Alternatively, promiscuity in signaling may also offer evolutionary advantages and enable different CLEs to work in combination to activate or switch off different RLK signaling pathways.CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) signaling through receptor-like kinases (RLKs) regulates developmental transitions and responses to biotic and abiotic inputs by communicating the physiological state of cells and tissues. CLE peptides have varying signaling ranges, which can be defined as the distance between the source, i.e., the cells or tissue that secrete the peptide, and their destination, i.e., cells or tissue where the RLKs that bind the peptide and/or respond are expressed. Case-by-case analysis substantiates that CLE signaling is predominantly autocrine or paracrine, and rarely endocrine. Furthermore, upon CLE reception, the ensuing signaling responses extend from cellular to tissue, organ and whole organism level as the downstream signal gets amplified. CLE-RLK-mediated effects on tissue proliferation and differentiation, or on subsequent primordia and organ development have been widely studied. However, studying how CLE-RLK regulates different stages of proliferation and differentiation at cellular level can offer additional insights into these processes. Notably, CLE-RLK signaling also mediates diverse non-developmental effects, which are less often observed; however, this could be due to biased experimental approaches. In general, CLEs and RLKs, owing to the sequence or structural similarity, are prone to promiscuous interactions at least under experimental conditions in which they are studied. Importantly, there are regulatory mechanisms that suppress CLE-RLK cross-talk  Thus, the ensuing cell to organ level effects vary as the downstream signal gets amplified and spreads through intercellular communication . This seProliferation is the increase in the number of cells with the same identity as a result of continuous symmetric cell divisions, whereas differentiation involves symmetric or asymmetric formative divisions resulting in cells with different identities. CLE-RLK signaling regulates proliferation and differentiation of meristems, the region that embodies pluripotent stem cells, which continuously supply proliferating cells in the plant body. Plant and non-plant CLE peptides also regulate primordium formation in the root during bacterial symbiosis and nematode infections. Such CLE-RLK mediated regulation of proliferation and differentiation have been reported largely at the organ and tissue levels . HoweverThere are several stages that lead to cell division and differentiation or proliferation at cellular level. When the need for proliferation and differentiation is evaluated, CLE peptides, in addition to other signaling factors, can be secreted to signal the decision. Then the stem cell that will undergo formative division stage 1— receivesMarchantia polymorpha encodes two CLE peptides: (1) MpCLE1, a TDIF-like peptide and (2) MpCLE2, a CLV3-like peptide, and two RLKs: MpTDIF RECEPTOR (TDR) and MpCLAVATA 1 (CLV1). The apical meristem, hosted by the apical notch, is located at the growing tip of the gametophyte body . The apical notch, apart from being responsible for the radial expansion of the thallus, also produces gametangiophores, which are the reproductive structures developed from the thalli  was resistant, implying that PpCLEs regulate proliferation through PpRPK2 (CLE peptides regulate both differentiation and proliferation in moss. h PpRPK2 . This inArabidopsis inflorescence meristem (IFM), two key CLE peptides, CLV3 and CLE40, are expressed in complementary regions: CLV3 is expressed in the central zone (CZ), where stem cells proliferate at a very slow rate. Stem cell daughters can shift to the peripheral zone (PZ) where the cells proliferate at a higher rate and start acquiring organ identities  in the PZ , thus indirectly promoting stem cell proliferation at the CZ  and its descendant cells in the PRM, and it has been shown to mediate both auto and paracrine regulatory signaling  and phloem pole pericycle (PPP) cells from differentiating into PSE likely through paracrine signaling. Cell files surrounding PSE have the ability to reprogram and acquire PSE identity. CLE45 from PSE cell file signals to the neighboring CC and PPP cell files expressing RPK2  to preveCLE25 is expressed in sieve elements of roots . This further led to defects in PSE differentiation during early seedling development. However, at later stages, these earlier PSE defects resulted in significant accumulation of starch in leaves due to compromised starch remobilization caused by defective phloem transport  have been shown to diffuse from phloem to procambium and promote proliferation of procambial/cambial cells by inducing WUSCHEL-related HOMEOBOX4 (WOX4) expression; in parallel, they also inhibit differentiation of cambial cells into xylem (PttPHLOEM INTERCALATED WITH XYLEM (PttPXY). PttCLE41 or PttPXY in hybrid poplar resulted in taller trees with increased stem diameter due to higher cambial cell proliferation and xylem cell formation  and suppresses these divisions by binding BAM1, which is expressed widely across the vascular zone including xylem precursors . BAM2 anvia the RLK HAESA-LIKE 1 (HSL1) expressed in MMCs, meristemoids and PCs likely in an autocrine manner (Meristemoid mother cells (MMC) are the stomatal lineage stem cells which give rise to the majority of the pavement cells (PC) in leaves by undergoing proliferative divisions and subsequent differentiation. An MMC first divides asymmetrically into a large stomata lineage ground cell (SLGC) and a smaller meristemoid cell, which in turn may undergo further rounds of asymmetric divisions forming more meristemoid cells. SLGC and meristemoid cells then differentiate into PCs and stomatal guard cells (GC), respectively . CLE9, ee manner . CLE9-HSe manner . Thus, Ce manner .Medicago truncatula, MtCLE12,-13 and -35 are expressed in the root in response to rhizobium and they mediate AON by signaling (by possibly binding) via the CLV1-like RLK SUPER NUMERIC NODULES (MtSUNN) expressed in the shoot. Thus, AON is likely the result of endocrine signaling from root to shoot , which is likely expressed in the leaf phloem  also resulted in hypernodulation. Moreover, LjKLV and LjHAR1 interact with each other , which suppresses excessive nodulation . In Medito shoot . Similarf phloem . Observaf phloem . Moreovedulation . Similarch other . This inMtCLE13 is already expressed in initial stages of nodule development. In the incipient and developing nodule primordium, MtCLE13 is expressed strongly in the dividing cortical cells and mildly in pericycle cells around the regions of bacterial infection. But in the later young, round nodule stage, it is expressed throughout. However, MtCLE12 is not expressed in the developing nodules, but in the later young, round nodule stage where, like MtCLE13, it is also expressed throughout the mature primordium  invade plant roots. The juvenile nematodes typically select a pericycle or procambial cell and inject several effector proteins including CLE peptide mimics into the cells through their stylet. The CLE mimics then reach the apoplast  and maniHsCLEB  length and LR numbers. It is hypothesized that CLE26 controls auxin transport, thus modulating the auxin distribution during LR initiation  fungi and rhizobia . Araya ear level  to influar level . Dong etondition . In summCLE6 is expressed at the bases of young rosette leaves and of floral organs, whereas CLE5 is expressed at the bases of young rosette leaves and of cauline leaves. CLE5 and 6 act downstream of leaf patterning factors in the leaf primordium to fine tune final leaf morphology. The mutants cle5 and cle5;cle6 show mild defects in leaf width, curvature and symmetry . It functions in parallel with CLAVATA signaling in mediating these processes. Several CLE peptides, including CLV3, and the RLKs BAM1, -2 and CLV1 are involved in this CLAVATA signaling pathway although how they interact to regulate flower primordium outgrowth and complete flower formation is not yet characterized. At higher temperatures, these processes are controlled by the transcriptional repressor EARLY FLOWERING 3 (ELF3) that bypasses CLV2/CRN signaling  and via other downstream regulators, including ABA. CLE9 overexpressing plants generally had smaller stomatal apertures and the leaves exhibited lower rate of water loss. Notably, unlike in the case of CLE25, BAMs are not the mediators of CLE9 signaling. Although the two CLEs might share the same downstream signaling components, the RLK that responds to CLE9 is not yet identified  and suppress strigolactone levels, thus reducing the rate of colonization  expressed in pollen. CLE45 signaling via SKM1 and SKM2, which are both expressed in pollen, have been shown to prolong pollen tube viability at high temperatures by maintaining its mitochondrial activity until it reaches the ovules  will provide a greater prospect of defining the ancestral role.2. The spatial dimension: Who initiates the CLE signal, who receives it and how?Understanding the spatial aspect of a signaling process is essential as it enables us to delineate the cells, tissue or organs that are in direct communication. For example, induction of stem cell proliferation in a given tissue could be a consequence of CLE peptide produced by the neighboring tissue  where rapid differentiation takes place.On the other hand, how the CLE signal reaches the destination is an essential part of the signaling process. The overall analysis of CLE signaling range show that auto- and paracrine CLE signaling is more common than endocrine. It is likely that establishing long-distance communication is challenging as it depends on several factors such as (1) controlling the expression level and concentration of the peptide which further depends on its stability , dilution during transport through xylem, duration, consistency and level of expression of the peptide (2) post-translational modification of the peptide (3) access to the transport system which includes expression pattern of both the peptide and RLKs, as they should be expressed in the vicinity of vascular tissue: CLE peptide for its extensive transport and RLK for conveniently binding the transported peptide molecules (4) conditions in the destination cells or tissue where further dilution or concentration of the peptide molecules can take place , 2016. HAn important attribute of the CLE-RLK interaction is promiscuity. Promiscuity is a broad term used in two main contexts :“Broader specificity”—This is when a protein can bind multiple ligands or interact with different proteins to mediate different effects. For example, “hub” proteins that contain disordered regions, which offer them conformational plasticity to serve as centers of signaling networks .“Physiologically irrelevant interactions”—This is when a protein interacts with a substrate in an artificial test scenario, which will not occur in the natural system. For example, a group of enzymes that are expressed as a part of an operon and function together will perform a particular process in the natural system. But one isolated enzyme, in a new environment, might perform an entirely new function owing to its active binding site that could bind another substrate .Promiscuous interactions among proteins in a given system could be influenced by: (a) Sequence and/or structural similarity among the possible binding partners, which decides the binding affinity to a specific interacting partner compared to other proteins , (b) Co-Promiscuity has been observed among CLEs and RLKs during their interactions—be it natural or forced, such as in experimental simulations. Here, we have illustrated some of the common observations with examples.Several CLE peptides (from the same or different species) causing the same effect—Examples: Exogenous application of CLE25, CLV3, CLE46, and TDIF upregulate NCED3 expression in leaves  CLV1 and BAM family of RLKs  and of its closest paralogue SlCLE9 in tomato SAM  and internal developmental signals trigger CLE/RLK expression, thus controlling their expression timing—CLE40 expression zone in the PZ in response to the proliferation/differentiation status of the SAM  controlled? Can it be achieved solely by regulating the CLE-RLK expression pattern and levels?What are the downstream signaling components of CLE-RLK and, by extension, the downstream effects that are conserved across clades? Understanding the differences in closely related non-angiosperm vascular plants will already be insightful.MN and RS conceived the idea. MN wrote the manuscript and made the figures. All authors contributed to the article and approved the submitted version.MN was supported by the DFG through a grant within the CRC1208 to RS.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.


Cannabinoids have the potential to be therapeutic for neurological and psychiatric diseases associated with neuroinflammation, neurodegeneration and pain . In Duncan et al., human neural precursor cell culture (ReN cells) were exposed to sublethal oxidative stress [tert-butyl hydroperoxide (tBHP)]—which is particularly associated with neurodegenerative diseases. They found that exposure to tBHP increased protein levels of cannabinoid receptors (CB1 and CB2) and of an N-acylethanolamine metabolizing enzyme, fatty acid amide hydrolase (FAAH), which metabolizes AEA, as well as the associated N-acylethanolamine molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) , a synthesizing enzyme for AEA. Intriguingly, they also found that the mild level of oxidative stress also increased neurite outgrowth. It is possible that because AEA can serve to promote neurite outgrowth  in FAAH (C385A) mouse model . They found that Raloxifene reversed the effects of mTBI on contrast sensitivity, light aversion, pupillary excessive dilation and optic nerve axonal loss. Furthermore, in the injured optic nerve, Honig et al., show that IBA1+ cell numbers are increased and have a normalized transcriptional profile after 10 mg/kg of Raloxifene following mTBI. Therefore, highlighting a further protective role for CB2 inverse agonism in the regulation of IBA1+ cells (predominantly microglia) following mTBI, which may contribute to the beneficial outcomes seen in visual behavior following mTBI. The protective effects of inverse agonism might seem counterintuitive, as inverse agonists suppress constitutive activity and activation of CB2 has shown a role in cytokine signaling and is generally considered anti-inflammatory (Young and Denovan-Wright, The remaining articles in this Research Topic discuss the role of CB2. Ferranti and Foster, which highlights a role for CB2 in schizophrenia, a disease becoming increasingly understood to possess inflammatory risk factors (Comer et al., Ferranti and Foster review reminds us that CB2 receptors have also been described on some neuronal populations. Therefore, a crucial next step in the field is to better understand the cellular distribution of central CB2 and the interplay between neuronal and non-neuronal CB2 under basal physiological conditions and across disease phenotypes.Our final article is a mini-review by Further understanding of the complex signaling of this system will hopefully lead to the generation or refinement of therapeutics for a host of neurological and psychiatric diseases and articles in this collection have contributed toward this goal.HV and VJ wrote the initial draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


Thromboelastography and rotational thromboelastometry are the viscoelastic point of care devices that use whole blood samples to assess coagulation and fibrinolysis. These devices give information from initiation of the coagulation cascade, activation of clotting factors to fibrin cross-linking, and contribution of fibrinogen and platelet to clot strength and clot lysis. Viscoelastic point of care tests are well established in hypocoaguable states like trauma, cardiac surgery, liver transplantation, and their use in critical care settings with coronavirus disease 2019 (COVID-19) is not so well-known. We performed a systematic review of studies on thromboelastography and rotational thromboelastometry and their modifications to assess their role in critically ill patients with COVID-19. Inclusion criteria were any kind of studies using thromboelastography or rotational thromboelastometry during coronavirus disease critical illness published in English. Ninety-three articles, from December 1, 2019, to August 31, 2020, were identified in the initial search, out of which 12 articles  satisfied the inclusion and exclusion criteria. Thromboelastography and rotational thromboelastometry were observed to detect the hypercoagulable changes and fibrinolysis shutdown associated with COVID-19. Hypercoagulability is associated with an increased risk of venous thrombosis and micro-thrombosis. This review identifies the role of thromboelastography and rotational thromboelastometry in studying the mechanisms contributing to coagulopathy and incidence of thrombosis in COVID-19. Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are able to detect a hypercoagulative state and fibrinolytic shutdown in coronavirus disease 2019 patients with findings of higher clot strength and shorter clot propagation time with a prolonged lysis time. A hypercoagulable state may be detected early by TEG/ROTEM with serial measurements being more informative in guiding about the trend of coagulation disturbance.Fibrinolysis shutdown as detected by viscoelastic tests may help in the prediction risk of thromboembolism.1 In addition to major venous thromboembolic events, arterial complications including myocardial infarctions have been reported in COVID-19.2 Thromboembolic complications may be a direct cause of death in COVID-19. Thrombotic microangiopathy was observed in an autopsy series of patients with COVID-19 in the United States.3The healthcare system all over the world has been challenged by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a series of 184 critically ill patients infected with coronavirus disease 2019 (COVID-19), the rate of venous thromboembolism was observed to be 26% and arterial thrombosis was observed to be 3.7%.5With the impact of change in coagulation homeostasis on a patient’s clinical condition, it is imperative to monitor coagulation and fibrinolytic system in patients with COVID-19. Prothrombin time (PT)/International normalized ratio (INR)/activated partial thromboplastin time (aPTT) measure the clotting activity of plasma, ignoring other contribution of platelets and fibrinogen whereas platelet count remains usually normal in COVID-19, with fibrinogen values giving only a quantitative measure, rather than a qualitative assessment.7,13 Rubulotta et al4 advised use of POC viscoelastic tests in all patients with COVID-19 with severe pneumonia as activation of coagulation and/or fibrinolysis may occur as part of host response. In addition, they postulated that technology such as the TEG could guide the clinician in understanding this disease by defining specific targets for treatment.Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are point of care (POC) viscoelastic hemostatic assays that provide an objective graphical evaluation of kinetics of all the stages of coagulation from initiation and propagation of clot formation till the time it reaches maximum strength and finally dissolves.We, therefore, conducted a qualitative systematic review of retrospective and prospective studies in critically ill patients with COVID-19 who underwent viscoelastic POC coagulation tests (TEG/ROTEM) and compared them with conventional coagulation tests (PT/aPTT/platelet count) to assess hypercoagulability.A comprehensive search strategy was developed following a consensus among the co-authors in collaboration with an external expert. Online databases were queried by pairing the keywords “COVID-19” Or “Coronavirus disease” OR “2019-nCoV” with “Thromboelastography” or “Rotational Thromboelastometry” along with their modifications. Three electronic databases Medline (Pubmed), EMBASE, and Google Scholar were searched from December 1, 2019, to August 31, 2020. Full text of all relevant articles was obtained and evaluated. The references of these articles were then hand searched manually. This systematic review was carried out in accordance with Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. Inclusion and exclusion criteria were established for studies to be suitable for inclusion in the systematic review to determine the relevance of data since we wanted to study how the TEG/ROTEM has been used in COVID-19.Inclusion criteria: (1) We included all publications in critically ill patients suffering from COVID-19 and the use of either TEG or ROTEM along with their modifications. (2) Title and abstract in the English language. (3) Retrospective and prospective observational and case-control studies were included where either TEG or thromboelastometry had been used to study coagulation system in patients with COVID illness.Exclusion Criteria: (1) Animal studies; (2) Viscoelastic tests other than ROTEM/TEG or their modifications; (3) Correspondence, case report, case series, editorial, erratum/correction regarding TEG/thromboelastometry; (4) Articles not in the English language.Three authors searched the electronic databases and screened all the titles and abstracts of the selected articles independently. Reference list of relevant studies was screened to identify any missing publications. Discrepancies were resolved by discussion and consensus. Full text of potentially eligible articles was then retrieved for further study. The full texts of selected articles were then screened. Quality assessment of observational and cross-sectional studies was done using the National Institute of Health (NIH) “Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies,” and any discrepancy was resolved by consensus opinion of the authors. The relevant information was extracted regarding the role of TEG and ROTEM in detecting changes in the coagulation system in critically ill COVID-19 patients admitted to intensive care unit.The search strategy revealed 93 articles from both manual and electronic databases. After removing duplicates, 79 articles were identified. Subsequent screening of titles and abstracts of these articles resulted in 17 potentially relevant papers describing hypercoagulability and thrombosis with the help of parameters of viscoelastic hemostatic assays. The full texts of these articles were assessed on the basis of inclusion and exclusion criteria regarding eligibility for inclusion in the systematic review.A total of 12 articles were included in the quantitative analysis. The details of the selection process and specific reasons for exclusion can be found in the PRISMA diagram . MaximumA total of 380 patients who participated in 12 studies during the period of February to August 2020 were assessed. Of the 12 included studies, 1 was cross-sectional, 6 were prospective, and 5 were retrospective observational study. All the included studies were single-center studies. According to the type of viscoelastic test used in the studies, 6 were based on TEG and 6 on ROTEM. The quality of observational and cross-sectional studies was analyzed using the NIH “Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies” and is presented in Modifications of ROTEM used in the studies were as follows: INTEM, evaluation of intrinsic system of coagulation pathway; EXTEM, evaluation of extrinsic coagulation pathways with tissue factor; FIBTEM, evaluation of fibrinogen contribution to blood clot with tissue factor and cytochalasin D which blocks contribution of platelets to maximum clot firmness leaving the impact of fibrin formation and polymerization; and modified TEM-tPA, exogenous tissue plasminogen activator is added to assess hypofibrinolysis.Definition of the parameters assessed in the TEG studies was as follows: R time (minutes), which is a measure of the clotting time (CT) from coagulation initiation to the appearance of the clot; K (minutes); α-angle, which is the velocity of clot formation; MA (mm), which is the maximal amplitude of the clot; and Lys-30 is the percentage decrease of clot amplitude at 30 minutes post-MA. Most of the included studies with TEG have defined hypercoagulability as decreased R time, K time, Ly30, and raised α angle as well as MA. Two of the studies have also included coagulation index as a measure of hypercoagulability and values more than 3 were considered significant. In ROTEM, the following parameters were analyzed: CT in seconds corresponds to the initiation phase of the clotting process from the beginning of coagulation cascade until the increase in amplitude to 2 mm; clot formation time (CFT) in seconds reflects the measure of the propagation phase of whole blood clot formation with an increase in amplitude starting from 2 to 20 mm; A5 and A10 reflect the clot strength at 5 and 10 minutes; maximum clot firmness (MCF) in mm is the maximum amplitude in millimeters reached in thromboelastogram giving a measure of clot stability; Ly30 is the fibrinolysis at 30 minutes, Ly60 is the fibrinolysis at 60 minutes; and maximum Lysis (ML%) is the measure of fibrinolysis. Most of the included studies in ROTEM have not clearly defined criteria of hypercoagulability. Most studies have suggested that values above or below the reference range will be used to distinguish hypercoagulability. Only one study has defined hypercoagulability as short CFT in EXTEM and high MCF in both EXTEM and FIBTEM. Fibrinolytic shutdown is a phenomenon defined as the presence of elevated D-dimer levels and low fibrinolytic activity. Ly30 < 0-0.8% is a parameter associated with fibrinolytic shutdown and high risk for thrombosis.Most of the studies with TEG and ROTEM have assessed the incidence of hypercoagulability in terms of clinical evidence of thrombosis  while some have assessed hypercoagulability only in terms of numbers of TEG and ROTEM parameters above the reference range. 17 Maatman et al15 have estimated that 58% of the patients have at least 1 TEG parameter above the reference range while 83% of the patients have 2 or more TEG parameters above the reference range. Two studies have assessed hypercoagulability in COVID patients directly in terms of hypercoagulable TEG parameters.18 The most common parameters associated with hypercoagulable TEG are high α-angle and MA and decreased Ly30. Mortus et al16 concluded that TEG MA has 100% sensitivity and negative predictive value.16 Fibrinolytic shutdown denoted by Ly30 <0.8% was found in 5 out of 6 studies18 . Clot formation time was assessed in 4 out of 6 studies, while MCF was assessed in 5 out of 6 studies showing hypercoagulable values.18 Fibrinolytic shutdown denoted by Ly30 = 0 was found in 1 out of 6 studies in EXTEM and FIBTEM assay14  remain within normal reference range and are not associated with an increased risk of thrombosis.17 with a 36% incidence of microthrombosis reported in 1 study.14 Five out of six studies in ROTEM have reported the incidence of thrombosis from 22% to 60%.24 However, none of the studies focused on the therapeutic use of TEG/ROTEM to guide treatment. Though we were able to assess our primary outcome of hypercoagulability, it was difficult to conduct a metaanalysis as data collection was at different time points in different studies.In this systematic review of 12 studies involving 380 patients with severe COVID-19-related critical illness, we observed hypercoagulable TEG/ROTEM findings as evidenced by higher clot strength and shorter clot propagation time in most studies. Another significant finding noted was the presence of fibrinolysis shutdown, which may be a very important contributor toward disease severity. The conventional laboratory tests of PT/INR, aPTT were observed to be within normal limits, with consistent presence of raised D-dimers and fibrinogen in all studies. Though the incidence of venous thrombosis was not analyzed in 3 studies with TEG, it was observed to vary from 25% to 43% in patients with hypercoaguable TEG in the remaining 3 studies,30 However, D-dimers test is non-specific and may be affected by age, gender, race, immobilization, hemodialysis, active malignancy, rheumatoid arthritis, sickle cell disease, and prior thromboembolic disease.31 In COVID-19 illness, especially in severe patients, the mechanisms of elevated D-dimer or thrombosis may include older age, chronic diseases, hypoxemia, hypercytokinemia, coagulopathy, and inevitable prolonged bed rest.32 In a recent meta-analysis, elevated D-dimer and fibrin degradation products (FDP), prolonged PT, and decreased antithrombin predict higher risk stratification and poorer prognosis in COVID-19.32 This ambiguity of conventional laboratory tests further sheds light on the significance of POC viscoelastic tests to study characteristics of coagulopathy.We observed that PT/INR and aPTT, which are frequently used to guide anticoagulant therapy or prognosticate, remained within normal limits in all studies though raised D-dimers and fibrinogen were consistently present. D-dimer indicates in vivo thrombus formation and plasmin-mediated degradation of cross-linked fibrin. Multisystemic manifestations of COVID-19 may be present with elevated circulating D-dimer levels.37 Endothelial damage was observed to be intimately linked with coagulopathy in severe sepsis as evidenced by an association between higher circulating biomarkers of sepsis and hypocoagulability.37 Johansson et al38 observed that hypocoagulability as evaluated by TEG was frequent at admission in general ICU patients and associated with a higher rate of ventilator treatment, higher rate of renal replacement therapy, and higher use of blood products. They found that hypocoagulability is an independent risk factor for 30-day mortality. Both procoagulant and anticoagulant states in disseminated intravascular coagulopathy (DIC), as indicated with thrombelastography, were demonstrated to have good correlations with clinically important organ dysfunction and survival. Compared with conventional coagulation tests, TEG/ROTEM can detect impaired fibrinolysis, which can possibly help to discriminate between sepsis and systemic inflammatory response syndrome.36Viscoelastic POC coagulation tests like TEG and ROTEM assess clot formation, strength, and dissolution by measuring the amount of continuously applied rotational force that is transmitted to an electromechanical transduction system and displays the result as a graph. The greatest advantage of POC Venous embolism (VE) assays is the assessment of qualitative function of multiple components of clot formation and lysis in a single rapid assay including platelets, fibrinogen, soluble coagulation factors, and other blood cellular components. Even in non-COVID era, POC viscoelastic assays like TEG and ROTEM have been used in critically ill patients.41 The coagulation pattern in severe COVID illness appears to be unique as evidenced by the presence of multiple fibrin thrombi within distended small vessels and capillaries and extensive extracellular fibrin thrombi in recent autopsy studies.42The coagulation disorder in COVID-19 is complicated. Various theories of increased macro- and microthrombotic complications in critically ill patients with COVID proposed include immune dysregulation, upregulation of ACE2 receptors with direct viral invasion, and perivascular inflammation causing endothelial injury and impairment of fibrinolytic system leading to a prothrombotic state.23 This was the reasoning proposed by Collet et al23 for the unlikely presence of fibrinolysis in COVID-19 patients with cytokine storm. Fibrin deposits are found in lungs due to dysregulation of coagulation and fibrinolytic systems. This approach has been used in few case series where thrombolytics with tPA were used in refractory cases of hypoxia, demonstrating improvement in PaO2/FiO2 ratio i.e partial pressure of oxygen to fractional inspired oxygen concentration ratio (P/F) ratios with single dose or prolonged infusions.44 Viscoelastic POC tests may help in monitoring the fibrinolytic activity. In contrast to the hypocoagulable state noted in sepsis in non-COVID patients, hypercoagulability was consistently noted in most studies using TEG and ROTEM in critically ill patients with COVID-19. The mechanism highlighted using viscoelastic tests include findings consistent with faster clot formation  and increased clot strength  along with the presence of fibrinolysis shutdown. So, the hypercoagulability is not just due to hyperfibrinogenemia but also due to impaired fibrinolysis. With the interplay between inflammation, cytokine storm, and subsequent activation of coagulation, SARS CoV-2 is likely to promote massive fibrin formation. Deposition of fibrin in alveolar and interstitial lung spaces along with microvascular thrombosis may worsen respiratory failure. Tissue plasminogen activators (tPAs) have a short half-life (3 minutes), whereas plasminogen activator inhibitor-1 levels are greatly increased.22 These findings were supported by Almskog et al21 who observed that coagulopathy is present early in the course of the disease. Atypical respiratory symptoms observed in COVID-19 may partly be caused by thromboembolism impairing lung perfusion. Rotational thromboelastometry variables may be affected earlier in disease course compared to D-dimers and may be of greater value as predictor of disease severity. In fact, patients with more severe disease may have a more pronounced coagulopathy in COVID-19 patients.21It is very important to identify the time of affliction of hypercoagulable state in COVID-19 to guide precise therapy. Viscoelastic tests suggest that coagulation disturbance may be remarkable in the early course of the disease. Pavoni et al studied the temporal association of ROTEM on the day of admission, fifth, and tenth day. They found that though the propagation of clot formation (CFT on INTEM) was more rapid in the first 5 days at admission, it normalized by the 10th day. However, the MCF remained high on INTEM, EXTEM, and FIBTEM even till the fifth and tenth day after admission with a significant decrease in ML% from admission to the tenth day, signifying a decrease in fibrinolysis. Severe COVID pneumonia was observed to be associated with hypercoagulation that persisted over time.16 observed that TEG MA provided 100% sensitivity along with 100% negative predictive value for thrombotic events, although there was no difference in prothrombin time, INR, aPTT, or platelets. Higher thrombotic rates were detected with TEG results outside of the normal reference range, with a 62% thrombosis rate. They also postulated that underdiagnosis or undertreatment of hypercoagulation may explain the high incidence of unexplained COVID-19 mortalities. Wright et al14 observed that 40% rate of venous thromboembolism (VTE) was reported in patients with fibrinolysis shutdown compared to 5% in those without. Further, α angle and D-dimer were significantly associated with new-onset need for dialysis. A D-dimer level above 2600 ng mL-1 was associated with higher need for new-onset dialysis (73% vs 17%). They found that a combination of 3 parameters of Ly30 of 0%, D-dimer >2600 ng mL-1, and need for hemodialysis was associated with VTE in 50% and need of hemodialysis in 80% of patients and may serve as a sensitive marker for patients most at risk of VTE and other thrombotic complications.14 Similarly, Maatman et al15 also suggested that serial use of TEG may provide useful insights to guide which patients are at higher risks for microvascular and macrovascular thrombosis. Thromboelastography may be used in prognostication and prediction of thrombotic episodes. Mortus et al21 suggested that if an individual at risk of developing COVID-19-related thrombosis is identified at an earlier stage, enhanced prophylaxis with low molecular weight heparin (LMWH) may decrease mortality in this group of patients. Viscoelastic tests like TEG or platelet function analyzer have also been recommended by the Chinese expert consensus on diagnosis and treatment of coagulation dysfunction in COVID-19. In the theory of the immune-thrombosis relationship where inflammation and thrombin formation are directly correlated, heparin could decrease the inflammatory response by blocking thrombin formation. The American Society of Haematology currently recommends standard prophylactic dose anticoagulation for all COVID-19 patients, reserving therapeutic anticoagulation for those with documented VTE.45 Viscoelastic tests of coagulation are easy single bedside tests that can evaluate different components and stages of coagulation and platelet function. Rubulotta et al4 have suggested that bedside technologies like TEG can guide clinician in exploring, learning about this new disease, and treating these patients to a more specific target.None of the included studies used TEG/ROTEM to guide therapy. However, Almskog et alThe quality of the studies included in the systematic review is suboptimal. Among the 12 studies included, 11 were of fair quality whereas 1 was of poor quality as analyzed using the NIH “Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies.” The different modifications of ROTEM like EXTEM, INTEM, FIBTEM, or TEM tPA are used in different studies. Most of the studies have only documented TEG at a single time point. However, this may not be the optimal measure of a patient’s coagulation profile over the duration of their ICU stay or hospitalization. Most studies have not mentioned the time gap between heparin and POC testing, which may have had an impact on the test result. Similarly, outcomes reported in different studies are different. Though some studies have reported the overall rates of thromboembolic events, true occurrence rates of pulmonary embolism are not known or underestimated as many patients have not undergone computed tomography scan due to logistic issues. This systematic review strongly establishes that TEG/ROTEM helps in the detection of hypercoagulation and fibrinolysis shutdown. Serial TEG/ROTEM over the course of illness may be more informative in guiding about the trend of coagulation disturbance. They can help in understanding the mechanism, time course, and prognostic value of coagulation disturbance in critically ill patients with COVID-19. It is prudent to understand that no test can work in isolation, and any tool is only as useful as the acumen of the clinician for its utilization, for prevention, diagnosis, or to guide therapy. Hence, constant vigilance is recommended. Further randomized controlled trials with more specific standardization of time of testing, repeated evaluation, and response to therapy so as in order to use these tests as a guide to therapy are the need of the hour. The relationship of TEG/ROTEM to thromboembolic events may be more instructive.


Mitochondrial and immune cell dysfunction contributes to the development of pulmonary arterial hypertension (PAH). We thus aimed to investigate mitochondrial respiration and mitochondrial gene expression patterns in the peripheral blood mononuclear cells (PBMC) of patients with idiopathic and hereditary PAH and their correlation to disease parameters. Mitochondrial respiration determined using high-resolution respirometry was not significantly different in PBMC when comparing an outpatient cohort of PAH patients with healthy controls. However, when directly comparing mitochondrial respiration to the hemodynamic parameters of an inpatient PAH cohort, mitochondrial respiration negatively correlated with pulmonary vascular resistance (PVR) and positively correlated with the cardiac index (CI). Furthermore, microarray analysis shows upregulation of mitochondrial erythroid-specific 5-aminolevulinate synthase 2 (ALAS2), as well as the regulation of genes involved in iron and heme metabolism, in the PBMC of patients with PAH, with ALAS2 upregulation in PAH patients being confirmed on the protein level. Multiple regression analysis with age and gender as confounders showed that both PVR and hemoglobin content negatively correlated with maximal respiration. Therefore, we conclude that mitochondrial function in the PBMC of PAH patients is affected by disease severity. However, further studies to investigate cell-type-specific alterations and functional consequences are necessary. Pulmonary arterial hypertension (PAH) is characterized by the abnormal remodeling and occlusion of pulmonary precapillary vessels, with a subsequent increase in pulmonary vascular resistance. Similar to cancer cells, in PAH, cellular metabolism is shifted from glucose oxidation to anaerobic glycolysis, which promotes proliferative pathways. Further mitochondrial alterations, including changes in mitochondrial dynamics (increased fission) and membrane potential (hyperpolarization), may also be important drivers of pulmonary vascular remodeling leading to PAH . PreviouPatient informed consent was obtained before the study commenced. This study was approved by the local authorities (AZ 17/18) and accords with the ethical principles for medical research involving human subjects set out in the Declaration of Helsinki.n = 10) to PAH patients from our outpatient clinic  to investigate alterations in mitochondrial respiration associated with the disease. (2) To correlate mitochondrial respiration with disease severity, we investigated a second inpatient group  with clinical indications for right heart catheterization so that sample collection and hemodynamic assessment could be performed at the same time point. We included patients with the diagnosis of IPAH and HPAH (according to the Nice classification) to exclude the effects of underlying conditions in PAH associated with other diseases  We compared the mitochondrial respiration of PBMC isolated from age- and sex-matched controls . The level expression of β-actin  protein was used as the loading control. The dilution of primary antibodies was 1:1000 for ALAS2 and 1:50,000 for β-actin. The dilution of corresponding secondary antibodies was 1:5000. The incubation time for primary antibodies was overnight at 4 °C, while for corresponding secondary antibodies, it was 1h at room temperature.Statistical analysis was performed with Jamovi (Version 1.6.23.0) using ANCOVA for the comparison of respiratory states in the patient groups (with age and gender as covariates), as well as GraphPad Prism 9 using a linear model for correlation analysis and multiple regression analysis. For Detailed Methods, please Refer to the The mitochondrial respiration and respiratory ratios were not significantly different in PAH patients and controls a,b. To fTo our knowledge, this is the first study to investigate mitochondrial respiration in the PBMC of PAH patients. We did not find a significant difference in mitochondrial respiration between a control group and two cohorts of IPAH/HPAH patients. However, our data provide evidence that mitochondrial respiration is decreased depending on disease severity in PAH patients, suggesting that multiple factors affect mitochondrial respiration in PAH. Since mitochondrial respiration decreased with increasing PVR, our findings accord with the concept of the metabolic switch in PAH, which includes decreased mitochondrial oxidative phosphorylation ,3,4,12. n-numbers that did not allow for analysis of the differences between IPAH and HPAH patients. Additionally, alterations detected in the microarray analysis were confirmed for the key enzyme of hemoglobin metabolism, ALAS2, but not specifically for other genes. Therefore, in future studies, a larger patient cohort should be investigated to confirm our findings and allow for a detailed analysis of different PAH subgroups.This study was conducted as a single-center study with limited Mitochondrial respiration in the PBMC of PAH patients correlates with disease severity and Hb levels. Further studies should investigate the contribution of FAO, mitochondrial biogenesis, and mitochondrial dynamics to alterations in mitochondrial function in PAH-PBMC, as well as their consequences for PBMC function.


Neurodegeneration is associated with defects in cytoskeletal dynamics and dysfunctions of the vesicular trafficking and sorting systems. In the last few decades, studies have demonstrated that the key regulators of cytoskeletal dynamics are proteins from the Rho family GTPases, meanwhile, the central hub for vesicle sorting and transport between target membranes is the Rab family of GTPases. In this regard, the role of Rho and Rab GTPases in the induction and maintenance of distinct functional and morphological neuronal domains (such as dendrites and axons) has been extensively studied. Several members belonging to these two families of proteins have been associated with many neurodegenerative disorders ranging from dementia to motor neuron degeneration. In this analysis, we attempt to present a brief review of the potential crosstalk between the Rab and Rho family members in neurodegenerative pathologies such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington disease, and amyotrophic lateral sclerosis (ALS). Despite their functional and morphological complexity, neurons share special requirements in intracellular trafficking between neuronal compartments involved in synaptic transmission, that include the soma, multiple dendrites, the axon, and the pre- and postsynaptic components , which are further divided into several subfamilies. In humans, the Rho family of GTPases contains 20 members and is divided into eight subfamilies  and Myosin Light Chin Kinase (MLCK) modulates actin stress fiber formation  chemokine used for stimulation  in rat hippocampal neurons , Picks disease (PiD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis , which are enriched with hyperphosphorylated Tau proteins  compared to Vav+/+ microglial cells  inhibitor  and neuronal-WASp (N-WASp). Cdc42 can also modulate the organization of microtubules by interacting with Cdc42-Interacting protein (CIP4). As a modulator of both actin and microtubule assembly, Cdc42 can induce both filopodia and membrane protrusions , and RhoJ  are associated with elevated expression of Cdc42 in the frontal cortex, compared to age-matched controls  are primarily restricted to neurons, but the A, B, and C isoforms can be found in endocrine tissue as well. The D isoform is expressed in adipose tissue, exocrine glands, and the endocrine pituitary. The GDP/GTP exchange cycle of Rab3A is required for the CaRab3 GTPases are predominantly localized to the presynaptic active zone where they regulate the priming and docking of synaptic vesicles Vieira, . InteresSeveral findings directly implicate Rab3 in synaptic dysfunction associated with Parkinson’s disease  via reducing the accumulation of downstream soluble toxic species  who escaped the disease, identified a rare Rab10 variant as a potential provider of molecular resilience against AD  identified Rab11 as a key regulator of Aβ production. The molecular mechanism involves Rab11-mediated recycling of BACE-1 to EE, followed by enhanced Aβ production , Par3, and Par scaffolding protein , wsp-1, was shown to play an important role in synaptic transmission at the neuromuscular junction . The WSP-1 protein was found to be co-localized with Rab proteins in the presynaptic terminal  and geranylgeranyl pyrophosphate (GGPP). FPP (a 15 carbon chain) and GGPP (a 20 carbon chain) are covalently bound to the C-termini of Rho and Rab family proteins (known as protein isoprenylation), assisting these proteins during their relocation to the plasma membrane. Levels of FPP and GGPP have been reported to increase in the aging rodent brain as well as in the human AD brain , a multidomain protein with scaffolding and GTPase GEF domains. ITSN has the ability to modulate Cdc42, Rac, and Ras GTPases. In the case of Ras, when it loses its GDP and before accepting GTP, it is in an in-between state referred to as “nucleotide free ras” (nfRas). Interestingly, nfRas can bind to PI3KC2b, preventing nfRas to bound GTP. ITSN can bind to PI3KC2b, which leads to the release of nfRas and its binding to GTP , and ITSN overexpression leads to repression of RabEP1-mediated endosomal aggregation and results in the degradation of RabEP1  disease, an axonal peripheral neuropathy (Saveri et al., Several studies have shown an interaction between Rab and Rac GTPases through their GEF and GAP proteins. Rac1 has a strong affinity for wild type Rab7, as well as for the constitutively active form of Rab7a. The activated form of Rac1 has also been shown to influence Rab7a activity through its GAP called Armus (Margiotta and Bucci, Cytoskeletal and intracellular trafficking pathologies are two major features of many neurodegenerative diseases. Thus, it would be significant that the modulators of these pathologies, i.e., Rab and Rho GTPases, physically and/or functionally interact to promote the disease progression. The further elucidation of these potential interactions would call for targeting both Rho and Rab GTPases individually or perhaps in combination. Rab GTPases are widely known to control vesicular transport through endosomal and lysosomal systems. While it is well-established that Rho GTPases control cytoskeletal organization and dynamics, recent research supports a role for Rho GTPases in the spatiotemporal control of intracellular membrane trafficking. Rho GTPases have been recognized to directly influence intracellular membrane sorting and trafficking (Olayioye et al., Rho and Rab GTPases play crucial roles in modulating neuronal cytoskeleton and vesicular transport, respectively. There is a body of literature emerging showing their potential interconnection physically and functionally. Understanding the crosstalk between Rho and Rab GTPases will provide greater insight into how the cytoskeleton is working together with the vesicular transport system to digest and export cellular waste, promote neuronal survival, and modulate synaptic remodeling. While it is important to apply a reductionist approach to investigate each family of these proteins as independent players, it is time to also consider them as team players trying to accomplish the common goals of maintaining neuronal homeostasis. To achieve better treatment options for neurodegenerative diseases, it is imperative we recognize the ability of neurons to move important components within and outside of the cell, as well as how it uses the cytoskeletal framework to accomplish this goal.All authors contributed to the writing of this review and agree to be accountable for the content of the work. All authors contributed to the article and approved the submitted version.


Airflow limitation with intractable progressive mechanisms is the main disease feature of chronic obstructive pulmonary disease (COPD). The pathological process of airflow limitation in COPD involves necroptosis, a form of programmed necrotic cell death with pro-inflammatory properties. In this paper, the correlations of small-airway disease and emphysema with airflow limitation in COPD were firstly reviewed; then, based on this, the effects of necroptosis on small-airway disease and emphysema were analysed, and the possible mechanisms of necroptosis causing airflow limitation in COPD were explored. The results showed that airflow limitation is caused by a combination of small-airway disease and emphysema. In addition, toxic particulate matter stimulates epithelial cells to trigger necroptosis, and necroptosis promotes the expulsion of cell contents, the abnormal hyperplasia of pro-inflammatory mediators and the insufficient clearance of dead cells by macrophages; these processes, coupled with the interaction of necroptosis and oxidative stress, collectively result in small-airway disease and emphysema in COPD. Necroptosis induces small-airway disease and acts as an emphysema-specific event rather than apoptosis.Necroptosis is implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD) through various signalling pathways.A complex link exists between necroptosis and apoptosis in COPD.Can necroptosis be ultimately targeted for therapeutic interventions in airflow limitation in COPD?Can the transformation between apoptosis and necroptosis be artificially controlled?Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally . The GloCurrently, bronchodilators  and anti-inflammatory drugs are the medications most commonly used to relieve restricted expiratory symptoms in patients with COPD and act by relaxing the bronchial smooth muscle or inhibiting inflammation. These drugs can delay the decline of lung function, facilitate more effective lung-emptying, remarkably improve movement tolerance and relieve dyspnoea, but they cannot prevent the progression of airflow restriction , 7. TherNecroptosis occurs after cell stress or injury and leads to necrotic morphology and the release of cell contents, inducing an inflammatory response. Necroptosis under normal circumstances is used to maintain homeostasis, but excess or deficiency can cause adverse effects, such as small-airway disease and emphysema. A recent study by Lu et al.  showed tThe respiratory process is divided into pulmonary ventilation and gas exchange. Pulmonary ventilation involves the passage of air in and out of the airways and alveoli. Maintaining adequate airflow during this period is the basis for normal breathing; otherwise, airflow limitation occurs. The main feature of COPD is the slow, progressive and irreversible expiratory airflow limitation. The Global Initiative for Chronic Obstructive Lung Disease uses the degree of airway limitation for COPD diagnosis and the staging of disease severity. Common indicators include forced expiratory volume in 1 second (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) . The pat+ T and CD8+ T lymphocytes and B lymphocytes [The airway acts as a passage for airflow. It branches gradually from the carina to the distal terminal bronchioles . Airway epithelial barriers as innate defence systems are disrupted by direct exposure to toxic particles, causing increased permeability and the release of large amounts of inflammatory cytokines and mucus. In COPD progression, the airway is blocked by inflammatory exudates containing mucus, particularly in small conducting airways (<2 mm in diameter) . A decrephocytes , 12. Besphocytes . Increasphocytes . Excess phocytes . Thus, sThe lungs are elastic. The potential energy generated by diaphragm contraction during inspiration is stored in elastic tissues and forms the elastic recoil of the lungs, which is released to constitute the impetus of lung-emptying during exhalation. The pathological changes in emphysema, a common manifestation of COPD, include irreversible destruction of alveolar walls, excessive dilation of the distal terminal bronchioles and reduced elastic recoil of the lung caused by disruption of the lung parenchyma. Elastic recoil measured at 90% of the total lung volume is associated with emphysema and FEV1 in patients with COPD, indicating that the loss of elastic recoil due to emphysema is one of the determinants of airflow limitation . EmphyseThe product of airway resistance and lung compliance provides the time constant for lung-emptying that is reflected in FEV1 and FEV1/FVC measurements, whereas airflow limitation is the result of the prolonged time constant of lung-emptying . Mao et Necroptosis is the programmed mode of active cell necrosis characterised morphologically by organelle swelling, plasma membrane rupture and cell lysis. When caspase (an apoptosis executive protein) is absent or inhibited, death receptors and Toll-like receptors on the cell membrane connect with extracellular ligands (e.g. tumour necrosis factor) to send an activation signal; then, receptor-interacting protein kinase 1 (RIPK1) activated by this signal phosphorylates RIPK3, and RIPK1 and RIPK3 together form an intracellular amyloid-like complex to assemble the necrosome, which acts as the necroptosis signalling transducer . RIPK3 mNecroptosis is morphologically distinct from other cell death modes except for necrosis and is heavily dependent on RIPK3 and MLKL in terms of pathways, which suggests the main detection methods of necroptosis, as shown in Fig. A recent study applied necroptosis-specific detection and found that patients with COPD have remarkably increased phosphorylated RIPK3 (p-RIPK3) and p-MLKL in lung tissue homogenates relative to those without airflow limitation; furthermore, the differences remained substantial after adjusting for age, gender and current smoking status of patients . This st−/−) and MLKL−/− mice were compared to normal mice under acute and chronic CS exposure, and the results revealed a remarkable decrease in inflammatory cells, chemokines, airway basement membrane thickness and collagen deposition in lung tissues, confirming once again that necroptosis is an upstream mechanism leading to airway inflammation and airway remodelling [Necroptosis induced by multiple common stimuli for COPD models promotes an abnormal increase in the inflammatory response and mucin levels, leading to airway inflammation, mucus overproduction and airway remodelling. Different types of epithelial cells  were exposed to CSE or PM to simulate the lung epithelia of patients with COPD who were directly exposed to toxic PM, and treatment with Nec-1 (a RIPK1 inhibitor)/GSK'872 (a RIPK3 inhibitor)/theaflavin-3,3′-digallate  remarkably reduced the expression of inflammatory cytokines and mucins that make up the airway mucus. The results indicate that necroptosis promotes inflammatory responses and mucin production at the cellular level , 27, 28.−/− mice were not substantial, which suggests that MLKL deletion is more effective in preventing emphysema than RIPK3 deletion, possibly because RIPK3 affects apoptotic and necroptotic pathways, whereas MLKL represents a necroptosis-specific effect. In conclusion, emphysema is a product of necroptosis.Apoptosis has long been believed to play an important role in lung structural remodelling and tissue destruction in COPD, and the rate of positive TUNEL staining is remarkably increased in the emphysema region . HoweverTable CFLAR (an encoding gene for transcribing the cell death regulator protein that heterodimerises with caspase-8 to inhibit necroptosis), which induces a gradual switch from apoptosis to necroptosis as the mode of lung epithelial cell death [Lung parenchymal epithelial cells located between environmental stimuli  and the organism have important functions in maintaining normal respiratory function, including barrier protection, fluid homeostasis, particle clearance, immune response initiation, mucus and surfactant production and post-injury repair. Abnormal lung epithelial cell damage and death are one of the initial events in COPD, as continuous exposure to toxic particles causes an increased incidence of cell death. Necroptosis, an important mode of lung epithelial cell death in COPD, causes the release of cell contents into the extracellular environment, including a large number of danger-associated molecular patterns (DAMPs), such as high-mobility group box-1 (HMGB1), S100 proteins, double-stranded DNA and ATP . The incll death –35 and pll death . After bll death .HMGB1, one of the DAMPs, can be used as a necrosis marker, is highly expressed in the epithelial cells and alveolar macrophages of patients with COPD and is negatively correlated with forced expiratory volume , 37. TheIn addition to inducing macrophages to produce large amounts of pro-inflammatory cytokines, necroptosis can recruit macrophages to the lung parenchyma and trigger the phagocytosis of necroptotic epithelial cells by macrophages, but the rate of clearance is slow , 25.During necroptosis, p-MLKL translocation to the cell membrane exposes phosphatidylserine to generate ‘find me’ and ‘eat me’ signals and releases extracellular vesicles containing phosphatidylserine, p-MLKL and other proteins to the surrounding environment, which drive the recognition and uptake of necroptotic cells by macrophages, but the overall phagocytosis efficiency is lower than that of apoptotic cells . KlöditzNecroptosis and oxidative stress interact with one another, and this circuit may be the autonomous mechanism for the intractable progression of airflow limitation in COPD. On the one hand, key proteins in the necroptosis pathway influence the onset of oxidative stress. The inflammatory mediator TNF-α is remarkably elevated in COPD, and TNF-induced reactive oxygen species (ROS) production is dependent on RIPK3, a key protein in the necroptosis pathway, which may be related to abnormal energy metabolism following activation of the key enzymes of the metabolic pathway by RIPK3 . After tRIPK1/RIPK3-related knockdown and the pharmacological inhibition of RIPK1 by Nec-1 can block ROS production and cell death in response to BV6/TNF-α stimulation, suggesting that RIPK1 and RIPK3 regulate ROS production; by contrast, ROS scavengers can block the interaction between RIPK1 and RIPK3, suggesting that ROS can promote the stabilisation of the RIPK1/RIPK3 necrosome complex to drive necroptosis signalling . Thus, aFigure Necroptosis as the primary form of cell death in the late stage of COPD leads to airway inflammation, mucus overproduction, airway remodelling and emphysema and ultimately causes expiratory airflow limitation in COPD. The related mechanism may involve plasma membrane rupture and the release of a large number of DAMPs into the extracellular environment after the necroptosis of lung parenchymal epithelial cells. The released DAMPs activate pattern-recognition receptors in neighbouring macrophages and induce the massive production of pro-inflammatory cytokines that give rise to immune responses and tissue remodelling. In addition, the increased number of necroptotic epithelial cells is difficult for macrophages to rapidly clear, which can also lead to pathological inflammation in the lung. Moreover, a positive feedback loop formed by ROS/RIPK1/RIPK3 exists in the interaction between necroptosis and oxidative stress, further prolonging airflow limitation in COPD. Therefore, studies on how necroptosis affects airflow limitation in COPD are beneficial to understanding the pathological mechanisms of COPD from a new perspective and may provide new ideas for the treatment of progressive airflow limitation in COPD.


Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a family of broad substrate specificity serine (Ser)/threonine (Thr) protein kinases widely expressed in many tissues that is capable of mediating diverse functional responses depending on its cellular and molecular microenvironment. This review briefly summarises current knowledge on the structure and regulation of CaMKII and focuses on how the molecular environment, and interaction with binding partner proteins, can produce different populations of CaMKII in different cells, or in different subcellular locations within the same cell, and how these different populations of CaMKII can produce diverse functional responses to activation following an increase in intracellular calcium concentration. This review also explores the possibility that identifying and characterising the molecular interactions responsible for the molecular targeting of CaMKII in different cells in vivo, and identifying the sites on CaMKII and/or the binding proteins through which these interactions occur, could lead to the development of highly selective inhibitors of specific CaMKII-mediated functional responses in specific cells that would not affect CaMKII-mediated responses in other cells. This may result in the development of new pharmacological agents with therapeutic potential for many clinical conditions. Other protein kinases have a broad substrate specificity so they can regulate a large number of proteins and functional responses. Such kinases, often referred to as multifunctional kinases, usually have multiple mechanisms of activation and regulation.Protein kinases regulate the function of proteins by phosphorylation at key locations where the negatively charged phosphate group induces conformational changes in the phosphorylated protein that alter its activity or ability to interact with other molecules. Some protein kinases have very narrow substrate specificity so they only regulate one, or very few, proteins and can only be activated in one way  [Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a family of multi-functional serine (Ser)/threonine (Thr) protein kinases , encoded by four genes ( CAMK2D) . One or CaMKII is expressed most highly in neurons and, consequently, its functional roles have been most extensively studied in neurons. The neuronal functions that CaMKII is critical in controlling include neurotransmitter synthesis and exocytosis, synaptic organisation, long-term plasticity, learning, memory consolidation and erasure following retrieval ,5,6,7,8.Herein, we provide an overview of the structure and regulation of CaMKII, with a focus on how the molecular environment, particularly the interaction of CaMKII with binding proteins, can control the function of CaMKII.Each isoform of CaMKII exhibits the same basic domain structure: an N-terminal catalytic domain, a C-terminal association domain, and a regulatory domain in between A. The reThe expression of CaMKII isoforms is highly variable across different tissues, and cells can contain more than one isoform. CaMKIIα and β are mainly expressed in nervous tissue , whereasThe majority of the CaMKII holoenzyme exists as a dodecamer consisting of two hexameric rings of subunits principally held together by the association domains which form an annulus with the association and catalytic domains forming two rings around the annular hub of association domains ,46 FiguC. The arCaMKII is regulated by multi-site phosphorylation and targeting to subcellular locations via interactions with specific binding proteins. Additionally, there is an interplay between these two regulatory mechanisms, as these interactions can be further modified by phosphorylation of both/either the kinase, and/or the binding protein. The interaction between the regulatory mechanisms of multi-site phosphorylation and molecular targeting through interaction with specific binding proteins, creates functionally different pools of CaMKII in different cells and different parts of the same cell. For this reason, the well-characterised behaviour of purified CaMKII in vitro often does not correctly predict the behaviour of CaMKII in vivo. It is also the existence of these functionally different pools of CaMKII that enables CaMKII to be a multi-tasking protein.All the CaMKII autophosphorylation sites are present in every CaMKII subunit and splice isoform and are conserved across species. We will review the functional effects of autophosphorylation at Thr286, Thr305/306 and Thr253, which are the three best-characterised sites.A 1000-fold increase in the affinity for calcium/calmodulin is induced, thereby prolonging the calcium/calmodulin-stimulated activity of CaMKII due to calmodulin ‘trapping’, which results in calmodulin dissociating more slowly once free calcium returns to basal levels .Induction of autonomous activity, which allows the enzyme to remain active even following calmodulin dissociation. Calcium/calmodulin binding disrupts the interaction between the autoinhibitory domain and the ATP and substrate binding sites, thereby activating CaMKII . Change the affinity of CaMKII binding to specific proteins that are located in specific subcellular sites . This alInitial enzyme activity of purified CaMKII requires the binding of both calcium and calmodulin. Following this, autophosphorylation of Thr286 in CaMKIIα  occurs quickly and produces the following outcomes:Under certain pathological conditions that result in periods of elevated reactive oxygen species, a pair of methionine residues in CaMKII (Met281/282), present in β, γ and δ, but not α isoforms, can become oxidised, resulting in an autonomous activation of CaMKII . This peRecent biochemical evidence also suggests that autonomous activity triggers the colocalization of subunits between holoenzymes ,49,55, aThr305/306 in CaMKIIα, and Thr306/307 in CaMKIIβ, γ and ∂, are a pair of secondary sites located within the calmodulin binding domain that can become phosphorylated once CaMKII is calcium-independent (autonomous) and can be phosphorylated under different conditions ,58. PhosThr253 was the first characterised CaMKII autophosphorylation site that controls CaMKII activity purely by regulating molecular targeting and has no effect on the activity of purified CaMKII in vitro . InitialOther sites, such as Ser279 and Ser314, can be phosphorylated both in vitro ,61,62 anThe importance, and mechanisms, of targeting in the regulation of CaMKII has recently begun to be appreciated.All four CaMKII genes undergo alternative splicing , which cThe importance of targeting and isoforms to the regulation of CaMKII is particularly highlighted in CaMKII-mediated cell proliferation. The inhibition of CaMKII can inhibit proliferation in a plethora of cell types ,74,75. OThe oldest evidence in support of CaMKII targeting regulating function comes from the evidence that CaMKII is concentrated in particular subcellular locations, such as the PSD, and that CaMKII can translocate from the cytoplasm . ThiCaMKII can also translocate to the cytoskeleton ,90. BindRegarding CaMKII function, CaMKII must be co-localised with the correct binding partner to produce the required cellular function. A variety of binding proteins have been identified that bind to CaMKII in a manner that varies with the phosphorylation state of CaMKII . Since tTargeting can achieve localised, or cell-specific, functions in one of two ways  can regulate CaMKII, via actions on both CaMKII and its binding partners. PP1, PP2A and PP2C, but not PP2B, can dephosphorylate Thr286 in vitro ,107,108.While PSD-associated CaMKII is primarily dephosphorylated by PP1 ,108,109,Changes caused by the molecular/cellular environment, largely due to the effects of CaMKII binding proteins, can bias the CaMKII autophosphorylation response to activation by calcium/calmodulin. The pattern of expression of CaMKII binding proteins varies with cell type and between nerve cells in different brain regions . Such ceAdditionally, the molecular environment can produce differential CaMKII functional responses by the stimulus-induced movement of CaMKII between different binding partners in protein complexes, such as those found in the PSD or the cytoskeleton . SubsequPanel A of Panel B shows how changes caused by a different signalling pathway can also alter CaMKII-mediated responses. At the top, non-phosphorylated CaMKII is shown bound to the same protein, in the same molecular environment, as in Panel A. A stimulus that does not involve CaMKII is shown resulting in the phosphorylation of a CaMKII binding protein in the complex that has a binding site specific for phosphorylated CaMKII and at a site that inhibits the protein’s ability to bind phosphorylated CaMKII. The initial stimulus-induced rise in intracellular calcium shown in Panel A allows the non-phosphorylated CaMKII to be activated by calcium/calmodulin and to produce the functional response ① A. HoweveComparisons of the CaMKII binding profiles of extracts from different cell lines, and tissues from different brain regions, revealed that different cell types express different CaMKII binding proteins and that treatment of tissue extracts with exogenous protein phosphatase greatly reduced CaMKIIα binding to many, though not all, of the proteins . It is wOnce the NMDA receptor has been activated by synaptic activity, opening its calcium channel and raising intracellular calcium concentrations in the post-synaptic spine and local dendrite, CaMKII in the vicinity can be activated by calcium/calmodulin. The resulting autophosphorylation of Thr286 allows CaMKII to bind to the GluN2B subunit of the NMDA receptor ,94. As lWhen the scaffold protein CASK binds to non-phosphorylated CaMKII, the interaction also induces a conformational change in CaMKII that autonomously activates it, but in a way that favours autophosphorylation at Thr305/306 . When in2+ influx into the dendritic spine activate CaMKII and autophosphorylation at Thr286. It also displaces DAPK1 that is bound to GluN2B and allows p-Thr286-CaMKII to bind to GluN2B, causing CaMKII to accumulate at the potentiated synapses. By contrast, the patterns of synaptic stimulation that produce LTD induce relatively lower levels of Ca2+ influx into the dendritic spine. This leads to calcineurin-mediated activation of DAPK1 and enhanced binding of DAPK1 to GluNR2B which, in turn, prevents the binding of CaMKII to GluN2B and CaMKII accumulation at depressed synapses [Another example of how stimulation-induced changes in interacting proteins can alter the functional outcomes of CaMKII activation is provided by the mechanisms of induction of LTP (long-term potentiation) and LTD (long-term depression)—two opposing forms of synaptic plasticity involved in learning, memory and cognition. DAPK1 (Death Associated Protein Kinase 1) is a calcium/calmodulin-dependent protein kinase that binds to the same region of the GluN2B subunit of the NMDA receptor as CaMKII, and, like CaMKII, is basally concentrated at dendritic spines. Patterns of synaptic stimulation that produce LTP induce high levels of Casynapses .Because CaMKII is involved in so many different cellular functions, including several pathological conditions, the ability to inhibit selectively some of these CaMKII-mediated functions offers potential therapeutic opportunities. However, precisely because CaMKII is expressed in so many different cells, direct inhibition of CaMKII kinase activity has been dismissed as unlikely to be therapeutically useful because of the high likelihood of unwanted side effects due to the inhibition of CaMKII in tissues, or functional responses, which are not involved in the pathological process that is the target of the desired therapeutic drug. The ability to indirectly inhibit CaMKII-mediated functional responses by specifically inhibiting the molecular targeting mechanism that controls the cell-specific CaMKII functional response offers new possibilities for the development of pharmaceutical agents with potential clinical application . Two popTwo examples of pathological conditions in which such specific inhibition of CaMKII targeting might have therapeutic application are neuronal cell death following a stroke and cell proliferation in cancer.Regions of the brain that are more sensitive to ischaemic damage show an increased phosphorylation of CaMKII at Thr253 following ischaemia in vivo, or excitotoxicity in vitro, but no difference in phosphorylation at Thr286 or Thr305/306 . CaMKII We have shown that phosphorylation of CaMKII at Thr286, but not Thr253, controls cancer cell migration and invasion , while pCaMKII located in different microenvironments in vivo can respond to stimuli differently—becoming phosphorylated at different sites resulting in differential interactions with binding proteins, translocation to different cellular locations and producing different functional outcomes. Future investigations should focus on characterising the molecular interactions responsible for the targeting of CaMKII in different cells in vivo. The structural features of the binding sites through which the targeting occurs could then be used to design inhibitors capable of selectively disrupting the interactions between CaMKII and specific binding partners in particular cells thereby interfering with the signalling pathways involved. This approach may then allow the selective control of the functions mediated by this multitasking protein in particular cells, with potential therapeutic applications in many clinical conditions such as ischaemic cell death following stroke .


The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases. Mitochondria are responsible for powering cell metabolism, calcium homeostasis and heat production. However, under pathologic conditions, mitochondria are also sources for reactive oxygen species (ROS) and cell death ,2. ThereMitochondrial diseases include both inherited defects and secondary dysfunctions associated with physiologic aging or pathologic conditions, ranging from neurodegenerative to oncologic diseases. They constitute a remarkable diagnostic and research challenge because of their significant heterogeneity in clinical symptoms and the wide range of enzymatic reactions, coenzymes and transporters involved in energy metabolism. Additionally, two different genomes  contain the different genes , all coding for mitochondrial proteins. For every patient or disease, a definite diagnosis requires the identification of the responsible gene associated with a specific biochemical, pathological and clinical profile, which comprises diagnosis that requires the interdisciplinary cooperation of clinicians, pathologists, biochemists and geneticists [Therefore, since mitochondrial dysfunction underlies numerous disorders, mitochondrial functional assays are often implemented in clinical and research settings to assess mitochondrial involvement in a physiologic or pathological condition. These assays can be performed in a multitude of biological samples and may include a wide range of parameters such as the quantification of ATP or ROS production ,7, oxygeIn humans, MRC is formed by four enzymatic complexes (I-IV) and two mobile electron transporters (coenzyme Q and cytochrome C), driving the electrochemical gradient and proton-motive force for energy production through ATP-proton synthase (complex V). The MRC function can be measured in tissue homogenates, cells or mitochondria-enriched fractions, and the obtained results must be compared to specific reference ranges. MRC analyses are routinely performed in energetic tissues with significant oxidative metabolism, mainly in skeletal muscle, although alternative measurements in less invasive samples such as fibroblasts or peripheral blood mononuclear cells (PBMC) are also measured in clinical settings . AlternaSample collection, tissue homogenization, freezing and storage methods are essential steps for the stability of the mitochondrial enzymes and may vary depending on the centers. Additionally, the spectrophotometric methodologies used for protein quantification and MRC enzyme determinations are very diverse. Differences in several key aspects have been found among different laboratories . In partCentro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), working in the diagnostic and research of mitochondrial diseases, started a coordinated program to establish common protocols for MRC enzyme determinations in all laboratories. The present work summarizes this experience and the reference values and adapted protocols to validate and standardize MRC assessment in mitochondrial diagnosis or research, with specific tips for the implementation of these protocols in other laboratories. The methods of this network for the standardization of MRC enzyme measurements were initially based on the protocols of French laboratories [In this context, seven Spanish laboratories belonging to the ratories  to widenratories . In this−1mg of protein−1) or normalized to citrate synthase (CS) activity, which is a widely considered biomarker of mitochondrial content [MRC complex activities were measured spectrophotometrically and were expressed either as absolute (units), specific enzymatic activities  sample collection; (ii) sample preparation ; (iii) protein measurement; (iv) MRC enzyme assessments; (v) CS quantification; and (vi) online database setup and result analyses .As in the Reagents section, only differences with previous procedures have been highlighted , althoug(i)Sample collection (expected time: 15 min)Human sample collection was carried out after written informed consent signature and ethics committee approval were obtained from each institution 63/2016 and C.I. 2768-N-21). Briefly, muscle, fibroblasts and PBMC samples were obtained for diagnostic purposes following standard medical procedures. These samples were obtained from subjects with clinical suspicion of a potential mitochondrial disease, and the results were used as normality ranges once the mitochondrial defect was ruled out.Muscle samples were obtained from a biopsy of skeletal muscle  and immediately frozen at −80 ± 5 °C until analysis. The characteristics of the individuals included in this study are summarized in For isolated cell measurements (fibroblasts or PBMCs), a collection of a minimum of 5 million cells is recommended. Fibroblasts (collected from a skin punch biopsy and grown in standard culture media) and PBMC (collected in EDTA-plasma tubes and isolated by Ficoll density gradient centrifugation) were washed twice with PBS and frozen at −80 ± 5 °C. For the quantification of MRC in mitochondrial-enriched preparations, fresh tissue or alive cells must be processed before cryopreservation.(ii)Tissue homogenization, cell lysate and mitochondria-enriched preparation (expected time: 15 min–1 h)Sample preparation is a critical step to obtain reproducible measurements. Tissue homogenate can be obtained by grinding or by using a polytron device in fresh or frozen tissue.Frozen muscle samples were quickly thawed on ice-cold mannitol (50 mg of tissue in 200 µL) followed by immediate fragmentation into small pieces using scissors and subsequent homogenization. Although glass–glass homogenizers had been previously recommended , in our For fibroblasts or PBMC, cells were thawed using 150 µL of ice-cold mannitol media for 5 million cells and then slightly sonicated on ice (twice for 5 s at 200 Watts). Cryopreservation is an optional step from collection to sonication. Both procedures (cryopreservation and sonication) enable the release of MRC complexes to allow proper enzymatic measurements .Mitochondria-enriched fractions from tissues or cells were obtained from fresh biological material (never frozen) through homogenization and sequential centrifugations ,16,19.(iii)Protein measurement (expected time: 1 h)Immediately after sample preparation and before MRC measurements, it is recommended to proceed with protein measurement and subsequent sample dilution to a final concentration of 2 mg/mL in mannitol solution to standardize reaction conditions (sample and solution volumes) and to meet the optimal kinetic requirements for MRC enzyme assays.Although both Lowry and bicinchoninic acid (BCA) protein assays are frequently used , in our After protein quantification, we recommend preparing aliquots with at least 500 µL of 2 mg/mL tissue homogenate, 200 µL of cell suspension or 100 µL of mitochondria-enriched suspension for further measurements of MRC. Samples should be immediately stored at −80 ± 5 °C in aliquots, if enzymatic assays cannot be performed within the same day to avoid extra freeze–thaw cycles.(iv)MRC enzyme assessment (expected time: 6 h)All enzymatic assays were performed using single-wavelength and temperature-controlled spectrophotometers, ideally with multi-cuvette carousel  and controlled software analysis . Although different temperature settings can be found in the literature ,16,17,18According to inter-laboratory and intra-laboratory results, enzyme assays with higher sensitivity to freeze–thaw cycles that should be performed on day 1 were quantified with complexes I, I + III and III, which showed lower activities in frozen homogenates (data not shown). Consequently, in this multicentric study, less sensitive complexes CII, CII + III, CIV and CS are recommended to be alternatively measured using frozen homogenates on day 2.Since all MRC enzyme measurements should include an internal control sample run in parallel, all participating centers used frozen aliquots from a common pool of porcine skeletal muscle homogenate at 2 mg/mL, which was also used for protein measurement.MRC activities were assayed as described previously  see Sup, except Although activity ranges and assay slopes were analyzed for each individual enzyme assay, we agreed to establish the previously recommended consensus analytic interval of 180 s for all MRC measurements , except The Complex V measurement (ATP-proton synthase assay) is not included in most MRC assay protocols ,16 becau(v)CS enzyme quantificationThe amount of mitochondria present in the samples is estimated by measuring the activity of the enzyme CS ,18,19. A(vi)Database, online work, quality controls and result analysisThe results of MRC enzyme activities of each participating center are systematically collected and updated online in cloud space where protocols, results and batches are recorded and shared among groups. Databases are routinely updated to gather reference control values, which include either the results of center-specific human samples or those of shared pork muscle aliquots used as common inter-group control samples see . The datThe present study provides data from seven independent centers summarizing all the experimental steps of MRC standardization using data from human muscle samples. The results of MRC enzymatic activities in human muscle homogenates after protocol standardization are shown in The results herein described are comparable to those obtained by the French laboratories , includiThe protocols herein contained and the methodological troubleshooting shared were the starting point of a diagnostic workflow. Despite this, we do not provide data/examples of specific patient’s diagnosis, as it is the daily practice of all laboratories participating in the present methodological standardization. We use the reference ranges provided in The pathological threshold for biochemical mitochondrial disease diagnosis should be set after analyzing the maximal number of control muscle samples available. However, due to invasiveness of muscle biopsies, diagnostic centers usually use those obtained from patients as control samples, when potential pathological mitochondrial conditions have been ruled out. On the other hand, the analysis of previously confirmed pathological samples is also very helpful to validate healthy reference values.The reference interval is usually established as one or two standard deviations from the average reference value, encompassing the 84th to 95th percentile of the healthy population, in the case of normal distributed parameters. However, alternative statistical calculations are also used .Regarding the source of variability for the reference ranges used in the different sites see , we did MRC enzyme deficiency may be suspected when enzyme activity is lower than this reference value, particularly if the patient shows clear symptoms of mitochondrial disease and additional evidence of mitochondrial disfunction such as elevated mitochondrial content (CS activity), abnormal anatomopathological findings (either ragged-red fibbers or COX−/SDH+ cells) or genetic variations.Of note, diagnostic thresholds depend on the source of samples analyzed, as well as reference values and the rate of false positive/negative values assumed. Sample analysis may vary, mainly, depending on size of the cohort, variable distribution (Gaussian or non-parametric) and composition , among others. The rate of sensitivity and specificity assumed  will determine the risk of ascertainment bias in false/positive diagnosis. Thus, each center establishes the pathological threshold based on mean values plus one or two standard deviations (for parametric parameters) or median values and percentiles (for non-parametric parameters), depending on all these considerations . AdditioIn our experience of multicentric protocol implementation, the network for communication in-person, online, by-phone or electronic mail among the members of all participating centers was essential for the work-in team in terms of troubleshooting and result comparisons. Additionally, the daily update of our online cloud-space databases was useful for the following: (i) record comparable information of control ranges, either from human or reference samples; and (ii) detect site-specific deviations, variations over time and batch-dependent changes. The annual exchange of calibration samples was routinely performed to detect potential bias.During the setup of MRC measurements, the comparison of our results with previously published data  was crucOne important source of variability affecting several MRC assays  was the cytochrome c reagent. Further research should be performed to normalize the basal redox balance of this commercial reagent to reduce derived batch-dependent variability.Despite all the above-mentioned complexities, the standardization of protocols for the validation of MRC measurements is an urgent need for all laboratories studying MRC defects, especially in diagnostic centers. It is difficult to avoid epidemiologic, logistic or sample collection differences between sites, which reflects the complexity that diagnostic and research laboratories face on a daily basis with real world circuits. However, it is possible to minimize the source of experimental variability in the laboratory analyses with standardized and robust experimental protocols.We strongly recommend setting up the herein reported protocols, as we found them to be reliable and sensitive, and they are specific methods for the study and diagnosis of mitochondrial dysfunction. These protocols are also recommended in the context of research settings, since they provide reliable methodological protocols for the measurement of MRC in humans not only in muscle biopsies but also in cell and animal models, setting the path for international standardization and the comparison of results in experimental networks or the scientific literature. In the case of MRC deficiency, the further association of mitochondrial defects with ROS production, proton leak, ATP decrease, heat imbalance, membrane potential disruption, dysfunctional mitophagy, altered calcium homeostasis or apoptosis may help to establish a link between organelle-to-cell dysfunction and disease.Mitochondrial respiratory chain enzymatic assays are relevant analytical tools in research and diagnosis with high inter-laboratory variability.Updated and functional standardized protocols, normality ranges and troubleshooting methods are herein provided to overcome the most common experimental challenges.This Spanish multicentric study joins the previous French and Italian initiatives to encourage other countries to establish common international analytical standards.Additional analyses of clinical, genetic and pathological measures should be performed to better understand the contribution of MRC defects to human disease.