Using 3 confirmatory cohorts we show that molBV is independent of the 16 S rRNA region and generalizable across populations. We use the score in a cohort without clinical BV states, but with measures of HPV infection history and immune markers, to reveal that BV-associated increases in the IL-1β/IP-10 cytokine ratio directly predicts clearance of incident high-risk HPV infection . Furthermore, we identify an alternate inflammatory BV signature characterized by elevated TNF-α/MIP-1β ratio that is prospectively associated with progression of incident infections to CIN2 + . Thus, BV is a heterogeneous condition that activates different arms of the immune response, which in turn are independent risk factors for HR-HPV clearance and progression. Clinical Trial registration number: The CVT trial has been registered under: NCT00128661.Bacterial vaginosis (BV) is a highly prevalent condition that is associated with adverse health outcomes. It has been proposed that BV’s role as a pathogenic condition is mediated via bacteria-induced inflammation. However, the complex interplay between vaginal microbes and host immune factors has yet to be clearly elucidated. Here, we develop  molBV, which they use to profile the inflammatory landscape of BV and predict progression of human papillomavirus infection to cervical pre-cancer.Here, Burk et al. develop an algorithm to diagnose bacterial vaginosis (BV) using the 16S rRNA gene, called  According to Centers for Disease Control and Prevention (CDC) and NHANES studies, the prevalence of BV is 29.2% amongst reproductive-aged women living in the United States5. Globally this condition is estimated to have an economic burden of approximately $5-billion per year7.Bacterial vaginosis (BV) is defined as vaginal dysbiosis with inflammation and accompanying symptoms including vaginal discharge8, increased risk for pre-term birth9, and low birth weights10. Moreover, an active state of BV is associated with an elevated risk for transmission of a variety of sexually transmitted infections (STIs) ranging from bacterial pathogens such as Chlamydia11 and Mycoplasma12, to viral agents including HIV14 and human papillomavirus (HPV)15. There is increasing interest in understanding the relationship between the cervicovaginal microbiome (CVM) and HPV natural history and progression to cancer20. In fact, differences in the CVM might explain why some high-risk HPV (HR-HPV) infections resolve, while others persist and progress. Lastly, BV is also associated with non-reproductive health issues such as obesity21.In addition to its ubiquity, BV is a urogenital condition that has been associated with adverse reproductive health outcomes including infertility22, which requires the presence of three out of four signs or symptoms: (1) homogeneous, thin, white discharge that smoothly coats the vaginal walls; (2) clue cells in a wet mount; (3) pH of vaginal fluid >4.5; and (4) a fishy odor from the vaginal discharge before or after addition of 10% KOH . Although commonly used, this approach has been widely criticized for a considerable rate of misdiagnosis23. An alternative to Amsel’s criteria is the Nugent score that creates a composite score based on counts of key bacteria morphologically identified on a Gram stain 24. Although this method is more sensitive than the Amsel criteria26, it has been shown to suffer from interobserver variability27 and its use has primarily been limited to research settings due to the amount of time, expertise, and costs required to perform the test28. The term, molecular BV has been introduced recently14 and there are various meanings depending on the system used for molecular detection and the correlation with clinical, bacteriologic, and/or microscopic BV28. It specifically refers to suboptimal states of the CVM that are usually associated with reduced levels of Lactobacillus as measured by molecular techniques.Clinical BV is primarily diagnosed using Amsel criteria32. A number of studies have reported the association of elevated IL-1β and BV32, whereas most immune markers associated with BV appear to differ across studies. It has been proposed that this variability may be due to small sample sizes, heterogeneity of study populations33, and/or differing microbial taxa within the CVM. It is important to identify the source of this variability since the pathogenic effects of BV appear to be associated with local inflammation36.Bacterial vaginosis has features of an inflammatory state and is associated with alterations of cervicovaginal cytokinesmolBV, that can reproducibly categorize BV using a Nugent-like 0-10 score across a variety of populations including those from the US and Africa. Using this molecular approach to identify BV, we report the association of a set of cervicovaginal cytokines with molBV-BV. In particular, we demonstrate that elevated levels of Lactobacillus iners may in part explain the detection of a BV-like inflammatory signature amongst molBV-BV negative women. Although there appears to be a predominant host immune response to molBV-BV, the CVM’s positive associations with alternative forms of inflammation are associated with specific microbial agents. We utilize these observations to explore risk factors for the rate of clearance and progression of oncogenic HPV. We provide evidence that an inflammatory cervical profile underlies the association of HR-HPV natural history with molBV-BV. Surprisingly, the alternative inflammatory pathway is associated with the progression of HR-HPV infections to neoplastic lesions. This study provides evidence of multiple host inflammatory pathways associated with the cervicovaginal microbiome that influence the outcome of cervicovaginal HPV infection and possibly other pathologic outcomes of bacterial vaginosis.In this study, we describe a 16S rRNA gene amplicon sequencing-based algorithm, called Initially, 30 young women with and without symptoms of BV were recruited for evaluation of Nugent, Amsel and 16S amplicon sequencing. Three samples were inadequate for study leaving a training set of 57 participants . Based on Amsel’s criteria, 22 were classified as BV-positive; whereas, Nugent score evaluations categorized 26 with BV, 8 as intermediate for BV, and 23 as inconsistent with BV (Supplementary Table 37. There was an average (SD) of 16,580 (487) 16S reads per sample. Fungal sequencing of an ITS1 region amplicon using recently validated primers38 resulted in an average (SD) of 16,290 (3486) ITS reads per sample. Following taxonomic assignments and clustering by Euclidean distances  or a state of polymicrobialism (n = 30). There was a highly significant tendency of the BV-positive samples to sort to the polymicrobial clade and BV negative samples to the Lactobacillus clade based on either the Amsel (p < 0.001) or Nugent BV diagnosis (p < 0.001). Hierarchical clustering using fungal communities revealed two primary clades: one dominated by Candida albicans, the other with a dominance of Malassezia restricta  and Amsel BV-positive samples .The 16S rRNA gene V4 region was amplified from cervicovaginal samples of all 57 participants, as it has been shown to robustly detect bacterial species from the cervicovaginal regionces Fig. , samplesp < 0.001) were highly associated with both Amsel and Nugent outcomes of BV  data that would maximize generalizability of such a metric. Thus, we limited the markers to those taxa present in > 80% of all samples at a relative abundance of ≥ 0.001% after subsampling to 10,000 reads. We identified 11 genera meeting these criteria including molBV was evaluated in publicly available datasets that included 16S rRNA gene amplicon NGS data and measures of bacterial vaginosis. One testing set contained 388 American women with available 16S data sequenced using the V1–V2 16S rRNA gene region (different from the V4 region used above) and clinical Nugent scores40. In addition, we identified two African populations, one collected in Cape Town (n = 90) and the other in Soweto (n = 78) that sequenced the V4 region of 16S and had Nugent measures of BV42. We ran the 16S amplicon NGS reads through the molBV pipeline to generate Nugent-like scores and observed a strong correlation between the clinical Nugent scores and the molBV scores in all three cohorts with ICC values between 0.71–0.81 43. The molBV score showed high AUC values (0.88–0.98) in all three datasets and outperformed other measures of the microbiome such as alpha diversity measures Chao1 and Shannon and the relative abundance of Lactobacillus  that had 32 cytokine proteins  quantitated from cervical secretions collected with a sponge (see Methods)45. Using three ordinal categories of BV derived from the molBV scores equivalent to Nugent BV negative , intermediate  and positive , we identified 13 cytokines significantly associated with a trend across the three BV states . Cytokine levels were also tested with respect to age, smoking and HPV16 status and did not show any significant associations , whereas IP-10 was inversely associated with BV . Supplementary Table molBV-BV in univariate analysis; 6 have been previously associated with BV and 7 additional cytokines are described in this report. Given that inflammation is a complex host response, we analyzed the interrelationships amongst the 32 cytokines to identify patterns of expression. A correlation network was constructed connecting those cytokines with a Pearson correlation >0.6  with BV  of BV negative women, had elevated IL-1β/IP-10 levels  in the absence of elevated IL-1β/IP-10.To determine whether women with 16. To evaluate the impact of bacterial vaginosis and directly test whether associated inflammation could be mediating the effect of BV on the natural history of HPV, we utilized the previously reported16 prospective cohort sub-study from the CVT trial. We utilized 16S NGS data from cervicovaginal DNA16 to calculate the molBV scores across two study visits (307/431 baseline participants had sequenced 16SV4 and cytokine data). Women who had sustained low levels of molBV vs. those that had sustained high molBV scores were more likely to clear HR-HPV over time . Briefly, sustained levels refer to women that had molBV levels above or below the cohort median for both of the measured visits (203/307 were included). Similarly, sustained high-levels of BV-associated inflammation vs. low, as determined by IL-1β/IP-10, were associated with lower rates of HR-HPV clearance . Sustained levels of this measure were also defined using stratification by the cohort median and agreement of the measure (above or below) at the two measured visits (183/307 were included). For detailed definitions of sustained-levels of molBV and IL-1β/IP-10 inflammation see the subsection “HPV natural history exposure/outcome definitions” in the “Methods” section of the manuscript.We previously reported that increased diversity of the cervicovaginal microbiome contributed to HPV natural historynce Fig. , p = 004molBV-BV and/or sustained BV-associated inflammation markers acted as independent risk factors for HR-HPV clearance, we used cox-proportional hazard models after covariate adjustment. Table molBV and IL-1β/IP-10 levels adjusted for age, smoking status and HPV16. Model 1 considered the dichotomized molBV states and found that compared to having sustained low levels of molBV (reference), the transition from high (V1) to low (V2) was a significant protective factor against clearing a HR-HPV infection . Model 2 considered the effect of both IL-1β/IP-10 and molBV states. In this cytokine-adjusted analysis, molBV levels were not associated with HR-HPV clearance, suggesting that IL-1β/IP-10 was an independent driver of HPV clearance . In this context, molBV states were not significant with the exception of the marginal signal from the group that became low at visit 2 . Given the strong correlation between molBV and IL-1β/IP-10 levels  vs. HR-HPV infection clearance with CVM components serving as predictors. Using values from V2, we first tested whether molecular BV was associated with progression to CIN2+ using a generalized linear model (Table molBV score was prospectively associated with increased risk for CIN2+ progression with the odds of progression increasing by 1.24 (95% CI: 1.02–1.55) per unit increase of molBV. However, the dominant BV IL-1β/IP-10 signature was not significant when molBV was also in the model  in women that might not have been evaluated for BV, but have an available cervicovaginal sample. Development of this method used a stringent diagnosis of BV including subjects concurrent for BV by both Nugent and Amsel criteria. The method was validated in three additional cohorts suggesting the generalizability of this particular molecular approach to generate a Nugent-like score. Although it is known that there is substantial variation between the cervicovaginal microbiome between African women and women with European ancestry53, use of the molBV algorithm in two African populations revealed high diagnostic AUCs for BV of 0.97 and 0.88 for Soweto and Cape Town sets, respectively. Moreover, the molBV diagnostic was also used to evaluate the local host inflammatory response in a population without available BV measures . We identified a total of 13 different cytokines associated with molBV-BV, of which 7, had not been previously reported (Supplementary Table 32). In addition, we utilize the molBV score and cytokine data to demonstrate the contribution of each to the natural history of HR-HPV infections using a prospective study design. Another interesting feature of this study was to demonstrate a prospective association of TNF-α with CIN2+ .In this study, we develop a relatively simple means to characterize cervicovaginal samples for BV using 16S rRNA gene amplicon next-generation sequencing (NGS) data. It is well known that features of the CVM are strongly associated with BVp-value = 3.67 × 10−48). IL-1β/IP-10 ratio was previously postulated to be a relevant signature for BV and the identification of women at higher risk of STI transmission47.Cytokine ratios were used in order to better address the interrelated data structure and we observed that increasing IL-1β/IP-10 ratios were strongly correlated with increasing molecular BV scores  would yield similar distributions of other cytokines given the correlation levels observed in Fig. 20. It is not currently known why certain high-risk infections clear while a small minority persists for years and eventually progresses to pre-cancer60. We utilized HR-HPV detection data within the Costa Rica vaccine trial cohort45 to evaluate cervical cytokine profiles, 16 S rRNA gene amplicon NGS data and the newly developed molecular BV states to interrogate possible mechanisms of HR-HPV infection clearance. Kaplan-Meier analyses revealed similar clearance patterns amongst women with sustained low vs. high IL-1β/IP-10 levels and low vs. high molBV scores +58. The molBV and the analyzed cytokines were considered in a linear context, other non-ordinal relationships might exist and are worthy of future investigation. Moreover, the developed molBV score was dependent on a relatively small set of bacteria and was based on “clean” BV diagnoses in which Amsel and Nugent’s tests agreed. This choice was made in order to facilitate the robustness of the measure. This appears to have been effective based on the associations seen within African populations but may present a limitation in populations with different structures of the CVM, especially ones that may occur in women where the two clinical scoring systems are discordant. Most of the women in the reported study are adolescents or young adults and we do not know if the analyses extend to women of all ages and geographic locations. Moreover, it is possible that other inflammatory signatures might exist in the cervicovaginal region that were not measured in the current report. Finally, in using the clinical Nugent score to guide our analyses we may have inadvertently missed important physiological phenomena of BV that are inherently diluted by this clinical score; future studies should utilize additional CVM reduction techniques such as the recently developed VALENCIA51, which produces community state types that are clinically agnostic.The currently reported analyses have several weaknesses that should be taken into account when interpreting the data. All of the analyses of the CVM utilized 16S rRNA gene amplicon sequencing. This method limits the taxonomic resolution of bacteria and other organisms constituting the microbiome. A deeper exploration of BV using techniques such as shotgun metagenomics may provide a more thorough explanation as to why there is significant heterogeneity in the local host inflammatory response to BV amongst different women. Additionally, although our core analysis utilized compositionally aware approaches, rarefaction was used when calculating beta diversity, which may bias the magnitude of the PERMANOVA result. The relationships between molBV score. We further demonstrate that this score was strongly correlated with a heterogeneous inflammatory landscape within the cervicovaginal region. Exploring these inflammatory markers further revealed a complex system of interactions between individual taxa, specific cytokines, and molecular BV states. In addition, we demonstrated that there is the potential for clinical relevance of the findings through the use of HR-HPV outcomes. We specifically show that different possible inflammatory states in BV are either associated with persistence of HR-HPV infection  or the progression of infection to precancer . In support of a role for TNF-α in the progression of HR-HPV infections, a recent report indicated that TNF-α was the main discriminatory biomarker associated with invasive cervical cancer63.Here we present a comprehensive molecular characterization of BV using 16S rRNA gene amplicon sequencing and a curated panel of cytokines. We demonstrate using multiple cohorts that 16S amplicon sequencing can be reliably used to diagnose BV employing the newly developed Whether the adverse health outcomes from BV are all based on the host inflammatory response remains to be rigorously evaluated. Deeper exploration of these associations is warranted using more robust techniques such as machine learning in order to further understand why certain women experience inflammation with BV, while others do not and how the host-microbiome relationships impact health. Lastly, the implication of BV inducing a local inflammatory response might imply signaling systemic inflammation, which was not assessed in this study. The role BV-induced inflammation might have in immune conditions more prevalent in women remains an interesting hypothesis that could have profound diagnostic and therapeutic ramifications.65. Cervicovaginal samples were collected from female patients, 15–25 years of age, with vaginal symptoms suggestive of BV (n = 30) or no symptoms (n = 30), both groups were recruited sequentially from the same clinic. Pregnant women were excluded. The parent study and BV sub-study were approved by the Institutional Review Board at The Icahn School of Medicine at Mount Sinai.This component of the study was conducted within an ongoing HPV study at Mount Sinai Adolescent Health Center (MSAHC) in New York City66. Vaginal swabs were collected for Nugent scores by carefully inserting a sterile swab into the vagina about two inches, gently rotating against the vaginal wall for 10–30 s, and then withdrawn without touching the skin to avoid contamination. De-identified swabs were placed in a plastic culturette tube and shipped overnight to an outside clinical laboratory for Nugent scoring following standardized criteria24. A composite score from 0 to 10 was generated with a diagnosis of Nugent BV assigned as follows: 0–3 was considered negative, 4–6 was considered intermediate, and 7–10 was considered diagnostic of BV.Subjects were evaluated for Amsel criteria by the examining physician and the presence of 3 out of 4 Amsel criteria established a diagnosis of BV® placed in PreservCyt transport medium . Samples were stored immediately at −20 °C until transport to the research lab at the Albert Einstein College of Medicine. In the lab, the samples were transferred to a 15 ml tube and gently centrifuged at 1500 RPM for 5 min. After removing the supernatant by decanting, the pellets were rinsed in 3 ml of TE . This solution was then vortexed and centrifuged at 1500 RPM for 5 min and the supernatant was removed by decanting. The remaining pellet and leftover solution (~150 µl) were used for DNA isolation via column processing with the QIAamp Mini spin column  following the manufacturer’s protocol. The purified DNA was eluted in 150 µl of elution buffer .Samples for microbiome analyses were collected using a Cytobrush68. All primers contained unique Golay barcodes to allow for dual indexing of each sample. PCRs were conducted in a 25 µl reaction with 2 µl input of template DNA, 16.75 µl of ddH20, 2.5 µl of Platinum 10× PCR buffer , 0.75 µl of MgCl2 , 0.5 µl of dNTP mix , 0.25 µl AmpliTaq Gold, polymerase , 0.25 µl of Platinum Taq DNA Polymerase , and 1 µl (5 µM) of each primer . Thermocycling conditions included an initial denaturation at 95 °C for 5 min, followed by 15 cycles of 95 °C for 1 m, 55 °C for 1 m, 72 °C for 1 m, followed by 15 cycles of 95 °C for 1 m, 60 °C for 1 m, 72 °C for 1 m, and a final extension at 72 °C for 10 min.PCR for bacterial communities was performed using forward (515F) GTGYCAGCMGCCGCGGTA and reverse (806R) GGACTACHVGGGTWTCTAAT primers which amplify the V4 hypervariable region of the prokaryotic 16S rRNA gene69. PCRs were conducted in a 25 µl reaction with 10 µl input of template DNA, 8.75 µl of ddH20, 2.5 µl of Platinum 10x PCR buffer (Invitrogen), 0.75 µl of MgCl2 , 0.5 µl of dNTP mix , 0.25 µl AmpliTaq Gold polymerase , 0.25 µl of Platinum Taq DNA Polymerase , and 1 µl (5 µM) of each primer . Thermocycling conditions included an initial denaturation at 95 °C for 5 mins, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. All PCRs were conducted in a GeneAmp PCR System 9700 (Applied Biosystems) and PCR products were verified by gel electrophoresis.PCR for fungal communities was performed using barcoded forward (48F) ACACACCGCCCGTCGCTACT and reverse (217R) TTTCGCTGCGTTCTTCATCG primers that amplify the ITS1 region of the prokaryotic ribosomal gene clusterPCR products for each sample were pooled by PCR assay (16S and ITS1) in approximately equal concentrations and 100 µl of the pooled products were loaded into a 3% agarose gel and run at 80 V for 3 h to separate the DNA fragments. The DNA fragment for each assay was excised and purified with a QIAquick Gel Extraction Kit (Qiagen) and quantified using a Qubit High Sensitivity dsDNA assay (Invitrogen). NGS library preparation was conducted on the purified pooled PCR samples from each assay with a KAPA LTP Library Preparation Kit  according to the manufacturer’s protocol. The library amplicons were validated on a 2100 Bioanalyzer  and sequencing of libraries was carried out on an Illumina MiSeq with 2 × 300 bp paired-end reads kit at the Genomics Core of the Albert Einstein College of Medicine.70. Reads were then demultiplexed using NovoBarcode based on unique dual Golay barcode combinations71.Illumina reads were initially right trimmed to remove bases that fell below PHREAD score 25 using PRINSEQ-lite72 was used to identify amplicon sequence variants using DADA273 for both the 16SV4 and ITS1 amplicon data. For 16SV4 amplicon sequence variants (ASVs) the naïve Bayesian classifier74 was used to assign taxonomy using the lab’s custom database that is comprised of GreenGenes 13.875, HOMD76, and vaginal reference sequences77. For fungal taxonomic assignments, BLAST78 was used with the UNITE database79. Taxonomic assignment was combined with the ASV data using custom bash scripts into a biome file80 and further processed with R81.QIIME2phyloseq82 package was used to import microbiome data into R83 and to calculate the Chao1, Fisher and Shannon alpha diversity measures as well as the Jensen–Shannon diversity index for beta diversity analyses. The vegan84 package was used to run the PERMANOVA. The pROC package was used for the AUC analyses85. All data visualization was achieved using the ggplot2 package86.The 87 was used for bacterial taxa  discovery. A linear model was used to determine significance of trends in the cytokine analyses and to extract ordinal ORs. Pearson coefficient was used for correlation analysis. The q-value88 package was used to correct the calculated linear trend p-values for multiple testing. Standard error of the mean was used to represent variation in sequencing depth between samples. Cox proportional hazard models were used in order to adjust the data for age, smoking, and HPV16 status in the survival analyses. The goodness of fit was assessed using the gof function from the survMisc package89 in all models and shown to have satisfactory performance at the 0.05 alpha threshold level. Age was treated as a continuous variable, HPV16 as binary (0 or 1) based on PCR results (V2), and smoking status as ordinal  all were taken from V2.The significance of belonging to taxon-specific hierarchical clusters in the heatmap analysis was assessed using Fisher’s exact test. Pairwise statistical significance in alpha diversity was determined using the Wilcoxon test. Significance in beta diversity was determined using PERMANOVA. ANCOM87 was used to determine which bacterial genera were associated with BV through the use of microbial reference frames46 . Out of the identified biomarkers only those that were present in ≥ 80% of samples with at least a 0.01% relative abundance after subsampling were retained for the calculation of molBV. These taxa included—Lactobacillus, Prevotella, Gardnerella, Megasphaera, Parvimonas, Clostridium, Porphyromonas, Adlercreutzia, Dialister, Atopobium, and Sneathia.ANCOMs46 Fig. . Only thLactobacillus and the markers elevated in the BV positive group . The log ratios were then analyzed using a robust regression with the Nugent scores serving as the outcome and each ratio as the predictor. The beta coefficients and intercepts for each of the ratios were extracted and are presented in Supplementary Table To create the microbial reference frames, log ratios were created using molBV, which is the imputed continuous Nugent score, the log ratios between Lactobacillus and BV specific markers were generated  and used, along with the data from Supplementary Table molBV:To calculate Xi represents the log ratio i, βi0 is the ratio’s corresponding intercept and βi1 is the beta coefficient. For an estimate to be valid for a given reference taxa, there had to be both Lactobacillus and a BV marker detected with a minimum of 1 read each. The final molBV score is the average of the valid log-ratio estimates that approximate the clinical Nugent score. Given the nature of regression prediction, molBV is not bound by the 0–10 range of the Nugent score and may take on non-integer values. To make the two scales more comparable, molBV was fit into the 0–10 range by using the following formula:In the formula molBV represents the raw score obtained from the above calculation, the max(molBV) is the highest calculated value in the cohort, the min(molBV) is the lowest calculated value in the cohort and molBV_scaled represents the final molBV score that falls into the desired 0–10 range.In the formula molBV score, are used to define BV status. A molBV score of 0–3 is considered negative for BV, 4–6 is considered intermediate, and a score of 7–10 is considered consistent with BV.Similar to Nugent scoring, ranges of the continuous molBV classifier. Full details about sample collection and processing can be obtained from the cited studies. The United States (USA) confirmation cohort was composed of 388 women with collection from three separate locations  with the women having a median age of 31 years40. The Cape Town cohort was composed of 90 women with a median age of 18 years44. The Soweto cohort was composed of 78 women with a median age of 18 years44.Three cohorts were used to confirm the 90. A customized panel including 32 cytokines, chemokines, and soluble receptors was quantitated using Luminex-based Milliplex Map Mulitplex Assays  as previously described91.Cervical sponge samples were collected from women participating in the HPV Costa Rica Vaccine Trial (CVT) using a Merocel sponge  as previously described45 were used to test the prospective association of molecular BV and cervicovaginal inflammation with HPV natural history stages. One analyzed outcome was time to clearance of high-risk HPVs . Women were selected from the CVT placebo arm where an incident high-risk HPV infection was detected (visit 1 sample this study). Clearance was recorded if a woman cleared all high-risk types detected at the incident visit within a 2-year observation window. The average time between collected-sample visits was 1.28 years. Data from the CVT trial45 was used to determine persistence status to ensure that the collected V2 sample fell within the observation window. The two core exposures were sustained high/low molBV and sustained high/low cervical inflammation as determined from IL-1β/IP-10 cytokine marker ratios. Inflammation and molBV sustained/persistent status were determined by median stratification; specifically the sustained status categories refer to a per-protocol approach where the exposure status had to be similar across the two analyzed visits. For example, the median molBV value across all study visits was 5.4 and if a women had a molBV value of 10 for visit 1 and 8 for visit 2, she would be placed in the sustained molBV high category. Participants with discordant molBV values were not included in this analysis in order to measure the per-protocol effect of sustained high/low molBV values in the context of HPV natural history . Excluded samples did not differ significantly from those retained in the analysis in terms of age, HPV16 positivity or smoking status (see Supplementary Tables DNA from cervical samples from the placebo arm of the Costa Rica vaccine trialFurther information on research design is available in the Supplementary InformationReporting Summary


The glass-ceramic particles with 74% crystallinity were used for this purpose as they can best act as both remineralizing and reinforcing agents. Strengthening mechanisms including crack deflection and crack-tip shielding were activated for the GICs containing 50–50 wt% bioglass and bioglass-ceramic as the optimum ratio. The progression of the GIC setting reaction at its early stages was also monitored and verified. We also discussed that our bimodal particle size distribution containing both micron- and nanosized particles may enhance the packing density and integrity of the structure of the cements after setting. In such GICs produced in this study, the toxic effects of Al are avoided while chemical bonds are expected to form between the cement and the surrounding hard tissue(s) through interfacial biomineralization and adhesion.Although the incorporation of bioactive glasses into glass ionomer cements (GICs) has led to promising results, using a bioactive glass as the only solid component of GICs has never been investigated. In this study, we developed an Al-free GIC with standard compressive strength using various combinations of 45S5 Bioglass Glass ionomer cements (GICs), also known as glass polyalkenoates, were developed and patented in 1969 at the Laboratory of the Government Chemist  , 3. GICs2+ and Al3+) leach out from glass particles resulting in the crosslinking of polyalkenoic acid chains [GICs are classified as acid-base cements consisting of an aqueous solution of a polyalkenoic acid and a powdered calcium aluminosilicate glass component with a basic nature . The pold chains , 8, whic2Ca2Si3O9) phase can improve the mechanical performance of GICs while enhancing their remineralizing properties [In addition to the field of dentistry, GICs have been considered as a potential substitute for polymethylmethacrylate (PMMA) bone cements for orthopedic applications due to their ability to chemically bond to hydroxyapatite (HA) . Unlike operties .GICs can be considered bioactive since they can release ions via dissolution of calcium aluminosilicate glass particles causing certain biomineralization within their clinical environment . Improvi®  is known to be the first commercial bioactive glass [® has similar characteristics to the common GIC glass component. For instance, it contains a high volume of network modifiers, CaO and Na2O, which can break the continuous Si–O–Si structural units resulting in the formation of non-bridging oxygens [+, Ca2+ with H+ from the solution [2+ ions may be able to react with the polyacrylate chains creating an ionically crosslinked polyacrylate matrix [43- is released from the Bioglass® as a result of reacting with the polyacid [2+ polyacrylate matrix [45S5 Bioglassve glass . 45S5 Bi oxygens . Upon insolution . When the matrix , 27, simpolyacid . It has e matrix . Formati® can be overreactive to the polyacid resulting in a premature setting. Alternatively, 45S5 glass-ceramic with appropriately lower bioactivity  and superior mechanical properties compared to those of the 45S5 Bioglass® may be used as the GIC glass component [Due to its high bioactivity index , 45S5 Biomponent .® might not have high basicity as other GIC glass compositions [® composition.Due to the lack of Al, 45S5 Bioglassositions . It has ositions , resulti® to its glass-ceramic as the glass component as well as polyacrylic acid aqueous solution. The new resulting GICs exhibited proper mechanical properties comparable to commercially available GICs. We also show that our 45S5 Bioglass®-based GIC not only meets the ISO compressive strength requirement but also may improve the remineralizing properties of cements.The main objective of this study was to develop a remineralizing, aluminum-free GIC consisting of an optimum ratio of 45S5 Bioglass® used in this research was prepared and donated by Dr. Robert Hill from the Queen Mary University of London. In short, calculated amounts of high-purity precursors were mixed and then melted in a platinum/rhodium crucible at 1380 °C for 1 h using an electric furnace. The resulting melt was then rapidly quenched in deionized water at room temperature. Next, the glass frit was dried and melted again for 30 min. followed by a grinding process using a vibratory mill [The 45S5 Bioglassory mill , 29, 31.® was annealed at 460 °C for 8 h under ambient atmosphere. X-ray diffraction  using Cu Kα radiation (10° < 2θ < 90°) was used to confirm that the amorphous nature of the material was completely retained.To eliminate any internal stress created during the grinding process, the as-received Bioglass® was heat treated through a controlled process [® are explained in detail in our previous study [® and isopropyl alcohol. Also, the morphology of the as-received glass and heat-treated glass-ceramic particles was examined using a scanning electron microscope .In order to prepare the 45S5 bioglass-ceramic containing combeite, the as-received 45S5 Bioglass process  to induc process . The heaus study . In brieus study . The degus study . The hea® and 45S5 bioglass-ceramics . In order to investigate the effect of incorporation of bioglass-ceramic, eleven powder mixtures containing 0, 10, 20, 30, 40, 50, 60, 70, 90 and 100 wt% bioglass-ceramic  in deionized water was used as the liquid component of the cement. The glass powder components were hand-mixed with the liquid component on a watch glass at different ratios. The powder to liquid ratio (P:L) with the best working and handling properties (P:L = 1:0.8) was found by trial and error among ten tested ratios ranging from 1:0.4 to 1:2.2. Note that all the samples were prepared at room temperature (21 ± 2 °C) throughout this process. Depending on the subsequent characterization technique, different setting procedures were followed as explained in the next section.n = 3) according to ISO 9917-1: dentistry-water-based cements [n = 6):The setting time of each cement was recorded ( cements . Compres cements . CylindrP is the maximum applied load (N) and D is the diameter of the sample (mm). The fracture surface of the cements was examined by SEM.where n = 6).A microhardness tester (Mitutoyo MVK-H1) was also used to measure the Vickers microhardness of all the cements by applying a 50 gf load on the surface of each cement for 30 s  were obtained and studied. To study the effect of immersion time, the C50 cements, which exhibited the highest mechanical properties among all compositions, were examined by FTIR after 1 d, 7 d, and 21 d immersion in DW. To investigate the early stages of setting, spectra were collected from the C50 composition at 1, 3, 5, 7, 10, 15, 20, and 25 min immediately post mixing (n = 3). To monitor the setting progress of the C50 cement, the band intensity ratios between the free carboxyls (COOH) absorption band at ≈ 1650 cm−1 to the Ca-polyacrylate (COO− Ca2+) absorption bands at ≈1550 cm−1 and 1410 cm−1 [− Ca2+/COOH band intensity ratios as a function of setting time  were plotted.The resulting cements were characterized by Fourier transform infrared spectroscopy  at room temperature over a wavenumber range of 500 to 4000 cm410 cm−1 –36 were ® and the heat treated bioglass-ceramic particles with 74% crystallinity are presented in Fig. ® has retained its amorphous nature after the stress-relief heat treatment at 460 °C as explained in section 2.1. Also, the XRD pattern of Fig. ® powder  and the SEM micrographs of both as-received Bioglassder Fig.  is in thder Fig.  shows a ®  ratios and setting times of all cements are presented in Table ®  resultin2+) in 45S5 Bioglass® glass network can be released upon reacting with PAA and take part in setting reactions [®-based GICs  leading to a high packing density  may interfere with the setting reaction which results in more unreacted particles remaining in the system  polyacrylate one. Formation of such inorganic network along with the aforementioned hard phase, leads to a gradual improvement in the strength, stiffness, and insolubility of GICs over time, even after the setting [In Fig.  setting . This is setting .Fig. 5Th® and bioglass-ceramic particles are submicron. Since these particles provide a large surface area to react with PAA, there may be an insufficient number of ionomer available to effectively hold a large number of submicron particles resulting in those particles remaining unreacted and unattached to the matrix of the cements which lower their final strength. This might be also the reason for the relatively low P:L ratio of the cements in this study (1:0.8) reported in Table By applying some adjustments, even higher mechanical properties may be obtained for these cements, particularly in longer term. According to the PSA results Fig. , more this study :0.8 repo®-based GICs are presented in Fig. −1 and 1550 cm−1 respectively correspond to the symmetric and asymmetric stretching vibration of Ca-polyacrylate complex (COO− Ca2+) and the peak at 1050 cm−1 is assigned to silicic acid (H2SiO3) formation [2+) leaching out of the glass particles [2 glass network when attacked by PAA [The FTIR spectra showing the effects of powder composition and immersion time on the structure of our Bioglassormation –36. Ca-pd by PAA .Fig. 6FT− Ca2+ and H2SiO3, among the three spectra, which may indicate a higher degree of crosslinking that is in line with its superior mechanical properties  and the COO− Ca2+ (1550 cm−1) peaks for our Bioglass®-based GIC (C50) over time. It appears that, the intensity of the COO− Ca2+ peak increases overtime at the expense of the COOH peak. This gradual time-dependent change can be an indication of the progression of the acid-base reaction until the GIC sets [2+ ions from the glass particles start to create salt bridges which crosslink the polyacrylate chains and lead to a decrease in COOH (1650 cm−1) peak intensity. Simultaneously, the intensity of peaks corresponding to the symmetric (1410 cm−1) and asymmetric (1550 cm−1) Ca-polyacrylate (COO− Ca2+) bonds increases. The progression of the GIC setting reaction is further assessed by plotting the COO− Ca2+/COOH ratios  over time  exhibited the highest combination of compressive strength and microhardness. Strengthening mechanisms such as crack deflection and crack-tip shielding were found to be activated enhancing the mechanical properties of this group of GICs. Also, cements with lower  and higher  bioglass-ceramic contents showed many loosely attached particles in their microstructure acting as crack initiation sites. It was also discussed that the bimodal particle size distribution of the solid component in these GICs may have contributed to their high packing density and structural integrity after setting where smaller particles mostly take part in the setting reaction while larger particles participate in strengthening mechanisms e.g., crack deflection. Supplementary in vitro and in vivo tests are required in the future to further study these 45S5 Bioglass®-based GICs and their long-term performance as dental restorative and/or bone cements.In this study, we developed an Al-free 45S5 Bioglass


The disjoining pressureof water was estimated from wicking experimentsin 1D silicon dioxide nanochannels of heights of 59, 87, 124, and1015 nm. The disjoining pressure was found to be as high as ∼1.5MPa while exponentially decreasing with increasing channel height.Such a relation resulting from the curve fitting of experimentallyderived data was implemented and validated in computational fluiddynamics. The implementation was then used to simulate bubble nucleationin a water-filled 59 nm nanochannel to determine the nucleation temperature.Simultaneously, experiments were conducted by nucleating a bubblein a similar 58 nm nanochannel by laser heating. The measured nucleationtemperature was found to be in excellent agreement with the simulation,thus independently validating the disjoining pressure relation developedin this work. The methodology implemented here integrates experimentalnanoscale physics into continuum simulations thus enabling numericalstudy of various phenomena where disjoining pressure plays an importantrole. Distances up to which a surfacecan affect liquid properties depends on the atomic composition: Ifeither atom is nonpolar, then the presence of only weak and short-rangevan der Waal’s force limits such changes to <5 nm;4 however, if both atoms are polar, then strong and long-range electrostaticforces can alter properties up to tens to hundreds of nanometers fromthe surface.7 The latter scenario often occurs in practical situationsinvolving water on various surfaces.A nanoscale thin liquid filmon a surface can have properties significantly different from thoseof its bulk form.8 Here, we usedisjoining pressure as the major variable governing the alterationin nanoscale liquid film, which also lays the foundation for advancingresearch on liquid–vapor phase-change heat transfer to developfuture thermal management devices. The pressure in such thin filmsis expected to be reduced based on the modified Young–Laplaceequation.9 Such a reduction, defined asdisjoining pressure,10 plays a fundamentalrole in a wide range of engineering and natural systems involvingbubbles,15 transpiration,17 emulsions,22 and membranes.23 The theoretical determinationof disjoining pressure of water on surfaces using extended DLVO theoryrequires fitted constants as the structural forces are usually unknown,and the surface potential cannot be measured directly for deionized(DI) water.29 However, numerical simulations such as molecular dynamics are currentlyinsufficient to computationally simulate large domains and accuratelycapture the intermolecular forces of water over larger distances.Likewise, the experimental estimation of sub-100 nm liquid films hasbeen severely limited to nonpolar films on solid surfaces31 primarily due to the evaporative and fluidic nature of nanoscalewater films. Here, we overcome these experimental challenges by characterizingwater’s wicking behavior in 1D silicon dioxide (SiO2) nanochannels of varying height, estimating the disjoining pressureof water from experimental data, and applying these disjoining pressurevalues in computational fluid dynamics (CFD) wicking and bubble nucleationsimulations.In this work, we focuson the pressure of nanoscale water films.The thermodynamic equilibrium of liquid/air inside the nanochannelsis dependent on several parameters including van der Waals forces,electrostatic forces, and structural forces; thus, it represents aGibbsian composite system.Supporting Information for sample fabrication). Due to thelong-range ordering (up to 50 nm away from surface) of water moleculeson silicon-dioxide surface shown in nuclear magnetic resonance studies,7 we fabricated four channels with heights of 59, 87, 124, and 1015nm  rectangular cross sectionchannel:32h and w are the height and width of the nanochannel, respectively;μis the fluid dynamic viscosity; and ΣP is thetotal pressure difference driving the wicking. The value of constant C .The wicking distance 37 If wicking is only driven by capillary pressure i.e., ΣP = Pc ≈ 2σ cosθ/h where σ is the surface tension, andθ is the contact angle, then 38t1/2 holds at nanoscale, Washburnequation is inconsistent in predicting the experimental wicking rate.Other published literature on wicking in rectangular cross sectionnanochannels include a similar observation42 and explained this inconsistency, primarily due to electro-viscouseffect45 or geometrical effect.47 However, these effectsdo not explain our observed deviation (please refer to the Supporting Information for a detailed explanation).Furthermore, a common major flaw in these prior studies is the contactangle θ used in 50 of the meniscusdue to the channel’s high aspect ratio . Thus, due to the rectangular cross section of the channel,we define two separate contact angles θtop and θside of the meniscus corresponding to the top and side views,respectively . The experimental datapresents two unique findings: (1) Despite as much as 50% fluctuationin the values of cos θtop, the wicking distance L is still proportional to t1/2  starts to diminish at distancesbeyond h/2 ∼ 60 nm from the surface, and isin good agreement with published studies7 which foundthe distance to be ∼50 nm using nuclear magnetic resonance.Next, we use the experimental data to calculate disjoining pressurein the nanochannels. to t1/2 3b; (2)Tg period3b are in51 However, as we only have four channel heights, disjoining pressure P̅d averaged over half the channel heightis estimated from experimental data using P = P̅d + Pc, the combination ofwhich can be rearranged intoC is the slope  in such confined channels will be differentfrom the bulk we still assume bulk properties as per the norm in literature46 and due to the fact that suchproperties have not yet been directly measured in experiments; infact, the disjoining pressure term is introduced in the modified Young–Laplaceequation30 to account for the lack of such propertiesat these scales. Thus, in side. However, as the heights of the channels are at nanoscale,it is not possible to experimentally measure θside. Considering the published experimental finding that water moleculeson a glass surface are highly ordered up to distances of 50 nm normalto the surface7 , we use molecular dynamics simulations to qualitativelystudy the effect of liquid structuring on contact angle by simulatingwicking of liquid argon in a hydrophilic nanochannel of two differentheights (please see the Supporting Information for simulation details and results). For the smaller channel heightwhere liquid structuring is prominent, θside is determinedto be ∼90°, whereas the larger channel height with mostlybulk liquid resulted in a θside value of ∼45°.Thus, using the same analogy for water in our SiO2 nanochannels,we assume θside = 90° for the smaller heightchannels (59 and 87 nm) and θside = θtop = 39.6° for the larger channel height (1015 nm) in P̅d × 106 Pa and b = 0.035 ± 0.007nm–1. The values of constants A and b are obtained from curve fitting the experimentaldata of Disjoining pressure is theoretically knownto continuously decreasewith increasing distance from the surface.ope from 2 of the ) in P̅d 4a. For te limits 4a. Pleasckness δ 4a which 52 to show the applicability of experimentally derived P̅d values into continuum simulations. Similar to the experiments,we simulate wicking of water in 59 and 1015 nm channels (please seethe Supporting Information for details)using a laminar multiphase volume of fluids (VOF) method.53 As the driving force of the wicking is ΣP = P̅d + Pc, Pc is achieved throughcontinuous surface force (CSF) modeling54 along with wall adhesion, and P̅d is invoked by a user-defined function (udf). The different θtop and θside values in the 59 nm channelare implemented by assigning the corresponding contact angles to theside walls and top/bottom walls of the channel, respectively. Wickingrates are found to be in good agreement with experimental data inboth channels . Thebubble nucleation temperature was determined to be 126.0 °C inthe CFD simulation , in excellent agreementwith CFD simulations. Thus, the obtained disjoining pressure expression(In order to quantitatively and independently validate the disjoiningpressure expression 4, we perequation 4 and impequation 4b. We siequation 4c. Initimulation 4c. Due tpression4 is indeTo summarize, we reporta fundamental study of estimating disjoiningpressure of water in silicon-dioxide nanochannels through experimentsof wicking in channels with heights of 59, 87, 124, and 1015 nm. Disjoiningpressure is found to be the primary driving force of wicking in smallerheight nanochannels, while capillary pressure dictates wicking inthe larger height nanochannel. The average disjoining pressure ofwater exponentially decreased with increasing film thickness, anda relation is derived by curve fitting the experimental data. Thedisjoining pressure relation is implemented in CFD simulations andis shown to capture the experimental wicking behavior. Such an implementationis then used to simulate bubble nucleation in a water-filled nanochannelwith a height of 59 nm. Simultaneously, nanochannels with a heightof 58 nm were fabricated, and bubble nucleation was achieved insidethe water-filled nanochannel by laser heating. The bubble nucleationtemperature measured in experiments was found to be in excellent agreementwith that obtained from CFD simulations, thus independently verifyingthe disjoining pressure model developed in our work.


The suitability of corticomotor inhibition and corticospinal excitability to measure brain health outcomes and recovery of sport-related head impact (concussion and subconcussion) depends on good inter-day reliability, which is evaluated in this study. Transcranial magnetic stimulation (TMS) reliability in soccer players is assessed by comparing soccer players, for whom reliability on this measure may be reduced due to exposure to head impacts, to generally active individuals not engaged in contact sport.TMS-derived corticomotor inhibition and corticospinal excitability were recorded from the rectus femoris muscle during two testing sessions, spaced 1–2 weeks apart in 19 soccer players (SOC—age 22 ± 3 years) and 20 generally active (CON—age 24 ± 4 years) healthy volunteers. Inter-day reliability between the two time points was quantified by using intra-class correlation coefficients (ICC). Intra-group reliability and group differences on actual measurement values were also explored.SOC = 0.61; ICCCON = 0.70) and corticospinal excitability  in both generally active individuals and soccer players routinely exposed to sport-related head impacts. Corticomotor inhibition showed lower coefficients of variation than excitability for both groups . No group differences between soccer players and generally active individuals were found on the corticomotor inhibition value (p > 0.05), but levels of corticospinal excitability were significantly lower in soccer players . Corticomotor inhibition also showed excellent inter-rater reliability (ICC = 0.87).Good inter-day reliability was evident for corticomotor inhibition (ICCCorticomotor inhibition and corticospinal excitability are stable and maintain good degrees of reliability when assessed over different days in soccer players, despite their routine exposure to head impacts. However, based on intra-group reliability and group differences of the levels of excitability, we conclude that corticomotor inhibition is best suited for the evaluation of neuromuscular alterations associated with head impacts in contact sports. The sequelae of repetitive impacts to the head in soccer and other contact sports are increasingly becoming a cause for concern.Non-invasive assessments of corticospinal excitatory and inhibitory mechanisms can help shed light on potential brain changes associated with subconcussive head impacts. However, no data exist on the reliability of such parameters in a contact sport context.The current study shows that parameters of corticospinal excitation and inhibition are stable and show good short-term test–retest reliability in soccer players. Inhibitory responses may be the better measure for assessments in a subconcussive context due to methodological limitations associated with corticospinal excitability.Injuries of a concussive (and more recently subconcussive) nature have been at the center of attention for researchers, clinicians and sporting bodies alike. Such injuries have been shown to be associated with both acute and chronic impairments to cognitive function –3, motorSingle pulse transcranial magnetic stimulation (TMS) allows researchers to quantify intracortical, corticospinal excitatory and inhibitory mechanisms governing motor skills . MeasureVarious factors may affect repeatability of TMS-based parameters, including the positioning of the coil over the scalp, the target muscle being investigated, number of stimuli applied over the primary motor cortex (M1) and the stimulator intensity . As suchWhether the test–retest reliability of TMS parameters depends on the population assessed is ambiguous. For example, whilst there is evidence suggesting that inhibitory and excitatory measures are reliable across the lifespan , other sThe aim of this study was to explore the inter-day reliability of excitatory and inhibitory mechanisms in soccer players, to establish whether these measures are suitable in detecting potential subconcussive-dependent changes to the brain-to-muscle pathways. Based on previous findings –17, we chttps://wnarifin.github.io/ssc/ssicc.html. Using a minimum acceptable reliability ICC of 0.6 (standard for “good” reliability), an alpha level of 0.05, 80% power and two repetitions per participant the recommended sample size is 18. Individuals participating in soccer were allocated in a group referred to as “SOC” , and the generally active control participants in “CON” . Participants in SOC ranged in terms of skills and abilities from recreational to playing for a club/University). At the time of recruitment soccer players headed the ball at least 15 times per week, whilst the control group were generally active individuals who did not partake in any contact sport. All participants were screened prior to taking part; potential candidates were excluded if they presented with any of the following: (1) history of brain injury resulting in loss of consciousness; (2) history of a neurological condition; (3) history of concussion in the 12 months prior to taking part; (4) family history of epilepsy; (5) current use of psychoactive recreational or prescription drugs; (6) do not normally head a soccer ball at least 15 times per week . The local Research Ethics Committee approved the study and procedures conformed to the guidelines set out by the Declaration of Helsinki. Written informed consent was obtained from all participants prior to taking part.A total of 39 participants were recruited for the study via advertisements on university noticeboards and social media. Sample size was determined using a web app developed by Arifin, 2018 [Participants were asked to refrain from vigorous physical activity, consuming alcohol, and caffeine or smoking for 24 h prior to each study session. Participants were also required to present to the laboratory fasted and were provided with a standardized breakfast. Prior to commencing data collection, participants reported to the laboratory for a familiarization session, during which they completed all outcome measures to acquaint them with the assessment procedures and minimize later learning effects. Following the practice session, participants reported to the laboratory for two further experimental days, spaced a minimum of one and a maximum of 2 weeks apart (mean follow up 11 ± 5 days).Maximal isometric voluntary contractions (MVC) were assessed from the quadriceps femoris of participants’ dominant leg (self-reported) at the beginning of each testing day. Participants first performed a series of warm up contractions (3 ×  ~ 50% (perceived) MVC and 3 ×  ~ 70% (perceived) MVC. Subsequently, participants were instructed to achieve peak torque and maintain the contraction for 5 s; all participants performed 3 contractions, with 60 s rest in between and consistent verbal encouragement was provided to ensure maximal effort. The highest torque output achieved was designated as MVC and stored .MVCs alongside all other electromyography (EMG) and TMS measures (see below) were recorded with participants sitting with their dominant leg secured to a calibrated load cell of an isokinetic dynamometer . Knee angle was set at 60° (0° being fully extended limb) and the arm of the dynamometer was set such that the axis of rotation was aligned with participants’ lateral femoral condyle.Electromyographic activity was recorded from the rectus femoris using a wireless system . Data were sampled at 2 kHz, and filtered using 500 Hz low and 1.0 Hz high band filters. Signals were analyzed offline . EMG activity was assessed using Ag/AgCl surface electrodes  with an intra-electrode distance of 2 cm positioned over rectus femoris; prior to electrode placement, the area of interest was shaved and abraded as per Surface Electromyography for the Non-Invasive Assessment of Muscles (SENIAM) guidelines . The posPeripheral stimulation of the femoral motor nerve was administered using an electrical stimulator . The stimulation site was identified by locating the femoral artery and placing a self-adhesive surface electrode (cathode) lateral to it, high over the femoral triangle, with the anode on the gluteus maximus. Single stimuli were delivered to the muscle while participants maintained a 20% MVC isometric contraction, and the intensity of stimulation was increased until a plateau in twitch amplitude and rectus femoris M-wave (Mmax) occurred. Supramaximal stimulation was delivered by increasing the final stimulator output intensity by a further 30%.2 unit, The Magstim Company Ltd., Whitland, UK) and a 110 mm double cone coil. Optimal coil location for generating MEPs was determined by placing the coil over the motor cortex, laterally to the vertex; the area where the largest MEP peak-to-peak amplitudes occurred was identified and marked on the scalp with ink [Motor evoked potentials (MEPs) were elicited in the rectus femoris of the dominant leg via single pulse TMS and assessed using electromyographic (EMG) recordings. Single magnetic stimuli of 1 ms duration where applied over the contralateral primary motor cortex using a magnetic stimulator  * 100; where σ is the standard deviation, and µ is the mean of the sample. CV differences between excitatory and inhibory responses were explored on scistat.com  using tests from Forkman [t test. Statistical significance was set at p ≤ 0.05; data are expressed as means (± standard deviation) unless otherwise stated.Data collected from the 2 laboratory visits were organized based on the timepoint at which they were recorded . Normality of the data was checked using the Shapiro–Wilk test, data were normally distributed. Reliability of the measures within each population was quantified using intra-class correlation coefficients (ICC) and coefficients of variation (CVs) computed by SPSS . ICC values for inter-day reliability were calculated using a 2-way random effect model , whilst ICC values for inter-rater reliability (for cSP) were calculated using a 2-way random effect model  with pooled data. All ICC values were interpreted as following: ≤ 0.39 = poor; 0.4–0.59 = fair; 0.6–0.74 = good; 0.75–1 = excellent, as outlined by Cicchetti . The CV  Forkman . AnalysiCON = 0.70) and in soccer players (ICCSOC = 0.61) (Table CON = 0.70) and in soccer players (ICCSOC = 0.59), respectively.Corticomotor inhibition appeared stable across timepoints, as ICC analysis showed good test–retest inter-day reliability both in generally active individuals   (Table p < 0.001).Intra-group analysis of corticomotor inhibition showed moderate CVs (CVp = 0.585) (Table p = 0.843).Inter-rater analysis for silent period duration showed excellent reliability (ICC = 0.87) with similar CVs between raters  (p = 0.305) and time points (p = 0.795) . In contrast, the excitatory parameter values showed differences between groups and timepoints  = 11.6; η2p = 0.32) , and at time point 2 . Corticospinal excitability in the soccer group at time point 2 (SOC 2) was significantly lower than in the generally active group at time point 1 , and at time point 2 .There were no differences between soccer players (116.8 ± 19.5 ms) and generally active individuals (120.7 ± 12.7 ms) when examining the actual cSP values, across both groups (p = 0.527) and time (p = 0.555), and no interaction effects (p = 0.443) , the soccer group was significantly lower than the controls  = 12.23; η2p = 0.145) , however soccer players were significantly lower than controls  = 11.90; η2p = 0.139) . CriticIt is furthermore important to highlight the inherent limitations of TMS that preclude a definite standpoint on its usefulness in a concussive/subconcussive context . One of With regards to methodological limitations, it should be noted that in the current study the values shown for corticospinal excitability are normalized to the Mmax of the same muscle, whilst previous studies presented raw values , 16. CorCorticomotor inhibition in the current study was quantified manually by two independent raters. Manual assessment of silent period duration is thought to be less objective than automated methods which may affect inter-rater reliability . Our finMore recently subconcussion in general, and soccer heading in particular, have become of societal importance due to the potential links between these head impacts and decreased brain function and an increased chance of neurodegenerative disease . As suchThis is the first study to demonstrate that parameters of cortical inhibition and excitability show good short-term test–retest reliability in soccer players and the general population alike. Therefore, these TMS-derived parameters are suitable in quantifying motor function in sports involving routine exposure to head impact. However, TMS-assessed excitation appears to be suppressed in soccer players as found in this study. Combined with the methodological limitations of TMS-assessed excitation seemingly resulting in increased variability across individuals in general, we conclude that corticomotor inhibition is the better measure for assessment and monitoring in the context of concussive and subconcussive injuries.


I read with interest the recent paper by Steinhausen et al. (2021).This paper reports the outcome of a retrospective review of bioactive glasscompared to autologous bone graft (ABG) in a staged surgical protocol fortreating established bone infection. The authors claim that bioglass isequally effective as ABG in eradicating infection but acknowledge that theirevidence is weak.The surgical treatment of chronic bone infection has been reported for wellover a century. In 1931, Jacob Kulowski published his classical series of 130cases of osteomyelitis, treated by Orr's method . Hissurgical debridement technique was similar to that described by Steinhausenet al. (2021), and he achieved a recurrence rate of 24 % (mean follow-up:19 months), in the pre-antibiotic era, with no defect fillers. In this newseries of 83 patients, the recurrence rate with bioglass was 29 % and19 % for ABG. Clearly the addition of bioglass or bone graft has notimproved outcome.The authors state that the longer follow-up period for the ABG group is nota source of bias because “most complications occurred within 12 months”.This same group previously published 50 of their 51 bioglass cases  with a mean follow-up of 12.3 months and a recurrence rate of14 %. This has increased to 29 % with a longer follow-up (mean 20.5 months). It would appear that the recurrence rate doubled in the 8 monthsafter the first year.There are no large randomized trials directly comparing the use of bioglasswith other options, and the authors correctly report that outcomes of smallstudies are inconclusive. However, there are two high-quality animal studies. In a rabbit model of MRSAosteomyelitis, Xie et al. (2009) reported that bioglass was no moreeffective than debridement alone (success rate 36 % for debridement and18 % for debridement and bioglass) but the addition of local antibioticsto calcium sulfate or bioglass increased success to 73 % and 81 %respectively. Boot et al. (2020) studied the effect of hydrogel and bioglassin an infected tibial nail model. They showed that bioglass or hydrogelwithout antibiotics produced no reduction in infection, butvancomycin-loaded hydrogel could eradicate established infection.It has been widely claimed that bioglass has the advantage of reducing therisk of antimicrobial resistance. This paper shows that cases treated withbioglass required significant additional systemic antimicrobial therapyafter repeated surgery or recurrence. It has been shown that repeatedsurgery promotes change in bacterial cultures .Recurrent, and often suboptimal, systemic therapy drives selection pressureand multi-drug resistance. There is no evidence that local antimicrobialtherapy contributes to this. The recent study of Bidossi et al. (2020)demonstrated that prolonged exposure to high-dose local antibiotics in aceramic carrier was not associated with any adaptations in bacteriaproducing antimicrobial resistance. In contrast to bioglass, the use oflocal antimicrobials may allow reduced systemic therapy and betterantibiotic stewardship .Steinhausen et al. (2021) also claim that “The tolerability of BAG is evendescribed as superior when compared to other bone substitutes”. We cannotfind any evidence for this statement in their paper. When comparing to thepaper they quote as evidence , the claim is notsupported. If we compare recurrence rate, reoperation rate, failure of bonehealing and amputation rate between the two papers, they report 29 %,47 %, 23 % and 6 % respectively. In our study of 100 cases, thefigures were 4 %, 3 %, 13 % and 0 %. We believe these outcomes wouldbe more tolerable for patients.Perhaps the most important conclusion from this paper is that serialdebridement, followed by defect filling without local antimicrobials, isnot a very effective treatment. We would not recommend use of a hip or kneeprosthesis with a 29 % failure rate within 2 years. This problem wasidentified in the 1970s and prompted the use of antibiotic-loaded PMMAbeads by Klemm and Buchholz . There is now encouraging evidencearound the use of single-stage surgery, facilitated by modern localantibiotic carriers, with few complications and high infection eradicationrates, even in the most complex cases .


The chemokine CXCL13 is used as complement to serology in the diagnostics of Lyme neuroborreliosis (LNB). We evaluated and compared the semi-quantitative, cassette-based ReaScan CXCL13 assay with the quantitative recomBead CXCL13 assay using a collection of 209 cerebrospinal fluid samples. The categorical agreement between results interpreted as negative, grey zone, and positive by the two methods was 87%. The diagnostic sensitivity was higher using the recomBead assay, whereas specificity was higher using ReaScan. Few manual steps, and a short turn-around time with no batching of samples makes the ReaScan CXCL13 assay an attractive complement to serology in the diagnostics of LNB.The online version contains supplementary material available at 10.1007/s10096-021-04350-y. Borrelia burgdorferi sensu lato complex in Europe [Borrelia antibodies  [Lyme neuroborreliosis (LNB), the most common form of disseminated infection by bacteria in the n Europe , typicaln Europe . Definite Bb AI) .The B cell-attracting chemokine CXCL13 has in previous studies been shown to be a useful and reliable complement to serology as it is elevated in the CSF of patients with very early LNB where the sensitivity of antibody tests may be low. Furthermore, CXCL13 has been proven useful in the monitoring of response to antibiotic treatment –7, and fThe main aim of this work was to evaluate the rapid and semi-quantitative ReaScan CXCL13 assays  performance and compare it to the quantitative recomBead CXCL13 assay , which was used in clinical routine in the laboratory in Jönköping as a complement to serology in selected cases and run once a week following batching of samples –17. Secon = 209) from definite LNB (n = 53), probable early LNB (n = 16) and non-LNB (n = 140) patients were retrospectively included from the Clinical Microbiology Laboratories in Region Jönköping County, Region Kronoberg, and Örebro University Hospital. Patients with symptoms strongly suggestive of LNB, such as subacute meningitis or peripheral facial palsy, and CSF pleocytosis, but, due to short symptom duration, with negative Bb AI, were in our study classified as probable early LNB [CSF samples , CXCL13 250–500 pg/mL (grey zone), and CXCL13 > 500 pg/mL (positive/suspected LNB), based on the instructions by the manufacturer.In the quantitative recomBead CXCL13 assay, based on the Luminex xMAP® technology, samples are analysed in batches. The range of measurement is 9–1000 pg/mL. The CXCL13 concentration is interpreted as CXCL13 < 190 pg/mL (negative), CXCL13 190–300 pg/mL (grey zone), and CXCL13 > 300 pg/mL (positive/suspected LNB), based on the instructions by the manufacturer.P value < 0.05 was considered statistically significant. The impact of repeated freeze thawing (− 20 °C) and cold storage at 4–8 °C for up to one week was compared with results based on fresh samples with ReaScan CXCL13.Both the ReaScan and recomBead CXCL13 assays are CE/IVD validated by their manufacturers. Their recommended cut-off values were applied in the study, since these are most likely the cut-offs used by laboratories using these assays. Grey zone results were interpreted as positives in the evaluation of sensitivity and specificity, based on the tradition in our laboratory. In the calculations, patients with probable early LNB were considered as LNB, as they were diagnosed with and treated for LNB by their physician. The correlation between variables was investigated by Spearman rank correlation analysis. A s = 0.76, P < 0.001.Overall, the categorical agreement between ReaScan CXCL13 and recomBead CXCL13 over the three CXCL13 concentration intervals was 87%. The reader values following analysis with ReaScan correlated with the CXCL13 concentration received after analysis with the recomBead assay; rThe categorical agreement between the two methods is presented in Table n = 209), was lower for the ReaScan CXCL13 assay (78%) compared with the recomBead assay (86%). However, the specificity of ReaScan CXCL13 was higher (95% vs. 91%), , including 10 patients with tick-borne encephalitis (TBE), the categorical interpretation of the results differed between the methods in six cases . However, samples over the measurement range of the recomBead CXCL13 assay were not further diluted in our comparison, and therefore the correlation may actually have been underestimated.The correlation between the reader values of ReaScan CXCL13 and the concentration of CXCL13 measured using recomBead was strong  if it is not possible to analyse the fresh CSF samples immediately. This observation is supported by the results presented by Hytönen et al. . They deThe time to an available test result using ReaScan CXCL13 was approximately 30 min, whereas the analytical turnaround time (TAT) for recomBead CXCL13 is approximately 4 h. Apart from the gain in TAT for the ReaScan CXCL13 assay, the possibility of immediate single-unit analysis of the samples after arrival to the laboratory, without batching of samples, which in our laboratory may be up to one week, is a major advantage for the ReaScan CXCL13 assay.In conclusion, our results demonstrate that the recomBead and the ReaScan CXCL13 assays show comparable performance in the identification of LNB. The advantages with fewer manual steps and shorter TAT with no batching of samples makes the ReaScan CXCL13 assay an attractive alternative suitable even for smaller laboratories, and the rapid test results may support clinicians in their treatment decisions.Supplementary file1 (DOCX 17.5 KB)Supplementary file2 (DOCX 13.7 KB)Below is the link to the electronic supplementary material.


The evolution of this interface throughout cycling involves multiscale mechanical and chemical heterogeneity at the micro- and nano-scale. These features are dependent on operating conditions such as current density and stack pressure. Here we report the coupling of operando acoustic transmission measurements with nuclear magnetic resonance spectroscopy and magnetic resonance imaging to correlate changes in interfacial mechanics (such as contact loss and crack formation) with the growth of lithium microstructures during cell cycling. Together, the techniques reveal the chemo-mechanical behavior that governs lithium metal and Li All-solid-state batteries are promising alternatives to Li-ion batteries. Here, the authors investigate the chemo-mechanical changes at the lithium metal/solid electrolyte interface via operando acoustic transmission and magnetic resonance imaging. The Li7La3Zr2O12 (garnet-type LLZO) solid-state electrolytes have ionic conductivities comparable to liquid electrolytes2, and are electrochemically stable up to ~3 V vs Li+/Li4. However, further optimization of the Li (s)–LLZO interface is required because lithium propagation through the LLZO electrolyte presents major issues at practical current densities6. Optimization of this interface is challenging because the transient chemical and mechanical mechanisms responsible for lithium microstructural growth in solid electrolytes are not readily observed, especially during battery operation. Apart from lithium filament protrusion on the plating side, prior work has also suggested that lithium stripping initiates a void-forming failure mode8. Poor interfacial contact from voids during stripping (discharge) leads to rate limitations, whereby lithium diffusion and creep towards the interface is unable to keep up with lithium depletion and migration into the electrolyte7. The formation of voids, or gaps between the stripping electrode and the solid electrolyte, leads to an increase in the interfacial resistance and an increase in the measured cell voltage, which we define as voltage polarization7.Solid-state electrolytes for high energy density lithium metal batteries exhibit a variety of improved electrochemical and physical properties compared to their liquid-state counterparts11. For example, Sharafi et al.10 characterized the Li (s)–LLZO interfacial kinetics using electrochemical impedance spectroscopy (EIS) to measure the various contributions to cell impedance as a function of temperature. Cheng et al.6 imaged surface cracks with scanning electron microscopy (SEM) and subsurface cracks with focused-ion-beam milling (FIB-SEM), along with surface chemical analysis with energy dispersive X-ray spectroscopy (EDS). These ex situ characterization techniques require cell disassembly for microscopy or spectroscopy, and provide no information on cell behavior during operation. The complex evolution of the Li (s)–LLZO interface during lithium plating and lithium stripping further benefits from operando characterization for an enhanced mechanistic understanding of coupled contact mechanics and microstructure formation heterogeneity.These hypotheses have primarily been supported by electrochemical analyses and post-mortem optical or microscopic observations12. Yet, operando characterization of the Li (s)–LLZO interface is challenging because of the inherent difficulties associated with probing buried solid–solid interfaces at the relevant length and timescales. In particular, synchrotron X-ray tomography provides high spatial resolution down to less than 50 μm, but X-ray resolution is directly related to elemental mass13. Lithium filaments and voids are, therefore, both X-ray transparent. Operando optical microscopy has been recently developed by Kazyak et al.14 utilizing a custom-designed cell with an optical viewing plane, which brings further visual insight to the evolving dynamics but also has a different cell geometry than the present study. Operando neutron depth profiling and nuclear magnetic resonance (NMR) both overcome the low sensitivity of lithium in the case of X-ray-based operando methods, by probing neutrons and nuclei, respectively18. Various operando characterization techniques for solid-state batteries have been well summarized in recent reviews by Strauss et al., Lou et al., and Tan et al.12,19,20.By observing the cell changes during operation , operando characterization techniques can unveil dynamic interactions within the cell more clearly than post-mortem analysis22. Acoustic transmission utilizes sound waves at ultrasound frequencies  which propagate through the cell layers, with sound speed and amplitude dependent on the mechanical properties of each layer23. Since solid/gas interfaces have poor acoustic signal transmission due to a high acoustic impedance mismatch, even minor contact loss at the stripped Li (s)–LLZO interface is expected to attenuate the acoustic amplitude24. In the presence of a solid/gas interface, propagating sound waves are mostly reflected back. Unlike conventional lithium-ion batteries with liquid electrolytes and phase changing graphite negative electrodes, lithium metal negative electrodes paired with solid-state electrolytes present a different medium for sound propagation. Instead of amplitude and sound speed changes due to bulk phase change, lithium plating and stripping increases the interfacial roughness between the lithium metal and the electrolyte. Contact loss due to lithium stripping forming voids decreases the intensity of the propagated wave, resulting in a loss of sound transmission intensity. This stochastic effect of interfacial roughness has been studied experimentally and computationally in other fields, but the effect of electrochemically induced nucleation, deposition, and stripping on acoustic transmission has not been thoroughly investigated26. Further explanation of the impact of interfacial roughness on acoustic wave transmission is provided in Supplementary Discussion In this work, we combine operando acoustic, NMR, and magnetic resonance imaging (MRI) techniques to probe the coupled relationship between interface mechanics, interphase chemistry, and microstructural formation at the mesoscale and microscale. Operando acoustic transmission can detect changes in cell modulus, density, and expansion in conventional lithium-ion cells27. We exploit this unique chemo-mechanical probe to evaluate the minimum stack pressure required to maintain conformal contact and interfacial stability (or lack thereof) over long voltage polarization steps. In particular, we apply stack pressures from 2 up to 13 MPa during lithium plating and stripping of symmetric Li (s)–LLZO cells to measure the effect of increasing stack pressure on void formation and creep recovery rate with acoustic transmission –LLZO interface and within the electrolyte, which encompasses interfacial voids and growth penetration through the solid-state electrolyte31. The combination of acoustics followed by ex situ MRI furthers understanding of the Li (s)–LLZO interface, because it correlates contact extent due to lithium stripping with lithium growth morphology near the buried interface. Besides the fixed pressure studies, we also design a fixed gap cell . This capability is useful for characterizing the mechanics and microstructure for the same cell under operation, without noise from cell-to-cell variations.Solid-state nuclear magnetic resonance (ssNMR) and MRI complement the acoustic measurements by simultaneously probing chemical composition and microstructure morphology/quantityion Fig. . The lowell Fig.  for cond7La3Zr2O12) electrolyte synthesized from the well-characterized rapid induction hot-press technique is used as a model system (further details in the “Methods” section)32. The resulting LLZO electrolyte pellets have been verified in a previous report for chemical stability, reproducibility, and low defect surfaces from intensive polishing protocols32. For Li/LLZO/Li symmetric cells, the typical galvanostatic charge current used in this study was 0.2 mA/cm2, lower than the critical current density (CCD) of 0.6–0.7 mA/cm2 measured at 2 MPa stack pressure and the CCD of 1.4–1.5 mA/cm2 at 7 MPa stack pressure. These values were validated in the fixed pressure cell with current ramp tests –LLZO interface is known to be unstable and induce Griffith-like crack propagation as first introduced by De Jonghe et al.34 and adopted by Porz et al.5 for LLZO. At current densities below the CCD at the given stack pressure, no lithium penetration within the electrolyte is expected according to Sharafi et al.10 Since practical solid-state electrolytes are designed with a sufficient CCD to cycle stably at relevant current densities, the present work primarily explores interfacial dynamics at values below the CCD. A practical solid-state battery would involve cathode loadings of 3–4 mAh/cm2, implying a 1 C charge and a discharge current rate of 3–4 mA/cm2, and a requirement for the CCD to be at least 3–4 mA/cm2 to mitigate the chance of short circuiting.The garnet LLZO –LLZO interface may be unstable at low stack pressures due to loss of interfacial contact from void formation on the stripped lithium foil side. When the rate of contact loss surpasses the rate of lithium creep, then the rise in overpotential due to loss of electrochemically active surface area may not recover2  increases to around 3000 Ω after the polarization step (orange), decreases back to ~500 Ω after the rest step, and then increases to over 6000 Ω after the second polarization step. The abnormal behavior of the low-frequency regime where a decrease in the real impedance portion is observed is attributed to an unstable interface, such that the electrochemical interfacial area changes within the time constant of the impedance test.Acoustic transmission is used to measure interfacial mechanical changes at the mesoscale in Li/LLZO/Li cells at 2 MPa stack pressure. A stack pressure of 2 MPa resulted in immediate acoustic amplitude attenuation after applying 0.2 mA/cm2 Fig. . This at2 Fig. . The ampmin Fig. . EIS con7Li MRI technique, to measure lithium growth inside the electrolyte ex situ  near the Li (s)–LLZO interface that extends into the solid electrolyte (7Li shift centered at ~0 ppm) after the polarization and repolarization test at 2 MPa. The distance of microstructural growth in the electrolyte is indicated by the position of the lithium microstructure peak on the y-axis of Fig. 7Li CSI shows new peaks at approximately −1 and 8 ppm , a itu Fig. . Lithium7 postulated that a sufficiently high stack pressure should drive lithium creep toward the interface resulting in minimal void formation. They observed, for 0.2 mA/cm2, minimal overpotential increase at stack pressures of 2 MPa and above (with 4 mAh/cm2 charge passed), some increase in overpotential at 1.6 MPa (with 5 mAh/cm2 charge passed), and a significant amount of overpotential increase at 1.2 MPa (6 mAh/cm2). Conversely, Zhang et al.28 presented a contact model taking into account factors other than stack pressure, including lithium elastoplasticity and creep, and found that at least 20 MPa is required for maintaining conformal contact. The assumed lithium metal yield strength of 14 MPa is substantially higher than the conventionally measured ~0.7–0.8 MPa for bulk lithium metal37, and is more in line with measurements conducted at the nanoscale by Greer et al.38. Results from our combined acoustic transmission and 7Li CSI analyses show that 2 MPa stack pressure results in an immediate loss of contact, supporting the hypothesis that the theoretical yield strength of ~0.7–0.8 MPa for bulk lithium is not an accurate indicator for interfacial contact stability   Fig. . While at = 217 h  than the first, and the cell polarized to 5 V in less than 3 h. After cell disassembly, a large crack was visible to the naked eye through the center of the electrolyte, which most likely resulted in the sudden amplitude attenuation event. The crack may have been related to in-plane stress caused by heterogeneous volume change in the electrolyte due to nearly 40 mAh of charge passed in the first step. 7Li CSI reveals the presence of microstructural lithium on both electrodes (250–260 ppm), which is attributed to microscale voids that persist on the stripped lithium side and lithium protrusions near the plated lithium side. At first glance, the 7Li CSI contour plot appears to show a short circuit as indicated by the crossover of the lithium microstructure signal from the plated side to the stripped side . Within the electrolyte, the presence of bulk lithium metal signal (240 ppm) instead of microstructural lithium signal (250–260 ppm) suggests that lithium microstructures adopt a planar morphology within the electrolyte. SEM imaging within the large crack through the middle of the LLZO  at the end of the test –LLZO interface over long durations of charge and throughout a wide range of stack pressure regimes. A relatively low stack pressure of 2 MPa resulted in amplitude attenuation to ~80% of the initial intensity and minimal recovery back to ~82% of the initial intensity during a 25 h rest step, along with significant microstructure buildup. Higher stack pressures of 7.4 and 13 MPa resulted in a mechanically stable interface as measured by the lack of acoustic amplitude attenuation, but still caused microstructure formation and a sudden amplitude attenuation event after passing a significant amount of charge. The enhanced interfacial stability at higher stack pressures results in over 40 mAh of charge passed until cell polarization. On the other hand, a higher current density of 0.5 mA/cm2 may increase the propensity for electrochemical shorting due to local lithium penetration. Lastly, these far-from-equilibrium operating conditions such as high voltage or high current densities may also lead to chemical degradation forming interphase compounds, as corroborated by 7Li CSI analysis of the electrolyte signal and EDS. DFT calculations by Thompson et al.42 suggest an electrochemical stability window of up to 5 V vs Li+/Li. In contrast, other studies have suggested a lower chemical stability window, including electrolyte oxidation to Li2O2, La2O3, Li6Zr2O7 at potentials as low as 3 V vs Li+/Li4. Probing these subtle chemical changes requires high resolution and localized chemical characterization, such as a recent study by Liu et al.39 using HRTEM and EELS that suggested bandgap reduction at LLZO grain boundaries and subtle changes in oxygen concentration. In the present study, the observable but minimal amount of chemical changes in CSI and EDS, along with the wide range of applied stack pressure, suggests that dominant changes are mechanical with some degree of chemical effects. Besides the minor deposition and growth of lithium within LLZO, the vast majority of the deposited lithium occurs at the electrode bulk, as observed by a roughened interface at the plated lithium side in cross-sectional SEM images –LLZO interface; conformal attachment of the copper foil to the LLZO surface is previously described by Wang et al.ppm Fig. . This inppm Fig. . We estippm Fig. .We demonstrate a proof-of-concept synchronized operando setup that can probe both the microstructure and cell mechanics of a buried interface during cycling. This is useful for investigating dynamic cell behavior free from cell-to-cell variations for all types of cell chemistries and materials held at relatively low stack pressure.2 or higher due to continuous void formation on the stripped lithium side, resulting in almost immediate acoustic amplitude attenuation and significant surface roughening. A stack pressure of 7 MPa and higher leads to creep-driven interfacial dynamics but does not indefinitely eliminate microstructure growth. 7Li CSI shows that the lithium microstructure formed within the electrolyte is oriented laterally to the electrodes. A high stack pressure of 13 MPa increases the likelihood of exceeding the local yield stress to the point of fracture and cell shorting. Sudden mechanical changes most likely due to crack propagation are detectable with operando acoustic transmission. In all cases, both electrochemical and acoustic signals show hysteresis despite a long OCV hold, indicating the lack of complete interfacial stability. The large swings in impedance ranging from short circuit to >6000 Ω indicate that the primary failure modes are mechanically driven due to the wide range of operating stack pressures, but local chemical heterogeneities may also be influential to some degree when driven to high overpotentials, and may be further studied with spatially resolved chemical analyses. A proof-of-concept synchronized operando acoustic/ssNMR characterization approach is used to measure both interfacial and microstructural changes at the same time. The non-invasive, non-destructive, and complementary usage of both acoustic transmission and ssNMR can be generalized for dynamically characterizing both mechanics and microstructural heterogeneity of other buried electrode and solid-state electrolyte interfaces. The operando chemo-mechanical data gathered at various stack pressure regimes can be incorporated into existing contact models, and further point toward the need for low stack pressure operation given the intrinsic degradation modes that are observed to arise at high stack pressures and long durations of charge passed.The results herein present a multimodal and dynamic mechanistic analysis of the Li (s)–LLZO interface for solid-state batteries. Changes in both interfacial contact and microstructural growth are characterized in operando using complementary acoustic transmission and ssNMR/CSI. The results indicate that a stack pressure of 2 MPa is insufficient for maintaining a conformal Li (s)–LLZO interface at 0.2 mA/cm32. Pellets were cut on a diamond saw to a thickness of ~1.3 mm for Li/LLZO/Li cells and ~2.0 mm for Li/LLZO/Cu cells. The LLZO pellets were polished with sandpaper (from 600 grit and progressively finer to 1200 grit) and a ~0.1 μm diamond paste, stored in an Argon glovebox  and heat treated at 400 °C for 3 h to remove Li2CO3 surface contamination. For Li/LLZO/Li cells, lithium metal foil  was pressed on both sides of the polished pellet surface under a load of 3.2 MPa and then heated at 170 °C under a load of ~1 MPa for optimal interfacial contact. Pressure was applied with a custom-built cell fixture using a spring gauge to control the applied load and an insulated hot plate to apply heat. For Li/LLZO/Cu cells, the metal current collector  was laminated to the polished LLZO surface by applying a pressure of 0.8 MPa at 900 °C for 5 min43. Battery-grade copper (10 μm) foils were purchased from Targray and used as current collectors. EIS and voltage polarization tests were conducted with a potentiostat (Gamry Instruments 3000). EIS was conducted in potentiostatic mode from 7 MHz to 500 mHz with 120 mV amplitude and 14 points per decade. The fixed pressure cell tests were conducted in a temperature chamber (Neware) at 30 °C.LLZO pellets were synthesized by rapid induction hot-pressing with details referenced in previous literature2, leading to delamination of the stripping side but an intact interface at the plating interface.SEM imaging was conducted after cell disassembly (Zeiss Sigma VP SEM). The tabs were removed, and the cell was placed on a sample stub within an Argon-filled glovebox. The stub was transferred to the SEM in transfer vessels to prevent exposure to ambient atmosphere. Most cycling conditions resulted in an exposed LLZO surface on the stripped side, either due to delamination at low stack pressures or complete stripping at high stack pressures. This exposed LLZO surface was then imaged at 12 kV. EDS was conducted with the attached Bruker EDS at 12 kV. SEM/EDS cross-sectional images and chemical analyses were conducted by cross-sectioning the cell with a stainless steel blade and mounting on a 90° angle on the stainless steel SEM stub. For example, Supplementary Fig. https://github.com/dansteingart/.22Two wide-band ultrasound transducers (Olympus) with central frequency of 2.25 MHz and 10 mm beam diameter were fixed in place within a double-piston pneumatic cylinder and spacers made of cross-linked polystyrene . The pneumatic cylinder is actuated with a solenoid controlled with an Arduino to apply constant pressure, and the spacers act as both force distribution plates as well as a fixed distance for acoustic transmission beyond the near-field. Acoustic signals were produced with an ultrasonic pulser  in transmission mode with 300 V impulse spikes of pulse width corresponding to the central frequency of the transducer, and measured with a high-resolution oscilloscope sampling at 1 GS/s (Picoscope). The physical hardware was controlled with Linux-based isolated and virtualized software operating systems (Docker) to ensure reliable and accurate fast data collection. Detailed explanation of the setup and technique is described elsewhere by Chang et al. and open source code can be found at 7Li ssNMR and CSI measurements were performed on a Bruker 400 Avance NEO spectrometer operating at 9.4 T (7Li frequency = 155.45 MHz) equipped with a 25 mm 1H/7Li Bruker MICWB40 probehead and a triple axis micro2.5 gradient system. 7Li chemical shifts were internally referenced to LLZO at 2.3 ppm. Prior to analysis, cells were sealed in an airtight vial under argon and oriented with the lithium metal electrodes perpendicular to the external magnetic field, B0.All 7Li CSI measurements were performed using a single phase-encoded spatial dimension, along z (B0), as reported previously44. After a 90° r.f.   pulse applied on the 7Li channel (34.6 μs at 200 W), a phase-encoding gradient was applied along the z-axis followed by a 180° r.f. pulse with a recycle delay of 0.5 s. One-dimensional 7Li ssNMR measurements were performed using a 90° pulse length of 78.9 μs at 30 W.matplotlib, with a separate package nmrglue used to interface between Topspin and Python.All NMR data were acquired and processed using Bruker Topspin 4.1.1 software. Data were plotted using the Python 3 package 2 < 0.5 ppm, H2O < 0.5 ppm) with thin strips of battery-grade copper mesh current collector  instead of nickel foil to minimize the amount of metal used. The ultrasonic transducers were attached to double-shielded type RG314 cables, and shielded electrochemical cables were attached onto the protruding copper mesh current collectors by using a minimal amount of tin solder between the exposed wiring and the edge of the copper mesh. The cell potential was checked with a multimeter to ensure good contact was achieved, before the operando tests. Typically, loss of electrochemical contact was due to tightening the cell threads and tearing the copper mesh foil inside the cell holder. Both the ultrasonic and the electrochemical cables were connected outside the magnet to a gold shielding plate with 15 MHz low-pass filters before connection to the corresponding instruments as a precaution, though we did not observe significant changes to the signal-to-noise with or without the filters. The primary change in reducing the signal-to-noise was threading the cabling on the bottom electrode down through the magnet, and the cabling connected to the top electrode up through the top of the magnet, such that no cabling crossed the electrolyte area.Two wide-band ultrasonic transducers (Olympus) with central frequency of 2.25 MHz and 10 mm beam diameter were firmly epoxied on either side of a machined polytetrafluroethylene (PTFE) cell holder, with acoustic gel couplant (Sonogel) placed between the transducer face and the PTFE cell surface Fig. . The fab7Li experiments are performed using a r.f. pulse power of 200 W to maximize sensitivity, we found that strong r.f. fields (>50 W) interfered with acoustic signal collection, causing intermittent noise or entirely disconnecting the acoustic pulser45. Resetting the r.f. pulse sequence with the transducers inside the magnet also appeared to interfere with acoustic data collection, and care was taken to start acoustic pulsing before moving the cell holder into the magnet. A custom script was used to continuously pulse acoustic waveforms at regular time intervals , and then read waveforms from the oscilloscope; starting the continuous pulse sequence while outside the magnet allowed for stable and uninterrupted acoustic data collection. Reducing the r.f. pulse power to 30 W also allowed for stable and uninterrupted acoustic data collection and NMR sensitivity sufficiently high for quantitative analysis. To clarify the order of each step, here is an example: the acoustic pulse sequence was started at t = 0 h with the cell inside the magnet probe and began to collect one averaged waveform every second, the probe was placed inside the NMR magnet at t = 0.1 h, the NMR pulse sequence was started at t = 0.5 h and began to collect one signal-averaged 1D NMR experiment every 17 min, and finally the electrochemistry was started at t = 5 h after some initial acoustic signal equilibration.While typical ex situ Supplementary Information