The study's goal was to develop a spore-based paper strip biosensor for detecting ß-lactam antibiotics in milk using the enzyme induction principle. A new spore-based paper strip biosensor has been developed after important operating parameters such as spore volume, substrate volume, exposure time and temperature, and incubation time and temperature were optimised. The limit of detection for various ß-lactam antibiotics, including amoxicillin, penicillin, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, cephalothin, cefalexin, cefoxitin, cefazolin, and cefuroxime, was determined in milk with detection sensitivity of 1 ppb, 2 ppb, 2 ppb, 10 ppb, 10 ppb, 10 ppb, 20 ppb, 10 ppb 1000 ppb, 10 ppb 300 ppb and 100 ppb, respectively. It was also tested with other contaminants such non-ß-lactam antibiotics, pesticides, aflatoxin, heavy metals, and other chemical contaminants, and no interference was found, indicating that the created biosensor had a low rate of false positive and negative results. In comparison to the AOAC-approved CHARM-ROSA ß-lactam strip test, which identified 7 raw milk and zero pasteurised milk samples positive for ß-lactam antibiotics, the sensor was further analysed and verified using 200 raw milk and 105 pasteurised milk samples. This indicates a perfect match between our biosensor and the AOAC-approved CHARM-ROSA ß-lactam strip test. The developed spore-based paper strip biosensors are expected to be useful in the rapid and cost-effective detection of ß-lactam antibiotic residues in milk samples at the dairy farm, reception dock, and production units, respectively. The notable work on enzyme electrodes by Clark and Lyons in 1962 denoted the beginning of the field of biosensors3. A biosensor is a device that combines a bio-recognition molecule with a transducer and transforms the recognition event into an analytical signal4. They have been used in a wide range of applications, including medical diagnostics, food quality assurance, environmental monitoring, and industrial process control, as well as the detection of biological agents5. Paper based biosensors have been intensively researched for building point-of-care (POC) devices and gaining popularity in diagnostics and pollution monitoring because they are biodegradable, biocompatible, and need less equipment to manufacture6. However, the innovation in biosensors is quite a challenging task in relations of its cost and shelf-stability of bio-recognition molecule.Paper based biosensors have become the backbone of the diagnostic industry. In recent years, paper has been employed as a platform for creating devices that detect certain analytes using functionalized sensors7. Many biological recognition molecules are existing naturally and based on their sensitivity and specificity, they can be used for the recognition of various components. Our research group at ICAR-NDRI used bacterial spores as a sensing molecule to detect antibiotics, pesticides and heavy metals in milk12.In today's world, many biological molecules are engaged in the sensing and recognition processes. These include cells, nucleic acids, enzymes, antibodies, proteins, peptides, etc.13.Till date, the biphasic transformation of spores from dormant to vegetative cell type and vice versa has been employed as the working principle for spore-based sensors. These sensors use a variety of biomarkers, including dipicolinic acid (DPA) and various enzymes produced during spore germination. The presence of a specific analyte (contaminant) can indicate a change in the germination process, which can be detected using any of the aforementioned germination biomarkers. Spores of several bacillus species have been employed as bio-sensing elements in the development of biosensors to detect a variety of biological and chemical pollutants14. Antibiotics given to dairy animals do enter the milk to some extent, and each antibiotic has a withdrawal (waiting) period during which the drug's concentration in the animal tissues decreases and is eliminated from the body. Antibiotics are the most regularly used antimicrobial drugs in the fight against mastitis-causing bacteria. The presence of ß-lactam antibiotics in the food chain is a major source of concern, as they can impede the expansion of starter cultures used in the dairy sector, cause allergic reactions in sensitive individuals, and modify the flora of the intestines15. The World Health Organization has classified the majority of these antibiotics as "critically important"16. Recently, FSSAI has specified the Maximum Residue Limits (MRLs) for these contaminants in milk with test methods involving conventional techniques like LC–MS, HPLC which requires complex sample processing steps, analytical procedure and skilled manpower for operation and therefore, availability of cost effective ready to use tests would be of great help to industry in regulatory compliance of these contaminants in dairy foods17.Antibiotics have long been used in conventional livestock production for treating disease, preventing disease, enhancing feed efficiency, increase growth and productivity in animals and animal products18. Our research group at ICAR-NDRI has developed rapid, novel spore based concepts /assays for detection of antibiotics20 and pesticides in milk11. DPA based kit is working on spore germination inhibition principle and detection of antimicrobial residues is based on development of yellow colour21. This test was further, transformed on paper strip for detection of antimicrobials based on release of marker enzyme after spore germination and development of blue colour on strip was indicative of absence of antimicrobials when incubated for 60 min at 64 °C22. The developed concepts are superior over existing prior-art in terms of sensitivity, selectivity, shelf-stability, cost per test, portable nature and requires minimal setup for testing. The concepts were validated from NABL accredited labs and technologies were commercialised to various entrepreneurs in India. In our lab, a chromogenic substrate based assay for specific detection of ß-lactam antibiotics in milk have also been developed based on substrate competition principle23, however, the test needs to be transformed on paper strip.In recent past, Paper Based Analytical Devices (PADs) have become popular because their fabrication is simple, low cost, portable, response time is fast, passive liquid transport and disposable in natureCurrent research describes a spore-based paper strip biosensor that uses the induction principle to detect the ß-lactam group of antibiotics in milk. The presence of ß-lactam antibiotics in milk poses a number of health hazards, including allergies, cancer, and microbial drug resistance. Antibiotics in milk must be detected using simple, low-cost technologies, particularly in remote locations where medical services are few. The device's design, manufacture, and operation are shown here, as well as the detection of ß-lactam antibiotics in milk samples. Finally, the device will aid in the prevention of pathogenic microorganisms developing antimicrobial resistance (AMR) Figure –S15.24. The stock solution of the chromogenic substrate nitrocefin was prepared in 500 µL organic solvent with the addition of 5 mg nitrocefin . ß-lactam antibiotics (HiMedia) solution was prepared by diluting standard stock solution in milk and stored at − 20 °C. Potassium phosphate buffer  (pH 6.8) was prepared by dissolving di-potassium hydrogen phosphate (K2HPO4) (0.174 g) and potassium di-hydrogen phosphate (KH2PO4) (0.136 g) in 100.0 mL of distilled water and stored at room temperature. Raw milk samples were collected from the dairy farms near by Karnal and pasteurized milk samples were collected from the local market of Karnal, stored under refrigerated conditions, without further treatment before test.The propagation medium was prepared by following the protocol in our recent workB. cereus ATCC 13061, B. licheniformis ATCC 12759, B.cereus ATCC 11778 were procured from American Type Culture Collection (ATCC) and B. cereus ATCC 10876 Virginia, USA.Different bacillus cultures namely Inoculation and reconstitution of spore carried out in Bio-safety Level-II cabinet . Incubator shaker  was used for the incubation of spore. Separation of pellet done in centrifuge . Spore stored in − 20 °C deep freeze . Lyophilization of spores were carried out in lyophilizer . Microbiological plate reader  used for adjustment of spore O.D. Immobilization of substrate on paper strip carried out using the Micro-pipette .B. cereus spores was carried out as per the protocol in our previous work25. Briefly, A single pure colony from streaked plate was transferred into 5.0 mL propagation medium and incubated at 37 °C for 24 h. 100 mL of growth medium was inoculated with overnight grown culture from propagation medium at the rate 1%, followed by incubation at 37 °C for 48 h. After incubation, culture from growth medium was further inoculated in 100 mL of sporulation medium at the rate of 7.5% for spore production. The incubation was carried out at 37 °C for 42 h followed by harvesting of spores by centrifugation at 10,000 rpm for 10 min. at 10 °C. The pellet containing spores was washed twice using potassium phosphate buffer  by centrifugation under similar condition. The spore concentration was standardised by optimising the critical spore concentrations and volume for better colour development on the paper strip in the shortest possible time period. The produced spores were lyophilized in micro-centrifuge tubes at the optimal concentration and volume for later usage.The production of Paper strips were fabricated by anchoring Whatman filter paper grade 3 on a plastic sheet with the help of two sided tape and the strips were cut in 3 cm × 0.5 cm (height x width). After cutting the strips, the working solution of nitrocfin (chromogenic substrate) was dispensed on to the paper and the strips were kept for drying at 37 °C for 30 min. Following drying, the strips were vacuum packed and stored at − 20 °C till further use Fig. .Germinant solution was prepared at the optimized concentration of nutrients and solution was autoclaved (121 °C/15 min/15 lbs pressure). The germinant solution was immobilized on paper discs and discs were dried at 64 °C for 30 min. After drying, discs were collected and filled in a container and stored at 4 °C till further use Fig. .26.Bacillus strains were screened for the induction of ß-Lactamase enzyme, when spores germinates in the presence of ß-lactam antibiotic residues, they will induce the production of ß-lactamase in germinating spores and this enzyme will hydrolyse the chromogenic substrate present on the strip and colour will change from yellow to red, which shoes the presence of ß-lactam antibiotic in the milk. On the other hand, when spores germinates in the absence of ß-lactam antibiotic, the induction of ß-lactamase will not occur and the chromogenic substrate will not be hydrolysed and colour will not change, which shoes the absence of ß-lactam antibiotic in the milk. Nitrocefin was used as a chromogenic substrate. As a cephalosporin, nitrocefin has a ß-lactam ring that can be hydrolysed by ß-lactamases enzyme. The degraded nitrocefin molecule quickly shifts from yellow to red after hydrolysis. Nitrocefin is a chromogenic cephalosporin, yet despite this, it doesn't seem to have any antibacterial characteristicsMilk sample was spiked with amoxicillin antibiotic at 10 ppb concentration to use as positive control and antibiotic free milk was used as negative control. Different bacillus strains were screened for the induction of ß-lactamase enzyme in the presence of ß-lactam antibiotics by using the protocol depicted in the Fig. B. cereus in the form of suspension (10–50 μL), quantity of ß-lactam antibiotic spiked milk sample (50–100 μL), quantity of chromogenic substrate (5–20 μL), exposure and incubation time (10–30 min.) were optimized for the development of spore based paper strip biosensor. The change in colour of the strip from yellow to red was taken as criteria for enzyme induction in presence of ß-lactam antibiotic residues in milk. The spore based paper strip biosensor was developed by optimizing all the essential operating parameters and assembled into a commercial kit prototype for its application in industries and under field conditions. The developed biosensor was screened for its limit of detection (LOD) against 12 different ß-lactam antibiotics such as amoxicillin, penicillin, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, cephalothin, cefalexin, cefoxitin, cefazolin and cefuroxime for evaluating the performance of sensor strain B. cereus by incubating with spiked milk samples at or below FSSAI/EU/Codex MRL27. To validate the results, all of the prepared antibiotic standards were evaluated with a newly developed spore based paper strip biosensor and the CHARM-ROSA test19. The results of validation of the sensor with 1 ppb, 2 ppb, 3 ppb, 4 ppb, and 5 ppb antibiotic standards (amoxicillin) spiked milk samples is depicted in the form of Fig. The spores of 2O2, formalin, Aflatoxin M1, and Pesticide) in working of the developed biosensor has been done. In which different non- ß-lactam antibiotics and other contaminants has been screened at their MRL level with and without combination of ß-lactam antibiotics. Raw milk (200 nos) collected from Karnal, Haryana were also tested with the developed biosensor. Amoxicillin spiked milk at > MRL in accordance with council regulation 37/2010/EU  was used as positive control while antibiotic free milk served as negative control. The result of spore based paper strip biosensor were compared with CHARM-ROSA test (Charm Science Inc. USA) and spore based assay21.Working efficacy of spore based paper strip biosensor was evaluated by interference study of non ß-lactam antibiotics  and other contaminants (Sodium hypochloride (NaClO), Disinfectant, HB. cereus ATCC 13061, B. licheniformis ATCC 12759, B. cereus ATCC 11778 and B. cereus ATCC 10876 were screened for the induction of ß-lactamase enzyme in the presence of ß-lactam antibiotics. Only one strain, B. cereus 10876 showed induction of ß-lactamase enzyme (colour of strip changed only in the presence of ß-lactam antibiotic), while the other three strains produced ß-lactamase enzyme constitutively even in the absence of ß-lactam antibiotic (colour of strip changed in the presence as well as in absence of ß-lactam antibiotic) as shown in Fig. B. cereus 10876 was chosen as the biological recognition molecule for the development of spore based paper strip biosensor for the rapid detection of the ß-lactam group in milk.Four different strains of bacillus species including B. cereus ATCC 10876 is inducible because the colour of the strip was changed only in the presence of ß-lactam antibiotic (test) while in the absence of ß-lactam antibiotic (control) the colour of the strip was remained yellow and the induction of ß-lactamase was also observed by Davies and his coworkers28. While in other strains of bacillus screened, the expression of ß-lactamase is constitutive, because the colour of the strip was changed in the presence as well as in the absence of ß-lactam antibiotic. The activity of ß-lactamase in unheated spores of B. cereus ATCC 10876 was achieved in real-time ranging from 50 to 60 min. However, activity of ß-lactamase in heated spores of B. cereus ATCC 10876 was not observed within 60 min of incubation time. Hence, based on early activity of marker enzyme, unheated spores of B. cereus ATCC 10876 was selected for further study and development of spore based paper strip biosensor for the specific detection of ß-lactam group in milk. Photographs of the results were taken with a smartphone in bright light, which may have contributed to colour variations on paper. Smartphones can include a variety of optical components, which can affect image quality and cause colour intensity to become more inconsistent29.Based on the results obtained during screening of different bacillus spores Fig. , it can In current investigation, optimization was carried out to get the best combination of all the key operating parameters for the development of the biosensor, including spore volume (10–50 µL), substrate volume (5–20 µL), sample volume containing antibiotic (50–100 µL), incubation temperature (30–45 °C), exposure time (0–30 min), and incubation time (10–30 min). All the key operating parameters were optimized by remaining others constant. The developed optimised spore based paper strip biosensor protocol for detecting ß-lactam antibiotics in milk involves two steps: exposure of bacillus spore to ß-lactam antibiotics for the induction of ß-lactamase enzyme and enzyme–substrate reaction. Each step's protocol is explained further down.B. cereus ATCC 10876 (O.D. = 1.0 ± 0.02) were lyophilized in micro-centrifuge tubes using Labconco free zone lyophilizer. Take two tubes containing lyophilized spores and add one nutrient disc in each tube, 80 µl of negative control and positive control was added into the tubes separately. Vortex for 15 s to mix the contents of each tube. After mixing, the tubes were incubated at 37 °C for 30 min to induce expression of marker enzyme in germination spores.20 µL spores of After exposure, the plastic strips functionalized with 10 µL of chromogenic substrate were added into each tube, which was then incubated for 30 min at 37 °C. The strips in each tube were inspected for red colour development after incubation for qualitative examination using the naked eye.The presence of ß-lactam antibiotics in milk was shown by the formation of red colour on the strip, whilst the absence of ß-lactam antibiotics was indicated by the yellow colour on the strip when examined with the naked eye.Limit of detection (LOD) is the lowest concentration of ß-lactam antibiotics that can induce the expression of the targeted enzyme in an enzyme induction based test. The optimized spore based paper strip biosensor was examined for LODs of 12 different ß-lactam antibiotics, The limit of detection (LOD) for various ß-lactam antibiotics, including amoxicillin, penicillin, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, cephalothin, cefalexin, cefoxitin, cefazolin, and cefuroxime, was determined after spiking these antibiotics in milk with detection sensitivity of 1 ppb, 2 ppb, 2 ppb, 10 ppb, 10 ppb, 10 ppb, 20 ppb, 10 ppb 1000 ppb, 10 ppb 300 ppb and 100 ppb, respectively. The minimum concentration of ß-lactam antibiotics that induced ß-lactamase enzyme induction was used to determine the LOD of that antibiotic. Milk samples were spiked with each antibiotic at concentrations below and above their MRL, then incubated at 37 °C/30 min with spores and nutrients to induce ß-lactamase production in germinating spores, followed by the addition of a chromogenic substrate functionalized strip and incubation at 37 °C/30 min as shown in Fig. Figure The selectivity of developed biosensor was confirmed by cross reactivity with other toxic and non-toxic contaminants like non ß-lactam antibiotics, pesticides, aflatoxin M1, detergents and disinfectants and other contaminants most commonly found in dairy food chain. These contaminants can potentially inhibit the spore germination or enzyme activity and can hinder the overall performance of the developed biosensor. Keeping this in view, the working performance of the developed biosensor was evaluated in presence of various contaminants using optimized protocol. The significant findings are shown in Figs. 30, who reported no effect of the other antibiotics on the sensors performance in detecting ß-lactam antibiotics in milk. B. cereus is resistant to many non- ß-lactam antibiotic31. The test was found working at FSSAI/CODEX/EU regulatory limits as well as lower regulatory limits with no false positive or negative results.Amoxicillin, kanamycin, trimethoprim, chloramphenicol, colistin, rifampin, amikacin, tetracycline, ciprofloxacin, vancomycin, gentamycin, and clindamycin were evaluated for cross-reactivity at their MRL. The results Fig.  showed t24. Cross-reactivity of pesticides such as chlorpyrifos, prefenofos, paraoxon methyl, dieldrin and endosulfan was tested at their MRL values in terms of inhibition of enzyme activity. The results  and the Codex Alimentarius Commission (CAC)36. Given the ubiquity of aflatoxin M1 in milk, the spore based paper strip biosensors performance was assessed in the presence of aflatoxin M1 in milk, both with and without ß-lactam antibiotics. Aflatoxin M1 had no effect on the germination and induction of B. cereus spores. Abdel-Shafi et al.37, confirmed aflatoxin M1 degradation by B.cereus37. Several research have been conducted to determine the presence of aflatoxin M1 in milk, and it has been determined that aflatoxin M1 is widely present in milk41. As a result, the spore based paper strip biosensor for detecting ß-lactam antibiotics in milk can be used in such circumstances without causing aflatoxin-M1 to interfere.Aflatoxins are a family of structurally related poisonous chemicals generated by 30, who found no effect of the aforementioned contaminants on the biosensor's performance.Other pollutants prevalent in milk, such as sodium hypochlorite, quaternary ammonium compound, hydrogen peroxide, and heavy metals, were evaluated with and without ß-lactam antibiotics to see if they had an inhibiting effect on the biosensor's performance. When sodium hypochlorite, quaternary ammonium compound, hydrogen peroxide, and heavy metals were used at their regulatory limits, there was no inhibitory effect on induction and enzymatic activity, as shown in Fig. 30, who found that the above-mentioned pollutants had no impact on the biosensor's performance in detecting ß-lactam antibiotics in milk.As a result of the findings on cross reactivity of other inhibitors on marker enzyme activity, such as non-ß-lactam antibiotics, pesticides, aflatoxin M1, sodium hypochlorite, quaternary ammonium compound, hydrogen peroxide, and heavy metals, further, it can be concluded that the induction and activity of marker enzyme is not inhibited in the presence of the aforementioned contaminants and is specific for the detection of ß-lactam antibiotic in milk. The findings are congruent with those of Kumar et al.42. In this study, 3.5% of samples were found to be contaminated with ß-lactam antibiotics, whereas data from Gaare et al.27 and Lejaniya et al.42 showed a 12% ß-lactam antibiotic contamination in raw milk.A total of 200 raw milk samples were collected from the un-organized cattle yards and evaluated for the presence of ß-lactam antibiotics using the standardized Spore-based Paper strip biosensor. The findings as showed in the Fig. 27 showed a 4% ß-lactam antibiotic contamination in pasteurized milk.A total of 105 pasteurized milk samples from various brands were purchased from local market of Karnal and tested for the presence of ß-lactam antibiotics using the standardized spore-based paper strip biosensor. The result findings revealed that none of the pasteurized milk samples tested positive for ß-lactam antibiotics Table . In comp43. In this part, validation of spore-based paper strip biosensor will be presented. The ß-lactam antibiotics spiked milk standards and developed spore-based paper strip biosensor was validated with CHARM-ROSA test for detection of ß-lactam antibiotics in milk . As a result, paper strips stored at − 20 °C are stable up to 9 months Fig. , whereasA spore-based paper strip biosensor that can detect the ß-lactam group of antibiotics in milk has been developed using an enzyme induction concept. The designed biosensor can detect twelve distinct ß-lactam antibiotics, including penicillin and cephalosporins. Antibiotics were discovered in milk, confirming the technology's potential for real-time analysis. This is also the first time that spore-based enzyme induction has been demonstrated to be a reliable detection method for the ß-lactam group in milk, contributing in the fight against microbial drug resistance. Because it can detect the ß-lactam group of antibiotics, in milk within one hour, the new spore-based paper strip biosensor has the potential to revolutionise the point-of-care diagnostics sector for animal healthcare and antimicrobial resistance prevention in humans. For the ß-lactam group of antibiotics suggested by FSSAI/CODEX, the sensitivity of the developed biosensor is below MRL. The working of the developed test has been found to be free of non-ß-lactam antibiotics and other pollutants. The spore-based paper strip biosensor is inexpensive and very sensitive to antibiotics in the ß-lactam class. By visual inspection, the current technique detects the ß-lactam group of antibiotics semi-quantitatively up to LOD level, which is highly useful for field application. The FSSAI has also recommended the harmonisation of CODEX criteria for antibiotic residues in milk, which will need testing of milk at many phases of production, processing, distribution, and storage. The spore-based paper strip biosensor created has commercial promise in the dairy industry because it is low-cost, sensitive, selective, repeatable, and real-time. It is a low-cost alternative to currently used conventional methods. The created technology is truly innovative on a worldwide scale, as it is unique and superior than prior art in terms of cost per test and infrastructure required for testing.Supplementary Information.


The bridging water molecules between Ca2+ and binding residue reduces the ruggedness of ligand exchange landscape by acting as a lubricant, facilitating the Ca2+ coupled protein allostery. Calcium-induced rotation of the helices in the EF-hands, with the hydrophobic core serving as the pivot, leads to exposure of hydrophobic sites for target binding. Intriguingly, despite being structurally similar, the response of the two symmetrically arranged EF-hands upon Ca2+ binding is asymmetric. Breakage of symmetry is needed for efficient allosteric communication between the EF-hands. The key roles that water molecules play in driving allosteric transitions are likely to be general in other metal ion mediated protein allostery.Allosteric communication between distant parts of proteins controls many cellular functions, in which metal ions are widely utilized as effectors to trigger the allosteric cascade. Due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding is intrinsically rugged. How metal ions achieve fast binding by overcoming the landscape ruggedness and thereby efficiently mediate protein allostery is elusive. By performing molecular dynamics simulations for the Ca 2+ sensor protein calmodulin as the model system, we revealed crucial roles of water-bridged interactions in the metal ion coupled protein allostery. The coordination of the Ca2+ to the binding site involves an intermediate in which the water molecule bridges the Ca2+ and the liganding residue. The bridging water reduces the free energy barrier height of ligand exchange, therefore facilitating the ligand exchange and allosteric coupling by acting as a lubricant. We also showed that the response of the two symmetrically arranged EF-hand motifs of CaM domains upon Ca2+ binding is asymmetric, which is directly attributed to the differing dehydration process of the Ca2+ ions and is needed for efficient allosteric communication.Natural proteins often utilize allostery in executing a variety of functions. Metal ions are typical cofactors to trigger the allosteric cascade. In this work, using the Ca CaM is composed of two nearly symmetric globular domains connected by a flexible central helix. Each domain consists of two helix-loop-helix motifs, termed EF-hands  with the negatively charged residues in the loop [2+, the helices undergo substantial rearrangement into a nearly perpendicular conformation, exposing the hydrophobic sites which enable recognition and subsequent activation of target proteins [Calmodulin (CaM), a versatile calcium 2+) sensiproteins , 12, 13.2+-triggered conformational changes in CaM is an example of allostery, which describes the structural changes that occur in enzymes and molecular machines at distances far from the site at which a ligand binds [2+ and Zn2+, have been widely utilized as effectors to trigger the allosteric cascade. However, due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding and unbinding is intrinsically rugged. Despite the biological importance of metal ion coupled protein allostery, two key questions remain elusive, including: i) What strategy do metal ions utilize to overcome the ruggedness of the binding landscape and achieve rapid binding/unbinding? and ii) What physical interactions enable the propagations of the metal ion triggered allosteric signal to regulate the downstream processes? Because of the availability of rich experimental data and the typical allosteric features, CaM is an ideal system for answering these questions computationally [In a more general context, Cand binds –16. Natund binds –27. Bindnd binds . Divalentionally , 32–47.2+ binding coupled conformational changes in CaM domains by explicitly modeling Ca2+ in the simulations. We extracted the free energy landscape of the Ca2+ binding coupled conformational changes of CaM domains at atomic level and demonstrated the full picture of the allosteric motions of CaM [2+ binding and protein allostery. The bridging water molecules between Ca2+ and binding residues reduce the free energy barrier of ligand exchange landscape, and therefore the landscape ruggedness, by acting as a lubricant. This enables efficient Ca2+-ligand coordination and allosteric motions in the CaM. We propose that the water-bridged coordination is a general mechanism utilized in metal-coupled folding and allosteric communication in proteins. We also showed that Ca2+ binding leads to rotation of the EF-hand helices with the hydrophobic core as the pivot, a structural change that should precede recognition by target proteins for signal transduction. In addition, the molecular simulations revealed obvious asymmetry in the allosteric coupling of the symmetrically paired EF-hands: the EF-hand 1 (EF1) binds Ca2+ in a sequential manner by chelating to the N-terminal residues followed by coordination with the central residues, and finally to the C-terminal residues. In contrast, chelation of Ca2+ to the EF-hand 2 (EF2) is initiated either by interactions with the residues in the N- and/or C-terminal residues followed by coordination to the central residues. Similar results were obtained for the cCaM. Such a symmetry breaking process is likely involved in larger multi-domain complexes, such as the bacterial chaperonin, GroEL, in which ligand binding induces asymmetric response in different subunits.Here, we used the bias-exchange metadynamics method  to invess of CaM , 49–51. 2+ binding and the conformational change. Quantitative analysis of the changes in these coordinates allows us to infer the mechanism of coupling between Ca2+ binding, the role of water, and the allosteric conformational changes in calmodulin. In particular, we use the “path collective variables” Sα , defined in the Methods and Materials section, to describe the conformational changes of the two EF-hand motifs. Small and large values of Sα correspond to open and closed states, respectively. In order to assess the consequences of Ca2+ binding, we use the native coordination numbers Nα (2+ in the native holo structure) that bind to Ca2+ during the allosteric transitions for the two EF-hand motifs, respectively. F and F, of the nCaM. For both EF1 and EF2, the conformational change of nCaM is tightly coupled to the extent of Ca2+ binding as indicated by the conformational distributions at different N1 and N2 values. When Sα (α = 1 or 2) is large and Nα (α = 1 or 2) is small, the closed state is more stable. Whereas when the native ligands are fully coordinated to Ca2+ the open structure is more stable (N1 = 6) to Ca2+, the EF1 samples a wide range of conformations as assessed by S1 fluctuations, suggesting the conformational plasticity of the open state. Conformational plasticity for the Ca2+ bound state, which was also observed in experiments, could be the major reason that the EF-hand motifs recognize and bind a variety of target proteins [We use well-tempered bias-exchange metadynamics , 52 and mbers Nα , represee stable . Interesproteins , 55.2+ binding. For example, when three or four ligands bind to Ca2+, the conformation of EF2 is poised to make a transition to the open-like structure, whereas EF1 remains in the closed-like structure. It is worth noting that the two EF-hand motifs were simulated as a whole in this work, and the conformational changes of the two EF-hands are not independent of each other. In the calculations of the free energy landscape of EF2, the EF1 is allowed to change its conformation. Therefore, the cooperativity between the two EF-hand motifs was included in the simulations. The free energy landscapes of the two EF-hand motifs shown in 2 is stabilized when EF1 is in the open conformation with most of the associated native ligands bound to Ca2+. Thus, the behavior of EF2 is affected by the conformation of EF1 through long range allosteric interactions or action at a distance between the two EF-hands, which will be discussed further in later subsection. The observed differences in the allosteric communication are reasonable since the two EF-hand motifs have different amino-acid sequences in the Ca2+ binding loop. Consequently, the ensembles of structures with three or four native ligands bound to the Ca2+ should exhibit substantial differences in the two EF-motifs. Such an asymmetry in the Ca2+ binding and conformational transitions of the symmetrically arranged EF-hand pair in CaM domains likely plays an important role in signaling.Although the two EF-hand motifs have similar structures, comparison of 2+ or Mg2+) due to the lack of explicit consideration of charge transfer, polarization, and protonation/deprotonation effects [ab initio quantum calculations for the Ca2+ coordination in calmodulin depend on the loop conformations [2+-formate affinity using the GROMOS96 and OPLS-AA force fields [2+ coupled conformational change of calmodulin domains. According to the discussions in Ref. [2+ mediated interactions and the barrier separating them in the free energy landscapes of this work may depend on the details of the force field parameters. However, as will be discussed in the end of this section, the qualitative features of the free energy landscapes are insensitive to the details of the used force fields.It is worth mentioning that conventional classic force fields often encounter difficulties in quantitatively reproducing the thermodynamic properties of the coordination bonds involving metal ions  play during the closed → open transition. NT (sum of the number of water molecules and the number of ligands bound to Ca2+), as a function of Nα  for the nCaM. At a low value of N1 , Ca2+ loses approximately two water molecules , only one water molecule is bound to the Ca2+, which makes the coordination number saturate at NT = 7. ; subsequently, the N-terminal part of the EF-loop plays a role in further coordination to Ca2+, establishing links between the incoming helix and the central β strands; finally, Thr26 and Glu31 are sequentially captured and bound to the Ca2+, leading to the native structure.Another possible reason for such a high free energy barrier is the dehydration of Caolecules , 65. Theand Ca2+ . In thisM mutant . When th2+, the most stable conformation of EF1 switches to the open form, suggesting the crucial role of the highly conserved Glutamic acid at the 12th position in the Ca2+ binding coupled conformational transition of nCaM [2+ coupled conformational motions of the cCaM [Interestingly, the results in Figs  of nCaM , 66. A rthe cCaM .2+ binding to EF2 is dramatically different , which is followed by the binding of the C-terminal residues Glu67 . Only at the final stage, it is bound to the central residues Asn60 and Thr62. Therefore, the Ca2+ binding to the EF2 follows the sequence N-terminal and/or C-terminal residues → central residues. The differing mechanism of coordination to Ca2+ probably arises from the divergence of their amino-acid sequences. For example, the “central” residues  in EF1 are more negatively charged compared to those in EF2. Consequently, coordination of these residues to Ca2+ occurs at a relatively early stage.The molecular process of Caifferent . Instead2+ binds to the central residues Asn60 and Thr62, a non-native ligand Gly59 is coordinated with Ca2+  in the nCaM (cCaM) is charged, whereas those in the EF2 (EF3) in the nCaM (cCaM) are neutral. The stepwise dehydration of the Ca2+ in the cCaM was also observed . In particular, coordination of the fourth (third) ligand to Ca2+, which corresponds to the binding of the terminal residues of the EF-hand loops to the Ca2+ by water bridged interactions (direct interactions) in EF1 (EF2), has the most prominent effect in changing the distance between the two close ends. Meanwhile, the dihedral angles formed by the two helices increase (red lines), indicating the rotation of the EF-hand helices. In the process of rotation, the hydrophobic cluster, which is stable during the Ca2+ binding, acts as the pivot , it switches to the open conformation. Due to the tight coupling between the two EF-hand motifs, Ca2+ coordination induced stabilization of the open conformation of the EF1 tends to promote the closed to open switching of the EF2 conformation, even though the coordination of the EF2 is not fully completed. Consequently, we observe that the major conformational switching occurs at N1 = 5 for the EF1, but it occurs at N2 = 3 for the EF2 as shown in the free energy profiles  the distance from oxygen of water molecule (Ow) to Ca2+ is less than 2.8Å; and (ii) the hydrogen bond is formed between water and the native ligand. For each residue listed in 2+-water-native ligand conformation exists at certain stages of the binding process with high probability. These results suggest that before the final chelation process, the native ligands already develop indirect interactions with Ca2+ mediated by water molecules. For example, after binding of Ca2+ to five of the native ligands of EF2, the last native ligand, Asn60 or Thr62, is not “free”, but has a probability of 0.65 to be bridged to the Ca2+ by a water molecule  of the residues in the holo structure that are coordinated to Ca2+. In terms of these native ligands, we define the collective variables, Nα (α = 1 and 2 (3 and 4) for EF1 and EF2 (EF3 and EF4), respectively, of the nCaM and cCaM) to describe the number of native ligands that are coordinated to Ca2+:The events driving the closed to open transition involve movement of a number of particles from distant parts of CaM domains. In order to capture such an allosteric movement, we need suitable reaction coordinates. Since the major events in the closed → open transition are triggered by Caith native ligand to the αth Ca2+. By native ligand we mean the oxygen atoms from distinct amino acids that are linked to Ca2+ and are present in the native (NMR) holo structure. We chose d0 = 2.5Å and r0 = 0.02Å. For all the Asp residues, the two oxygens from the same residue were considered “degenerate”, namely, when more than one oxygen is coordinated to Ca2+, we consider only one of them to be a native ligand. However, for the Glu residues in the 12th position of the EF-loops of nCaM and cCaM, two oxygens are treated as individual ligands. We treated coordination of Ca2+ to Asp differently from binding to Glu because in the holo structure of the nCaM and cCaM  Ca2+ is coordinated to both oxygen atoms of Glu but only to one of the Asp oxygen atoms.In α = 1, 2, 3, and 4 dictates the αth EF-hand, and i = 1) and open (i = 2) structure for a given conformation X(t). A small value of Sα corresponds to closed structure and a large value of Sα corresponds to open structure. We used λ = 20 in our simulations.We also used the “path collective variables” calculated using,N1, N2, S1, and S2 for the nCaM simulations, and N3, N4, S3, and S4 for the cCaM simulations. Exchange between replicas was attempted every 10,000 MD steps, and each replica lasts for 800ns. In the well-tempered algorithm, we used a bias factor of 200 for the rescaling of the Gaussian height, and 0.3kJ/mol as the initial Gaussian height. More detailed descriptions of the simulation procedure can be found in To use the bias-exchange method with metadynamics, we designed 5 replicas. In the first replica, there is no bias in the CVs. In each of the other four replicas, a bias was applied to one of CVs, including 2+ for the nCaM. The minimum distance between the Ca2+ and two oxygen atoms of Glu31 is chosen as the collective variable D [ri  is the distance between each oxygen and the Ca2+, and σ = 5000Å. The well-tempered rescaling factor is 12, and the initial Gaussian height is 0.2kJ/mol.We also used well-tempered metadynamics to study water-bridged coordination of Glu31 to the Cariable D , which w2+ and simultaneously within 3.5Å distance of any of the two oxygen atoms of Glu31, the electrostatic interactions between the water and the side-chain heavy atoms of Glu31 are turned off. This was accomplished by modifying the source code of GROMACS package.In the control simulation, once a water molecule comes into the first ligand shell of CaD = 6.5Å to limit the region of the phase space accessible during the simulation:K = 50kJ/mol/Å2 and Dwall = 6.5Å. We performed 20 independent 100ns simulations for each system, and then calculated the ensemble average to obtain the free energy reported in 2+.To achieve better convergence we use a restraining potential at The numerical data used in all figures are included in S1 Text(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


In contrast, it has been elusive how astrocytes respond to DA via Ca2+ signaling and regulate synaptic transmission and reward systems. Previous studies suggest various molecular targets such as MAO-B, D1R, or D1R–D2R heteromer to modulate astrocytic Ca2+ signaling. However, which molecular target is utilized under what physiological condition remains unclear. Here, we show that DA-induced astrocytic Ca2+ signaling pathway switches during development: MAO-B is the major player at a young age (5–6 weeks), whereas DA receptors (DARs) are responsible for the adult period (8–12 weeks). DA-mediated Ca2+ response in the adult period was decreased by either D1R or D2R blockers, which are primarily known for cyclic AMP signaling , suggesting that this Ca2+ response might be mediated through Gq pathway by D1R–D2R heterodimer. Moreover, DAR-mediated Ca2+ response was not blocked by TTX, implying that this response is not a secondary response caused by neuronal activation. Our study proposes an age-specific molecular target of DA-induced astrocytic Ca2+ signaling: MAO-B in young mice and DAR in adult mice.Dopamine (DA) plays a vital role in brain physiology and pathology such as learning and memory, motor control, neurological diseases, and psychiatric diseases. In neurons, it has been well established that DA increases or decreases intracellular cyclic AMP (cAMP) through D Dopamine (DA) is a neurotransmitter that functions in the brain to regulate neural processes, including the reward system, motor control, cognition, and memory . The mai2+ signaling is critically involved in various gliotransmitter releases such as glutamate [d-serine [2+ influx in astrocytes, a Gq-coupled G-protein coupled receptor (GPCR) is the predominant form of Ca2+ elevations in astrocytes via IP3R2 in the endoplasmic reticulum (ER) [2+  via Gq-GPCR pathways [2+ signaling by oligomerization of Gi-coupled GABAB receptors [Astrocytes are the most abundant cell types in the brain and play a vital role in both physiological –8 and palutamate , GABA [1lutamate , ATP [16d-serine , which alum (ER) . There apathways . On the eceptors .2+ in response to DA in vitro [2+ signaling are (1) monoamine oxidase B (MAO-B) pathway [3R2-mediated Ca2+ release from the endoplasmic reticulum (ER). On the other hand, we have previously demonstrated that MAO-B in adult striatum is responsible for GABA synthesis, but not DA degradation [2+ signaling in astrocytes is not well characterized but expected to share the IP3R2-mediated Ca2+ release from ER. Previous reports suggest that in nucleus accumbens, astrocytic D1R [2R [2+ response. Another possibility is D1R-D2R heteromerization observed in artificial in vitro systems [In contrast to neurons, a recent study reported that DA does not modulate cAMP level of astrocytes . Insteadin vitro , ex vivoin vitro , 23, andin vitro  in vario pathway  and (2)  pathway , 21, 23.radation . Meanwhiytic D1R  or neuro D1R [2R  can indu systems , 27. How2+ signaling in mouse mPFC astrocytes. Using genetically encoded calcium indicator (GECI), GCaMP6f, we have found that DA-induced astrocytic Ca2+ signaling switches from the MAO-B pathway (5–6 weeks) to the DAR pathway (8–12 weeks). Our findings show the age-dependent switching of DA-induced astrocytic Ca2+ signaling during development and suggest an age-specific target to modulate astrocytic Ca2+ signaling.In this study, we investigate the age-dependent change of DA-induced CaYoung and adult C57BL/6J mice were provided by IBS Research Solution Center. MAO-B KO mice  were maintained on a heterozygote. MAO-B WT and MAO-B KO mice were obtained from mating cages of heterozygous males and females. The animals were kept on a 12 h light–dark cycle with controlled temperature and humidity and had free access to food and water. Mice were kept in group-housed (4 to 5 mice together). Experimenters and animal managers took care of mice following National Institutes of Health (NIH) guidelines. Every animal experimental procedure was approved by the Institutional Animal Care and Use Committee of the Institute for Basic Science .12) was packaged by the IBS virus facility . Before injection, the virus was diluted by 3 times with saline.The AAV containing GfaABC1D-GCaMP6f , haloperidol , SCH-23390 , and TTX  were bath applied for 15 min. The concentration of each chemical is indicated in each experiment.For ex vivo astrocyte CaMice were deeply anesthetized with isoflurane (3% for induction and 1.5% during surgery) and placed on a stereotaxic device . After an incision of the scalp, the skull was cleaned and matched the dorsoventral level of bregma and lambda for accurate targeting of a brain region. The holes were drilled bilaterally in the skull targeting the mPFC using the following coordinates: AP, 1.8 mm; ML, ± 0.4 mm; DV, − 2.3 (young) or − 2.5 (adult) mm from the bregma. A glass capillary  was pulled by a vertical puller  and was connected to a microinfusion pump  through polyethylene tubings . The AAV-GfaABC1D-GCaMP6f virus was loaded into the glass capillary and slowly infused into mPFC at a rate of 0.1 μL/min with 0.5 μL of total volume in each hemisphere. The injection needle was left in place for an additional 10 min before the withdrawal. For young mice, the virus was injected at postnatal day 25. After at least 2 weeks of recovery, the mice were sacrificed and used for imaging experiments.3, 10 d-glucose, 5 MgCl2, 3 KCl, 1.25 NaH2PO4 and 0.1 CaCl2; pH 7.4, saturated with 95% O2 and 5% CO2. To obtain mPFC brain slices, the brain was attached to the chamber of a vibratome  and 300-μm-thick coronal slices were collected. Slices were then transferred to an incubation chamber filled with artificial cerebrospinal fluid (aCSF) (in mM): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1.5 CaCl2, 1.5 MgCl2 and 10 glucose; pH 7.4, saturated with 95% O2 and 5% CO2, at room temperature. All imaging experiments were conducted after 1 h of stabilization.Mice were deeply anesthetized with 3% isoflurane and decapitated. The brain was quickly removed from the skull and submerged in cold sucrose-based dissection buffer (in mM): 212.5 sucrose, 26 NaHCO2+ imaging, a blue light  was applied to brain slices under a fluorescent upright microscope  with a 63 × water objective (Zeiss). Drugs were treated by bath application which is controlled by a valve controller . To measure Ca2+ response, the peak of GCaMP6f signals after drug treatment was normalized by its baseline (ΔF/F0 ratio). Analyzed ROIs were chosen based on the response from the 1st (or before drug) DA treatment. Image acquisition and ROI analysis were performed using Imaging Workbench (INDEC Biosystems) and ImageJ (NIH).After stabilization, obtained brain slices were moved to a recording chamber where aCSF continuously flowed over slices at around 1 mL/min. To perform CaAll data are given as mean ± SEM. Statistical comparisons were performed using an unpaired Student’s t-test or a one-way ANOVA test using Prism 9 (GraphPad). Statistical significance is indicated as follows: ***p < 0.001; ****p < 0.0001; ns, not significant.2+ in response to DA [2+ imaging  and adult mice (66.89 ± 2.852), which are consistent with previous findings , whereas KDS2010 significantly increased in adult mPFC astrocytes  and D2R antagonist  simultaneously. In contrast to the result from MAO-B inhibitor condition, DAR antagonists significantly reduced DA-induced Ca2+ response in an adult (27.92 ± 2.591) but not in young mPFC astrocytes  and D2R . DA-induced Ca2+ response in adult mPFC astrocytes was almost completely blocked by both SCH-23390 (10.20 ± 2.394) and haloperidol (9.185 ± 2.412) compared to control  mice. DA-induced astrocytic Ca2+ responses were not significantly different between MAO-B WT (79.07 ± 10.52) and MAO-B KO (80.94 ± 8.578), suggesting that MAO-B is not a mediator of DA-induced Ca2+ signaling in adult astrocytes  period, whereas DAR-mediated Ca2+ signaling is the major form during the adult (8–12 weeks) period. It is intriguing that DA response is age-dependently switched from MAO-B to DAR pathway within 2 weeks of the juvenile period.In this study, we have investigated the astrocytic Ca2O2) is produced and it might evoke ER Ca2+ release [2+ release might be crucial for the expression of synapse-regulating genes and synaptic development [2+ signaling in adult mPFC astrocytes possibly induces a release of various gliotransmitter such as glutamate, D-serine, GABA, and ATP. It would be interesting to investigate which gliotransmitter is released in future studies.One possible explanation for this switching phenomenon might be due to a change of astrocytic role in response to DA. There is a previous report that astrocyte is critical for DA homeostasis in the developing prefrontal cortex . Based o release . This Caelopment . On the 2+ response is majorly through MAO-B in mPFC of young mice. However, in adult mice, MAO-B-dependent Ca2+ elevation in response to DA disappeared. This is an interesting phenomenon because MAO-B expression level is known to increase along with aging in most structures of rodent brains [2+ response and MAO-B in adulthood. Then what could be the role of MAO-B in the adult mPFC? As we have recently demonstrated, MAO-B is responsible for synthesizing GABA in adult striatal astrocytes, rather than DA degradation [In this study, we found that DA-induced astrocytic Cat brains . The pret brains . Howevert brains . Based oradation . Based o


Rabbit meat is popular with consumers mainly for its high-quality protein. Among the main factors influencing the characteristics of rabbit meat proteins to appertain the breed, genotype, carcass part and age. Conventional production of rabbit meat in many European countries is mainly ensured by intensive production systems, when commercial meat-type albinotic rabbit crossbreds are used. However, spotted and solidly coloured lines of meat rabbit breeds have begun to be used in rabbit breeding schemes as some consumers have begun to negatively perceive meat from albinotic coloured rabbits. The aim of the present study was to assess the effects of the sire genotype, sex and carcass part on the composition of meats of rabbits fattened under conditions where no synthetic drugs were used. Crossbreeding of Mecklenburger Schecke sires with a commercial dam line of HYLA rabbits resulted in a worse nutritional quality of meat proteins in progeny. These findings point to a possible risk of alterations in the nutritional quality of meat proteins when using different rabbit sire genotypes than those originally intended for the specific commercial crossbreeding scheme.Longissimus thoracis et lumborum (LTL) muscle as compared to hind leg meat (p ˂ 0.001). Significant effects of the rabbit sire genotype and the genotype x sex interaction on proportions of some AA in meat were found (p ˂ 0.001). Crossbreeding of the Mecklenburger Schecke (MS) sires with a commercial dam line of HYLA rabbits resulted in a lower proportion of the total AA and all EAA monitored in meats of MS sired males as compared to MS sired females and HYLA rabbits (p ˂ 0.05). The sex-related effect on AA profile was not so noticeable in final commercial crossbreds of HYLA rabbits when compared to MS sired progeny. These findings point to a possible risk of alterations in the nutritional quality of meat proteins when using different rabbit sire genotypes than those originally intended for the specific commercial crossbreeding scheme. However, on the contrary, higher contents of magnesium (p ˂ 0.05), manganese (p ˂ 0.001) and zinc (p ˂ 0.05) were found in meats of MS sired progeny as compared to HYLA rabbits.The aim of the study was to assess the effects of the sire genotype, sex and carcass part on the composition of the meat of rabbits, which were fattened under conditions where no synthetic drugs were used. As for carcass parts, the higher content of both total amino acids (AA) and all essential AA (EAA) monitored was found in the  RabbiB6, B12) . It is lRabbit meat is often popular with consumers mainly for its high-quality protein, which shows a higher digestibility value compared to other meats such as beef or pork ,8. The qConventional production of rabbit meat in many European countries is mainly ensured by intensive fattening of commercial meat-type rabbit crossbreds . In addikk) and spotted- (genotype Kk) coloured rabbits excel in important production traits. When they are crossed with white-coloured rabbits, they pass a spotted or solidly dark colour of coat on their progeny [We found in our previous study that the use of the Mecklenburger Schecke (MS) males as terminal sires in a rabbit crossbreeding scheme led to favourable growth performance and some of the carcass characteristics in their progeny. The MS breed is medium-sized, and these rabbits show a well-muscled cylindrical body, with wide fore- and hindquarters. The breed displays three typical colour genotypes, which also differ in growth performance and meatiness. In this regard, solidly-  × CD dams. The experimental group  consisted of MS sired progeny, when the semen of MS males was inseminated to females of the same HYLA CD line as in the H group. The used MS sires (n = 6 sires) originated from small-scale hobby breeding stocks; all of them were kk or Kk genotypes. Females of the HYLA CD line were inseminated with the heterospermic insemination dose, which contained spermatozoa of all involved MS sires. The study was performed in the commercial Centre of HYLA rabbits . On the farm, which focuses mainly on the production of breeding HYLA crossbred rabbits used in other commercial farms, neither synthetic allopathic veterinary drugs nor synthetic anticoccidial drugs are used. A total of 112 crossbred rabbits  were used in the experiment. The control group  consisted of albino crossbred rabbits of the HYLA combination AB sires (2 per rabbit. The cage size was 90 cm × 40 cm × 35 cm (length × width × height). The lighting period was 12 h light/12 h darkness, temperature ranged from 17 °C to 20 °C and relative humidity was 55% to 60%. Rabbits were fed ad libitum by commercial compound pelleted feeds  and the grower (K-Optimum) and finisher (K-Finisher) diets were used from the 35th to 64th day of age and from the 65th day of age to slaughter, respectively. The nutrient composition of the diets used is shown in The experimental design of the study was conducted according to , which rLTL muscles and hind legs were dissected. Then, both hind legs were deboned. Finally, samples of the LTL and hind leg meat were packed and stored at −20 °C until analysed.At the end of fattening (108 days of age), 24 rabbits per genotype , were randomly selected (one rabbit per cage), weighed and slaughtered after a previous 12 h fasting in the abattoir. The rabbits were mechanically stunned with a captive bolt gun and bled, then the skin, distal parts of the tail, gastrointestinal and urogenital tracts and the distal part of the legs were removed according to the methodology described by Blasco and Ouhayoun  for rabbAll samples were analysed in triplicate. The dry matter (DM) of the samples was determined by weight upon drying the sample at 105 °C under the prescribed conditions. The Kjeldahl method using a Buchi analyser  was performed to determine the crude protein (CP) content. A Soxhlet method was used to determine the ether extract by a Soxtec apparatus . The ash was determined by weighing the sample after incineration at 550 °C. Water/protein (W/P) ratio was calculated from the formula: (1000–dry matter content)/CP content.AA contents were determined following acid hydrolysis in 6 N HCl at 110 °C for 24 h using the Automatic Amino acid Analyzer AAA 400 , based on the colour-forming reaction of AA with the oxidative agent ninhydrin according to procedures used by Straková et al. . The AA p ˂ 0.05. All statistical procedures were performed by the STATISTICA CZ version 10 software.The arithmetic mean and standard error of the mean (SEM) were determined for all assessed traits in respective evaluated groups. A Shapiro-Wilk test was used to test the normality of data distribution within the assessed groups. The normality was found in all the variables. Statistical evaluation of data followed basic procedures . DiffereLTL was related to the higher CP content and lower W/P ratio (p ˂ 0.001) as compared to hind leg meat (LTL muscles (p ˂ 0.001). The higher dry matter content of rabbit leg meat . The hinp ˂ 0.001; p ˂ 0.001), hind leg meat and LTL muscles (p ˂ 0.05). On the contrary, H rabbits displayed a higher hind legs yield as compared to the MS sired rabbits (p ˂ 0.001). The sex of rabbits affected the carcass dressing and a higher level was found in males compared to females (p ˂ 0.05). Concerning basic indicators of the proximate chemical composition of meat in both assessed carcass parts, the rabbit genotype influenced only the ash content in the hind leg meat; the higher value of ash content was observed in H rabbits (p ˂ 0.01). The sex of rabbits affected only the content of ether extract in LTL, the lower value of ether extract was found in females compared to males (p ˂ 0.05). In addition, a significant effect of genotype x sex interaction on the content of CP and ash (p ˂ 0.001) and on the W/P ratio (p ˂ 0.01) in hind leg meat was found.When compared to the control , a higher slaughter weight of MS sired progeny at 108 days of age (p ˂ 0.001) monitored was found in the LTL muscle as compared to hind leg meat. In particular, a higher proportion of Leu, Iso, Thr, Arg, His, Val, Met, Ser, Asp and Tyr was found in the CP of LTL compared to the CP of hind leg meat (p ˂ 0.001). By contrast, higher proportions of Gly (p ˂ 0.001) and Phe (p ˂ 0.01) were found in the CP of hind leg meat than in LTL meat.As for carcass parts assessed , the higp ˂ 0.001) as well as all EAA (p ˂ 0.05) was found in the CP of hind leg meat of H rabbits as compared to MS sired rabbits. Similarly, in the CP of LTL meat, the higher proportion of total AA (p ˂ 0.05) and all NEAA monitored (p ˂ 0.01) was found in H rabbits as compared to MS sired rabbits. As for the proportion of particular AA in the CP of hind leg meat, higher values of Leu, Iso, Thr, Pro, Gly and Tyr (p ˂ 0.001), of Val and Ser (p ˂ 0.01) and also of Asp and Glu (p ˂ 0.05) were found in H rabbits as compared to MS sired rabbits. Concerning the proportion of particular AA in the CP of LTL meat, higher values of Gly and Tyr (p ˂ 0.001), then of Leu, Thr, Val and Ser (p ˂ 0.01) and also of Iso, Asp and Glu (p ˂ 0.05) were observed in H rabbits than in MS sired rabbits.Concerning a rabbit genotype , a highep ˂ 0.01) and the total AA monitored (p ˂ 0.05), in particular of Phe (p ˂ 0.001) and also of Lys, His and Ala (p ˂ 0.01) was found in females as compared to males. By contrast, there was a higher proportion of Met and Tyr (p ˂ 0.01), as well as Gly (p ˂ 0.05) in the CP of male hind leg meat as compared to female hind leg meat. In addition, a significant effect of genotype x sex interaction on the proportion of all EAA (p ˂ 0.001), total AA (p ˂ 0.01), as well as NEAA (p ˂ 0.05) monitored in CP of hind leg rabbit meat was found; specifically, on the proportion of Lys, His, Phe, Tyr, Glu, Val, Asp and Gly. Within LTL, the rabbit sex influenced the proportion of all EAA monitored (p ˂ 0.05), of Tyr (p ˂ 0.001) and of Ala, Lys and Arg (p ˂ 0.05) in CP of this meat; the higher proportion of total EAA, Ala, Lys and Arg and the lower Tyr proportion was found in females. Moreover, the rabbit genotype x sex interaction had a considerable effect on the proportion of the total AA and all EAA (p ˂ 0.001) and of all NEAA monitored (p ˂ 0.01) in LTL meat; specifically, on the proportion of Arg, His, Phe, Tyr, Lys, Val, Asp, Glu, Gly, Thr and Ser. Regarding the effect of gender , in the p ˂ 0.01) and lower K and Cu (p ˂ 0.01) and Zn (p ˂ 0.05) level was observed in hind leg meat as compared to LTL meat , Zn (p ˂ 0.01) and K (p ˂ 0.05) in hind leg meat was found in MS sired rabbits as compared to H rabbits. In LTL meat, a higher level of Na and Mn (p ˂ 0.001), Mg and Zn (p ˂ 0.05) was observed in MS sired rabbits than in H rabbits. Regarding a rabbit genotype , the higp ˂ 0.05) and lower level of Mg (p ˂ 0.01) was found in the hind leg meat of females as compared to males. A significant effect of genotype x sex interaction on the content of Na (p ˂ 0.001), Ca and Fe (p ˂ 0.01) in this meat was also found. In LTL meat, a higher content of K was found in males compared to females (p ˂ 0.05). In addition, the rabbit genotype x sex interaction had a significant effect on the content of Fe (p ˂ 0.05) in LTL. Concerning a sex effect , a higheLTL meat, a higher yield of hind leg meat was found in fattened H rabbits than in MS sired rabbit progeny . This fact is demonstrated by the higher muscularity of hind legs in final commercial crossbred H rabbits. The hind leg meat yield of rabbit genotypes in the present study was similar to that reported for the Belgian Giant Grey and Termond White rabbits [LTL muscles, their yield was also slightly higher in H rabbits than in MS sired rabbits  and a higher LTL yield was found in H females than in MS sired females . In the present study, the lower meat yield of the assessed rabbit groups was found in MS sired female progeny. Additionally, slightly lower values for the LTL yield (6.9 to 7.1%) were found in 78-day-old rabbits [A higher SW of MS sired progeny at 108 days in the present study is similar to that found in 100-day-old New Zealand White rabbit males  and cons rabbits . In the  rabbits , as comp rabbits . Within  rabbits ,31. This rabbits . LTL muscle was associated with the higher CP content and lower W/P ratio as compared to hind leg meat. This finding is in agreement with findings reported in three-month-old rabbits [LTL, furthermore the DM content was also higher in LTL meat of the five and a half-month-old New Zealand White rabbits [LTL of the present study favours this rabbit meat for subsequent meat processing, since it contributes to a higher product yield due to lower loss of its own water and better ability to hold added water [LTL, a higher intramuscular fat (IMF) level was found in hind legs in the present study, furthermore its content is inversely associated with the content of CP and water in rabbit meat [LTL. By contrast, the opposite trend for the ash level was found in the Pannon White rabbits [Regarding the proximate chemical composition of meat in the present study, the assessed traits were influenced mainly by a carcass part and the higher DM content of the  rabbits ,33. By c rabbits . The lowbit meat . This fibit meat ,33; howebit meat . Higher  rabbits ; their S rabbits ,33.A rabbit genotype in the present study influenced only the total mineral content in hind leg meat, its higher value was found in H rabbits. Metzger et al.  mention LTL; leaner meat was found in females. Similarly, Ortiz Hernández and Rubio Lozano reported lower IMF content in 70-day-old females of the New Zealand White breed compared to their males; nevertheless, they found the opposite trend in the Californian breed [LTL meat was higher in males of the present study, which also was observed (p ˃ 0.05) by North et al. [LTL of meat rabbit genotypes. It seems that male rabbits may in some cases deposit more IMF into the LTL muscle with advancing age. Based on the IMF content of hind leg meat in the present study, MS sired females were most mature at the given slaughter age.In the present study, a rabbit’s gender affected only the IMF level in an breed . It is kan breed . In the h et al.  in the LLTL as compared to hind leg meat in rabbits of the present study was presented by a higher proportion of total AA (+2.9%) and particularly all EAA assessed (+11.4%). On the other hand, in hind leg meat, a higher proportion of Phe (+9.2%) and Gly (+7.9%) was found. In particular, the higher proportion of seven out of nine EAA assessed and of three NEAA was found in LTL meat. The order of representation of individual EAAs was almost identical in both evaluated rabbit carcass parts, with the following order starting from the highest proportion: Lys → Leu → Arg → Val → Iso. Regarding NEAA, the order of the individual AAs was again almost identical in both evaluated carcass parts, the highest proportions were demonstrated for Glu → Asp → Ala → Gly. In general, meat AA composition is influenced by different syntheses of AA as related to different biological stages of animals . For conLTL of 70-day-old rabbits of different genotypes [LTL of the 105-day-old Ira rabbits [LTL meat considerably increased with an advanced rabbit age [The above stated findings of the present study are in agreement with those found by Migdal et al. and Bivolarski et al. in the three-month-old New Zealand White rabbits ,40 and benotypes  and also rabbits . Resultsbbit age . LTL and especially in hind legs, the higher nutritional value of meat CP was found in H rabbits as compared to MS sired rabbits. In the case of hind leg meat, a higher content of both total AA (+5.9%) and EAA monitored (+5.3%) was found in H rabbits than the MS sired rabbits and a higher proportion of four out of nine EAA and higher proportion of six out of seven NEAA monitored was found in the CP of hind leg meat in H rabbits. However, the lowest values of these EAA and three out of 7 NEAA displayed the MS sired males, furthermore the MS sired females did not show such a decrease in quality of hind leg proteins when compared to H rabbits. In particular, the most significant variation in this meat was found for Tyr (27.5%), Thr (12.3%), Leu (10.8%), Val (10.3%) and Iso (10.2%). As for LTL, similarly higher content of total AA (+4.3%) was found in H rabbits than in MS sired rabbits and a higher proportion of four out of nine EAA and a higher proportion of five out of seven NEAA monitored was found in the CP of LTL meat in H rabbits. When compared to MS sired progeny, the largest difference in the content of Tyr (+23.5%) and in the content of Gly (+13.6%) and Val (+9.2%) was found again in the CP of LTL meat in H rabbits. A genotype effect on the AA profile in LTL rabbit meat was recently observed as well [In the present study, a surprisingly significant effect of sire genotypes on the AA composition in both evaluated carcass parts was found. Both in , Leu 10.%, Val  and of three out of seven NEAA . In LTL, the sex affected proportions of 2 EAA  and of two NEAA . However, it is necessary to note that generally higher proportions of a majority of above-mentioned AA in females were demonstrated because of lower proportions of these AA in MS sired males. As demonstrated, the rabbit genotype x sex interaction played the very important role for these traits in the present study.Due to higher proportion of EAA in both carcass parts assessed, a higher nutritional value of meat proteins was found in females than in males in the present study and the proportion of total NEAA in the meat CP content was not influenced by a rabbit sex. The sex had a more considerable effect on the AA profile variation in hind leg meat than in In contrast to the findings of the present study, no sex effect on the EAA profile in rabbit hind leg meat was observed , who evaFurthermore, no sex differences in AA profile of meat were reported recently: neither in pigs , cattle The taste of food is one of the main factors determining food preference and eating habits ; simultaA large variation in the content of minerals in rabbit meat was reported in recent studies. LTL meat. Furthermore, Ca content of hind leg meat differed between rabbit sexes, mainly due to its considerably higher level in H females. Calcium helps develop and maintain strong bones and teeth, increases the utilization of other minerals  and its adequate intake contributes to the prevention of cardiovascular diseases [LTL meat of the present study is similar to that reported [In the present study, the higher Ca content and lower K, Cu and Zn content was found in rabbit hind leg meat as compared to diseases ,54. The reported .LTL, a higher level was found in males than in females. In general, rabbit meat has a higher K concentration than the meat from other animal species [LTL was found in the MS sired genotype. The levels of Na in meat in the present study are similar to those observed in [Although a lower ash content in hind leg meat was found in MS sired rabbits in the present study, the content of four out of nine assessed minerals in this meat was higher than in H rabbits. Similarly, a higher content of 4 monitored minerals was also found in the LTL of the MS sired genotype. Thus, a higher content of natural forms of some minerals, mainly of Mg, Mn and Zn was observed in the meat of the MS sired progeny. Regarding K level, a genotype affected its value only in hind leg meat, the higher level of K was found in the MS sired progeny. Moreover, in  species ,56, whicerved in ,56. Rabberved in . Apart ferved in ,56. In aerved in ,56.Regarding trace elements in meat of the present study, Fe was the most abundant microelement, followed by Zn, which is in agreement with the finding  observedLTL meat displayed better dietetic properties than hind leg meat due to its lower IMF content and higher proportion of total AA, mainly of all essential AA.A rabbit Surprisingly, substantial effects of sire genotype and genotype x sex interaction of rabbits on the AA profile of meat were found. Crossbreeding of MS sires with a commercial dam line of HYLA rabbits resulted in a worse nutritional quality of meat proteins in fattened MS sired males. These findings point to a possible risk of alterations in the nutritional quality of meat proteins when using different sire genotypes than those originally intended for the specific commercial crossbreeding scheme.On the other hand, the content of natural forms of some minerals was higher in the meat of MS sired progeny.


We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+ loading. The loss of PN mitochondrial Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signalling and alleviation of altered excitation–transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling.Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in CaThis article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. The coordinated regulation of Ca2+ cycling under the conditions of different workload demand is achieved by the close physical proximity of the main Ca2+ cellular store, the sarcoplasmic reticulum (SR), and mitochondria distributed throughout the cell. At the junction between the SR and mitochondria, a space known as the subcellular microdomain, local Ca2+ fluxes of high magnitude lead to mitochondrial Ca2+ uptake , which enhances tricarboxylic acid cycle dehydrogenases, leads to faster NAD(P)H reduction and finally increases ATP generation by feeding the electron transport in the respiratory chain .2+ uptake is compromised in HF  in failing cardiomyocytes at increased stimulation frequencies. Therefore, normalization of mitochondrial Ca2+ regulation may be a novel therapeutic approach to restore altered Ca2+-mediated transcription and prevent adverse cardiac remodelling.Accumulating knowledge on mitochondrial dysfunction in HF has been recently reviewed  and it c. 2https://doi.org/10.5061/dryad.pvmcvdnn9. Materials and methods are described in detail in the electronic supplementary material.The data supporting findings of this study are available from the Dryad Digital Repository: All procedures involving animals were carried out in accordance with the Federal Act on the Protection of Animals  or the NIH Guide for the Care and Use of Laboratory Animals (UC Davis) and were approved by the Institutional Animal Care and Use Committee. Human hearts (from patients and organ donors whose hearts could not be used for transplantation) were acquired via collaboration with the Division of Cardiac Surgery . The use of human samples was approved by the Ethical Committee of the Medical University of Graz, and all experimental procedures were carried out in accordance with the Declaration of Helsinki.. 3 (a)a), and in the space between the two nuclei in typically binucleated ventricular cells (figure 1c). Quantification of mitochondrial size and shape by individual tracing of mitochondria defined as organelles enclosed by a double contoured membrane in cytoplasmic and PN spaces showed that mitochondrial perimeter and aspect ratio are significantly different in the two subpopulations (figure 1b), with PN mitochondria being smaller and more spherical. Interestingly, IF mitochondria in TAC-operated mice at seven weeks post-intervention appeared smaller and rounder, and were increased in number per area when compared with controls (figure 1d), while these parameters remained unchanged for PN mitochondria. These data suggest a shift of typical IF mitochondria towards a phenotype more resembling that of PN mitochondria during the development of HF. As PN mitochondria are critically involved in mitochondrial genesis and turnover, this phenotypic shift could be a result of premature mitochondrial recruitment and/or delayed degradation with important functional consequences for cardiomyocyte bioenergetics.To address potential changes in the organization and function of mitochondrial subpopulations during pressure overload-induced HF, we first performed confocal and electron microscopy (EM) imaging of intact adult ventricular cardiomyocytes and mouse myocardium preparations, respectively. As previously observed in EM images of rabbit hearts , ultrastfigure 1 (b)a,b). However, myocyte growth was even greater, resulting in a reduced mitochondrial volume as a fraction of cell volume (figure 2c). This reduced relative mitochondrial volume was already decreased substantially for IF mitochondria at one week post-TAC, with similar values obtained at seven weeks post-TAC. These values are in remarkable agreement with a previous study using the same model, in which an advanced three-dimensional stereology method for quantification of cardiomyocyte composition was used  to assess mitochondrial Ca2+ uptake in IF and PN mitochondria from control and failing mice (c–e). In agreement with previous work  studied. TAC caused a dramatic reduction in mitochondrial Ca2+ uptake in both IF and PN mitochondria at each of the three values of [Ca2+] studied . Again, functional capability to sequestrate Ca2+ was disproportionally impaired in PN mitochondria, which also exhibited a higher threshold level of [Ca2+] for uptake compared with IF mitochondria . The seemingly faster Ca2+ uptake observed in TAC versus sham PN mitochondria at higher [Ca2+] (2 and 10 µM) is likely due to already saturated mitochondrial [Ca2+] after exposure to 1.35 µM Ca2+ in sham mice.Compromised mitochondrial Caing mice c–e. In aous work , we obsechondria c,e; howe studied c,d. Agai2+ uptake by mitochondria plays an important role in (sub)cellular Ca2+ cycling, and its dysregulation has a profound effect on excitation–contraction coupling and cytoplasmic [Ca2+] in HF. As PN mitochondria are particularly functionally impaired in TAC animals and failing human hearts, this raises an interesting question as to whether altered Ca2+ uptake by PN mitochondria may have a measurable effect on nucleoplasmic Ca2+ homeostasis in HF.Ca (e)2+ uptake on nucleoplasmic CaTs in ventricular myocytes isolated from healthy mouse hearts (a–c). Cells were simultaneously labelled with the Ca2+ indicator Fluo-4 for quantification of subcellular CaTs and TMRM for detection of mitochondrial localization in the absence and presence of the specific MCU inhibitor Ru360 (10 µM) [2+ uptake to the extent that mitochondrial Ca2+ uptake on a time scale of action potential-induced CaTs is essentially blocked [2+] and high TMRM signal surrounding the nucleus on both poles , compatible with the idea that PN mitochondria may buffer Ca2+ around the nucleus during electrically stimulated CaTs, therefore shaping the nucleoplasmic-to-cytoplasmic [Ca2+] gradients in cardiomyocytes during the cardiac cycle. Similar regions were observed in cardiomyocytes from non-failing human hearts (d), hence strengthening their general applicability and clinical relevance.To address this question, we first tested the effect of pharmacological inhibition of mitochondrial Cae hearts a–c. Cell (10 µM) ,30. Ru36 blocked . In the th poles a,b, compn hearts d, hence 2+ levels and amplitude . Notably, increased Ca2+ cycling in PN regions also translated into higher nucleoplasmic CaTs, in terms of both diastolic [Ca2+] and amplitude of CaTs. These data raise the possibility that the lack of PN mitochondrial Ca2+ uptake as observed in TAC cardiomyocytes may lead to more rapid propagation of CaTs from the cytoplasm to the nucleus, and moreover, to the disproportional increase in nucleoplasmic versus cytoplasmic CaTs in diseased myocytes, especially at higher pacing rates where mitochondria are expected to take up more Ca2+ [2+] in the nucleoplasm to a much larger extent than in the cytoplasm, leading to the activation of nuclear CaMKII and consequential nuclear export of the transcriptional regulator HDAC4  in failing cardiomyocytes, especially in the nuclear compartment. Notably, Ru360 had no effect on cytoplasmic [Ca2+] in either sham- or TAC-operated mice. The reduction in nuclear CaT amplitude seen in TAC  is explained by the dramatic rise in diastolic nuclear [Ca2+] in failing TAC myocytes , which limits nuclear Ca2+ efflux between beats that is caused by altered Ca2+-regulating proteins and nuclear envelope structure  by healthy PN mitochondria, which is revealed by Ru360 treatment, is necessary to accelerate nuclear [Ca2+] decline, lower diastolic nuclear [Ca2+] and lower nuclear CaT amplitudes to limit Ca2+-dependent transcriptional signalling. The loss of this Ca2+-buffering ability in failing heart PN mitochondria is why Ru360 no longer makes things worse .We conclude that the normal buffering of PN [Ca. 42+ uptake across a range of different [Ca2+], reduced mitochondrial membrane potential and increased ROS production in failing cardiomyocytes exposed to increased frequency of pacing. Depressed Ca2+ uptake in PN mitochondria has a direct effect on nucleoplasmic CaTs and may play a causal role in disturbed excitation–transcription coupling and progression of maladaptive cardiac remodelling. These data identify a novel subcellular mechanism of functional decline during HF development that could help the advancement of new cardioprotective strategies and/or better understanding of molecular mechanisms underlying the beneficial effects of current cardiometabolic therapies.The results of the present study indicate that PN mitochondria are functionally altered to a higher degree than IF mitochondria in failing cardiomyocytes, as manifested by the severely depressed Ca (a)et al. [2+] rise in PN mitochondria Ψm, Ca2+ uptake and ATP production [2 autofluorescence under conditions of increased cell work induced by high-frequency pacing. Quantitative analysis in TAC cardiomyocytes revealed nearly maximal FAD/FADH2 ratio (oxidized) at basal conditions, and the TAC cardiomyocytes were significantly more oxidized than sham cardiomyocytes. In addition, it has recently been demonstrated that pressure overload induced by TAC resulted in a significant increase in the expression of Nox2 oxidase subunits and a more oxidized state overall as assessed by the GSH/GSSG ratio [+, Ca2+ and the mitochondrial redox environment, a concept comprehensively elaborated upon by Cortassa et al. [2 ratio with minimal ROS generation in sham cardiomyocytes shifted to depressed Ca2+ uptake, and severely oxidized FAD/FADH2, with a concordant increase in ROS generation in TAC mice.IF mitochondria retained beyond their regular turnover rate would be expected to show slightly decreased functional properties and be especially vulnerable to stress in hypertrophic and failing cardiomyocytes. In addition, those sent to be recycled in PN spaces would show more pronounced signs of functional exhaustion, with potential consequences to overall cardiomyocyte vulnerability including functional and structural outcomes. Pressure overload-induced HF has been shown to result in increased mitochondrial oxidative stress and ROS generation and reduced Δoduction ,39, obseoduction  demonstrSG ratio . It is aa et al. . This coThe most novel finding here is that, while IF mitochondria showed signs of exhaustion mostly under stress, PN mitochondria from failing myocytes showed signs of functional deterioration already at baseline and high frequency of pacing pushed them into even more oxidative stress and functional decline figures  and 4. T2+, especially at higher cytoplasmic Ca2+ levels. In HF, diminished SR Ca2+ release and increased intracellular Na+ levels depress mitochondrial Ca2+ uptake, which impairs their capacity for sustaining optimal matrix NAD(P)H redox potential. This in turn boosts oxidative stress, especially upon increased workload. Here, we found severely diminished IF mitochondrial Ca2+ uptake in failing cardiomyocytes, and notably, even more depressed Ca2+ uptake by PN mitochondria  protected mice from developing a full-blown HF phenotype [2+ uptake had clear whole-cell functional outcomes in isolated rabbit atrial myocytes [2+ uptake with the polyamine compound spermine.Another important parameter of mitochondrial fitness with implications in a variety of cellular functions is their ability to take up Cachondria . Althougerfusion , recent  [et al.  showed thenotype . In addimyocytes . Specifi (c)2+ uptake in preventing cardiac remodelling and its transition to HF could be their ability to specifically influence (peri)nuclear Ca2+. This idea is supported by the prominent mitochondrial accumulation in PN regions and the space between the two nuclei in characteristically binucleated ventricular cells , which can phosphorylate histone deacetylase 4 (HDAC4) and drive its nuclear export. This process mediates the de-repression of Mef2-dependent transcription, implicated in the development of HF [2+ levels may, on the other hand, ensure a pool of active PN CaMKII which will keep rephosphorylating any exported HDAC4 that becomes dephosphorylated after leaving the nucleus. This could prevent HDAC4 from reentering the nucleus and reinforce the transcriptional signalling of activated CaMKII.The increase in (peri)nuclear Cagulation . For exant of HF . Increas2+ crosstalk occur in the slower disease progression observed in animal models and patients who suffer from HF with preserved ejection fraction.One important aspect to consider when interpreting the present data is the variety of HF phenotypes in regard to left ventricular ejection fraction, pathophysiology, underlying triggers , mechanisms of progression and response to treatment. The development of powerful techniques for comparative transcriptome analysis of cardiomyocytes and non-myocytes on a single cell level identified numerous aetiology-specific alterations in gene expression, pointing to different underlying mechanisms of the disease progression . This hi2+ levels—contribute to the development and progression of hypertrophy and HF. Normalization of mitochondrial Ca2+ regulation may therefore be a novel therapeutic approach to restore altered Ca2+-mediated transcription and prevent adverse cardiac remodelling.Taken together, the present study implicates fitness and functionality of PN mitochondria as an important determinant of cardiac remodelling that may—via shaping nucleoplasmic Ca


The title of this essay is as much a question as it is a statement. The discovery of the β-lactam antibiotics—including penicillins, cephalosporins, and carbapenems—as largely (if not exclusively) secondary metabolites of terrestrial fungi and bacteria, transformed modern medicine. The antibiotic β-lactams inactivate essential enzymes of bacterial cell-wall biosynthesis. Moreover, the ability of the β-lactams to function as enzyme inhibitors is of such great medical value, that inhibitors of the enzymes which degrade hydrolytically the β-lactams, the β-lactamases, have equal value. Given this privileged status for the β-lactam ring, it is therefore a disappointment that the exemplification of this ring in marine secondary metabolites is sparse. It may be that biologically active marine β-lactams are there, and simply have yet to be encountered. In this report, we posit a second explanation: that the value of the β-lactam to secure an ecological advantage in the marine environment might be compromised by its close structural similarity to the β-lactones of quorum sensing. The steric and reactivity similarities between the β-lactams and the β-lactones represent an outside-of-the-box opportunity for correlating new structures and new enzyme targets for the discovery of compelling biological activities. Streptomyces [The discovery of antibacterial antibiotics revolutionized the practice of medicine ,2. Amongptomyces ,14,15,16ptomyces ,18, so aptomyces ,20.The structural focus is the β-lactam ring—the four-membered cyclic amide sub-structure—of penicillin . The β-l®) is currently in late-stage clinical evaluation for brain cancer [The marine antitumor antibiotic salinosporamide A  offers an cancer ,26. Its n cancer ,28. An en cancer . The bioIn this essay, we address the following questions. Do marine organisms biosynthesize notable inhibitors of bacterial cell-wall biosynthesis? Do marine organisms biosynthesize classical antibacterial β-lactams? Do marine organisms biosynthesize (other) β-lactams? Is there a relationship between the β-lactam functional group and the β-lactone functional group, and the observation from the marine environment that marine bacteria have numerous β-lactam-hydrolyzing enzymes?At present, no marine structure having both the efficacy and safety to justify its clinical use has been identified. None of the structures shown in Streptomyces [Staphylococcus aureus) antibacterial and antimycobacterial (Mycobacterium tuberculosis) activities . In addition, the abyssomicins show human cell-line cytotoxicity. The enzyme target of the abyssomicins is 4-amino-4-deoxychorismate synthase (PabB), the enzyme catalyst for the synthesis of 4-aminobenzoic acid from chorismite [The structures in ptomyces ,52,53,54orismite . Loss oforismite ,53. PabBorismite . Intramoorismite . This meStreptomyces  [−1 against all strains of S. aureus [Sorangium cellulosum on a >150 L scale secured multi-gram quantities of chlorotonil. Its chemical stabilization (by reductive mono-dechlorination) followed by bis-epoxidation (to improve the water solubility to 16 µM) gave structure 2b-Epo, retaining essentially the full breadth of biological activities [−1 against several Gram-positive bacteria (including an MIC90 of 0.1 mg·L−1 against methicillin-resistant S. aureus), oral safety in mice at 50 mg·kg−1 attaining serum concentrations above the MIC for the activity for 24 h, and efficacy in the S. aureus mouse neutropenic thigh infection assay at i.v. 2 × 5 mg·kg−1 dosing [Anthracimycin is a macrolide isolated from marine abolite) ,58. Anthabolite) ,60,61,62. aureus . Notwithtivities . Structu1 dosing . Its mec1 dosing . Its own1 dosing ,67. Equi1 dosing ,69 and s1 dosing ,70. Mari1 dosing . The pro1 dosing ,73,74,75The structures in Streptomyces [S. aureus , as an inhibitor of the FEM enzymes unique to S. aureus [−1 cyslabdan, the MIC of imipenem is reduced from 16 mg·L−1 to 0.015 mg·L−1). The potentiation of the MICs of β-lactams was much less  [Streptomyces) across a panel of Gram-positive and Gram-negative bacteria. The basis for the synergy was attributed (in part) to the inhibition of the β-lactam-hydrolyzing (β-lactamase) activity of the panel [−1), dual mechanisms of action (inhibition of both peptidoglycan and protein biosynthesis) with low frequency of resistance mutation, and accessibility to synthetic modification, with the possibility of expansion of their antibacterial activity to Gram-negative bacteria [Labdanes are secondary metabolites of both terrestrial and marine ptomyces ,89,90. A. aureus , cyslabdsporins) , implicahe panel . Remarkahe panel . Lipoxazhe panel ,96. Theybacteria . The relStreptomyces) is a weak (IC50 = 225 µM) inhibitor of Gram-positive β-lactamases [Ki = 6 µM) of the clinically much more relevant AmpC β-lactamases of Gram-negative bacteria [The two final structures in ctamases . Halisulbacteria . The cryCephalosporium acremonium fungus, which biosynthesizes cephalosporin C. Notwithstanding the singular importance of his discovery, no antibacterial β-lactam has been isolated since from a marine source. The subsequent β-lactam sub-families—nocardicins, clavulanic acid, monobactams, and carbapenems—discovered over the course of ensuing decades are secondary metabolites of terrestrial bacteria. While numerous biologically active secondary metabolites are isolated from marine Penicillium fungi, none is a β-lactam [Kallichroma tethys identified two genes (pcbAB and pcbC) encoding proteins homologous to the penicillin biosynthetic enzymes of Acremonium chrysogenum [It is uncertain whether marine organisms biosynthesize classic β-lactam antibiotics. A momentous event in the history of the β-lactams was the isolation by Brotzu in 1945—from the Mediterranean Sea, near a sewage outfall located at Cagliari, Sardinia—of the β-lactam . A 2003 ysogenum . While cysogenum ,103,104.Streptomyces bacterium [Pseudomonas bacteria [Chartella papyracea. It is not described as yet as having a biological activity [Hymeniacidon sp. sponge [Plasmodium falciparum parasite [Marine organisms biosynthesize other β-lactams. However, the structural exemplification is sparse . Antibioacterium . Its Grabacteria ,107, doebacteria ,109. Chaactivity . Monamph. sponge . The isoparasite . The molA prominent role for marine-biosynthesized, β-lactam-containing enzyme inhibitors is not yet supported. To this date, three β-lactam-containing natural products are identified as biosynthesized by marine organisms. None is an inhibitor of bacterial cell-wall biosynthesis. To date, marine organisms biosynthesize several compounds that are adjuvants of the antibiotic activity of the terrestrial β-lactams , and several structures that inhibit in vitro the resistance enzymes, which hydrolytically degrade terrestrial-derived β-lactam antibiotics. These few examples could be interpreted to signify that the β-lactam functional group lacks significance within marine biology. Such an interpretation might follow the expectation that within the marine environment, the enzyme catalysts capable of the hydrolytic degradation of the terrestrial β-lactam antibiotics are not necessary and thus are uncommon. The evidence that this conclusion is incorrect is overwhelming. The oceans teem with such enzymes.Two reasons support the prevalence of β-lactam-degrading enzymes in marine environments. The first reason is the copious and undisciplined use of antibiotics in human and animal medicine. The result is a profound “anthropogenic pollution” of the marine environment . AntibioVibrio bacteria [The basis for a second reason begins with the reminder that a principal basis for the resistance of bacteria to β-lactam antibiotics is the production of hydrolytic enzymes. These enzymes divide between those using an active-site serine nucleophile  and those using zinc-ion catalysis . The serine β-lactamases are related evolutionarily to the penicillin-binding proteins of cell-wall biosynthesis, again with an ancient evolutionary separation of the β-lactamases from the penicillin-binding proteins ,123. Thebacteria ,132. QuoH)-one—while having lower intrinsic reactivity due to the absence of ring strain—is the ring system of the N-acylhomoserine lactone (AHL) class of quorum-sensing elicitors [N-acyl moiety is bacterial species-dependent (the AHL depicted in Panel B in Vibrio bioluminescence), the homoserine lactone ring is common to the AHL class of structures. One common transformation to abolish AHL signaling (a process termed quorum quenching) is the hydrolysis of the AHL amide with the release of the fatty acid. A second common transformation to achieve quorum quenching is hydrolytic ring-opening of the lactone. This reaction is catalyzed by the AHL lactonases  ,134,135.licitors ,132,144.licitors ,146. WhiOne example merits further comment. A class A β-lactamase produced by a bacterium at 1050 m below the surface of the Pacific Ocean turns over penicillins at or near the diffusion limit. This level of catalytic competence cannot be adventitious, implying a directed evolution for the purpose . In lighPseudomonas aeruginosa quorum sensing [Vibrio quorum sensing, but also as a result of enzyme inactivation within the fatty-acid biosynthetic pathway and not within quorum pathways [Pseudomonas fluorescens) [The intrinsic reactivity of β-lactones has been exploited to identify other antibacterial enzyme targets ,154. Sev sensing . A seconpathways . Antibacpathways ,158,159,pathways ,160,161.rescens) . The Grarescens) ,164. Therescens) . To our In this essay, we discuss evidence to support the possibility that promiscuity with respect to the substrate—β-lactone or β-lactam—for marine β-lactonases/β-lactamases may explain (in part) a diminished advantage for a marine fungus or bacterium to produce a β-lactam antibiotic. A similarity in the chemical reactivity for the β-lactone and β-lactam may suggest value to examining marine β-lactones as new inhibitors of cell-wall biosynthesis. Both suggestions fit within “outside-the-box” approaches  to ensur


Excessive Ca2+ release from the internal stores is followed by a subsequent extracellular Ca2+ influx through the Orai1 Calcium Release Activated (CRAC) channel, significantly contributing to a sustained Ca2+ overload under pathological conditions. Ca2+ overload evokes irreversible changes in the pancreas, whereas blockade of such Ca2+ elevation has been shown to prevent the development of pathological cellular changes, effectively protecting against experimental AP  remains one of the few untreatable inflammatory diseases that requires hospitalization and has a remarkably high mortality rate in severe cases. AP is primarily caused by gallstones and excessive alcohol consumption, but it can also be a side effect of medical treatments, for example with asparaginase  and Ca2+ store reuptake (SERCA) mechanisms in a situation with severely reduced ATP production , pancreatic acinar cells (PACs), stellate cells (PSCs) and pancreatic macrophages (PM) is shown in Fig. 2+ overload in different cell types of the pancreas are undoubtedly our best hope of developing a cure for the main causes of AP. Another promising development is the use of the energy supplement galactose, which provides cells with additional ATP under AP conditions  (grant MR/J002771/1) and Children with Cancer UK (grants 17/248 and 19/288).Peer Review HistoryClick here for additional data file.