Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes secreted by many organisms and viruses. LPMOs catalyze the oxidative cleavage of different types of polysaccharides and are today divided into eight families  within the Auxiliary Activity enzyme class of the CAZy database. LPMOs minimal architecture encompasses a catalytic domain, to which can be appended a carbohydrate-binding module. Intriguingly, we observed that some LPMO sequences also display a C-terminal extension of varying length not associated with any known function or fold. Here, we analyzed 27,060 sequences from different LPMO families and show that 60% have a C-terminal extension predicted to be intrinsically disordered. Our analysis shows that these disordered C-terminal regions (dCTRs) are widespread in all LPMO families (except AA13) and differ in terms of sequence length and amino-acid composition. Noteworthily, these dCTRs have so far only been observed in LPMOs. LPMO-dCTRs share a common polyampholytic nature and an enrichment in serine and threonine residues, suggesting that they undergo post-translational modifications. Interestingly, dCTRs from AA11 and AA15 are enriched in redox-sensitive, conditionally disordered regions. The widespread occurrence of dCTRs in LPMOs from evolutionarily very divergent organisms, hints at a possible functional role and opens new prospects in the field of LPMOs. LPMOsdatabase . LPMO-lidatabase ,13.2 or H2O2 to hydroxylate the polysaccharide substrate [The catalytic domain of all LPMOs hitherto characterized adopts an immunoglobulin-like β-sandwich fold . The actubstrate ,16. Likeubstrate ,18,19.Due to structural constraints required for catalysis, enzymes adopt a globular and folded conformation. However, there are growing evidences of enzymes  possessing functionally-active intrinsically disordered regions (IDRs) . IDRs doDuring our previous investigations, we noticed that some LPMOs display an intriguing C-terminal extension, which was not encountered in other CAZymes. These C-terminal regions are commonly removed during expression construct design prior to LPMO characterization. In this study, we used bioinformatics to analyze these uncharacterized C-terminal extensions occurring in different LPMO families. The objectives were to (i) predict their degree of disorder, (ii) investigate their distribution across LPMO families and organisms and (iii) analyze their molecular features.LPMO amino-acid sequences were retrieved by BlastP  searchesSubsequently to curation, LPMO sequences with a C-terminal region of unknown function longer than 30 residues (UNK) were extracted and analyzed with MobiDB-lite 3.0 . We chosMobiDB-lite uses eight different predictors  to derive a consensus that is refined to remove short disordered regions and keep only those that consist of at least 20 consecutive residues predicted as disordered . Since IUPred and DisEMBL use a 20-residue sliding window, to avoid biases due to the analysis of partial sequences, for each sequence we reconstituted the sliding window by adding the ten amino acids that precede the UNK sequence and used this enlarged sequence for disorder prediction. However, disorder statistics only considered residues strictly belonging to the UNK region, where the boundaries of the latter were inferred as described above. A residue is considered as disordered if at least five out of the eight predictors predict it as disordered. A C-terminal sequence was considered to be disordered (and hence classified as dCTR) if MobiDB-lite 3.0 predicts at least one LDR. The fraction of disordered residues is calculated by dividing the number of residues predicted to be disordered by the length of the dCTR. The properties of the dCTRs were derived from their primary sequence through the toolkit localCIDER . These phttp://cprofiler.org/cgi-bin/profiler.cgi) [dCTR), with the corresponding content in the PDB Select 25 database (CPDB), as (CdCTR-CPDB)/CPDB. PDB Select 25 is a data set containing PDB structures with less than 25% sequence identity [The amino acid compositional bias was estimated by the Composition Profiler webserver (ler.cgi) , by compidentity .The prediction of redox-sensitive, conditionally disordered regions was generated by the stand-alone version of IUPred2A, keeping default parameters . The metThe prediction of Disordered Binding Sites (DiBS) was generated by the stand-alone version of ANCHOR2 in IUPred3 . ANCHOR2p-value for multiple comparisons. Analysis and data processing were conducted by in-house developed Python and R scripts for plotting and performing statistical analyses. The significance of multiple comparisons was evaluated with the non-parametric Kruskal-Wallis test. When the null hypothesis was rejected, we applied the post-hoc Dunn’s multiple comparison test to test for differences among the LPMOs families. The Bonferroni Correction was used to adjust To analyze the C-terminal regions of LPMOs, we parsed 27,060 LPMO sequences, belonging to seven distinct families . The C-terminal regions of LPMOs (of at least 30 residues in length) were segregated into different categories A: (i) LPWe then analyzed the LPMO UNK C-terminal regions using MobiDB-lite 3.0 to identify predicted disordered regions. The choice of the MobidDB-lite 3.0, over other disorder predictors, was dictated by the consensus-based approach able to capture different disorder “flavors” and by the implementation of a filtering step that guarantees that the predicted IDR covers at least 20 consecutive amino acids , www.cazy.org), this finding is in agreement with previous studies that have found that prokaryotic proteins are less enriched in IDRs compared to proteins from eukaryotic species [Considering all LPMOs sequences in the different families C, we not species ,53. StriThe modularity of CAZymes is known to play an important role in the function of the enzyme . In lighNext, we looked at the peculiarities of the dCTRs (length and amino-acid composition) in the six LPMOs families . While dCTRs can have varying length ranging between 30 , to over 1000 amino acids A, the mep < 0.01–0.001, Dunn’s test), while AA10, AA14 and AA16 dCTRs have a median disorder content of 70%. AA11 and AA15 dCTRs have a lower disorder content, with a median value of disordered residues of 55% and 45%, respectively. Looking at the fraction of disordered residues as a function of the dCTR length, we observed a general trend, whereby disorder content decreases with the length of the dCTR  different from those observed in AA11 and AA15 families . These Ω values suggest that charged residues are relatively well mixed along the dCTR sequence, thus favoring extended conformation. By contrast, an increase in Ω, as in the dCTRs of the AA16 family, reflects segregation of Pro, Lys, Asp, Arg, and Glu, and favors more compact conformations, as indicated by the representation in the diagram of state , the net charge per residue (NCPR) and the position of dCTRs on the diagram of states, as defined by Das et al.  Figure . NCPR coof state A.In light of the redox activity of LPMOs and of the enrichment in Cys in dCTR sequences of the AA11 and AA15 families, we sought to assess whether dCTRs can be redox-regulated, conditionally disordered regions. To explore this aspect, we used a tool implemented in IUPred2A to capture redox-sensitive regions that are likely to undergo disorder-to-order transitions as a function of the redox state of their cysteines . The metp < 0.0001, Dunn test) than that of the other dCTRs (The function of IDRs is often related to binding to other proteins and/or ligands via short recognition elements in disordered binding sites (DiBS). The residues belonging to DiBS have a propensity to form energy favorable interactions with globular partners, instead of intrachain interactions, resulting in at least partial folding of the DiBS. Here, we used the ANCHOR2 predictor to investigate the presence of DiBS in the dCTRs. er dCTRs . Consideer dCTRs , a findiStreptomycetales), characterized by a mycelial morphology. Among the viral LPMO-dCTRs, we noticed the presence of fusolin proteins (AA10 LPMOs), notably studied for their insecticidal activity [As LPMOs are widely distributed in living organisms (and viruses), we investigated the distribution of LPMO-dCTRs across the different kingdoms . LPMO-dCactivity .Aspergillus nidulans, and quantified their transcript levels on different constituents of plant cell wall  [Podospora anserina, a total of seven AA9 out of the 33 AA9 LPMOs encoded by its genome were identified in the secretomes [PaLPMO9D, which was secreted in the sugar beet pulp condition, bears a 90 amino acids-long dCTR. After heterologous expression in Pichia pastoris, PaLPMO9D did not show any activity on cellulose [Gloeophyllum trabeum are the only example of LPMOs characterized from brown rot fungi, so far. The genome of G. trabeum displays four genes encoding five AA9 enzymes, due to alternative splicing of the gene lpmoA. In particular, Kojima et al. [GtLPMO9A-2, which contains a C-terminal region, herein confirmed to be a dCTR. The enzyme showed a broad specificity and demonstrated a high efficiency in depolymerizing xyloglucans. Interestingly, the authors described four conserved cysteines in the dCTR region, which make this sequence susceptible of redox regulation. In that work, they also described the enzyme GtLPMO9D presenting a low complexity C-terminal region, herein identified as dCTR. The phylogenetic analysis revealed that this enzyme is part of a different clade quite distant from other AA9 characterized so far [To get more insight into the role of LPMO-dCTRs, we parsed available experimental data from the literature to know whether genes encoding LPMO-dCTRs are regulated and/or LPMO-dCTRs are secreted. We report in glucans) . Interescretomes . Interesellulose . LPMOs fa et al.  characted so far . OverallIn this study, using an in-silico approach, we discovered that about 60% of LPMO members, from most of the LPMO families, possess an intrinsically disordered C-terminal region that had hitherto gone unnoticed. Within glycan-processing enzymes, the presence of these dCTRs has so far only been observed in LPMOs. Indeed, to our knowledge, no dCTR has ever been observed in other classes of CAZymes  nor in other types of oxidoreductases . Data from the literature report that IDRs can provide several advantages to enzymes, such as ability to tune the affinity for different substrates , fast anO-glycosylation, which is known to occur in fungal and actinomycetes CAZymes [O-glycosylation of linkers in CAZymes is known to provide resistance to proteases and to impart extended conformations, thus extending the operating distance of the enzyme thanks to excluded volume effects [The disordered nature of dCTRs mainly arises from an enrichment in Ser, Thr and Pro suggesting PTMs, in particular  CAZymes ,57,58. T effects . It has  effects . Yet, liStreptomyces. While some Streptomyces species efficiently degrade plant biomass, these bacteria also share common morphological traits with fungi, i.e., they form a complex mycelium and sporulate under adverse conditions [We noticed that bacterial LPMO-dCTRs are mostly found in Actinobacteria from the genus nditions . Even ifnditions . Interesnditions . Considenditions . TherefoPhytophthora infestans. The authors observed that the AA17 LPMO catalytic domain is followed by a polypeptide predicted to be intrinsically disordered, which is variable in length and amino-acid composition and rich in Ser, Pro, and Thr residues [P. infestans pathogenic cycle, although the contribution of the dCTR in the pathogenic mechanism has not yet been established.It is also worth considering that LPMO-dCTRs could play a role in pathogenicity although in different contexts. Recent investigations in plant pathogen oomycetes, unveiled a pectin-active LPMO (AA17 family) from residues . This AAAnother intriguing example of a possible connection with pathogenesis, concerns fusolins, which are produced by insect viruses, and are examples of LPMO-dCTRs within the AA10 family. The C-terminal region in this particular AA10 LPMO (with a putative chitin activity) is responsible for spindle formation, i.e., proteinaceous crystalline inclusion bodies which enhance virus infectivity . The cryRecently, LLPS has also been shown to play a role in the extracellular milieu, as exemplified by galectin-3 . One of In conclusion, using computational approaches, we have shown that intrinsically disordered C-terminal regions are very common in most LPMO families. The variable length and amino-acid composition of LPMO-dCTRs and their presence across different domains of life (and viruses) may reflect either different functions or a common function conferred by distinct sets of physico-chemical properties that would result from host-specific adaptation. Based on all these in-silico analyses, we call the CAZyme community to include these extensions when characterizing LPMOs in a biological and/or biotechnological context. Further functional investigations are needed to experimentally assess the degree of disorder of these C-terminal regions and provide insights into their role.


Studies evaluating strategies for the rapid development, implementation, and evaluation of clinical decision support (CDS) systems supporting guidelines for diseases with a poor knowledge base, such as COVID-19, are limited.We developed an anticoagulation clinical practice guideline (CPG) for COVID-19, which was delivered and scaled via CDS across a 12-hospital Midwest health care system. This study represents a preplanned 6-month postimplementation evaluation guided by the RE-AIM  framework.The implementation outcomes evaluated were reach, adoption, implementation, and maintenance. To evaluate effectiveness, the association of CPG adherence on hospital admission with clinical outcomes was assessed via multivariable logistic regression and nearest neighbor propensity score matching. A time-to-event analysis was conducted. Sensitivity analyses were also conducted to evaluate the competing risk of death prior to intensive care unit (ICU) admission. The models were risk adjusted to account for age, gender, race/ethnicity, non-English speaking status, area deprivation index, month of admission, remdesivir treatment, tocilizumab treatment, steroid treatment, BMI, Elixhauser comorbidity index, oxygen saturation/fraction of inspired oxygen ratio, systolic blood pressure, respiratory rate, treating hospital, and source of admission. A preplanned subgroup analysis was also conducted in patients who had laboratory values  present. The primary effectiveness endpoint was the need for ICU admission within 48 hours of hospital admission.P<.001). Adherence with the anticoagulation CPG was associated with a significant reduction in the need for ICU admission within 48 hours  on multivariable logistic regression analysis. Similar findings were noted following 1:1 propensity score matching for patients who received adherent versus nonadherent care .A total of 2503 patients were included in this study. CDS reach approached 95% during implementation. Adherence achieved a peak of 72% during implementation. Variation was noted in adoption across sites and nursing units. Adoption was the highest at hospitals that were specifically transformed to only provide care to patients with COVID-19  and the lowest in academic settings (47%-55%). CPG delivery via the CDS system was associated with improved adherence (odds ratio [OR] 1.43, 95% CI 1.2-1.7; Our institutional experience demonstrated that adherence with the institutional CPG delivered via the CDS system resulted in improved clinical outcomes for patients with COVID-19. CDS systems are an effective means to rapidly scale a CPG across a heterogeneous health care system. Further research is needed to investigate factors associated with adherence at low and high adopting sites and nursing units. COVID-19, caused by SARS-CoV-2, has infected millions of people worldwide. This disease has shown many unique attributes including a hypercoagulable profile . COVID-1CDS technology solutions offer a mechanism that in support of the learning health system (LHS) facilitates long-term process and quality measure improvements ,12. CDS On April 9, 2020, our institution developed and disseminated a 3-tiered CPG for anticoagulation in COVID-19 in collaboration with national experts . Given tThis study represents a preplanned 6-month implementation evaluation guided by the RE-AIM  framework  of the aA COVID-19 evidence-based medicine (EBM) team was created in March 2020 to rapidly review, catalogue, and publicly disseminate evidence related to proposed COVID-19 therapeutics including anticoagulation management . Due to conservative treatment approach) due to concerns for bleeding and disseminated intravascular coagulopathy, and some studies suggesting tissue plasminogen activator infusions (aggressive treatment approach) for patients with severe respiratory failure [Guided by the EBM team’s recommendations, system hematology service line leads (S Shah and MR) oversaw the development of a CPG in collaboration with national hematology experts . This CP failure ,18. Ulti failure , we incoThe COVID-19 anticoagulation CPG CDS system was developed by the M Health Fairview clinical informatics development team (KK and PW) in collaboration with the Associate Chief Medical Informatics Office for CDS (S Switzer) in May 2020. Recovered COVID-19 was built into the EHR.The interruptive best practice advisories were essentially “safety checks” to surveil if patients were on venous thromboembolism (VTE) chemoprophylaxis on admission or if the criteria for VTE risk changed  and were only triggered for providers with ordering privileges. To ensure the best practice advisory would not trigger for patients who have recovered from COVID-19, an infection status of Development, dissemination, and implementation followed our system protocol SCALED  for CDS user-centered design, pilot testing, scaling, and evaluation. Prior to pilot testing, the CDS underwent iterative user interface/user experience improvement during May 2020. The CDS was piloted on May 14, 2020, and scaled on May 24, 2020. To support adoption and usability, a discipline-specific CDS dissemination strategy was carried out in May 2020 . To ensuProvidersThe clinical decision support (CDS) system was presented to intensivist, hospital medicine, and primary care practice groups via formal didactic methods.Our system utilized a daily workflow document for intensivists and hospital medicine providers caring for COVID-19 patients representing best practices, recent publications, and ongoing trials. The anticoagulation CDS was integrated into this workflow document and remained as a constant on this document throughout the implementation period.In the university setting, CDS was presented at grand rounds on divisional, departmental, and medical school platforms.PharmacyThe CDS system was presented routinely at the System-wide Anticoagulation Committee.AllOur system utilized a COVID-19 intranet COVID-19 resource page. This CDS was placed within the system guidelines for management of COVID-19 patients.The CDS was also posted on the University of Minnesota evidence-based medicine COVID-19 website, on a public facing webpage for COVID-19 evidence-based practice geared toward clinicians.Members of the study team  developed a COVID-19 data mart to facilitate near real-time evaluation of the CDS. Structured query language was used to automate daily export of COVID-19 EHR data into the data repository. A preprocessing pipeline was developed and implemented using Python 3.7.3. (CreateSpace) and Stata 16 (StataCorp) to generate a flat file for each patient, including patient anticoagulation risk stratification, tier of anticoagulation received, reach, adherence, clinical outcomes , comorbidities, home medications, inpatient medications received , daily laboratory and vital data, and demographics. LogicStream Health , an analysis platform for EHR content, was utilized to evaluate order set, passive, and interruptive alert utilization.A preplanned 6-month implementation evaluation was conducted with guidance from the RE-AIM framework.Reach was defined as the number of patients admitted each month, who received CDS (CDS reach) or appropriate anticoagulation (CPG reach) (numerator) over the number of patients admitted each month with COVID-19 (denominator). These 2 definitions of reach were used to facilitate internal performance monitoring. For example, the state where CPG reach is high but CDS reach is low represents integration of the CPG into normal workflow without the need for CDS.Adoption was defined at the implementation site and nursing unit level as the number of patients admitted each month, who received guideline-concordant anticoagulation therapy (numerator) over the number of patients admitted each month with COVID-19 (denominator). It was not possible to define adoption accurately at the provider level as we were unable to assign a single provider responsible for a patient’s initial care. Patient’s receive orders from a variety of provider types, including house staff, advanced practice providers, or attending physicians either within the emergency department or the inpatient team, and thus we are unable to assign a single provider responsible for CPG adherence.implementation evaluation was conducted to investigate the effect of various CDS alert methods  on anticoagulation CPG adherence. Adherence was defined at the patient level as receiving guideline-concordant care within 24 hours of admission. Additionally, CPG fidelity was evaluated for each VTE risk stratification  with clinical outcomes was assessed via multiple methods.To evaluate First, multivariable logistic regression was used for binary dependent variables and negative binomial regression was used for continuous variables with a skewed distribution  using all 2406 patients who either received adherent care (n=1650) or did not receive adherent care (n=853). All models were risk adjusted using the confounding variables included below.Second, 1:1 nearest neighbor propensity score matching was used to create cohorts of patients who received CPG-adherent care (exposure or treatment of interest). Univariate logistic regression was then used to compare the need for ICU admission within 48 hours (primary outcome) for patients who received (vs did not receive) CPG-adherent care on admission (exposure). Kaplan-Meier curves were also estimated via a time-to-event analysis  and compared using the log-rank test. Propensity scores were estimated with logistic regression using the confounding variables listed below. Two evenly matched groups were formed with the common caliper set at 0.01. Following matching, there were 1342 patients included (671 patients in each propensity-matched cohort). Standardized difference was evaluated prior to and after propensity matching to ensure that the standardized difference was <0.1 in the propensity-matched cohort for each confounding variable . The disThird, to account for the competing risk of death prior to ICU admission by 48 hours, which occurred in 5 patients, a competing risk regression model  was used in the propensity-matched cohort. Cumulative incidence curves were also estimated. Due to the importance of age as a confounding variable, age-stratified cumulative incidence curves were also generated.The primary clinical outcome for the above models was the need for ICU admission within 48 hours of hospital admission. This endpoint was chosen clinically as the primary outcome because adherence with anticoagulation best practices is hypothesized to reduce microthrombosis and macrothrombosis events, and minimize progression of the disease and critical illness.Secondary outcomes of interest for the above models were also evaluated, including all-cause in-hospital mortality, the need for ICU admission at any time during hospitalization, the need for mechanical ventilation, hospital length of stay, and the development of VTE or bleeding complications. Additionally, a binary composite outcome metric was developed and coded as positive if a patient had all-cause in-hospital mortality, required ICU admission, required mechanical ventilation, or required a hospital length of stay greater than 7 days.The exposure or treatment of interest for the above models was defined as a binary variable if patients received guideline-adherent care on hospital admission.With regard to confounding variables for the above models, variables known to be associated with the outcome of more severe COVID-19 infection  were included as confounding variables for all analyses. This list of variables was developed by our team of subject matter experts with clinical and research expertise managing patients with COVID-19. All models were risk adjusted to account for patient-level baseline demographics , the moWith regard to subgroup analysis, of the 2503 patients, initial D-dimer, C-reactive protein, creatinine, and absolute neutrophil to absolute lymphocyte ratio data were present for 1181 patients. As these laboratory values have been shown on admission to be predictive of worse clinical outcomes -23, a seWith regard to data missingness, overall missingness was low . Given the low rate of missingness, imputation was deemed unnecessary .P value <.05.Statistical analyses were performed using Stata MP, version 16 (StataCorp). Statistical significance was defined as a The data underlying this article were provided by M Health Fairview  with permission from M Health Fairview Research and IT. Data will be shared on request to the corresponding author with the permission of M Health Fairview.A total of 2503 patients required in-hospital admission during the study period, with polymerase chain reaction–confirmed COVID-19 . The medCPG reach improved following implementation of the CDS system. CPG reach peaked during piloting and scaling of the CDS system with an adherence rate of 74.4%. In the 6 months since scaling, adherence averaged 67% .primary hypothesis tested was if adherence with the anticoagulation CPG on hospital admission was associated with a reduced need for ICU management by 48 hours. Adherence with the anticoagulation CPG was independently associated with reduced need for ICU admission within 48 hours of hospital admission on multivariable logistic regression analysis  . A time-to-event analysis was also conducted. Patients who received adherent care on admission (vs patients who did not) were more likely to not require ICU admission by 48 hours . Cumulative incidence functions are provided in The  P<.001) . In the P<.001) and reduced all-cause in-hospital mortality . Adherence with the anticoagulation CPG was independently associated with reduced bleeding complications , but not VTE complications . Adherence with the anticoagulation CPG was independently associated with an increased hospital length of stay . This effect persisted on excluding patients who had in-hospital death . None of the other secondary analyses reached statistical significance  or the need for ICU admission at any time during hospitalization  (P=.006). Adherence with the anticoagulation CPG was independently associated with an increased hospital length of stay . This effect persisted on excluding patients who had in-hospital death . None of the other secondary analyses reached statistical significance  . Adherenificance .To investigate adoption rates across the system, we evaluated adoption by hospital. Our system includes 12 hospitals, 2 university settings that include resident and fellow trainees, 2 COVID-19 cohorted hospitals  staffed P<.001).Adherence was evaluated in the context of CDS. Adherence when CDS was delivered was 70% as compared to 62% without CDS  workflow, during the rounding navigator, and within the general EHR order environment. Adherence with anticoagulation was the highest when these passive elements were integrated within the admission (75%), rounding (75%), or transfer (80%) navigators  than wheWe then sought to investigate the relationship between adherence and passive versus interruptive CDS intervention formats. Overall, 1423 (56.9%) patients had no CDS elements that were passive or interruptive. On the other hand, 699 (27.9%) patients had passive-only CDS delivered to providers, 111 (4.4%) patients had interruptive-only CDS delivered to providers, and 270 (10.8%) patients had a combination of passive and interruptive CDS delivered to providers. The combination of passive CDS and interruptive alerts was associated with the highest adherence with the anticoagulation CPG .Variation in adherence was noted across baseline risk groups. Patients in the moderate-risk group were less likely to receive adherent care  compared with patients in the high-  and low-risk groups . Following implementation, wash-out maintenance stabilized at 67% in months 5 to 6 (October to November 2020).This study represents the completed iteration of a continuous LHS cycle . AdherenThis study provides an important and early example of the real-world application of the LHS during COVID-19, a period with surged clinical resources and uncertain evidence base. Critical to our success was the early development of a COVID-19 data mart that included highly granular structured and unstructured patient-level data. Integration with an EHR analysis solution  facilitated near real-time evaluation of CDS alert activities by providers.Our health care system has a rigorous and validated protocol for the development, implementation, scaling, and evaluation of user-centered CDS systems with over 20 use cases implemented each year overseen by various enterprise CDS committees. Typically, the process of development, implementation, and scaling requires months, and in this case, it occurred in a matter of weeks. COVID-19 provided a heightened sense of urgency and purpose in health care research and quality improvement that resulted in rapid progress in CDS development. The dedicated EBM team facilitated prompt CPG updates in response to rapidly changing evidence. Augmented stakeholder engagement and buy-in from the informatics development team were also critical elements for success. The combination of expedited access to fully preprocessed and analyzable EHR data updated daily along with extraordinary team engagement and stakeholder support were critical for rapid implementation.Despite these successes, the room for optimization was identified from this analysis. First, in an attempt to minimize alert fatigue, the CDS system was initially designed to only trigger for patients with COVID-19 but not on anticoagulation. While it was successful for the months of June and July in achieving near 95% reach, based on the data presented in this study, we hypothesize that providers became comfortable attempting to order anticoagulation independent of the order set, resulting in patients being on incorrect anticoagulation and preventing corrective triggering of the CDS system. Others have shared this experience, where an attempt to develop a user-centered CDS system minimizing alerts resulted in a system that was overly passive and could not change behavior . DespiteSecond, we identified a large variation in adoption across hospitals and nursing units. Our academic health system is unique in the sense that we created specialty cohorted hospitals for COVID-19 patients  needing Third, following each LHS evaluation cycle (practice to data), it is imperative that positive findings are disseminated widely. We were surprised that adherence did not improve following our interim effectiveness analysis in August 2020, which identified a significant and independent improvement in clinical outcomes with anticoagulation. The unified theory of acceptance and use of technology posits that a key construct affecting technology use intention  is performance expectancy . EssentiIn the early phase of the pandemic, there was widespread confusion about the underlying pathogenic mechanism and its implications for patient outcomes, leading to highly variable practice in the medical community. To date, limited data exist on the management approach for COVID-19–associated coagulopathy, particularly in high-risk critically ill populations and for patients who are either managed as outpatients or posthospital discharge patients ,30. At tCOVID-19 is a global emergency; given the lack of robust/consistent guidelines from leading societies, institutions had to develop a local approach to create a uniform plan of care and an approach for its implementation. This is specifically problematic in larger systems with multiple hospitals. Our health system was particularly vulnerable to this issue owing to significant heterogeneity resulting from a recent merger . As described above, there were concerns for an increased risk of bleeding, and many individual practitioners in our system were apprehensive to order anticoagulation in patients with COVID-19, leading to variable VTE prevention strategies and adverse patient outcomes.Our study has several limitations. First, our institutional preference for implementation evaluation is typically a mixed-methods approach. However, due to contact precautions surrounding COVID-19 and significantly increased provider workload, it was not feasible to perform a qualitative analysis of staff. Thus, this represents a quantitative-only approach, which may not fully discern specific trends. To expand on hypotheses that arose from this research, a future direction includes a voluntary survey of health care providers  surrounding their familiarity with COVID-19 institutional guidelines and their experiences interacting with the CDS system. Survey question development was guided by unified theory of acceptance and use of technology constructs for technology acceptance. Additionally, while we identified an association with adherence and the effectiveness of anticoagulation, it is important to not misconstrue this analysis. We did not evaluate anticoagulation versus no anticoagulation and the association with outcomes, but rather we evaluated adherence with the guideline versus nonadherence with the guideline. Thus, nonadherent patients could have been receiving either more or less aggressive anticoagulation than the comparison group. In our institution, adherence was associated with improved clinical outcomes; however, this may not be generalizable to other institutions with different baseline cultural practices for anticoagulation management. VTE and bleeding complications were extracted using structured EHR data. These events may be underreported. Our relatively small cohort size (n=2503) and single health care system represent additional limitations of the study.This study provides an early example of the real-world application of the LHS during COVID-19. With or without a pandemic, there is a need for the implementation of evidence-based practice that is most up-to-date. Traditionally, the largest barrier to this effort has been the need for making major changes in the workflow. With the widespread use of EHRs and increasing consolidation of health care systems, the application of a CPG through the use of a CDS system can offer an easy tool for implementation without adding confusion related to workflow changes, thus bringing uniformity in care at every level in the system and influencing the quality of care and patient outcomes.


Since the end of 2020, several vaccines have become available as part of the global efforts to contain the adverse health outcomes of the coronavirus disease 2019 (COVID-19) pandemic. Although research has confirmed their safety on large scales, several post-marketing reports have revealed some rare cardiovascular side effects. Towards the end of the first half of 2021, multiple reports indicate possible links between COVID-19 vaccines (both mRNA-based vaccine and vector-based vaccines) and myopericarditis. Nevertheless, cardiac conduction disease in this context has only rarely been reported.In this report, we present two cases of probable vaccination-induced cardiac conduction disturbances along with a thorough literature review. In addition, we discuss probable pathophysiological mechanisms and insights into the suggested areas for future research. To our knowledge, these are the first published cases to result in permanent pacemaker implantation. In March 2020, the World Health Organization (WHO) announced coronavirus disease 2019 (COVID-19) the second pandemic of the 21st century . Since tSince the end of 2020 and the beginning of 2021, several vaccines have been found promising in terms of efficacy and safety -7. HowevAlthough numerous reports have linked COVID-19 vaccination to myopericarditis , to dateCase OneAn 89-year-old fit and independently mobile Caucasian female was admitted to the Coronary Care Unit (CCU) one day after she had received her first COVID-19 AstraZeneca (ChAdOx1 nCoV-19) vaccine. She had a remote history of total knee replacement but otherwise did not have a significant previous cardiac history (apart from previous electrocardiograms [ECGs] showing a bifascicular block with no symptoms). The sequence of events started a few hours (estimated to be three to four hours) after taking the vaccine. She started to experience headaches, nausea, non-specific body aches, fever, and chills. She was not sure if she had chest pain but reported probable constricting chest discomfort as part of the generalized body aches. A few hours after the resolution, these symptoms were followed by expressive dysphasia and pre-syncopal symptoms, for which she was rushed to the emergency department as a possible stroke presentation. Upon arrival, all her symptoms had completely resolved. A computed tomography (CT) scan of the brain was unremarkable. However, she was noted to be bradycardic with a heart rate of approximately 30 beats per minute. The rest of her observations included a blood pressure of 182/79 mmHg, oxygen saturation of 96% on room air, the highest recorded temperature of 37.8°C, and a respiratory rate of 16 breaths per minute. Cardiovascular and respiratory examinations were unremarkable. Initial laboratory investigations were within normal parameters. These included full blood counts, renal function tests and electrolytes, C-reactive protein, coagulation profile, liver function tests, thyroid function tests, HbA1c, calcium, magnesium, phosphate, and blood gas analysis. Serum troponin-T was mildly elevated at 14 ng/L (using high-sensitivity assay). This was the only sample taken and there were no serial measurements of this test as this was deemed to be below the threshold of significance for acute coronary syndrome as per the hospital protocol. An initial ECG  performed the next day showed normal ejection fraction (>55%) with only grade one diastolic dysfunction which was insignificant and was attributed to the patient’s age. Otherwise, there was neither evidence of regional wall motion abnormalities nor any significant interventricular dys-synchrony.As the patient remained in the above-mentioned rhythm (2:1 AV conduction block) overnight with some episodes of probable complete heart block (CHB) on telemetry, a decision was taken the next day to proceed with implanting a dual-chamber permanent pacemaker (PPM).It is worth mentioning that the patient was not on any negative chronotropic agents and did not have any obvious modifiable risk factors or causes for bradyarrhythmia. Cardiac magnetic resonance imaging (MRI) was neither feasible nor safe at the time (due to the acuity of the presentation). Moreover, in our view, it would not have had any impact on the management plan.The patient was discharged home the same day after PPM insertion. She continued to have pacing follow-up appointments. The first one was two months after discharge. At the two-month follow-up, a well-functioning pacemaker was observed. Further investigation showed an underlying rhythm of intermittent 2:1 AV block. The atrial pacing percentage was 1.7% while the ventricular pacing was 37%. Another pacing investigation after four months showed an underlying sinus rhythm with non-pacing-dependent ventricular activity. The patient also underwent a carotid Doppler at this time as an outpatient study, which did not show significant lesions.Case twoAn 85-year-old Caucasian male was known to have depression, anxiety, glaucoma, and a history of right forehead basal cell carcinoma excision six years ago. He had good functional baseline and normal mobility. His regular medications included mirtazapine 15 mg once a day, Timolol eye drops, and atorvastatin 20 mg once a day. He was allergic to penicillin with an undisclosed nature.He presented to our emergency department with gradually worsening breathlessness, effort intolerance, and episodes of dizziness at rest over a course of 10 days after taking his first dose of the COVID-19 Pfizer-BioNTech (BNT162b2) vaccine. On direct questioning, the patient denied having had any fever, chills, or body aches immediately after the vaccination.His initial observations upon arrival were a regular heart rate of 36 beats per minute, a temperature of 36°C, a respiratory rate of 17 breaths per minute, and a blood pressure of 180/82 mmHg. He had normal cardiovascular and chest examinations. He did not have lower limb edema, and his jugular venous pressure was not raised. Laboratory investigations included full blood counts, renal function and electrolytes, C-reactive protein, cardiac enzymes, coagulation profile, liver function tests, thyroid function tests, HbA1c, calcium, magnesium, phosphate, and blood gas analysis, which were largely unremarkable.A chest X-ray did not show any obvious abnormalities. He underwent a 12-lead ECG . Another follow-up at four months from the date of the PPM insertion confirmed the same findings.The cardiac conduction system of the heart is a specialized system that primes organized and coordinated cardiac muscle contraction resulting in effective systole and diastole. This system consists of the sinoatrial SA node, the AV node, the bundle of His, and the ventricular conduction system  . CardiacMyocarditis, one of the acquired causes of CCD, is defined as cellular inflammatory infiltration with or without necrosis of the myocytes . The EurLazaros et al. postulated a cytokine-mediated immune response as the culprit for COVID-19 vaccine-induced myocarditis . AlthougAs mentioned above, our first patient presented with prodromal symptoms shortly after vaccination suggesting a probable “cytokine storm.” Although it was unfortunate that an MRI scan was not feasible, the presence of chest discomfort and mild troponin leak (above the 99th centile of the reference range) might arguably point towards a myocarditic process. In the literature review, we identified a few cases of CCD in the context of vaccination-induced myocarditis. In the first one, among several cases, Marshall et al. reported the case of a 16-year-old male who developed fever, fatigue, and loss of appetite two days after his second dose of the COVID-19 Pfizer-BioNTech (BNT162b2) vaccine. The authors reported evidence of junctional escape rhythm with AV dissociation. There was also evidence of ST-segment elevation. The patient eventually recovered completely . The secIt is interesting to note that although both of our patients ended up needing PPM insertion, subsequent pacing follow-up revealed almost complete pacing dependence only in our second patient. Therefore, It is unclear whether this represents the potential reversibility of the inflammatory process with the recovery of the conductive system function or the co-existence of a progressive CCD  particularly in the second case. Hence, the question of whether vaccination-induced CCD is a matter of “triggering” versus “confounding factor” remains to be answered. Certainly, our first patient had previous ECGs confirming background CCD.In the context of the current pandemic, we realistically acknowledge that the benefits of the COVID-19 vaccination outweigh the risks reported so far. On a clinical level, however, we suggest taking into consideration individual patient factors that might prove significant in the risk-benefit clinical reasoning . These should not be viewed as contraindications to vaccination but rather should raise awareness about such potential side effects.As per our case presentation and discussion, we believe that CCD holds the potential of being a rare complication of COVID-19 vaccines, especially in the context of worldwide vaccination inertia. It is yet unclear what underlying mechanisms play a role (whether immune-mediated or otherwise). We acknowledge that COVID-19 vaccination programs remain vital to hinder the progression of the pandemic. However, physicians should have a high degree of vigilance and awareness of potential side effects. In the context of myocarditis and CCD, it might be worthwhile paying attention to some factors such as baseline cardiac conduction and old ECGs. Further research is required to clarify this further.


Although many laboratories worldwide have developed their sequencing capacities in response to the need for SARS-CoV-2 genome-based surveillance of variants, only a few reported some quality criteria to ensure sequence quality before lineage assignment and submission to public databases. Hence, we aimed here to provide simple quality control criteria for SARS-CoV-2 sequencing to prevent erroneous interpretation of low-quality or contaminated data. We retrospectively investigated 647 SARS-CoV-2 genomes obtained over 10 tiled amplicons sequencing runs. We extracted 26 potentially relevant metrics covering the entire workflow from sample selection to bioinformatics analysis. Based on data distribution, critical values were established for 11 selected metrics to prompt further quality investigations for problematic samples, in particular those with a low viral RNA quantity. Low-frequency variants (<70% of supporting reads) can result from PCR amplification errors, sample cross contaminations, or presence of distinct SARS-CoV2 genomes in the sample sequenced. The number and the prevalence of low-frequency variants can be used as a robust quality criterion to identify possible sequencing errors or contaminations. Overall, we propose 11 metrics with fixed cutoff values as a simple tool to evaluate the quality of SARS-CoV-2 genomes, among which are cycle thresholds, mean depth, proportion of genome covered at least 10×, and the number of low-frequency variants combined with mutation prevalence data. The epidemics of COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) reported in Wuhan City (China) in December 2019 rapidly spread to other regions in China and then to all continents, causing the 2020/2021 pandemic. In December 2020, southeastern England experienced a surge of new SARS-CoV-2 infections with the identification of the first variant of concern (VoC), named B.1.1.7 , in BrazThe rapid dissemination of these variants elicited concerns over a deterioration of the fragile epidemiological situation and an increased number of infections, adding significant pressure on the health care system. In this context, the European Centre for Disease Prevention and Control  and the TC) value below 30 cycles  were extracted using our automated molecular diagnostic platform with a MagNA Pure 96 instrument . All samples were processed with the CleanPlex SARS-CoV-2 15 panel and CleanPlex dual indexed (Paragon Genomics number 918011 ) accordihttps://github.com/metagenlab/GENCOV), a modified version of CoVpipe (https://gitlab.com/RKIBioinformaticsPipelines/ncov_minipipe). Briefly, reads were filtered with fastp (NC_045512.2 with bwa (https://github.com/metagenlab/GENCOV/releases/tag/1.0). The frequency of individual mutations was calculated in this study based on all samples from 10 sequencing runs. To monitor the quality of the workflow, an internal control was included in the runs. The sequences always passed quality controls and were assigned to the correct lineage (B.1.258.17). The same 30 mutations were identified in all 5 controls. One sample exhibited an additional mutation that was only supported by 10 reads.Illumina reads were processed using GENCOV  with project number While SARS-CoV-2 genome sequencing and analysis are performed in many laboratories worldwide for epidemiological purposes, no clear criteria have been proposed to assess the quality of SARS-CoV-2 sequences. Critical thresholds or control limits , 18 are Twenty-six metrics  were collected for their potential to evaluate the quality of SARS-CoV-2 genome sequencing, from DNA extraction to bioinformatics analyses. To reduce redundancy while retaining the ability to evidence problems along the entire workflow, 11 metrics were selected based on expert knowledge in genomics and excluding redundant metrics . AlthougUpper and lower control limits were established based on the distribution of the 11 selected metrics , with ta3 up to 109 copies/ml , significantly affecting many of the selected quality metrics , and the sequences were rejected in the initial manual validation of the samples . This cutoff optimized the sequencing success in our setting but should be adapted to other workflow specificities, according to study questions and sample characteristics. Although limited by the number of mouth swabs (n = 31), we observed that nasopharyngeal swabs (n = 586) resulted in an overall better sequencing quality, likely linked to lower viral loads in mouth swabs or to the presence of PCR inhibitors in saliva .Clinical specimens contain a diverse range of viral load, from 10opies/ml . As clinKey to the reporting of accurate results is the detection of potential contaminations that could hamper variant calling, affecting cluster analyses and leading to inaccurate lineage definition. The presence of low-frequency variants, defined as positions with variable nucleotides in 10% to 70% of mapped reads , could rTC) or with a low library quantification . However, some samples harbored a large number of unique low-frequency variants (Table S3), likely as a result of PCR amplification errors. In such cases, the consensus genome sequence can still be used for lineage assignment. Finally, the occurrence of multiple low-frequency variants observed in other samples of the sequencing run is suggestive of a possible cross contamination. This highlights the importance of carefully validating all sequencing metrics. This quality analysis was critical in the following two cases to identify cross-contaminations.TC of 32, identified as B.1.1.7 lineage by genomic analyses. However, it did not show the characteristic S gene dropout signature of this variant by RT-PCR  and the other sample as B.1.160 . A close examination of the three sequences showed the presence of several low-frequency variants in samples A (Table S4) and B (Table S5)  but not in C (Table S6). Further analyses of the prevalence of low-frequency variant calls showed that 20 positions and 9 positions for cases A and B, respectively, were observed in multiple other genomes, which suggested contamination. Low-frequency variants were used here to prevent erroneous lineage assignment. To provide more empirical evidence, we have included additional relevant examples that were identified in samples posterior to this analysis (Table S7).Case 1 included a first nasopharyngeal swab  with a y RT-PCR  and presTC of 30 can be used as a preanalytical criterion to exclude samples, but depending on the scientific question, higher TC could be investigated and quality rigorously assessed. Nasopharyngeal swabs might be preferred to mouth swabs , and lower respiratory samples can be investigated as prolonged RT-PCR detection of viral RNA in lower respiratory specimens was documented . On the other hand, PCR errors are expected to arise randomly throughout the genome and should be unique to each sample. Therefore, we strongly recommend the evaluation of the proportion of low-frequency variants, their prevalence in other SARS-Cov-2 samples from the same sequencing run, and their prevalence in all SARS-CoV-2 sequenced to date as a technical control before data submission to public health agencies and public databases.


The effect of ezetimibe, Niemann-Pick C1-like 1 inhibitor, on liver fat is not clearly elucidated. Our primary objective was to evaluate the efficacy of ezetimibe plus rosuvastatin versus rosuvastatin monotherapy to reduce liver fat using magnetic resonance imaging-derived proton density fat fraction (MRI-PDFF) in patients with non-alcoholic fatty liver disease (NAFLD).A randomized controlled, open-label trial of 70 participants with NAFLD confirmed by ultrasound who were assigned to receive either ezetimibe 10 mg plus rosuvastatin 5 mg daily or rosuvastatin 5 mg for up to 24 weeks. The liver fat change was measured as average values in each of nine liver segments by MRI-PDFF. Magnetic resonance elastography (MRE) was used to measure liver fibrosis change.p = 0.020). There were significant reductions from baseline to study completion by MRI-PDFF for both the combination and monotherapy groups, respectively . Individuals with higher body mass index, type 2 diabetes, insulin resistance, and severe liver fibrosis were likely to be good responders to treatment with ezetimibe. MRE-derived change in liver fibrosis was not significantly different . Controlled attenuation parameter (CAP) by transient elastography was significantly reduced in the combination group , but not in the monotherapy group .Combination therapy significantly reduced liver fat compared with monotherapy by MRI-PDFF .The trial was registered at ClinicalTrials.gov (registration number: The online version contains supplementary material available at 10.1186/s12916-022-02288-2. The incidence of non-alcoholic fatty liver disease (NAFLD) is increasing globally and is closely related to metabolic syndrome features including obesity, insulin resistance, dyslipidemia, and diabetes mellitus (DM). Cardiovascular disease, which is closely related to dyslipidemia, is a major cause of mortality in patients with NAFLD . FurtherCholesterol alone can contribute to the progression of non-alcoholic steatohepatitis (NASH) , and theHowever, the effects of ezetimibe have not been clearly elucidated in human studies. Ezetimibe did not significantly reduce liver fat in a previous randomized controlled trial by Loomba et al. . This stThe ESSENTIAL  study was an investigator-initiated, randomized, open-label, prospective, active-controlled clinical trial to examine the efficacy of ezetimibe 10 mg/day orally combined with rosuvastatin 5 mg/day versus rosuvastatin 5 mg/day orally for up to 24 weeks to improve hepatic steatosis as measured by magnetic resonance imaging-derived proton density fat fraction (MRI-PDFF). Participants of the study were recruited from Severance Hospital from May 2018 to June 2019 in Seoul, Korea.ClinicalTrials.gov (registration number: NCT03434613). All participants provided written informed consent and the Ethics Committee of the Yonsei University College of Medicine approved this study (4-2017-1168), which conforms to the ethical principles of the 1975 Declaration of Helsinki.The trial was registered at Patients were required to meet all the following criteria for study inclusion: 19–80 years of age, hepatic steatosis documented by abdominal ultrasound, hyperlipidemia indicated to treat by the domestic dyslipidemia treatment guideline , diagnosAll patients underwent a baseline assessment before undergoing randomization, including medical history and physical exam. For those who had undergone abdominal ultrasonography within the past 1 year, test results were reviewed for the presence of hepatic steatosis. Otherwise, abdominal ultrasonography was performed for screening purposes.A random allocation sequence was computer-generated elsewhere, and participants were assigned in a 1:1 ratio to receive treatment with ezetimibe plus rosuvastatin or rosuvastatin alone. To equalize the proportion of type 2 diabetes in both groups, patients were stratified according to the presence of diabetes before randomization.The primary outcome was change in liver fat by MRI-PDFF quantified as the average in colocalized regions of interest (ROI) within each of nine liver segments . SecondaFor hepatic fat quantification, MRI-PDFF sequence (mDIXON Quant) was used, and a fat fraction map was automatically generated by the manufacturer’s console. For liver fibrosis measurement, MRE was performed using a 2-dimensional gradient-echo sequence and a passive driver placed on the participant’s right upper abdomen  and compared by α and 0.05 with β assuming study completion by ≥29 participants per arm.We expected the ezetimibe treatment group to have a liver fat reduction of >10% compared to baseline and the statin monotherapy group to have a <5% reduction compared to baseline. We also expected a dropout rate of 15–20%; thus, we planned to enroll 70 participants to achieve power of at least 80% with 2 or more, under 30 kg/m2), presence of type 2 diabetes, sarcopenia  <2 standard deviations below the gender-specific mean for the Korean population: SMI (%) <29.0 in men and <22.9 in women) [Additional analyses for treatment response were conducted across multiple subgroups to examine whether the effect of ezetimibe differed on the basis of patient demographics or clinical characteristics. Characteristics for subgroup analysis included age , sex , body mass index (30 kg/mn women) , HOMA-IRn women) .All statistical analyses were performed using IBM SPSS Statistics version 26.0 .n = 34) or rosuvastatin monotherapy (n = 36). Three subjects in the combination group and 3 patients in the monotherapy group were excluded from the final analysis  and the monotherapy group  (Table p = 0.020)  (Table p = 0.898).Compared to baseline, there were no significant differences at end of treatment by MRE in either the combination group or monotherapy group , but not for the rosuvastatin alone group . In terms of hepatic fibrosis, there was no significant difference in LSM from baseline to end of treatment in the combination group  or the monotherapy group .We also evaluated changes in liver fat and fibrosis by TE. A significant decrease in CAP at end of treatment was observed for ezetimibe plus rosuvastatin . However, changes in all variables including BMI, alanine aminotransferase, aspartate aminotransferase, fasting glucose, HbA1c, and homeostatic model assessment insulin resistance were not significantly different between the two groups .Changes in anthropometric and biochemical variables are summarized in Table p > 0.05).To explore drug mechanisms, additional tests were performed on several values known to be inflammation markers of NASH progression. Significant improvement was reported in IL-18 in the combination group and in tPAI-1 and IL-18 in the monotherapy group, but there was no significant difference in treatment effect between groups , presence of T2DM, presence of sarcopenia, increased HOMA-IR , and increased baseline MRE   at baseline. Two participants in each group were discontinued due to protocol violations. There was no significant adverse event reported in this study.This randomized controlled clinical trial showed ezetimibe with rosuvastatin was significantly better than rosuvastatin monotherapy for reducing liver fat as measured by MRI-PDFF. Consistent with this finding, a significant reduction of liver fat was also confirmed in end-of-treatment CAP measured by TE, but only for the combination group. Ezetimibe combination treatment did not significantly decrease fibrosis as measured by MRE or TE. In subgroup analyses, ezetimibe combination treatment was more effective in subjects with higher BMI, presence of T2DM, presence of sarcopenia, increased HOMA-IR, and increased baseline MRE.Ezetimibe inhibits the absorption of dietary fat, including cholesterol, via NPC1-L1 . SeveralOur study demonstrated that significant improvement in hepatic steatosis occurred after treatment with the ezetimibe combination, and there may be several reasons for this result. First, this study was conducted in combination with a statin. Unlike statins, which inhibit cholesterol synthesis in the liver, ezetimibe targets gastrointestinal cholesterol absorption within the small intestine. In this process, ezetimibe can inhibit an increase in dietary cholesterol absorption, which can arise from the use of statins . NPC1-L1Second, differences in the characteristics of the current study population may have the potential to influence the ezetimibe effect. Subgroup analysis showed that combination treatment was more effective in improving hepatic steatosis in individuals with higher BMI, presence of T2DM or sarcopenia, higher HOMA-IR, and increased baseline MRE . Inhibiting absorption of dietary cholesterol by ezetimibe can contribute to improvement in inflammation and insulin resistance . ConsideAlthough this study cannot fully explain its contributory mechanisms, there is evidence that ezetimibe changes the bile acid pool or microbiome . Bile acProgression from simple steatosis to NASH or fibrosis is of clinical importance. It is known that oxidative stress plays an important role in the progression of inflammation and fibrosis . EzetimiIn this study, we also report improvement of hepatic steatosis in the monotherapy group. Previous studies of statins have shown inconsistent results regarding their potential contribution. Dongiovanni et al. reported the protective role of statins in individuals at risk of non-alcoholic steatohepatitis . HoweverThere are several limitations in this study. Baseline hepatic steatosis was marginally lower in the monotherapy group compared to the combination group. Liver biopsy, the gold standard to measure hepatic steatosis or fibrosis, was not performed. In this study, it was confirmed that co-administration of ezetimibe with statin contributed to the improvement of hepatic steatosis. However, further evidence should be implemented to confirm clinically relevant effects of ezetimibe on hepatic fibrosis or clinical outcomes in NASH patients. The small sample size and short observation period of 6 months were not sufficient to determine whether concomitant use of ezetimibe could lead to improvement in hepatic fibrosis. We enrolled patients with hepatic steatosis documented by abdominal ultrasound, and one subject in each group excluded having no documented hepatic steatosis (<5% on MRI-PDFF) at baseline. We performed the final analysis as a per-protocol analysis, which may lead to an increased risk of selection bias. The open-label design implies that the interpretation of the results of this study should be made with caution. On balance, there are several notable strengths. First, we used the MRI-PDFF method, recognized as a highly reliable method of measuring hepatic steatosis , to evalIn conclusion, the use of ezetimibe in combination with rosuvastatin significantly improved hepatic steatosis in patients with NAFLD. Individuals with higher BMI, T2DM, insulin resistance, and severe liver fibrosis were likely to be good responders to ezetimibe treatment. These data indicate that ezetimibe plus rosuvastatin is a safe and effective therapeutic option to treat patients with NAFLD and dyslipidemia.Additional file 1: Appendix 1. Detailed Exclusion criteria. Appendix 2. MRI-PDFF for fat quantification and MRE for liver fibrosis quantification. Figure S1. MRI map. Table S1. Pulse sequence parameters for magnetic resonance imaging. Table S2. Longitudinal Changes in Hepatic Steatosis and Fibrosis Using Fibroscan.


Up to 80% of reproductive-aged women experience premenstrual symptoms. Premenstrual Dysphoric Disorder (PMDD) is a severe form, affecting 2-5% of women. Combined oral contraceptive pills (COCPs) are used in the treatment of PMDD. Clinical practice suggests that a newer COCP containing nomegestrol acetate (2.5mg) and 17-beta estradiol (1.5mg), may be a suitable treatment for mood symptoms in PMDD.This was a clinical follow-up feasibility study of women who had attended the Monash Alfred Psychiatry research centre, Women’s Mental Health Clinic, with a diagnosis of PMDD. 67% of the sample also had concurrent cPTSD, 29% co-morbid anxiety, and 20% depression. They were recommended treatment with nomegestrol acetate/17-beta estradiol. Eligible women were contacted by telephone to answer a questionnaire to assess women’s subjective response to nomegestrol acetate/17-beta estradiol, acceptability and the Depression, Anxiety and Stress Scale-21 (DASS-21) after being recommended nomegestrol acetate/17-beta estradiol. The paired-sample t-test was used to determine if there were any statistically significant differences in the DASS-21 scores over the study observation period (before and after taking nomegestrol acetate/17-beta estradiol).p<0.05) in the overall DASS-21 scores from before women commenced nomegestrol acetate/17-beta estradiol and after commencement of treatment.35 (74.5%) women reported a subjective positive mood response to nomegestrol acetate/17-beta estradiol, 31 (63.3%) adhered to the medication, and only 10 (20.4%) women reported side effects as the main reason for discontinuing nomegestrol acetate/17-beta estradiol. There were statistically significant reductions (This preliminary study supports the acceptability and effectiveness of nomegestrol acetate/17-beta estradiol as a treatment for mood symptoms in PMDD. Further research, particularly a randomized controlled trial, is required to elucidate the effect of nomegestrol acetate/17-beta estradiol treatment on mood in PMDD. The prevalence of premenstrual symptoms in women of reproductive age is reported to be up to 80% . At the via genomic and non-genomic activity at their respective receptors  have been shown to influence neurochemistry, brain function and the activity of neurotransmitters gamma-aminobutyric acid (GABA), serotonin and dopamine eceptors . A receneceptors .There is also evidence that estrogen is neuroprotective, in many different brain regions, including the brain stem, with concomitant improvements in mood symptoms .Progesterone, however, can contribute to worsening of mood symptoms in susceptible women, evidenced by worsening of mood symptoms in women who use progesterone-only forms of contraception such as the progesterone-only pill and the levonorgestrel intrauterine device . DepressManagement options for PMDD include non-pharmacological treatments, hormonal treatments such as the combined oral contraceptive pill (COCP), estrogen patches and micronized progesterone, as well as antidepressants either intermittently or continuously, or a combination of treatments. If these pharmacological treatments are unsuccessful, gonadotropin-releasing hormone (GnRH) analogues and surgery  with menopausal hormone therapy may be required .Given that fluctuations in hormones clearly contribute to PMDD symptom on and off-set, management with hormonal treatment such as the COCP to stabilize the gonadal hormones is an appropriate option. COCPs have mixed evidence in the management of mood symptoms. While studies suggest some COCPs worsen mood symptoms , 9, otheStudies of newer generation COCPs containing antimineralocorticoid and antiandrogenic progestins such as drospirenone are promising , 14, 17.Nomegestrol acetate (2.5mg) and 17-beta estradiol (1.5mg)  is a newer COCP that has been successful in clinical practice for the off-label treatment of mood symptoms in PMDD. Nomegestrol acetate/17-beta estradiol is a monophasic preparation with an extended regimen of 24 active pills followed by four placebo pills .Nomegestrol is structurally similar to progesterone and has strong antigonadotrophic and moderate antiandrogenic activity and no effect on estrogenic, glucocorticoid or mineralocorticoid activity . A pooleCurrently, drospirenone containing COCPs are the recommended COCPs for the treatment of PMDD . To dateThis study was undertaken to determine the acceptability and the subjective opinion of nomegestrol acetate/17-beta estradiol treatment on mood in women with PMDD and to determine the changes in the DASS-21 scores before and after nomegestrol acetate/17-beta estradiol treatment in women with PMDD.rd, 2020.This study was a single centre clinical follow-up study that included a questionnaire formulated by the authors (ER and JK) to assess the acceptability and women’s subjective response to nomegestrol acetate/17-beta estradiol and the clinical outcomes using self-report questionnaire the DASS-21. Project 285/17 WMHC Database was approved by the Alfred Hospital Ethics Committee on July 3Study participants were identified from the Monash Alfred Psychiatry research centre (MAPrc) Women’s Mental Health Clinic (WMHC) database. The WMHC provides second opinion consults by expert psychiatrists and psychologists for women with a range of psychiatric disorders. The clinic carefully considers the impact of hormonal changes in the management of mental illness. Women who had attended the WMHC at MAPrc from January 2018 to June 2020, had a clinic diagnosis of PMDD, and had been recommended nomegestrol acetate/17-beta estradiol were included. The diagnosis of PMDD was based on a modified clinical assessment using the central 4 items of the Caroline Premenstrual Assessment Scoring System (C-PASS)  - the seOnly women who had previously completed the WMHC database Participant Consent Form were eligible for inclusion. Women were excluded if they had never taken the recommenced treatment of nomegestrol acetate/17-beta estradiol or taken nomegestrol acetate/17-beta estradiol for less than one month. If women had previous self-harming behavior or a suicide attempt soon after taking nomegestrol acetate/17-beta estradiol they were excluded for safety reasons. Confidentiality was ensured and verbal consent gained from the participants at the start of the follow-up phone call.Data  was collected as part of the participants’ clinical appointment at the WMHC. Menopausal status  was determined by the treating clinician at the clinic appointment based on a comprehensive assessment, including questions about the menstrual status and menopausal symptoms. Follow-up phone calls were made to all eligible participants (those patients with PMDD who have been recommended nomegestrol acetate/17-beta estradiol), and were approximately 20 minutes in length. During this follow-up phone call, the participants completed the DASS-21 scale and were asked questions about their use of nomegestrol acetate/17-beta estradiol, if they take it continuously, side effects of nomegestrol acetate/17-beta estradiol, their subjective opinion of the effect of nomegestrol acetate/17-beta estradiol on their mood and other treatments that they were previously or currently taking.The DASS-21 is a validated and reliable self-report questionnaire to measure the negative emotional states of depression, anxiety and stress , 32. In p>0.01). To determine if there were any statistically significant differences in DASS-21 scores between the first visit and the follow-up call, the paired-t test was used. Given the sample size was relatively small and collected from a more heterogeneous patient population, and to reduce any potential bias in the data, we used bootstrapping technique to reduce its impact by generating a bias-corrected (BCa) confidence interval. Thus, using bootstrapping, 1000 random samples were generated to determine the BCa confidence interval to further support the robustness of the results.The analysis was performed using the IBM statistics program, SPSS . To determine if continuous data  were normally distributed, Shapiro-Wilk test for normality was used and histograms were generated. The skewness and kurtosis were within the acceptable range and the overall normality results showed no major violation of normality , smoking, alcohol use, concurrent anxiety, depression, and complex post traumatic stress disorder (cPTSD), taking a selective serotonin reuptake inhibitor (SSRI), antidepressant, antipsychotic, benzodiazepine, anticonvulsant, current use of estrogen patch, and menopausal status .The mean age of the 49 women included in this study was 36 years (sd=8.07). The number of clinic visits during the study period ranged from one to four with a median of two clinic visits Table 1.The clinical characteristics of the participants are summarized in The current and past hormonal treatment and the patients’ opinion on its effectiveness on mood were reported in All women had taken nomegestrol acetate/17-beta estradiol at some point after their first clinical appointment at WMHC. High adherence was reported, with 31 (63.3%) women still currently taking nomegestrol acetate/17-beta estradiol. Nearly half (46.9%) of the women had taken nomegestrol acetate/17-beta estradiol for more than one year with most women (71.4%) taking it continuously. Out of 18 (36.7%) women who had discontinued nomegestrol acetate/17-beta estradiol, side effects were the most commonly provided reason (55.6%), 4 (22.2%) women reported that nomegestrol acetate/17-beta estradiol did not help with their mood, 5.6% of women stated medical reasons for stopping treatment and 5.6% of women were attempting to conceive or pregnant.The most common side effects reported were breakthrough bleeding (40.8%) and decreased libido (24.5%). Thirty five (74.5%) women reported a subjective positive effect of nomegestrol acetate/17-beta estradiol on their mood, 6 (12.8%) reported a neutral effect and 6 (12.8%) reported a negative effect Table 4.p<0.05). In addition, series of sensitivity analyses were conducted on specific subgroups of participants for some key baseline characteristics or potential cofounders one by one. The sensitivity analyses showed similar significant mean differences for groups with no alcohol use, no smoking, no concurrent depression, anxiety or cPTSD, no current use of an estrogen patch, no use of an SSRI, other antidepressant, antipsychotic, benzodiazepine, and anticonvulsant users. Similar significant findings on overall DASS-21 scores were found for women at reproductive stage, with comorbidity of cPTSD, younger than 39 years old and those who used nomegestrol acetate/17-beta estradiol continuously during the study period.To evaluate the secondary outcome of the study; the clinical effectiveness of nomegestrol acetate/17-beta estradiol, a paired-sample t-test was used to assess the mean differences of the DASS-21 overall scores. The mean, SD and SE of DASS-21 scores from the clinic visit and phone call are displayed inIn our pilot study, we evaluated the feasibility and clinical effectiveness of nomegestrol acetate/17-beta estradiol in women with PMDD. Over two-thirds of women adhered to nomegestrol acetate/17-beta estradiol during the study period, while around 20% of them reported discounting of the medication due to side effects. The number of women reporting a positive mood response to nomegestrol acetate/17-beta estradiol was much higher than for previously used COCPs and there was a significant reduction in their self-reported overall DASS-21 score.The individual differences in response to a COCP could be due to the type of COCP and if the COCP was taken continuously or intermittently. The most common previously used COCP in our group was ethinylestradiol and levonorgestrel. Levonorgestrel is an older synthetic form of progesterone with androgenic properties and therefore may worsen mood symptoms in women with PMDD . The impEighteen women in our study discontinued nomegestrol acetate/17-beta estradiol, with side effects being the most commonly provided reason. The rate of reported side effects from nomegestrol acetate/17-beta estradiol in our study appears higher than what has been previously reported by Mansour et al.  and WestMore women in our study reported weight gain (18.4%) compared to the 7.9% reported by Mansour et al  and the The secondary outcome in this study focused on clinical outcomes. This follow-up study suggests that nomegestrol acetate/17-beta estradiol may be an effective management option for mood symptoms in women with PMDD. The statistically significant improvements in the overall DASS-21 scores before and after treatment with nomegestrol acetate/17-beta estradiol, support our findings, that the majority of women reported a subjective positive mood response to nomegestrol acetate/17-beta estradiol.Of note, there is a high prevalence of trauma in our population. 89.8% of women in our study had a history of trauma with cPTSD being a concurrent clinic diagnosis in 67.3% of women.cPTSD occurs in individuals who have experienced prolonged traumatization and is characterised by emotional dysregulation, dissociation, somatization and poor self-esteem . It is uLimitations of our study include our small sample size and the study design; in that it was an observational study with no comparison group. Given the retrospective nature of this study, there is a risk of recall bias in the women reporting their subjective mood response to nomegestrol acetate/17-beta estradiol and side effects, particularly more so for the women who were no longer taking nomegestrol acetate/17-beta estradiol. The DASS-21 score is not specific to PMDD and we did not specify the phase of the menstrual cycle women were at when they first completed the DASS-21 at the clinic, prior to commencing nomegestrol acetate/17-beta estradiol. We excluded women who had self-harming or suicidal behavior soon after taking nomegestrol acetate/17-beta estradiol for safety reasons which could have resulted in an overestimation of the positive effect of nomegestrol acetate/17-beta estradiol given that we excluded women who had a negative mood response. We also excluded women who used nomegestrol acetate/17-beta estradiol for less than one month, as it usually takes at least 1 month to see treatment effects. This may have introduced selection bias as there is a possibility the women who did not initially tolerate nomegestrol acetate/17-beta estradiol stopped taking the treatment after a few weeks. Furthermore, we did not take other factors into consideration that may influence mood and thus may change response to nomegestrol acetate/17-beta estradiol.This is the first study to investigate the feasibility and clinical effectiveness of nomegestrol acetate/17-beta estradiol treatment in the management of PMDD mood symptoms. Our findings support what we have seen in clinical practice and suggest that nomegestrol acetate/17-beta estradiol could be well tolerated by women suffering from PMDD and could be an effective first line treatment option for women with PMDD. Future research is required, particularly a randomized controlled trial of a large sample comparing nomegestrol acetate/17-beta estradiol to other recommended first line treatments for PMDD, either a drospirenone containing COCP or an SSRI.The manuscript has been submitted solely to this journal and is not published in the press or submitted elsewhere.jayashri.kulkarni@monash.edu.The data analyzed in this study was obtained from routinely collected information as part of standard treatment in the Women's Mental Health Clinic, Alfred HREC project 285-17 ], the following restrictions apply: 1) That the aim/ purpose for the data access is clearly stated and must be for a genuine research purpose, 2) That the name and affiliation of the person/s requesting data access must be clearly stated 3) That the requesting individual/s must declare all and any conflict of interest - including past & current employment and any commercial involvement. 4) That any patient identifying details are kept confidentialRequests to access these datasets should be directed to the corresponding author: Professor Jayashri Kulkarni AM, The studies involving human participants were reviewed and approved by The Alfred Ethics Committee. The patients/participants provided their written informed consent to participate in this study.This clinical follow-up study was conceived by JK and CT. JK, consultant psychiatrist, had ultimate patient care responsibility. CT, endocrinologist, provided endocrine specialist care for patients. The initial questionnaire, phone calls, data collection, and data entry were completed by ER. NT assisted ER with the implementation of the objective DASS-21 scale and data analysis. In the revision and resubmission, LK, biostatistician, contributed enormously to the reanalyses and other work as per the reviewers’ suggestions. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Low-frequency SCS works by activating large-diameter Aβ fibers in the lateral discriminatory pathway  at the dorsal column (DC), creating paresthesia-based stimulation at lower-frequencies (30–120 Hz), high-amplitude (3.5–8.5 mA), and longer-duration/pulse-width (100–500 μs). In contrast, high-frequency 10 kHz SCS works with a proposed different mechanism of action that is paresthesia-free with programming at a frequency of 10,000 Hz, low amplitude (1–5 mA), and short-duration/pulse-width (30 μS). This stimulation pattern selectively activates inhibitory interneurons in the dorsal horn (DH) at low stimulation intensities, which do not activate the dorsal column fibers. This ostensibly leads to suppression of hyperexcitable wide dynamic range neurons (WDR), which are sensitized and hyperactive in chronic pain states. It has also been reported to act on the medial pathway (drives attention and pain perception), in addition to the lateral pathways. Other theories include a reversible depolarization blockade, desynchronization of neural signals, membrane integration, glial–neuronal interaction, and induced temporal summation. The body of clinical evidence regarding 10 kHz SCS treatment for chronic back pain and neuropathic pain continues to grow. There is high-quality evidence supporting its use in patients with persistent back and radicular pain, particularly after spinal surgery. High-frequency 10 kHz SCS studies have demonstrated robust statistically and clinically significant superiority in pain control, compared to paresthesia-based SCS, supported by level I clinical evidence. Yet, as the field continues to grow with the technological advancements of multiple waveforms and programming stimulation algorithms, we encourage further research to focus on the ability to modulate pain with precision and efficacy, as the field of neuromodulation continues to adapt to the modern healthcare era.Since the inception of spinal cord stimulation (SCS) in 1967, the technology has evolved dramatically with important advancements in waveforms and frequencies. One such advancement is Nevro’s Senza Chronic pain is known to negatively impact patients’ social relationships, well-being, and work productivity ,7,8,9,10Diagnosing chronic pain is challenging. Patients usually present with pain and evidence of loss of function that persists beyond the expected course, usually greater than six months duration, refractory to conservative treatment, and often a focal pathoanatomical etiology cannot be found, but rather a combination of complex biopsychosocial factors is discovered. Excessive dependence on healthcare providers and family, withdrawal from social activities and work, physical deconditioning due to fear avoidance behavior, and development of psychosocial sequelae that impair function and/or recovery are identifiable patterns in patients suffering from chronic pain. When chronic low back pain is identified from a detailed history and physical examination, further evaluation with diagnostic imaging and assessment tools such as the Fear-Avoidance Behavior Questionnaire, Brief Pain Inventory, and other measures can be utilized to access the biopsychosocial complexity of chronic low back pain ,25,26. T® SCS System  for HF10, which received Food and Drug Administration (FDA) approval in 2015 and is commercially available in Europe, Australia, and the United States. Its product is an implantable high frequency spinal cord stimulation system which is thought to target the dorsal horn with HF10 therapy at 10,000 Hz or the dorsal column with lower frequency stimulation.Spinal cord stimulation (SCS) is a minimally invasive intervention utilizing electrical current to modulate pain signals that has been considered an adjunctive option in the management of chronic refractory pain. Shealy first introduced SCS implantation in 1967 based on the “gate control theory” proposed by Melzack and Wall ,31,32. SSCS has become a frequently performed surgical procedure for the treatment of chronic pain around the world, and now the use of SCS is considered one of the most advanced interventional methods in the treatment for chronic pain. In the United States, failed back surgery syndrome (FBSS) has been the most common reason for implantation followed by complex regional pain syndrome (CRPS), while in Europe the most common indications include intractable angina and painful peripheral vascular disease ,34. MultThe instrumentation of SCS, as well as our understanding thereof, have been rapidly evolving with new theories, different waveforms, and programming options, in addition to emerging high-quality clinical studies discussing the role of SCS in chronic pain. Compared to conventional treatment options, SCS offers a cost-effective and reversible means for pain reduction .A spinal cord stimulator is implanted either by percutaneous or laminotomy approach. This therapy is trialed first, typically via percutaneous leads placed into the epidural space and secured to the skin, before proceeding with the surgical implant. Importantly, the surgical implant is reversible and can be removed if necessary. The implant procedure is performed in the sterile environment of an operating room with the patient prone, often using monitored anesthesia care or with general anesthesia and intraoperative neuromonitoring. Utilizing fluoroscopic guidance, the desired entry point is visualized and an incision is made just medial to the pedicle 1–2 levels below the intended interlaminar entry point. The incision is typically 1–2 inches in length. Dissection with strict hemostasis is carried until the lumbar fascia is reached and two needle introducers are directed towards the desired epidural entry point with the needle points towards the midline. Upon entering the epidural space, the SCS leads are inserted through the needles and advanced under fluoroscopy to the desired vertebral level in the dorsal epidural space. Optimal placement is confirmed fluoroscopically, and the leads are anchored to the lumbar fascia. In addition to percutaneous electrode implantation, a small laminotomy can be performed and a paddle electrode can be surgically implanted. Next, a subcutaneous pocket is created for the implantable pulse generator (IPG). The leads are tunneled to the pocket site. The incisions are copiously irrigated, hemostasis is obtained, and the leads are connected to the IPG. The SCS system is tested intra-operatively for full functionality. Lastly, the IPG is safely placed into the pocket site with any extra length of the SCS lead coiled underneath, the incisions are closed in a layered manner, and a sterile dressing is applied.Historically, traditional low-frequency SCS (LF-SCS) has required maximum overlap of stimulation-induced paresthesias with painful areas to achieve pain relief . As suchHigh-frequency 10 kHz SCS works with a proposed different mechanism of action that is paresthesia-free with programming at a frequency of 10,000 Hz, low amplitude (1–5 mA) and short-duration/pulse-width (30 μS) . High-frHigh-frequency 10 kHz SCS offers several distinct advantages over LF-SCS. First, it applies a single waveform at 10,000 Hz frequency at a subthreshold level to provide pain relief without paresthesia, thus, eliminating the need for paresthesia overlap upon which LF-SCS relies. Second, a growing body of evidence demonstrates clinical superiority in favor of high-frequency 10 kHz SCS over LF-SCS ,51,52,53p < 0.001) of high-frequency stimulation in contrast to low-frequency stimulation at six months and twenty-four months. A total of 78.7% of patients had greater than 50% back pain reduction, and similar results were reported for leg pain responders at 12-month follow-up in the 10 kHz SCS treatment arm, in contrast to 51.3% in the LF-SCS treatment arm. At 24-month follow up, 76.5% and 72.9% of patients had greater than 50% reduction in low back pain and leg pain, respectively, in the 10 kHz SCS treatment arm, compared to 49.3% in the LF-SCS treatment group. In 2020, Al-Kaisy et al. performed a sub-analysis of pooled data from the previous two prospective studies and concluded that 10 kHz SCS reduced opioid use, disability, and improved pain control in patients with non-surgical refractory back pain . Th. Th91]. Additional animal studies have begun to elucidate the biochemical and neuromodulatory bases by which high-frequency 10 kHz SCS ameliorates chronic pain. Mitogen-activated protein kinases (MAPKs) are thought to play an important role in neural plasticity and inflammatory response following painful stimuli. Three MAPK proteins that have been established as important mediators of these responses with effects on hypersensitivity, nociception, and allodynia include extracellular signal-related protein kinases (ERKs), p38, and c-Jun N-terminal kinases (JNKs) ,99,100. n = 10 in each group) of animals: a naive control group, a sham group with SCS lead implantation without electrical stimulation, a sham group with high-frequency 10 kHz SCS, an SNI group with lead implantation without electrical stimulation, and an SNI group with high-frequency 10 kHz SCS. The animals were tested for SNI-induced mechanical and cold allodynia, cerebrospinal fluid (CSF) levels of glutamate, glutamate transporter activity, and miniature excitatory postsynaptic current (mEPSC) transmission. High-frequency 10 kHz SCS treatment in rats was found to decrease both mechanical and cold allodynia in SNI-induced neuropathic pain. Additionally, spinal CSF levels of glutamate were found to be increased following sciatic nerve injury, and these increases were reversed by treatment with 10 kHz SCS. Furthermore, high-frequency 10 kHz SCS treatment resulted in increased glutamate transporter activity following SNI, but did not result in changes in expression of glutamate transporter. Miniature excitatory postsynaptic currents in lamina II neurons were found to be increased following SNI regardless of treatment group. Finally, 2-methyl-6-(phenylethynyl) pyridine (MPEP), a metabotropic glutamate 5 receptor (mGluR5) allosteric inhibitor, and (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG), a mGluR5 agonist, were injected intrathecally and resulted in significant alterations to 10 kHz SCS induced analgesia. Injection of MPEP resulted in an antinociceptive effect on allodynia, while CHPG resulted in a pronociceptive effect on allodynia.In addition to MAPKs, glutamate receptors have been studied in the molecular pathogenesis of neuropathic pain and pain secondary to nerve injury. The process of central sensitization following nerve injury involves neuroplastic changes in response to nociceptive stimuli, with a key component of this plastic process being changes in glutamate receptors . AccordiThese studies examining changes in neurotransmitters in response to high-frequency 10 kHz SCS begin to elucidate the biochemical basis by which it produces pain relief without producing paresthesias and furthermore provides evidence to support its use to modulate the neurochemical process leading to pain.Human studies have begun to elucidate the anatomical mechanisms by which high-frequency 10 kHz SCS affects the experience of pain. De Groote et al. examined these mechanisms using resting state functional magnetic resonance imaging (rsfMRI) . In thisAdditionally, high-frequency 10 kHz SCS appears to lead to changes in volume of specific brain regions . De GrooThese studies demonstrate that high-frequency 10 kHz SCS modulates the experience of pain not only at the neurochemical level, but also at the neuroanatomical level, leading to changes in activity in areas of the brain associated with chronic pain. The ability to modulate or modify these anatomical pathways may serve as a basis for understanding why high-frequency 10 kHz SCS produces superior pain relief versus LF-SCS in many patients.Studies have also begun to use 10-channel electroencephalogram (EEG) to analyze the differences in neural patterns generated when using 10 kHz SCS versus 60 Hz tonic SCS. Telkes et al. investigated these differences in nine patients undergoing SCS implantation . This stHigh-frequency 10 kHz SCS involves a paresthesia-free paradigm as stimulation occurs below the sensory threshold, in contrast to LF-SCS, which relies on paresthesia production and pain overlap. Several working hypotheses for the mechanisms of pain relief with 10 kHz SCS have been proposed ,107,108,The body of clinical evidence regarding high-frequency 10 kHz SCS treatment for chronic pain continues to grow as the technology in spinal neuromodulation evolves. There is high-quality evidence supporting is use in patients with persistent back and radicular pain, particularly after failed spinal surgery. Kapural et al. published a multicenter randomized clinical trial of patients with heterogeneous diagnoses, and compared high-frequency 10 kHz SCS therapy with LF-SCS. Data suggested that the HF-SCS group had a greater response rate relative to LF-SCS and greater pain reduction in both back and leg regions at 24-month follow-up (8). The SENZA series of trials introduced robust data regarding the use of high-frequency 10 kHz SCS and provides substantial evidence that √ is not only safe, but also statistically and clinically effective with demonstrated superiority of high-frequency 10 kHz SCS in contrast to LF-SCS in the treatment of chronic back and neuropathic pain, with level I evidence supported by real-world data studies and clinical experience ,110,111.Our understanding of the 10 kHz SCS MOA has expanded, and there is high-level, well-established evidence that high-frequency 10 kHz SCS is a safe, minimally invasive, cost-effective treatment option with high patient satisfaction in multiple chronic pain conditions. High-frequency 10 kHz SCS has been shown to improve pain at short- and long-term follow-up, reduce disability, improve functional scores, decrease opioid utilization, and improve quality of life; therefore, it is feasible to consider earlier use of this intervention in the chronic pain treatment algorithm.The future of SCS is bright with significant technological advancements in waveforms and programming algorithms, in addition to the creation of devices with the ability to deliver multiple stimulation settings. The expanded options offer the ability to modulate pain with precision and efficacy in a variety of ways, as the field of neuromodulation continues to adapt in the modern healthcare era. As the field continues to grow, we encourage further research regarding potential changes in pain biomarkers with the use of 10 kHz SCS and additional applications of this therapy in other chronic pain conditions and spinal cord diseases, such as spasticity and motor control after spinal cord injury.