Trypanosoma equiperdum is the causative agent of dourine, a parasitic venereal disease of equids. In this work, rabbits were infected with T. equiperdum strain OVI; serological tests , used for the diagnosis of dourine in horses, were applied to study rabbit humoral immune response and to characterise T. equiperdum antigen pattern recognised by antibodies from infected rabbits. Moreover a protein extract of T. equiperdum strain OVI was produced and tested in skin tests on infected rabbits to detect the cell-mediated response induced by T. equiperdum, in order to evaluate its use in the field diagnosis of dourine. Sera of infected rabbits recognized in immunoblotting Trypanosoma protein bands with molecular weight below 37 kDa, providing a serological response comparable with that already observed in dourine infected horses. Moreover the trypanosome protein extract was capable to produce in vivo delayed-type hypersensitivity (DHT Type IV) in rabbits and proved itself to be non-toxic and non-sensitizing. Trypanosoma equiperdum . Other members of the subgenus are T. evansi, the causative agent of surra in a wide range of mammals , and T. brucei, the causative agent of sleeping sickness in humans. T. brucei affects also domestic  and wild animals1.Dourine is a sexually transmitted disease affecting equids and caused by the protozoan T. equiperdum is not transmitted by an invertebrate vector and it is primarily a tissue parasite that is rarely detected in the blood. Dourine is sexually transmitted; transmission by artificial insemination has not been confirmed, but it could potentially occur since T. equiperdum is present in the seminal fluid and in the genital tissues. The average mortality in horses is about 50%; donkeys and mules are more resistant and may remain unapparent carriers. To date, infected equids are the only known reservoir of the parasite3. No vaccines for dourine are available and pharmaceutical therapy is not recommended; the eradication strategy currently applied imposes slaughtering of seropositive animals4. This policy, however, is not economically feasible to be applied in developing countries, where horses are extensively used for transport and agriculture5.In contrast with other trypanosomes, 3 and has been also reported in Mongolia and Kazakhstan6. Most countries where dourine and other trypanosomoses, such as surra, are endemic do not regularly notify these diseases, so their spatial distribution remain not well known7. Recent outbreaks of trypanosomosis in Europe, as dourine in Italy8 and surra in Grand Canaria, Spain and France10 caused, respectively, by imported infected horses and camels, highlight the risk of importation of equine trypanosomosis into non-endemic countries. In spite of important economic losses caused by equine trypanosomoses, they can still be considered as neglected diseases11. Epidemiological investigations highlighted that knowledge of pathogenesis of dourine and host–pathogen interactions is still limited. Moreover the lack of vaccines, the inability of drugs to cure the neurological stage of the disease, and the limitations of current diagnostics make more difficult the control of the disease12. In addition, differential diagnosis is extremely difficult in geographical areas where T. equiperdum coexists with T. evansi, because the two parasites are genetically and antigenically related and the clinical outcome of the two diseases in horses is very similar3.Dourine is endemic in Africa and South America14. T. equiperdum is able to infect rabbits, showing that this species could be used in the research on dourine16 and other trypanosomoses. Small mammals (rodents and lagomorphs), are preferred for laboratory research instead of other mammals as horses, cattle and sheep, due to their lower maintenance costs, shorter reproduction time and availability of transgenic models. Moreover, small mammals could be useful for the study of diseases that affect larger mammals.A better comprehension of the host immune response following infection could allow to identify targets that could improve the diagnosis of the disease. Despite the availability of in silico models or the development of alternative methods, such as cellular cultures that aim to reproduce tissues and organs, animal experimentation is still necessary to understand the pathogenesis of diseases and to develop and evaluate new therapeutic strategiesT. equiperdum strain OVI and their humoral response was studied in vivo using serological tests  currently used for the diagnosis of dourine in horses.In this work, rabbits were experimentally infected with T. equiperdum strain OVI was produced and used as skin test antigen on rabbits in order to evaluate its performances and its safety. The skin test antigen could be used in the field diagnosis of dourine in horses, in particular in endemic areas, just as brucellin and tuberculin are used in the diagnosis of, respectively, brucellosis and tuberculosis in cattle.Moreover, a protein extract of 17 implementing Directive 2010/63/EU of the Council of the European Communities on the protection of animals used for scientific purposes18. Ethical approval was obtained from the Italian Ministry of Health .Animal experimentation was carried out in compliance with Italian national law (Legislative Decree 26/2014)T. equiperdum, 9 rabbits were used as negative controls, 5 rabbits and 2 guinea pigs were used to assess toxicity and sensitization effects of the skin test antigen.Healthy adult immunocompetent male rabbits, weighting 2.5–3.0 kg, and healthy female guinea pigs, weighting 250 g each, were used. In particular, 18 rabbits were experimentally infected with 19.Animals were kept in an enriched environment, considering the needs of behavioural biology, and were caged individually but ensuring eye contact. Animals were maintained in a conventional facility with 12-h light/dark cycle, at 15 ± 5 °C for rabbits and at 20 ± 4 °C for guinea pigs; free access to regular food and tap water were allowed. Social and food enrichment were guaranteedT. equiperdum strain, provided by Onderstepoort Veterinary Institute, Pretoria, South Africa (OVI T. equiperdum), was produced in rats according to the World Organisation for Animal Health (2018)3, and stored at − 80 °C until use. Nine rabbits were infected intrascrotally15 with cryopreserved T. equiperdum in order to amplify the parasite and to adapt it to the rabbit. When a marked scrotal edema appeared, rabbits were sacrificed; then the scrotum of each rabbit was removed and the edematous material was taken, homogenized and diluted in 0.01 M phosphate-buffered saline, pH 7.5 (PBS), to get 104 live trypanosomes/ml. Trypanosomes were counted by Bürker counting chamber. This homogenized antigen was inoculated intrascrotally in other 9 rabbits . Blood samples of infected rabbits were collected from the marginal ear vein, after applying local anaesthesia, to verify the presence of antibodies versus T. equiperdum before the infection (T0) and at times 7, 14, 21, 28 and 35 days post infection . As negative control, nine rabbits were inoculated with PBS only and blood samples were taken at time from T0 to T35. Sera were stored at – 20 °C until use. The second group of 9 inoculated rabbits and negative controls were used also to test the skin test antigen.3. Briefly, the antigen was produced in rats using the OVI T. equiperdum, according to the OIE Manual3. Sera were diluted five-times with veronal buffered saline, and those showing more than 50% of fixation level at the dilution 1:5 were considered as positive and tested again to the end point using twofold dilutions.Complement fixation test (CFT) was performed according to the method described for equids in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals20. Briefly, rabbit sera were diluted 1:100 in PBS containing 0.05% Tween 20 and 0.1% BSA (dilution buffer); as secondary antibody, a peroxidase-conjugated goat Anti-Rabbit IgG (H + L)  diluted 1:30,000 in dilution buffer was used. Results were expressed as percentage of positivity (PP), calculated using the following formula:Indirect-ELISA (i-ELISA) was performed according to Bonfini et al.T. equiperdum15 and the serum of a healthy rabbit were used. The cut-off value of the ELISA was calculated by adding 4 times the standard deviation to the mean PP value of sera collected from the 9 rabbits in the control group at times from T0 to T3521. Sera showing a PP value greater than the cut-off value were considered positive.As positive and negative controls, a lyophilized serum obtained from a rabbit experimentally infected with OVI 22, using rabbit sera diluted 1:5,000 and the peroxidase-conjugated goat Anti-Rabbit IgG (H + L) (Bio-Rad) diluted 1:3,000. Image acquisition was carried out with Chemidoc MP (Bio-Rad) using Image Lab Software version 4.0.1 (Bio-Rad). Sera reacting with bands with molecular weight (MW) equal or less than 37 kDa were considered as positive for Trypanosoma antibodies23.The immunoblotting assay was performed according to Luciani et al.T. equiperdum3 and containing approximately 90% live trypanosomes, as assessed by a Bürker counting chamber. After washing with PBS at 2000 rpm for 20 min at 4 °C, the supernatant was discarded and the buffy coat containing trypanosomes was collected and, after further washings to remove red cells, the pellet was collected, resuspended in PBS and lysed by freeze/thawing cycles in liquid nitrogen. The lysed cells were filtered through 0.8 µm, 0.45 µm and 0.22 µm filters . The protein concentration of the antigen was determined using the BCA Protein Assay Kit  and adjusted to 1.4–2.0 mg/ml with PBS.The antigen was produced from blood collected from rats that had been experimentally infected with OVI T. equiperdum, the 9 infected rabbits and the negative controls were shaved at two rectangular areas of about 3.0 × 9.0 cm on both flanks. Each of the two areas was divided into 3 equal squares. T. equiperdum skin test antigen (0.2 ml) was inoculated intradermally into 3 squares according to Latin square design and physiological solution (0.2 ml) into the remaining 3 squares as control. Skin reactions (diameters of erythema) were measured 24 h after antigen injection. The reaction was considered positive if an erythema of 8–25 mm of diameter was present.Six weeks after infection with OVI Toxicity and sensitization effects of the skin test antigen were evaluated using, respectively, guinea pigs and rabbits.T. equiperdum skin test antigen and observed for 7 days to evaluate toxicity effects .Two guinea pigs were inoculated subcutaneously with 0.5 ml of T. equiperdum skin test antigen. Twenty days after the third inoculation, another dose was injected into the same rabbits and into other 3 rabbits, previously not-inoculated with T. equiperdum skin test antigen (control group). Animals (test group and control group) were observed from the first inoculation until the end of the experiment (48 h after the last inoculation) to check for signs of sensitization (erythema and/or edema) at the point of injection.Six rabbits divided into 2 groups (test group and control group) were used to evaluate skin sensitization effects of the antigen. Three rabbits (test group) were inoculated intradermally 3 times at 5-day intervals with 0.1 ml of Statistical analysis was performed with the calculation of the percentage of reactor animals (observed or presumed) and the relative uncertainty, according to a Bayesian approach and using a Beta distribution, at 95% confidence interval, considering the percentage of rabbits that gave a skin test positive reaction, and taking into consideration two types of reactions: rabbits that showed positive reactions in all the skin treated areas and rabbits that showed positive reaction in at least one skin treated area.T. equiperdum became positive after 7 days from inoculation (T7) (CFT titres ≥ 1:40) and maintained high antibody titers (CFT titres ≥ 1:320) for the entire duration of the experiment  and gradually increased till 83.7–116.7% at time T35. Calculated cut-off was 9.2%; according to this value, at time T7 rabbits #1, #5, #6, #7 and #8 tested positive and rabbits #2, #3, #4 and 9 tested negative, while at time T14 all the rabbits tested positive. PP values for the rabbits of the control group were less than 6.9% throughout the experiment  25.Phylogenetic analysis has suggested an evolution of 3. These tests are not specific for T. equiperdum, due to antigenic similarities among T. equiperdum, T. evansi and other Trypanosoma species26.OIE-prescribed serological tests for dourine are CFT and indirect fluorescent antibody test (IFAT); the latter is currently used to confirm infection or resolve inconclusive CFT results. Other serological tests used in the diagnosis of dourine are ELISA, radioimmunoassay, counter immunoelectrophoresis, agar gel immunodiffusion (AGID), immunoblotting and a card agglutination testT. equiperdum infected rabbits and to compare the results with the horse humoral response. Infected animals tested positive for anti-Trypanosoma antibodies at CFT after 7 days post infection (p.i.) and at i-ELISA and immunoblotting at times between 7 and 14 days p.i.. Moulton et al.16 and Hiepe et al.27 observed an increase in antibody titres after, respectively, 7 and 9 days p.i. in rabbits infected with T. equiperdum. In another work of Bishop et al.28, antibodies versus T. equiperdum were detected after 14 days p.i. in experimentally infected rabbits and horses. According to Luciani et al.22 a mare, experimentally infected by tranfusion of blood collected from dourine naturally infected horses , became positive at CFT and immunoblotting after 14 days post infection (p.i.), and tested positive also at IFAT 3 days later (17 days p.i.). Hébert et al.29 carried out experimentally infections on horses to validate a model for assessing drug efficacy against T. equiperdum infection: 9 horses, intravenously inoculated with OVI T. equiperdum , became seropositive at CFT after 7–9 days p.i., while among 3 horses intravaginally infected with different doses of the parasite, only one became positive at CFT at day 23 p.i.. In a work of Hagos et al.5 6 stallions were intravenously infected with T. equiperdum Dodola 834/940 strain ; seroconversion was observed after 32 days p.i. by CATT/T. evansi.Three serological tests, developed for the diagnosis of dourine in horses, were applied in the present study to evaluate the humoral response of OVI T. equiperdum infections varies depending on the dose and route of inoculation, and the strain of the parasite.So, just as for other pathogens, the time of seroconversion in T. equiperdum antigenic profile with low molecular weight bands (< 37 kDa), just as observed in dourine positive horses23. These results seem to confirm the hypothesis that T. equiperdum low molecular proteins are important in the humoral immunitary response against this pathogen both in horse and rabbit and could represent possible candidate diagnostic antigens for the development of more specific tests for dourine.Moreover, immunoblotting results obtained in the present work demonstrated that antibodies from infected rabbits, in contrast to antibodies from healthy rabbits, specifically recognise a T. cruzi or other Trypanosoma species using a delayed type hypersensitivity test34. Mansfield and Kreier33 described positive skin test reactions of the Arthus type (type III hypersensitivity reactions) in rabbits infected with T. congolense. Delayed hypersensitivity reactions were observed in rabbits and mice infected with T. brucei and T. rhodesiense27. Thé et al.34 described a DTH response in a skin test performed on mice infected with T. cruzi. Regarding the cell-mediated response induced by T. equiperdum in horse, to date no scientific data are available in the literature.Some authors evaluated the cellular immune response of laboratory animals  infected with Mycobacterium bovis in cattle; serological tests are used in parallel or serial testing to improve the detection of infected animals and confirm the results of intra-dermal skin tests. The use of tuberculin testing and stamping-out of reactors has eliminated M. bovis infection from farmed cattle in some countries36. Brucellin skin test, performed using a standardised and s-LPS-free antigen preparation, is used for the screening of unvaccinated cattle in the diagnosis of brucellosis. The brucellin skin test has high sensitivity and specificity and is also used to detect Brucella infections in unvaccinated sheep, goats and pigs. Moreover, the test may aid when false positive serological reactions due to cross-reacting bacteria are suspected, as false positive reacting animals always give negative results in the skin test37, and being independent of circulating antibodies, it might improve the diagnosis of brucellosis in latent carriers of Brucella39.Delayed hypersensitivity reactions are usually used to detect infections caused by T. equiperdum was also produced and used as skin test antigen on T. equiperdum infected rabbits in order to evaluate its ability to produce an in vivo delayed type hypersensitivity, to obtain a diagnostic tool for the use in the field for dourine and other diseases caused by trypanosomes of the subgenus Trypanozoon, just as it is commonly done for brucellin and tuberculin in the detection of Brucella spp. and M. bovis infections in ruminants. Skin test antigen produced a delayed-type hypersensitivity  response and the diameter of skin reactions obtained was in a range 8–25 mm, similarly to what can be observed in animals treated with other kind of allergens, namely tubercolin or brucellin. Moreover skin test antigen proved to be non-toxic and non-sensitizing, when tested, respectively, on guinea pigs and rabbits. These encouraging results support further studies to standardize the manufacturing process and chemical characteristics of the new skin test antigen described in this paper, in order to guarantee reproducibility of its diagnostic performances. It is also necessary to evaluate the cell-mediated response induced by T. equiperdum in healthy and infected horses. Since the dourine cases reported in Italy in 2011, no dourine outbreaks have been detected in Italy and in other European countries, so to date it has not been possible to test skin test antigen on naturally infected horses. However, since skin test antigens do not interfere with serological diagnosis, being independent from humoral immunitary response, they could be employed in parallel with serology to improve the evaluation of the health status of equids when dourine infection is suspected. Moreover, the lack of vaccines for dourine and surra reduces false positive results that could be obtained using skin test on vaccinated animals. Further studies should be done to evaluate diagnostic performances of skin test antigens on T. equiperdum infected horses in areas where dourine is endemic and to compare the cell-mediated response induced by T. equiperdum in both rabbits and horses. OVI T. equiperdum skin test antigen should also be tested on T. evansi infected horses to evaluate possible cross-reactions.In this work a protein extract of OVI According to the results obtained from this study, the rabbit model could be used to increase the pathogenic knowledge of dourine, and it could help to develop more effective diagnostic tools and possibly new strategies for dourine control and prophylaxis.Supplementary Information.


Objective: To study GRB7 protein expression in normal human tissues and breast and ovarian cancers, and determine its clinical significance.Results: GRB7 protein was expressed in multiple tissues, including myoepithelial cells of normal breast and fibroadenoma. Cytoplasmic GRB7 expression was seen predominantly in HER-2 positive and, to a lesser extent, triple negative breast cancer. Membrane localization of GRB7 was present in a subset of breast cancers with high cytoplasmic GRB7 expression. Univariate and multivariate analysis found that cytoplasmic GRB7 expression was associated with a negative progesterone receptor status, while membrane GRB7 expression was associated with a negative axillary nodal status. Membrane associated GRB7 expression was present in a subset of ovarian cancers with high cytoplasmic GRB7 expression. Membrane GRB7 expression displayed a trend towards improved recurrence free survival (RFS). Landmark analysis suggested an RFS advantage for ovarian cancers that had GRB7 membrane expression and survived beyond 27 months; GRB7 membrane expression in two or more cores (out of three) predicted an improved RFS. Membrane expression of GRB7 protein was observed in breast cancer cell lines with high GRB7 protein expression in vitro.Conclusion: GRB7 protein membrane expression may be associated with a better prognosis in breast and ovarian cancers. The favorable prognostic value of GRB7 protein membrane expression and its underlying mechanism is worthy of further investigation.Methods: Immunohistochemistry of normal human tissues, breast tissues of various pathologies, and clinically annotated ovarian cancers was performed to correlate the patterns of GRB7 expression with biomarkers or clinical outcome. Growth factor receptor-bound protein-7 (GRB7) protein is a 532 amino-acid adaptor molecule that lacks intrinsic enzymatic activity, but mediates signal transduction from multiple cell surface receptors to specific downstream signaling pathways inside the cell. The GRB7 protein contains an amino-terminal proline-rich region, a central GM (Grb/Mig) segment with a pleckstrin homology (PH) domain to bind phospholipids which could recruit GRB7 to the plasma membrane, and a carboxyl-terminal src-homology 2 (SH2) domain involved in the recruitment of host proteins to activated upstream target receptors , 2. Consin vivo and in vitro [The GRB7 gene is located in close proximity to the HER-2/erbB2 gene, forming the HER-2-amplicon core, and is commonly, though not always, co-amplified and overexpressed with HER-2 in human breast cancers –10. Althin vitro , 14. Resin vitro . The proGRB7 protein expression has been reported in cytoplasm, focal adhesions, stress granules, and the nucleus (reviewed in ). DeletiGRB7 overexpression was found to be significantly correlated with metastatic cancers. In esophageal and pancreatic carcinomas, GRB7 was positively correlated with the presence of lymph node metastases , 20. HigIn the current study we report the tissue distribution of GRB7 protein by IHC in normal human tissues, including benign breast diseases. Additionally, we performed GRB7 IHC on both breast and ovarian cancer TMAs. We find GRB7 membrane expression in both ovarian and breast cancers that are associated with high cytoplasmic expression of GRB7. Membrane expression of GRB7 protein is observed in breast cancer cell lines with high GRB7 protein expression by IF and IHC. GRB7 membrane expression is associated with less nodal involvement in breast cancer and a trend towards improved recurrence free survival in ovarian cancer.GRB7 has distinctive expression patterns among multiple human tissues as shown by immunohistochemistry (IHC). GRB7 is abundantly expressed in tissues such as bone marrow, salivary glands, brain , heart, prostate and kidney (cortex), yet the amount of GRB7 is variable in liver, skeletal muscle, endometrium, stomach and colon. Lower GRB7 expression is seen in breast, fallopian tubes and pancreas, while almost no GRB7 expression was detected in the esophagus, lung, ovary or spleen (see n = 30) with GRB7 antibodies revealed that in 33%  of the cases, GRB7 expression was found in the myoepithelial cells surrounding the mammary gland ducts, while there was no observable GRB7 staining in the rest of the samples   see , 1B.IHC analysis of the normal ovarian tissue samples showed that GRB7 was highly expressed in the cytoplasm of surface epithelial cells, while the ovarian parenchyma and stroma were negative for GRB7 staining. In addition, the fallopian tubes showed weak cytoplasmic GRB7 expression in the columnar epithelium lining the tubes see –1E.n = 30) were benign, 80.91% (n = 339) were invasive ductal carcinoma (IDC), 6.68% (n = 28) were ductal carcinoma in situ, 3.10% (n = 13) were invasive lobular carcinomas, and the remainder  included atypical hyperplasia, mucinous adenocarcinoma, adenoid cystic carcinoma and Paget’s disease.Commercially obtained TMA slides containing normal and cancerous breast tissue samples were screened. Of the 505 sample cores, we removed 21 samples due to duplication, 27 due to lost tissue, and 8 due to sections being different from breast glandular tissue  in 19.15% of the samples, medium expression (score of 2+) in 15.14% of the sections, weak expression (score of 1+) in 25.17% of the samples and the absent of staining (score of 0) in the rest (40.53%). Interestingly, in some cases, GRB7 was accentuated in the membrane in addition to being found in the cytoplasm . For a more detailed summary of the GRB7 expression levels and location for the different histologies, see n = 15; mean age 29.50 ± 11.35 years) expressed GRB7 in the myoepithelial cells, while no GRB7 expression was seen in the fibrocystic changes samples   of the fibroadenoma samples (ars) see .n = 339) is 18 to 86 years, with an average age of 49.32 ± 11.39, and a median of 49  samples. The age range of the study subjects , with breast cancer risk factors that are known to be associated with clinical outcomes. Overexpression of GRB7 was found to be associated with HER-2, ER and PR markers , while no association was observed for age, tumor size and lymph node involvement. Breast cancer risk factors were then further analyzed using a multivariate logistic regression method in order to identify independent prognostic factors , and that PR negative status has a 1.818 times larger odds having high GRB7 expression as PR positive status . factors . Here wep-values of 0.0269, 0.0002 and 0.0458, respectively). Multivariate logistic regression showed that negative lymph node status has 2.294 times higher odds of having cytoplasmic GRB7 expression and membrane accentuation as positive lymph node status , and that HER-2 positive status has 10.880 times larger odds of having cytoplasmic GRB7 expression and membrane accentuation as HER-2 negative   .p-value < 0.0001, When we looked at the possible association between GRB7 expression levels and membrane staining, we found that there was a significant association (chi-square test n = 48), we found 4 with medium (2+) GRB7 expression, 10 with weak (1+) GRB7 expression, and 34 samples with no GRB7 expression (0). These results are consistent with our prior findings that a fraction of breast cancers that are TNBC have GRB7 expression [n = 137), and found 1 with strong (3+) and membrane-accentuated GRB7 expression, 6 with medium (2+) GRB7 expression, 42 with weak (1+) GRB7 expression, and 88 samples with no GRB7 expression (0).When we analyzed the sections with triple negative breast cancer , while 80.35% (n = 139/173) had adjuvant chemotherapy. The histology types of the ovarian cancers were 21.39% (n = 37/173) for non-high-grade serous carcinoma (non-HGS), and 78.61% (n = 136/173) for high-grade serous ovarian cancer (HGSOC), as previously reported [A summary of the Clinical parameters analyzed for this cohort of patients is summarized in reported .p-value < 0.0001, Similar to the GRB7 expression in breast IDC sections, IHC analysis of the ovarian tissues showed that GRB7 was mainly expressed in the cytoplasm of cells, ranging from no GRB7 expression  to weak  to medium  and strong  GRB7 expression, for a total of 73.41% patients expressing GRB7 in the cytoplasm see –4D. MembThe clinical events in our ovarian cancer analysis are defined as Recurrence Free Survival  and Overall Survival (OS). An overview of the characteristics of these ovarian cancer patients can be found in the vs. no expression (0) , weak (1+) or none (0) , or when we compared high GRB7 expression (3+) with less GRB7 expression as a group (0 to 2+) . See When we looked at the cytoplasmic expression of GRB7, we found no significant differences in the length of RFS nor OS for expression  p-value of 0.1709 and 0.1952 respectively). On a closer look of the data, there appeared to be a delayed effect for GRB7 membrane expression and RFS, and performing a landmark analysis using 27 months (810 days) as our cut off mark, patients that survived after 27 months had a significantly better RFS if they also had GRB7 membrane-associated expression than if they did not show membrane staining , suggesting a result of clinical significance. No significant results were found for GRB7 membrane-associated staining for OS. See We then analyzed the membrane-associated GRB7 expression, and found that there was no significant difference in RFS nor OS . There was no effect on the OS  with those with fewer or none expressing, we found that there was an overall significant lengthening of the RFS in these patients  or a moderate (2+) level of GRB7 protein expression in the cytoplasm is present in 19.72% and 16.90% of breast cancers, respectively. A high level of GRB7 expression (3+) is highly associated with HER-2 protein over-expression (3+), but as previously reported by us and others only a fraction of breast cancer specimens with HER-2 over-expression (3+) has high GRB7 protein expression (3+). A low (1+) to moderate (2+) level of GRB7 protein expression is noted in triple negative breast cancers as described earlier [Most notably, we find that when GRB7 protein is expressed in the cell membrane, it is often associated with high (3+) GRB7 protein cytoplasmic expression, though 3 + GRB7 cytoplasmic staining also occurs in the absence of membrane accentuation. Univariate followed by multivariate analysis finds that GRB7 cytoplasmic expression is associated with high HER-2 protein over-expression and negative progesterone receptor status. GRB7 membrane expression however is associated with HER-2 protein over-expression and negative axillary node status. The negative prognostic value of GRB7 cytoplasmic expression was consistent with prior findings , 11. TheMembrane expression of GRB7 protein has been observed in ovarian cancer samples. As with breast cancer, membrane expression of GRB7 protein is present in ovarian cancer samples with high GRB7 expression (3+) in the cytoplasm but only a fraction of ovarian cancer specimens with high cytoplasmic expression of GRB7 protein (3+) exhibit GRB7 membrane expression. GRB7 protein and RNA expression have been associated with high grade ovarian cancers . We findThough intracellular localization of GRB7 protein had been the subject of prior studies, membrane accentuation had not been previously reported. This could be due to the antibody reagents used in prior studies, or overexpression of epitope tagging of GRB7 protein in experimental systems , 17. EpiIn conclusion, our study finds that membrane-affiliated expression of GRB7 may be associated with a better prognosis in both breast and ovarian cancers. GRB7 protein expression in the cytoplasm and membrane in fact, carries the opposite prognostic significance for breast cancer. The above may reflect the outcome of diverse signaling events in the cells. Further study is warranted to understand the signaling complexities that GRB7 emanates from different cellular locations. This knowledge is essential for development of GRB7 directed therapeutics and understanding of its involvement in breast and ovarian cancer biology.All patients were recruited from the OHSU Knight Cancer Institute under approved institute protocols. All patients underwent ovarian cancer staging and/or debulking surgery when this was considered an effective option. Tumor subtypes were classified according to 2014 World Health Organization criteria and we utilized binary grading system . MedicalThe multiple normal tissue and breast cancer microarrays (TMA) were obtained from commercial suppliers .Ovarian cancer tissue samples  were obtained from the Oregon Ovarian Cancer Registry and Tissue Repository (OOCRTR) at the Oregon Health and Science University, and paraffin-embedded and analyzed as previously described , 26. BriAnti-human GRB7, a monoclonal antibody produced in rabbit, was purchased from Abcam .TMA embedded in FFPE blocks were serially cut into 6 μm-thick sections using a microtome, directly transferred on SuperFrost Plus glass slides, and incubated for 2 h at 60° C. Slides were deparaffinized in xylene, followed by a rehydration in an ethanol series  and kept in water until the next step. Antigenity was enhanced by boiling the sections in antigen retrieval buffer  at 95° C for 20 min, followed by a step cool down . Endogenous peroxidases were blocked with Bloxall  for 10 min at RT. Slides were then incubated in blocking buffer (0.5% BSA in 0.1M Tris/0.15M NaCl) for 30 min at RT, followed by incubation with the primary antibody  for 48 h at 4° C in a humid chamber. Slides were rinsed in 0.1M Tris/0.15M NaCl, incubated with an anti-rabbit secondary antibody coupled to peroxidase according to standard protocols with the Vectastain Elite ABD HRP kit , and developed using the ImmPACT DAB chromogen  kit. After rinsing slides in 0.1M phosphate buffer, sections were counterstained with Hematoxylin according to standard protocols. Sections were then dehydrated , cleared in xylene and mounted in Permount Mounting Medium . As a negative control, block sections were also labeled without exposure to primary antibody.For image analysis, subcellular localization and expression levels were determined for both cytoplasmic and membrane fractions. The extent of immunochemical reactivity was graded on a scale of 0-3 based on intensity as follows: background (0), light (1), moderate (2), and strong (3) GRB7 stainings. The grading was performed manually by an experienced Gynecologic Oncology pathologist (P M-F). The grading was blinded to the clinical outcome or other associated biomarkers. Images were taken on an AXIO imager.A2 microscope , attached to an Axiocam ERc 5s camera, using the ZEN2-blue software.Breast cancer cell lines MDA-MB-231, HCC1954 and SKBR3 were grown in RPMI-1640 , supplemented with 10% Fetal Bovine Serum . All cells were grown to approximately 80% confluence prior to splitting/harvesting. All breast cancer cell lines were initially obtained from ATCC.anti-human GRB7, rabbit monoclonal antibody ; anti α-tubulin, mouse monoclonal antibody ; anti HER-2/ErbB2, rabbit polyclonal antibody .2EDTA, 1 mM EGTA, 1% Triton X-100) supplemented with HALT protease and phosphatase inhibitor cocktail , incubated on ice for 15 min, centrifuged for 10 min at 10,000 rpm at 4° C, and the supernatant transferred to a new tube. Total protein concentration was quantified using the Pierce BCA protein assay kit . 40 μg of protein were denatured in reducing buffer, heated to 95° C for 10 min, and run on a 7.5% gel . Following SDS/PAGE gel electrophoresis, proteins were transferred to nitrocellulose membranes, which were probed for GRB7 (1:1000), HER-2 (1:1000) and α-tubulin (1:2000). Fluorescent protein-conjugated secondary antibodies  were then used for signal detection. Visualization was carried-out with the LI-COR Odyssey Infrared Imaging System Scan, with a resolution of 169 μm (LI-COR).Breast and ovarian cell lines were lysed in Cell Lysys Buffer (20 mM Tris-HCl (pH 7.5), 150 mM, NaCl 1 mM, Na488 secondary antibody .anti-human GRB7, rabbit monoclonal antibody ; goat anti-rabbit-Alexa Fluor®II 8-well chamber slide systems  were fixed for 20 min with 4% paraformaldehyde/4% sucrose in PBS at RT and followed by washes in PBS. Cells were then permeabilized with 0.3% Triton X-100 in PBS for 5 min at RT, and washed again in PBS. Slides were then incubated in blocking buffer (0.5% BSA in 0.1M Tris/0.15M NaCl) for 30 min at RT, followed by the incubation with the primary antibody  for 24 h at 4° C in a humid chamber. Slides were then developed in two ways: 1) IHC, using the Vectastain Elite ABD HRP kit as described above), and 2) Immunofluorescence, described here. Following three washes with PBS, cells were blocked again for 30 min, washed three more times with PBS and incubated with an anti-rabbit secondary antibody coupled to Alexa Fluor488. Cells were washed with PBS, and incubated for 3 min with 5μg/ml WGA-Alexa-Fluor647-  and 300nM DAPI  to label the plasma membrane and the nuclei. Following three more washes with PBS, cells were mounted using Everbrite mounting medium . Cells were imaged using an inverted epifluorescent Zeiss Axio Observer Z1 microscope, a 20× objective, and an Andor Luca EMCCD camera . Fluorescence for Alexa Fluor488 and Alexa Fluor647 was detected using 485 nm excitation/535nm emission and 640 nm excitation/680 nm emission wavelengths respectively, DAPI was detected using 350 nm excitation/430 nm emission . In all experiments, cells were imaged at middle height of the cell, with exposure times of 200 ms. Merged images were prepared using FIJI software [Cell cultures grown on Lab-Teksoftware .Chi-squared tests were used to test for associations between GRB7 expression and clinical covariates. A multivariate logistic regression was used to identify independent prognostic factors, and a simple kappa coefficient estimate was used to test for agreement between variables. Survival curves were generated using the Kaplan-Meier method, with significance evaluated using the log-rank test of significance, and in some cases a Wilcoxon test was used as comparison. All analyses were performed with SAS software version 9.4 , and graphs were generated by R software and Graphpad Prism.


Astrocytes, once thought to be passive cells merely filling the space between neurons in the nervous system, are receiving attention as active modulators of the brain and spinal cord physiology by providing nutrients, maintaining homeostasis, and modulating synaptic transmission. Accumulating evidence indicates that astrocytes are critically involved in chronic pain regulation. Injury induces astrocytes to become reactive, and recent studies suggest that reactive astrocytes can have either neuroprotective or neurodegenerative effects. While the exact mechanisms underlying the transition from resting astrocytes to reactive astrocytes remain unknown, astrocytic calcium increase, coordinated by inflammatory molecules, has been suggested to trigger this transition. In this mini review article, we will discuss the roles of astrocytic calcium, channels contributing to calcium dynamics in astrocytes, astrocyte activations along the pain pathway, and possible relationships between astrocytic calcium dynamics and chronic pain. Under physiological conditions, astrocytes occupy non-overlapping regions and function in discrete areas  and inhibitory γ-aminobutyric acid (GABA). The majority of Glu in the extracellular space is absorbed (80–90%) by excitatory amino-acid transporter (EAAT)-1 and 2, which are predominantly expressed in astrocytes  activity, on the other hand, is directly regulated by astrocytic Ca2+. Transient receptor potential (TRP) channel subfamily A (TRPA), a cation channel shown to be important in maintaining basal astrocytic Ca2+ concentration in astrocytes, controls the expression of GAT in a Ca2+-dependent manner , a structure of fine astrocytic processes wrapped around neuronal synapses, affect astrocytic buffering ability and provide spine stability in an astrocytic Ca2+ affects astrocytic lactate secretion is unknown, but the control of vascular smooth muscle tone, which is another important way of providing nutrients and oxygen, is directly regulated by astrocytic Ca2+  between synapses , but they could be more appropriate for providing sustained signaling in cases such as protecting tissues after an injury during recovery. While some alterations in astrocytic Ca2+ signals have been associated with dysfunctions in the CNS such as chronic pain, what kind of astrocytic Ca2+ changes lead to pathological conditions is still unclear. Since astrocytic Ca2+ signals triggered by different sources seem to have different functions, different sources of astrocytic Ca2+ are discussed in the next section.As described above, astrocytics Ca2+ sources contribute to astrocytic Ca2+ dynamics. Ca2+ releases from intracellular stores and influx from the extracellular space both contribute to astrocytic Ca2+ changes, albeit with different patterns. Intracellular sources of astrocytic Ca2+ include inositol triphosphate receptor (IP3R), ryanodine receptor (RyR), and mitochondrial permeability transition pore (mPTP). Ca2+ Channels that contribute to Ca2+ influx into astrocytes from the extracellular space include Orai channels, ligand-gated ion channels , TRP channels, sodium/calcium exchangers (NCX), and voltage-gated Ca2+ channels.Several Ca2+ increase occurs by activation of IP3R expressed on the endoplasmic reticulum (ER). Activation of IP3R leads to a robust Ca2+ increase in the cell body. Ca2+ dynamics become nearly absent in the cell body of astrocytes with inhibition of IP3R or knockout of IP3R2, a major type of IP3R in astrocytes, but some Ca2+ activity in astrocytic processes remains  generated by mitochondria and the increase in Ca2+ released from the ER, the mitochondrial membrane depolarizes via the opening of the mPTP, which ultimately leads to the necrotic death of astrocytes  on the ER, which sense Ca2+, cluster to activate Orai channels  could also contribute to astrocytic Ca2+ elevation. Several types of voltage-gated Ca2+ channels—L, N, R, and T types—are expressed in astrocytes. Although astrocytes are not electrically excitable, cultured astrocytes responded with an increase in intracellular Ca2+ to electrical stimulation  in astrocytes prevented astrocyte activation, reduced inflammation in the brain, and promoted myelin regeneration  and affective  aspects, which are separately processed by discriminatory and the affective pain pathways. Interestingly, in chronic pain models, reactive astrocytes are found not only at the site of noxious stimulation or injury but also along both pain pathways . Astrocya et al.  found tha et al. .Numerous studies have suggested that reactive astrocytes in the spinal cord are associated with chronic pain. Astrocyte activation, indicated by GFAP upregulation, is reported in various pain models , suggesting that reactive astrocytes in the thalamus may be the underlying cause  resulting from nerve injury , the region important for identifying the intensity and location of pain, is also enhanced by nerve injury. GFAP expression and frequency of astrocytic CaAstrocytes in brain regions known to process the cognitive-affective aspect of pain are also activated with chronic pain. Sciatic nerve ligation significantly enhanced GFAP expression in the cingulate cortex  subtype Cx43  or diffusion of extracellular ATP .While some studies suggest that aberrant changes in astrocytic Ca2+ increased in the S1 and the ACC following nerve injury  release and synapse formation, indicating that rewired circuits could cause neuropathic pain , which remodel synapses. Altered connectivity between neurons and associated hyperactivity may be the underlying cause of chronic pain. Since reactive astrocyte exhibits prominent Ca2+-dependent gliotransmitter release, enhanced Glu released by reactive astrocytes that excessively excite neurons may also contribute to chronic pain. Changes in neurotransmitter buffering activity could also contribute to the changes in neuron–astrocyte interaction ensuing chronic pain. Buffering of the inhibitory transmitter—GABA—by astrocytes via GAT is directly affected by astrocytic Ca2+, with more GABA taken up with increasing Ca2+. Although GAT activity was shown to enhance the synaptic efficacy of an inhibitory synapse by reducing ambient GABA that desensitize the inhibitory synapse  and the A2 (neuroprotective; Liddelow and Barres, 2+ is unknown. It is possible that astrocytic Ca2+ induced by different channels may drive reactive astrocytes to be one type or the other. Therefore, investigating how different astrocytic Ca2+ channels contribute to different types of reactive astrocytes in regard to chronic pain development will provide a deeper understanding of the roles of astrocyte in pain and may offer better targets for pain control.Astrocytic CaJC and YH contributed equally to the manuscript. All authors contributed to the article and approved the submitted version.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.


Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca2+ ([Ca2+]cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca2+ influx channels involved in eATP-induced [Ca2+]cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca2+ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca2+]cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes. Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of  Arabidopsis thaliana, eATP can act as a damage-associated molecular pattern (DAMP) and activates immunity signalling through the plasma membrane purinoreceptor AtDORN1  [2+ ([Ca2+]cyt) [2+ as a second messenger to changes in transcription due to eATP. It may be that the nuclear Ca2+ increase that follows eATP-induced [Ca2+]cyt increase [2+-CAM interaction. Understanding how eATP causes [Ca2+]cyt increase is, therefore, relevant to downstream responses. Extracellular ATP (eATP) is implicated as an apoplastic signal molecule in the abiotic and biotic stress responses of plants, their cellular viability, growth and stomatal regulation ,3,4,5,6.as P2K1) . A plasmas P2K1) . eATP-deas P2K1) ,10, poina2+]cyt) ,12,13,14a2+]cyt) ,16. Evena2+]cyt) ,18,19. Mincrease  activate2+]cyt increase in Arabidopsis roots requires plasma membrane Ca2+ influx channels but the molecular identities of these channels remain unknown, as is also the case for other organs [Arabidopsis annexin 1 protein (AtANN1) could be involved in mediating plasma membrane Ca2+ influx [2+]cyt increase when expressed in Xenopus oocytes [2+ channel in the root [Ca2+]cyt response to salinity stress, hyperosmotic stress, and oxidative stress [2+]cyt signalling pathway has been tested using an Atann1 loss of function mutant constitutively expressing cytosolic (apo)aequorin as a luminescent [Ca2+]cyt reporter [2+]cyt [2+]cyt response towards both eATP and eADP in roots. It affects the spatial extent of intracellular ROS accumulation in roots and influences their eATP-induced transcriptional response.It is known that eATP-induced cyt ,12,14,212+]cyt elevation was found to be wholly reliant on the AtDORN1 receptor [Atann1 (loss of function mutant) and Col-0 (expressing cytosolic (apo)aequorin under the 35S CaMV promoter) were excised and assayed individually to measure [Ca2+]cyt in the presence of eATP. Addition of control solution after 35 s of measurement evoked a monophasic [Ca2+]cyt increase in response to mechanical stimulation (“touch response”) before returning to the basal level (Atann1 roots was similar to Col-0 in terms of the amplitude (“touch peak”) and the total accumulation of [Ca2+]cyt   and although the first eATP peak was lower in Atann1 it was not significantly different to Col-0 (p > 0.05) (2+]cyt peak response of Atann1 was significantly lower than Col-0 (p < 0.0001)  d. The se 0.0001) e. The to2+]cyt increase in roots [Atann1. As shown in p > 0.05) or the overall [Ca2+]cyt (p > 0.05) between Col-0 and Atann1 (Atann1 produced a biphasic [Ca2+]cyt increase following the touch response. No significant difference was found between Col-0 and Atann1 in the touch peak response (p > 0.05)  (p < 0.0001) (2+]cyt compared to Col-0 (p < 0.0001) (AtDORN1 expression cannot explain the impairments in Atann1′s response to extracellular nucleotides as no significant difference in the receptor’s expression between Col-0 and Atann1 roots was found in either control conditions (p > 0.5) or in the presence of 1 mM ATP (p > 0.5)  d. Unlike 0.0001) d and a n 0.0001) e. Overal 0.0001) e. Lowerep > 0.5) f. Theref2+]cyt increase. The ATPγS used was a tetralithium salt whereas the ADPβS was a trilithium salt. A LiCl treatment (4 mM for ATPγS and 3 mM for ADPβS) was carried out alongside the non-hydrolysable analogues as a lithium control. Seven-day-old individual whole roots responded with the biphasic [Ca2+]cyt increase when tested with different concentrations of eATPγS, or the eATP/LiCl salt control (p > 0.5). The Col-0 first [Ca2+]cyt peak did not require eATP hydrolysis (no significant difference between 1 mM eATPγS and 1 mM eATP/LiCl) and indeed was already “saturated” at 0.1 mM eATPγS   and 0.2 mM eATPγS (p < 0.001)  (2+]cyt accumulated where there were significant differences between Col-0 and Atann1 (p < 0.0001) in the AUC for every concentration tested  and ADP analogue  were then used to confirm that the agonists acted as signal molecules rather than as energy sources that drive the [Ca control a. As see eATPγS) c. The fiM eATPγS c. As in nificant c. Howeve< 0.001) c. These and 1 mM d. AtANN1< 0.001) d. This wn tested e.Atann1 generated the transient biphasic [Ca2+]cyt elevation after the touch peak response when challenged with different concentrations of eADPβS or 1 mM eADP with 3 mM LiCl (Atann1 produced the same level of touch peak response for each treatment (p > 0.5) (Atann1 in the first peak [Ca2+]cyt response regardless of the concentration of eADPβS tested (p < 0.001) (Atann1 was found to produce lower [Ca2+]cyt responses than the Col-0 in the second peak (p < 0.001) (p < 0.001) (Both Col-0 and  mM LiCl a. Both Cp > 0.5) b. Just lp > 0.5) c. In con< 0.001) c. Consis< 0.001) d and in < 0.001) e for allArabidopsis roots that requires Ca2+ influx and is largely dependent on AtRBOHC activity cyt in response to eATP [2+]cyt elevation by 1 mM eATP of seven-day-old cotyledons of Col-0 and Atann1 was compared. Consistent with previous results on Col-0 true leaves cyt accumulated   d.2+]cyt response at 0.1 mM   d. Thus, 2+]cyt elevation in roots, with consequences for eATP-induced ROS accumulation and gene expression. Mechanical or “touch” stimulus can cause accumulation of extracellular ATP by Arabidopsis root tips but much less so in older regions of the root [2+]cyt  or root cell-specific GCaMP3 cyt a but it receptor . For eATthe apex ,36,37,38the apex , showingthe apex ,34 so it mM eATP c but not mM eATP c suggestc GCaMP3  is now nArabidopsis roots, eATP (but not eADP) causes intracellular ROS accumulation, which relies on Ca2+ influx and the AtRBOHC NADPH oxidase [2+ influx acts downstream of AtDORN1 and upstream of AtRBOHC/AtRBOHD. It is not known why eADP is ineffective. The pharmacological block that effectively eliminates eATP-activated Ca2+ influx across the plasma membrane only inhibits half of the intracellular ROS in roots caused by eATP [2+]cyt elevation in Atann1 roots in response to eATP could therefore have been sufficient to cause the observed normal level of ROS accumulation, particularly at the apex  to be discovered.AtANN1 is now known to be involved in cyt was determined according to the calibration formula given in cyt in response to both eATP and eADP. It is postulated that AtANN1 might operate as an ROS-activated plasma membrane Ca2+ channel, downstream of AtDORN1. Since loss of AtANN1 does not completely abolish the [Ca2+]cyt increase, there are clearly other channels in the pathway. AtANN1 appears to regulate the spatial extent of eATP-induced intracellular ROS in the root. At the gene expression level, AtANN1 is involved in eATP-induced up-regulation of AtACS6 and AtWRKY40, thus potentially directing eATP signalling towards ethylene production and salt stress tolerance.This study has identified AtANN1 as a component in mediating the root increase of [Ca


The disturbance of IP3R1 is related to several neurodegenerative diseases. This study investigated the mechanism of IP3R1 in myocardial ischemia/reperfusion (MI/R). After MI/R modeling, IP3R1 expression was silenced in myocardium of MI/R rats to explore its role in the concentration of myocardial enzymes, infarct area, Ca2+ level, NLRP3/Caspase-1, and pyroptosis markers and inflammatory factors. The adult rat cardiomyocytes were isolated and cultured to establish hypoxia/reperfusion (H/R) cell model. The expression of IP3R1 was downregulated or ERP44 was overexpressed in H/R-induced cells. Nifedipine D6 was added to H/R-induced cells to block Ca2+ channel or Nigericin was added to activate NLRP3. IP3R1 was highly expressed in myocardium of MI/R rats, and silencing IP3R1 alleviated MI/R injury, reduced Ca2+ overload, inflammation and pyroptosis in MI/R rats, and H/R-induced cells. The binding of ERP44 to IP3R1 inhibited Ca2+ overload, alleviated cardiomyocyte inflammation, and pyroptosis. The increase of intracellular Ca2+ level caused H/R-induced cardiomyocyte pyroptosis through the NLRP3/Caspase-1 pathway. Activation of NLRP3 pathway reversed the protection of IP3R1 inhibition/ERP44 overexpression/Nifedipine D6 on H/R-induced cells. Overall, ERP44 binding to IP3R1 inhibits Ca2+ overload, thus alleviating pyroptosis and MI/R injury.Intracellular ion channel inositol 1,4,5-triphosphate receptor (IP3R1) releases Ca Ischemia/reperfusion (I/R) injury, as a complicated pathological process, includes nonspecific damage caused by ischemia and injury induced by reactive oxygen, nitrogen species, and inflammatory cytokines during reperfusion2. Myocardial I/R (MI/R) injury induces myocardial cell apoptosis, and contributes to adverse cardiovascular outcomes, heart dysfunction, and structural damage3. Intracellular Ca2+ ([Ca2+]i) overload is a key cause of cellular injury and myocardial apoptosis4. Strong activation of Ca2+ channels may be an essential mechanism for neuronal death after cerebral I/R injury5. Cardiomyocyte cell death in response to I/R results from the increases in Ca2+ 6. I/R injury activates endoplasmic reticulum (ER) stress in cardiomyocytes, and persistent ER stress with an increase in [Ca2+]i concentration from the ER through the inositol 1,4,5 trisphosphate receptor (IP3R), leads to cardiomyocyte apoptosis7. These references provide a novel perspective for I/R treatment that degrading [Ca2+]i concentration in view of ER stress and IP3R may be beneficial to relieve MI/R injury.It is widely accepted that ischemic disease represents a major health threat as it leads to numerous mortality every year across the world8. Increased expression of IP3R1 is also observed after hypoxia/reperfusion (H/R) injury of spinal cord dorsal column in vitro9. IP3R, a [Ca2+]i release channel is predominately expressed on the ER membranes10. Endoplasmic reticulum resident protein 44 (ERP44) binds to IP3R1 and regulates [Ca2+]i concentrations by modulating IP3R1 activity11. ERP4 has also been shown to participate in Ca2+ homeostasis, maturation of secretory proteins within ER, cardiac development, control of blood pressure, and ER stress activation13. But little is known about the role of ERP44 in MI/R injury.Elevated levels of IP3 in adult cardiomyocytes are associated with the development of cardiac hypertrophy, arrhythmias, and heart failure16. Pyroptosis is characterized by activation of nod-like receptor protein-3 (NLRP3) inflammasome and Caspase and the release of interleukin (IL)-1β and IL-1818. The role of pyroptosis in cancers, cardiovascular diseases, and microbial infection-related diseases is a hot spot in recent years20. As has been verified, cell pyroptosis is also closely involved in MI/R injury21. Inhibited pyroptosis helps to attenuate renal I/R injury22. Importantly, Ca2+ signaling is critical for NLRP3 inflammasome activation to induce pyroptosis23. However, the role of IP3R/ERP44 in pyroptosis in MI/R injury remains unknown.Pyroptosis is a type of inflammatory programmed cell death induced by pro-inflammatory mediators and exhibits morphological characteristics that are different from apoptosis and necrosis2+ overload and pyroptosis through the NLRP3/Caspase-1 pathway and thus plays a protective role in MI/R injury has not been reported at home and abroad. Therefore, the aim of this study is to study the protective mechanism of ERP44 binding to IP3R against MI/R injury and to provide a new theoretical basis for the management of MI/R injury.Given the former analyses, whether ERP44 binding to IP3R can regulate Cap < 0.05; Fig. p < 0.05; Fig. p < 0.05; Fig. We established the MI/R model in SD rats by surgery, and found that IP3R1 expression in cardiomyocytes of rats after I/R treatment was significantly increased , and the levels of inflammatory factors were significantly elevated  were downregulated. This effect was similar to that of H/R cells treated with calcium channel blocker Nifedipine D6  in cardiomyocytes of MI/R rats was inhibited and the release of inflammatory factors (IL-1β and IL-18) were alleviated after inhibition of IP3R1. NLRP3 inflammasomes consist of NLRP3, ASC, and procaspase-132. It is well accepted that Caspase-1 and GSDMD are pyroptosis-related proteins33 and GSDMD controls the release of IL-1β and IL-1834. Elevations IL-1β and IL-18 contribute to cell pyroptosis35. Inhibiting NLRP3-mediated pyroptosis is neuroprotective against cerebral I/R injury36. Taken together, inhibiting IP3R1 expression can reduce the levels of pyroptosis markers induced by MI/R and alleviate myocardial injury. When IP3R1 expression was decreased using siRNA, the activity of H9C2 cells was increased and the levels of pyroptosis markers and inflammatory proteins were decreased. The results were consistent with the results in vivo, suggesting that IP3R1 downregulation inhibited intracellular pyroptosis to alleviate the H/R injury.IP3R1 expression is increased markedly in pigs exposed to high stress2+ overload which can lead to apoptosis9. During the process of MI/R injury, the [Ca2+]i concentration continues to increase and leads to cardiomyocyte apoptosis and myocardial damage37. After downregulating IP3R1 expression in myocardial tissue of MI/R rats using shRNA, the Ca2+ level in mitochondria was clearly decreased; similar results were found in H/R-challenged cells. Sustained global elevation of [Ca2+]i eventually results in zymogen activation and inflammatory injury38. After silencing IP3R1, the inflow of Ca2+ and the release of [Ca2+]i stores are reduced in cells with TNF-α-induced [Ca2+]i overload39. To reduce [Ca2+]i and maintain Ca2+ homeostasis can protect cardiomyocytes from I/R injury4. These results suggested that inhibiting IP3R1 expression can inhibit the [Ca2+]i overload induced by MI/R. Then we investigated how did IP3R1 inhibition inhibit the [Ca2+]i overload. Besides, ERP44 could directly inhibit the activity of IP3R125 and regulate the Ca2+ signal of cardiomyocytes24. Therefore, we detected ERP44 expression in the myocardium of MI/R rats and in the cells treated with H/R. After MI/R treatment and H/R treatment, the expression of ERP44 was notably decreased. In addition, immunoprecipitation showed that ERP44 could directly bind to IP3R1. ERP44 directly interacts with IP3R1 and inhibits IP3-induced Ca2+ release40. IP3-induced Ca2+ release is decreased in HeLa cells overexpressing ERP44 through IP3R channels40. The level of Ca2+ in H/R-induced mitochondria was decreased clearly after overexpression of ERP44, indicating that the binding of ERP44 to IP3R1 inhibited the MI/R-induced mitochondrial Ca2+ overload.Increased activity of IP3Rs causes mitochondrial Ca41. Ca2+ influx is required for NLRP3 activation and cytokine release42. Moreover, we evaluated whether ERP44/IP3R1-mediated [Ca2+]i level monitors the pyroptosis. After ERP44 overexpression, the activity of H/R-challenged cells was increased, the levels of pyroptotic markers  were reduced, and IL-1β and IL-18 were downregulated. Inhibition of IP3R channel has been reported to attenuate Ca2+ mobilization and NLRP3 inflammasome activation43. This effect of ERP44 overexpression was similar to that of H/R cells treated with calcium channel blocker Nifedipine D6. Blocking Ca2+ mobilization inhibits activation of the NLRP3 inflammasome complex23. In brief, overexpression of ERP44 could inhibit the transport of Ca2+ by binding to IP3R1, thus reducing mitochondrial Ca2+ overload in H/R cells and alleviating cardiomyocyte pyroptosis. To verify that the [Ca2+]i overload regulated by the binding of ERP44 and IP3R1 is controlled by the pyroptosis mediated by the NLRP3/Caspase-1 pathway, we added Nigericin (an activator of NLRP3) to H/R cells overexpressing ERP44 or silencing IP3R1. Activation of NLRP3 reversed the protective effect of overexpression of ERP44 or inhibition of IP3R1 on H/R-induced cells. The NLRP3/Caspase-1 axis is a typical pathway of cell pyroptosis44. A study has reported that the inhibition of pyroptosis can mitigate cerebral ischemia-induced brain injury45. These results further confirmed that the increase of [Ca2+]i level regulated by ERP44 and IP3R1 can cause H/R-induced cardiomyocyte pyroptosis through the NLRP3/Caspase-1 pathway, thus regulating MI/R injury.Perturbation of intracellular ion homeostasis is a key signal for activation of NLRP3 inflammasome and subsequent pyroptosis2+ overload, thus alleviating pyroptosis, inflammation, and MI/R injury. These results discovered a novel therapy for patients with MI/R. In the future, we shall carry out more prospective trials to refine our clinical guidance for the treatment of MI/R.All in all, the current study supported that ERP44 could bind to IP3R1 and inhibit CaThe study got the approval of the Clinical Ethical Committee of Affiliated Hospital of Guangdong Medical University. Informed consent was signed by each eligible participant. All experimental procedures were implemented on the Ethical Guidelines for the Study of Experimental Pain in Conscious Animals.Sprague-Dawley (SD) rats (180–200 g)  2017-0033) were anesthetized using 2% pentobarbital sodium  and ventilated with rodent respirators  at 80 °C in volume control mode, once a minute. The left axillary was exposed by left thoracotomy at the edge of the left sternum between the 4th intercostal space. The left anterior descending (LAD) coronary artery was ligated about 2 mm from its origin using 6-0 surgical sutures. Occlusion was confirmed by observing the pale myocardium in the left ventricle below the suture line. The rats in the sham group were anesthetized, and the suture was not occluded under the LAD. The rats in the I/R group were ligated with LAD for 30 min and then reperfused for 2 h.7 TU/mL, while rats in the sh-IP3R1 group were subjected to an intracardiac injection of 30 μL p-LV-sh-IP3R1 (Thermo Fisher), with the virus titer at 2 × 107 TU/mL. All rats were euthanized 48 h after operation  and the cardiac tissues were rapidly removed. The cardiac tissues of three rats in each group were used for area at risk (AAR) measurement, 6 for heamatoxylin and eosin (HE) staining and immunohistochemical staining, 6 for Ca2+ determination and 6 for other indicators. Each SD rat was selected for any experiment by the researchers who did not participate in the study.All 84 SD rats were numbered according to the weight and were randomly assigned by random number method to sham group, I/R group, sh-NC (short hairpin RNA-negative control) group, and sh-IP3R1 group, with 21 rats in each group. Three days before the I/R surgery, rats in the sh-NC group were subjected to an intracardiac injection of 30 μL p-LV-sh-NC , with the virus titer at 2 × 10AAR/LV (left ventricle) reflects the degree of myocardial ischemia, while infarct size (INF)/AAR reflects the level of dead myocardium. After the I/R surgery, the suture under the LAD was removed. To identify AAR, rats were perfused with 2 mL 2% Evans blue  through the inferior vena cava. The hearts were removed, washed with phosphate-buffered saline (PBS) solution and frozen at −20 °C. Then, the heart was sliced into five sections at 1 mm, incubated at 37 °C for 15 min with 1% 2,3,5-triphenyltetrazolium chloride (TTC) and PBS solution, and fixed for 2 h with 10% formalin. Image J software analyzed the imaging and calculated the percentage of INF to AAR using a weight-based method.Blood samples were collected from the inner eye angle vein of rats. Serum components were stored at −80 °C for subsequent analysis. Myocardial enzymes creatine kinase isoenzyme MB (CK-MB) and lactate dehydrogenase (LDH) were determined using a semi-automatic biochemical analyzer .The left ventricle of heart tissue was fixed in 10% formaldehyde, dehydrated with increasing concentrations of ethanol, embedded in paraffin, and cut into sections at 4 μm. The sections were stained with HE  after dewaxing, and observed under the light microscope .46, the cardiomyocytes were isolated by enzyme digestion and loaded with Fluo 4-AM  at 37 °C for 20 min. Fluorescence changes of Fluo 4-AM were recorded by confocal microscopy at 25 °C.According to a previous study2+, Rhod2 AM  was cultured in a phenol-free Dulbecco’s modified Eagle’s medium (DMEM) containing 0.03% pluronic acid at 37 °C for 40 min. Then, the cells were washed to remove Rhod2 and degreased in phenol-free DMEM containing 10% FBS. Fluorescence signals were recorded by 561 nm excitation wavelength and 588 emission wavelength under a confocal microscope.In order to measure mitochondrial CaThe heart sections were dewaxed and hydrated. After antigen repair, the sections were immersed in 3% hydrogen peroxide for 10 min to block the endogenous peroxidase, and blocked with 5% bovine serum albumin (BSA), and incubated with the primary antibody GSDMD  at 4 °C overnight. After that, the sections were incubated with the secondary antibody IgG  for 1 h, and stained with hematoxylin after 2,4-diaminobutyric acid staining. After dehydration and clearing, the sections were observed.−ΔΔCt method, with GAPDH as the internal reference.The total RNA content from cells was extracted using the one-step method of TRIzol kit . High-quality RNA was confirmed by UV analysis and formaldehyde deformation electrophoresis. According to the qRT-PCR kit (ThermoFisher), fluorescent qPCR was performed. PCR primers Table  were desThe total protein content from myocardial tissue or cells was extracted for measurement of protein concentration using the bicinchoninic acid assay (ThermoFisher). The extracted protein was added into the loading buffer and boiled at 95 °C for 10 min. Next, the protein sample (30 μg) was separated by 10% (w/v) electrophpresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked using 5% BSA for 1 h and cultured with the primary antibodies at 4 °C overnight. After being washed by tris buffered saline tween (TBST), the membranes were incubated with secondary antibody for 1 h and then washed by TBST before enhanced chemiluminescence developing and visualization using Bio-Rad GelEZ imager . Image J software  was utilized to analyze the gray value of target bands. The antibodies used were IP3R1 , IgG , NLRP3 , ASC , GSDMD , GAPDH , and ERP44 .According to the manufacturer’s instructions of the Caspase-1/ICE colormetric assay kit , the activity of Caspase-1 in rat heart tissues or cells was detected.2. When the cell confluence reached above 90%, the cells were passaged. The cells were detached using trypsin, resuspended, and adjusted to 1 × 104 cells/cm2, and then incubated at 37 °C in the incubator containing 5% CO2. Cells at 3–6 generations were used for the experiment.H9C2 cells  were cultured in DMEM supplemented with 1% penicillin, streptomycin, and 10% FBS at 37 °C and 5% COsi-IP3R1 and si-NC were synthesized by GeneChem . ERP44 cDNA was cloned into pcDNA3.1 (Invitrogen) and transfected with Lipofectamine 2000 (Invitrogen) according to the instructions. Nifedipine D6 and Nigericin were purchased from MedChemExpress, and the cells were treated.H9C2 cells were allocated to blank group , H/R group , H/R + si-IP3R1 group, H/R + si-NC group, H/R + NC group, H/R + ERP44 group , H/R + Nif group (H/R-treated cells were added with 50 μM Nifedipine D6), H/R + si-IP3R1 + Nig group, and H/R + ERP44 + Nig group .3, 1 mM NaH2PO4 H2O, 2.5 mM CaCl2 2H2O, 0.5 mM sodium EDTA 2H2O, 20 mM sodium lactate, 16 mM KCl (pH = 6.2)]. After being pre-inflated with 95% N2 and 5% CO2 for at least 5 min, ischemia buffer was added to the cells, and then the cells were placed in a sealed chamber containing deoxygenation reagent. The AnaeroPack system  was used to produce near-anaerobic conditions. The cells were incubated at 37 °C for 1 h in the incubator containing <1% O2 and ~5% CO2. Then cells were exposed to near-aerobic conditions for 2 h and incubated under normal conditions (reperfusion) for 24 h. The control cells were cultured in complete medium without ischemia and H/R. The same method was used for isolated adult cardiomyocytes, but the hypoxia time was reduced to 45 min.H9C2 cells were maintained in serum-free DMEM for 2 h and treated with ischemia buffer [118 mM NaCl, 24 mM NaHCOBefore hypoxia, H9C2 cells and adult cardiomyocytes were loaded with Rhod2. Fluorescence was observed under a microscope. Cell density was determined by crystal violet staining to standardize the intensity.Co-IP of IP3R1 and ERP44 was performed as per the instructions of the kit (Thermo Fisher). The cells were dissolved in IP buffer, and the supernatant was incubated with protein A/G agarose beads  at 4 °C for 3 h with anti-IP3R1 antibody or anti-ERP44 with IgG antibody as a control. The agarose beads were washed with cold PBS and the binding protein was eluted. The precipitated protein was determined by western blot analysis.Cell viability was evaluated by CCK-8 assay . Cardiomyocytes or H9C2 cells were seeded into 96-well plates at 5000 cells/well. After proper treatment, the medium was replaced with 100 μL CCK-8 solution (containing 90 μL serum-free DMEM and 10 μL CCK-8 reagent). The absorbance at 450 nm of each well was measured.The levels of IL-1β and IL-18 in tissue or cells were detected according to Rat IL-1β/IL-1F2 Quantikine ELISA kit  and Rat IL-18 ELISA kit .p value was obtained from a two-tailed test, where a value of p < 0.05 was indicative of a statistically significance.Data analysis was introduced using the SPSS 21.0 . Kolmogorov–Smirnov test was employed to assess whether the data were in normal distribution. Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) or two-way ANOVA were applied for the comparisons among multiple groups, and Tukey’s multiple comparison test was applied for the post hoc test after ANOVA. The


Non-equilibrium and equilibrium thermodynamics of an interacting component in a relativistic multi-component system is discussed covariantly by exploiting an entropy identity. The special case of the corresponding free component is considered. Equilibrium conditions and especially the multi-component Killing relation of the 4-temperature are discussed. Two axioms characterize the mixture: additivity of the energy momentum tensors and additivity of the 4-entropies of the components generating those of the mixture. The resulting quantities of a single component and of the mixture as a whole, energy, energy flux, momentum flux, stress tensor, entropy, entropy flux, supply and production are derived. Finally, a general relativistic 2-component mixture is discussed with respect to their gravitation generating energy–momentum tensors. The treatment of multi-component systems is often restricted to transport phenomena in chemically reacting systems, which means the mixture consisting of different components is shortly described by 1-component quantities such as temperature, pressure and energy that are not retraced to the corresponding quantities of the several components of the multi-component system. That is the case in non-relativistic physics ,3 as welConsidering a multi-component system, three items have to be distinguished: one component as a member of the multi-component system which interacts with all the other components of the system; the same component as a free 1-component system separated from the multi-component system; and the multi-component system itself as a mixture. which is composed of its components. Here, all three items are discussed in a covariant-relativistic framework. For finding out the entropy-flux, -supply, -production and -density, a special tool is used: the entropy identity which constrains the possibility of an arbitrary choice of these quantities ,10,11,12Equilibrium is defined by equilibrium conditions, which are divided into basic ones given by vanishing entropy-flux, -supply and -production and supplementary ones such as vanishing diffusion flux, vanishing heat flux and zero rest mass production ,12. The The paper is organized as follows: After this Introduction, the kinematics of a multi-component system is considered in the next two sections for introducing the mass flux and the diffusion flux densities. The energy–momentum tensor is decomposed into its (3+1)-split, and the entanglement of the energy and momentum balances are discussed, followed by non-equilibrium thermodynamics of an interacting component of the mixture and that of the corresponding free component. The equilibrium of both is considered. Thermodynamics of the mixture starts with three axioms: additivity of the mass flux densities, of the energy momentum tensors and of the 4-entropies of the components resulting in those of the mixture. Entropy, and entropy-flux, -supply and -production are found. The paper finishes discussing the gravitation generated by a special general-relativistic 2-component system: one component equipped with a symmetric energy–momentum tensor, a mass density and vanishing external force density, the other one with a skew-symmetric energy–momentum tensor, an external force density and vanishing mass density. A summary and an appendix are added.Z components. The component index Z. Each component has its own rest frame We consider a multi-component system consisting of In general, the components have different 4-velocities, namely Here, Equation 2 is the The mass production term has two reasons: an external one by mass supply and one internal one by chemical reactionsThe external mass supply As each component, the multi-component system also has a mass density Mixture Axiom: The balance equation of a mixture looks like the balance equation of a one-component system. ■ ■ Especially here, the mixture axiom is postulated for the balance equations of mass, energy–momentum and entropy. According to the mixture axiom, the mass balance of the mixture looks according to 2(4)Nk;kNow, the question arises: Which quantities of the components of the mixture are additive? Obviously, neither the mass densities For the present, mass density Equation . ContracEquation 2,3 resulThe mass density quations  and 5)44ϱ and thEquation 1 also a mponents In Equation 1 appearsg factor fA:=1c2uquations 1 and 5)1 appearsA comparison of Equation (10) with Equation 4 resultsEquation 2, thus dAccording to Equations 1 and (1)The sign of From Equations 3 and 9)3 and (9)Introducing the diffusion flux densityBy introducing the projectorsAccording to Equation 2, the diEquation 1.The energy–momentum tensor Here, Consequently, the interaction of the The (3+1)-split of the in general non-symmetric energy–momentum tensor of the This energy–momentum tensor of the material theory is not induced by a Lagrangian or a variational problem. It presents a quantity on its own. An example is the energy–momentum tensor which is needed for describing liquid crystals or polar fluids: it is non-symmetric because the stress tensor Equation  is used The (3+1)-components of the energy–momentum tensor in Equation  are the and (30)The (3+1)-split of tensors is a usual tool in relativistic continuum physics. The (3+1)-components—generated by the split—have physical significance which originally is hidden in the unsplit tensors. Thus, we generate by (3+1)-splitting the following covariant quantities of the The symmetric part of the energy–momentum tensor in Equation  is(32)TThe stress tensor is composed of the pressure For interpreting the (3+1)-components, we now consider their physical dimensions. According to Equations  and 9)119)1, we By taking Equations , 35)1 a1 a35)1 a1 and  into accThe (3+1)-split in Equation  of the eThe energy–momentum tensor in Equation  is that Z components: that is the mixture which consists of these Z interacting components. Because this interaction is already taken into account by the (3+1)-split-components, the energy–momentum tensors of the components are additive without additional interaction terms. Consequently, the energy–momentum tensor We now consider the equivalent-system composed of the Multiplication with quations 1 and (41Equation  results For a 1-component system (Starting with Equation (41), we obtainAccording to Equations  and 42)42)1, theEquation  followsEquation 1 resultiFrom Equation  followsEquation 2 resultiFrom Equation  followsFrom Equation (47)The additivity of the energy–momentum tensors in Equation  results The 4-velocity Equation 4.The influence of the additivity of the energy–momentum tensors on the balance equations of energy and momentum is investigated in the next section.If the energy-momentum tensors of the quations  and (27)The balances in Equations 3 and (60quations  and (27)The (3+1)-split of the divergence of the energy–momentum tensor of the Equation 1 in(61)If the component index Equation , we obtaEquation  and (60)The additivity of the energy–momentum tensors in Equation  results Taking Equations 2 and (27Equation  with um Inserting Equations  and (27)As Equation  indicateIn addition, if the total force density supply and the total momentum supply are zero according to Equation 2,(68)TkEquation 1,2(69)∑As expected, the supplies of energy and momentum remain entangled in a system of vanishing total force and momentum densities. The entanglement vanishes for such isolated systems for which the force and momentum supplies for all The (3+1)-split of the spin tensor of an Equation 1 into(7Introducing the following covariant abbreviationsEquation  results By Equations  and (73)Analogously to Equations  and (41)Taking Equations  and (73)Analogously to Setting II, we introduce the spin tensor of the mixture as the sum of the spin tensors of the According to the mixture axiom, the spin tensor of the mixture is defined by Equation 1 as spinEquation  by settiFrom Equation 1, we obtEquation  into accThe (3+1)-components of the spin tensor of the mixture result from Equations  and (73)Equation 4 or EquaEquation  and EquaIf there exists an external angular momentum densityAccording to Setting III,entropy identity ,39A-compThe basic equations of General-Covariant Continuum Thermodynamics (GCCT) of a mixture  Equatio–(217) anIn GRT, as a consequence of Einstein’s equationsquations  and 28)(253)Rab−quations 2,3) be iong time ,41,42. TProposition [■ position : The genEquation 2,3 and tquations 2 and . AccordThe balance equations defining the plain component (P) are according to Equations , 25) an an25) anBy definition, the plain component is characterized by a symmetric and divergence-free energy–momentum tensor and vanishing spin. According to Equations  and 256–(261), s2 and (262)ΘPaThe situation changes, if the plain component is regarded as a mixture component of a 2-component mixture whose second component is introduced in the next section.The balance equations defining the ghost component (G) areBy definition, the ghost component is characterized by vanishing mass density ΘGaSurprisingly, a “non-material” system such as the ghost component has a gravitational effect.Plain and ghost components form a 2-component mixture, which is discussed in the next section.According to the settings in Equations , 42) an an42) anFor fitting the GCCT plain-ghost mixture into the GRT, the Mathisson–Papapetrou equations (Equations  and (256Equation  results According to Equations (260)Equation .The gravitation generating energy–momentum tensor of the plain-ghost mixture is according to Equations , (269)2 quations  and  situation: An observer takes the plain-ghost mixture for a 1-component plain mixture because the ghost component is “dark”: no additional mass density and no spin can be detected from the point of view of the plain component. Erroneously, this observer supposes that Equations –(261) arEquation  does notEquation . This siEquation –(265), tOne question arises: Does a ghost component exist in nature and what is its physical essence? It is evident according to Equation  that theEquation  of the eA multi-component system is formed by its components, which are characterized by own individual quantities, such as velocity, density, chemical potential, stress tensor, temperature, heat flux, entropy flux densities, entropy production, supply and further items. All these individual quantities determine those of the multi-component system, which is described as a mixture. Individual temperatures of the components result in multi-temperature relaxation towards the corresponding equilibrium generating a common temperature of all components and of the mixture. A temperature of the mixture in multi-temperature relaxation non-equilibrium depends on the used thermometer and cannot be defined unequivocally.Starting out with the rest mass densities of the components of the multi-component system, the mass flux densities of the components are defined by introducing their different 4-velocities. The mixture of the components is characterized by several settings. The first one is the additivity of the component’s mass flux densities to the mass flux density of the mixture. In combination with the mixture axiom, this setting allows defining mass density and 4-velocity of the mixture and the diffusion fluxes of the components. The non-symmetric energy–momentum tensor of one component interacting with the mixture is introduced, and its (3+1)-split together with the component’s mass and diffusion flux densities are generating the entropy identity , which rThe exploitation of the entropy identity requires additional settings, which result in physical interpretations of entropy density, flux and supply inducing the entropy production from the entropy identity. By use of the entropy identity, Lagrange multipliers are introduced concerning the constraints taken into account. These Lagrange multipliers are temperature, chemical potential and an additional non-equilibrium variable which characterizes the considered component to be a part of the mixture. Beside the classical irreversible processes—diffusion, chemical reactions, heat conduction and friction—an additional irreversible process—multi-temperature relaxation—appears due to the embedding of the considered component into the mixture. Different from the classical case, the mass production term, the heat flux density and the viscous tensor are modified by so-called effective quantities.Equilibrium is defined by equilibrium conditions, which are divided into necessary and supplementary ones ,12,45. TThe corresponding free component is defined by undistinguishable component indices (that is not the mixture which is a multi-component system). This 1-component system represents the easiest classical case, serving as a test of whether the interacting component in the mixture is correctly described. The vanishing of the entropy production in equilibrium is shortly investigated: the so-called Killing relation of the vector of 4-temperature is neither a necessary nor a sufficient condition for equilibrium. In addition, the statement that materials are perfect in equilibrium cannot be confirmed.Finally, a special 2-component mixture is considered in the framework of General Relativity resulting in: a matter-free spin component influences the gravitation of the matter component.


However, the application of the original version of the nanomachine to investigate the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them one at a time, was prevented by the impossibility to preserve [Ca2+] as a free parameter. Here, the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined both in the absence and in the presence of Ca2+ . The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, the generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine made of an ensemble of eight heavy-meromyosin (HMM) fragments of myosin from rabbit psoas muscle, carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca In the sarcomere, the structural unit of the striated  muscle, two antiparallel arrays of the myosin II motor domains extending from the thick filament pull the nearby thin, actin-containing filaments from the opposite extremities of the sarcomere toward the center a. The in2+] induced by cell membrane depolarization, Ca2+ binding to troponin in the thin filament induces a structural change in the regulatory complex (troponin and tropomyosin)  b that maomyosin) b are stiCell studies, on the other hand, are complicated by the large ensemble of motor proteins and filaments and by the confounding contribution of the sarcomeric  proteins. Single-molecule mechanics use purified proteins and thus allow for the definition of the pure motor protein action and the comparison between different isoforms or native and mutant or genetically modified proteins. Single-molecule mechanics, however, suffer from the intrinsic limit in that they cannot investigate the properties that uniquely emerge from the collective motor within the architecture of the half-sarcomere. 2+-regulated actin-binding protein, [Ca2+] of 0.1 mM . These limits have been overcome with the realization of a one-dimensional nanomachine titrated to contain the minimum number of motor molecules, which, upon interaction with an actin filament, are able to mimic the half-sarcomere performance, generating steady force and constant velocity shortening . In the nditions ) is necenditions . SettingF-V) relation of the nanomachine powered by eight HMMfragments of myosin purified from rabbit psoas in 2 mM ATP are determined either in the absence or in the presence of 0.1 mM Ca2+ (the concentration required for BTA preparation with gelsolin). At the same temperature (~23 °C), all the relevant mechanical parameters (development of the maximum force in isometric conditions (F0), maximum shortening velocity (the unloaded shortening velocity V0) and maximum power (Pmax)) are the same independent of the [Ca2+] and the protocol used for BTA assemblage. Moreover, a kinetic model simulation based on the performance of fast mammalian skeletal muscle is able to fit all the relevant mechanical parameters of the nanomachine, once the methodological limits of the in vitro mechanics are taken into account, simply scaling down the number of motors available for actin interaction. The nanomachine with BTA assembled using the Ca2+-insensitive gelsolin fragment TL40 preserves [Ca2+] as a free parameter, and the development of a multiscale model for interfacing data at different hierarchical levels of the striated muscle opens new avenues for the investigation of the Ca2+-dependent function/dysfunction of any specific sarcomere protein.The force developed under isometric conditions and the force–velocity  or in Ca2+-free solution (BTA prepared with the gelsolin fragment TL40), as described in the Materials and Methods  , following the formation of the actin–myosin interface, the force rises to a maximum steady value (F0) of ~ 16 pN. At this point, the control is switched to force (it 2) and then a staircase of force drops to 11 (it 3), 7 (it 4) and 3 pN (it 5) is imposed, which elicits shortenings at constant velocity progressively larger as force decreases. When the control is switched back to position clamp (it 6), the force recovers a maximum steady value identical to the initial F0, demonstrating that the number of motors available for attachment to the actin filament remains the same throughout the whole interaction.The experiment in 2+ (light gray dashed bars), the frequency distributions of F0 almost superimpose and the mean values are 15.41 ± 0.73 pN in 0.1 mM CaCl2 and 15.88 ± 0.66 pN in Ca2+-free solution. The difference is not significant according to the t-test (p > 0.1). Thus, there is no significant effect of Ca2+ on the isometric force. As shown in F-V relation in either condition is plotted in 2, light gray symbols in Ca2+-free solution), showing that the F-V points lie along the same relation irrespective of the presence/absence of Ca2+. The dashed lines are the fits to the respective F-V data using Hill’s hyperbolic equation  or the gelsolin fragment TL40 in Ca2+-free solution. The use of the TL40 fragment totally preserves the nanomachine performance while making [Ca2+] a free parameter, opening the possibility for studies of the Ca2+-dependent mechanisms of the other sarcomere proteins. The power–force , the moi) isometric tetanic force F0 250 kPa, (ii) unloaded shortening velocity V0 8.6 μm s−1 per half-sarcomere and (iii) F-V relation as in a/F0 (the parameter that estimates the curvature of the relation) of 0.36. The density of force per cross-sectional area can be translated to force per half-thick filament (htf), taking into account the thick filament density  relation, calculated from the F-V relation, shows a maximum power (Pmax) of 460 aW per htf attained at ~1/3 F0 , already described in detail in the previous paper on the nanomachine , are:  is the minimum  increases, due to the increase in the rate of motor detachment following the execution of the working stroke  as a free parameter. The problem is solved here by devising a new protocol in which the BTA is prepared using the Ca2+-insensitive gelsolin fragment TL40. The effect of Ca2+ on the performance of the nanomachine in its simplest version (eight HMM fragments from the fast isoform of rabbit myosin II pulling on an actin filament) is tested, demonstrating that none of the relevant mechanical parameters, the isometric force F0 (16.8 pN), the unloaded shortening velocity V0 (3.5 μm s−1) or the maximum power Pmax (5.5 aW), depends on the presence/absence of Ca2+. Thus, the use of the TL40 fragment to prepare the BTA totally preserves the nanomachine performance while making [Ca2+] a free parameter, opening the possibility for studies of the Ca2+-dependent mechanisms of the other sarcomere proteins.In perspective, the nanomachine allows for the role of the sarcomeric (accessory and regulatory) proteins to be investigated by integrating them in the system one at a time, but in the original system , this peA simplified mechano-kinetic scheme a is usedF-V relation  to the orientation in the opposite direction (red continuous line). It must be noted that the observed F-V relation  in the marginal ear vein, in accordance with the Italian regulation on animal experimentation (Authorization 956/2015-PR) in compliance with Decreto Legislativo 26/2014 and EU directive 2010/63. Two rabbits were used for the experiments.Adult male rabbits , provided by Envigo, were housed at Centro di servizi per la Stabulazione Animali da Laboratorio , under controlled conditions of temperature (20 ± 1 °C) and humidity (55 ± 10%), and were euthanized by injection of an overdose of sodium pentobarbitone (150 mg kgHMM fragment of myosin was prepared from rabbit psoas muscle as reported previously . The funActin was prepared from leg muscles of the rabbits according to . Polymeri) in the conventional way  was 100 μg mL−1 [The mechanical apparatus is described in detail in . Briefly2, 5 mM MgCl2, 10 mM DTT and 2 mM ATP) [w/v) [−1 glucose oxidase, 20 µg mL−1 catalase, 5 mg mL−1 glucose and 0.5% w/v methylcellulose, 400 cP) was flowed at 10 µL min−1. The presence of methylcellulose inhibited the lateral diffusion of F-actin [w/v methylcellulose did not affect the mechanical or kinetic properties of the nanomachine [The nanomachine was hosted in a flow chamber which had two separate compartments in which actin and myosin were flowed separately, thereby preventing uncontrolled protein interactions: one compartment was used to introduce the bead-attached actin, the other compartment, in which the support for the myosin motors was mounted, was used to introduce HMM dissolved in buffer A  [w/v) . The fib F-actin , therebyV, µm s−1) in the responses to the reduction of force to F values (pN) below the isometric value (F0) was measured by the slope of the displacement trace (the red trace interpolated with black lines for clarity in F-V data were fitted with the hyperbolic Hill’s equation [F + a) · (V + b) = (V0 + b) · a, where a and b are the distances of the asymptotes from the ordinate and abscissa, respectively, and V0 (the ordinate intercept) estimates the maximum or unloaded shortening velocity. Here, a is a parameter that is used to express the degree of curvature of the relation, has the dimension of a force (pN) and, when normalized for F0, is an index of the relative maximum power that can be delivered at intermediate forces [−1 · pN = 10−18 W = aW. Dedicated programs written in LabVIEW  and Origin 2015 (OriginLab Corporation) were used for the analysis. Experimental points are reported as mean ± SEM unless otherwise stated.The velocity of shortening (equation : (F + a)e forces . The powt (= 10−5 − 10−6 s), depends on the stiffness of the system, which is dominated by the trap compliance. Both the force generated by the motors, transmitted through the BTA, and the force of the optical trap act on the trapped bead. The motion of the bead was simulated with over-damped dynamics by using a drag coefficient calculated from the bead radius and the viscosity of the medium (Stokes’s law). The results were numerically simulated by a stochastic model already published  that est