoma cell line, were obtained from the European Collection of Authenticated Cell Cultures (ECACC No. 94062922). Cultivation of DH82 cells for grafting was carried out in standard conditions as previously described ,34,45,466 DH82 cells in 100 µL FBS- and NEAA-free medium were injected once into the subcutis of each animal’s left flank using single-use pen needles . Subsequently, tumor development was monitored every second to the third day by measuring tumor width and length with an analog vernier precision caliper . Tumor volume was calculated as ((shortest diameter2 × longest diameter)/2) [A total number of 96 female severe combined immunodeficiency (SCID) mice (CB17/Icr-Prkdcscid/lcrlcoCrl) were obtained from a commercial breeder  at 4 weeks of age. Mice were kept in groups of 6 in individually ventilated cage systems  and had an adaptation period of 2 weeks. Mice were subcutaneously xenotransplanted with DH82 cells as previously described ,34. Brieeter)/2) . Further3), 3 groups of mice (n = 6 per group and time point) were injected ten times every second day with (1) 100 µL intratumoral injections of CDV, strain Onderstepoort  or (2) UV-inactivated CDV-Ond (DH82-UV-CDVai xenografts) or (3) medium (DH82-medium xenografts) in the same amount and following the same treatment scheme as controls. Non-treated DH82 xenografts (n = 6 per time point) served as additional controls (non-infected DH82 xenografts). Tumor growth was monitored as described above. In addition, mice were controlled for changes in body weight and signs of adverse reactions. Necropsies were performed, and tumors were sampled on days 44, 54, 63 and 77 after transplantation (dpt). Animals were preliminary sacrificed when tumors exceeded a volume of 1.7 cm3 .Starting 35 days after xenotransplantation when tumors exhibited a diameter of ~0.5 cm  were either stained with hematoxylin–eosin (HE) for standard histology or used for immunohistochemical analysis.Immunohistochemistry (IHC) was performed by applying the avidin-biotin-peroxidase complex  procedure as previously described ,49,62,63For negative controls, specific primary antibodies were replaced by ascitic fluid from non-immunized BALB/cJ mice (for CDV and MMP-2), an anti-rat IgG1 isotype antibody (for Mac3), rat serum from non-immunized rats (for CD44) and serum from non-immunized rabbits . The dilution of negative controls was chosen according to the protein concentration of replaced primary antibodies.2. Afterwards, the percentage of necrotic tumor area was calculated by dividing necrotic area by total tumor area and multiplying the result by 100.HE-stained tumor slides were morphometrically analyzed to determine total tumor area and to quantify necrosis. Therefore, HE stained slides containing a cross-section of the complete tumor diameter were digitized , and morphometry was performed using the Aperio ImageScope software . The percentage of necrotic tumor areas was determined by manually contouring the boundaries of each neoplasm to adjacent subcutaneous tissue, as were the areas of necrosis within the tumor. Calculation of corresponding areas was stated in µmSimilarly, immunohistochemically stained slides using antibodies directed against CDV, Mac3 (CD107b/LAMP2) and cleaved caspase-3 were digitized. Morphometry was performed using the Aperio ImageScope software .2 total tumor area. Thresholds of positive pixel count v9 algorithm for each staining were manually adjusted according to staining intensity.The area of chromogen expression was determined within the total area of the grafted tumor tissue (“region of interest”) and stated as positive pixels/µmFor MMPs and TIMP-1, pictures from each xenotransplant were taken at a magnification of ×400 in 10 randomly selected areas of tumor center and periphery, respectively, using the Cell-D imaging software . Obtained pictures were analyzed using the positive pixel count algorithm of the Aperio ImageScope viewer . Only areas containing neoplastic tissue were analyzed. Tumor-free regions, necrotic areas and artifacts  were excluded from further analysis. For the positive pixel count algorithm, hue value of 0.1 and hue width of 0.5, color saturation threshold of 0.04 were used, and any intensity of staining was considered positive. The number of positive pixels was divided by the total number of pixels (negative and positive) in the analyzed area and multiplied by 100 to receive the percentage of positive pixels. Values from each section were averaged.2 tumor area.Microvessel density analysis within the xenografts was performed by manually counting the number of CD31-positive structures containing a definite lumen as described before . Twenty-p < 0.05. Analyses were performed with the Statistical Analysis System, Version 9.3 .Statistical analysis of tumor volume development, histological and immunohistological evaluation was calculated using the Wilcoxon rank-sum test with the significance level Analysis of immunohistochemical data for MMPs, TIMP-1, including the difference in MMP and TIMP-1 expression between different groups and between central and peripheral areas of the xenotransplanted histiocytic sarcomas was performed using the Wilcoxon signed-rank test with a Tukey–Kramer post hoc adjustment.Graphical illustrations were designed with GraphPad Prism, Version 8.0.1 .


Myocardial infarction is the leading cause of cardiovascular mortality, with myocardial injury occurring during ischemia and subsequent reperfusion (IR). We previously showed that the inhibition of protein kinase C delta (δPKC) with a pan-inhibitor (δV1-1) mitigates myocardial injury and improves mitochondrial function in animal models of IR, and in humans with acute myocardial infarction, when treated at the time of opening of the occluded blood vessel, at reperfusion. Cardiac troponin I (cTnI), a key sarcomeric protein in cardiomyocyte contraction, is phosphorylated by δPKC during reperfusion. Here, we describe a rationally-designed, selective, high-affinity, eight amino acid peptide that inhibits cTnI’s interaction with, and phosphorylation by, δPKC (ψTnI), and prevents tissue injury in a Langendorff model of myocardial infarction, ex vivo. Unexpectedly, we also found that this treatment attenuates IR-induced mitochondrial dysfunction. These data suggest that δPKC phosphorylation of cTnI is critical in IR injury, and that a cTnI/δPKC interaction inhibitor should be considered as a therapeutic target to reduce cardiac injury after myocardial infarction. Myocardial infarction (MI) is the leading cause of cardiovascular death ,2, with d ~ 5 nM; To determine the effects of cTnI phosphorylation by δPKC, we employed our method to rationally design a peptide inhibitor of protein–protein interactions using a homology search to identify similar regions in the otherwise non-related proteins, δPKC and cTnI A. We pre30minR60min) in a Langendorff apparatus led to a greater than four-fold increase in the phosphorylation of cTnI, as compared to a heart subjected to perfusion under normoxic conditions (90 min perfusion). We found that this increase in cTnI phosphorylation was prevented by treatment with ψTnI at the time of reperfusion, similar to the pan-sensitive δPKC inhibitor (δV1-1) , had no biological activity, demonstrating the specificity of the peptide  A. ψTnI w (δV1-1) B. Import (δV1-1) A. Direct (δV1-1) B, to a s peptide A,B.Mitochondrial dysfunction is a key feature of IR injury ,20,21,22Cardiac troponin I regulates cardiomyocyte contraction in conjunction with troponin C and troponin T via the calcium-mediated interaction between actin and myosin. In an ex vivo model of cardiac IR injury, we observed a decrease in myocardial damage, as evidenced by a reduction in creatine kinase release and infarct size when the δPKC-mediated phosphorylation of cTnI was selectively inhibited by a novel protein–protein interaction inhibitory peptide, ψTnI; the effect of ψTnI was similar to the benefit observed when treating with the pan-inhibitor of all δPKC phosphorylation, δV1-1. In addition to attenuating IR-induced myocardial injury, blocking the phosphorylation of cTnI by δPKC ameliorated the mitochondrial dysfunction associated with IR, as evidenced by the increased mitochondrial oxygen consumption and decreased mitochondrial oxidant production. Myocardial infarction is the leading cause of cardiovascular mortality, with injury occurring during ischemia and subsequent reperfusion. Most therapeutics are targeted towards re-establishing and maintaining perfusion, and there are currently no established therapies to mitigate reperfusion injury. We previously found that δPKC is a key mediator of IR injury, and the inhibition of δPKC activity, with the pan-inhibitor peptide δV1-1, at the time of reperfusion attenuates injury in animal models of IR, and in patients with acute myocardial infarction ,9. δPKC 2O2 from isolated cardiac mitochondria after IR. As expected, cardiac IR decreased mitochondrial function (as measured by oxygen consumption), and increased H2O2 production. Interestingly, treatment with ψTnI was sufficient to prevent the IR-induced increase in mitochondrial oxidant production and the decrease in oxygen consumption. This improvement in mitochondrial function was paralleled by an improvement in tissue viability in IR hearts treated with ψTnI. An IR event leads to critical changes in mitochondrial function, bioenergetics, and redox balance in the cardiomyocyte ,37. ThesWe rationally designed and utilized a selective peptide inhibitor of this phosphorylation event as a tool to assess the role of cTnI phosphorylation in IR injury. The advantage of our approach is that peptides have good biodistribution, with cell entry facilitated by the conjugated TAT sequence; peptides are water-soluble and consist of naturally occurring amino acids . FurtherBetween 10- and 12-week-old wild-type male Sprague Dawley rats (250–300 g) were purchased from Charles River; only male rats were used to reduce the potential confounding factor of cardioprotective estrogen effects. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30–90 mg/kg) and sodium heparin (1000 units/kg). After 4–5 min, pain sensitivity was performed via a toe pinch, and the hearts were removed by a terminal surgical procedure. The Administrative Panel on Laboratory Animal Care at Stanford University (protocol #10363) and the Ethic Committee on Animal Use of the Institute of Biomedical Sciences at University of Sao Paulo (protocol #60/2017) approved all animal protocols.MU peptide were synthesized on solid support using a fully automated microwave peptide synthesizer , using a homology sequence, conjugated to TAT47-57 carrier peptide, a short positively charged peptide that is used as a carrier for the delivery of the peptide into the cell, using a Gly-Gly spacer. The peptides were synthesized by SPPS (solid phase peptide synthesis) methodology with a fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) protocol. The final cleavage and side chain deprotection was performed manually without microwave energy. Peptides were analyzed by analytical reverse-phase high-pressure liquid chromatography (RP-HPLC)  and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), and purified by preparative RP-HPLC .Peptides were synthesized as previously described . In bried. Kd values were also calculated by measurement of the Kon and Koff values at a single concentration. This was performed by fitting the binding curve to a double exponential function, and the first rinse to a single exponential, using a single concentration. Results are from three independent experiments.In vitro binding assay was performed as previously described . PeptideMU (TAT-DWAANIDA) was administered at 1μM at the time indicated in Isolated rat hearts were hung on a Langendorff apparatus in the Mochly-Rosen laboratory at Stanford University, as we previously described . Only raPDB ID: 4Y99 was loaded into the Molecular Operating Environment (MOE) 2019.0102 software; 4Y99.C chain (cTnI) was kept and other chains were deleted. 4Y99.C was prepared using the QuickPrep functionality in MOE with default settings. QuickPrep optimizes the H-bond network and performs energy minimization on the system.In order to better visualize the degree of conservation of cTnI across evolution, a heat map was constructed. A BLAST query was performed on the ψTnI sequence and the top 50 sequences, scored by similarity, were collected. Then, orthologs for each species were collected. For each gene, the number of identities compared to the human ψTnI sequence was enumerated. These data were then expressed as a heatmap. To further examine the degree of conservation of the ψTnI sequence, a PSI-BLAST (Position-Specific Iterative BLAST) was performed using human cTnI (UniProt ID: P19429) and human δPKC (UniProt ID: Q05655) as the query sequences. PSI-BLAST was utilized as it utilized more general protein “features” to identify homologies; therefore, it is better suited to examine distant evolutionary relationships [g 15 min at 4℃). Samples were boiled with Laemmli buffer containing 2-mercaptoethanol, loaded on SDS–PAGE, and transferred to PVDF membrane, 0.45 μm . Membranes were probed with the antibody of interest, and then visualized by ECL , 1.25 mM 3-aminophthalhydrazide  in 1 M Tris pH 8.5. Antibody specificity was validated based on the target protein molecular weight on Western blot. The following antibodies were used in this study: cardiac troponin I ; troponin I-C antibody (D-12) ; phospho-troponin I (Cardiac) (Ser23/24) antibody ; phospho-IRS1 ; phospho-Drp1 ; phospho-MARCKS ; phospho-STAT ; mouse IgG HRP linked whole Ab ; rabbit IgG HRP linked whole Ab .A Bradford assay (Bio-Rad Protein Assay Dye Reagent Concentrate (#5000006)) was used for protein quantification, after samples were lysed in 1x lysis buffer, and lysate was separated from cell debris , containing succinate 2 mM , malate 2 mM  and glutamate 2 mM  substrates, with continuous stirring at 37 °C. ADP 1 mM  was added to induce state 3 respiratory rate. Mitochondrial O2 consumption was monitored using a computer-interfaced Clark-type electrode (OROBOROS Oxygraph−2k). Mitochondrial H2O2 release was measured using Amplex Red 25 μM (Molecular Probes A12222) horseradish peroxidase 0.5 U/mL (Sigma P8125) system, and detected using a fluorescence spectrophotometer (ʎex = 563/ʎem = 587 nm) (F-2500 Hitachi—Hitachi).Heart mitochondrial function was assessed as previously described . Brieflyp < 0.05 (GraphPad Prism).Values are presented as mean ± standard deviation relative to the average value for the control group, unless otherwise stated. Group differences were assessed by one-way ANOVA with Tukey’s correction for multiple comparisons with statistical significance established at We rationally designed and utilized a selective peptide inhibitor of δPKC phosphorylation of cTnI as a tool to assess the role of cTnI phosphorylation during IR injury. The inhibition of cTnI phosphorylation by δPKC with ψTnI attenuates myocardial IR injury and prevents IR-induced mitochondrial dysfunction.


Current research aimed to explore the therapeutic values of different earthworms as antibacterial, anticoagulant, and antioxidant agents. Amynthas corticis, Amynthas gracilis, Pheretima posthuma, Eisenia fetida, Aporrectodea rosea, Allolobophora chlorotica, Aporrectodea trapezoides, Polypheretima elongata, Aporrectodea caliginosa, and Pheretima hawayana, were collected and screened for biological activities. Antibacterial effect analysis of earthworm species was done against fourteen bacterial pathogens, i.e., Escherichia coli, Serratia marcescens, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa (1), Salmonella typhimurium, Shigella flexneri, Enterobacter amnigenus, Serratia odorifera, Pseudomonas aeruginosa (2), Staphylococcus warneri, and Lactobacillus curvatus, via agar well diffusion, crystal violet, MTT, agar disc diffusion, and direct bioautography assays. Antioxidant potential was evaluated through ABTS and DPPH assays. Lipolytic, proteolytic, and amylolytic assays were done for lipase, protease, and amylase enzymes confirmation. In vitro anticoagulant effects were examined in the blood samples by measuring prothrombin time.  Ten different earthworms, i.e., P. aeruginosa (1), P. aeruginosa (2), S. warneri, and L. curvatus. The maximum zone of inhibition of E. coli was recorded as 14.66 ± 0.57 mm by A. corticis, 25.0 ± 0.0 mm by P. posthuma, 20.0 ± 0.0 mm by E. fetida, and 20.0 ± 0.0 mm by A. trapezoid. Cell proliferation, biofilm inhibition, the synergistic effect of extracts along with antibiotics, and direct bioautography supported the results of agar well diffusion assay. Similarly, P. hawayana, A. corticis, A. caliginosa, and A. trapezoids increase the prothrombin time more efficiently compared to other earthworms. A. corticis, A. gracilis, A. rosea, A. chlorotica, P. elongata, and A. trapezoides showed maximum DPPH scavenging potential effect.  Results revealed that all earthworm extracts showed the inhibition of all tested bacterial pathogens except  The coelomic fluid of earthworms possessed several bioactive compounds/enzymes/antioxidants that play an important role in the bacterial inhibition and act as anticoagulant agents. Therefore, the development of new therapeutic drugs from invertebrates could be effective and potential for the prevention of the emergence of multidrug-resistant bacteria. The ABTS. epidermidis, E. coli, and K. pneumonia) was freshly prepared and was homogenized with nutrient agar and decanted over a chromatogram as a thin layer. These plates were kept at room temperature for 5 min and then incubated overnight at 37°C. The growth inhibition zones were measured around the active chromatogram spots. The antibacterial activity of constituents present in the spot was further confirmed by spraying TBTB solution (Thiazolyl blue tetrazolium Bromide) on Petri plates and these plates were incubated at 37°C for 4 h.The agar overlay technique was used for the measurement of direct bioautography with slight modifications as described by Roopalatha and Vijay-mala . The TLCIn vitro anticoagulant effect of earthworm extracts were observed in the blood samples by evaluating prothrombin time [bin time . About 1http://easycalculation.com/statistics/standard-deviation.php). The statistical significance was evaluated by one-way analysis of variance (ANOVA) at p ≤ 0.001 and MS Excel program was also used to plot graphs with error bars of standard errors of the means (SEM). For sensitivity tests, (0) was used for no sensitivity, ∗(>1–5 mm) for low sensitivity, ∗∗(>5–10 mm) for moderate sensitivity, and ∗∗∗(>10–25 mm) for high sensitivity.Each experiment was repeated in triplicate. Mean ± standard deviation from absolute data was calculated using an online calculator  and Eisenia fetida, Allolobophora chlorotica, Aporrectodea rosea, Aporrectodea trapezoides, and Aporrectodea caliginosa (Lumbricidae) were identified by Prof. Dr. Jorge Domínguez (Spain) and Dr. Fatima Jalal (Pakistan) and further used for screening biological activities of their extracts.A. corticis, A. gracilis, P. posthuma, E. fetida, A. rosea, A. chlorotica, A. trapezoides, P. elongata, A. caliginosa, and P. hawayana) was analyzed against fourteen bacterial pathogens such as E. coli, S. marcescens, S. pyogenes, S. epidermidis, S. aureus, K. pneumoniae, P. aeruginosa (1), S. typhimurium, S. flexneri, E. amnigenus, S. odorifera, P. aeruginosa (2), S. warneri, and L. curvatus through agar well diffusion method, biofilm inhibition, and cell proliferation inhibition assays. MICs results revealed that bactericidal effect of earthworm extracts was increased with increase concentration , S. pyogenes (7.0 ± 0.0 mm), S. odorifera (9.0 ± 0.0 mm), K. pneumoniae (7.66 ± 0.57 mm), and S. typhimurium (6.33 ± 1.15 mm). Amynthas gracilis showed moderate inhibition of E. coli (9.0 ± 1.0 mm), S. odorifera (7.0 ± 1.73 mm), and K. pneumoniae (5.66 ± 0.57 mm)  . Pheretiectively .E. fetida extract showed the maximum inhibition of E. coli, S. marcescens, K. pneumoniae, S. aureus, E. amnigenus, S. odorifera, and S. flexneri , respectively, while moderate inhibition of S. pyogenes (8.0 ± 0.0 mm) and S. typhimurium (6.0 ± 0.0 mm) was recorded. Aporrectodea rosea extract showed maximum inhibition of E. coli, S. marcescens, K. pneumoniae, S. pyogenes, and S. aureus as 18.0 ± 0.0 mm, 16.0 ± 0.0 mm, 19.0 ± 0.0 mm, 13.0 ± 0.0 mm, and 15.0 ± 0.0 mm. On the other hand, a moderate zone of inhibition was recorded in the case of P. aeruginosa and S. epidermidis with 10.0 ± 0.0 mm and 10.0 ± 0.0 mm zone of inhibition.Similarly, Aporrectodea trapezoides showed the maximum inhibition of E. amnigenus (11.0 ± 0.0 mm). Bactericidal effect of Allolobophora chlorotica indicated the maximum inhibition of E. coli, S. marcescens, S. aureus, E. amnigenus, and S. odorifera  while moderate sensitivity was measured against K. pneumoniae (10.0 ± 0.0 mm), S. pyogenes (8.66 ± 0.57 mm), S. typhimurium (6.0 ± 0.0 mm), and S. flexneri (6.0 ± 0.0 mm) ( 0.0 mm) .P. elongata showed the maximum growth inhibition of K. pneumoniae, E. amnigenus, and S. flexneri as 10.33 ± 0.57 mm, 13.33 ± 0.57 mm, and 10.33 ± 0.57 mm while moderate inhibition of E. coli, S. marcescens, S. pyogenes, and S. aureus were recorded . Aporrectodea caliginosa extract showed the moderate zones of growth inhibition against E. coli, S. marcescens, K. pneumoniae, S. typhimurium, S. flexneri, and E. amnigenus  . On the other hand, it was observed that all earthworm extracts showed the lowest growth of inhibition of P. aeruginosa (1), P. aeruginosa (2), S. warneri, and L. curvatus  . Similarcurvatus .P. aeruginosa, K. pneumoniae, S. pyogenes, S. epidermidis, and S. aureus . Likewise, gentamycin indicated the maximum inhibition of P. aeruginosa, K. pneumoniae, S. pyogenes, S. epidermidis, and S. aureus . Similarly, Sulfamethoxazole showed the maximum inhibition of P. aeruginosa and S. epidermidis , while the lowest inhibition was recorded for other tested bacterial pathogens. Amoxicillin showed moderate inhibition of P. aeruginosa, K. pneumoniae, and Staphylococcus epidermidis .Ciprofloxacin showed the maximum inhibition of α) means the effect of two compounds is equal to the sum of the effect of two compounds taken separately. Antagonistic effect (β) shows the effect of two compounds is less than the sum of the effect of two compounds taken individually of each other  indicates the greater effects of two compounds when taken together than the sum of their separate effect. Additive effect  synergistic effect against K. pneumoniae, P. aeruginosa, and S. epidermidis. On the other hand, Ciprofloxacin showed antagonistic effect against E. coli when combined with extract. Moreover, Ciprofloxacin showed a significant (p ≤ 0.001) synergistic effect against E. amnigenus, P. aeruginosa, S. marcescens, K. pneumoniae, S. odorifera, and S. pyogenes. Similarly, the synergistic effect of gentamycin was also observed against K. pneumoniae, E. amnigenus, S. pyogenes, S. aureus, S. odorifera, and P. aeruginosa when combined with extract (at p ≤ 0.001). Amoxicillin along with extract showed an additive effect against S. epidermidis. On the other hand, the antagonistic effect of Sulfamethoxazole was recorded against P. aeruginosa and S. epidermidis (p ≤ 0.001) synergistic effect of amoxicillin was recorded against E. amnigenus, S. odorifera, and S. flexneri.Three types of interactions were recorded when ibiotics . When thdermidis . The sigE. coli and S. odorifera when all antibiotics were applied with A. chlorotica extract at p ≤ 0.001  and positive (Chloramphenicol) controls . The valAmynthas corticis extract significantly reduced the biofilm of S. epidermidis, K. pneumonia, S. pyogenes, S. odorifera, and L. curvatus. Amynthas gracilis extract reduced the biofilm of S. marcescens, S. aureus, and S. pyogenes. Pheretima posthuma extract reduced the biofilm of E. coli, S. marcescens, K. pneumonia, and S. typhimurium. Eisenia fetida extract inhibits the biofilm of P. aeruginosa (a) and S. warneri. Aporrectodea rosea showed the biofilm reduction of E. coli, S. marcescens, and P. aeruginosa (b). Allolobophora chlorotica indicated the reduction of biofilm of K. pneumonia, P. aeruginosa (b), and S. warneri. Aporrectodea trapezoides extract showed the biofilm reduction of E. coli, S. marcescens, S. odorifera, S. typhimurium, and S. warneri. Polypheretima elongata reduced the biofilm of S. epidermidis and P. aeruginosa (b). Pheretima hawayana reduced the L. curvatus biofilm only. Aporrectodea caliginosa showed the biofilm reduction of almost all tested bacterial pathogens.Biofilm inhibition results revealed that all the earthworm extracts reduced the biofilm formation compared to the control (nutrient broth having bacteria growth) and tested antibiotics . AmynthaA. corticis, A. gracilis, A. rosea, A. chlorotica, P. elongata, and A. trapezoides showed maximum DPPH scavenging potential effect. On the other hand, P. elongata indicated the same DPPH and ABTS scavenging potential. The highest value of antioxidant potential was recorded for P. elongata as 85% for both ABTS and DPPH scavenging assay. For A. corticis maximum scavenging potential was recorded as 80.74% and 77.5% for ABTS and DPPH, respectively. For A. gracilis 72% scavenging potential was recorded while 19% was recorded in the case of DPPH assay. P. posthuma indicated 81.57% potential through ABTS while indicating 19% potential through DPPH assay. E. fetida extracts showed 54.4% and 56% potential for ABTS and DPPH scavenging assay, respectively. In the case of A. rosea, 49.53% and 76% potential were measured through ABTS and DPPH scavenging assay. A. chlorotica indicated 10.45% and 70% values for ABTS and DPPH assay, respectively. A. trapezoids showed the lowest potential at 8.88% and 41% for ABTS and DPPH scavenging assay. A. caliginosa also showed minimum scavenging potential as 13.82% and 18% through ABTS and DPPH assay. For P. hawayana ABTS and DPPH scavenging potential were measured as 15.5% and 48%, respectively.The antioxidant potential effect of all earthworm extracts was screened via DPPH and ABTS scavenging assays. S. epidermidis, E. coli, and K. pneumonia which showed good sensitivity towards P. posthuma, E. fetida, and A. rosea extracts. Bioautography revealed clear zones of bacterial growth inhibition on each TLC strip after treatment with TBTB with purple background indicating one or more bioactive antimicrobial compounds in earthworm extracts. It was observed that lipid biomolecules have a potent antibacterial effect against these tested bacterial pathogens.The chemical constituents present in earthworm extracts were evaluated by thin-layer chromatography (TLC) by using precoated silica gel plates. To get the maximum efficient separation of components, five different solvent systems were used. Only the lipid biomolecules were indicated through TLC-developed plates in the form of movement of fat droplets in solvent system A, B, and C and not indicated in solvent system D and E. Bioautography was performed against P. hawayana extracts prolonged the clot formation time more efficiently than all other species at 15 min : 18 sec and A. corticis also increases the prothrombin time more efficiently which was recorded as 12 min : 34 sec. A. trapezoids extracts also increased the clotting time as 11 min : 30 sec while A. caliginosa showed clotting time as 10 min : 41 sec. P. posthuma and A. rosea indicated a moderate increase in prothrombin time measured as 6 min : 18 sec and 6 min, respectively, while E. fetida, P. elongata, and A. chlorotica indicated low prothrombin time as 5 min : 50 sec, 4 min : 45 sec, and 4 min, respectively.Results showed the mean coagulation time of plasma when treated with earthworm extracts. Negative control has a mean coagulation time of 2 min: 20 s. The findings showed that the coagulation time of plasma increases with the addition of earthworm extract. The extract of earthworm species has been used for the treatment of various diseases including anti-inflammatory, antioxidant, antitumor, and antibacterial diseases, as a food ingredient , in Traditional Chinese Medicine, and in Japan, Vietnam, and Korea , 67. EarEisenia fetida extract showed the maximum inhibition of E. coli, S. marcescens, K. pneumonia, S. aureus, E. amnigenus, S. odorifera, and S. flexneri, Polypheretima elongata showed the maximum growth inhibition of K. pneumoniae, E. amnigenus, and S. flexneri, Allolobophora chlorotica indicated the maximum inhibition of E. coli, S. marcescens, S. aureus, E. amnigenus, and S. odorifera, Aporrectodea rosea extract showed maximum inhibition of E. coli, S. marcescens, K. pneumoniae, S. pyogenes, and S. aureus, Pheretima posthuma extract indicated the maximum growth inhibition of E. coli, S. marcescens, K. pneumonia, S. aureus, S. pyogenes, S. odorifera, and S. flexneri, and Amynthas corticis extract showed the maximum inhibition of E. coli, S. marcescens, and E. amnigenus. Our findings agree with the outcomes of previous literature that the coelomic fluid of earthworms contains bioactive compounds that participate in various biological activities [The antibacterial results revealed that the bactericidal effect of earthworm species varied against both Gram (+) and Gram (−) bacterial isolates. Even some earthworm extracts did not affect tested bacteria. This variation may be due to the type of bacterial isolates, cell wall composition of tested bacteria, concentration and quality of extracts used, nature and presence of the bioactive compound in the extract, and the type of extract used. But one thing that is interesting is that the antibacterial activity of earthworm species is attributed to the presence of bioactive substances existing in the earthworm extracts. Results revealed that different earthworm species showed the maximum inhibition of tested bacterial isolates; for example, tivities –73.Pheretima guillelmi earthworm [Eudrilus eugeniae. The outcomes of the current research are consistent with the findings of previous literature as Vasanthi et al. [Eudrilus eugeniae against S. aureus; Kathirewari et al. [Lumbricus rubellus extracts against E. coli, S. aureus, Salmonella pullorum, and P. aeruginosa; Verma and Verma [P. posthumous had maximum antibacterial activity against E. coli (19.00 mm); Chauhan et al. [Eudrilus eugeniae; and Bhorgin and Uma [A. hydrophila.Lumbricin-PG, the antimicrobial peptide, was identified from arthworm . Bansal arthworm  also demi et al.  presentei et al.  found thi et al.  studied nd Verma  found thn et al.  illustra and Uma  showed tE. coli and S. aureus. Biofilm inhibition assay, cell proliferation inhibition assay, synergistic effect, and TLC-bio autography supported the results of agar well diffusion assay. Therefore, we can say that the development of new techniques, i.e., uses of enzymes with antibiotics, antimicrobial peptides production from vertebrates, invertebrates, and other microbes, and hybrid antibacterial drug production to prevent the emergence of bacterial pathogens are too effective, operative, and active [Hence, the current research sustained the findings of previous researchers that earthworm species possessed potential bioactive compounds, i.e., enzymes and antioxidants that play a key role in the growth inhibition of infectious pathogens. In the current research, it was observed that various earthworms possessed proteolytic, lipolytic, amylolytic, and antioxidant activities. Therefore, we can say that these bioactive substances (enzymes) and antioxidants may act as an antibacterial agent having various mechanisms/modes of actions , such asd active , 37, 38.d active  due to tEisenia fetida, Pheretima posthumous, Lumbricus rubellus, Eudrilus eugeniae, and Pheretima guillemi [P. hawayana, A. corticis, A. caliginosa, and A. trapezoids increase the prothrombin time more efficiently while P. posthuma and A. rosea indicated a moderate increase in prothrombin time. On the other hand, E. fetida, P. elongata, and A. chlorotica indicated low prothrombin time. So, we can say that the bioactive components of coelomic fluid of earthworm species may have interfered with both intrinsic and extrinsic pathways of coagulation; namely, they inhibit the activities of VII, II, IX, X, thrombin (thrombin IIa and prothrombin II), and Xa factors and played a key role in the fibrin degradation (Blood circulation is essential for human survival . During guillemi –93. Thesguillemi . In the radation . Our outin vivo studies should be needed to explore the inhibition mechanisms of coagulation by using earthworm extracts.We concluded that all earthworm species have antibacterial, antioxidant, and anticoagulant activities, and these findings can be used as a basis for the prevention of multidrug-resistant emergence, and production of animal-based anticoagulant agents. Furthermore,


Skillful sea-ice prediction in the Antarctic Ocean remains a big challenge due to paucity of sea-ice observations and insufficient representation of sea-ice processes in climate models. Using a coupled general circulation model, this study demonstrates skillful prediction of the summertime sea-ice concentration (SIC) in the Weddell Sea with wintertime SIC and sea-ice thickness (SIT) initializations. During low sea-ice years of the Weddell Sea, negative SIT anomalies initialized in June retain the memory throughout austral winter owing to horizontal advection of the SIT anomalies. The SIT anomalies continue to develop in austral spring owing to more incoming solar radiation and the associated warming of mixed layer, contributing to further sea-ice decrease during late austral summer-early autumn. Concomitantly, the model reasonably reproduces atmospheric circulation anomalies during austral spring in the Amundsen-Bellingshausen Seas besides the Weddell Sea. These results provide evidence that the wintertime SIT initialization benefits skillful summertime sea-ice prediction in the Antarctic Seas. Efforts have been made to understand physical processes underlying interannual variability of the Antarctic sea ice. Both the atmospheric teleconnections from El Niño-Southern Oscillation (ENSO) and the Southern Annular Mode (SAM)4 are considered as key drivers for interannual sea-ice variations in the Antarctic Ocean through changes in regional atmospheric circulations6.Antarctic sea ice plays a key role in regional and global climate through changes in atmospheric and oceanic circulations over a wide range of spatiotemporal scales. Antarctic sea ice reaches its maximum extent in September and its minimum in March, and undergoes large interannual variations through remote atmospheric forcing and local air-sea-ice interaction. Most of the Antarctic Seas do not show any statistically significant trend in the sea-ice extent (SIE). The only exception is the increasing trend during the satellite observation period in the Ross Sea7 conducted sensitivity experiments and reported that recent sea-ice increase in the Ross and Amundsen-Bellingshausen Seas between 2000 and 2014 has a close link with a deepening of the Amundsen Sea Low through atmospheric teleconnection from the negative phase of the Interdecadal Pacific Oscillation (IPO)8. Furthermore, a positive phase of the SAM contributes to a negative trend of wind stress curl in the Southern Ocean, which enhances upwelling of warm water through Ekman suction and causes significant retreat of the Antarctic sea-ice after 20169.Using an atmospheric general circulation model, a previous study10 performed two reforecast experiments with/without sea-ice initialization and concluded that sea-ice initialization adds almost no values to prediction skills of the Antarctic SIE except during austral summer. The prediction skills of the Antarctic SIE are generally lower than the persistent skills using the observed sea-ice concentration (SIC) anomalies. Since the Antarctic SIC anomalies are spatially inhomogeneous with positive and negative signs, some parts of the Antarctic Ocean may show improvement of sea-ice prediction skills with sea-ice initialization. In fact, a long-term simulation of CGCM11 showed that there are regional differences in the sea-ice persistence predictability, with high predictability in the Indian Ocean sector and Amundsen-Bellingshausen Seas and low predictability in the Weddell and Ross Seas, respectively. Furthermore, a recent study12 has pointed out that the SIC initialization during austral winter using the observational data improves representation of the SIC anomalies in the Weddell Sea during austral spring, probably induced by local air-sea-ice interactions13. However, it remains a challenging issue to further extend the lead time of skillful sea-ice prediction beyond a season.Atmospheric circulation changes associated with ENSO and the SAM are key sources of sea-ice predictability in the Antarctic Ocean, but the mechanisms behind these regional sea-ice predictabilities are not yet well established. In fact, a limited number of studies have reported low prediction skills of SIE in the Antarctic Ocean. For example, using global climate models, a previous study14 was made to correct the springtime SIT distribution with the observational data and to confirm that the model with the SIT initialization better estimates the summertime Arctic SIE than that without the SIT initialization. Using global climate and statistical models, subsequent studies18 have drawn similar conclusions, suggesting that the SIT initialization is crucial for extending a lead time of skillful sea-ice prediction in the Arctic Ocean, particularly during boreal summer. Recently, a few studies have made further attempts to utilize the CryoSat-2 satellite observation data for the SIT initialization21 and provided solid evidence for improvement of the Arctic sea-ice prediction during austral summer.To tackle this unresolved issue, initialization of sea-ice thickness (SIT) using observational data or reanalysis product has received much attention as a potential way forward to performing the long-ranged sea-ice prediction. Using a regional ice-ocean model, the first attempt22  with/without the initialization of SIT from C-GLORS ocean reanalysis22 see “”. Compar22 and GIOMAS23; see “Spatial pattern of the Antarctic sea ice shows a spatially inhomogeneous structure on seasonal-interannual timescales. Seasonal mean of the satellite SIC during late austral summer-early autumn (January–March) is large near the coasts of the Antarctica Fig. a, with t24 has discussed large uncertainties in the SIT observation data as there are large systematic and random measurement errors  shows meridionally maximum extent in most of the Antarctic Ocean Fig. a, with lea Figs. d, f, wheea Figs. b. AlthouSince the Weddell Sea shows the large seasonal-interannual sea ice variability in the Antarctic Ocean, we choose it to examine the impact of SIC and SIT initializations on sea-ice predictability using a CGCM see “”. FigureStandardized root mean squared errors (RMSs) for the SIC anomalies based on the observation Fig. a show laTo provide a broad perspective on the impact of the wintertime SIT initialization on the summertime SIC prediction in the Antarctic Ocean, we discuss spatial patterns of the ACCs for the January–March mean SIC anomalies in the whole Antarctic Seas Fig. . The perTo quantify the degree to which the prediction skills of the SIC anomalies are improved with the SIC and SIT initializations, we show time series of the SIC anomalies during January–March averaged in the western Weddell Sea . This is a region where the large difference in the ACCs between the STR and CTR experiments is found Fig. . We obtaTo explore the physical processes underlying the improved SIC predictions in the STR experiment, we conducted composite analysis for low sea-ice years in the Weddell Sea based on the time-series analysis of Fig. The differences in the SIC anomalies have a close link with those in the SIT anomalies. Time series of composite SIC anomalies averaged in the western Weddell Sea  show almost no difference between the CTR and STR experiments during July–September Fig.  in “MethThe vertical processes in the SIT balance involve both contributions from the overlying atmosphere and the ocean below sea ice. Time series of composite anomalies in the net surface heat flux and its component over the sea-ice/ocean Fig. c show thFurther analysis of mixed-layer temperature balance , composite anomalies of SLP and horizontal winds at 10 m show that northeasterly wind anomalies associated with negative SLP anomalies prevail over the Weddell Sea Fig. a. The neea Figs. b. The STea Figs. c. The diea Figs. d, the SI12, the SIT initialization extends the lead time of skillful sea-ice prediction across two seasons. Furthermore, the SIT initialization improves representation of atmospheric circulation anomalies in the Weddell and Amundsen-Bellingshausen Seas through modulation of atmospheric internal variability. Physical processes underlying the improvement of the atmospheric circulation anomalies remain unresolved and require further research using an atmospheric circulation model.This study has highlighted the importance of sea-ice thickness (SIT) initialization on skillful sea-ice prediction in the Antarctic Ocean, particularly in the Weddell Sea. The SIT anomalies initialized during austral winter using the C-GLORSv7 ocean reanalysis can be sustained via horizontal advection of the SIT anomalies and developed through air-sea ice-mixed layer feedback during austral spring. This leads to skillful prediction of the SIC anomalies during late austral summer-early autumn (January–March) with a lead time of 6–8 months. Compared to the SIC initialization that improves the spatial distribution of the SIC anomalies with a lead time of 1–3 months10 with sea-ice initialization, but the differences in the ACC skills between the CTR and STR experiments show positive values added most effectively during December–April 37, which includes the Louvain-la-Neuve Sea Ice Model 2 (LIM2) sea ice model38 and has 0.5° × 0.5° horizontal resolution of ORCA tripolar grid (ORCA05) with 31 vertical levels. The SINTEX-F2 model exchanges the atmospheric and oceanic fields every two hours to compute air-sea momentum, heat and fresh water fluxes by means of the Ocean Atmosphere Sea Ice Soil 3 (OASIS3) coupler39.To evaluate sea-ice prediction skills in the Antarctic Seas, we performed two reforecast experiments using a CGCM, called the Scale Interaction Experiment-Frontier Research Center for Global Change 2 (SINTEX-F2)40. Then, we spun up the SINTEX-F2 model with monthly climatology of the observed SST during 1950–1981. After that, we initialized the model SST with the observed SST every month during 1982–1985. To initialize the model SST, we nudged the model SST to the observed one by applying three negative feedback values  to the surface heat flux. For the initialization data, we used the weekly OISSTv2 dataset31 with a horizontal resolution of 1° × 1° and the daily OISSTv2 dataset41 with a horizontal resolution of 0.25° × 0.25°. Furthermore, we adopted two vertical mixing schemes to represent the impact of small vertical scale structures within and above the thermocline of the tropical oceans34. These differences in the initial conditions lead to generation of 12 ensemble members (Table 22, then integrated the model over 1 year starting from each month’s 1st of 1986–2017. In the STR experiment, we initialized the model SST with the observed one from the OISSTv2 dataset and the SIC and SIT with the ones from the ocean reanalysis, then integrated the model over 1 year for each month of 1986–2017. To initialize the model sea ice, we adopted a relaxation timescale of 5 days to nudge the model SIC and SIT to those from the ocean reanalysis. To calculate monthly anomalies, we subtracted monthly climatology and linear trend from the model output during the analysis period of 1986–2017.We initialized the oceanic component of the SINTEX-F2 model using the mean temperature and salinity of World Ocean Database 1998, starting from the rest staters Table . From 19To explore the physical processes on the SIT variability, we calculated SIT tendency balance using a following equation:42:43, −3 compared to that at 10 m depth, To investigate temporal evolution of mixed-layer temperature that impacts on sea-ice growth/decay from the sea-ice bottom, we also calculated a balance of mixed-layer temperature tendency using the following Eq. Supplementary Information.


The efficacy assessment of human anti‐IgE monoclonal antibodies (mAbs) in animal models before clinical trials is hampered due to the lack of cross‐reactivity of anti‐IgE mAbs between species.We developed CRE‐DR , an anti‐IgE mouse mAb that recognizes canine and human IgE, and then examined its IgE specificity and cross‐reactivity between three animal and human species.282NTNDWIEGETYYC294), we generated a hybridoma producing CRE‐DR. The CRE‐DR purified from the ascites of hybridoma‐inoculated mice was used for ELISA and Western blot analysis to examine reactivity to dog, human, and rodent IgEs as well as recombinant bovine serum albumin (BSA)‐conjugated to canine, human, and rodent IgE amino acid peptides corresponding to the immunizing sequence. We then performed enzyme‐linked immunosorbent assays (ELISAs) for dog IgE using sera from dogs with atopic dermatitis (AD) after inhibition with canine IgE and IgG. The amino acid sequence recognized by CRE‐DR was identified by ELISA using synthetic peptides.After mouse immunization with a synthetic peptide derived from canine IgE (CRE‐DR is a monoclonal mouse IgG1κ specific for dog IgE, and the ELISA values in atopic dog sera were inhibited by dog IgE, but not dog IgG. The binding of CRE‐DR to human IgE was relatively maintained, but not to rodent IgEs, which results were confirmed with the BSA‐conjugated IgE peptides of the various species. The CRE‐DR reactivity was supported by the comparison of amino acid sequence of CRE‐DR epitope, DWIEGETYYC, in dog IgE; one, two, and three amino acids were substituted in the human, rat, and mouse IgE epitopes, respectively.CRE‐DR is a mAb cross‐reactive to dog and human IgEs, which can allow the use of a dog model of allergy to test the efficacy of a CRE‐DR‐derived anti‐IgE therapeutic mAb before human clinical trials. In contrast, the dog has been long known to suffer from spontaneous IgE‐mediated reactions that result in clinical disease manifestations, such as atopic dermatitis (AD).Animal models of allergy could be used to reduce the risk of failure before testing anti‐IgE therapeutic mAbs in human clinical trials. Mice transgenic for the human FcεRIα gene have been used to evaluate the inhibition of anti‐IgE humanized antibodies on recombinant human IgE bound to the neo‐expressed human FcεRIα 424HLP426 of human IgE—the IgE binding region to FcεRIα—Since the amino acid sequence between IgEs of humans and animals is not highly homologous,In this study, we generated a unique anti‐IgE mAb, CRE‐DR , cross‐reactive between dogs and humans, against a peptide well‐conserved between species. The reactivity of CRE‐DR was investigated against canine, human, and rodent IgE. Finally, the CRE‐DR epitope was mapped to confirm its cross‐reactivity.22.1The peptide for mouse immunization consisted of a 13‐mer peptide whose NTNDWIEGETYYC sequence corresponds to the position 282–294 segment of the dog IgE heavy chain (GenBank No. AAA56797.1), because the site was protruded outside in the IgE structure , human IgE (Merck KGaA), rat IgE , mouse IgE (BD Pharmingen), and dog IgG (Cappel) were used for fluorescence ELISAs with biotinylated CRE‐DR (EZ‐Link NHS‐PEG12‐Biotin) (Thermo Fisher Scientific), using the Gemini XPS microplate reader  and SoftMax Pro software (Molecular Devices), as reported previously.2.3SDS‐PAGE was performed with a polyacrylamide linear gradient gel (5%–20%) for IgE reagents of dog (Bethyl Laboratories), human (Merk KGaA), mouse (BD Pharmingen), dog IgG (Cappel), and canine serum albumin  at 2 μg/lane for Coomassie Brilliant Blue staining and 0.1 μg/lane for Western blot analysis under both reduced and nonreduced conditions. For Western blot analysis, the gel was transferred to a PVDF membrane (Bio‐Rad Laboratories) and reacted with biotinylated CRE‐DR at 1 μg/ml in 10‐fold diluted blocking buffer  after blocking. The membrane was washed with TBS‐0.05% Tween 20 (TBS‐T), and bands were visualized with streptavidin‐conjugated alkaline phosphatase  and 1‐Step™ NBT/BCIP Substrate Solution (Thermo Fisher Scientific).2.4Escherichia coli BL21star (DE3) transfected with a pET‐15b vector carrying an N‐terminal His‐tag sequence followed by a thrombin site and the cloning sites of NdeI and XhoI, between which a synthesized DNA was inserted. From the translated amino‐ (N‐terminus) to the carboxy‐terminus (C‐terminus), this synthesized DNA encoded a segment of bovine serum albumin (BSA) , a GS‐linker (GGSGGTGGSGS), and the dog IgE peptide used in the mouse immunization. After the His‐tagged recombinant protein was purified by nickel resin, the final concentration in PBS was determined by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Similarly, we synthesized DNAs for the 433GTRDWIEGETYQC445 (GenBank No. AAB59424) and 411VAKDWIEGYGYQC423 (GenBank No. BAQ55489.1) peptides, which correspond to the 282NTNDWIEGETYYC294 immunizing dog IgE sequence in the human and mouse IgE, respectively, used as recombinant human and mouse proteins . All of these procedures were outsourced to the GenScript Biotech Corporation. The cross‐reactivity of CRE‐DR to the peptides and to recombinant BSA  was examined by ELISA.We produced a recombinant protein with a molecular weight (MW) of 71 kDa by expression in 2.5Recombinant silkworm‐expressed Der f 2 was prepared as previously reported .Inhibition ELISAs were performed with dog IgE (Bethyl Laboratories) and dog IgG (Cappel) as inhibitors.2.6The original peptide used for mouse immunization, and seven N‐ or C‐truncated fragments of this peptide, were synthesized by a manufacturer . Each peptide was conjugated to a 6x His‐tagged GGSGGS‐peptide at its N‐terminus and a GGSGGS linker. To design a capture ELISA, the anti‐His‐tag mAb  was immobilized onto the plates to capture the peptides that were then detected by the biotinylated CRE‐DR, as described above.33.1Only one hybridoma cell line produced a mAb reactive to both dog and rat IgE. The mouse IgG subclass of CRE‐DR was IgG1κ. CRE‐DR specifically recognized dog IgE, but not dog IgG Figure ,C, confiThe CRE‐DR binding to the BSA‐dog peptide increased in a dose‐dependent manner, while that to BSA alone remained low Figure . The maxIn the data of Western blot analysis Figure , CRE‐DR 3.2Der f 2‐specific IgE in the four canine sera was quantitatively measured after conversion of the fluorescence units (FU)s to IgE concentrations in nanograms per milliliter, using a standard curve, as in Figure 3.3ELISA reactivity of CRE‐DR to human IgE was reduced only to lesser extent than that to canine IgE, but the reactivity to rat IgE was largely lost, and that to mouse IgE was completely disappeared Figure . The tenThe cross‐reactivity of CRE‐DR to human IgE was also confirmed with Western blot analysis using BSA‐peptides Figure , showing3.4The CRE‐DR reactivity disappeared when four amino acids from the N‐terminus (Peptide No.3) and at least one amino acid from the C‐terminus (Peptide No. 4–8) were deleted from the original peptide (Peptide No. 1) Figure . The reaOne amino acid difference was found between the canine and human CRE‐DR epitopes: at position 293 in the dog IgE sequence, the tyrosine (Y) is replaced by a glutamine (Q) in the human one (GenBank No. AAB59424) Table . There a4The anti‐IgE mouse IgG1κ mAb CRE‐DR was specific for dog IgE—but not dog IgG—and cross‐reactive with human, but not rodent IgE, which were confirmed by ELISAs and Western blot analysis. The DWIEGETYYC amino acid sequence of the CRE‐DR epitope on dog IgE is well‐conserved with that of human IgE, as there is only one amino acid difference  showed the same amino acid sequence of CRE‐DR epitope as dog IgE, presumably recognized by CRE‐DR. Cow IgE constant region (GenBank: ANN46381.1) showed one amino acid substitution  in the dog peptide. Cat IgE (GenBank: AAD20023.1) showed two amino acid substitutions  in the dog peptide. The substitution of one amino acid will not be considered as a big obstacle for CRE‐DE cross‐reactivity, since Iso and Val are similar in physiological properties of the amino acids. An amino acid substitution in the dog peptide from Tyr to Lys at 293 was observed in horse IgE (GenBank: CAC44498.1), which physiological properties are not similar, affecting the binding of CRE‐DR. It is suspected hat CRE‐DR may lose the reactivity to the horse IgE in some degree.The conformational structure of human IgE PDB, 1F6A17 showeThere is some value in evaluating if the CRE‐DR, as a mAb whose epitope is clearly identified, would recognize the IgE of other mammalian species. Previously, we created the CRE‐DM, a rat mAb cross‐reactive between dog and mouse IgE,Due to the unique ability of CRE‐DR to recognize both human and canine IgE, a dog model of IgE‐mediated allergy could be utilized to investigate the efficacy of anti‐IgE therapeutic mAbs derived from the CRE‐DR. A caninized CRE‐DR mAb could be first tested in dogs with spontaneous IgE‐mediated  AD to detect if this mAb could prevent or reduce clinical signs. Then, the results in dogs would be informative as preclinical data for the development of a humanized antibody derived from the CRE‐DR. Due to the closeness of IgE‐mediated allergic reaction between canine and human AD,In summary, we generated CRE‐DR, a unique mAb specific for dog IgE that also recognizes human IgE; such cross‐reactivity was confirmed by the recognition of both IgE and its derived recombinant peptides in dogs, humans, and mice, and the identification of the CRE‐DR epitope. An anti‐human IgE therapeutic mAb derived from the CRE‐DR could be developed after validation of the efficacy of a caninized CRE‐DR mAb on a canine model of IgE‐mediated AD. The caninized CRE‐DR itself will also be the first antibody drug for IgE in dogs. Thus, the cross‐reactivity of CRE‐DR should be highly considered to be useful for the treatment of allergy in both human and animalsK. Masuda is a stockholder and Chief Executive Officer of the Animal Allergy Clinical Laboratories, Inc.; K. Masuda and T. Saito are inventors in the patent application related to the CRE‐DR.There is no human clinical study. All mouse‐based experiments had been approved beforehand by the Animal Care and Use Committee of the companies performing the contract research.Performed experiments and analyzed data: Akiko Kumagai. Performed experiments: Takuya Nara, Mizuho Uematsu, and Yoko Kakinuma. Analyzed and interpreted data, contributed the final writing: Takashi Saito. Conceived the original idea, planed and directed all the experiments, supervised the project, analyzed and interpreted data, wrote the manuscript: Kenichi Masuda.


Serine/arginine protein kinases (SRPKs) phosphorylate Arg/Ser dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. The existence of a large internal spacer sequence that separates the bipartite kinase catalytic core and anchors the kinases in the cytoplasm is a unique structural feature of SRPKs. Here, we report that exposure of HeLa and T24 cells to DNA damage inducers triggers the nuclear translocation of SRPK1 and SRPK2. Furthermore, we show that nuclear SRPKs did not protect from, but on the contrary, mediated the cytotoxic effects of genotoxic agents, such as 5-fluorouracil (5-FU) and cisplatin. Confirming previous data showing that the kinase activity is essential for the entry of SRPKs into the nucleus, SRPIN340, a selective SRPK1/2 inhibitor, blocked the nuclear accumulation of the kinases, thus diminishing the cytotoxic effects of the drugs. ATR/ATM-dependent phosphorylation of threonine 326 and serine 408 in the spacer domain of SRPK1 was essential for the redistribution of the kinase to the nucleus. Substitution of either of these two residues to alanine or inhibition of ATR/ATM kinase activity abolished nuclear localization of SRPK1 and conferred tolerance to 5-FU treatment. These findings suggest that SRPKs may play an important role in linking cellular signaling to DNA damage in eukaryotic cells. Caenorhabditis elegans to humans ATP and 0.1 μg activated Akt or 42 units (0.5 μL) DNA-PK for 30 min at 30 °C. Recombinant active Akt1 and DNA-PK were purchased from Upstate Biotechnology  and Promega, Madison, WI, USA, respectively. The reaction buffer for the Akt assays contained 12 mM Hepes pH 7.5 and 10 mM MgCl2, while DNA-PK was used as a kinase source, where the assay buffer contained 50 mM Hepes pH 7.5, 50 mM KCl, 10 mM MgCl2, 0.2 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and 80 μg/mL BSA. Double-stranded DNA was omitted in the DNA-PK assay since it did not enhance the SRPK1 phosphorylation. A similar DNA-independent mode of action has previously been reported for the phosphorylation of adenoviral L4–33K protein by DNA-PK [Kinase assays were carried out in a total volume of 25 μL containing GST-SRPK1 or GST-SRPK1 mutants (1 μg each) as the substrate with 25 μM ATP, 1 μCi of [γ-2O2 (10 mM), which resulted in almost complete nuclear translocation of SRPK1 , which is a motif–profile scoring algorithm that takes into consideration not only the phosphorylation motif but also the influence of the neighboring residues [We next wished to probe the mechanism driving the nuclear import of SRPK1 after treatment with genotoxic agents. Taking into account the fact that the nuclear translocation of the kinase occurs rapidly and phosphorylation has already been implicated in this process , we atteresidues , a threoresidues . Accordiresidues . In addiresidues .Therefore, we mutated both Thr326 and Ser408 to alanine, either individually or in combination, finding that a mutation of either of these two residues prevented the relocation of the kinase from the cytoplasm to the nucleus in response to 5-FU treatment B. NeitheThis evidence indicates that both these sites appeared to be induced by genotoxic agents; thus, both were critical for the nuclear translocation of SRPK1. As the effect of phosphorylation can be imitated by introducing negatively charged residues, we substituted Thr326 and Ser408 with aspartic acid. While the phosphorylation-defective mutants  were almost completely restricted to the cytoplasm, the phosphorylation-mimicking mutants  were partially localized in the nucleus in the absence of 5-FU treatment, with the relocation being more notable for the double mutant . In accoAlthough Asp is singly charged, whereas pThr and pSer are doubly charged at physiological pH, and therefore, the partial nuclear translocation may be due to reduced negative charge of the double phosphomimetic mutant as compared to phosphorylated SRPK1, these data may also imply that the phosphorylation of Thr326 and Ser408 was necessary but not sufficient for the nuclear translocation of SRPK1 and an additional modification(s) is (are) required.In a subsequent step, we sought to identify the kinase responsible for targeting Thr326 and Ser408. Since activated Akt has previously been reported to induce SRPK1 autophosphorylation on Thr326 , we firsThr326 and Ser408 are within the S/TQ motifs that are preferred phosphorylation sites for PI3K-like kinases. According to Scansite 4.0, the predicted kinase on these sites was DNA-PK, which belongs to the PI3K-like family of serine/threonine protein kinases and plays an essential role in DNA damage signaling . HoweverTaking into consideration the fact that Thr326 and Ser408 are within a consensus targeted by PI3K-like kinases and DNA-PK was not the upstream kinase, we next sought to investigate whether ATM and/or ATR, which are the two other main members of the PI3K-like family, were responsible for phosphorylating these two residues. In this respect, PhosphoNET, another predictor of human phosphorylation sites, hints toward ATR as being the main candidate kinase for targeting Thr326 and Ser408. As shown in According to our previous data, preventing the nuclear translocation of SRPK1 via pharmacological inhibition of ATR/ATM should exert a beneficial effect on 5-FU-treated cells. To address this issue, we tested the viability of HeLa and T24 cells upon treatment with 5-FU or ATR/ATM inhibitors alone, or in combination . The treHow genotoxic agents convey signals to the nucleus of cancer cells to mediate their action is not fully understood. Here, we provide evidence that SRPKs are part of these signaling pathways. The nuclear translocation of SRPKs closely correlates with the appearance of γH2AX foci and mediates 5-FU and cisplatin sensitivity.SRPKs have been reported to translocate into the nucleus upon the stimulation of cells with growth factors  and hormSRPIN340 significantly prevented the nuclear translocation of SRPKs in 5-FU and cisplatin-treated cells, thus diminishing the cytotoxic effects of the drug . The impThe increased nuclear levels of SRPKs were primarily, if not exclusively, associated with the increased phosphorylation of SR proteins, leading to alterations of the splicing machinery. Even in the report by Jang et al., cyclin D1 upregulation was mediated by increased phosphorylation of SC35 . HoweverIn line with the analysis performed by Boeing et al. , we provZhou et al. also identified Thr326 as a site that was phosphorylated in an Akt-dependent manner after EGF treatment of HeLa cells , even thOne intriguing issue arising from our data and the data reported in the literature is why growth factor/hormone-mediated nuclear translocation of SRPKs is beneficial to the cell, while, in contrast, stress-induced nuclear accumulation of SRPKs is associated with toxic effects. In our opinion, the answer relies on the balance between the cytoplasmic and nuclear levels of SRPKs, which is critical for the cell. This balance is probably achieved by how and to what extent SRPKs are post-translationally modified. Growth factors and/or hormones induce the regulated nuclear translocation of a rather limited number of SRPK molecules, which then mediate the corresponding biological responses, such as well-defined alterations in the splicing machinery and/or partial reorganization of chromatin, leading to the necessary transcriptional changes. In this respect, sub-stoichiometric levels of phosphorylation, e.g., Akt-induced SRPK1 phosphorylation, while of low importance in in vitro assays, may be functionally significant in the cellular context. On the other hand, genotoxic/stress signals induce stoichiometric phosphorylation that results in the massive nuclear accumulation of SRPKs. It is also possible that additional post-translational modifications, such as acetylation, are also implicated in the nuclear accumulation of SRPKs. The lack of complete nuclear localization of the double-phosphomimetic mutant (FLAG-SRPK1326/408D, Overall, our findings strongly suggest a key role for SRPKs in the DNA damage response, with the kinases being actively involved in the genotoxic agents- induced cell death. Yet, more extensive research is required to fully characterize the modifications and the implicated enzymes that dictate the nuclear translocation of the kinases, as well as to unravel the nuclear functions of SRPKs that mediate the downstream signaling events of genotoxic agents.Summarizing our results, we propose a model for SRPK1 activity and function in relation to its subcellular localization . In untr


The HU values of pedicles were insignificantly less in loosened than intact screws . All patients had clinical improvements. In conclusion, the HU values, as a surrogate for BMD, were unrelated to screw loosening in DDS. Therefore, patients with compromised BMD might be potential candidates for dynamic stabilization rather than fusion.Although osteoporosis has negative impacts on lumbar fusion, its effects on screw loosening in dynamic stabilization remain elusive. We aimed to correlate bone mineral density (BMD) with screw loosening in Dynesys dynamic stabilization (DDS). Consecutive patients who underwent 2- or 3-level DDS for spondylosis, recurrent disc herniations, or low-grade spondylolisthesis at L3-5 were retrospectively reviewed. BMD was assessed by the Hounsfield Unit (HU) in vertebral bodies (VB) and pedicles with and without cortical bone (CB) on pre-operative computed tomography (CT). Screw loosening was assessed by radiographs and confirmed by CT. HU values were compared between the loosened and intact screws. 176 patients and 918 screws were analyzed with 78 loosened screws found in 36 patients (mean follow-up: 43.4 months). The HU values of VB were similar in loosened and intact screws ( There is an increasing popularity of such quantification since the correlations between HU and BMD have been established3. The adaptation of the HU on pre-operative CT scans allows a swift evaluation of patients’ BMD and can serve as an alternative to dual-energy X-ray absorptiometry (DEXA), which has been a standard assessment for BMD pre-operation. Recent reports have demonstrated the application of the HU values in the prediction for the risks of pseudoarthrosis, instability of implants, and bone density-related complications in spinal fusion surgery5.During the past decade, bone mineral density (BMD) reportedly has been assessed by measurement of the Hounsfield Unit (HU) on computed tomography (CT) scans in spinal surgery9. In reports of short to mid-term follow-up, pedicle-screw based dynamic stabilization systems, such as the Dynesys  dynamic stabilization (DDS), has demonstrated similar clinical success rates to the standard lumbar fusion surgery11. However, durability remains the most frequent concern of these non-fusion constructs, especially in patients with inadequate BMD. The fatigue failure at the interface between the bone and screws, which frequently would cause screw loosening, remains the biggest challenge for DDS due to the need for continuous movement in such motion-preservation device12. The incidences of screw loosening in DDS were approximately 5% per screw and 20% per patient in 2–5 years post-operation16, although there were no associated adverse effects on the clinical outcomes. Since the demands of mechanical strength and durability for these dynamic screws are likely higher than the conventional screws designed for fusion18, it is reasonable to infer that the better the BMD, the lower rates of screw loosening and the higher chances of clinical success in the dynamic stabilization surgery of the lumbar spine. Nevertheless, there are scant reports on the correlation of BMD and success of DDS.Dynamic stabilization has also gradually gained acceptance in the past decade as an option of surgical management for lumbar spondylosisSince it remains elusive in the literature how BMD would affect the success of DDS in lumbar spine surgery, the current study aimed to evaluate BMD by HU values and correlate with the incidences of screw loosening in a series of patients who underwent surgery. To date, this is the first study to address the correlation between HU and screw loosening in DDS.16.A consecutive series of patients who underwent surgical decompression and stabilization with 2- or 3-level DDS at L3 to L5 from 2007 to 2015 were retrospectively extracted from a prospectively collected database for analysis. The inclusion criteria were symptomatic spinal stenosis with hypertrophic ligamentum flavum, facet hypertrophy, minimal or less than grade I spondylolisthesis, degenerative disc disease, and recurrence of herniated intervertebral discs (HIVD) that were refractory to medical treatment. The clinical manifestations included neurogenic claudication, radiculopathy, or low back pain. The exclusion criteria were Meyerding grade I or II spondylolisthesis, coronal or sagittal plane deformity, and osteoporotic vertebral fractures. Patients who did not complete the follow-up protocols for more than 2 years were also excluded from the study. All patients had posterior midline incisions for laminectomy and pedicle-screw based DDS stabilization through a paramedian fascia incision for screw insertion via the Wiltse approach. The surgical technique was identical to that detailed in our previous publicationsThe present study received institutional review board (IRB)/ethics approval in our institute . All study procedures were performed in accordance with relevant guidelines/regulations. A waiver of informed consent was approved by the IRB.The clinical outcome parameters were prospectively collected and reviewed retrospectively. The parameters included visual analog scales (VAS) for back and leg pain, Oswestry Disability Index (ODI) scores, and modified Japanese Orthopaedic Association (JOA) scores. Clinical data were collected by two special nursing assistants during admission at pre-operation, and post-operative 1.5, 3, 6, 12 and 24 months at outpatient clinics.The Hounsfield Unit value was determined on pre-operative CT scans within 6 months prior to surgery in all patients. The region of interest (ROI) was calculated on PACS systems  with a similar area size. The HU value of the vertebral body was measured on an axial plane at the pedicle level where the screws’ tips were usually set Fig. . The ROIPost-operative follow-up of radiographic films was undertaken at each time point in the outpatient clinic. Post-operative MRI and CT scans were arranged at approximately 18 to 24 months after surgery. Screw loosening was defined as a double-halo sign (radiolucency around the screw for more than 1 mm wide) on radiographic films or CT scans  was used for all statistical analysis. Paired t-tests and independent t-tests were used for continuous variables. Chi-squared tests were done for categorical variables. Statistical significance was determined by a IRB approved .Waived by IRB.p = 0.72), sex distribution , and follow-up length  were all similar between the loosening and non-loosening groups of patients. In regard to comorbidities, the number of smoking patients, or with hypertension or diabetes were not different between the two groups .Demographic data are shown in Table p < 0.001 in all clinical parameters, Supplementary Table Collected clinical outcome parameters included VAS for back and leg pain, ODI scores, and modified JOA scores. The final follow-up data demonstrated significant improvement compared to pre-operative data in the entire cohort  by screw, and 20.5% (36 out of 176 patients) by patient. We found that the VB HU was not significantly different between the loosened screws and the intact screws. The HU of pedicles, whether including or excluding CB, was also similar between the loosened screws and the intact screws. Our data suggested that BMD may not play an important role in screw loosening for dynamic stabilization.20. It is widely accepted that osteoporosis has a huge impact on pedicle screw loosening, and screw loosening is associated with a high risk of revision in lumbar fusion surgery22. Screw loosening is also a well-known complication in pedicle screw-based dynamic stabilization. There were nearly 5% of screws found with loosening in about 20% of patients undergoing pedicle screw-based DDS in the existing literature. The risk factors included diabetes and old-age7. However, it has never been investigated if osteoporosis is a risk factor for screw loosening in pedicle screw-based DDS.Pedicle screw loosening is a common complication after lumbar fusion surgery, especially in osteoporotic patients. The incidence of screw loosening can reach 60% among osteoporotic patients in long-term follow-ups after pedicle screw placement23. Although most academic spine surgeons have recommended the evaluation of BMD before spine surgery, less than 50% of them routinely arranged DEXA for their patients pre-operatively24. Alternative methods to evaluate BMD have been advocated in addition to DEXA. The Hounsfield Unit value, measured on computed tomography, is another common tool which can help in detecting osteoporosis in lumbar spine instrumentation25. Studies have shown that the CT HU value is closely related to BMD27. Several studies also demonstrated that the CT HU value is able to predict pedicle screw loosening after lumbar screw fixation29.Dual energy X-ray absorptiometry (DEXA) is considered the standard method in assessing bone mineral density (BMD) to evaluate the severity of osteoporosis based on World Health Organization (WHO) criteriap = 0.14) in pedicle screw-based DDS. Some authors have advocated that there may be a stronger association between HU values of pedicle and screw loosening than HU values of VB in lumbar fusion surgery31. Due to a more significant relevance of biomechanical stability and a stronger trabecular architecture, there are reports suggesting that screw stability depends more on the pedicular part than the VB33. Our data showed different findings: that neither the VB CT HU nor the pedicle CT HU value was related to screw loosening in pedicle screw-based DDS , although the loosened screws had insignificantly lower HU. However, screw loosening did not lead to worse clinical outcomes in our patients undergoing DDS, which is compatible to our previous research7.Our study is the first to adopted the CT HU value as a surrogate for BMD, and investigate the relationship between the CT HU value and screw loosening in pedicle screw-based DDS. We found that the CT HU value of VB is similar between loosened and intact screws  in total screws, and 20.5% (36 out of 176 patients) in total patients. The loosening rate was slightly different from our published data 29. Therefore, it is more precise to say that the CT HU value, as a surrogate for BMD, is not associated with screw loosening in our patients undergoing pedicle screw-based DDS.The primary strength of our study is that we have measured the CT HU value at every VB and pedicle occupied by screws. Approximately 170 patients, 900 screws and all relevant CT HU values were measured and analyzed. A comprehensive data set of CT HU values was acquired in our cases, compared to previous studies that only measured VB or pedicle CT HU values at a certain segment as a representative for all segments of HU values when both BMD and HU values can be very different at each segment among one patientThere were also limitations to our study. First, this is a retrospective study from a cohort of patients who underwent dynamic stabilization. Further prospective studies are warranted to validate our findings. Second, although the CT HU value and DEXA T-scores are common tools for detecting osteoporosis or osteopenia in the lumbar spine, the ideal definition and quantification method for the bone quality of the lumbar spine remain undetermined. The measurement of the CT HU value may not be a perfect reflection of bone quality, but is still currently an acceptable proxy for assessing osteoporosis or osteopenia.In this series of DDS, the clinical outcomes were not affected by screw loosening. The pre-operative CT HU values of the VB and pedicles were not different between the loosened and intact screws. Also, the HU values, as a surrogate for BMD, were not correlated with screw loosening in DDS. Therefore, patients with compromised BMD might be potential candidates for dynamic stabilization.Supplementary Information.