Proteins are social beings. Especially disordered proteins like company, they hardly act without interacting with another macromolecule. In most known cases, these other macromolecules are other proteins, sometimes nucleic acids and very seldom something else. Disordered proteins are rather newcomers in protein science. The first papers on these proteins came out in the fourth quarter of the last century. What is more, they were hardly recognized before the great paper of Wright and Dyson published in J. Mol. Biol. in 1999 ,3 providPaper of two merged special issues on the same topic: “Functionally relevant macromolecular interactions of disordered proteins” are summarized and listed in the order of their online publication date. Research, review and concept papers listed separately, starting with the oldest one in this Editorial.In the first paper of this series, a study on the effect of acetylation on the phase separation tendency of Tau protein was reported by Ferreon et al. It is well known that intrinsically disordered protein Tau is involved in Alzheimer’s disease. Recently it was shown that Tau is capable of undergoing liquid-liquid phase separation, which involves weak protein-protein interactions and it is considered as an initiation of Tau aggregation observed in Alzheimer’s disease. In this work, it was shown that acetylation disfavors phase separation and aggregation of Tau, therefore, acetylation prevents the toxic effects of liquid-liquid phase separation dependent aggregation .Srivastava et al. tried to decipher RNA-recognition patterns of IDPs in the next paper. They analyzed the protein-RNA complexes which undergo disordered to ordered transition (DOT) during binding. The DOT region is small and positively charged, like the binding sites in globular proteins. However, for DOTs of IDP have significantly higher exposure to the water, than their counterpart in structured protein. These findings can help to develop tools for identifying DOT regions in RNA binding proteins .Contreras et al. studied a Protein (LrtA protein of Synechocystis sp. PCC 6803) which is oligomeric and folded in solution, but the single-chain is only folded and stable in their N terminal half of the polypeptide (residues 1–100) while the other half (101–197) is very unstable and rather disordered with chameleonic sequence properties. While disordered protein which undergoes mutual synergistic folding upon binding to each other, this is a rather rear case, when it happens only with half of the protein. The other half remains folded before and after self-association .Arabidopsis thaliana at 22 and 37 °C, and they compare both the amino acid compositions and levels of intrinsic disorder of heat-induced and heat-repressed proteins. Heat-induced proteins are enriched in intrinsically disordered regions and depleted in hydrophobic amino acids in contrast to thermophile prokaryotic proteins [The origin of the thermal stability of eukaryotic proteins was studied and compared with that of thermophilic and mesophilic proteins of prokaryotes by Alvarez-Ponce et al. The eukaryotic model system was proteins .A decision-tree based meta server to predict disordered parts of proteins and their residues involved in binding motifs has been developed by Zhao and Xue. The meta server is based on four predictors: DisEMBL, IUPred, VSL2, and ESpritz. The meta server provides higher accuracy than each of these independent predictors .2P2). They concluded that down-regulated genes in stromal cell-adherent lymphoma cells encode proteins that are characterized by elevated levels of disorder [Arvidsson and Wright applied a protein disorder approach characterizing differentially expressed genes analyzing cell adhesion regulated gene expression in lymphoma cells. They checked if predicted protein disorder was differentially associated with proteins encoded by differentially regulated genes in lymphoma cells. Intrinsic disorder protein properties were extracted from the Database of Disordered Protein Prediction  protein are capable of RNA binding. They explored the RNA binding capability of two; uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein They demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not, are capable of binding to different RNA constructs in vitro .1H NMR signals is presented in the next paper. Tompa et al. also provided a description of key elements of the procedure conceived for the thermodynamic interpretation of such results. The results enable a quantitative description of the ratio of ordered and disordered parts of proteins, and the energy relations of protein–water bonds in aqueous solutions of the proteins [A method to characterize the hydration of proteins based on evaluating two-component wide-line proteins .Homma et al, studied the evolution rate structural domains (SDs) and intrinsically disordered regions (IDRs) of immune-related mammalian proteins. IDRs are generally subject to fewer constraints and evolve more rapidly than SDs. However, it turned out that for immune-related proteins in mammals, the evolution rates in SDs come close to those in IDRs .Moosa, M.M. et al. applied direct single-molecule observation to study sequential DNA bending transitions by the SoxSox2 is a transcription factor which assumed to achieve its regulatory diversity via heterodimerization with partner transcription factors. However, single-molecule fluorescence spectroscopy suggests that Sox2 alone can modulate structural landscape of the DNA in a dosage-dependent manner .Synechocystis sp. PCC 6803 in separated form with various physical-chemical techniques. At physiological conditions isolated C-LrtA intervened in a self-association equilibrium, involving several oligomerization reactions. They concluded that C-LrtA was an oligomeric disordered protein [In a paper which was a follow-up of the Contreras, L.M. et al. paper , Neira e protein .Mishra et al. extended their one-bead-per-amino-acid model for intrinsically disordered proteins to account for phosphorylation in studying the effect of phosphorylation on nuclear pore complex selectivity. The simulations show that upon phosphorylation the transport rate of inert molecules increases, while that of nuclear transport receptors decreases. The models provide a molecular framework to explain how extensive phosphorylation decreases the selectivity of the nuclear pore complexes .Walter et al. studied the hydrodynamic properties of the intrinsically disordered potyvirus genome-linked protein, VVPg), of the translation initiation factor, eIF4E, and of their binary complex (VPg)-eIF4E. N-terminal His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study revealed the His tag contribution to the hydrodynamic behavior of proteins .The role of intrinsically disordered linkers in the confinement of binding domains in enzyme actions was studied in the following paper. By statistical physical modeling Szabo et al. show that this arrangement results in processive systems, in which the linker ensures an optimized effective concentration around novel the binding site(s), favoring rebinding over full release of the polymeric partner. By analyzing 12 enzymes they suggest a unique type of entropic chain function of intrinsically disordered proteins, that may impart functional advantages on diverse enzymes in a variety of biological contexts .Machulin et al. studied the contribution of repeats in ribosomal S1 proteins into the tendency for intrinsic disorder and flexibility within and between structural domains for all available UniProt S1 sequences. Using charge–hydrophobicity plot cumulative distribution function (CH-CDF) analysis they classified 53% of S1 proteins as ordered proteins, the remaining proteins were related to molten globule state. According to the FoldUnfold and IsUnstruct programs, relatively short flexible or disordered regions are predominant in the multi-domain proteins. Their results suggest that the ratio of flexibility in the separate domains is related to their roles in the activity and functionality of S1 .Pseudomonas aeruginosa. The results show an increase of the secondary structure content of DBD concomitantly with a decrease of its conformational heterogeneity upon its binding to Azurin [The decrease of disorder level of p53-DBD upon interacting with the anticancer protein Azurin by mean of Raman spectroscopy was monitored by Signorelli et al. This technique was found to be suitable to elucidate the structural properties of intrinsically disordered proteins and was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from o Azurin .Structural and functional properties of a capsid protein of dengue and related flavivirus. Dengue, West Nile and Zika have very similar viral particle with an outer lipid bilayer containing two viral proteins in the nucleocapsid core were studied by Faustino et al. Using dengue virus capsid protein as the main model, the protein size, thermal stability, and function with its structure/dynamics features were correlated. Their findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA .α –helices. The WH2 motif consensus sequences “LKKV” subsequently binds to actin through large conformational changes of the disordered domain V [Chan-Yao-Chong, et al. investigated the early steps of actin recognition of Neural Wiskott–Aldrich Syndrome Protein (N-WASP) domain V. Using docking calculations and molecular dynamics simulations, their study shows that actin is first recognized by the N-WASP domain V regions which have the highest propensity to form transient domain V .Mentes et al.’s paper is the follow-up of the Magyar’s paper  of this Conformational ensembles of alpha-Synuclein were studied using single-molecule force spectroscopy and mass spectroscopy by Corti et al. This work applies single-molecule force spectroscopy to probe conformational properties of α-synuclein in solution and its conformational changes induced by ligand binding. This analysis provides support to the structural interpretation of charge-state distributions obtained by native mass spectrometry and helps defining the conformational components detected by single-molecule force spectroscopy .The topic of the Mészáros et al. paper is closely related to the ones of the Mentes et al’s. paper  and the Three Rett syndromes (RTT) treatment-related genes MECP2, CDKL5 and FOXG1 in silico by evolutionary classification and disordered region assessment were reported in this paper. Fahmi, M. et al. provided insight into the structural characteristics, evolution and interaction landscapes of those three proteins. They also reported the disordered structure properties and evolution of those proteins which may provide valuable information for the development of therapeutic strategies of RTT .Sánchez-López et al. report about the structural determinants of the N-terminus of the prion protein and the effect of binding copper ions in their review. They discuss the current knowledge of how mutations can impact the copper-binding properties of prion protein both in health and disease progression .In their review paper Ciemny et al. first introduced the technique of Monte Carlo and other simulation for predicting possible structures of unstructured protein, protein-peptide complexes and unfolded states of globular proteins. They presented several case studies on various disordered proteins. They also proposed the use of the CABS coarse-grained model with Monte Carlo sampling scheme. They also show that CABS can be combined with the use of experimental data too .The literature information on the alteration of disordered proteins in neurodegenerative and other diseases reported in this review. Martinelli et al. discussed how the misfolded proteins can be involved in Alzheimer’s, Parkinson’s and other diseases. The most common form of misfolding IDPs is the formation of neurotoxic amyloid plaques. The review discusses important special cases of beta-amyloid, alpha-synuclein, tau etc. They also show drug candidates for later use in to treatment of diseases caused by misfolded IDPs .This is the first review from the Greb-Markiewicz lab in which Kolonko and Greb-Markiewicz summarized our current knowledge on helix-loop-helix/Per-ARNT-SIM (bHLH–PAS) proteins, considering their structures and intrinsic disorder nature based on NMR and X-ray analysis. Currently, all determined structures comprise only selected domains (bHLH and/or PAS), while parts of proteins, comprising their long C-termini, have not been structurally characterized yet since these regions appear to be intrinsically disordered. These intrinsically disordered parts contribute a lot to the flexibility and function of these proteins .The second review from the same lab is the paper of Tarczewska and Greb-Markiewicz which is a follow-up publication of the review paper of Kolonko and Greb-Markiewicz , the curFinally, in the last review paper of this collection, Owen and Shewmaker summarized our current knowledge on “The Role of Post-Translational Modification in the Phase Transition of Intrinsically Disordered Proteins”. They pointed that intrinsically disordered regions are critical to the liquid–liquid phase separation that facilitates specialized cellular functions and discuss how post-translational modifications of intrinsically disordered protein segments can regulate the molecular condensation of macromolecules into functional phase-separated complexes .In his concept paper, Rikkerin, E.H.A. considers the “first line response” role of disordered protein in the protection against pathogens and disease. He presents several examples of how disorder and post-translational changes can play in the response of organisms to the stress of a changing environment. He proposes that some disordered proteins enable organisms to sense and react rapidly as the first line responds .


Despite numerous trials investigating robot-assisted therapy (RT) effects on upper-extremity (UE) function after stroke, few have explored the relationship between three-dimensional (3D) reach-to-target kinematics and clinical outcomes. The objectives of this study were to 1) investigate the correlation between kinematic parameters of 3D reach-to-target movements and UE clinical outcome measures, and 2) examine the degree to which differences in kinematic parameters across individuals can account for differences in clinical outcomes in response to RT.. Potential associations between individual differences in kinematic parameters and differences in clinical outcomes were examined.Ten chronic stroke survivors participated in a pilot RT intervention (eighteen 1-h sessions) integrating cognitive skills training and a home-action program. Clinical outcome measures and kinematic parameters of 3D reach-to-target movements were collected pre- and post-intervention. The correlation between clinical outcomes and kinematic parameters was investigated both cross-sectionally and longitudinally . Changes in clinical outcomes and kinematic parameters were tested for significance in both group and subject-by-subject analysesModerate-to-strong correlation was found between clinical measures and specific kinematic parameters when examined cross-sectionally. Weaker correlation coefficients were found longitudinally. Group analyses revealed significant changes in clinical outcome measures in response to the intervention; no significant group changes were observed in kinematic parameters. Subject-by-subject analyses revealed changes with moderate-to-large effect size in the kinematics of 3D reach-to-target movements pre- vs. post-intervention. Changes in clinical outcomes and kinematic parameters varied widely across participants.Large variability was observed across subjects in response to the intervention. The correlation between changes in kinematic parameters and clinical outcomes in response to the intervention was variable and not strong across parameters, suggesting no consistent change in UE motor strategies across participants. These results highlight the need to investigate the response to interventions at the individual level. This would enable the identification of clusters of individuals with common patterns of change in response to an intervention, providing an opportunity to use cluster-specific kinematic parameters as a proxy of clinical outcomes.NCT02747433. Registered on April 21st, 2016ClinicalTrials.gov,  Every year, about 795,000 people suffer a new or recurrent stroke in the United States  leading A large body of literature based on motor learning theories has shown that high-intensity, high-dosage rehabilitation interventions can facilitate sensorimotor recovery in stroke survivors –9. GuideResearchers and clinicians have investigated the use of kinematic parameters of UE movements as a proxy for clinical outcome measures after stroke –18. ThisKinematic parameters derived via tracking of 2D arm reaching movements moderately correlated with FMA-UE scores, WMFT and WMFT-FAS scores, and self-reports of the amount of use (MAL-AOU) and quality of movement (MAL-QOM) of the hemiparetic limb –21. ModeBecause they are more relevant from a functional point of view than 2D movements, several authors have focused their efforts on the analysis of 3D movements and the associations between their kinematic parameters and clinical outcome measures. Seminal work by Cirstea  showed aTo that end, we analyzed pilot data collected during 3D reach-to-target movements in chronic stroke survivors who received a novel robot-assisted therapy protocol to 1) investigate the correlation between kinematic parameters of three-dimensional (3D) reach-to-target movements and UE clinical outcome measures, both cross-sectionally and longitudinally, and 2) examine the degree to which differences in kinematic parameters across individuals can account for differences in clinical outcomes in response to RT.ClinicalTrials.gov (NCT02747433). All participants provided written informed consent according to the Declaration of Helsinki.The study was approved by Partners Healthcare Institutional Review Board and registered on Ten chronic stroke survivors participated in the study . The stuStudy volunteers participated in the Active Learning Program for Stroke (ALPS) in which one-on-one sessions with an occupational therapist consisted of cognitive strategy training and individualized home programs aimed to facilitate the transfer of robot-trained UE movements to functional use of the paretic arm and hand during everyday tasks. In this development of concept study, the repetitive movement therapy was delivered using two commercially available devices: the ArmeoSpring®  and the Amadeo® . All participants received 18 one-hour sessions of therapy over a period of 6–7 weeks. Additional details concerning the intervention are reported elsewhere .Subjects were assessed at baseline and at the end of the intervention using clinical outcome measures across domains of the International Classification of Functioning, Disability and Health (ICF) . A singlA 10-camera motion capture system  was used to track reflective markers placed on the upper body during reach-to-target movements  was used to derive the movement kinematics from the position data of the UE reflective markers. The start and end of the reach-to-target movements were identified using the marker positioned on the metacarpal head of the index finger; events were manually verified. Joint angles were estimated using the Nexus 2.8.1 software via a standard biomechanical model in which anatomical joints are represented as universal joints.The data was then imported to MATLAB  and custom scripts were used to derive the eleven kinematic parameters listed in Table The marker positioned on the metacarpal head of the index finger was also used to derive the following six parameters. The trajectory directness  and the Spearman rank tests were used to assess correlations between the kinematic variables and clinical outcome measures. These analyses were performed both cross-sectionally and longitudinally. Cross-sectional analyses (i.e. pre- and post-intervention assessments) examined whether kinematic parameters were correlated with the severity of motor impairments and functional limitations captured by the clinical outcome measures. Longitudinal analyses (i.e. pre- vs. post-intervention assessments) were performed to determine if the changes in the kinematic parameters and clinical outcome measures following the intervention were correlated. These correlation tests were carried out both for total clinical scores  and for scores associated with the assessment of proximal body segments , as the evaluated kinematics did not measure specific movements of distal body segments . Correlation coefficients were labeled as high (0.70 to 1.00), moderate 0.50 to 0.70), low (0.30 to 0.50) or negligible (0.00 to 0.30), according to the “rule of thumb” proposed by Hinkle et al. [ to 0.70,Paired t-tests were performed at the group level to determine if the clinical outcome measures and kinematic parameters collected during the study significantly changed in response to the intervention. Because of the small sample size, we used bootstrapping with 1000 samples and percentile-corrected confidence intervals for these analyses.d effect size values were calculated for each comparison using the pre-intervention standard deviation as the denominator in the calculation of Cohen’s d. We used Cohen’s conventions of d = 0.20, 0.50, and 0.80, representing small, medium, and large effect sizes, respectively [In addition, subject-by-subject analyses were performed for both the kinematic parameters and clinical outcome measures to examine changes occurring at the individual level using paired t-tests with bootstrapping. Cohen’s ectively . Clinicaectively –54.p-values were adjusted for multiple comparisons using a Holm correction [Statistical tests were performed using SPSS . Significance was set a priori at α = 0.05 and rrection . In the Both cross-sectional and longitudinal correlation analyses examined associations between kinematic parameters and clinical outcome scores. Table rs < 0.70) were observed for the majority of the comparisons. Although statistically significant associations were identified between many kinematic parameters and clinical outcome measures, high correlation coefficients (rs > 0.70) were only found between elbow flexion-extension, thorax rotation, and torso excursion and a clinical measure of UE impairment (FMA-UE).The upper panel (A) of Table rs < 0.50) were found between the changes in the kinematic parameters and clinical outcome measures following the intervention, except for a negative moderate association between the FMA-UE and shoulder flexion-extension.The lower panel (B) of Table Paired t-tests revealed significantly improved motor function with a large and medium-to-large effect size for all clinical outcome measures, except for the performance time (WMFT-Time) which displayed a small effect size Table . DetaileT%Vmax) was marked by a medium-to-large effect size and the change in log dimensionless jerk (LDJ) was marked by a small-to-medium effect  and torso excursion (TExc) as seen in Fig Figure At the individual level, all subjects displayed changes in kinematic parameters, some of which exhibited medium to large effect size. The first row of data in Table Examination of subject-by-subject changes in clinical outcome measures collected during the study also showed remarkably different responses to the intervention across individuals. Table This pilot study examined changes in kinematic parameters of 3D reach-to-target movements in response to an intervention that combined cognitive strategy training and individualized home programs to facilitate the transfer of robot-trained UE movements to functional use of the paretic arm and hand during everyday tasks. Two fundamental questions were addressed via group and subject-by-subject analyses: 1) What are the associations between the kinematic parameters of 3D reach-to-target movements and UE clinical outcome measures? and 2) Can differences in kinematic parameters across individuals account for differences in clinical outcomes in response to RT?The kinematic analysis methods utilized in this pilot study provide the methodology to explore group and individual differences in response to interventions and to identify kinematic parameters that may be used as a proxy for clinical outcome measures. These methods may be used in a future larger clinical trial aimed to further differentiate the key treatment elements  and personal characteristics  associated with the recovery of motor function after stroke. The low to moderate correlation between individual kinematic parameters of 3D movements and clinical scores observed in this pilot study may be attributed to the three factors discussed below.High variability in the response to UE intervention among these chronic stroke survivors likely contributed to the lack of statistically significant correlation as well as unexpected associations between clinical outcome measures and kinematic parameters at the group level. For instance, Table Furthermore, by examining Fig. Importantly, these results highlight the shortcomings of group analyses as the ones reported in Table Although statistically and clinically significant improvements were apparent on clinical outcome measures after only 18 sessions of therapy Tables  and 7, cWould interventions marked by a higher-dosage and delivered over a longer time period have resulted in greater changes in motor strategies reflected in the kinematic parameters of arm reaching movements? While high dosage UE intervention studies after stroke , 57 may The kinematic parameters reported in this study were derived from a single 3D reach-to-target motor task, whereas clinical outcome assessments typically measure the functional use of the paretic arm and hand during a variety of movement tasks. This may contribute to a mismatch between the changes in motor abilities indicated by clinical outcome assessments and those that are kinematically derived from a single motor task. For instance, the kinematics of reach-to-target movements primarily measure the control of proximal body segments. In contrast, changes in the clinical outcome measures employed in the study also reflect changes in the control of distal body segments (i.e. manipulation tasks), which were not investigated by our kinematic analysis. Recent recommendations highlight the need for utilizing a battery of five motor tasks to assess changes observed in movement kinematics during clinical trials with a focus on sensorimotor recovery in stroke survivors . AdditioThe results of this pilot study provide motivation to further explore the mechanisms of recovery during rehabilitation interventions in chronic stroke survivors. The small number of participants did not allow us the use of more complex statistical analyses and did not enable the identification of clusters of individuals who responded consistently to clinical interventions. In larger studies, analyses could be carried out separately for each cluster or at the group level using more complex regression models to examine possible relationships among the variables, which may be or not be linear. These models could include factors that may contribute to the relationship between kinematic variables and clinical outcomes, such as age, handedness, stroke lesion and chronicity. Larger studies would also allow for the kinematic investigation of 3D reach-to-target movements on a target-by-target basis (see plots in Further investigation of UE kinematics across an array of motor tasks that better reflect important clinical changes in the control of movement may contribute to greater understanding of motor function recovery. The use of standard sets of tasks and kinematic parameters to test motor capacity across studies will allow for better comparison across studies and a means to aggregate kinematic data for meta-analytic reviews , 58. TheThe results of this development-of-concept study showed large variability in the response to rehabilitation in a small sample of individuals with chronic motor impairments after stroke. Although statistically significant correlations were identified between kinematic parameters and clinical outcomes, correlation coefficients were not high. The high variability across kinematic parameters suggests no consistent pattern of change in UE motor strategies across participants. It is unclear if interventions marked by larger motor gains would display a more consistent change in kinematic behaviors. Nonetheless, our results support the need to further investigate the impact of interventions at the individual level. This would enable the identification of clusters of individuals with common patterns of change in kinematic parameters in response to an intervention, providing an opportunity to use cluster-specific kinematic parameters as a proxy of clinical outcomes.Additional file 1: Figure S1A. Variance observed across targets in the kinematic variables of efficiency in the non-paretic and paretic arm. Figure S1B. Variance observed across targets in the kinematic variables of speed and planning in the non-paretic and paretic arm. Figure S1C. Variance observed across targets in the kinematic variables of smoothness in the non-paretic and paretic arm. Figure S1D. Variance observed across targets in the kinematic variables of trunk posture in the non-paretic and paretic arm. Figure S1E. Variance observed across targets in the kinematic variables of arm posture in the non-paretic and paretic arm.


The global pandemic secondary to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading to unprecedented global morbidity and mortality. With a bewildering array of complications, renal involvement in various forms is common, including serum electrolyte derangements. Hypokalaemia secondary to SARS-CoV-2 was common in a reported Chinese cohort. Here we review the emerging evidence on hypokalaemia and SARS-CoV-2 infection, the potential pathophysiological mechanisms based on early clinical and histopathological data and important clinical implications. Mechanisms of hypokalaemia are multifactorial and so the electrolyte disturbance can be difficult to avoid. We provide further support to the theory of renin-angiotensin-aldosterone (RAS) activation, discuss the strengths and weaknesses of implicating RAS involvement and highlight the importance of calculating the transtubular potassium gradient to identify those at risk of hypokalaemia and its complications. The most frequent serious manifestation of infection is pneumonia with 15% of patients developing serious manifestations such as hypoxia, dyspnoea, extensive pulmonary involvement and acute respiratory distress syndrome (ARDS)5. A recent meta-analysis including 50,466 patients with SARS-CoV-2 infection described an ARDS incidence of 14.8%6. Anecdotal data and clinical observation from patients with the World Health Organization named disease coronavirus disease 2019 (COVID-19) has highlighted a bewildering array of presentations and pathologies in those infected with SAR-CoV-2 many of which, it should be stressed, are rare. These include additional respiratory complications 7, cardiovascular complications (acute cardiac injury (7%8), cardiomyopathy (1/3 patients9), cardiac tamponade, heart failure, dysrhythmias (17%8) and venous thromboembolic events (20%10))11, neurological complications , Guillain-Barre syndrome  and encephalopathy)13, acute liver and/or pancreatic injury (29% and 17% respectively in one cohort)14, cytokine storm syndrome, septic shock, DIC, diarrhoea, Kawasaki-like disease14 and renal complications 15.The global pandemic secondary to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading to unprecedented global morbidity and mortality. Common symptoms include fever (43% of patients), cough (50%) and dyspnoea (29%) but other features such as myalgia (36%), diarrhoea (19%), anosmia and hypogeusia (10%) are also common16. Here we aim to review the evidence, potential clinical implications and possible mechanisms of electrolyte disturbances and kidney injury in patients with SARS-CoV-2 infection.A few small studies have demonstrated that SARS-CoV-2 can affect the kidneys directly but it is unclear if this is a key mechanism in acute kidney injury seen in patients with SARS-CoV-2 infection. Involvement of the renin-angiotensin-aldosterone (RAS) pathway has been speculated as a mechanism to lead to more severe COVID-19 disease and patients with severe disease were more likely to have hypertension, chronic kidney disease (CKD), diabetes mellitus and cardiovascular disease than those with milder disease. This is, however not confirmed and is preliminary data that may be the subject of bias with disregard of age as a potential confounding factor17. ACE2 is crucial to counter-regulate RAS and shares approximately 60% homology with ACE. RAS pathway components are co-expressed in most organs and tissues in the body and communicate via both paracrine and autocrine signalling. ACE2 converts angiotensin-II into angiotensin-(1-7), which acts on the Mas receptor expressed on a variety of tissue cell lineages relevant to cardiovascular disease in addition to type 2 alveolar epithelial cells. It lowers blood pressure through vasodilation and promotion of renal sodium and water excretion and it also attenuates inflammation by producing nitric oxide18. Meanwhile, ACE converts angiotensin-I to angiotensin-II which has directly opposing effects to signalling mediated by ACE2. Angiotensin-II acts at the type 1 angiotensin receptor (AT1R) to increase blood pressure by the induction of vasoconstriction and renal sodium and water reabsorption. This also creates oxidative stress which promotes inflammation and fibrosis. The balance between these two opposing pathways determines potential tissue injury, predominantly in the heart and kidneys17. Not only has the ACE2 receptor found to be used for host cell entry by the SARS-CoV virus, but the affinity for SARS-CoV-2 is 10-20-fold higher18. During host cell entry, proteolytic cleavage of ACE2 by transmembrane serine protease 2 (TMPRSS2) occurs which is thought to suppress ACE2 expression. This in theory would lead to a reduction in angiotensin-(1-7) generation and angiotensin-II levels would increase resulting in oxidative-stress mediated tissue damage and hypertension17. This mechanism resulting in lung parenchymal injury has already been demonstrated in mice injected with SARS-CoV virus19.Firstly, it is important to highlight how transmission of the virus occurs in humans. The SARS-CoV-2 spike protein enters host cells via the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of pulmonary type 2 alveolar cells17.Involvement of the RAS pathway has therefore been speculated as a mechanism to lead to more severe COVID-19 disease and patients with severe disease were more likely to have hypertension, chronic kidney disease (CKD), diabetes mellitus and cardiovascular disease than those with milder disease. This is, however not confirmed and is preliminary data that may be the subject of bias with disregard of age as a potential confounding factor20. In fact, the incidence of ventricular fibrillation has been shown to be fivefold higher in patients with hypokalaemia compared to those with hyperkalaemia21. Furthermore, inadequate management of hypokalaemia has been identified in 24% of hospitalised patients22, reiterating that we need to be even more vigilant with the management of this life-threatening condition during a pandemic where resources are even more scarce.Hypokalaemia is known to cause muscle weakness, paraesthesia, thirst, muscle cramps and weakness, but hypokalaemia is an important complication of any disease due to it being a potentially life-threatening condition23. This was released around a similar time to concerns around RAS inhibitors and SARS-CoV-2 where speculation that RAS inhibitors may increase risk of SARS-CoV-2 infection and its severity due to the knowledge that ACE2 is the viral binding site for host cell entry. It has been stressed by multiple organisations globally that RAS inhibitors should be continued unless otherwise medially indicated as the risks of stopping these important drugs have numerous serious complications to the patient. This study claimed that hypokalaemia was prevalent amongst SARS-CoV-2 infected patients, affecting 108 of 175 patients (62%) that had a serum potassium of <3.5 mmol/l and only 10 patients had a serum K >4.0 mmol/l which is the value required for those with myocardial dysfunction. Of the patients, 22% had severe hypokalaemia (serum potassium <3.0 mmol/l). In total, 11% of all patients and 28% of those with severe hypokalaemia displayed metabolic alkalosis (pH >7.45) compared to 4% with normokalaemia suggesting the possibility of RAS activation. The study suggested that the hypokalaemia was not due to gastrointestinal (GI) loss of potassium as only a small proportion of patients had GI symptoms, there was no significant difference between serum potassium in those with or without diarrhoea and significant urinary potassium wasting was shown. A total of 20 patients had a spot urine potassium/creatinine ratio and there were significantly higher urinary potassium/creatinine ratios in those with hypokalaemia compared with normokalaemia. The study reported that the degree of hypokalaemia correlated with severity of SARS-CoV-2 symptoms and they suggested that hypokalaemia can be difficult to correct as seen in two patients because the renal potassium wasting persists until clinical recovery from the virus. This study is relatively small, limited to a Chinese population and did not disclose medications or diuretic use which could be a significant confounding factor here. As diuretics  are used to improve oxygenation in patients with ARDS, some of these patients may well have been prescribed them. It was postulated that hypokalaemia in these patients is due to the effects of increased angiotensin-2 resulting from the proteolytic cleavage of ACE2 from virion invasion of the host although this conclusion cannot be made without measuring components of the RAS system in these patients. Further studies including declaration of medications/diuretic use and measurement of components of the RAS pathway are necessary to make conclusions about aetiology of hypokalaemia in SARS-CoV-2 infection although hypokalaemia was clearly a clinical risk in these patients that should be communicated to the medical community.The release of a pre-print retrospective Chinese study initially sparked interest into hypokalaemia as a potentially prevalent biochemical disturbance in SARS-CoV-2 infection24. Hypokalaemia was present in 41.3% of RT-PCR positive patients and was reported as a ‘common laboratory finding’. It was also suggested in this paper that the hypokalaemia was due to the increased effects of angiotensin-2 but, again, the RAS components were not measured in patients. In contrast, the largest SARS-CoV-2 case series so far (1099 patients included) did not demonstrate any major difference in serum potassium between those with mild and those with severe disease2. Serum potassium was mostly reported as normal in this cohort. SARS-CoV was found in the urine in one patient but urine was not routinely tested for the virus in this cohort.A study performed in Hong Kong during the 2003 SARS-CoV epidemic compared symptoms and laboratory findings in SARS-CoV RT-PCR swab-positive patients to negative patients25. However, this study did not acknowledge if any of the patients were taking diuretics or other medications that could alter serum potassium or sodium concentration.A small Chinese, retrospective cohort of 12 patients who were hospitalised with varying degrees of severity of SARS-CoV-2 infection were studied to comprehensively assess renal function, risk factors for, and incidence of early renal injury. 50% of patients had hypokalaemia and 50% also had hyponatraemia27 and is done by aldosterone in response to low or high serum potassium i.e. NCC is maximally phosphorylated when serum potassium is <2.5 mmol/l and not at all phosphorylated when serum potassium is >5.2 mmol/l28. In states of decreased circulating volume, ENaC activity is increased due to the effects of excess aldosterone . Whether this generates an effect on potassium or not depends on the integrated activity of the entire distal nephron. In a state of low circulating volume, NCC in the early DCT is ‘switched on’ which results in increased reabsorption of sodium and chloride in the early DCT and, along with sodium reabsorption in the PCT, a net increase of sodium reabsorption from the kidney occurs. As a result, sodium delivery to the aldosterone sensitive distal nephron (collecting duct) is decreased which offsets the effects of ENaC and potassium excretion is therefore unchanged. This results in a state of hyperaldosteronism without hypokalaemia28. It should be noted that, although the renal potassium ‘switch’ mechanism is a popular theory, it has never been proven although there are case reports of patients with normokalaemic primary hyperaldosteronism that develop hypokalaemia with a thiazide diuretic29.The theory of RAS activation causing hypokalaemia is popular, however it is recognised that hyperaldosteronism does not always cause hypokalaemia. This is explained by the ‘renal potassium switch’ mechanism , which i30. Although SiADH is frequently associated with atypical pneumonia and pneumonia is a frequent complication of SARS-CoV-2 infection, the literature does describe this phenomenon in the absence of respiratory symptoms, fever and any alternative explanation for SiADH31. It is therefore prudent to consider testing for SARS-CoV-2 infection in cases of SiADH without a clear aetiology. Additional studies are required to ascertain incidence and pathogenesis of SiADH in SARS-CoV-2 infection.Various case reports describe the onset of Syndrome of inappropriate anti-diuretic hormone secretion (SiADH) in some patients with SARS-CoV-2 infection32. 75% of patients admitted to hospital for mild, moderate or severe respiratory failure due to SARS-CoV-2 infection had acute incomplete renal Fanconi syndrome. Proteinuria was always associated and often with severe renal phosphate leak . Renal associated hypokalaemia and renal tubular acidosis was not analysed in this study due to many patients requiring potassium supplementation in addition to the effects of mechanical ventilation, GFR alterations and other factors. The authors suggest that the development of incomplete renal Fanconi syndrome could be used as a predictor of acute kidney injury (and a prognostic marker) as 88% of patients with severe stage 2 and 3 KDIGO acute kidney injury experienced proximal tubule injury prior to acute kidney injury onset. None of these patients had significant haemodynamic instability or had high mean SOFA scores to suggest an alternative pre-renal aetiology for their acute kidney injury. A quarter of the patients were given Lopinavir/ritonavir but this is rarely associated with acute kidney injury and not associated with renal Fanconi syndrome.One retrospective cohort study of 42 laboratory-confirmed COVID-19 patients without a history of chronic kidney disease have shown a high incidence of novel acquired incomplete renal Fanconi syndrome, often preceding acute kidney injury and/or resolving during the clinical recovery phase of the infection34. The first demonstrated prominent proximal tubule injury  with frank necrosis observed in some specimens and three patients had high creatinine phosphokinase and pigmented casts on light microscopy, probably representing rhabdomyolysis. There was occasional distal tubule and collecting duct swelling with some interstitial oedema and virus particles were seen in seven of the specimens on electron microscopy in proximal tubule epithelial cells and the podocyte, both of which are the only sites of renal ACE2 expression33. The second post-mortem case series in (currently in pre-print) examined six post-mortem renal histopathology samples of patients who died with SARS-CoV-2 infection34. All specimens had histopathological evidence of acute tubular necrosis, two patients had severe lymphocytic infiltration of the tubulointerstitium and SARS-CoV-2 nucleocapsid antigens were observed in renal tubule cells. Transmission electron microscopy was used in samples from two different patients, both of which demonstrated the presence of virions and virus-like particles in renal cells and these cells were markedly swollen with mitochondrial, lysosomal and endoplasmic reticulum expansion. These finding suggest that SARS-CoV-2 directly infects kidneys and specifically infects and damages kidney tubules. Furthermore, strong CD68+ macrophage presence and C5b-9 deposition seen in the tubulointerstitium of all samples (yet absent in the rest of the kidney tissue and very little in the glomeruli and capillaries in two patients) suggest that further tubular damage occurs as a consequence of macrophage recruitment and C5b-9 activation and deposition. It would be logical to suggest that histopathological evidence of damage to tubular cells would result, clinically, as a tubulopathy, of which we know urine electrolyte wasting is a recognised complication and these case series are histopathological evidence to support this suggestion.There are two studies so far (one in pre-print form) to look at renal histopathology on post-mortem findings in SARS-CoV-2 patients who died of the disease 35. It is therefore assumed that this is one way in which the kidney is ‘infected’ by the virus and reiterates the probability of direct virus-induced tubular damage. Various case series have demonstrated the virus in urine37 and it is unclear as to how it enters the tubular lumen and whether this is via the apical membrane of tubule cells. However, one study did look at the presence of SARS-CoV-2 in bodily fluids over time and did not detect the virus in urine in any of the nine patients studied38. A couple of case reports have demonstrated that the virus may affect the kidney in other ways which may be an alternative explanation for the entry of virus into the urine40. Collapsing focal segmental glomerulosclerosis (FSGS) has been described twice in the literature so far in patients of African or African American origin and both of these patients demonstrated severe tubular injury. It was noted that severe acute tubular necrosis (ATN) was observed in the absence of haemodynamic compromise and severe pulmonary involvement suggesting that the tubulopathy was not ischaemic and more likely to be because of either direct viral toxicity or cytokine-mediated damage.Finally, we know that TMPRSS2 primes SARS-CoV-2 to gain cellular entry and TMPRSS2 is only detectable (in low levels) at the S3 section of the proximal convoluted tubule in the kidneyVarious clinical and histopathological studies have demonstrated evidence of hypokalaemia, hyponatraemia, SiADH, incomplete Fanconi syndrome and tubulopathy in patients with SARS-CoV-2 infection.21.The data from China and evidence from the SARS-CoV case series provide good clinical justification to monitor potassium levels in patients infected with SARS-CoV-2. We do need to see this clinical finding replicated in multi-centres to be able to make definitive conclusions about this clinical sequela. Although hyperkalaemia can occur for many reasons in patients with SARS-CoV-2 infection, the incidence of ventricular fibrillation has been shown to be five-fold higher in patients with hypokalaemia compared to those with hyperkalaemia41. The main premise underlying the TTKG is the absence of significant solute transport in the collecting duct so that any change in urinary potassium concentration only occurs due to medullary water reabsorption and the TTKG does not overestimate the gradient for collecting duct potassium secretion. However, we now know that some urea is reabsorbed in the late cortical collecting duct and this urea recycling aids the tubular secretion of potassium so it should be noted that the TTKG should not be used as a diagnostic tool in hyperkalaemia aetiology as it is not reliable42. In situations of dilute urine and high urine flow rate, the TTKG also underestimates potassium secretory capacity in the hyperkalaemic patient43. However, we believe that the utility of the TTKG is still of value in hypokalaemic patients to differentiate between renal and extra-renal potassium wasting providing the aforementioned criteria necessitating its accuracy is adhered to.Based on evidence of significant urine potassium wasting which can be prolonged, we suggest checking the transtubular potassium gradient (TTKG) in patients with SARS-CoV-2 infection. The TTKG adjusts the urinary potassium for the concentrating effects that occur in the collecting duct where water is removed from urine. This would help identify those at risk of severe or prolonged hypokalaemia and prompt pre-emptive cautious electrolyte monitoring and replacement which should help reduce the risk of hypokalaemic complications which can be fatal. The TTKG is easily measured and only requires measurements of serum and urine osmolality and potassium (urine potassium/serum potassium)/. A high TTKG in the setting of hypokalaemia suggests that potassium wasting is of renal origin. This, for example, may be due to hyperaldosteronism, pseudohyperaldosteronism or a potassium wasting tubulopathy. The validity of this measurement depends on three assumptions: (1) few solutes are reabsorbed in the medullary collecting duct, (2) potassium is neither secreted or reabsorbed in the medullary collecting duct and (3) the osmolality of fluid in the terminal collecting duct is iso-osmolar to plasma. As water is reabsorbed in the medullary collecting duct, urine potassium concentration is not an accurate index evaluating distal potassium secretion because the effect of water is not taken into account. The urine to plasma osmolality ratio adjusts for the degree of medullary water reabsorption which increases urine potassium concentration as more water is reabsorbed. Therefore, TTKG is relatively accurate providing the urine is not dilute, that the osmolality of urine is greater or equal to the osmolality of plasma  and that urine sodium concentration is >25mmol/l so that distal sodium delivery is not limiting44 so future studies determining RAS system involvement of SARS-CoV-2 infection should, as well as TTKG, also consider urine pH as an early marker of RAS system activation.Measuring components of the renin-angiotensin-aldosterone pathway would help us to identify if there really is involvement of the SARS-CoV-2 virus; however, measuring these components in the acute setting has its limitations. The RAS system is heavily influenced by a multitude of factors. Many of the patients with SARS-CoV-2 infection are critically unwell and often present to hospital in states of low cardiovascular volume. It has been recommended that patients with ARDS also be kept in a relative state of volume depletion as to not worsen pulmonary interstitial oedema and thus oxygenation. Both of these factors would typically result in RAS activation and could be very misleading when determining virus-related RAS pathway involvement. Some studies have shown that low urine pH (causing acidic urine) can be a predictor of RAS activation45. This is particularly relevant for a couple of reasons. First, many of the drugs currently undergoing clinical trials in SARS-CoV-2 patients prolong the QTc interval  and some drugs also risk hypokalaemia which may worsen pre-existing electrolyte disturbance . Second, cardiac involvement in SARS-CoV-2 is high (44.4% of infected patients admitted to ICU experienced an arrhythmia). Induction of arrhythmias can be due to multi-factorial aetiologies of cardiac injury in SARS-CoV-2 patients such as hypoxia-mediated, direct tissue damage, cytokine-storm syndrome, worsening coronary perfusion and the direct effects of medications47. Both of these factors really emphasise the importance of maintaining normokalaemia in these patients to reduce morbidity and mortality.Potassium levels <3.0 mmol/l can be arrhythmogenic and specifically can cause QTc interval prolongation, torsade de pointes, ventricular fibrillation and sudden cardiac death+-K+-2Cl- co-transporter (NKCC2) in the thick ascending limb of the loop of Henle which results in failure of sodium, potassium and chloride to be reabsorbed into the concentrated medullary interstitium . This enhances distal tubular concentration of sodium, reduced hypertonicity of the surrounding interstitium and less water reabsorption in the collecting duct. Distal sodium delivery increases potassium loss via the Na+/K+-ATPase pumps at the apical membrane of the principal cell of the collecting duct as there is more sodium to be exchanged with potassium for excretion. Thiazide diuretics can cause hypokalaemia by the same principle of enhanced distal sodium delivery as they block the sodium-chloride co-symporter (NCC) in the distal convoluted tubule. Furthermore, loop diuretics also block NKCC2 at the macula densa which  induces renin secretion, making RAS system measurement inaccurate if patients are on diuretics. If diuretics are to be used, it would be wise to consider potassium-sparing agents in hypokalaemic patients to reduce the cardiac complications of worsening hypokalaemia that can occur with those that are not potassium sparing.In addition to the arrhythmogenic effects of both the SARS-CoV-2 cardiac sequelae and various clinical trial drugs, many patients are being given diuretics to improve the oxygenation in ARDS, which also risks hypokalaemic complications. Loop diuretics can induce hypokalaemia by blocking the Na48. Tubular injury seems to be multifactorial in these patients; ATN from SIRS/Cytokine Storm Syndrome, peritubular congestion, virion invasion, drug toxicity and pigment-cast nephropathy from rhabdomyolysis and therefore difficult to avoid.It would be logical that with clear histopathological data suggesting tubular injury, hypokalaemia is to be expected in patients infected with SARS-CoV-249. The strong association of hypokalaemia with arrhythmia and sudden cardiac death reiterates the importance of vigilance for detection and treatment of this electrolyte disturbance during and after admission given such poor outcomes when spontaneous circulation is lost.The data on outcomes in cardiac arrest for SARS-CoV-2 patients is unfortunately poor. Chinese data suggests that in a series of 136 patients who underwent resuscitation efforts, return of spontaneous circulation was only achieved in 18 (13.2%) and only four patients were alive at 30 daysTo truly know the extent of involvement of the RAS pathway as a potential cause of hypokalaemia in SARS-CoV-2 patients, further studies require measurement of all components of the RAS pathway. Until we know this, we cannot make definite conclusions about the mechanisms of hypokalaemia in these patients outside of the histopathological evidence of tubulopathy published, of which multiple aetiological factors are likely.No data are associated with this article. Summary This is a narrative review of hypokalaemia experienced in SARS-CoV-2 infection.Strengths The paper addresses available literature. The paper correctly critiques the level of evidence included, which for the most part includes small and poorly conducted observational studies.Major weaknesses NoneMinor WeaknessesPlease broaden abstract to include electrolyte disturbances in addition to hypokalaemia, reflecting the article as a whole.In the first paragraph, there is quite a lot of text dealing with the many complications of COVID infection, which is a bit irrelevant.1.“…proximal tubule epithelial cells and the podocyte, both of which are the only sites of renal ACE2 expression” This is not true; see The Human Protein Atlas or for example“A few small studies have demonstrated that SARS-CoV-2 can affect the kidneys directly, but it is unclear if this is a key mechanism in acute kidney injury seen in patients with SARS-CoV-2 infection”? Reference. It would be useful to include a comment on the prevalence of acute kidney injury in SARS-CoV-2 patients.“Hypokalaemia is known to cause muscle weakness, paraesthesia, thirst, muscle cramps and weakness, but hypokalaemia is an important complication of any disease due to it being a potentially life-threatening condition”. Please rephrase – hypokalaemia is important, but at the same time rare (does not occur with ‘any disease’).22, reiterating that we need to be even more vigilant with the management of this life-threatening condition during a pandemic where resources are even more scarce”. Please reference, or clarify. Management of hypokalaemia is not usually resource-intensive, and I have not seen evidence that potassium replacement is difficult to come by in the pandemic.“Furthermore, inadequate management of hypokalaemia has been identified in 24% of hospitalised patients“This study claimed that hypokalaemia was prevalent amongst SARS-CoV-2 infected patients, affecting 108 of 175 patients (62%) that had a serum potassium of 4.0 mmol/l which is the value required for those with myocardial dysfunction.” Hypokalaemia affected 62% of patients with a K less than 3.5? Or hypokalaemia as defined by K<3.5 affected 62% of the cohort.“only 10 patients had a serum K >4.0 mmol/l which is the value required for those with myocardial dysfunction”. Please clarify what is meant by this phrase.“Principal cell” does not warrant capitalisation.[21].” This data is from the setting of acute myocardial infarction, not SARS-CoV-2, and therefore may not be applicable. Please add a qualifying statement.“Although hyperkalaemia can occur for many reasons in patients with SARS-CoV-2 infection, the incidence of ventricular fibrillation has been shown to be five-fold higher in patients with hypokalaemia compared to those with hyperkalaemiaYou recommend measuring the transtubular potassium gradient. Does this significantly alter management in these patients, outside of a research context?“It has been recommended that patients with ARDS also be kept in a relative state of volume depletion as to not worsen pulmonary interstitial oedema and thus oxygenation.” This advice has since been dropped in the context of SARS-CoV-2 – please make this clear.I do not think the full explanation of how loop diuretics/thiazides can cause hypokalaemia materially adds to the paper. Some aspects of the writing were difficult to followIs the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?YesReviewer Expertise:Renal tubular physiology.We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read the revised review. It is much improved. I would accept without further revision.Is the topic of the opinion article discussed accurately in the context of the current literature?PartlyAre arguments sufficiently supported by evidence from the published literature?PartlyAre all factual statements correct and adequately supported by citations?PartlyAre the conclusions drawn balanced and justified on the basis of the presented arguments?PartlyReviewer Expertise:I am an expert on molecular underpinnings of potassium balance and electrolyte disorders. I coined the "potassium switch"I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. As pointed out by this review, the evidence for hypokalemia in COVID-19 is weak, promulgated by a single unpublished article that was not corroborated by larger series studies. My chief concern about the review, is that it amplifies the myth. Furthermore, the frequent use of loop diuretics in ARDS, which cause hypokalemia, makes a causal connection between the development of hypokalemia and COVID-19 precarious. At the very least, the title should be changed to minimize the connection between hypokalemia and COVID-19.   Paragraph 1 is somewhat misleading.  Although COVID-19 does have an array of different sequela, acute respiratory distress syndrome (ARDS) seems to be the most common serve form of the disease. The first paragraph should provide an incidence of this and the other sequela. Reference to large studies from China, Europe and North American should be cited. Paragraph 2 The statement “SARS-CoV-2 may affect the kidneys directly and indirectly by both renal and renin-angiotensin-aldosterone system (RAS) involvement,” has no factual basis. There are a few small studies that demonstrate that SARS-CoV-2 can infect the kidney but it is presently unclear if this is a key mechanism of AKI in COVID19. Paragraph 3 Provide a reference to Mas receptor on type 2 alveolar epithelial cells as stated. Paragraph 3 last sentence. This is evidence for SARS-COV, not SARS-COV2 Provide a reference to “hyperaldosteronism does not always cause hypokalaemia.” Although “Switch” activation has been a popular explanation for this, it has never been proven. Perhaps, the authors could cite clinical studies, demonstrating the development of severe hypokalemia in normokalemic PA patients following thiazide administration as evidence. The limitations of TTKG should be articulated.Is the topic of the opinion article discussed accurately in the context of the current literature?PartlyAre arguments sufficiently supported by evidence from the published literature?PartlyAre all factual statements correct and adequately supported by citations?PartlyAre the conclusions drawn balanced and justified on the basis of the presented arguments?PartlyReviewer Expertise:I am an expert on molecular underpinnings of potassium balance and electrolyte disorders. I coined the "potassium switch"I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.


Gut microbiota and the host exist in a mutualistic relationship, with the functional composition of the microbiota strongly influencing the health and well-being of the host. In addition to the standard differential expression analysis of host genes to assess the complex cross-talk between environment (diet), microbiome, and host intestinal physiology, data-driven integrative approaches are needed to identify potential biomarkers of both host genes and microbial communities that characterize these interactions. Our findings demonstrate that the complementary application of univariate differential gene expression analysis and multivariate approaches such as sparse Canonical Correlation Analysis (sCCA) and sparse Principal Components Analysis (sPCA) can be used to integrate data from both the healthy infant gut microbial community and host transcriptome (exfoliome) using stool derived exfoliated cells shed from the gut. These approaches reveal host genes and microbial functional categories related to the feeding phenotype of the infants. Our findings also confirm that combinatorial noninvasive -omic approaches provide an integrative genomics-based perspective of neonatal host-gut microbiome interactions. Early microbial colonization in infants is critically important for directing neonatal intestinal and immune development and is especially attractive for studying the development of human-commensal interactions ,2. HenceUsing exfoliated cells, we applied novel transcriptome-based methods to identify the best single-gene, two-to-three gene combinations (biomarkers) to distinguish between dietary treatments, e.g., fingerprint/classify breast- (BF) vs. formula-fed (FF) term infants . The besAs part of an ever-expanding effort to generate predictive genomic network data, our interdisciplinary team has pioneered molecular and systems biology methodologies for simultaneously monitoring host gastrointestinal exfoliome gene expression and the gut microbiota . In termBased on these initial findings, the goal of this study is to examine the multivariate structure of host intestinal exfoliome expressed genes related to immunology , microbiOur proof-of-principle approach provides novel insight into the structure of each data type  in isolation and suggests potential host-microbiome interactions following the introduction of dietary substrates in the early neonatal period. This approach extends our previous work  and incoThe source of the human samples analyzed herein was previously described . Briefly+ mRNA were previously described [Methods for isolating and quantitating stool microbial DNA and host polyAescribed ,11. For escribed . Metagenescribed . Metagenescribed . The genTo assess the impact of diet on microbiota functional characteristics, microbial data were aligned, as previously described in  using MGNormalized host gene expression values were used to test for significant differences in expression (DE) when comparing BF vs. FF infants . All proShort chain  and branched chain  fatty acid concentrations were analyzed by gas chromatography, as previously described  and are We elected to compare the performance of two multivariate analyses methods, sPCA and sCCA, on both synthetically generated data and on our infant dataset. sPCA and sCCA are recent modifications of the classical Principal Component Analysis (PCA) and Canonical Correlation Analysis (CCA) ,28. PCA The metagenome sequence data have been deposited in the European Bioinformatics Institute’s Short Read Archive (ERP001038). The human exfoliome data have been deposited in the NCBI Gene Expression Omnibus (GSE31075).p-values levels of 0.05.Analysis of VFA concentrations in feces demonstrated differences in both short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA) between BF and FF infants . AlteratThe composition of exfoliated intestinal epithelial cells may directly contribute to alterations in gene expression, thus the anatomic origin of the host gene expression signature derived from the exfoliated cells was determined. The relative expression of genes previously identified and expressed predominantly in specific anatomic locations  were quantified a. SignatSince the application of either CCA or PCA to multi-omic data in situations where the number of samples is much smaller than the number of measured variables presents challenges related to the mathematical operations on the data matrixes, we used optimized sparse versions of CCA and PCA: sCCA and sPCA respectively ,18. As ap-value < 0.05) in expression between the two infant feeding phenotypes. In addition, sCCA analysis did not detect any correlative structure between SFCA host genes and data from SEEDLevel2 microbial categories  and multivariate approaches (sPCA and sCCA) to assess host-microbe crosstalk in healthy human infants. Importantly, testing for DE genes did not detect many strongly DE genes after the appropriate FDR correction of the raw p-values . These fWe have previously reported that the exfoliated transcriptome reflects the tissue-level transcriptome in a mouse model of NSAID enteropathy . AlthougWhile sPCA does not aim to detect potential interactions between host genes and the microbial SEEDLevel2 categories, it can detect structures in each individual data type that might not be related to mutual correlative relationships between these two data types. Therefore, we applied sPCA to evaluate the general structure of each one of the three prior knowledge gene lists  and the CCA is a statistical method for exploring the correlative relationships between two multivariate sets of variables . The canGenes identified by the multivariate analysis include free fatty acid receptor-2 (FFAR2 or GPR43) and FFAR3 (GPR41) . This isCLDN1) was identified by sCCA as a barrier function gene associated with microbiome virulence characteristics. IL-22 is a homeostatic cytokine preserving the integrity of boundary organs and tissues, and is only occasionally proinflammatory [We have previously demonstrated that virulence characteristics of the microbiome exhibit differential sensitivity to breast milk as compared to formula . Therefoammatory . IL-17 aammatory . Unexpecammatory . Additioammatory . We obseCLCF1 (Cardiotrophin-like cytokine factor 1), which stimulates B-cell proliferation and Ig production [Genes related to T- and B-cell function were identified by our analyses. For example, the transcription factor CEBPB (CCAAT/enhancer-binding protein beta), which reduces proliferation and promotes expression of differentiation-related genes in T-cells , was up-oduction  and CD22oduction . Taken tACTG1) and A4 (ACTA4), which were up- and down-regulated in BF vs. FF, respectively. The G actins assemble into polarized filaments that form networks impacting the cytoskeleton and generate force to support internal cell motility [With respect to intestinal barrier function, fewer genes were identified, but included claudin 1 (CLDN1) and claudin-4 (CLDN4). Claudins are a family of small transmembrane proteins which, along with occludin, are the most important components of the tight junctions . Claudinmotility . Chemokine ligand 2 (CXCL2) expression was higher in BF infants, which seems counterintuitive to the general lower expression of inflammatory markers. This barrier function gene, also designated macrophage inflammatory protein 2-alpha, has been associated with inflammatory diseases and is chemotactic for neutrophils . NeutropOf the 27 genes identified by sCCA, NR3C1, LTBP4, and CTNND1 showed the greatest difference in expression between BF and FF infants . The NR3Another host immune related gene that was associated with microbial virulence gene expression was LTBP4, or latent transforming growth factor beta binding protein 4. The protein encoded by this gene binds TGF-β as it is secreted and targeted to the extracellular matrix. This protein controls TGF-β activation by binding to the latency-associated peptide, which is located in the regulatory chain of the growth factor and regulates integrin-dependent activation of TGF-β . Little Catenin delta 1 is encoded by the gene CTNND1, which was one of the barrier related genes associated with the SEEDLevel2 microbial genes. This protein, also known as p120, is a major component of multiprotein cell-cell adhesion complexes containing other catenins and epithelial cadherin (E-cadherin) . It is aThis study uses a combination of univariate and multivariate statistical approaches to identify shifts in postnatal developmental patterns in the early neonatal period. Specifically, we have demonstrated that both sCCA and sPCA can be used in support of the formulation of hypothesis-based patient-powered precision medicine studies via its ability to identify candidate genes that might be active in the host gut epithelium as well as SEEDLevel2 commensal microbiome categories that reflect the different feeding types in neonates. Our results show that these two multivariate approaches complement the testing for significant difference in host gene expression and can provide a deeper insight of the structure present in data (sPCA and sCCA) as well as identify potential interaction between the host gut epithelium and the commensal microbiota (sCCA). Furthermore, we propose that our ability to use host exfoliated cell mRNA instead of biopsy or autopsy material, in combination with microbiome-derived DNA, RNA and metabolites, will enable the development of novel predictive computational models describing host-microbe interactions associated with healthy gastrointestinal development of infants.


Background: The regulation of melanoma by noradrenergic signaling has gain attention since pre-clinical and clinical studies suggested a benefit of using beta-blockers to control disease progression. We need to confirm that human melanoma recapitulates the mechanisms described from pre-clinical models. Methods: The sources and targets of norepinephrine in the microenvironment of 20 human melanoma samples was investigated using immunostaining. The effect of an exposure to beta-blockers on immune cell type distribution and expression of immune response markers was assessed with immunostaining on 212 human primary melanoma. A statistical analysis explored the effect of an exposure to beta-blockers on progression free survival, melanoma related survival, and overall survival on the 286 eligible patients. Results: Tumor cells and macrophages may be a source of norepinephrine in melanoma microenvironment. Tumors from patients exposed to wide spectrum beta-blockers recapitulate the increased infiltration of T-lymphocytes and the increased production of granzyme B observed in pre-clinical models. An exposure to beta-blockers is associated with a better outcome in our cohort of melanoma patients. Conclusion: This study shows the association between an exposure to wide spectrum beta-blockers and markers of an effective anti-tumor immune response as well as the protective effect of beta-blockers in human melanoma patients. There is growing evidence that the catecholamine norepinephrine (NE) plays a role in melanoma progression and the associated immune suppression. One of the hypotheses is that the sympathetic nervous system releases norepinephrine in lymphoid organs and at the tumor site promoting both tumor progression and immune suppression ,2. In viThe sources of norepinephrine, the different receptors involved as well as the local versus systemic regulations induced by catecholamines in human melanomas is still a matter of debate ,6,7,8. IUsing a cohort of melanoma patients from our archived tissue bank, we assessed the effect of the exposure to various types of beta-blockers on the local immune pattern and melanoma progression. We completed our study by a survival analysis assessing the association between exposure to beta-blockers and melanoma related death or disease progression. Our findings in melanoma patients who are on a beta-blocker treatment largely recapitulate our previous observations in animal models.We investigated the presence of noradrenergic sources at the tumor site. The common hypothesis is that nerve fibers, coming from the peripheral nervous system innervating the skin, brings a noradrenergic input that triggers tumor development. Comparing human samples from normal skin with nevi (benign and dysplastic) and melanoma, we observed a significantly higher innervation of all melanocytic lesions A. These On a series of 20 human melanomas, we used immunohistochemistry to assess the expression of norepinephrine in the tumor tissue. Norepinephrine was only detected in tumor cells and macrophages . In macrIn order to identify putative targets for beta-blockers, we assessed the expression of the two main adrenergic receptors ADRB1 and ADRB2. We observed the expression of ADRB1 receptors on macrophages and on mast cells A,B. MelaSince some beta-blockers may interact with serotonergic signaling ,10,11, wThese results indicate that norepinephrine is produced in the tumor microenvironment and that targets for beta-blockers are expressed in situ.286 patients diagnosed as having primary melanoma were included in the study between 1 January 1987 and 29 April 2014 at the Department of Dermatology of the University Hospitals of Geneva, Switzerland. We limited our selection to patients diagnosed with melanoma at least 5 years ago. We therefore benefit from a significant follow-up period to assess the effect of an exposure to beta-blockers on the risk of death or disease progression. Selection strategy is presented in the n = 230 in the control group, n = 41 in the cardioselective beta-blockers group and n = 15 in the wide-spectrum beta-blockers group). The two groups of patients exposed to beta-blockers at melanoma diagnosis were significantly older than patients of the never exposed group. We did not find any differences among groups regarding other characteristics or clinical variables.We distinguished three study groups based on their exposure to beta-blockers. n = 144) infiltration by neutrophils was scarce but a subset of the samples in each study group show a large neutrophil infiltrate (more than 50 cells per mm2).Patients incidentally exposed to any type of beta-blocker  had a significantly lower intra-tumor blood vessel density, as assessed by CD34 staining. Lymph vessel density assessed by D2-40 (podoplanin) staining was not significantly different across the three groups of exposure to beta-blockers . Only paWe assessed the presence of mast cells by staining for mast cell tryptase antigen. The exposure to cardio-selective beta-blockers only is correlated with a higher density of mast cells in the tumor microenvironment. We did not observe any significant difference in MHC class II molecules expression between each study group.The expression of TNF alpha was not significantly different in our three study groups despite a trend towards a higher production of TNF alpha in the tumors of patients exposed to wide spectrum beta-blockers. Likewise, the expression of inducible nitric oxide synthase (iNos) was not significantly different among the three study groups despite a trend towards a lower production of iNos in the tumors of patients exposed to wide spectrum beta-blockers.Finally, we observed a higher density of cells expressing IL-10 in samples from patients exposed to wide spectrum beta-blockers but not cardio-selective type.Only one patient exposed to wide spectrum beta-blocker experienced a progression event and none of them died from melanoma related death, this result in a lack of statistical power due to the lack of event. We pooled cardioselective and wide spectrum beta-blocker into one exposed group. We considered two types of exposure to beta-blockers: (1) exposure before the diagnosis of melanoma (no exposure vs. exposure with cardioselective or non-cardioselective beta-blockers), and (2) exposure to beta-blockers before the diagnosis of melanoma and during follow-up (never users vs. users before the diagnosis of melanoma vs. users after the diagnosis of melanoma). Among 229 patients never exposed to beta-blockers before diagnosis, 24 patients experienced an exposure to beta-blockers after the diagnosis of melanoma (21 under cardioselective and 3 under wide spectrum beta-blocker therapy). In this group exposed to beta-blockers after primary melanoma resection, the exposure to beta-blockers began 47.2 +/− 59.5 months  after the resection of the primary tumor. These patients secondarily exposed to beta-blockers were significantly older (mean age 70.4 ± 11.3 years) than patients in the control group. Our primary endpoints were progression free survival and melanoma related survival. p = 0.086), but the group of patients exposed to beta-blockers before diagnosis and during follow up has a significantly lower hazard of disease progression compared to non-exposed patients (p = 0.042). A greater Breslow index and ulceration were associated with a higher risk of disease progression in univariate and multivariable model. The use of beta-blockers was significantly associated with progression-free survival in univariate analyses. The use of beta-blockers before diagnosis and during follow up was significantly associated with a lower risk of disease progression; there was a difference in the hazard of disease progression between beta-blocker users depending on the time of introduction of the beta-blocker (before or after diagnosis). In multivariable analyses, the overall use of beta-blockers was not significantly associated with disease progression (p = 0.034), in particular the hazard was lower among patients exposed to beta-blockers before diagnosis as compared to non-exposed patients (p = 0.023). In multivariable analyses, only Breslow index remains significantly associated with a higher risk of melanoma related mortality.The risk of melanoma related death was significantly increased with a greater Breslow index and ulceration of the tumor in univariate analyses. The use of beta-blockers was significantly associated with a lower hazard of melanoma related death (p = 0.039). The hazard of all-cause mortality was also significantly and independently associated with a greater Breslow index and age.The use of beta-blockers is not significantly associated with all-cause mortality but the hazard of all-cause death was significantly lower in patients exposed to beta-blockers after the diagnosis of melanoma compared to never exposed patients , or compThe repurposing of beta-blockers as anti-tumor agents is being considered in several cancers. On the one hand, pre-clinical studies showed that beta-blockers can block cell proliferation, migration, invasion, resistance to therapy, and metastasis ,8,12. OnWe first investigated whether the effect of beta-blockers rely on systemic regulations only, or can be partially attributed to local actions. We showed that norepinephrine and the main adrenoceptors ADRB1 and ADRB2 are present in the tumor microenvironment. Despite the hypothesis that norepinephrine is delivered locally by sympathetic innervation, we observed that nerve fibers surrounding the tumors are negative for norepinephrine and catecholamines synthesizing enzymes. The increased nerve fiber density around all melanocyte lesions may be related to the Schwann cell origin of melanocytes. Schwann cells are necessary for peripheral nerve survival and melanocytes like Schwann cells may release factors increasing these fibers survival in the microenvironment of melanocyte lesions  In contrInterestingly, we observed the expression of ADRB1 on macrophages and mast cells, two cell types known to predominantly express ADRB2 in the literature. Both were negative for ADRB2, which suggests that macrophages and mast cells could be of a particular phenotype in the microenvironment of melanoma. Altogether, these results show that norepinephrine and its receptors are present locally in the tumor and its microenvironment. The wide expression of beta-adrenoceptors in the tumor environment and the presence of norepinephrine provide local targets for beta-blockers. Affecting tumor cells, immune cells and keratinocytes, noradrenergic signaling may rule different aspects of tumor progression. We next assessed the expression of different biomarkers of melanoma progression on a cohort of patients exposed or not exposed to beta-blockers at the time of diagnosis. We distinguished the exposure to cardio-selective beta-blockers from the exposure to wide spectrum beta-blockers. Despite the small size of our sample, we could observe some significant differences between our three study groups. We have to note that among our patients exposed to wide spectrum beta-blockers, 9 were treated with eye drops and 6 per os without any major difference in the histopathological data. Eye drops of beta-blockers are known to induce systemic effects ,20 and oPre-clinical studies in animal models of melanoma suggested that exposure to the wide spectrum beta-blocker propranolol results in an increased infiltration of T-lymphocytes and an increased cytotoxic activity . This inSince the observation of the dramatic effect of propranolol on the involution of infantile hemangiomas ,24, betaFurthermore, we observed that the exposure to both types of beta-blockers is associated to a reduced density of CD34+ stromal fibroblasts (vimentine +). These cells are suggested to be involved in angiogenesis, tumor stroma formation, matrix remodeling and immunosuppression . Beta-blThe role of mast cells in cancer initiation and progression is highly debated ,27,28,29Altogether these results suggest that wide spectrum beta-blockers are promising agents in adjuvant therapy for melanoma. Only wide spectrum beta-blockers are associated with biomarkers of an increased anti-tumor immune response and a decreased tumor growth. Nevertheless, cardio-selective beta-blockers may affect partially tumor progression modulating tumor angiogenesis and CD34+ stromal fibroblast density. n = 15) only one patient experienced a progression event and none of them died from melanoma. In light of this limitation and the lack of power related to the low number of events we pooled both type of beta-blockers into one group for survival analyses. Our results indicate that exposure to beta-blockers before melanoma diagnosis and during the follow up period significantly decreases the risk of disease progression.We have to note that among patients exposed to wide spectrum beta-blockers  and were registered under project n° 15-092.Retrospective study of patients diagnosed with a first primary melanoma, in a single center, with a clinical record for at least five years from primary melanoma diagnosis.286 patients diagnosed as having primary melanoma were included in the study between 1 January 1987 and 29 April 2014 at the Department of Dermatology of the University Hospitals of Geneva, Switzerland. A subset of 212 patients was considered for immuno-histochemical analyses, when the primary tumor sample was available and the remaining tumor tissue reliable with the original recorded Breslow index. Our primary endpoints were the effect of an exposure to beta-blockers on progression free survival and melanoma related death. The effect of beta-blocker exposure on overall survival was assessed as a secondary outcome.The original records and the selection strategy are presented in Tissue blocks were sectioned in 7 µm slices using a standard microtome and mounted on superfrost plus slides . The tissue sections were oven-dried overnight at 52 °C. Before starting staining protocols, the slides were dewaxed in four baths of Ultraclear  for 5 min each. The slides were rinsed in four baths of absolute ethanol for 3 min each and kept in distilled water until use.Prior to all immunostainings presented in this manuscript, an epitope recovery using citrate buffer pH6 was performed. Citrate buffer was prepared by diluting 1.92 g of anhydrous citric acid  in 1 L of distilled water. The acidity was adjusted to pH6 with 5N NaOH under constant monitoring with a pHmeter . The citrate buffer solution was then warmed to boiling point using a standard microwave oven at power 900 Watts. The tissue slides were dived in the boiling buffer and kept in microwave oven for 16 min at 350 Watts. The bucket containing the citrate buffer and the slides was allowed to cool down at room temperature for 30 min. The slides were then rinsed in distilled water 2 times for 1 min and transferred to phosphate buffer saline  for further staining procedures.Primary antibodies . The quantification of cell populations and the quantification of markers’ expression was performed on wide pictures acquired with an axioscan Z1 slide scanner . The quantification of markers and cell population was obtained with Definiens Tissue studio software . The quantification of nerve fiber density was done on ImageJ using Feature J hessian plugin.Automated image analyses need quality control steps to guarantee the reliability and accuracy of the estimation based on image processing. This is particularly true when a large amount of pictures from different datasets (or experiment replications) are compared. Our preliminary work consisted in creating quantification routines to establish guidelines for batch analyses and standardize the work of the experimenter. Three subgroups of patients have been exposed to different types of beta-blockers before melanoma diagnosis and during the follow-up period. Cardio selective beta-blockers are selective antagonists for beta-1 adrenoceptors as opposed to wide spectrum beta-blockers which are non-selective antagonists of beta-1 and beta-2 adrenoceptors. We distinguished three study groups based on their exposure to beta-blockers: a control group consisted in patients who were never exposed to any type of beta-blockers at diagnosis of melanoma, and two groups of patients exposed to cardioselective beta-blockers or wide spectrum beta-blockers respectively. We used Chi-2 or Fischer’s exact tests for the comparisons of categorical variables by groups of exposure to beta-blockers, and Kruskal–Wallis nonparametric test for comparisons of continuous variables (by groups of exposure to beta-blockers). For the comparisons of histological characteristics, we performed pair-wise post-hoc comparisons applying the Nemenyi test. p values < 0.05 were regarded as significant. We used STATA (version intercooled 16.0) for all analyses.Competing risk models were performed to assess the association between exposure to beta-clockers and progression free survival, melanoma related survival or overall survival. We performed three different survival analyses: (1) Progression free survival . The follow-up started on the date of melanoma diagnosis and ended on the date of the first event of recurrence, death for any cause or on the date of last information ; (2) melanoma-related survival where we considered other cause of death or death of unknown cause as competing risk events; and (3) overall survival where the follow-up started on the date of melanoma diagnosis and ended on the date of death for any cause or on the date of last information . A Cox proportional hazards model was used to evaluate the effect of beta-blocker treatment on overall mortality, independently of other significant prognostic factors. Competing risks Cox regression models were performed using other cause of death or death of unknown cause as competing risk events to evaluate the effect of beta-blocker treatment on melanoma-related mortality then on disease-free progression, independently of the same significant prognostic factors. The potential prognostic factors tested were: Breslow thickness, age (< vs. ≥ 60 years), and ulceration. All tests were two-tailed and All raw data for histopathological and survival analysis are available as Taken together, the apparent cytotoxic switch of the local anti-tumor immune response in the wide spectrum beta-blockers exposed group and the better outcome of patients exposed to beta-blockers suggest that wide spectrum beta-blockers are promising agents to prevent disease progression. This highlights the need for wide scale prospective studies investigating the efficacy of wide spectrum beta-blockers as an adjuvant treatment for melanoma.


The paper aimed to analyze and evaluate the present literature data on the clinical effectiveness of using the bacillus Calmette–Guérin (BCG) vaccine in protecting against the novel coronavirus disease 2019 (COVID-19).Several novel clinical data have shown a relationship between the vaccinated population with the bacillus Calmette–Guérin (BCG) vaccine and the severity and mortality rate from coronavirus disease 2019 (COVID-19). However, the linkage between the BCG vaccine and COVID-19 infection mortality and morbidity rate is still ambiguous. The BCG has been protected previously from many other respiratory viral infections. The efficacy of the BCG vaccine in the protection against COVID-19 depends on various factors including social, economic, cultural norms, mitigation efforts, health infrastructure, and demographic differences between countries.Thus, the literature analyses show a noticed difference between the countries that follow national vaccination programs than in countries that do not follow such programs . However, there are not any recommendations for using BCG in the protection against severe cases of COVID-19. The severity of COVID-19 maybe depends on the age, immune state of the patient, and the level of vaccine coverage. The possible reason for BCG protection is trained immunity in both diseases. The novel COVID-19 pandemic is a global emergency that required a worldwide response to control the progression of the novel infection and try to limit its expansion. The COVID-19 is an acute lower respiratory system infection results after getting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) virus. The first reported case of COVID-19 was on 30 December 2019 in Wuhan, China . The BCGMycobacterium tuberculosis (Mtb) infection since 1921 in children, but unfortunately, it is not effective in adults [Nature journal explained the reasons for the ineffectiveness of BCG vaccine in young and adults, due to the failure in the control of the infection and delayed response of the mucosal dendritic cells (DCs) in presenting antigen of the Mtb to the CD4+ T cell which allow the bacteria to divide (time factor), and not due to a weak response. Moreover, the external supply of activated Mtb antigen-primed DCs into vaccinated mice, at the time of Mtb exposure have successfully induced CD4+ T cell response and eliminated early Mtb growth [Mycobacterium bovis infection. BCG vaccine induces an immune response against Mycobacterium tuberculosis via CD4+ T helper cell and CD8+ T cytotoxic cell [Bacillus Calmette–Guérin (BCG) vaccine is used to protect from n adults . Recentlb growth . The tubb growth . Variablxic cell . This rexic cell –13. The xic cell . TherefoUndeniably, the BCG contributes to the protection and lowering of the severity of the novel COVID-19 infection through triggering innate immunity particularly via enhancing macrophage activity and CD4+ T cells. Several studies presented a correlation between BCG vaccination and COVID-19 mortality under different scenarios . Using oTo date, most of the published studies show an association between the BCG vaccination and the severity of COVID-19 infection –32. The After the presence of effective vaccinations against COVID-19, now the studies turn to whether the BCG vaccine enhances the induction of immune response against COVID-19, if combined with the novel COVID-19 vaccines or not? Hypothetically, it is not surprising that BCG will induce the effect of vaccine efficacy through its non-specific effects. But for proofing, it required extra clinical data and research.Now it is understood that the BCG vaccine exerts a wide range of effects not only against TB but also on other non-TB-related pathologies through its capacity to modify cells of the innate immune system to create a memory for the pathogen without antibodies.