Live-attenuated SARS-CoV-2 vaccines received relatively little attention during the COVID-19 pandemic. Despite this, several methods of obtaining attenuated coronaviruses are known. In this systematic review, the strategies of coronavirus attenuation, which may potentially be applied to SARS-CoV-2, were identified. PubMed, Scopus, Web of Science and Embase databases were searched to identify relevant articles describing attenuating mutations tested in vivo. In case of coronaviruses other than SARS-CoV-2, sequence alignment was used to exclude attenuating mutations that cannot be applied to SARS-CoV-2. Potential immunogenicity, safety and efficacy of the attenuated SARS-CoV-2 vaccine were discussed based on animal studies data. A total of 27 attenuation strategies, used to create 101 different coronaviruses, have been described in 56 eligible articles. The disruption of the furin cleavage site in the SARS-CoV-2 spike protein was identified as the most promising strategy. The replacement of core sequences of transcriptional regulatory signals, which prevents recombination with wild-type viruses, also appears particularly advantageous. Other important attenuating mutations encompassed mostly the prevention of evasion of innate immunity. Sufficiently attenuated coronaviruses typically caused no meaningful disease in susceptible animals and protected them from challenges with virulent virus. This indicates that attenuated COVID-19 vaccines may be considered as a potential strategy to fight the threat posed by SARS-CoV-2. The COVID-19 pandemic continues to be a pressing global public health problem ,3, the pSARS-CoV-2 is an etiologic factor of COVID-19. As of 10 April 2022, 496,507,539 cases have been diagnosed globally, and the disease led to 6,177,354 deaths ,9. The vVaccine research and development in the case of SARS-CoV-2 and COVID-19 is focused on the spike (S) protein because it is a target of neutralizing antibodies. Its receptor binding domain (RBD) has the highest concentration of neutralizing epitopes, and some vaccines contain the RBD instead of a full S protein ,13. IndeAttenuated viral vaccines are often regarded as somewhat unsafe due to attenuated virus’ ability to spread, mutate and recombine with wild-type (WT) strains . NeverthAttenuated coronaviruses are a subject of a relatively large volume of research. Moreover, vaccines against several veterinary coronaviruses were commercialized, including infectious bronchitis virus (IBV) , which aAttenuated coronaviruses may be obtained through the old approach, which is serial passage in cell culture . IBV canOn rare occasions, an attenuated strain of a coronavirus may occur naturally. A good example is the porcine respiratory coronavirus (PRCV), which is a deletion mutant of TGEV with altered tropism. It usually causes a mild respiratory disease instead of a lethal enteric one ,51,52.Escherichia coli [A newer approach is to create the recombinant attenuated coronavirus with specific mutations through the use of reverse genetics systems . Coronavhia coli ,56, makehia coli . Methodshia coli ,58, in-fhia coli , circulahia coli  or assemhia coli . Coronavhia coli ,62. It mhia coli . It alsohia coli . Howeverhia coli .Reverse genetics allows for the creation of coronaviruses that could not be obtained during serial passage in cell culture due to reduced fitness, which precludes them from appearing in culture in significant proportion. Some chimeric coronaviruses  and coroSARS-CoV-2 emerged in 2019 in Wuhan, China  and is oThis systematic review aimed to identify mutations that have been applied to—or could be applied to—SARS-CoV-2 to attenuate it based on a literature search. The obtained results were analyzed to determine the potential features of attenuated SARS-CoV-2 vaccines. Particular focus was paid to the efficacy and safety of such vaccines. In order to increase the quality of the reported evidence, and to reasonably limit the number of research papers included in this review, while simultaneously taking into account the absence of the published results from trials involving humans, the analyzed studies were limited to those presenting in vivo data on attenuated COVID-19 vaccines from animal models. It was concluded that the furin cleavage site (FCS) deletion is the most promising strategy, although it perhaps needs to be used in combination. It was also determined that a sufficiently attenuated SARS-CoV-2 virus is unlikely to cause any significant pathology but is likely to be sufficiently immunogenic to protect from both severe and mild disease.The article is written in English;The article describes an actual and practical creation, acquisition or use of a coronavirus;Alternatively: That coronavirus lacks characteristics described in criterion no. 3, but its other properties stemming from some identified mutations are useful in the context of LAV;That coronavirus shows an attenuated phenotype in vivo such as decreased mortality, reduced symptoms, viral titer or load after infection relative to a virus from which it is derived. The infection may be either in a natural host of the virus or a suitable animal model;The described coronavirus can be propagated with a measurable viral titer and no passage limit in permissive cell line or embryonated chicken eggs;The nucleotide sequence of the coronavirus or amino acid sequence of its proteins are known and can be compared to the parental virus;The attenuating mutations can be applied to SARS-CoV-2. That is, the differences between parental and attenuated viruses affect amino acid residues, whole proteins or nucleotide sequences present in SARS-CoV-2. In case of doubt, sequence alignment and literature data will be used to decide if the attenuation method is applicable;The article presents data from in vivo studies.To identify eligible articles that describe attenuated coronaviruses, Scopus , Web of The article describes the generation or acquisition of a coronavirus insufficiently, precluding replication of the results;The article describes sequencing or genotyping of a coronavirus in infected humans or animals but not its isolation;The described virus must be pretreated  before it can be used as a vaccine. That is, the specimen was only tested as a component of partially or fully inactivated vaccine;The article is a review;The article is a preprint;The article presents only the data not useful in the context of this study, such as sequencing and structural data.The exclusion criteria were as follows:The focus of this study is to identify suitable animal studies, but human studies were not explicitly excluded should any been found. Coronavirus-derived replicons that can be packaged in coronavirus-like particles were also allowed. Sequence alignment described in the inclusion criterion no. 6. was performed using Unipro UGENE v. 41   and the Mutations present relative to the WT virus;Titers at the time the WT virus reached peak titer;Results of in vitro studies;Any determinants of pathogenicity of the viruses observed in vivo; mortality and weight loss reported as significant were extracted quantitatively;Results pertaining to the immunogenicity of the attenuated viruses observed before challenge, quantitatively;Any results of the studies aimed at studying the reversion to virulence;Results of challenge studies; mortality and weight loss reported as significant were extracted quantitatively.The data were extracted from relevant articles by one researcher. WebPlotDigitalizer 4.5  was used to extract data from plots . AlthougFor the purposes of review, the data were imputed into separate columns within a spreadsheet. Data regarding the same coronavirus from different articles were grouped together. The most important outcomes are presented in A total of 2314 articles were retrieved from the Scopus database , 1906 arThere were a total of 101 attenuated coronaviruses described in the included papers, and 83 of them were tested in vivo. They were derived from seven viruses, namely: 29 viruses were derived from SARS-CoV, 28 viruses from SARS-CoV-2, 27 viruses derived MHV, six viruses from MERS-CoV, six viruses from IBV, three viruses from PEDV and two viruses from HCoV-229E, although none of the attenuated HCoV-229E viruses were tested in vivo. While several strains of SARS-CoV-2 were used as the starting point for attenuation, MHV-A59, MHV-JHM, SARS-CoV MA15  and SARSA total of 27 attenuation strategies, which can be or were applied to SARS-CoV-2, were identified. A full list is presented in The viruses were evaluated in several animal models; the most important ones are listed in The most promising strategy for SARS-CoV-2 attenuation was the deletion of the FCS. FCS is consistently lost when the virus is passaged in Vero E6 cells, but it can also be deleted using reverse genetics ,131,132.The reverse genetics techniques were used to introduce several attenuating mutations into coronaviral genomes. They mostly centered around abrogating innate immunity evasion and disabled such functions as the NSP1 host protein translation inhibitor ,97, NSP3Interestingly, two types of attenuating mutations that did not result in altered amino acid sequences were identified. First, the synonymous codons within a defined region of the viral genome may be exchanged. This leads to suboptimal codon pairs in a process known as codon pair deoptimization. The resulting virus exhibits less efficient protein synthesis and reduced viral fitness and pathogenicity ,119,121.Based on the results of the animal studies, the attenuated SARS-CoV-2 virus would be a highly effective COVID-19 vaccine. The intranasal route of administration appears to be the most suitable, as it was utilized in most of included studies involving SARS-CoV-2. Sufficiently attenuated SARS-CoV MA15 and SARS-CoV-2 viruses caused only minor inflammation in vaccinated animals . The immThe role of LAV in the context of the COVID-19 pandemic primarily needs to be to prevent transmission to the most susceptible individuals, who are not eligible for LAVs themselves. Other benefits may include the reduced impact of isolation of the diseased patients on the economy and reduction in rarely occurring severe cases of COVID-19 in individuals without significant risk factors. It must also be remembered that LAV would need to be rapidly developed in the pandemic setting.There are generally two methods of obtaining attenuated coronaviruses: reverse genetics and serial passage. Regarding the former, it was reported that a SARS-CoV-2 clone could be obtained through reverse genetics within a month or less . When itWhile, based on some published research, it is evident that attenuated SARS-CoV-2 virus can be rapidly created, its potential safety profile requires a more careful examination. It must be remembered that COVID-19 is non-fatal for the majority of individuals . TherefoBased on the information presented in the previous paragraph, animal models might be used to predict the properties of an attenuated COVID-19 vaccine. However, a large subset of studies regarding attenuated coronaviruses did not concern SARS-CoV-2. If the data collected using animal infection models with different coronaviruses are to be applied to SARS-CoV-2, it is necessary to understand some crucial characteristics of those models. Some of the mutations in the included viruses, other than those derived from SARS-CoV-2, affected highly conserved functions regulating the evasion of innate immunity. These consistently yielded attenuated phenotypes in different viruses. More caution is perhaps to be required if the attenuation stems from altered tropism. MHV-A59 expressing an MHV-JHM spike showed markedly reduced neurovirulence after the spike L1114R mutation was introduced, especially in combination with Q1067H and Q1094H ,118. FurDespite the broad tropism of MHV, most attenuated viruses derived from it were not very pathogenic. On the other hand, PEDV, an enteric virus, appeared to be less sensitive to attenuating mutations than the aforementioned viruses ,108. It −/− mice. Interestingly, they most often died but did not lose weight upon MHV-A59 infection. Both weight loss and mortality were not noted when the virus had the NSP3 N515A mutation. This may suggest that the attenuated SARS-CoV-2 vaccine would be safe for patients receiving TNF inhibitors [−/− mice were able to clear the NSP3 N515A MHV-JHM IA mutant and NSP16 D130A SARS-CoV MA15 mutant, but others died or developed chronic infection with the revertant virus [While the included studies described experiments in young and aged animal models, there were no relevant immunosuppression models except for TNFhibitors . As far hibitors ,150,151.hibitors . Some RAnt virus ,110. ThiAs the models of coronavirus infections were discussed in the previous paragraphs, the following part will focus on the targets of attenuation methods.Coronaviruses encode a few conserved proteins involved in preventing the detection of viral pathogen-associated molecular patterns and the disruption of interferon signaling or the antiviral functions of interferon-stimulated genes. Most of the attenuation strategies in this review involved mutations that disabled those functions. Betacoronavirus NSP1 is a protein primarily involved in inhibiting the translation of and degrading host mRNA . This alCoronaviral NSP14 has two enzymatic activities. Its proof-reading exonuclease activity is not dispensable in SARS-CoV-2 . HoweverNSP15 and NSP16 are yet other proteins involved in coronaviral immune evasion. NSP15 has endonuclease activity, which prevents dsRNA sensing ,159,160,The next target of attenuating mutations was the envelope protein, which was intensively studied in SARS-CoV. Disruptions in the E protein ion channel and PDZ-binding motif (PBM) were attenuating, as was the deletion of the whole envelope protein. The exact role of the ion channel activity of the SARS-CoV E protein is not very well understood, but its involvement in inflammasome activation was well-proven and may be a mechanism involved in the virulence of the virus ,161. SevRegarding the FCS mutants, it was established that they are less fit in TMPRSS2-expressing cells ,115,116.Based on the arguments presented in The final protein targets that were altered in attenuated mutants are accessory proteins. Their deletions are only weakly attenuating in SARS-CoV-2 . They diThe strategies mentioned above alone did not entirely prevent the propagation of the viruses in vaccinated animals. However, the development of an attenuated or attenuated-like COVID-19 vaccine for immunocompromised patients that mimics the properties of highly attenuated MVA appears feasible. Simultaneous deletion of ORF3a and E proteins in SARS-CoV-2 leads to packaging-deficient replicons that require complementation with E and ORF3a proteins in trans to produce infectious progeny and cannot propagate in normal mammalian cells ,95. TheyInterestingly, based on animal studies, it has been shown that the SARS-CoV-2 variant of concern Omicron, lineage BA.1  is As it has been demonstrated, several attenuating mutations can be introduced into the SARS-CoV-2 genome through the use of reverse genetics. However, safe use of the vaccine will not depend solely on the lack of pathogenicity of the vaccine virus. New variants may arise if the WT and vaccine SARS-CoV-2 are allowed to recombine in human host cells, as it was in the cases of other coronaviruses. In fact, recombination between the vaccine and WT strains of IBV seems to increase the diversity of circulating strains ,39,40,41In reference to the existing data from human studies, there was one SARS-CoV-2 virus for which such data were available. An interesting isolate that displayed a minimal cytopathic effect was found in an asymptomatic patient who presented a high viral load. Vero E6 cells infected with this virus were viable and could be passaged. Such a persistently replicating variant would allow for obtaining more vaccine doses from a single batch of cells .As mentioned before, combining two or more attenuation strategies may be desirable to increase the degree of attenuation. This was performed for only five of the included viruses; FCS deletion was combined with codon deoptimization while PBM was deleted from ORF3a and E protein simultaneously ,82. No cThe next issue discussed herein will be the probable safety and efficacy profile of the attenuated SARS-CoV-2 vaccine. Attenuated SARS-CoV and SARS-CoV-2 usually caused mild lung inflammation and sometimes minimal lung damage in animals, as evidenced by histopathological examinations. While certainly not desirable, it does not preclude the applicability of the attenuated SARS-CoV-2 vaccine. The attenuated influenza vaccine was also noted to cause pulmonary inflammation in some ferrets in preclinical studies . Any pro50 [Considering the immunogenicity of attenuated coronaviruses, it was usually high and similar to the WT virus . A notab50 .An interesting finding related to immunogenicity is that SARS-CoV Urbani and SARS-CoV MA15 with the E protein deletion and SARS-CoV Urbani with TRS replacement were more immunogenic in aged animals ,119. ThiRegarding the efficacy of the attenuated COVID-19 vaccine, the most convincing reports come from challenge studies . SARS-Co6 plaque-forming units (PFU), may be easily used to vaccinate individuals, it is unclear if such an amount of SARS-CoV-2 or other coronaviruses can actually be encountered during real-life exposure. Meanwhile, in the challenge studies, the vaccine and challenge doses may be similar. One reason for this is to increase the severity of the disease and mortality in the particular animal model. An already mentioned study did not find SARS-CoV Urbani with the envelope deletion to be protective when the challenge dose was 50 times higher than LD50 [50  was sufficient to infect 53% healthy, SARS-CoV-2-naïve individuals [Nevertheless, challenge studies have a peculiar feature related to infectious doses ,72,119. han LD50 . Nonetheividuals . The exaividuals . The 6–8ividuals .An interesting aspect of the attenuated COVID-19 vaccine may be the choice of the route of administration. Intranasal appears to be the most obvious. It mimics the natural mode of infection and delivers the virus to the tissues where it can efficiently replicate. In fact, the human challenge study of virulent SARS-CoV-2 used nasal drops to deliver the challenge virus . It is aComparing the potential profile of attenuated SARS-CoV-2 and currently used COVID-19 vaccines leads to an important issue that has not been answered by using animal models so far. Almost all currently used COVID-19 vaccines require more than one dose for full efficacy and booster doses due to waning immunity . MeanwhiFinally, according to the limitations and implications of this review, it must be stated that attenuating mutations may have different effects in different coronaviruses even if the amino acid residues they affect are part of a clearly conserved sequence. Several examples of this are known in the literature; for example, NSP14 proof-reading exonuclease is redundant for replication but necessary for virulence in SARS-CoV or MHV ,184. On Since no attenuated COVID-19 vaccine is currently marketed, the results from clinical trials are not available and cannot be discussed. Instead, it seems clear that the potential of attenuated SARS-CoV-2 as a vaccine was largely neglected. Although, at this point, a new COVID-19 vaccine may not have a significant impact, it may be worth it if the development of LAVs against emerging pathogens would receive higher priority in the future. Perhaps a range of attenuating mutations may be identified in different viral taxa to facilitate the rapid development of LAVs through reverse genetics. Nevertheless, if another pandemic coronavirus emerges ,188,189,In conclusion, attenuated COVID-19 vaccines had considerable potential to alter the course of the COVID-19 pandemic. Animal studies have demonstrated the potential for the safety and efficacy of attenuated SARS-CoV-2 vaccines with the additional benefits of being one-dose and needle-free, allowing for intranasal administration and reducing transmission. Attenuated SARS-CoV-2 would likely cause transient, asymptomatic pneumonia at most. FCS disruption seems to be the best strategy for achieving SARS-CoV-2 attenuation because it was proven to be hard to revert, transmission-preventing and immunogenicity non-altering, while maintaining high titer and stability in Vero E6 cells. Another promising strategy is TRS replacement, which has the potential to prevent recombination between the vaccine and WT viruses. Other strategies are usually aimed at preventing the evasion of innate immunity by the virus. A combination of attenuation methods to achieve a vaccine virus that is safe, immunogenic and does not revert is probably the most desired. Reverse genetics seems to be the method of choice for obtaining an attenuated COVID-19 vaccine because of its predictable results. SARS-CoV-2 passaged in cell culture usually loses FCS, which is a result that was also achieved through reverse genetics. Other mutations may also arise in them, the exact importance of which was not thoroughly studied. Notably, the recently emerged and highly infectious SARS-CoV-2 variant of concern, Omicron, may be perceived as a weakly attenuated SARS-CoV-2 virus.


ABCD1 have been explored. Here, we studied a Chinese patient with clinical features similar to those of early-stage CALD but with a negative molecular diagnosis and a sibling who had presumably died of CALD. Trio-based whole-exome sequencing (trio-WES) and RNA sequencing (RNA-Seq) revealed a novel hemizygote NCSS variant c.901-25_901-9 del in ABCD1 intron 1, resulting in a complex splicing pattern. The in vitro minigene assay revealed that the c.901-25_901-9 del construct contained two aberrant transcripts that caused skipping of exon 2 and a small 48-bp deletion on left of the same exon. We identified a novel NCSS variant, that extends the spectrum of the known ABCD1 variants, and demonstrated the pathogenicity of this gene variant. Our findings highlight the importance of combining RNA-Seq and WES techniques for prompt diagnosis of leukodystrophy with NCSS variants.Cerebral adrenoleukodystrophy (CALD) is a fatal genetic disease characterized by rapid, devastating neurological decline, with a narrow curative treatment window in the early stage. Non-canonical splice-site (NCSS) variants can easily be missed during genomic DNA analyses, and only a few of them in  ABCD1 mutations  was identified in both HEK293 and MCF-7 cells. In contrast, the minigene of the proband’s variant (c.901-25_901-9 del) of ABCD1 produced two aberrantly spliced products in both HEK293 and MCF-7 cells that were shorter than the wt minigene. PCR product I [exon A–exon B] was 389 bp and skipped the entire exon 2, whereas product II [exon A–△exon 2 (133 bp)–exon B] was 522 bp with a 48-bp deletion on the left of exon 2. Of the two products, product I was mainly obtained.To test the sequencing analysis results, we performed functional in vitro experiments to study the splicing events. Different splicing patterns were observed between the c.901-25_901-9 del mutant and wt . The minAfter CALD diagnosis, the proband received allogeneic hematopoietic stem cell transplantation three months after the first admission, without major adverse events. Eighteen months after treatment, the proband was going back to school. Until the last visit, 30 months after treatment, the boy had achieved smooth school performance without major functional disabilities. Cerebral demyelination was gradually arrested as determined using the brain MRI. The extended family members of the mother are healthy ; they reABCD1 in the proband. The proband’s mother, who carried heterozygous variants of ABCD1, was asymptomatic. RNA-Seq combined with the minigene assay confirmed that exon 2 skipping was the splicing pattern of ABCD1 with the c.901-25_901-9 del variant.In the present study, we performed trio-WES and RNA-Seq in members of a Chinese family with two different clinical phenotypes of CALD and an uninformative multi-gene panel test. We identified the novel hemizygous VUS c.901-25_901-9 del of In our study, leukodystrophy was considered based on family history and the sibling’s symmetrical lesions of the white matter of the central nervous system. Leukodystrophy comprises a large group of rare genetic disorders primarily affecting the white matter of the central nervous system. Disease prognosis is poor, as there is no cure in most cases. There are only a few leukodystrophies that can be cured, and this is only in the initial stages of the disease, emphasizing the need for a rapid diagnosis . The proIn some cases, other peroxisomal disorders, such as Zellweger spectrum disorder , acyl-CoAs the proband was in the early stage of the disease, genetic tests needed to be optimized urgently to study the disease etiology, as the CALD therapeutic window is narrow. NGS genetic testing, including gene panel testing, WES, or whole genome sequencing (WGS), is the most successful approach for diagnosing rare diseases with the highest diagnostic yield ,37. In cABCD1. This variant was not detected in the leukodystrophy multi-gene panel test, which proves the importance of combining these sequencing methods in uncertain cases that might be linked to gene mutations. As of September 2022, only 43 ABCD1 variants located in NCSSs have been reported in the ABCD1 Variant Database . According to the ACMG standards and guidelines, NCSS variants are considered VUSs unless their effect on splicing has been validated [ABCD1 . However, to the best of our knowledge, only a few mRNA studies have explored these NCSS variants in ALD [ABCD1 splicing, thereby causing a premature termination of transcription and degradation of defective transcripts.The majority of noncoding variants are involved in the production of aberrant transcripts through exon skipping, cryptic site use, or intron retention . RNA-Seqs in ALD . In our ABCD1 variants and provide a basis for the early diagnosis of ALD in clinical practice for the improved management of the disease to prevent negative outcomes. A limitation of the study is that we could not identify the variant and determine the VLCFA level in the extended family members of the mother. Procuring this information in the future may help confirm the pathogenicity of the variant. Another limitation is that our findings are based on a single-family study, and studies involving more cases with this variant will further validate our conclusions. The combination of comprehensive DNA sequencing and RNA-Seq, and careful clinical, radiological, and biochemical evaluations, holds promise for both expediting the diagnostic process and reducing the number of unresolved leukodystrophy cases. By evaluating the pathogenic effects of the splicing variant, we gained crucial information for the assessment of a yet-to-be identified NCSS. Our findings expand the genotypic spectrum of


This case report presents the management of a 69-year-old gentleman with acute coronary syndrome in the setting of an incidentally detected hepatocellular carcinoma with intra-tumoral bleed. Initially, the patient presented with fever, cough, and sudden onset of dyspnea on rest accompanied by angina, after which he was diagnosed with non-ST segment elevated myocardial infarction complicated with congestive cardiac failure. His laboratory and radiological investigations were suggestive of a possible infective etiology which, in an era of COVID-19, was investigated further with a high-resolution CT scan of the chest, which was suggestive of features of pulmonary edema along with an incidental discovery of liver lesions on the abdominal cuts. A further workup with a dedicated triple-phase computed tomography scan abdomen demonstrated features of undiagnosed hepatocellular cancer with intra-tumoral bleeding. Therefore, a mesenteric celiac angiogram followed by trans arterial bland embolization of the bleeding vessel was performed. In the same setting, for the simultaneous management of the acute coronary syndrome, coronary angiography performed revealed a triple vessel disease which was immediately followed by a percutaneous transluminal coronary angioplasty. There is a rising trend in the incidence of liver cancer, with an expected incidence of more than one million cases by the year 2025, out of which hepatocellular carcinoma (HCC) accounts for approximately 90% of cases . The incWe report the case of a 69-year-old gentleman who presented to the emergency department of our hospital, with fever, cough, and sudden-onset breathlessness on rest, accompanied by angina, after getting transferred from another hospital on non-invasive ventilation. The patient initially presented to an external hospital where he was vitally unstable with a temperature of 100.2F, Saturation of peripheral oxygen (SpO2) measuring 84% on room air, and blood pressure (BP) of 90/50mmHg, after which he was vitally stabilized and transferred to our hospital on non-invasive ventilation. On admission, he had a heart rate of 130bpm, BP of 100/70mmhg, and SpO2 measuring 93% on room air. An electrocardiogram (ECG) coupled with a high sensitivity troponin T(hs-TnT) value of 217ng/L  done at the hospital, was suggestive of a non-ST segment elevated myocardial infarction (NSTEMI), complicated with clinical features suggestive of congestive cardiac failure (CCF) [Given his symptoms in an era of the COVID-19 pandemic, a nasopharyngeal swab was collected and tested for COVID-19, whose results arrived a day after admission and were negative. His blood sample was collected and sent for routine laboratory investigations. The results of laboratory results are summarized in Tables In light of the COVID-19 pandemic, we performed a further workup with a high-resolution computed tomography (HRCT) of the chest which was suggestive of features of fluid overload Figure , along wA further workup with a dedicated triple-phase contrast-enhanced computed tomography (CECT) scan of the abdomen was hence performed, which showed a well-defined heterogeneously hypodense round mass, measuring 7.3x8.8x7.1cm, closely approximated to the diaphragmatic surface of the liver, with hyperdense areas within, which represented blood contents in segment VIII of the liver along with an incidental finding of a hydatid cyst measuring 4.5x4.8x4.2cm in the right lobe of liver segment VIII Figure . The lesThe dilemma of managing NSTEMI in the presence of an intra-tumoral bleed led to performing a mesenteric celiac angiogram, that showed an abnormal hypervascularity with tumor blush from the hepatic artery coursing through the tumor in segment VIIII Figure . Of noteAdditionally, the CAG performed through the femoral route revealed a triple vessel disease, with a diffusely diseased right coronary artery (RCA) in its whole course, 60% stenosis of the distal left main artery, 90% ostial involvement of the left anterior descending artery (LAD), and an 80% ostial involvement of the left circumflex artery (LCX) Figure .Therefore, in the same setting, a PTCA was performed with a 3.5x24mm drug-eluting stent (DES) in the left main artery through the right femoral access Figure .The patient was taken off bilevel positive airway pressure the following day and discharged after two days, following complete stabilization. He was further planned for endoscopic ultrasound-guided-fine needle aspiration (EUS-FNA) of the nodal metastasis with sorafenib therapy on follow-up -12.This case is a suitable illustration of the multifaceted diagnostic workup and management of a 69-year-old male, with acute coronary syndrome and an incidental finding of hepatocellular carcinoma with active intra-tumoral bleeding. Incidental findings usually come with the challenge of deciding whether the further treatment for which the diagnostic workup has been performed, should be continued. In our case, multiple specialties were involved in deciding the further line of management. The patient’s acute presentation of an NSTEMI and CCF required immediate management of the patient and a diagnostic CAG was the next best step, for which DAPTs and anticoagulation had to be administered, but the coexisting tumoral bleed could have aggravated in that case. Hence, embolization of the hepatic artery along with administering diuretics, DAPTs, and anticoagulation seemed the next best approach.Since there are no standard guidelines so far due to the rarity of such cases, the most up-to-date approach was hence followed. After thoroughly searching PubMed, PubMed Central, and Google Scholar, two cases with similar associations of acute coronary syndrome and hepatocellular carcinoma were found. One of the cases shows the occurrence of acute coronary syndrome occurring in a patient with hepatocellular carcinoma secondary to hepatitis C cirrhosis . This diThis is a rare case of simultaneous management of an acute coronary syndrome by performing a left heart catheterization which requires anticoagulation, in the setting of an incidentally detected probable hepatocellular carcinoma with active intra-tumoral bleeding. This case also highlights the importance of considering the possibility of a malignancy, even in the absence of any symptoms and underlying risk factors. The simultaneous occurrence of an acute coronary syndrome with a probable hepatocellular carcinoma and a hydatid cyst encourages further research to study the possibility of an association between them. The above report is a good reminder for physicians to be vigilant of the abdominal cuts on chest scans.


The results show five-times larger terrOC release from the Eurasian than from the American Arctic. Most of the circum-Arctic terrOC originates from near-surface soils (61%); 30% stems from Pleistocene-age permafrost. TerrOC translocation, relative to land-based terrOC stocks, varies by a factor of five between circum-Arctic regions. Shelf seas with higher relative terrOC translocation follow the spatial pattern of recent Arctic warming, while such with lower translocation reflect long-distance lateral transport with efficient remineralization of terrOC. This study provides a receptor-based perspective for how terrOC release varies across the circum-Arctic.Arctic change is expected to destabilize terrestrial carbon (terrOC) in soils and permafrost, leading to fluvial release, greenhouse gas emission and climate feedback. However, landscape heterogeneity and location-specific observations complicate large-scale assessments of terrOC mobilization. Here we reveal differences in terrOC release, deduced from the Circum-Arctic Sediment Carbon Database (CASCADE) using source-diagnostic (δ This synthesis of carbon isotope data in circum-Arctic shelf sediments provides a holistic, receptor-based perspective of how carbon release and transport vary between Arctic soils, peat and permafrost deposits amongst the different Arctic regions. These systems contain terrestrial organic carbon (terrOC) stocks equivalent to twice the carbon pool in the atmosphere. Arctic permafrost soils hold about 1000 Pg OC in the top 3 m2. Another 200 Pg OC are stored in old Pleistocene-age permafrost such as coastal Ice Complex Deposits (ICD)3 that occur primarily in northeast Siberia and Alaska, and are particularly prone to collapse5. Furthermore, peatlands in boreal soils outside the permafrost zone are estimated to contain an additional 230 Pg OC6. Climate warming may cause release of terrOC from these pools by, e.g., deepening of the seasonally thawed active layer of permafrost soils, thermokarst, thermal collapse and erosion of ice-rich ICD, or degradation of peatlands. This may expose large amounts of terrOC to fluvial transport and to microbial degradation, with subsequent emissions of greenhouse gases (foremost CO2 and CH4), climate-carbon feedback8 and Arctic ocean acidification9. However, large uncertainties exist regarding the dynamics and processes of large-scale terrOC release in the Arctic.The Arctic is warming twice as fast as the global average11.Robust projections of the future Arctic carbon-climate feedback require a quantitative understanding of large-scale terrOC release. Yet, the overarching picture of terrOC release across the circum-Arctic rests on a scattered collection of location-specific observations. State-of-the-art permafrost models retain large uncertainties of (i) permafrost area, (ii) depth of the active layer, (iii) frequency and spatial extent of thermokarst; (iv) net carbon exchange between terrOC systems and the atmosphere, as well as (v) lateral leakage of carbon to aquatic conduits. Overall, non-linear terrOC release pathways are particularly challenging to predict, such as the collapse of ICD, thermokarst formation, tundra and forest fires, as well as fluvial release of OC; processes that may all affect the net ecosystem carbon balance15. Arctic rivers may shed light on terrOC release on a drainage basin scale and resolve the seasonal variation of different source compartments17. By contrast, the seven Arctic shelf seas have the advantage to serve as natural integrators of terrOC release from the river drainage basins through the sequestration of riverine OC from dissolved and particular forms in their sediments upon aggregation and settling with increasing salinity18. Moreover, shelf seas are recipient of OC from erosion of coastal permafrost deposits (ICD), which is suggested to be the dominating vector of terrOC release to the extensive Laptev and East Siberian Seas19.Lateral leakage of terrOC through aquatic conduits is a key component of the carbon cycle in the often water-logged circum-Arctic. The large rivers and coastal shelf seas act as nature’s own integrator and permit studies of terrOC release from different source compartments as well as geographically distinct drainage basins and coastal regions21 and uses an inverse receptor-based approach to deduce terrOC release from different source compartments in the circum-Arctic regions  allows to calculate the relative contribution of different sources to the terrOC sequestered in the sediments. Further, fluxes of terrOC release from different terrestrial compartments are quantified using 210Pb-based mass accumulation rates (n = 164) in the circum-Arctic shelf seas. Finally, the relative vulnerability of the different terrOC compartments to be released was investigated by comparing shelf terrOC accumulation fluxes with the corresponding catchment-specific terrOC stocks; i.e., OC in permafrost22 and non-permafrost surface soils (SurfSoil)23, ICD3, and peat6. To this end, an Integrated Carbon Release Index is introduced (I-CRI) to contrast geographical and source-specific differences of terrOC release across the circum-Arctic.The present study leverages off the recently established Circum-Arctic Sediment Carbon DatabasE (CASCADE)13C/Δ14C) provided by CASCADE allows us to distinguish between different terrOC sources and OC produced by marine phytoplankton . Accordingly, OC from surface soils to a maximum depth of 100 cm was applied as end member in the entire circum-Arctic, with the exception of the Beaufort Sea where also deeper layers and peatlands were included due to overlapping isotopic compositions (δ13C/Δ14C) and limitation to three end members in the mixing model. Pleistocene age OC in ICD occurs in northeastern Siberia, Alaska and western Canada, and was hence considered as second terrOC end member in the Laptev, East Siberian, Chukchi and western Beaufort Sea shelves. Further, deep peat below 100 cm depth was considered as pre-aged terrOC end member for the Kara Sea, which has the world’s largest peatland in its catchment. Petrogenic OC released from rock weathering was used as 14C-depleted terrOC end member in the Canadian Arctic and the Barents Sea, to account for the significant release of petrogenic OC in these regions25 . By contrast, the 13C end member was assumed to stay constant as previous studies indicated continued terrestrial 13C signatures despite major cross-shelf aging of terrOC26. Previous research also suggested aging of marine OC during cross-shelf transport29. However, the source location and the transport dynamics of marine OC are uncertain and the scale for marine OC aging in continental shelf seas is unknown, albeit likely much less than for terrOC due to both longer transports distances and lower recalcitrance.TerrOC end members were also corrected for aging during cross-shelf transport. Cross-shelf aging of terrOC was previously quantified by 31. Moreover, the partitioning of terrOC release correlates with the relative distribution of the land-based terrOC stock in the circum-Arctic  source apportionment of OC reveals that most of the terrOC was released from surface soils  and routes via rivers17. Yet, Pleistocene-aged permafrost OC accounts for less than a fifth of the dissolved and particulate OC export from, e.g., the Lena and Kolyma rivers that drain extensive ICD landscapes17, which points at a stronger coupling of ICD-OC release with erosion of the vast and exposed coastline. Based on a large-footprint dataset, the present study suggests that coastal erosion drives the large fluxes of ICD-OC release in the circum-Arctic.The large portion of terrOC released from ICD emphasizes the particular importance of collapsing Arctic coastlines in northeastern Siberia, Alaska and the US Beaufort Sea Fig. . While I36. Further, the concentrations of petrogenic OC in Beaufort Sea sediments  are also similar to the range of petrogenic OC concentrations in fluvial suspended material from within the Mackenzie basin (0.12–0.63%)36, which thus is broadly consistent with the results of the dual-isotope source apportionment and indicates that petrogenic OC in Beaufort Sea sediments is largely attributed to rock weathering and river export from the Mackenzie river and its tributaries36. Less certain, however, is whether petrogenic OC—likely to be highly recalcitrant—contributes to any notable emissions of CO2 and thus affects the active carbon cycle, which is why petrogenic OC contributions were not considered terrOC release in the forthcoming part of the present study.In addition to terrOC from soils, permafrost, and peat, petrogenic rocks release considerable amounts of OC in the catchments of the Canadian Arctic and off Svalbard. For the Canadian Arctic Archipelago and the Barents Sea, petrogenic OC accounted for 37 ± 26% and 16 ± 11% (mean ± s.d.) of their total OC releases, respectively. For the Beaufort shelf, we find 51 ± 13% of the OC in sediments to originate from petrogenic sources, which agrees with the large range (19–88%) suggested by previous studies specifically addressing this region23 that accumulates in sediments over the course of 100 years  to five times higher relative release in the basins of the Canadian Arctic Archipelago (1.0 ± 0.7) and the East Siberian Sea , and in the East Siberian Sea (0.8 ± 0.3). The I-CRI clearly shows differences in terrOC release propensities across the seven Arctic shelf sea catchments and between the terrOC source compartments.Terrestrial OC accumulation in the recipient shelf seas can be compared with the vast terrOC stocks of the circum-Arctic source compartments to provide a metric and perspective on regional differences in large-scale terrOC releases. To this end, the Integrated Carbon Release Index (I-CRI) represents the percentage of the total terrOC, SurfSoil-OC, ICD-OC, and Peat-OC stock in each drainage basin  than in larger drainage basins that extend further to the south . We hypothesize that the large differences in relative terrOC release to the Arctic Ocean receptors reflect a combination of processes, including regional differences of Arctic climate change, degradation during transport via hydrological conduits, and the thermal state of the ground. Key hypothesized mechanisms are further addressed below.Comparing the I-CRI with environmental properties of the circum-Arctic drainage basins offers an approach to explore possible drivers behind regional differences in terrOC release. First, the variability in I-CRI015 Fig. , d37,38.38. This caused notable warming of permafrost soils, particularly those situated in the continuous permafrost zone39. One of the effects of Arctic change is the deepening of the seasonal permafrost active layer, which is suggested by an increasing number of yet-scattered records41. Furthermore, first signs of accelerated coastal erosion in the circum-Arctic have been reported43.The amplified rise in Arctic temperatures may be an important driver of terrOC release from circum-Arctic permafrost soils. For the period 1960–2015, circum-Arctic average summer temperatures (May-October) increased by 1.4 °C in the Beaufort Sea catchment and up to 2.2 °C in the Canadian Arctic Archipelagoterr  and I-CRISurfSoil  with the average summer warming trends 1960–2015 in the circum-Arctic , and in the drainage basins of the East Siberian (19%) and Chukchi seas 44, which is also supported by the increased discharges of rivers in northeastern Siberia46. While this may suggest a connection between terrOC release and recent warming trends across the circum-Arctic, the range in warming is only 50% while the range in I-CRI is a factor of 5–7. In addition, recent Arctic warming, terrOC release and translocation may operate over different time scales. It therefore seems unlikely that temperature increase alone can explain the large differences in terrOC release across the circum-Arctic pinpointed in this study.Geographical variability in the temperature increase across the circum-Arctic might contribute to the regional differences in terrOC release. This is supported by the spatial correlation of the I-CRItic Fig. . These o47. This may be particularly anticipated for the circum-Arctic, where large parts of the catchment area are water-logged, and freshwater runoff governs terrOC release and degradation10. The I-CRI of the different shelf seas varies by a factor of five for total terrOC (I-CRIterr), and by a factor of seven for surface soils (I-CRIsurfsoil), showing generally higher source-receptor fluxes for smaller high-latitude basins, and lower fluxes in larger, southward-extending, catchments. Hence, the I-CRI is spatially correlated with the southward extent 49 and show high I-CRISurfSoil values, while the two southernmost basins (Beaufort and Kara seas) host only 43% and 17% of continuous permafrost  specifically, CO2 emissions are up to nine times higher than the ocean export54, with the highest CO2 emissions in the more southerly, non-continuous permafrost and peat-dominated part of the catchment. By contrast, the Laptev Sea catchment—to 80% underlain by continuous permafrost—reveals CO2 evasion only twice that of the fluvial terrOC export. The basins of the Chukchi and East Siberian Seas, which are smaller and more northerly, also associate with notably less CO2 evasion relative to the terrOC export (CO2 evasion three and four times higher than export) than for the Kara and Beaufort Seas basins. This further supports the argument of stronger terrOC degradation in southerly and permafrost-free areas. In addition, this suggests the importance of efficient remineralization of released terrOC as part of the carbon cycle and constitutes a potentially significant source of greenhouse gas upon permafrost thaw and terrOC mobilization in the circum-Arctic.Comparing terrOC release with riverine COeas Fig. 31,50–52.This study provides a large-scale receptor-based assessment of carbon releases from potentially climate-sensitive terrestrial deposits around the entire circum-Arctic. Dual-carbon isotope source apportionment and recipient fluxes in the circum-Arctic shelf seas reveal large differences in propensity for terrOC remobilization both from different source compartments and between the different Arctic Ocean drainage basins. The release of terrOC from the Eurasian-Arctic sector is about five times larger than from the American-Arctic sector. Most of the released terrOC originates from Surface Soil (61 ± 39%), with Ice Complex Deposit permafrost as the second largest source (30 ± 10%). The index metric of receptor-based terrOC fluxes to inland terrOC stocks (I-CRI) provides a perspective on the relative terrOC release propensity along the entire circum-Arctic. The geographical patterns of the I-CRI suggest that one driver of terrOC release may be Arctic warming and the progressive permafrost thawing over the past half century, which broadly shows a spatial pattern similar to normalized terrOC release in the circum-Arctic. However, the normalized terrOC release varies by a factor of five between larger and smaller catchments, which also suggests efficient degradation of terrOC as part of long-distance freshwater transport and underway remineralization of terrOC to greenhouse gases. Taken together, this large-scale perspective on terrOC release patterns informs about the functioning of climate-relevant carbon remobilization across the circum-Arctic, and provides a benchmark for future investigations of terrOC release in the Arctic during climate change.13C and Δ14C values of OC were taken from CASCADE21 (https://bolin.su.se/data/cascade). Only data from surface sediments was considered in this study, which are here defined as the surface layer at the water-sediment interface and not deeper than 5 cm.Total OC concentrations as well as δ13C, 14C) mixing model55 to distinguish fractions derived from the major OC sources  using the δ13C and Δ14C values of OC from 260 stations distributed across the Arctic Ocean . We also tested the sensitivity of the source apportionment and the OC accumulation rates to the cross-shelf transport correction. Source fractions calculated without considering cross-shelf transport revealed terrOC fractions 2 ± 4% larger , while the relative uncertainties of the OC source fraction estimates were 11% higher. As this mostly affected samples at outer shelf locations with low mass accumulation rates there was no acknowledgeable effect to the mass accumulation budget. We further tested the effect of any post-depositional OC aging to the source apportionment. Based on 210Pbxs sedimentation rates in circum-Arctic shelf sediments , and the corresponding 14C depletion of marine OC over the 0–2 cm depth interval, we find resulting OC fractions to shift <1%  compared to source fractions for which the end member remained unchanged. Even under very low sedimentation rates (0.004 cm/yr), the (oldest) petrogenic OC fraction in Beaufort and Barents seas sediments is only around 1% smaller, which is much lower than the uncertainty estimates of this fraction. Hence, any post-depositional OC aging can be ruled out and will not significantly affect the source apportionment.The effect of the cross-shelf transport correction was largest for the SurfSoil-OC end member. For instance, SurfSoil-OC transported to a location in the outer Laptev Sea 500 km away from the Lena river mouth would be affected by aging 2750 yearsThe results of the final source apportionments for all stations are provided in Supplementary Table 210Pb dating of 152 shallow sediment cores. In addition, we performed gap-filling 210Pb dating for 12 cores, of which ten were located in the Laptev and East Siberian seas, and two in the Kara Sea  / OC (wt%) / 100. In total, our flux calculations are based on 164 cores dated using 210Pb profiles . For calculating fractional accumulation rates, OC fractions were interpolated and projected to the North Pole Lambert Azimuthal Equal Area coordinate system, which is a projection that is area conservative across the latitudes. The resulting interpolated layers were multiplied with interpolated total OC concentrations from CASCADE as well as with the gathered and interpolated MAR data.22 using spatial OC densities for the active layer and permafrost to a depth of 100 cm. This dataset was amended by the Harmonized World Soil Database (HWSD)23, which also includes non-permafrost soils at a vertical extent of 0–100 cm depth. To calculate the OC stock in ICD permafrost in northeastern Siberia, Alaska and northwestern Canada, we calculated the ICD stock for each drainage basin using the fraction of the spatial coverage of ICD in the whole circum-Arctic and then allocated the total ICD-OC stock of 212 Pg C according to that distribution3. The OC stocks in circum-Arctic peatlands were calculated using the maps provided by Hugelius et al.6. It should be mentioned that peat overlaps with the surface soil 0–100 cm depth interval in areas of high peat coverage, such as for the Kara and Beaufort seas. All mapping was carried out in ArcGIS 10.6  and calculations were done in Matlab R2018a.To compare circum-Arctic accumulation of OC from terrestrial sources we calculated the stock of OC stored in surface soils (of both permafrost and non-permafrost soils), ICD and peatlands for each shelf sea watershed. Surface soils were defined as soils to a depth of 1 m. The calculation of the stock of OC in permafrost surface soils was facilitated by the NCSCDf, i.e., SurfSoil, ICD, Peat) represents the percentage (%) of the respective terrOC stock that is translocated to the shelf receptor per century (100 years). Hence, it is used to reflect the efficacy of terrOC release relative to the inland terrOC stock:f is the mass of the fraction (of surface soil or ICD) accumulated to the sediments of each shelf sea, and OCf is the corresponding land-based OC inventory (of OC in surface soil or ICD). We also calculated the I-CRI for total terrOC using the sum of surface soil, ICD, and peat for MAR, and the total terrOC pool as OC inventory.Comparing the fractional OC accumulation in shelf sediments with the partitioning of the OC stored in the respective catchment in each of the circum-Arctic shelf seas informs about the tendencies of OC stores to be laterally released to the coastal sediments. The Integrated Carbon Release Index (I-CRI) per terrOC fraction (Linear regression analysis was used to investigate the correlation between the I-CRI and the environmental characteristics of the drainage basins.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Persistent pain is a key symptom in people living with knee osteoarthritis (KOA). Infra-slow Neurofeedback (ISF-NF) training is a recent development focusing on modulating cortical slow-wave activity to improve pain outcomes. A parallel, two-armed double-blinded, randomized sham-controlled, feasibility clinical trial aimed to determine the feasibility and safety of a novel electroencephalography-based infraslow fluctuation neurofeedback (EEG ISF-NF) training in people with KOA and determine the variability of clinical outcomes and EEG changes following NF training. Eligible participants attended nine 30-min ISF-NF training sessions involving three cortical regions linked to pain. Feasibility measures were monitored during the trial period. Pain and functional outcomes were measured at baseline, post-intervention, and follow-up after 2 weeks. Resting-state EEG was recorded at baseline and immediate post-intervention. Participants were middle-aged (61.7 ± 7.6 years), New Zealand European (90.5%), and mostly females (62%) with an average knee pain duration of 4 ± 3.4 years. The study achieved a retention rate of 91%, with 20/22 participants completing all the sessions. Participants rated high levels of acceptance and “moderate to high levels of perceived effectiveness of the training.” No serious adverse events were reported during the trial. Mean difference (95% CI) for clinical pain and function measures are as follows for pain severity , pain interference , pain unpleasantness  and physical function . EEG sources demonstrated frequency-specific neuronal activity, functional connectivity, and ISF ratio changes following NF training. The findings of the study indicated that the ISF-NF training is a feasible, safe, and acceptable intervention for pain management in people with KOA, with high levels of perceived effectiveness. The study also reports the variability in clinical, brain activity, and connectivity changes following training. Persistent pain is a significant presenting complaint in knee osteoarthritis (KOA), leading to activity limitation and participation restrictions . AvailabElectroencephalography-based neurofeedback (EEG-NF) is a non-invasive neuromodulatory technique in which cortical electrical activity is measured and fed back in real-time to the individual to facilitate self-regulation of the cortical activity to influence specific behavior or clinical outcomes . EEG-NF A two-arm, parallel-group, double-blinded, randomized sham-controlled feasibility trial was conducted in Dunedin, New Zealand. The trial is reported according to the Consolidated Standards of Reporting Trials (CONSORT) extension for randomized pilot and feasibility trials . The desStudy participants were recruited from the Dunedin community using convenience sampling. Periodic advertising was carried out through Newspapers, Facebook (Sponsored), an e-mail invitation to the University of Otago (UO), displaying study flyers at the Public Hospital, School of Physiotherapy clinic, and other UO departments. Interested volunteers contacted the first author via telephone or email and underwent screening using the Qualtrics online survey platform  . EligiblAdults aged 44–75 years, with a clinical diagnosis of KOA with pain severity of at least ≥ four on an 11-point numerical rating scale for a minimum duration of 3 months were eligible to participate in the study .Participants were excluded if they had one of the following situations or conditions: underwent surgery or other invasive procedures in the last 6 months and any surgical procedures scheduled within 8 weeks after screening; undertaken any steroid injections to the knee joint in the past 3 months or on oral steroids in the previous month; current intake of centrally acting medications or intention of taking new medications in the next 8 weeks; neurological conditions or diseases; soft tissue injuries of the knee in the last 3 months; cognitive impairments; difficulty or inability to read or understand English or provide informed consent; and pregnancy or 6 months post-labor. A paper-based Mini-Mental State Examination (MMSE) was carried out for screening volunteers with cognitive impairments. The maximum MMSE is scored out of 30 points, and volunteers scoring a total score of 24 or below were excluded from the study.Participants were randomized into either active ISF-NF or sham ISF-NF groups (in a ratio of 1:1) using an open-access block randomization program by the department research administrator not involved in the assessments, allocation, or interventions. The allocations were concealed until after the initial assessment was completed. The NF provider (first author) opened the sealed envelope before the first NF training session.Participants and the outcome assessor were blinded to the group allocation. The group was disclosed to the participant after completing the follow-up questionnaire after 2 weeks after the post-intervention assessment session. Every week, all the participants were asked at the end of the third training session, “Which training condition do you think you received?” to determine the blinding integrity .recruitment rate was determined by the number of participants attending the screening assessment. The randomization rate was assessed by the ratio of the number of participants randomized into the trial from amongst those eligible. The retention rate was determined by the number of sessions attended by the participant as per the initial appointment schedule. The drop-out rate was measured as the number of participants who dropped out from each group and expressed as a percentage of the total number of participants enrolled in the study.The Acceptability is a “multi-faceted construct that reflects the extent to which people receiving a healthcare intervention consider it appropriate, based on anticipated or experienced cognitive and emotional responses to the intervention” . All theThe subjective likelihood that the ISF-NF training will have a persuasive impact on the participant . ParticiAn adverse effect is described as any harmful sign or symptom resulting from the treatment, which could be related to the ISF-NF training. All the participants were instructed to complete a Discontinuation-Emergent Sign and Symptom (DESS) inventory to record worsening or improving side effects compared to the status before every training session. DESS has been used in previous NF studies to record adverse events associated with NF training . DESS isPain, function, and psycho-social constructs were collected using validated questionnaires. The multi-dimensional constructs were chosen based on the biopsychosocial model of pain literature. Based on the recommendations by Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) consensus II, Pain , physical and emotional functioning measurement tools were included . A summaQuantitative Sensory Testing (QST) procedures including pressure pain threshold (PPT), mechanical temporal summation (MTS), conditioned pain modulation (CPM), vibration perception threshold, cold hyperalgesia, tactile acuity, and body schema integrity  are detaResting-state EEG was obtained in a quiet room while the participant was sitting upright in a comfortable chair by an independent researcher. EEG was collected using the Mitsar EEG system sampled at 500 Hz with WinEEG software at baseline (T0) and immediately post-intervention (T1) . The EEGTwo weeks following the final training session, all participants were contacted by a phone call or email to complete the online survey on pain  and adverse events using the DESS tool.Eligible participants attended nine sessions  of NF training at the School of Physiotherapy, UO. The intervention was provided by a researcher (JM) who was not involved in the outcome assessment and randomization process. Two NF experts trained the researcher who provided the intervention in the trial. Before the trial, the researcher conducted NF training sessions on 10 healthy people to get familiarized with standardized training protocols . The exp® Core™ i7-6700HQ CPU @ 2.60GHZ) produced by the ASUSTek computer INC. The Comby EEG lead cap with sensors (Ag/AgCl) with appropriate size was fixed to the individual’s head, with reference electrodes placed at the mastoids. A sparse amount of EEG gel was applied to each electrode using a syringe with caution to prevent bridging between adjacent electrodes. The tip of the syringe was used to move the hair beneath each electrode, and a mild abrasion of the scalp was performed. This was to ensure proper electrode placement on the scalp and to acquire quality EEG recording.During each session, participants were asked to sit on a chair with their back supported and stay relaxed for 10 min, allowing the trainer to prepare the participant for the NF training. Both active ISF-NF and sham ISF-NF were administered using a 21-channel DC-coupled amplifier produced by BrainMaster Technologies, Inc. The amplifier was connected to a high-end laptop  . The ideEach participant received auditory feedbacks delivered by BrainMaster Technologies Software for an approximate of 60–80% in real-time when the participant’s brain activity met the desired infraslow (0.0–0.1 Hz) threshold at the targeted ROIs.The software calculated the ratio in real-time in the ISF band, and the feedback was given when the ratio was > 1 based on satisfying the following equation:As the dACC and SSC combine two current densities and the pgACC only one, the current density of the pgACC was doubled . EffortsConditions for the sham ISF-NF group was the same as the active ISF-NF group, except the participants received sound feedback according to someone else’s pre-recorded session. To ensure this, we have trained healthy participants with an active ISF-NF program for nine sessions. We captured the feedback sound using Audacity software, a free and open-source digital audio editor and recording application . ParticiUsing WinEEG advanced software, raw EEG data were sampled at 500 Hz for 19 channels, filtered from DC to 50 HZ, plotted, and exported in American Standard Code for Information Interchange (ASCII) format ., SSC right (S1Rt) , pgACC , and dACC  (The exported files were then batch imported into the EEGLAB (MATLAB R2020a). Each file was resampled to 128 Hz, bandpass filtered from 0.01 to 0.1 Hz and the first 4 s of data were truncated. Subsequently, Average Fourier cross-spectral matrices  were com 16, 32) .Each EEG file was carefully inspected in ICoN for eye blinks, muscle artifacts, perspiration, and body movements, and the artifacts were manually rejected from the file. ICoN performs fast blind source separation (BSS) on multiple EEG time-series using second-order statistics (SOS). The ICoN allows source estimation, data filtering , and source localization of separated sources through the LORETA-Key software . The sofExact low-resolution brain electromagnetic tomography (eLORETA) software  was usedWe also computed the ISF band ratios between the ROIs as below and descriptively compared between the groups.Similar eLORETA techniques were applied to compute the FC between the ROIs . FC is aAll the graphs and calculations were performed using GraphPad Prism software version 9.1.0 for Windows .As this was a feasibility trial, a formal sample size estimation was not conducted, and feasibility outcomes were reported based on the feasibility trial recommendations . Hypothen = 11) or sham ISF-NF (n = 11). Participants were middle-aged (61.7 ± 7.6 years), New Zealand European (90.5%), and mostly females (62%) with an average knee pain duration of 4.0 ± 3.4 years. The demographic and clinical characteristics of the participants at baseline are summarized in Twenty-two participants with KOA underwent baseline assessment and were randomly assigned to receive either active ISF-NF  were randomized to receive either active ISF-NF training (n = 11) or sham ISF-NF training (n = 11).None of the participants indicated any objection or raised concerns regarding the randomization process. All the participants who underwent baseline assessment sessions . However, 85.7% rescheduled their training sessions at least once during the NF training period due to various personal reasons.Due to the COVID-19 lockdown in New Zealand , one parBoth active and sham group participants reported ISF-NF training as an acceptable intervention with high levels of perceived levels of effectiveness .Participants in both the groups had reported higher levels of motivation, mood, and engagement with the training sessions .Eight participants from the active group correctly identified their allocation group at the end of the training sessions. The results are presented in No serious adverse effects were reported during the trial period. Also, none of the reported symptoms was worsened during the training period . Two shaParticipants in both groups have shown changes in pain severity, pain interference, and unpleasantness . IndividAnother observation includes the improvements in pain severity during immediate and follow-up assessments when compared to baseline. The sham group tends to taper off on pain severity after the training period, whereas the active training group effect is either maintained or further improved over time . Also, pThe log transformed CSD values were averaged across all voxels belonging to the dACC, pgACC, S1Rt, and S1Lt for the ISF band .The balance between the areas of interest  for the ISF band was calculated and illustrated in The strength of the ISF band FC between the four ROIs for both the groups are represented in To our knowledge, this study is the first to assess the feasibility, safety, and acceptability of ISF-NF training in a musculoskeletal pain population using a randomized, double-blinded, sham-controlled design. The trial results suggest that a future fully powered clinical trial to evaluate the effectiveness of ISF-NF in KOA is feasible, safe, and acceptable, with participants rating high levels of perceived effectiveness. The overall willingness to participate in the trial was high. The results demonstrate a 100% treatment fidelity with all the NF training components, including dosage, duration, and frequency of intervention was delivered and completed as per the study protocol . The stuAs planned, we recruited the desired number of participants in 5 months of the recruitment period . While tThe majority of the participants were New Zealand European and middle-aged females, consistent with the previous NZ-based studies on KOA . HoweverAcceptability and perceived levels of effectiveness are two important domains in the design, evaluation, and implementation of health care interventions . Our resParticipants in both groups also reported moderate to high levels of perceived effectiveness of the training. The domain is understudied in health care interventions, and it is important to conduct standalone research or incorporate it to add further value to future clinical trials. In the current study, we have used only a single-item measure to rate the level of the acceptability and perceived levels of effectiveness with NF training. In the future, studies need to consider incorporating a qualitative methodology approach to investigate participants’ perception of the NF training to understand the depth of the perspective component associated with the intervention.Participants did not report difficulties or concerns with the assessments, and training procedures, and follow-up. Although all participants completed their training sessions, some of the training sessions were rescheduled within the same week. The main reason for rescheduling is participants’ unavailability to undergo training. Another challenge was that participants were not comfortable returning to work/home with dried electrode gel. Although participants were offered the option to wash their hair at the study location, none did so due to other commitments/personal reasons following the training session. Future research could consider incorporating dry electrodes for training .Evidence from previous studies has reported various side effects associated with NF training . It appeThe level of motivation and mood may impact the participants’ engagement with the NF training . RecentlWe found that most of the active ISF-NF participants could predict their group allocation correctly. A potential reason could be due to reduction of their symptoms. On the other hand, most sham group participants indicated that they were in the active group, as they believed and were hopeful of being in it. This could potentially contribute to the non-specific effect observed in the sham group and needs further investigation. Future trials must incorporate methodologies to investigate any relationship between the subjective pain measures observed in the sham group with their associated motivational or belief neural network . The faiThis study demonstrated changes in the clinical outcomes in both active and sham ISF-NF groups. It is noteworthy that the active ISF-NF group showed greater improvements in pain unpleasantness and pain interference scores. This study also observed a group difference in the time course effect on the symptoms during training, post-training (immediate and follow-up) assessments. The time course effect of NF is discussed in previous literature , and theConsidering the nature of the study design, we haven’t performed any statistical analysis for the EEG changes. However, the descriptive EEG data will help design and perform sample size calculations for future studies investigating the efficacy of ISF-NF training inducing neural changes in the chronic pain population. Future trials also need to consider a full band activity (>0.1 Hz) and connectivity EEG analysis to explore the effects of ISF-NF and its relationship with change in clinical pain outcomes. The results also demonstrated a positive trend in the balance between the ascending (SSC and dACC) and descending (pgACC) pain pathways in the active group. Moreover, the findings support the concept of the brain imbalance model of chronic pain , and thuOur findings should be interpreted with consideration of the study’s limitations. We recruited a small sample of KOA individuals with a homogeneous sex and race distribution, which restricts the generalizability of the study findings. The feasibility of long-term follow-up assessment and a higher number of NF training sessions was not studied in this trial. Despite the limitations, this study provides an important foundation for designing and implementing future clinical trials investigating ISF-NF protocols. Our study outcomes regarding the feasibility, safety, and acceptability provide validation for such future studies to replicate and explore the effectiveness of ISF-NF for chronic musculoskeletal pain conditions. We recommend a fully powered RCT to investigate the ISF-NF balance training protocol’s effectiveness with a justifiable sample size calculated based on the reported means and standard deviations for detecting differences between groups of at least medium effect size (Cohen’s d = 0.5) with 80% statistical power.In conclusion, this feasibility clinical trial suggests that a fully powered clinical trial is feasible and safe to be conducted in people with KOA. Moreover, people with KOA reported high levels of acceptability and perceived high levels of effectiveness of training and higher levels of motivation and engagement with the NF training. The study provides variability in the clinical and EEG measures, thus informing the future studies in ISF-NF for the estimation of sample size for a fully powered clinical trial.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by Health and Disability Ethics Committee (HDEC), New Zealand (19CEN182), and the cultural consultation was obtained from the Ngāi Tahu Research Consultation Committee (5733_21392). The patients/participants provided their written informed consent to participate in this study.DD, DA, RM, and JM provided the cortical areas to be targeted, their coordinates, and the desired training protocol. MS developed the ISF-NF programme based on the provided regions and coordinates. JM conducted the participant recruitment, data collection, EEG data processing, analysis, and wrote the first draft. All authors contributed to the interpretation of the results, manuscript revision and reviewed the final version making the necessary changes, approved the submitted version, and agreed to be accountable for the content of the work.


The COVID-19 pandemic has led to significant loss of life and severe disability, justifying the expedited testing and approval of messenger RNA (mRNA) vaccines. While found to be safe and effective, there have been increasing reports of myocarditis after COVID-19 mRNA vaccine administration. The acute events have been severe enough to require admission to the intensive care unit in some, but most patients fully recover with only rare deaths reported. The pathways involved in the development of vaccine-associated myocarditis are highly dependent on the specific vaccine. COVID-19 vaccine-associated myocarditis is believed to be primarily caused by uncontrolled cytokine-mediated inflammation with possible genetic components in the interleukin-6 signaling pathway. There is also a potential autoimmune component via molecular mimicry. Many of these pathways are similar to those seen in viral myocarditis, indicating a common pathophysiology. There is concern for residual cardiac fibrosis and increased risk for the development of cardiomyopathies later in life. This is of particular interest for patients with congenital heart defects who are already at increased risk for fibrotic cardiomyopathies. Though the risk for vaccine-associated myocarditis is important to consider, the risk of viral myocarditis and other injury is far greater with COVID-19 infection. Considering these relative risks, it is still recommended that the general public receive vaccination against COVID-19, and it is particularly important for congenital heart defect patients to receive vaccination for COVID-19. The COVID-19 pandemic has left its mark across the globe with a reported mortality rate of approximately 0.13% with around six million total COVID-19 deaths reported worldwide ,3. This Myocarditis is a potentially deadly condition most often caused by infectious diseases, though hypersensitivity reactions and autoimmunity have also been implicated ,14,15. MHowever, only a few viruses, including COVID-19, directly infect cardiac myocytes ,34,35,36The most significant contributing pathways for myocarditis development are those from inflammation. Viral particles are recognized by toll-like receptors (TLR), nucleotide oligomerization domain (NOD)-like receptors (NLR), and intracellular pattern recognition receptors (PRR) ,39. ActiViral myocarditis is caused by the amalgamation of many of these pathways depending on the specific pathophysiology of the inciting virus. In the case of enteroviruses, a very important initial pathway is the direct infection and disruption of cardiac myocytes . In addiAnother etiology of myocarditis linked to viral infections is systemic inflammatory or autoimmune syndromes. One of the most common syndromes associated with viral infections that causes cardiac dysfunction and myocarditis is Kawasaki disease in children . While KMyocarditis in COVID-19 infection has contributed to the severity and mortality of the disease in some patients. While COVID-19 is primarily a respiratory virus, infection affects other organs, including the heart, leading to symptomatology and, in some cases, death . ArrythmMany cohort and case studies have recorded incidents of cardiac involvement and myocarditis among hospitalized COVID-19 patients, including those with severe COVID-19 infection. One study estimated that COVID-19 myocarditis had a prevalence of 2.4 per 1000 hospitalized COVID-19 patients but increased to 4.1 per 1000 when unconfirmed myocarditis cases were included . Of the The overall mortality rate of patients with COVID-19 myocarditis has been reported to be 14% by some studies . Others As a result of the COVID-19 pandemic, the benefits and risks of the COVID-19 vaccines have become an important topic. While predominantly a viral respiratory infection, COVID-19 has effects on multiple organ systems, including the cardiovascular system. As mentioned before, the mortality rate has been reported around 0.13% with an estimated six million reported deaths ,3. The aSafety reports have shown that cases of myocarditis after COVID-19 vaccination were as many as 4.4 events per million recipients . Other rThe suspected pathways involved in vaccine-associated myocarditis are varied and highly dependent on the individual vaccine. Cytokine hyperstimulation is a general term that encompasses responsible pathways that are vaccine and individual specific. The causes of cytokine hyperstimulation are believed to be due to differences in genetic variants of the cytokines, cytokine receptors, and/or the types of cells involved in the specific immunologic pathways accounting for variable patient symptoms because of the elevated cytokine levels. Even when viral components are attenuated to have significantly reduced immunogenicity, these genetic differences can modify the reactivity to the vaccine in some subjects, causing adverse inflammation and even organ damage, resembling true infection . While tAnother similar reaction is eosinophilic myocarditis. The specific mechanism of cardiac damage caused by eosinophilic myocarditis is variable as well . Some stSimilar to the eosinophilic myocarditis autoantigen pathway is molecular mimicry. While the antigens used in vaccines are carefully chosen to avoid cross reactivity, some have molecular similarities with normal human tissue. The antibodies formed against these antigens have the risk of recognizing and damaging healthy tissue that is erroneously identified as foreign . DependiThe COVID-19 vaccines are not the first vaccine associated with myocarditis. Rare cases have been reported with the pneumococcal, hepatitis B, smallpox, and human papilloma virus vaccines, and some formulations of the influenza vaccine ,143,144.Due to the public prominence of the COVID-19 vaccines, much interest has been raised regarding the association of the vaccines with myocarditis. Several different mechanisms have been implicated in the development of COVID-19 vaccine-associated myocarditis . SimilarOnce myocarditis develops, several different outcomes can occur depending on the mechanism of injury. In many cases, vaccine-associated myocarditis is self-limited and will resolve on its own. In more severe cases, or in viral myocarditis, sustained damage leads to disease progression. When continued cytokine production or autoantibodies from molecular mimicry is involved, the increased damage to the heart ultimately leads to dilated cardiomyopathy . If the The pathway by which myocarditis stimulates fibrosis is an intricate network of immunological cascades that can have many inciting factors. In the case of coxsackieviruses, profibrotic cytokines such as TGF-β, TNF-α, IL-1, and IL-4, had elevated activity . TGF-β iTNF-α is another active inflammatory cytokine that has been found elevated in mice cardiac tissue with myocarditis and has been implicated in autoimmune myocarditis ,186. TNFTNF-α binds two receptors on cardiac myocytes, tumor necrosis factor receptor I (TNFRI) and tumor necrosis factor receptor II (TNFRII) . TNFRI iViral myocarditis can be from either direct cardiac tissue damage or from the immune response to the virus as previously discussed. If untreated, continued cardiac dysfunction and chronic infection leads to fibrotic cardiac remodeling. The degree of remodeling can be correlated with cardiac fibrosis observed on cardiac magnetic resonance imaging (MRI) ,161,162.Congenital heart defects are estimated to affect as many as 1% of live births in the US . As surgThe effects of COVID-19 infection in CHD patients are not well understood with limited data of outcomes in specific defects. Reports differ in their conclusions about the risk of COVID-19 infection severity and mortality in CHD patients ,214,215.Similarly, the effects of the COVID-19 vaccine on adult CHD patients are not well known. One study regarding COVID-19 vaccination in 208 adult CHD patients found no increase adverse events compared to a control patient population. Of these 208 patients, only one patient developed pericarditis and recovered after standard management with non-steroidal anti-inflammatory drugs (NSAIDs) . OfficiaThe COVID-19 pandemic is an unprecedented crisis, leading to the similarly unprecedented approval of the COVID-19 mRNA vaccines. Though the vaccines have been shown to be safe and effective, they have adverse effects, just as all vaccines do. Of particular concern has been the increasing number of reports of vaccine-associated myocarditis, of which the COVID-19 vaccines now account for the majority of reports . The spe