P < 0.05) amounts of Zn than males. Animals younger than 3 years old have lower (P < 0.05) levels of Na than those between 3 and 5 years old. Horses used for reproduction had lower (P < 0.05) Mg concentrations than animals used for running and working. Tensile strength was similar between demographic characteristics. Horses from Santa Gertrudis military ranch had higher (P < 0.05) levels of Ca, Se, and Na compared with horses from other sampling location. Copper was higher (P < 0.05) in horses from racecourse. Potassium was higher (P < 0.05) in horses from the Presidential General Staff. Overall, there was no evident connection between sex, ages, breeds, and riding disciplines from different regions of Mexico and the mineral composition of the hoof or its tensile strength. Further research should focus on the relation on specific feeding regimes, horse individual characteristic, hoof mineral contents, and tensile strength.Hoof mineral profile is important as it could affect locomotion. Factors such as body weight, sex, age, and riding disciplines affect hoof mineral profile. In Mexico and globally, studies are needed on the characterization of mineral profile of hooves and tensile strength, as this could help to prevent overgrowth or microfractures. Therefore, in the present survey, 165 samples of equine hoof cuttings from different sex, ages, breeds, and riding disciplines from different regions of Mexico were analyzed for their mineral composition, and a universal testing machine was used to measure tensile strength. More than half of the samples were from males (63%) and aged 3 to 5 years (52%). Most samples were obtained from horses used for reproduction (36%) and working (29%) purposes. The most preponderant minerals were K , Na , and Ca (631 μg/g). Tensile strength ranged from 1.2 to 45 N. Females had higher ( Equidae family represent the result of a digitigrade evolution, as they rest on a finger, protected by a horn tissue covering called a hoof  Chihuahua , and (3) Zacatecas   without apparent locomotion problems and clinically healthy, aged 4.8 ± 3.1 years, with a mean body weight of 441 ± 68 kg. Samples were taken from three locations: (1) Mexico City (CDMX) [private ranch, mounted police, Presidential General Staff (PGS), racecourse, and military school,  n = 50) .Age was divided in categories using 33% and 66% percentiles ; thus, three categories were obtained: <3 years, 3–5 years, and >5 years. Most of the animals chosen for the present study were young animals because it is the age range used for surveillance or working, and the surveyed horses belong to this riding discipline. Demographic data from surveyed horses is shown in Equine hoof samples (65 ± 21 g) were obtained from hoof trimmings routinely performed on horses during shoeing and/or trimming. Each sample was obtained from the right forelimb, from the area of the wall, from the bearing surface. Before trimming, each hoof was cleaned with a wire brush, then scrubbed with deionized water. Upon collection, clippings from each hoof were placed into labeled plastic bags and stored for subsequent analyses .® Illinois, USA) to reach 2 mm of thickness. Samples that were not broken during notching were not considered for analysis because they did not have enough thickness for analysis. The rest of each hoof wall clipping was repackaged into labeled plastic bags and stored at room temperature until processed for mineral analysis  is held at the ends and subjected to tension until it breaks  with a load capacity of 5 kilonewtons (KN)  and an at breaks . All anaIn order to determine the association, the hoof mineral elements  were measured from hoof trimming samples. Firstly, organic matter was removed, and then samples were pulverized and digested with nitric acid at 70°C for 12 h  and thenP < 0.05) among means were compared with Bonferroni test.Depending on the type of variables, descriptive statistics  were obtained for the mineral elements, tensile strength, and demographic characteristics of surveyed horses. To test for normality, the Shapiro–Wilk test was performed. A fixed-effects generalized linear model (GzLM)  was usedPrincipal component analysis (PCA) with the Varimax criterion and canonical discriminant analysis (CDA) were performed for mineral elements and tensile strength –19. Due 2 (CI: 14.56–19.63 N/mm2)  . Due to P < 0.05) amounts of Zn than males. Animals younger than 3 years old have lower (P < 0.05) levels of Na than those between 3 and 5 years old. Horses used for reproduction had lower (P < 0.05) Mg concentrations than animals used for running and working. Horses from Chihuahua had higher (P < 0.05) levels of Ca, Se, K, and Na compared to horses from other sampling locations, but lower Zn. Due to amounts of sample, Cu was only analyzed from CDMX, and the values were higher for racecourse (2.22 ± 0.99 μg/g) and the lowest for the mounted police (1.59 ± 1.29 μg/g) (P < 0.05). Tensile strength (P > 0.05) was similar between all demographic characteristics. Horses from Santa Gertrudis military ranch had higher (P < 0.05) levels of Ca, Se, and Na compared to horses from other sampling locations. Potassium was higher (P < 0.05) in horses from the Presidential General Staff.The PCA resulted with two significant components where the Kaiser test explained 56.62% of total variation. P < 0.05)  . In canoFor the breed trait, canonical 1 explained 74.7% of the total variation, and the most discriminating elements were as follows: K and tension strength having a positive correlation, and in canonical 2, the most discriminating elements were Ca and Mg having a negative correlation, and 23.80% of the total variation was explained. For the riding disciplines, canonical 1 described 73.30% of the total variation, with K as the most discriminating mineral with a positive correlation. Canonical 2 explained 20.20% of the total variation, with Mg as the most discriminating mineral with a positive correlation. Canonical 3 explained 5.20% of the total variation, with Fe as the most discriminating mineral with a positive correlation. Canonical 4 described 1%, being Fe has a positive correlation and is the most discriminating mineral, while canonical 5 explained 0.30% of the total variation, having the following as the most discriminating minerals: Zn with a positive correlation and Na and Ca with a negative correlation. The hoof structure is very important for horse welfare and performance, and therefore, results obtained in this study seek to expand the knowledge and existing values on the mineral contents of horses' hooves, in addition to tensile strength values –22. UntiWith regard to the concentration of minerals determined by age, horses under 3 years of age had higher levels of Zn compared to horses over 3 years of age, but Na levels were lower in younger horses. Zinc is a key mineral for keratinization process. The keratin cells of the upper part of the hoof lose their nucleus and organs; during this process, the cells die and form the corneal structure of the hoof . Zinc coAnother factor that could affect hoof structure is the working hours that horses are subjected to, which implies physical and, therefore, physiological wear and tear. Not meeting nutritional requirements can lead to bone or tissue pathologies of hooves , 27. In In the present study, sampling location was a factor that affected some mineral content in horse hooves. For example, Zn was higher in the Santa Gertrudis military ranch compared to the rest of the sampling locations, while Na was lower from samples obtained from a private ranch located in Mexico City. According to FAO, in arid and semi-arid areas such as the Santa Gertrudis military ranch located in Chihuahua State, there is usually an accumulation of exchangeable Na in the soil, which clearly increases Na content of the pastures in those regions . In thisAlthough in the present study no significant differences were found using a generalized linear model between breeds, when using CDA, it was observed that the canonical variables Ca, Mg, Fe, Zn, K, Na, and the tensile strength were separated between breeds, mainly associated with Spanish breed. Other studies have shown that hoof mineral contents vary between breeds and between species. For example, sole contents of Ca were higher in Anglo-Arab horses  compared with Monterufoli Pony (373 ± 144 ppm) . In anotIt is important to consider that when carrying out this type of studies, the specific hoof area for sampling can yield to differences in mineral levels. For example, Mg levels obtained in this study were 181 ± 98 μg/g, while de Souza et al.  reportedIn this study, K and Na were the most preponderant minerals in hoof samples. In this study, Na was 2,242 ± 1,102 μg/g, while other studies  have repCalcium is a mineral to which more attention must be paid, as it helps to create cross-links of sulfur between the proteins of the hoof, allowing cohesion between cells . The strIn the case of Fe, a value of 114 ± 145 μg/g was obtained, which is below the values (290.58 ± 256.26 ppm) reported by de Souza et al. . With reOn the other hand, the metabolic inter-relationship (antagonistic or synergistic relationship) that exists between mineral elements must be considered . For exa2)   and thosreported . Some of2 and coronary edge tensile strength from 52.6 to 62.9 N/mm2. This indicates that the hoof has areas that deform differently with the horse's gait; therefore, it is important to try to have these structures in the best possible state, since poor conformation can cause growth abnormalities and development, which predisposes to hoof microfractures. Several studies (post mortem using tendons, ligaments, and cartilage (When performing PCA and CDA, the tensile strength was introduced as a variable, obtaining positive correlations in both tests. This suggests that there is a relationship between these variables and mineral elements. In this regard, Zenker et al.  reported studies –37 have artilage –40, whilLastly, carrying out these types of studies is difficult due to the sample amounts that can be obtained per animal. It must be considered that the animals have different conditions such as feeding regime, handling, and geographical location, and that is why it is necessary to have a well-defined inclusion criterion, which will lead to a greater number of samples and more robust results for mineral contents in horse hooves.Results from this survey offer a first profile and established a reference for mineral profile from Mexican horses with different characteristics. Overall, there was no evident connection between sex, ages, breeds, and riding disciplines from different regions of Mexico and the mineral composition of the hoof or its tensile strength. Further research should focus on the relation on specific feeding regimes, horse individual characteristic, hoof mineral contents, and tensile strength.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because samples were collected from private owners and no animal manipulation was involved. Written informed consent for participation was not obtained from the owners because samples obtained were when horse hooves were trimmed, owners just allowed us to remove the small pieces from trimmed hooves.RR-M contributed to conception and design of the study. GR-C organized the database and made the sampling and processing of the studied materials. AH-G performed the statistical analysis. GR-C and EV-B-P wrote the first draft of the manuscript and sections of the manuscript. FT-T is the main tutor of the study and he reviewed the results obtained and the manuscripts. All authors contributed to the article and approved the submitted version.This study was funded by the Universidad Nacional Autónoma de México, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Nutrición Animal y Bioquímica.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pathologies, such as cancer, inflammatory and cardiac diseases are commonly associated with long-term increased production and release of reactive oxygen species referred to as oxidative stress. Thereby, protein oxidation conveys protein dysfunction and contributes to disease progression. Importantly, trials to scavenge oxidants by systemic antioxidant therapy failed. This observation supports the notion that oxidants are indispensable physiological signaling molecules that induce oxidative post-translational modifications in target proteins. In cardiac myocytes, the main driver of cardiac contractility is the activation of the β-adrenoceptor-signaling cascade leading to increased cellular cAMP production and activation of its main effector, the cAMP-dependent protein kinase (PKA). PKA-mediated phosphorylation of substrate proteins that are involved in excitation-contraction coupling are responsible for the observed positive inotropic and lusitropic effects. PKA-actions are counteracted by cellular protein phosphatases (PP) that dephosphorylate substrate proteins and thus allow the termination of PKA-signaling. Both, kinase and phosphatase are redox-sensitive and susceptible to oxidation on critical cysteine residues. Thereby, oxidation of the regulatory PKA and PP subunits is considered to regulate subcellular kinase and phosphatase localization, while intradisulfide formation of the catalytic subunits negatively impacts on catalytic activity with direct consequences on substrate (de)phosphorylation and cardiac contractile function. This review article attempts to incorporate the current perception of the functionally relevant regulation of cardiac contractility by classical cAMP-dependent signaling with the contribution of oxidant modification. The sympathetic nervous system represents the most powerful regulator of cardiac contractile function. Thereby, the monoamine neurotransmitters epinephrine and norepinephrine innervate postsynaptic β-adrenoceptors on the surface of cardiac myocytes allowing canonical intracellular signal transduction. This involves the generation of the intracellular second messenger 3′-5′-cyclic adenosine monophosphate (cAMP) and subsequent activation of its principle cellular effector enzyme cAMP-dependent protein kinase (PKA). Incessant investigations are focusing on understanding the isoform-specific, spatiotemporal and structural contributions as well as disease-specific alterations in PKA signaling. PKA is “the kinase” in cardiac myocytes that links neurohumoral extracellular signals to changes in contractile function. This process termed excitation-contraction coupling occurs via phosphorylation of key substrate proteins ,2 Figur.PRKACG (Cγ) and PrKY have been described as retrotransposons [PKA is a heterotetrameric enzyme that is composed of a dimeric regulatory (R) and two catalytic subunits. Several PKA-C isoforms are expressed in human tissue: Cα, Cβ and PrKX. Cβ isoform expression appears to be predominantly in the nervous and the immune system and PKA-Cα is ubiquitously expressed ,4. In hunsposons ,6. Assocnsposons . Importansposons  or even nsposons  excess onsposons . Also, tnsposons . Those s2+ release and uptake [For both, RI and RII regulatory subunits, α and β isoforms have been described . Generald uptake . All regd uptake . The twod uptake ,19 allowd uptake .2+-cycling. PKA-mediated phosphorylation of the Ca2+ channel inhibitor Rad (Ser25/38/272/300), a Ras-like GTP-binding protein family member, relieves its constitutive inhibition on CaV1.2. This increases channel open probability and thus contributes to the inotropic response [2+ into the SR and its release into the cytosol by the ryanodine receptor (RyR2). PKA-mediated phosphorylation of SR membrane-associated phospholamban (PLN) at Ser16 abolishes its inhibitory effect on the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2), resulting in accelerated Ca2+ import into the SR during diastole and, in consequence, positive lusitropy [2+ during systole and enhances contractility [2+ sensitivity that has been attributed to direct myofilament protein phosphorylation. Thereby, Ca2+ desensitization of the myofilaments is reduced by PKA-mediated phosphorylation of cardiac troponin I (cTnI) at Ser23/24, a component of the heterotrimeric troponin complex [2+, further enhancing the PKA effect on sarcomere Ca2+ sensitivity and cardiac lusitropy [PKA substrates in cardiac myocytes that regulate contractile function involve both, myofilament proteins and those involved in intracellular Caresponse . At the usitropy ,23,24. Aactility ,27. An i complex ,29,30 an complex ,32,33. Pusitropy ,35,36.It is commonly accepted that PKA represents the main effector for intracellular cAMP and that binding of the second messenger activates the kinase. Interestingly, already de-cades ago in the 1980s, researchers described oxidation of PKA. However, only recently increasing research efforts were undertaken to pinpoint the nature and functional importance of PKA oxidation on specific cysteinyl thiols. This timely delay might be due to the former lack of availability of sophisticated knock-in animal models and techniques to study protein oxidation in complex homogenates . FurtherOxidative post-translational modification of type I PKA regulatory subunit (RI) was first described by Potter and Taylor in 1980 , who inv2O2 [2O2. These results suggested the following order of events: oxidant-mediated disulfide formation in RI releases PKA-C, without the necessity of prior cAMP-binding. Altogether, these data provided solid indication for oxidative PKA-RI dimerization as a novel activatory mechanism of PKA. Thereby, activation might occur independently from classical catecholamine-evoked β-AR signaling with impact on phosphorylation-regulated cardiac contractility.An important advancement to answer this important issue was provided by the work of Brennan et al. in 2004, who identified PKA-RIα among the proteins that underwent oxidative dimerization in response to treatment with the disulfide-inducing agent diamide in rat ventricular cardiac myocytes by unbiased two-dimensional SDS-PAGE . This fi2O2 . Protein1-AR anta-gonist atenolol suggested that the effects occurred independently from the classical receptor-mediated activation pathway. Surprisingly, oxidative PKA-RI dimer formation was also detectable after exposure to H2O2, which failed to evoke detectable effects on protein phosphorylation in our experiments. All tested compounds resulted in reducing agent-reversible disulfide crosslinking of PKA-RI subunits and highlighted that in cardiac myocytes oxidative PKA-RI dimerization alone was not sufficient to activate the kinase. It is unlikely that exposure of cardiac myocytes to different oxidants could result in the formation of different kind of disulfide bonds between PKA-RI protomers with a potentially differential impact on kinase activity. Previous work had convincingly determined the orientation of PKA-RI protomers within the dimer, supporting the conception of the antiparallel Cys17-Cys38 bond [A recent study performed by our group partially supported the above-described observations, but pointed to a more complex scenario regarding the functional outcome of PKA oxidation in a cardiac myocyte context. When comparing the impact of an array of model oxidants on PKA oxidation and cardiac myocyte function , we obses38 bond ,41. Mores38 bond . Activats38 bond ; quaterns38 bond .Ito and IK1 [A valuable tool in the delineation of the physiological consequences of PKA-RI interdisulfide formation in vivo represented the PKA-RIα Cys17Ser knock-in mouse model . “Redox  and IK1 . Althoug2O2-exposed cardiac myocytes. This allowed to formulate the hypo-thesis that the interdisulfide bond may alter the affinity of PKA-RI for certain AKAPs that guide oxidant-mediated PKA activity to particular cellular compartments. This assumption was indeed well founded, as the N-terminal D/D-domain of PKA-R that in the type I isoform harbors Cys17 and Cys38 does not only mediate regulatory subunit dimerization, but also the interaction with AKAPs , and both are susceptible to oxidation ,63,64 FA. Cys199sequence . Althougsequence ,67,68. Isequence . Experimsequence . Intradisequence ,66. Inhisequence . The autIn this context, it may seem contradictory that the oxidation of PKA-RI is ascribed to kinase activation, although the same modification exerts an inhibitory effect on the catalytic subunit of PKA. The spatiotemporal order of events might play a role in this scenario. Holoenzyme assembly protects PKA-C Cys199 from oxidative modification . OxidatiAlthough PKA is considered one of the best characterized protein kinases in the heart, plenty aspects remain unknown. This involves the impact of oxidative post-translational modifications on holoenzyme conformation, kinase activation, activity and cellular functions. The oxidant-mediated impact on PKA at a given time and cellular location is the end-product of orchestrated events based on the susceptibility of both, regulatory and catalytic PKA subunits for thiol oxidation. In cooperation, these modifications have a significant influence on the phosphorylation of critical cardiac myocyte proteins and thus cardiac contractility, but also other cellular PKA functions. Thorough research will be required in the coming years to gain a better understanding of the (patho)physiological PKA redox regulation, its interplay with the classical cAMP-dependent activation pathway, the consequences for cardiac performance and how this knowledge can be applied to develop novel therapies for cardiac diseases.In humans, the 518 genes of the kinome that were identified to putatively encode for protein kinases are opposed by only 189 genes encoding for protein phosphatases ,72. Whil2+ channel [2+-cycling and cardiac contractility. Moreover, PP2A is an important regulator of mitogen-activated protein kinase (MAPK) signaling and thus plays a critical role in cardiac remodeling, development, physiological function and the manifestation of cardiac pathologies [PP2A is the major serine/threonine phosphatase in eukaryotic cells and highly abundant in the heart . It is i channel ,76, cMyB channel , cTnI [7 channel , most li channel ,79,80,81 channel . Due to hologies ,87,88,89The heterodimeric PP2A core enzyme consists of a catalytic (PP2A-C) and a scaffold subunit (PP2A-A), which both exist as an α or β isoform ,91. PP2A+/K+-ATPase and Na+/Ca2+-exchanger [Combination of the core enzyme with one of at least 18 regulatory B subunits (PP2A-B) forms the fully active PP2A holoenzyme B. The rexchanger ,99,100. xchanger . In linexchanger . NotablyIn addition to holoenzyme composition itself, numerous post-translational modifications (PTM) have been identified that additionally impact on subunit assembly and PP2A-mediated substrate dephosphorylation. Several members of the B’/B56 group of regulatory PP2A subunits have been identified as phosphoproteins ,103,104.Also, the PP2A catalytic subunit is prone to PTM. Nitration, methylation and phosphorylation of PP2A-C occur within its conserved C-terminus and affect PP2A activity by altering holoenzyme assembly. Various studies described PP2A-C to be susceptible to NO-mediated nitration at Tyr284, which is assumed to elevate PP2A activity by inducing the dissociation of PP2A-C from its respective scaffolding subunit ,110,111.2O2 and oxidized glutathione exerted an inhibitory effect on PP2A, while the activity was restored by exposure to reducing agents [Another PTM that has not yet received much attention in the regulation of PP2A activity is the reversible oxidation of PP2A-C cysteine thiols. A number of studies showed that PP2A-C activity was inhibited by treatment with various thiol-modifying agents including maleimide and mercurial compounds ,125. Addg agents ,130,131.g agents ,134,135.g agents . In lineIn rat brain cerebral cortex, Foley et al., further highlighted the high reactivity of PP2A-C thiols by demonstrating that spontaneous oxidation without the addition of an oxidizing agent was sufficient to induce disulfide bond formation ,136. FurOnly little is known about the physiological functions of PP2A-C oxidation in cardiac myocytes. In our recently published study on the effects of oxidants on the phosphorylation of cardiac myocyte proteins, we observed that HNO donor compounds induced protein phosphorylation via oxidation-dependent translocation of PKA towards critical myo-filament substrates . Further2O2-treated murine ventricular cardiac myocytes, but no differences were observed in cardiac fibroblasts [As outlined before, phosphorylation and methylation of the PP2A-C C-terminus alter PP2A activity via modulation of the interaction with PP2A regulatory subunits. Accor-ding to these observations, it has been suggested that PP2A-C oxidation may exert its inhibitory effect via an influence on the well-established PTM of the C-terminus . Al-thouroblasts . This suroblasts . This prroblasts ,134,135,roblasts .2O2 reportedly induced PP2A oxidation in brain [2O2 may not be able to oxidatively inhibit PP2A-C in cardiac myocytes. Pharmacological treatment of adult rat ventricular myocytes by our group showed, in contrast to HNO donors and diamide, no H2O2-mediated (100 µM) alterations in the phosphorylation status of the PP2A substrate proteins cMyBP-C, cTnI or PLN [2O2. They showed that overall PP2A activity remained unchanged in cells treated with H2O2. They even reported increased activity for a PP2A moiety that was precipitated in association with its substrate ERK [2O2 [2O2 to reproducibly induce oxidative PP2A-C inhibition in cardiac myocytes at the concentrations applied in these studies. A potential explanation could be that the inhibitory effect was masked by other H2O2-induced cellular events.Interestingly, the susceptibility of PP2A to oxidative inhibition varies between cell types, which is probably due to differences in their antioxidant capacity. While Hin brain , Caco-2 in brain  and fibrin brain , H2O2 maI or PLN . Two pubrate ERK  or PP2A rate ERK , respectrate ERK . In contERK [2O2 , highligIn our experiments, oxidation-dependent translocation of PP2A into the myofilament-containing fraction of cardiac myocytes occurred . It, howTo unravel the actual role of PP2A-C and PP2A-B oxidation on PP2A subunit affinity, holoenzyme localization and phosphatase activity as well as the underlying regulatory mechanisms, more detailed research employing animal and cell models expressing redox-deficient phosphatase subunits are needed. Thus far, the available data suggest that PP2A subunit oxidation represents a previously underrated extension in the spectrum of PTM that contributes to the fine-tuned spatiotemporal regulation of PP2A activity in the heart and, most likely, many other tissues.Protein phosphatase 1 (PP1) is a highly conserved Ser/Thr-phosphatase, whose action is essential for unperturbed cardiac function ,146,147.1-3, PP1β  and the PP1γ splice variants γ1 and γ2 [2 is predominantly expressed in testis [2+, are coordinated by six amino acids , which are highly conserved among eukaryotic protein phosphatases [The PP1 holoenzyme consists of a catalytic subunit that assembles with a multitude of regulatory proteins, which determine phosphatase substrate specificity, regulate subcellular targeting and were also proposed to impact on protein stability ,154,155.1 and γ2 ,157,158.n testis , the othn testis ). PP1 can testis ,161. Thin testis . As a cophatases .An estimated amount of over 100 different proteins can interact with the PP1 catalytic subunit to assemble the PP1 holoenzymes. Although a regulatory role for many of these interacting proteins has been discovered, for some it remains unclear, whether they possess a regulatory function, represent PP1 substrates or both ,155. The2+- and cAMP-dependent signaling [Two prominent examples for PP1 regulatory proteins are inhibitor-1 (I-1) and inhi-bitor-2 (I-2) which, according to their name, inhibit PP1 activity, but with opposing dependence on phosphorylation. While I-1 requires phosphorylation to exert its inhibitory function, phosphorylation of I-2 temporarily abolishes its inhibitory effect on PP1 . Mouse mignaling . Additioignaling ,174, it ignaling ,177,178.2O2 on PP1 from SK-N-SH cell lysates, indicating that PP1 may be susceptible to direct oxidative modification [2O2-mediated phosphatase inhibition. This finding gave rise to the hypothesis that the inhibitory effect of H2O2 on PP1 may be caused by a combination of cysteine modifications and the oxidation of metal ions located within the catalytic center of PP1. A study published shortly after reported that H2O2-treatment of differentiated PC12 cells reduced PP1 activity and simultaneously resulted in enhanced phosphorylation levels of the PP1 substrate eIF2α [2O2 by Nox4 leading to in vivo inhibition of PP1. Neither DTT nor GSH could restore PP1 activity in this study, while ascorbic acid dose-dependently reversed PP1 inhibition. Biophysical experiments further supported the hypothesis that the oxidation of catalytic Mn and/or Fe ions may be the underlying mechanism for PP1 inactivation. Finally, the replacement of amino acid residues Asp64 or Asn124, which participate in the coordination of the cofactor metal ions, for Asn or Asp, respectively, increased the H2O2-induced inhibitory effect. Importantly, the inhibition of PP1 activity by Nox4 was also shown to play a role in the heart, as this process was identified to limit infarct size in murine hearts after ischemia/reperfusion. Another observation that may be interpreted in support of thiol modification-independent inhibition of PP1 is the absence of a cysteine residue corresponding to Cys269 in PP2A, which mediates PP2A inactivation by several inhibitory agents [iK) of OA and is likely to partially explain the higher affinity of OA for PP2A [In addition to the myriad of regulatory proteins that regulate PP1 function in the cell, PP1 activity is also modulated by oxidative modification. In 2002, Sommer et al.  investigfication . An impote eIF2α . This efte eIF2α . The daty agents ,134,135.y agents . Interesfor PP2A ,182. Thu2O2 in juvenile cells was reversible by DTT, β-mercaptoethanol and NAC, arguing for the involvement of thiol oxidation. However, a lack in discrimination between PP1 and PP2A activity in these experiments did not allow to definitely assign the effect to one or the other phosphatase. Using recombinant PP1α, a combination of DTT and ascorbate resulted in profound restoration of activity after H2O2-mediated inactivation, suggesting that metal ion oxidation, but also the modification of cysteine thiols was responsible. PP1α contains 13 cysteine residues and only slight deviations exist between isoforms. By mass spectrometry analysis of recombinant PP1α, Cys62 and Cys105 were detected in the oxidized sulfonic acid (-SO3H) state. Although this supported the assumption of H2O2-mediated PP1 thiol oxidation, it is a surprising result as sulfonic acid modification is considered to be irreversible and indicates over-oxidation, damage and dysfunction.Despite the fact that experimental evidence exists that points towards the metal ions of PP1 in the catalytic center as the site of inhibitory oxidation, there is also data that convincingly supports the existence of oxidative cysteine disulfide bond formation. PP1 isolated from rabbit skeletal muscle could be inactivated by alkylating, oxidizing and mercaptide-forming thiol-reactive reagents . Analysi2O2 [In brain, oxidative stress had been associated with neuronal damage and schizophrenia in earlier studies ,184. An 2O2 . Both ty2O2 . In this2O2 , may pla2O2-induced inactivation when Cys127 and Cys273 were exchanged to Ser in recombinant PP1 [2+ ions were present in the mass spectrometry experiments argues for the active center metal ions as the major target for oxidation [It is currently not possible to definitively conclude, which oxidative modifications of PP1 occur under physiological conditions in cardiac myocytes, but the importance of disulfide formation is doubtful. Cys127 was the main site involved in interdisulfide bonds reported by Singh et al. . Howevernant PP1 . A protenant PP1 ,179,180.xidation . It is axidation  have notxidation . In summ2O2-induced oxidative stress was shown to inhibit PP1 [2+-cycling at the SR. Interestingly, although PLN Ser16 phosphorylation after AngII-exposure was shown to rely on PKA, no other phosphorylation site of PP1/PKA substrate proteins displayed alterations. It was therefore speculated that only a small PLN-associated subpopu-lation of PP1 was susceptible to inhibition via Nox2. This matched with the release of ROS by Nox4, which was shown to occur locally at a microdomain level [Substantial evidence for PP1 oxidation as a physiological regulatory process in the heart is provided by the fact that it can be induced by in vivo-generated ROS. An inhibitory effect on Ser/Thr phosphatases via ROS was determined in senescent human diploid fibroblasts . Other pibit PP1 . Interesibit PP1 . This efin level . The two2O2-exposure in a previous study [An indication that PP1 oxidation may also mediate subcellular localization is provided by experiments showing DTT-reversible PP1 accumulation in the myofilament fraction of cardiac myocytes after exposure to the HNO donor NCA . Whetherus study .2O2 in neonatal rat cardiac myocytes [During β-AR stimulation, PKA signaling is tightly coupled to the regulation of its opposing phosphatases. Phosphorylation of the PP1 inhibitor I-1 or the B56δ regulatory subunit of PP2A by PKA represents regulatory feedback mechanisms that can further modulate the phosphorylation of PKA/PP1/PP2A substrate proteins such as PLN or cMyBP-C in cardiac myocytes ,171,189.myocytes . In adulmyocytes ,186,188.The overarching question remains whether pro-oxidative modulation of the PKA signaling cascade might be exploited as a druggable target with therapeutic implications. Since increased ROS production is associated with adverse development of cardiovascular disease, administration of antioxidants was presumed as a therapeutic strategy. Des-pite promising observational studies and preclinical results , clinica2+-reuptake into the SR and an acceleration of cross-bridge cycling by disulfide crosslinking of proteins localized to the SR—PLN and SERCA2a—and the myofilament—tropomyosin and the myosin light chains—compartment [Interestingly, compounds that induce protein oxidative modifications have been proposed as a potential therapy for the treatment of cardiovascular diseases. A prominent example is HNO. The HNO donor BMS-986231 , a pro-drug of CXL-1020, is currently tested in clinical trials for the treatment of patients with acute decompensated heart failure ,199,200.partment ,207,208.partment . HoweverAn alternative, but undoubtedly promising strategy for the inspiration of novel therapeutics is the development of selective compounds that may target exclusive sites within the protein kinase “cysteinome” ,210. TheIn order to apply therapeutic strategies to modulate the highly important PKA signaling network either by global pro-oxidative intervention or selective cysteine modification, detailed research is invaluable to better understand the regulatory processes underlying oxidation-mediated PKA, PP1 and PP2A activity and to determine the specific contributions of each of the enzymes to oxidant-induced changes in protein phosphorylation and cardiac contractility.The physiological role of ROS in the modulation of the PKA signaling pathway to ascertain unperturbed physiological functions is a relatively new concept. It took over 40 years to pinpoint the inducible character of PKA-RI oxidation that not merely is a purification artefact or a constitutive modification, but occurs dynamically inside the reducing cellular environment upon controlled ROS production. Thus far, the exact signaling scenario that impacts for example on excitation-contraction coupling—whether RI oxidation activates the kinase, how and if it triggers the cAMP-independent release of the catalytic subunits or whether alteration in AKAP affinity represents the main clue of RI interdisulfide formation—remains the topic of intense future investigations. In addition, oxidant susceptibility of the other signaling molecules that are players in the PKA-signalosome, namely the phosphatases PP2A and PP1, the respective AKAPs and even the catalytic subunit itself complicates the picture. When and whether this knowledge on reversible oxidation of the PKA signalosome will (ever) result in an innovate therapeutic treatment strategy, still requires a long and arduous scientific effort to gain a deeper understanding involving the generation of sophisticated experimental models. But, nevertheless, the outcome might represent the right avenue to tread for significant benefit in the therapy of heart failure and other diseases involving the PKA signaling pathway in the future.


This mismatch points to the importance of the role ice albedo feedback plays in the investigated EOT-like transition, a component that is not captured in the laboratory model. Our conclusion is supported by numerical simulations performed in a global climate model (GCM) of intermediate complexity, where both “closed” and “open” configurations were explored, with and without active sea ice dynamics. The GCM results indicate that sea surface temperatures would change in the opposite direction following an opening event in the two sea ice dynamics settings, and the results are therefore consistent both with the laboratory experiment (slight warming after opening) and the paleoclimatic data (pronounced cooling after opening). It follows that in the hypothetical case of an initially ice-free Antarctica the continent could have become even warmer after the opening, a scenario not indicated by paleotemperature reconstructions.Pronounced global cooling around the Eocene–Oligocene transition (EOT) was a pivotal event in Earth’s climate history, controversially associated with the opening of the Drake Passage. Using a physical laboratory model we revisit the fluid dynamics of this marked reorganization of ocean circulation. Here we show, seemingly contradicting paleoclimate records, that in our experiments opening the pathway yields  The Eocene–Oligocene transition (EOT) is marked by a major climatic change on the global scale, with paleotemperature records indicating a rapid drop of global mean temperature. The EOT also coincides with the largest extinction event in the Cenozoic3, and extraterrestrial impact events are also known from this interval4.The opening of major gateways in the Southern Ocean, namely the Drake Passage and the Tasmanian Seaway around the Eocene–Oligocene boundary ca. 34 Ma, is widely regarded as a key contributor to the thermal isolation of Antarctica  and the glaciation of the continent5. In their seminal paper DeConto and Pollard6 argued that the opening of gateways was merely a secondary factor for AIS development and falling atmospheric CO2 level was the main driver of EOT. However, significant gaps and contradictions exist in the available CO2 proxy data in the most relevant time interval between 40 and 30 Ma7, thus setting a precise timeline of events , and hence to establish their causal links remains controversial. Here we revisit this problem with a focus on the climate impact of Drake Passage opening, using a novel combination of a laboratory experiment and numerical simulations.The opening of the southern seaways enabled the development of the Antarctic Circumpolar Current (ACC) which inhibits warm surface currents to transport heat from the Tropics to the Antarctic continent. The details of the role that the onset of ACC played in the growth of continental-scale Antarctic ice sheet (AIS) are still far from being fully resolved8 making it the largest ocean current—is facilitated by the unique geographical boundary conditions of the Southern Ocean, the only ocean basin on the planet that is not confined by meridional continental boundaries. The “thermal wind balance” facilitated by this flow tends to maintain tilted density interfaces  sloping northward, yielding baroclinic instability in the ACC10. Analogously to atmospheric weather systems the instability generates ubiquitous eddies in the Southern Ocean, with a typical scale of 13. Blocking this circumpolar zonal pathway with a continental barrier would deviate the flow to form meridional boundary currents which in turn would lead to the buildup of a zonal (east-west) gradient in sea surface temperature and a significantly larger meridional heat transport between the equatorial and polar regions than in the “open” configuration (i.e. without the barrier)15.The present-day ACC is dominantly driven by wind stress and the meridional buoyancy contrast (yielding quasi-geostrophic flow) of the ocean itself, whose sea surface temperature (SST) field exhibits a strong temperature difference of per se. Our experimental and numerical findings suggest that if we do not incorporate sea ice dynamics in our models, approximating the case of an initially ice-free Antarctica, then the expected global cooling does not occur, somewhat contradicting the traditional reasoning. However, if the continent is cold enough, thus sea ice is present, then the opening can indeed enhance further glaciation and cooling. These findings, although admittedly conceptual in nature, may contribute to the better understanding of the timing of the various EOT-related events.Here we do not aspire to accurately model neither the actual paleoceanographic processes in their vast complexity, nor the present-day Southern Ocean dynamics that cannot be fully understood without considering, e.g., water exchange with the Atlantic and internal mixing. Instead, inspired by the significance of the Drake Passage opening, the present study investigates the underlying physical basics of the heat exchange dynamics in a topologically similar, but radically simplified system. The widespread claim which we intend to critically address is that an opening event resulting in the formation of a circumpolar pathway could, in itself, lead to the thermal isolation (and glaciation) of the polar region and, eventually, the decrease of global temperature, via blocking meridional heatflow. If so, this reasoning would suggest that Antarctica may not have necessarily been covered with a permanent ice sheet before the formation of the circumpolar pathway. It is also possible, however, that due to other reasons the glaciation preceded the Drake Passage opening, and the latter merely accelerated the further cooling. Thus, regarding the physical basics of the problem, the key question we are focusing on is whether or not the topological reorganization of the ocean currents, facilitated by an opening event, is sufficient to yield cooling 19: the so-called differentially heated rotating annulus, introduced by Raymond Hide in the early 1950s20. The setting captures the two most relevant factors in the formation of ACC-like geostrophic jets and baroclinic surface vortices, namely the meridional temperature gradient and planetary rotation . In panel (b) the WST field of the “open” configuration subject to the same forcing is presented, exhibiting irregular baroclinic (Rossby) waves and much less pronounced temperature fluctuations  and skewness (Sk) of the first (closed) and second (open) leg of the WST signals from the 12 infrared channels (“latitudes”) in 7 experiments is presented in Figs. ΔTr see “” sectionons Fig. b and—as ons Fig. c.14, in this configuration the radial advective heat transport is independent of rotation rate Our fluid dynamical interpretation of the above observations is the following: in the “closed” configuration the barrier facilitates the buildup of a marked azimuthal  pressure gradient associated with strong radial  flow which enhances full-depth overturning. According to the “conjecture” of Raymond Hide21, for a differentially heated rotating annulus within the Rossby number range investigated here, it is expected that these eddy effects are responsible for the largest part (at least 60%) of the total advective meridional heat transport, in analogy with the situation of the present-day Southern Ocean, where overturning is also primarily eddy-driven.In the “open” case, where a mean zonal pressure gradient would not get established, the advective heat transfer in the meridional direction shows a nontrivial dependence on the rotation rate—or, more precisely, on the thermal Rossby number—of the system.  With the increasing angular velocity the radial heat transport steadily decreases, up until the point where the rotation rate is large enough—i.e. the thermal Rossby number is small enough—for the onset of baroclinic instability. Then, with the emergence of eddies, two new ways of meridional heat and mass transport emerge and become increasingly dominant: direct eddy flux, and the perturbation of the zonally symmetric meridional transport by the action of the eddies. These eddy-related contributions compensate for the decreasing transport attributed to Ekman layer transport so that the total meridional heat flow remains almost constant, but still smaller than in the non-rotating (or the “closed”) case. According to a combined numerical and experimental analysisThe stronger overturning—characteristic to the “closed” configuration—yields a reduced temperature contrast between the near-surface and deeper domains of working fluid. Hence, in this case, it is reasonable to assume that the average surface temperature is closer to the volumetric average of the whole water body. In the “open” setting, however, the dominantly eddy-driven overturning (turbulent or wave) yields weaker heat transport and, hence, larger vertical temperature gradients  of intermediate complexity, the so-called Planet Simulator (PlaSim), developed at the University of Hamburg25 for the Late Eocene–Early Oligocene COInstead of implementing the realistic paleogeographic shorelines and the (mostly unknown) paleobathymetry of the Eocene epoch, the “closed” configuration was modeled simply by a full-depth meridional barrier (“dam”) in the Drake Passage connecting the tip of South America and the Antarctic peninsula, while otherwise keeping the present-day continental arrangement unchanged, as sketched in Fig. As an adjustable setting, the so-called ice module of PlaSim could be switched on or off. When active, the module computes the extent and thickness of sea ice dynamically as the temperature field changes. When turned off, however, even when the water temperature decreases below freezing point in a given grid cell, the formation of ice shelves is inhibited and, hence, so are the resulting effects on the system . The latter setting therefore approximates the laboratory experiment where sea ice dynamics is also absent. Fig. Panels (a) and (b) of Fig. The spatial patterns associated with the (imaginary) opening event’s impact on the time-averaged surface temperatures of the Southern Ocean and Antarctica are demonstrated in the maps of Figs. per se does not cause such cooling, in agreement with the findings from the laboratory experiment.To summarize, sea ice dynamics—most notably ice-albedo feedback—appears to play a key role in the chain of mechanisms that connect the opening of the Drake Passage and the overall decrease of temperature. Without sea ice dynamics the opening Our results indicate that the classic interpretation of the EOT global cooling, and the narrative of Antarctic glaciation in particular, needs to be reconsidered. The findings from a conceptual laboratory model as well as numerical simulations in a GCM suggest that under constant thermal forcing conditions the opening of a meridional barrier does not necessarily yield an overall decrease of surface temperatures. Moreover, it may even have the opposite effect—a slight warming in the investigated region—if sea ice formation is inhibited. If, however, the conditions are such that they allow for ice coverage to be present in the “closed” and “open” configurations alike, then indeed—in line with the paleoclimate record—the South polar region gets markedly colder in the “open” case than in the “closed” one.26 also indicated that closing the present-day Drake Passage alone would not necessarily affect the mean SST in the Southern Ocean significantly. Instead, the observed differences between the closed and open configurations were found to be very sensitive to the CO2 concentration and, hence, the sea ice cover in the region. Moreover, another work using a fully coupled atmosphere-ocean model with a mid-Oligocene geography27 suggests that the development of ACC appears to be more of a consequence than a driver of the global cooling, and as such it rather acted as a feedback mechanism in the EOT. More recent numerical studies29 also emphasized the amplifying role of the ice-albedo feedback in the process, in agreement with our findings.Earlier numerical simulationsThe question arises, however, of why we observe increased water surface temperatures in the laboratory experiment after the opening, whereas the Southern Ocean’s mean surface temperature remains practically unaffected by changing the configuration of the Drake Passage in the ice-free  GCM simulations Fig. . First, Our laboratory experiment is a radically simplified, abstract representation of the Southern Ocean. The setting does not capture wind stress or interior deep-ocean mixing, just to name two crucial factors that propel and shape the present-day ACC. Beta effect would also play a key role in a more realistic closed configuration, as the northward flow at the closed passage would likely form a western boundary current. Nor does our laboratory model involve the effects of other continental boundaries and heat and material exchange with the ocean gyres. Nevertheless, the lessons learned here on the heat transfer of quasi-geostrophic flows in the two most basic topological configurations are relevant and insightful. Removing the barrier allows the formation of an ACC-like meandering circumpolar jet, and blocks the development of a mean zonal pressure gradient, thus reducing meridional heat transport. In this “open” setting the mainly eddy-driven remaining overturning is expected to yield increased vertical temperature contrast in the system, which manifests in the increase of surface temperatures.5. With sea ice already present at the continent, the opening could indeed catalyze further ice formation  and could eventually lead to the thermal isolation of Antarctica and the strengthening of the ACC. However, in the hypothetical case of an initially sea ice-free Antarctica the continent could have become even warmer following the opening, a scenario not indicated by paleotemperature reconstructions. The proposed story line is consistent with the interpretation of DeConto and Pollard6, who argued that the falling atmospheric CO2 level was the primary cause for the glaciation of Antarctica, and consequently, this event could possibly predate the opening of the gateway.These findings, even though they may seem counterintuitive, provide circumstantial evidence in favor of the assumption that the Antarctic coastal waters may already have been, at least partially, ice-covered before the opening of the Drake PassageL is the characteristic horizontal length-scale (e.g. basin size), H denotes the fluid depth, Ta which quantifies the relative importance of viscous effects:The desired hydrodynamical similarity of the flow in the experiment to the actual ocean circulation requires certain nondimensional numbers (similarity parameters) to match, at least to the order-of-magnitude. In case of thermally driven rotating flows that are studied here, the most important similarity parameter is the thermal Rossby number  For our experiments two differentially heated rotating annuli were used, one located at the von Karman Laboratory of the Eötvös University  and another at the Fluid Centre of the Brandenburg University of Technology . The geometrical parameters of the Budapest tank are as follows: the radius of the inner (cooled) cylinder is Seven of the experiment runs were conducted in the Budapest tank, which is equipped with a mentclass2pt{minimTa—calculated with The temperature setpoints and the corresponding values of mentclass2pt{minim19, therefore the fact that the nondimensional numbers do not match precisely are not relevant from a qualitative point of view.The studied parameter range falls in the same geostrophic turbulent dynamical regime as the aforementioned values  it proved to be an appropriate numerical tool to investigate possible behaviour of the climate system on global scale.In the present study the climate model, Planet Simulator (PlaSim)30, i.e., the horizontal resolution of the simulations is T21, which yields a grid of approximately 5.6° × 5.6°. As a non-standard aspect of our model setup, the PlaSim atmosphere is coupled to a large scale geostrophic (LSG) ocean 24. The atmospheric dynamics are described by primitive equations that represent conservation laws, thermodynamics and the hydrostatic approximation. Through parameterization, the model accounts for numerous unresolved processes, including sea ice formation, which is a key feature in our study. The so-called sea ice module is based on the zero layer model of Ref.33. This model computes the thickness of the sea ice from the thermodynamic balances at the top and the bottom of the sea ice. The zero layer model assumes the temperature gradient in the ice to be linear and eliminates the capacity of the ice to store heat. For each marine cell on the grid, sea ice is allowed to form when the surface temperature drops below 271.25 K (–1.90 °C). If a grid cell is covered by sea ice, snowfall is accumulated on top of the ice. Snow is converted to sea ice if there is sufficient snow to suppress the ice/snow interface below the sea level. The typical sea ice thickness for the fully ice-covered sea is around 1 meter.For our purposes, we make use of the same atmospheric setup as in Ref.24. This LSG ocean model was originally proposed by34, and is described more fully by35, and it has been used in a number of climate and paleoclimate studies . In Maier-Reimer et al. 1993 the LSG ocean model was investigated in details. It has been showed that the simulated mean ocean circulation for appropriately chosen surface forcing fields adequately and realistically reproduces the principal water mass properties, residence times, and large-scale transport properties of the observed ocean circulation within the constraints of the model resolution. We used the default resolution of 3.5 x 3.5 degrees and 22 non-equidistant vertical layers along with a realistic present-day bathymetry. The typical maximum basin depth is 5500 meters. We mention that the LSG model was also applied previously to investigate the role of the closed Drake Passage in ocean dynamics38. In our model setup the original LSG model is modified, the “closed” configuration was modelled simply by a full-depth meridional barrier (“dam”) in the Drake Passage connecting the tip of South America and the Antarctic Peninsula, otherwise keeping the present-day continental arrangement unchanged. The initial state and other properties of the LSG model ocean are the default ones24. The LSG ocean is spun up with present geography and bottom topography for a period of 10000 years to reach steady state. After the spin-up we compute a 1000 year long period to investigate the impact of the closed/open Drake Passage.The LSG ocean model is based on the observations that for large scale ocean circulation models designed for climate studies, the relevant characteristic spatial scales are large compared to the internal Rossby radius throughout most of the ocean. At the same time the characteristic time scales are large compared with the periods of gravity modes and barotropic Rossby wave modes. The LSG ocean model was developed by Maier-Reimer and Mikolajewicz in early 1990sSupplementary Information.


Ruppia sinensis habitats with annual and perennial populations were surveyed in the Yellow River Delta (YRD). Biomass and seasonal seed bank size were used to evaluate population status and potential recruitment capacity. Sediment geochemical parameters including moisture, sulfide, Chl a, carbohydrate, OM, TOC, TN, and TP were measured to compare sediment nutrient composition and variability. The results revealed a higher biomass and larger seed bank in the annual R. sinensis population compared with the perennial population. The P levels in sediments between the two R. sinensis populations were similar; while the N level in the sediment of the annual population was significantly higher than the perennial population, which might support the recruitment of vegetative shoots when a large amount of seeds germinated during wet periods. The annual population exhibited greater resilience after habitat desiccation, with the population recovering rapidly once water appeared. The results of this study add to the knowledge of R. sinensis populations and their sediment geochemical characteristics, and can be used as a reference for Ruppia population conservation and management.Annual and perennial populations commonly occur for the same submerged aquatic angiosperm species, yet relationships between population types and sediment characteristics are poorly understood. In the current study two  Phragmites australis, Suaeda salsa, and Tamarix chinensis being the dominant species  is the broadest, and best conserved wetland ecosystem in temperate China . Rich sa species . Increas species . These cst China . Meanwhi storage .Ruppia, a genus of submerged marine angiosperms, commonly inhabit shallow systems, such as coastal lagoons and saltmarshes  elucidate the main factors resulting in R. sinensis populations being differentiated as annual or perennial and (2) assess the effect of R. sinensis absence on habitat sediments. The results of this study could serve as simple records for Ruppia populations in YRD, further our understanding of annual and perennial Ruppia characteristics and provide more information to inform Ruppia management.In the current study, we selected two 2 . The water level at Site 1 fluctuates seasonally, becoming dry in winter. Site 1 has an annual R. sinensis population. Site 2 occurred around a brackish water pond  and was approximately 5,000 m2 in area. The salinity at the two sites ranged from 7.2–11.6 to 9.3–16.7 psu, respectively, and there was little anthropogenic influence at either site.The Yellow River Delta (YRD) is a wetland ecosystem in the warm temperate zone of China covering an area of approximately 5400 km2 . The YRD2 . Yu and km apart , comprisR. sinensis populations over a period of 2 years. This observation period included an unusual extreme desiccation event from March 2018 to May 2018 , showers or thunderstorms , moderate rain or snow , and heavy rain or rainstorm . We then quantified the monthly precipitation for each of these four precipitation categories.We investigated the two May 2018 , which rR. sinensis populations were observed from October 2016 to July 2018 . We randomly sampled four R. sinensis cores  from May to August 2017 to compare the biomass of the two populations. The recruitment capacity of the two populations was assessed using seed bank size. To do this, we surveyed dynamic seed bank changes at the two sites using four replicate core samples .The vegetative status of both R. sinensis populations. For each deep sediment core, the upper 40 cm was analyzed, which was cut into 2 cm segments  using a cutting ring and packaged into ziplock bags. The seasonal differences in the sediments at the two sites were investigated through four shallow sediment cores  collected at five sampling periods, May, October, and December 2017, and May and July 2018. Each shallow core (upper 20 cm) was divided into 5 cm segments  and packaged into ziplock bags. All of the sediment samples were transported to the laboratory within 2 days (stored on ice to avoid the water in the samples evaporating during transportation), where they were stored at −20°C prior to sample analysis.Sediment cores at two different depths  were collected from the two sites. Four deep sediment cores  were collected at each site in May 2017 and 2018, respectively, to investigate the vertical sediment characteristics in the two a (Chl a), moisture content, carbohydrate, OM, TOC, total nitrogen (TN), and total phosphorus (TP) were measured in every second segment of the deep sediment cores (10 segments from each deep sediment core). Sediment grain size was determined by pooling five segments, i.e.,10 cm/sample, with three replicates. The sulfide content was determined using the iodometric method  of the sediment samples was determined using the following equation:Sulfide content, Chlorophyll c method , and Chlr method . PigmentW represents the fresh weight (g) of the initial sediment, and MD represents the weight (g) of the dried sediment. The dried sediment samples were sieved through a 250-μm sieve, after being ground by hand with a mortar, to remove coarse debris and stones. A number of analyses were then conducted to determine the physical and chemical properties of the sediments. Carbohydrates were analyzed using the phenol-sulfuric acid method . The general differences between the two sites and the monthly differences of each site in terms of biomass (2017: May to August) and seed density  were compared using a one-way ANOVA. The grain sizes including medium diameter (D50) and the clay/silt ratio at every sediment depth of the two populations were compared with one-way ANOVA. Principal component analysis (PCA) was used to assess the relationship between the sediment characteristics and the variables investigated including sample time, sediment depth, and sample site. PCA was performed using the “prcomp” function in the R software program to determine the multivariate ordination of the 11 sediment parameters for seasonal and vertical sediment assessment, and PCA plots were constructed using the FAC-TOEXTRA package  was employed to compare the general effects of population type (sample site), sediment vertical distribution (sample depth), and season (sample time) on the sediment indexes including moisture content, TP, TN, OM, carbohydrate, TOC, Chl  package  in the RR. sinensis at both study sites differed in terms of population type . Although the monthly biomass at Site 1 was higher than at Site 2, these differences were not statistically significant, with the exception of May 2017 . In 2017, the surface sediment contained more water than the deeper layers. In contrast, the deeper layers of sediment were wetter than the surface sediment in 2018, which was the dry year  and rainy value (precipitation), were similar . The higdry year .p = 0.466, p < 0.001). In 2018 TN was lower than 2017 levels at both sites (p < 0.001). TN in the surface sediment of Site 1 was higher than that of the other sediment layers; however, this pattern was not observed at Site 2 , indicating that 6–14 cm was the most suitable sediment layer for sulfide accumulation.Similar levels of TP were found at both sites . During the regular year (2017), the Chl a level in the sediment at Site 1 was higher than at Site 2, which was correlated with the biomass differences between the two sites . There were seasonal changes in the three sediment carbon parameters , and these changes differed between the two sites . Carbohydrate levels at Site 1 showed significant seasonal differences, which were twice as high as Site 2 (p < 0.001). The highest carbohydrate level at Site 1 was observed in May 2018, when water was present again and new R. sinensis seedlings were growing. No significant changes in carbohydrate values were found at Site 2 at this point.Both Chl wo sites . The Chlwo sites . While sbalanced . The TOCbalanced . Moreovebalanced . In mostThe analysis results of the deep sediment cores with eight sediment assessment parameters  and three classifying parameters  explained 64.4% of the variation in the first two components . The firRuppia sinensis is a commonly distributed seagrass in the YRD. Information relating to population types and the relationships between sediment characteristics and vegetative status are essential for population management. Similar to R. cirrhosa and R. maritima (R. sinensis populations in the YRD in the current study. Temperature and light availability are two key environmental parameters impacting the phenology of seagrass (R. sinensis seeds collected from Site 2 germinated immediately under optimal germination conditions, and the dormancy of the remaining seeds could be broken by low temperatures (R. sinensis seeds at Site 1 germinated immediately when surface water appeared, while no seedlings appeared at Site 2 (maritima , annual seagrass . The preseagrass . In our eratures . Howevert Site 2 . It appeRuppia species are more reliant on clonal growth for population regeneration, both annual and perennial populations flower and produce seeds in the reproduction season (R. sinensis annual population in the current study suggested that this population placed more reproductive energy into producing seeds than the perennial population (Ruppia habitats in the Everglades-Florida Bay ecotone (R. sinensis life cycles. The TN levels in the sediment at Site 1 were higher than Site 2, which likely benefited the growth of shallow shoots, increasing the capacity for clonal growth within the population (R. sinensis at Site 1 might also result in more TN input after decomposition of plant material at the end of the life cycle. The higher nutrient levels in the sediment of the annual R. sinensis population might support the recruitment of vegetative shoots when a large amount of seeds germinated during wet periods. In contrast, the nutrient conditions in the sediment of the perennial population were relatively stable.Although perennial populations of other n season . The larpulation . However ecotone , TP in tpulation . In addia content of sediments is a good representation of the abundance of primary producers such as living algae and undegraded macrophyte tissues (a levels below depths of 18 cm in the sediments could imply limited presence of algal and R. sinensis detritus at this depth. However, in the shallow sediment layers, higher Chl a values were found when more R. sinensis was growing, which may be a result of more R. sinensis detritus settling in sediment and more suitable conditions for algae growth at this time. Moreover, a previous study indicated that Ruppia not only provided organic carbon directly to the biogeochemical cycle but also provided physical support for the attachment of other macrophytes, which also amplified the production and diversity of the system (R. sinensis biomass occurred at both sites after the dry spell, which may have resulted in a decrease in sediment sulfide levels. The results of the current study also showed a peak in the vertical sulfide gradient at a depth of 6 cm, indicating that the sediment conditions at this depth were suitable for sulfide accumulation (Detritus from aquatic macrophytes is one of the most important endogenous sources of nutrients in wetlands . The Chl tissues . Low Chle system . Sedimenmulation .Ruppia detritus supports a complex food web through bacterial decomposition (R. sinensis shoots, and then increased significantly when surface water appeared in May 2018, when R. sinensis seedlings germinated at Site 1. Although there was a similar trend in OM at Site 2, there was no germination of R. sinensis seeds. This variation indicated the presence of algae, which emerged quickly when surface water appeared and is also an important resource of sediment OM (R. sinensis were rapidly growing. However, the carbohydrate levels in the sediment at Site 2 were always low (The organic carbon from position . Organicposition . The thriment OM , 7. TOC iment OM . Howeveriment OM , 8, the iment OM . Meanwhiiment OM . The cariment OM . This vaways low .R. sinensis population types in the YRD exhibited different resilience strategies under extreme desiccation conditions. An annual R. sinensis population was present at Site 1. Higher levels of TN were observed at this site which could benefit R. sinensis seedling growth, and promote population recovery when the water re-accumulated after drying up in winter. The appearance of R. sinensis was also accompanied by more algal growth. This not only increased primary productivity, but also increased carbon deposition and enriched the sediment. The appearance of water was the key factor resulting in the two different R. sinensis population types, which could be represented through sediment characteristics such as water moisture content. Of the different carbon parameters used to evaluate sediment carbon deposition in the current study, TOC was the most indicative in explaining the differences between the different R. sinensis populations. There were higher TOC levels recorded in the annual R. sinensis population compared with the perennial population. The results of this study provided a useful reference for the conservation and management of both annual and perennial Ruppia populations. This study also provided an example of the sensitivity of different carbon parameters in assessing the relationships between vegetation and sediment carbon.In summary, the two different The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RG did the conceptualization, investigated the data, and composited the original manuscript. XS performed the methodology. YZ carried out the funding acquisition, supervised the data, and reviewed and edited the manuscript. ScX, SX, SY, YuZ, and XZ investigated the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Preliminary molecular dynamics simulations on the apo form of Mpro were performed taking into account both the hydrogen donor and acceptor natures of the Nδ and Nε of His41, a member of the catalytic dyad. The potential energy surfaces for the formation of the Se–S covalent bond mediated by EBS and EBS-OH on Mpro are discussed in detail. The EBS-OH shows a distinctive behavior with respect to EBS in the formation of the noncovalent complex. Due to the presence of canonical H-bonds and noncanonical ones involving less electronegative atoms, such as sulfur and selenium, the influence on the energy barriers and reaction energy of the Minnesota hybrid meta-GGA functionals M06, M06-2X and M08HX, and the more recent range-separated hybrid functional wB97X were also considered. The knowledge of the inhibition mechanism of Mpro by the small protease inhibitors EBS or EBS-OH can enlarge the possibilities for designing more potent and selective inhibitor-based drugs to be used in combination with other antiviral therapies.The inhibition mechanism of the main protease (M Coronaviridae family as the Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) the shared sequence similarity is ~80% [pro) and a main protease (Mpro) also known as 3-Chymotrypsin-like protease (3CLpro) [pro acts in the immune regulation and cleavage of the polyprotein at eleven different sites generating nonstructural proteins, such as RNA polymerase, endo- and exo-ribonuclease, which are relevant in the replication process of the virus [pro is only found in the virus rather than in the host cell, this protein represents an interesting target for the development of new promising anticoronavirus therapeutic agents [pro forms a homodimer (protomer A and B) and each protomer consists of three domains: domain I (residues 8–101), domain II (residues 102–184), and domain III (residues 201–303) which are connected by a loop region (residues 185–200) [pro, the catalytic dyad consisting of His41 and Cys145 is located in the cleft between domains I and II [pro, the cysteine target of ebselen is crucial for its protease activity [pro and PLpro [pro of SARS-CoV-2 virus with the concentration required to produce 50% of the maximum possible effect and indicating antiviral activity in cells, the EC50, equal to 4.67 µM; superior to that of N3 (16.77 µM) [pro, induced us to investigate the Mpro inhibition mechanism by both EBS and EBS-OH inhibitors [pro inhibition mechanism has been studied by using Michael acceptor and peptidomimetic inhibitors at the QM-MM level of theory [pro and stimulate the design of other similar drugs.Since early 2020 the whole world has been trying to face the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,2. With  is ~80% . The mor is ~80% . Human i is ~80% . Followi(3CLpro) ,7. Mpro c agents ,7. Struc185–200) ,7. In MpI and II ,10. The I and II . The preI and II ,13,14,15I and II ,17,18,19I and II . In factI and II ,23. EBS I and II  by covalactivity . In thisactivity ,27 and hactivity . EBS inhactivity ,30 and tactivity ,34,35,36nd PLpro ,38. Repuhibitors ,39. The hibitors ,41 and ahibitors . So far f theory ,45,46,47pro enzyme [pro in both the Nδ and Nε protonated states of His41 to better evaluate their eventual influence on the covalent inhibition process by EBS. For clarity, Mpro in the Nδ form will in the whole manuscript be named Mpro-HID and Mpro in the Nε form will be Mpro-HIE. To shed light into the inhibition mechanism of EBS and EBS-OH, the potential energy surfaces (PES) for the formation of the covalent complex between SARS-CoV-2 Mpro and the two considered inhibitors have been calculated and analysed, considering the effects of the dielectric value, ε = 80 and ε = 4, for the water and protein environments, respectively. In the case of EBS-OH only the inhibition mechanism by Nδ–His41 was performed. The calculated PES for the two inhibitors can be used to determine whether, and to what extent, a covalent inhibitor is reversible or not. This knowledge is important in order to provide in the near future, additional leads for covalent inhibitors obtained by incorporating small-molecules, such as EBS and EBS-OH, into hybrid molecules which also have few side effects. In addition, the ability of other DFT functionals  as single points on the B3LYP-D3 optimized geometries were tested to obtain more accurate data for the energy barriers and reaction energy of the inhibition mechanism driven by the sulfur–selenium covalent bond formation. Moreover, stimulated by the most recent literature regarding the inhibition mechanism of Mo enzyme ,45,46,47o enzyme ,48,49. Tpro, many works have focused on classical Molecular Dynamics simulations with [pro-HID and Mpro-HIE represent a preliminary step useful to obtain comparative structural and dynamic properties to deeply describe EBS and EBS-OH inhibition mechanisms. In an effort to better understand the molecular mechanisms involved in the inhibition of Mons with ,48,49,50ons with ,51 inhibpro has been well characterized by crystallography. So far about 1000 entries of X-ray crystal structures for SARS-CoV-2 Mpro are present on the Protein Data Bank in an apo form or a complexed one [SARS-CoV-2 Mexed one ,53,54,55exed one  was usedexed one , these iexed one ,57.pro-Nδ and Mpro-Nε, several properties were taken into account: root mean square deviation (RMSD); RMSD-based clustering; root mean square fluctuation (RMSF), solvent accessible surface area (SASA), radial distribution function (RDF), volume of the binding pocket, salt bridges and the H-bond between residues. Results are reported in To compare the dynamic behavior of MThe analysis of the calculated RMSD for the residue pair Cys145-His41 in both the Nδ and Nε forms shown in  allows uA different behavior may also be noticeable in the trend of RMSF see  where itpro-HID and HIE  in Mpro-HID and a gauche-like one (50.69°) in Mpro-HIE.From the superposition of the most representative structure derived from RMSD-based clustering  of the M and HIE , it is ppro HID. Moreover, the catalytic pocket volume changes dependently on Mpro-HID or Mpro-HIE , in which the binding free energy (ΔGbind) depends only on noncovalent interactions and is related to the inhibition constant Ki. In the second stage a chemical reaction transforms the EI complex generating the final covalent complex (E-I). In the case of EBS this mechanism is proposed by MS/MS study [−2 will be zero  of the two molecules, shown in 2) becomes more negative (−0.661 e vs. −0.602 e in EBS) and the oxygen (O3) decreases its charge (−0.597 e vs. −0.618 e in EBS). Furthermore, the charges on C3′, C4′ and C5′ reflect the effect of OH as an activating group on C5′ (ortho) and C3′ (para), while as a deactivating one on C4′ (meta) with respect to EBS.It is possible to remark that the presence of an OH moiety on the position ortho of the N-phenyl ring of EBS introduces a redistribution of the charge that can be fruitful in the course of the inhibition mechanism. The NBO charge values collected in −1) to the totally planar one  are engaged in an extensive network of H-bonds between them and with the His41 imidazole ring. Such behavior confirms that due to the presence of a catalytic dyad (Cys145 and His41) in Mpro [From the water radial distribution function (RDF) analysis, obtained as a function of the distance between the water oxygen and side chains of Cys145 and His41  thiolate, is now able to perform the nucleophile attack on the selenium (TS1) with the formation of a new selenylsulfide bond and the concomitant ring opening, also as result of the water-mediated proton delivery on behalf of His41, finalizing the covalent inhibition (E-I). In this way the highly nucleophilic thiolate anion is formed. This step is very crucial for catalysis performed by CPs . The forContrary to what occurred in previous theoretical studies on proteolysis and on the inhibition of other CPs ,71,72 anpro residues comes from pKa values predicted using H++ computations [Further indications on the protonation states of the Mutations   with broader surfaces indicative of stabilizing vdW interactions, as a consequence of the planar conformation. In addition, we underline the presence of an interaction between selenium with the thiol group acting as donor of electron density typical of the chalcogen bond. This was more pronounced in the HID form (3.54 Å) than in the HIE one (4.00 Å) as also evidenced by the more extended isosurfaces of the NCI (pro-HID and HIE the water molecules trapped inside the active site span the distance from the Nε (δ) of His41 and nitrogen of the selenyl amide moiety with typical H-bond values as shown in In fact, the related noncovalent interaction (NCI) plots  evidence the NCI . These fpro-HID, the EBS assumes a less accentuated twisted conformation (Φ −42.7°) than that in EBS-OH, but more evident than that in EBS in Mpro-HIE (Φ −16.8°).When EBS-OH is considered, the torsion angle Φ between two planes twists the biphenyl-like system to be perpendicular (Φ 84.5°), driven also by the formation of an H-bond between the OH substituent with the carbonyl side chain of the Asn142 present in the catalytic pocket see . NCI anapro-HID will be deeply discussed, while details referring to Mpro-HIE are given in the pro-HID) from the acid counterpart (thiol group of Cys145), S--H: 1.47 Å in HID. Analysis of the obtained imaginary frequency  and 7.4 kcal mol−1 in water (ε = 80) for the Mpro-HID   than the corresponding species in Mpro-HIE in both considered environments (see Looking at εH+) see . The shoents see .The Se–N “scissile” bond suffers a sensible elongation (2.02 Å for both HIE and HID) making it more prone for its next cleavage. pro [−1 and 8.6 kcal mol−1 for ε=80 and ε = 4, respectively  accounts for the arrangements observed in TS1, where the S–Se bond is formed (2.43 Å), the Se–N is now completely broken (3.38 Å) and the neutral His41 is restored . The “se−1 and 9.3 kcal mol−1 in ε = 80 and ε = 4, respectively that is comparable with those obtained in the case of EBS  accompanied by a consequent elongation of the selenium–sulfide bond (2.05 Å)  in a water (protein) environment; related to the starting complex with the neutral Cys145 and His41  . The tignd His41  and, as pro-HID inhibition promoted by EBS, the process continues with the nucleophilic attack of the Sγ atoms on the selenium one and the proton transfer from the catalytic histidine to the nitrogen atom is also mediated by water molecules as depicted in the TS1  using ε = 80 (ε = 4), are related to the previous noncovalent complex EI’  [The inhibition rate constant of SARS-CoV-2 protease by these compounds has not been experimentally determined so it is not possible to compare the calculated barrier with any observed data. From the inhibitory potency investigated for EBS-OH with PL0.67 μM) . On the As can be seen from PESs , and theThat said, some deviation occurred in the case of ε = 4 that proposes for both inhibitors a minor exothermicity of the covalent product see . Probablpro-HID so far, our analysis leads us to propose [−1 [Since the reversibility of the reaction depends on the barrier of the reverse reaction, and with the inhibition process promoted by EBS and EBS-OH showing comparable activation barriers, the process with more exothermic results will also be more irreversible. On the basis of the described behavior of EBS and EBS-OH in M propose  EBS-OH tpose [−1  emphasizpro-EBS system and the whole benchmark was limited to the ebselen inhibitor. In fact, in the case of EBS-OH, the single point calculations with the above mentioned functionals were performed only on the EI and EI’ complexes due to the marked behavior of the non-covalent complex (EI’) with respect to that of EBS, with its implications for the future design of possible inhibitors with improved kinetics.Starting from the principle that no universal rule exists for the choice of the “best” functionals, the B3LYP results for the inhibition process were benchmarked against the latest-generation density functionals, such as M06-2X, ωB97X, M06, M06-2X and M08HX in order to point out an adequate level of theory to use in future investigations on the inhibition promoted by ebselen derivatives. Results are collected in In general, it is noteworthy that the first step of the reaction leading to the noncovalent complex is the most sensitive to the used functionals, in particular for M08HX and ωB97X, in terms of barrier heights which become low enough to suggest that the activation for obtaining the zwitterionic catalytic dyad takes place more easily and regardless of the dielectric constant value see . All theThe same does not apply for the second step of the reaction, since from the results reported in The ωB97X functional  results In the case of EBS-OH, the exothermicity of the formation of the EI’ complex is confirmed by the used functionals see  corroborAs far as the molecular dynamics simulations are concerned, a detailed description is given in the pro-HID inhibition process promoted by EBS for benchmarking, such as M06 [pro-HIE are reported in the Other latest-generation functionals explicitly developed for systems with long-range electron correlations were employed on the Mh as M06 , M06-2X h as M06 , ωB97X [h as M06 , and M08h as M06 ,99,100. h as M06 . ωB97X bh as M06  and was h as M06 ,102. Howh as M06 ,62,65,81h as M06  analysispro. This enzyme plays a central role in the SARS-CoV-2 viral life cycle and for this reason became an attractive drug target against the COVID-19 disease. The inhibition process occurs in two steps: Cys145 is activated by His41, forming the ion pair Cys-His. The formed nucleophile species (deprotonated Cys145) performs the nucleophile attack on the selenium with the formation of the selenylsulfide bond, and takes place in concert with the ring opening promoted by the water-mediated proton delivery by His41, leading to the stable covalent enzyme-inhibitor complex. The S–Se covalent bond formation, the focus of covalent inhibition and a common step of the two examined inhibitors, implies a series of the chemical events that are caught well in the stationary points intercepted through potential energy surfaces. For both inhibitors, the process result is irreversible even if it is more accentuated in the case of EBS-OH. Our outcomes allow us to suggest that, as already found in PLpro SARS-CoV-2, in Mpro SARS-CoV-2, EBS-OH may result in being a more potent drug. Our calculations have provided deeper insights on the observed inhibitory potency of EBS-OH with respect to EBS as presented in the greater stability of the EI’ complex relative to that of EBS. The cluster used appears to be adequate for the examined inhibition, providing information that should be exploited to generate good candidate SARS-CoV-2 inhibitors. Our results could contribute to increase the current knowledge of small-molecule covalent inhibitors and stimulate the design of drugs obtained by their incorporation into new hybrid non-Michael acceptor inhibitors. Although the winning strategy such as vaccines is available against the virus, the resource of antiviral drugs represents a helpful approach to reduce the signs of the disease caused by SARS-CoV-2 or by its variants.In the present work, the covalent inhibition of the main protease from SARS CoV-2 by repurposing organoselenium compounds, such as ebselen and its derivative on the position 2 of benzisoselenazol-3(2H)-one ring, was investigated at the QM level in the framework of density functional theory. By using a large cluster of 314 atoms our quantum chemical computations have clarified, at an atomistic level, the inhibition mechanism of two ebselen-like non-Michael acceptor covalent inhibitors of M


The GTP-binding protein, Rho, plays a significant role in the cellular pathology of Parkinson’s disease. The downstream effector of Rho, Rho-associated kinase (ROCK), performs several functions, including microglial inflammatory response and enhanced Parkin-mediated mitophagy. Its inhibition shows neuroprotective effects in carried studies. Parkinson’s disease pathology also rests on incomplete removal of damaged mitochondria, leading to neuronal impairment. ROCK has different isoforms, inhibition of which have been shown to decrease the adverse changes in microglia. There has also been evidence of a decreased release of inflammatory cytokines and a reduction in degradation of dopaminergic neurons on the addition of ROCK inhibitors. Additionally, ROCK inhibitors have recently been shown to increase the activity of hexokinase 2 (HK2), relocating it to mitochondria, and therefore leading to upregulated mitochondrial targeting. Understanding the cellular basis of ROCK activity and its inhibition may help us advance in creating new strategies for the treatment of Parkinson’s disease. Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by several neuronal symptoms, including rigidity, tremors, bradykinesia, and postural instability. Neuropathological hallmarks include loss of dopaminergic neurons in the substantia nigra, leading to decreased transmission of dopamine ; and intRho-associated kinase (ROCK) is a downstream effector of the small GTP-binding protein Rho . ROCK haAdditionally, there has been increasing evidence suggesting loss of mitochondrial function and its insufficient degradation as central in PD. This happens by ubiquitin ligase Parkin and protein kinase: PTEN-induced kinase 1 (PINK1) mediated mitophagy -11. AmplIsoforms of Rho-associated kinaseROCK is a serine-threonine molecule with diverse functions in neurons as can be seen in Figure Microglial response to Rho-associated kinase activityMicroglia are phagocytic cells with migratory potential. For such cells, cytoskeletal activity is significant and not only enables them to migrate but also to remove cellular debris. This cytoskeletal activity is dependent on ROCK. Interestingly, ROCK also causes the release of cytokines and chemokines, which is initiated by ligand binding to toll-like receptors and specific receptors, releasing superoxides and nitric oxide . ROCK inEvidence of neuronal degeneration in response to Rho-associated kinaseOver the years, several studies have demonstrated neuronal cell death after induction with dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and MPTP metabolite 1-methyl-4-phenylpyridinium (MPP+). Treatment of mice with MPTP, mediated degeneration of dopaminergic neurons and its terminals in the substantia nigra compacta (SNc). However, a simultaneous reduction was observed in this process on the administration of ROCK inhibitors, indicating its role in dopaminergic neuroprotection . FurtherROCK inhibition also suppresses the release of inflammatory cytokines (interleukin- ß and tumor necrosis factor-a) from microglial cells. The microglial inflammatory response is also mediated by the interaction of ROCK with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, producing high concentrations of reactive oxygen species to kill invading organisms and ROCK inhibitors prevent this activation .Therefore, ROCK involvement in Parkinson’s disease is multifactorial, involving several pathogenic processes. It suggests ROCK inhibition being a promising candidate for dopaminergic neuron protection.Mitophagy during normal circumstancesMitophagy is a complex process as seen in Figure Interestingly, once relocating to the OMM, Parkin then ubiquitylates numerous target proteins, including HK1 and HK2, indicating a possibility of this step being required for Parkin recruitment. This reflection also states that Parkin activity may be terminated by turnover of HK1/2 after ubiquitylation. Loss of hexokinase activity would leave the PINK1/Parkin process inactive .Role of ROCK inhibition in PINK1/Parkin-mediated mitophagyAs stated above, PINK1 induces the translocation of Parkin, an E3 ubiquitin ligase, to the impaired mitochondria for their removal via autophagy (mitophagy). The ROCK inhibitors are evidenced to activate the Parkin-mediated mitophagy pathway thereby causing 399 neuroprotective effects in paraquat-treated flies .Studies have identified that MYO6 (myosin VI), moves towards the minus end of actin filaments, interacts with Parkin, and is recruited to the insulted mitochondria via its ubiquitin-binding domain. This myosin then initiates the formation of F-actin cages, which encapsulate the injured mitochondria . F1-actiROCK2 also has a role in phosphorylating cofilin and stabilizing the actin cytoskeleton. Therefore, a recent study has stated the possible effect of ROCK inhibitor in mitophagy by destabilizing the actin cytoskeleton. This facilitates the formation of F-actin cages around depolarized mitochondria, which leads to their degradation. Moreover, the study speculates the effect attributed to increased levels of mitochondrial HK2 . An addiThis was evidenced in an additional screen performed, showing Parkin recruitment to depolarized mitochondria, identifying HK2 activity as required for this recruitment with shared effects of HK1. Further, knockdown of both HK1 and HK2 showed stronger inhibition of Parkin recruitment than HK2 alone. This explains that HK isoforms are required for Parkin relocation and targeting of mitochondria .This provides us with facts that Hexokinase could be stated as an essential component in mitochondrial targeting and is a potential target for the treatment of Parkinson’s disease. This can be done by ROCK inhibition, which is shown to increase hexokinase activity.Rho-associated kinase inhibitorsSeveral types of ROCK inhibitors have been reported. Fasudil (isoquinoline derivative) is one. It blocks ROCK competitively at the adenosine triphosphate (ATP) binding site of the kinase. Both ROCK1 and ROCKII are inhibited by fasudil with IC50 values of 0.26 μM and 0.32 μM, respectively. Hydroxyfasudil .Y-27632 is another type of ROCKI and ROCKII inhibitor that competitively binds to the ATP site . A more Safety profileROCK inhibitor, Fasudil has not been evidenced to cause severe side effects on patients with cerebral vasospasm. Moreover, it was well-tolerated in clinical trials, including patients with conditions of stable angina or on the treatment of acute ischemic stroke. They showed no unsafe effects or adverse changes in heart rate and blood pressure readings .Nevertheless, studies share that ROCK1 or ROCKII knockout mice demonstrated developmental irregularities, including extreme prenatal defects in mouse embryos on the administration of Y-27632, indicating less understood functions of ROCK during the developmental stages of the embryo. Further, the administration of Y-27632 exhibited behavioral changes in mice on emotional tests. More ocular side effects were seen on a high dosage of Y-39983 in eyes of rabbits and monkeys, showing punctate subconjunctival hemorrhages. The above-stated side effects in tested animals suggest that more in-depth assessments are required on ROCK inhibitors to have a better understanding of their safety profile .Local effects on the eye include blepharitis and blurred vision. Systemically, ROCK inhibition was evidenced to induce an adjustable drop in lymphocyte counts in limited individuals .LimitationStudies conducted on the effect of ROCK inhibition on increasing hexokinase-mediated mitophagy by PINK1/Parkin process amplification specifically are recent. This article provides only details of 2 ROCK isoforms with little depth given to the effects of their inhibition individually.Evidenced studies elaborate on ROCK functions and provide ample suggestions on how its inhibition can make therapeutic contributions in the treatment of Parkinson’s disease. There are collective indications of increased dopaminergic activities in cells with inhibited ROCK and, similarly, dopaminergic deterioration when subjected to MPTP, supported by rising levels of ROCK noticed. Parkin relocation to damaged mitochondria and subsequent mitophagy is also indicated to be enhanced on ROCK inhibition, which causes an increase in HK2 activity: required for Parkin relocation, mediating mitochondrial engulfment.These collective pieces of evidence are significant in establishing a cellular basis of Parkinson’s disease. It is essential that these molecular processes are thoroughly understood in order to create therapeutic advances in the near future. However, this was limited to insufficient studies on different isoforms of ROCK. Also, minor clarifications remain on the potential side effects of ROCK inhibition and is yet to be explored. Although ROCK inhibition has been shown to improve the process of axonal degeneration, functional recovery and remyelination remain unclear. The long-standing effects and properties of ROCK inhibition will have to be assessed, even though first preclinical long-term studies display adequate tolerability and safety. Further contributions to its side effect profile or its reassessment in translational trials in patients of PD could bring emerging conclusions about the impact of ROCK inhibitors and their efficacy and safety in neurodegenerative diseases.