We aimed to investigate the association of circulating retinol-binding protein-4 (RBP4) levels with long-term cardiometabolic risk profiles and whether sex disparity mattered.We included 784 non-diabetic participants aged 40 years and above from a well-defined community-based cohort at baseline in 2005 and they were invited to attend the on-site follow-up examination for two consecutive times with 3-year intervals in 2008 and 2011, respectively. Serum RBP4 was measured at baseline, and the anthropometry and biochemical measurements were performed at each visit. Generalized estimating equation models were used to assess the association of serum RBP4 levels with the dynamic changes in adiposity and glucolipid profile.10RBP4) were significantly associated with waist circumference , fasting, and 2-h post-loading glucose , serum triglycerides , total cholesterol , P<0.0001), and marginally with body mass index , P=0.046], in total participants, after adjusting potential confounders. The association of RBP4 with 2-h post-loading glucose was stronger in women than that in men . The analysis of change in Z-score of cardiometabolic profiles corresponding to each 1-unit increment in log10RBP4 showed consistent results.Based on all the baseline and the 3- and 6-year follow-up data, baseline serum RBP4 levels (each 1-unit of logHigher RBP4 levels are associated with longitudinal increase in adiposity and deteriorated glucolipid profile defined by repeated measurements. The associations differ in sex regarding to the 2-h post-loading glucose. Retinol-binding protein 4 (RBP4), a retinol transporter in circulation belonging to the lipocalins family , has beeSexual dimorphisms in cardiometabolic disease features including prevalence, progression, and outcome were reported in the epidemiological and clinical setting. Recently, sex difference has been reported to play a pivotal role in the pathogenesis of several cardiometabolic disease –13. A reIn current longitudinal cohort study with metabolic traits repeatedly measured and followed up for two consecutive times with 3-years’ interval, we investigated the association of baseline serum RBP4 with long-term dynamic metabolic profile changes including adiposity, blood glucose, and lipid metabolism. Meanwhile, we lay a particular emphasis on sex disparities in the relationship between baseline RBP4 levels and the metabolic profiles.The study participants were from a two-step blood glucose survey started at 2004, performed in an urban community of Shanghai. The study protocols were approved by the Institutional Review Board of the Ruijin Hospital, and written informed consent was provided by each participant. The detailed study design and protocols of this community-based cohort have been described in previous literature , 15. AllAll the participants were invited to attend the follow-up visits for two consecutive times with a 3-year interval. Similarly, a 75-g OGTT and blood sampling were also performed in the subsequent follow-up visits. There were 1267 participants without diabetes at baseline, 918 and 784 individuals attended the follow-up visit and with blood sampled in 2008 and 2011, respectively. For the present study, 784 individuals without diabetes at baseline and attended the baseline and the two follow-up visits during a period over 6 years were included in the final analysis. A flow chart showing the study procedure is shown in Information on sociodemographic characteristics, lifestyle, disease, and medication history was collected by questionnaire at baseline and the subsequent visits. Current smokers or drinkers were defined as individuals who smoked or drank regularly in the past 6 months. Anthropometry traits like body height, body weight, and waist and hip circumferences were measured by well-trained examiners, when participants were dressed in light clothes and with bare feet. Body mass index (BMI) was calculated as body weight in kilograms divided by height squared in meters. Blood pressure was measured at the right arm three times consecutively with 1 min intervals after at least 5 min of rest in the seated position, with an automated electronic device .Overnight fasting and 2-h post glucose-load plasma glucose concentrations and fasting serum concentrations of triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol, uric acid, and creatinine were measured using an automatic analyzer . Serum insulin concentrations were measured by radioimmunoassay . The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as the following formula : HOMA-IRSerum RBP4 levels were measured at baseline in duplicate by enzyme linked immunosorbent assay developed in-house, using polyclonal and monoclonal antibodies generated against recombinant human RBP4 protein and purified using affinity chromatography . The assContinuous variables in normal distribution were reported as means ± SDs, while the skewed ones are presented as medians (interquartile ranges). Categorical variables are presented as frequencies (%). The comparison of the metabolic traits for the three measurements at baseline, 3-year, and 6-year follow-up was conducted by using the analysis of covariance after adjustments for age and sex.Generalized estimating equations (GEE) model, accounting for correlation over time instead of assuming independence, is often used to analyze longitudinal data . The GEETo further detect whether sex affects the association between serum RBP4 levels and plasma glucolipid profile, we performed a subgroup analysis according to men vs. women and tested the significance of the multiply interaction with sex. Metabolic traits were also standardized to Z score to facilitate comparison of RBP4 effect sizes.P values of less than 0.05 were considered statistically significant for all the analyses. SAS software version 9.4  was used to perform statistical analysis.The general characteristics of 784 participants who attended three visits are summarized in The median RBP4 level at baseline was 16.4 (inter-quartile range 12.1-21.7) ug/mL. Notably, women had lower serum RBP4 levels compared with men .2  increment of BMI and 3.12 cm increment of waist circumference . Additional adjustment for BMI, smoking status, and drinking status did not change the associations between baseline serum RBP4 levels and fasting plasma glucose, OGTT 2-h plasma glucose, serum triglycerides, and total cholesterol concentrations. When further adjusted for anti-diabetes drug usage in glucose metabolism traits or lipid-lowering drug usage in the lipid profile (model 3), RBP4 was significantly associated with fasting and 2-h post-loading glucose , serum triglycerides , and total cholesterol . We did not find any significant associations between serum RBP4 levels and fasting insulin, HOAM-IR, HOMA-β, LDL cholesterol, and HDL cholesterol.The associations between baseline serum RBP4 levels and adiposity indexes, glucose metabolism traits, and lipid profile are presented in P values for significance between RBP4 and metabolic traits were presented in To track the change in association between RBP4 and metabolic traits, multiple linear regression analyses were performed at baseline and two follow-up visits. The dynamic 10-RBP4) were significantly associated with waist circumference in men  but not in women  (P for interaction=0.31). Baseline serum RBP4 was significantly associated with OGTT 2-h plasma glucose in women  but not in men  (P for interaction=0.001). Both in men and in women, baseline serum RBP4 was significantly associated with serum triglycerides and total cholesterol (both P ≤0.01).For adiposity indexes, Z score standardized analysis revealed a little bit stronger association of baseline serum RBP4 levels with waist circumference than BMI Table 4.In the present study, we systematically assessed the association of baseline RBP4 levels with a longitudinal dynamic cardiometabolic profiles which were evaluated at baseline and two consecutive times of follow-up with 3-years’ interval. We found that in the middle aged and elderly population without diabetes at baseline, baseline higher RBP4 levels were associated with increased BMI, waist circumference, fasting and OGTT 2-h plasma glucose triglyceride, and total cholesterol. Furthermore, sex disparities matter in the relationship of RBP4 with OGTT 2-h plasma glucose, in which RBP4 was associated with OGTT 2-h plasma glucose in women, but not in men.Elevated circulation RBP4 levels were identified in obese subjects. However, RBP4 levels were associated with BMI in few epidemiological studies. Instead, more studies indicated RBP4 levels were positively associated with visceral body fat or abdominal fat mass –21 in inIn the current study, we found plasma glucose concentrations in non-diabetes were associated with RBP4 levels as expected. Interestingly, OGTT 2-h plasma glucose concentrations had a more robust association with RBP4 levels than fasting plasma glucose concentrations, which might suggest OGTT 2-h plasma glucose as one main contribution through which increased RBP4 levels confer a higher risk of type 2 diabetes. It has been reported that OGTT 2-h plasma glucose level was regulated by intestine-derived incretin hormone besides glucose-stimulated insulin secretion . IntestiRare literature reported sexual difference in the association between baseline RBP4 levels and glucose metabolism . In the Sex hormones have pronounced effects on body composition, adipose tissue mass distribution, and metabolic homeostasis. Men have relatively more energy storage in the abdominal and intra-abdominal adipose tissue depots, while premenopausal women store the excess energy in subcutaneous adipose tissue , 30. ThiIn this study, there was no association for RBP4 and fasting insulin levels, HOMA-IR, or HOMA-β. These discrepancies within literature may lie in several aspects including that the study populations varied widely in study type, sample size, health condition, and ethnicity. Several studies also declared null association between RBP4 and insulin resistance –33. AnotRBP4 has also related to an unfavorable lipid metabolism , 35–37. The main strength of this study is the longitudinal design with systematic and repeated monitoring for metabolic traits. Systematic metabolic traits including adiposity index, glucose metabolic traits, and blood lipid profile made it practicable to get a comprehensive knowledge about the association of RBP4 on cardiometabolic risk profile. Besides, the repeated measurements of metabolic traits also reduced the bias derived from a single cross-sectional survey. Another strength is the novelty of the current study, as we identified the sexual difference in the association between RBP4 levels and metabolic traits, especially for the 2-h post-loading glucose, which implies an import issue of sex disparities in the disease pathophysiology.Severe limitations should be acknowledged. First, serum RBP4 was not repeatedly measured at follow-up, which prohibited us to track a long-term trajectory of circulating RBP4 levels. One previous study aimed to assess the biological variation of adipokines RBP4 within individuals by measuring the level of RBP4 twice, approximately 4 months apart . The intIn conclusion, the baseline serum RBP4 level is robustly associated with a longitudinal profile of waist circumference, fasting and OGTT 2-h plasma glucose, triglyceride and total cholesterol, and marginally associated with BMI. The association of RBP4 levels on metabolic traits differ in sex, with stronger association with OGTT 2-h plasma glucose level in women. The results imply an import issue of sex disparities in the disease pathophysiology. Further experimental investigations are required to better understand the molecular mechanisms underlying the sex-specific deterioration of metabolic traits due to elevated RBP4 levels.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Each patient/participant provided the written informed consent to participate in this study.MX, YB, and JW contributed to the concept and design. JX, HD, YH, and QW contributed to the analysis of data. JX drafted the manuscript. MX edited the manuscript. TW, ML, ZZ, JL, MD, DZ, YX, GN, WW, YB, and MX contributed to collecting and the acquisition of data. All authors made important contributions to critically revising the manuscript for important intellectual content. MX guarantees this work, has full access to the data, and takes responsibility for the integrity of the data. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Science Foundation of China , the Shanghai Municipal Education Commission–Gaofeng Clinical Medicine Grant Support [grant number 20152508 Round 2], the Shanghai Shenkang Hospital Development Center . T.W., M.L., Z.Z., J.L., Y.X., W.W., G.N., J.W., Y.B., and M.X. are members of the Innovative Research Team of High-Level Local Universities in Shanghai.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Results. The study revealed minimal troponin levels in GDM, and healthy groups equal to 0.02 ng/mL and 0.028 ng/mL, respectively. The GDM group is defined by an interquartile range of hs-TnT higher than the healthy group. This study confirms previously reported upper levels of troponin in healthy children. There are possible health problems that can appear during infancy and influence the further development of a child affected by GDM.This study is aimed at testing the hypothesis that serum analysis of high-sensitivity troponin T in neonates may serve as a diagnostic tool to predict the risk of gestational diabetes mellitus (GDM). 86 newborns were studied and stratified into two groups: healthy group; newborns with body weight ≥ 10th percentile, born in good condition (APG 8-10pts) of pregnancy not complicated by diabetes, and the GDM group; neonates born to mothers with type 1 or type 2 diabetes.  Gestational diabetes mellitus (GDM) is one of the most common complications during pregnancy. Both gestational diabetes and pregestational diabetes are responsible for several complications in the perinatal and neonatal periods. Therefore, early diagnosis of GDM and early treatment are the main elements of prevention of complications such as birth defects , macrosoThe troponin complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). In cardiac and skeletal muscles, troponin C binds calcium that coordinates the Ca2 + -regulatory system of the muscle, troponin T controls interactions between actin and myosin, and troponin I exerts inhibitory properties. Troponins I and T have been found mainly in cardiac muscles, whereas Troponin C is present in cardiac and skeletal muscles . CardiacThe study was carried out according to the Declaration of Helsinki of the World Medical Association (WMA) . The stuN = 86 newborns were studied and stratified into two groups: 52 healthy , that is, newborns with body weight ≥ 10th percentile, born in good condition (APG 8-10pts) from pregnancy not complicated by diabetes, and 34  born to mothers with type 1 or type 2 diabetes. The statistical description of the study group is shown in In the study, we used data from blood examinations of newborns whose mothers were hospitalized in the Department of Neonatology, Department of Obstetrics, Feminine Diseases, Gynecology Oncology, and Regional Hospital Bródnowski, Warsaw, Poland. A total of Heparinized plasma of blood from the neonates was collected by venepuncture in a tube with lithium and heparin. The collected samples were chilled to 4°C and centrifuged at 1500 rpm, and the serum was collected. The serum collected was stored at -20°C until laboratory tests were performed.The concentration of cardiac troponin T hs was measured using the Cobas Elecsys assay in neonatal blood serum on a Cobas 8000 analyzer. The assay consisted of two steps: in step 1, a complex was incubated, for which a biotinylated antigen was used with a monoclonal antibody specific for cardiac troponin T, and a monoclonal antibody specific for cardiac troponin T that was labeled with a ruthenium complex. In step 2, incubation with particles coated with streptavidin. In this step, the complex is bound to the solid phase as a result of the affinity of biotin and streptavidin.The mixture was then transferred to a measuring chamber in which the tropomyosin T concentration was measured by the electrochemiluminescence level. The tropomyosin concentration was expressed in ng/mL.All statistical analyses were performed using the R package . The norSince there is a lack of statistical differences in studied parameters between the genders and neonates, the results are analyzed only as a function of the experimental group, that is, GDM—children born from diabetic mothers and neonates born from healthy mothers. The statistical description of the groups studied is shown in The box-plot representation of troponin levels stratified by study group is shown in Analysis of the current literature indicates that an increase in cardiac troponin T and I (cTnT and cTnI) is not limited only to myocardial infarction . For exaThere is a difference between the median troponin levels in healthy neonates reported in this study (0.12 ng/mL) and those reported by Trevisanuto et al.  (0.17 ngHowever, the results of this study confirmed the previous study that indicated that hs-cTnT levels do not conform to a Gaussian distribution in children older than two days . FurtherIdentifying marker that allow for the prediction of the health of newborns is of paramount importance from a medical point of view. This study tested the hypothesis of the potential diagnostic applicability of serum troponin levels in neonates. The observed differences, although statistically insignificant, between healthy newborns and those born to diabetic mothers can be indicative of possible health problems that can appear during childhood and may influence the future development of a child. However, follow-up studies are required to verify this hypothesis.


In univariable analysis, only higher tTau was significantly correlated with poor neurocognitive performance . Among PWoH, no CSF biomarkers were significantly associated with worse NCI. Predicted residual error sum of squares (PRESS) analysis showed no evidence of overfitting. Poorer neurocognitive performance in aging PWH was associated with higher CSF tTau, a marker of age-related neuronal injury, but not with biomarkers of amyloid metabolism. The findings suggest that HIV might interact with age-related neurodegeneration to contribute to cognitive decline in PWH.We evaluated whether biomarkers of age-related neuronal injury and amyloid metabolism are associated with neurocognitive impairment (NCI) in people with and without HIV . This was a cross-sectional study of virally suppressed PWH and PWoH. NCI was assessed using a validated test battery; global deficit scores (GDS) quantified overall performance. Biomarkers in cerebrospinal fluid (CSF) were quantified by immunoassay: neurofilament light (NFL), total Tau (tTau), phosphorylated Tau 181 (pTau181), amyloid beta (Aβ)42, and Aβ40. Factor analysis was used to reduce biomarker dimensionality. Participants were 256 virally suppressed PWH and 42 PWoH, 20.2% female, 17.1% Black, 7.1% Hispanic, 60.2% non-Hispanic White, and 15.6% other race/ethnicities, mean (SD) age 56.7 (6.45) years. Among PWH, the best regression model for CSF showed that higher tTau (The online version contains supplementary material available at 10.1007/s13365-022-01081-4. As people with HIV (PWH) age on virally suppressive antiretroviral therapy (ART), they become susceptible to age-related neurodegenerative disorders such as Alzheimer’s disease (AD). The biomarker phenotype of AD as compared to HIV-associated neurocognitive disorders (HAND) is distinct, supporting differences in the underlying pathophysiology. For example, one study reported that cerebrospinal fluid (CSF) total Tau (tTau) was increased in a group with AIDS dementia complex (ADC) compared to unimpaired PWH and younger PWoH, but lower than in an Alzheimer’s disease comparison group  may contribute to altered APP processing. Thus, combination ARV treatment of SweAPP N2a cells increased Aβ generation and markedly inhibited microglial phagocytosis of FITC-Aβ1-42 peptides in murine microglia  Tau, and CSF Aβ42. Results are mixed, with some studies finding increases in PET amyloid ligand uptake  and at academic centers in southern California participating in the California NeuroAIDS Tissue Network (CNTN). Inclusion criteria were PWH with viral suppression and PWoH; all underwent lumbar puncture. Exclusion criteria were active neurological illnesses other than HIV and active psychiatric or substance use disorders that might interfere with completing study evaluations. Individuals with non-HIV-related neurocognitive confounding conditions likely to explain neurocognitive impairment were excluded using a previously described method  to 5  and then averaged across tests to obtain a Global Deficit Score (GDS)  who answered standardized questions on the impact of cognitive impairment on activities of daily living were classified with respect to the HIV-associated neurocognitive disorders (HAND) system  was used to measure biomarkers in CSF, including neurofilament light (NFL), total tau (tTau), phosphorylated Tau 181 (pTau181), amyloid beta (Aβ42), and Aβ40. For NFL, the assay used the Quanterix NFLight Advantage kit  at the manufacturer’s recommended 1:100 dilution for CSF. Aβ42, Aβ40, and tTau  to reduce platform time at room temperature to < 3 h to minimize Aβ42, and Aβ40 loss ; for tTau, Aβ42, and Aβ40 the variability averaged < 7% CV, 4% CV, and 7%CV, respectively; and for pTau181 the variability averaged < 4% CV. The intra-assay variability of the duplicate measurements averaged < 20% CV . Lower and upper limits of quantitation for CSF assays were: NFL 68.6, 40,000 pg/mL; tTau 8.8, 575 pg/mL; Aβ42 16.8, 3624 pg/mL; Aβ40 60, 9520 pg/mL; pTau181 4.8, 352 pg/mL. We also calculated ratios of Aβ42:Aβ40, tTau:Aβ42, and pTau:Aβ42.t-tests, and Wilcoxon tests, as appropriate. Biomarker values were log10-transformed to improve the normality of their distributions for parametric analyses. Univariable associations between GDS and biomarker levels were analyzed using Pearson r values. Interactions between HIV serostatus and biomarker levels were examined. Confounds known to influence the selected biomarkers or neurocognitive outcomes were evaluated using multivariable modeling, and included demographics. Multivariable associations used stepwise, mixed regression (p to enter/exit 0.1). To assess the possibility that the final selected model was overfit, we calculated the predicted residual error sum of squares (PRESS). Analyses were repeated while stratifying by HIV serostatus. Secondary analyses related the biomarkers and their ratios to potential confounds . Analyses were conducted using JMP Pro® version 15.0.0 .Participant characteristics were summarized using means, standard deviations, percentages, p = 0.0002).Participants were 256 virally suppressed PWH and 42 PWoH, 20.2% female, 17.1% black, 7.1% Hispanic, 60.2% non-Hispanic white, and 15.6% other race/ethnicities, mean (SD) age 56.7 (6.45) years. Among PWH, median (interquartile range [IQR]) current and nadir CD4 + T cells were 458 cells/μL  and 57 cells/μL , respectively; all took ART and were virally suppressed. PWH and PWoH were demographically similar , and showed a trend for PWoH . Older age correlated with higher CSF tTau for both PWH  and PWoH . Older age significantly correlated with higher CSF pTau181 values in PWH , while factor 1 was not . Among the biomarkers comprising factor 2, both higher levels of tTau and NFL were each significantly correlated with worse neurocognitive performance  while pTau was not   had higher CSF NFL and tTau values and CSF tTau:Aβ42 ratios than NP normal . The three HAND subgroups had similar biomarker and ratio levels for all other biomarkers. PWH with HAND had higher values than those without, and the HAD subgroup had the highest values.As shown in Table Supplementary Table 4 shows that, for all participants, each of the CSF biomarkers was strongly positively correlated with the others.r = 0.303, p = 8.82e-7), CSF tTau , and CSF pTau181 , but not CSF Aβ42  or CSF Aβ40 , but not the other biomarkers. Among PWH, sex was not significantly related to any of the CSF biomarkers (ps > 0.20). None of the biomarkers was significantly associated with ethnicity or education (ps > 0.05).Among PWH, increasing age was not related to neurocognitive performance. Among PWH, increasing age correlated with higher CSF NFL , but was not related to the other biomarkers or biomarker factors. Duration of ART was not significantly related to GDS . The most common regimen type taken by participants was a combination of a protease inhibitor (PI) with 2 nucleoside reverse transcriptase inhibitors (NRTIs), comprising 40.3% of the total. Additional regimen types, in order of frequency, were: a non-nucleoside NRTI plus 2 NRTIs (23.6%), a 3-class regimen of a PI an NNRTI and two NRTI (21.5%), an integrase inhibitor (II) with two NRTIs (9.87%), a PI plus an II (0.0858%), a PI with an NNRTI (0.0858%), and a regimen with a PI, an NNRTI, an II and 2 NRTIs 3.00%).Among PWH, GDS was not related to any of the following common comorbidities: hypertension, diabetes, hyperlipidemia, cardiac disease, tobacco smoking, hepatitis C virus serostatus, chronic pulmonary disease, myocardial infarction, renal disease, congestive heart failure, or head injury. Biomarkers and biomarker ratios also did not relate to any of these comorbidities. All PWH took ART and were virally suppressed at the time of assessment. The median (IQR) total duration of exposure to ART was 108  months. Longer duration of ART exposure correlated with lower CSF Ab42 levels  together with lower pTau181  best-predicted poor neurocognitive performance. In univariable analysis, only higher tTau was significantly correlated with poor neurocognitive performance . Age was not significant in an adjusted model (p = 0.293). To evaluate goodness of fit and possible overfitting, we compared the predictive R2 (0.0401) to the model R2 (0.0267). The similar values indicate no evidence of overfitting.Among PWH, the best regression model showed that higher tTau (p = 0.225). Together, both HIV serostatus and CSF tTau were significant .In PWoH, the multivariable model found that no CSF biomarkers were significantly related to neurocognitive performance. In a multivariable model, the interaction of HIV serostatus with CSF tTau was not significant  in CSF in virally suppressed, cognitively unconfounded PWH, but not in PWoH. No correlations were found with biomarkers of amyloid metabolism, though a caveat here is that the number of PWoH was relatively small, limiting statistical power. These results highlight the emerging role of age-related neuronal injury in the aging population of PWH. As anticipated based on prior studies, we found higher levels of CSF NFL, a marker of axonal injury, correlated with poorer neurocognitive performance in PWH.With the rapidly expanding intersection of long-term HIV and age-related neurodegenerative disorders, the field is increasingly interested in characterizing how, and to what extent, neurodegenerative disorders might contribute to the persisting high prevalence of neurocognitive impairment in HIV Below is the link to the electronic supplementary material.


Key proteins involved in cardiac dyad function are bridging integrator 1 (BIN1), junctophilin 2 and caveolin 3. The work presented here aims to reconstruct the evolutionary history of the cardiac dyad, by surveying the scientific literature for ultrastructural evidence of these junctions across all animal taxa; phylogenetically reconstructing the evolutionary history of BIN1; and by comparing peptide motifs involved in TT formation by this protein across metazoans. Key findings are that cardiac dyads have been identified in mammals, arthropods and molluscs, but not in other animals. Vertebrate BIN1 does not group with members of this protein family from other taxa, suggesting that invertebrate BINs are paralogues rather orthologues of this gene. Comparisons of BIN1 peptide sequences of mammals with those of other vertebrates reveals novel features that might contribute to TT and dyad formation. The analyses presented here suggest that the cardiac dyad evolved independently several times during metazoan evolution: an unexpected observation given the diversity of heart structure and function between different animal taxa.Cardiac dyads are the site of communication between the sarcoplasmic reticulum (SR) and infoldings of the sarcolemma called transverse-tubules (TT). During heart excitation–contraction coupling, CaThis article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. This isThe current review will focus on the evolution of a subcellular domain involved in efficient excitation–contraction coupling (ECC) in mammalian cardiomyocytes: the dyad junction. This will involve examination of ultrastructural and molecular evidence for the presence of these membrane contact sites among distinct metazoan  groups. This information will be used to gain insights into the evolution of cardiac dyad junctions.. 2The evolution and comparative physiology of cardiac ECC has been extensively summarized in previous reviews ,51. The  (a)special issue, dyad junctions of mammalian cardiomyocytes are the interface between TT of the sarcolemma and terminal cisternae of the sarcoplasmic reticulum (SR). These membrane contact sites contain voltage-sensing L-type Ca2+-channels  in the TT and type 2 ryanodine receptor Ca2+-release channels (RyR2) in the SR, together forming Ca2+-release units (CRUs). During ECC, sarcolemmal depolarization causes a conformational change in the LTCC, gating its channel and allowing Ca2+-influx, which is amplified by opening of RyR2, in a process termed Ca2+-induced Ca2+-release (CICR), 2+-release overcomes buffering to allow the Ca2+ to spread from the dyadic nanodomains to the bulk sarcoplasm, activating the contractile apparatus, leading to force production. Similar junctions are present in mammalian skeletal muscle but are triadic (two TC-SR contacts per TT); possess a distinct type of LTCC (α1S or Cav1.1); and Ca2+-release channel (RyR1); and operate by a conformational coupling mechanism, rather than by CICR. Within mammalian cardiomyocytes, peripheral couplings between the sarcolemma and SR also contain CRUs and contribute to ECC. The mechanism of dyad formation is uncertain, but probably involves invagination of the sarcolemmal and recruitment of addition TT membranes, either pre-coupled to the SR or delivered independently of it [Danio rerio (zebrafish) skeletal muscle, genetic manipulations, microscopy and mathematical modelling indicate that TT are formed by an endocytic capture mechanism [As described in detail in other articles in this ly of it . In Daniechanism .The ultrastructure of the mammalian TT system varies between species ; during Mus musculus, [Eidolon helvum, [Oryctolagus cuniculus, [Balaena mysticetus, [Elephas maximus, [Felis catus [Canis lupus familiaris, [Sus scrofa, [Equus caballus, [2+ release of large magnitude is triggered by a small influx of through LTCCs in the ventricles of the common shrew, Sorex araneus [Didelphis virginiana, a marsupial. In Di. virginiana, it was noted that TT were first detectable at postnatal day 43 and was fully developed by day 105 (fully weaned joeys) [Platypus and Echidna.Ultrastructural evidence for the presence of cardiac dyads has been reported in most orders of mammals. Among eutherian mammals, these include rodents ; bats ; lagomoniculus, ); cetaceticetus, ); probosmaximus, ); carnivis catus  and dog iliaris, ); artiod scrofa, ) and peraballus, ). The pr araneus  supporti (b)Columba livia) [Amazilia tzacatl) [Gallas gallas) and the zebrafinch [2+ buffering by several mechanisms [2+ entering via LTCCs and released from peripheral SR only needs to diffuse short distances to activate the contractile machinery. The ventricular myocytes of zebrafinch and chicken possess extensive peripheral SR and extra-junctional or corbular SR, enriched in RyR channels [2+ signals deep within these cells via a CICR mechanism [2+ accumulating capabilities of SR from the atria and ventricles of Japanese quail (Coturnix japonica) exceed those of their mammalian counterparts, supporting this CICR ‘relay station’ role [Neither TT nor dyads have been detected in the heart tissues of diverse avian species, including in pigeon (a livia) ; the ruftzacatl) ; the chibrafinch . In termchanisms . Avian cchannels . Corbulaechanism . The Ca2on’ role , which fTrachemys scripta elegans) [Varanus exanthematicus) [Amyda sp. [V. exanthematicus normal ECC appears to occur predominantly through Ca2+ influx through the LTCCs. During times of greater demand, such as stimulation by cathecholamines, increased Ca2+ influx through the reverse mode of sodium–calcium exchangers (NCX) and enhanced CICR underpin enhanced ECC [Fossil evidence indicates that birds diverged from other theropod dinosaurs during the Jurassic Period, 165–150 Myr . Unlike elegans) , in snakelegans) , nor in maticus) . Howevermyda sp. . Whethernced ECC .Rana pipiens, are very narrow (less than 5 μm in diameter) and are devoid of TT [Bufo marinus, about two-thirds of the Ca2+ signal elicited by depolarization is owing to Ca2+ release from the SR. This CICR mechanism requires Ca2+ influx, largely via LTCCs and by NCX transporters operating in reverse mode [2+ influx to trigger CICR from the SR.Despite their importance in the history of scientific discovery of ECC in skeletal muscle, there are few reports describing the ultrastructure of the myocardium in amphibians. Ventricular myocytes from the northern leopard frog, id of TT . In sinurse mode . These oPleuronectes platessa) [Oncorhynchus mykiss) [Gadiculus thori, fourbeard rockling, Enchelyopus cimbrius, and the haddock, Melanogrammus aeglefinus) [Esox lucius) and mackerel (Scomber scombrus) [Cyprinus carpio) [Clarias gariepinus) [Thunnus alalunga) and the Pacific bluefin tuna  [Da. rerio) [2+-storage capacity of the SR is enhanced [Of all teleost fishes  studied, none are reported to possess TT or dyads in their cardiomyocytes. This has been found in many teleost species: in the myocardium of European plaice (latessa) ; the ven mykiss) ; the atrlefinus) ; the myocombrus) ; the atr carpio) ; the veniepinus) ; the venentalis) ,31; and . rerio) . In seve. rerio) ,31. In a. rerio) . Howeverenhanced .Raja eglanteria), the sandbar shark (Carcharhinus plumbeus) and the smooth dogfish (Mustelus canis)), did not detect the presence of TT in atrial or ventricular myocytes. In these species, inhibition of SR fluxes (by a combination of thapsigargin and ryanodine) decreased net force and increased relaxation time in strips of atrial and ventricular myocardium, suggesting a role of CICR [Among Chondrichthyes (cartilaginous fishes), ultrastructural investigation of elasmobranchs (clearnose skate ( of CICR .Myxine glutinosa (order: Myxiniformes) has a cardiovascular system with six main contractile organs: a branchial (or main) heart, which transports blood from the gills to the body; a portal heart which conveys blood from the gut to the liver; two caudal hearts and two cardinal hearts. In this organism, peripheral SR couplings were found in both the portal heart and in the ventricle of the branchial heart, but no TT were reported [Lampetra fluviatilis) belongs to the order Petromyzontiformes, which have a well-developed heart, with a single atrium and ventricle as contractile components. Myocytes from the La. fluviatilis heart are long, but unlike most other ectothermic vertebrates, are relatively wide (a diameter of about 12 μm for ventricular myocytes). Ultrastructurally, lamprey cardiomyocytes contain an extensive SR, interacting with the sarcolemma at peripheral junctions, but TT were not observed. Functionally, cardiac ECC in La. fluviatilis depends on LTCC, with some force production requiring SR Ca2+ stores that are sensitive to ryanodine and caffeine (modulators of RyR gating) [2+-release might serve to propagate Ca2+ signals in these relatively large cardiomyocytes, by CICR.The Atlantic hagfish reported . The riv gating) . This SR (c)In this section, the term ‘invertebrates’ refers to non-vertebrate metazoans. Studies on the ultrastructure of invertebrate heart contractile cells have been comprehensively reviewed , althougCorella willmeriana contain peripheral couplings, but no TT [Branchiostoma spp.) and Hemichordatea . The cardiovascular systems of echinoderms  are anatomically simple compared with other deuterostomes. The muscle cells of the sea-cucumber Parastichopus tremulus dorsal hemal vessel lack Z-discs and TT, but possess an SR system with peripheral couplings [Deuterostomes are a superphylum of animals in which, during embryonic development, the blastopore (first opening) becomes the anus. This group includes chordates , hemichordates and echinoderms. Among tunicates, cardiomyocytes of the ascidiacean ut no TT . There aouplings .Helix aspersa [Mytilis edilis [Lepidopleurus asellus and Tonicella marmorea. No peripheral couplings between the SR and sarcolemmal were observed in these chitons [Busycon canaliculatum) are reported to lack typical TT, and have an extensive SR system, with peripheral couplings and interconnected extra-junctional segments deep within the myoplasm [2+ signal propagating function to the corbular SR of bird and mammalian hearts. Busycon canaliculatum cardiomyocytes possess multiple shallow invaginations of the surface membrane, that were termed ‘sarcolemmal tubules’. Whether these sarcolemmal tubules are a rudimentary TT system, or if they represent caveolae, awaits further experimental investigation.Together with the deuterostomes, protostomes comprise the majority of bilaterian animals. Protostomes were once defined as having the blastospore forming the mouth during development, but the embryogenesis of different taxa within this group is more diverse. The lophotrochozoans are a major superphylum of protostomes which includes molluscs, annelids, brachiopods, bryozoans and others. Most molluscan taxa possess an open circulation with a simple heart tube. Molluscan cardiomyocytes are variable in diameter (2–28 μm), depending on species and anatomical location. TT and dyads have been reported in cardiomyocytes of some molluscs, such as the snail,  aspersa  and the s edilis ; but not chitons . Cardiommyoplasm . These wSiboglinum fiordicum [Eisenia foetida [Arenicola marina [Like mammalian smooth muscle, the contractile cells of annelid hearts, or pseudohearts, are non-striated ,24. TT hiordicum , to inte foetida , to extea marina . PeripheThe ecdysozoans are a superphylum of protostomes that includes nematodes, priapulids, onychophorans, loriciferans, kinorhynchans, tardigrades and arthropods. Of these, only onychophorans are reported to possess cardiovascular systems. However, it should be noted that the body-walls of nematodes contain striated muscles, with TT that form dyad and triad junctions with the SR .Limulus polyphemus [Penaeus japonicas [Oniscus asellus and the pond slater Asellus aquaticus [Nephila clavata [Drosophila melanogaster [Tmetonyx cicada [Sympetrum danae [Lepidurus arcticus [Dyad and triad SR junctions with the TT have been reported in the cardiomyocytes of most classes of arthropod, including the horseshoe crab lyphemus ; the Kuraponicas ; the comquaticus ; the orb clavata  and the nogaster . In addix cicada ; the draum danae ; and thearcticus .Beroe ovata: it is homogenously distributed in the radial fibres but is located more centrally in the longitudinal fibres. Invaginations of the surface membrane were not observed [Mnemiopsis leydii were not detected [The historical term Radiata refers to non-bilaterian animals. These include coelenterates: the cnidaria  and the ctenophores (comb-jellies). Although they lack hearts, coelenterates do possess smooth muscle cells. Two distinct populations of SR have been identified in the two types of giant smooth muscle fibres of the ctenophore observed . Similardetected .Other metazoan groups, such as the poriferans (sponges) and placozoans, do not possess smooth or striated muscle. Consequently, it is unlikely that these animals contain SR or TT, but their genomes encode precursors of the molecular machinery necessary to produce such membrane contact sites .. 3v1.2 in heart dyad physiology have been extensively evaluated in previous publications [Even in mammalian cardiomyocytes, the molecular machinery required for the formation of dyad junctions has not been completely elucidated. However, three proteins known to participate in dyad formation and function will be analysed in the current work: the bridging integrator 1 (BIN1), junctophilin 2 (JPH2) and caveolin 3. The evolution and roles of RyR2 and Caications ,51, figuications . (a)Homo sapiens BIN1 is available at the Ensembl 2021 website, release version 105  [Saccharomyces cerevisiae possesses six homologues, whereas the ecdysozoans Caenorhabditis elegans and Dr. melanogaster have one. The tunicates Ciona intestinalis and Ciona savignyi have 2 BIN1 homologues, which on the basis of their protein sequence identities, are likely to be BIN1 orthologues. The hagfish Eptatretus burgeri, the lamprey, Petromyzon marinus, and the coelacanth, Latimeria chalumnae, all have three BIN1 homologues. Most of the vertebrates investigated possess four BIN1 homologues, but there is gene family expansion in teleost fishes, with many taxa encoding six homologues, salmonids between 9 and 11, and the goldfish Carassius auratus, having 15. There appear to be BIN gene losses and gains in other vertebrate groups: such as the blue-ringed sea krait, Laticauda laticaudata, having one homologue and the sloth, Choloepus hoffmanni, two; in contrast to the white-tufted-ear marmoset, Callithrix jacchus, which has seven. The consequences of such expansion or loss of BIN1 homologues on cardiac dyad function awaits characterization.BIN1 has several pseudonyms, including amphiphysin 2 and myc box-dependent-interacting protein 1. These names reflect the functional diversity of this protein: it is involved in both the generation and sensing of the curvature of biological membranes, in processes including endocytosis, synaptogenesis, tumour suppression, regulation of the actin cytoskeleton and TT formation. This pleiotropic functionality reflects structural diversity, resulting from alternate messenger RNA splicing of multiple exons. BIN1 is a member of the BAR  domain, involved in dimerization to form a banana-shaped structure; for review, see . Multiplv1.2 to cardiac TT, thereby enhancing ECC. This effect was dependent of the C-terminal 172 amino acid residues of BIN1 and not on its N-terminal BAR domain [v1.2 trafficking are reduced [2+ transients [v1.2 interactions at CRUs. In contrast to an earlier study, it was found that all five spice-variants of BIN1 present in rat could generate functional TT, with those containing the PI-binding motif being particularly effective at this activity [Early heterologous expression studies indicated that of isoforms of BIN1 present in mammalian skeletal muscle, those containing exon 11 promoted membrane infolding. This exon encodes a phosphoinositide-binding domain, located near the centre of the protein, which recruits BIN1 to the surface membrane where these lipids are enriched . The rolR domain . In card reduced . In mousansients . The majansients . In humaactivity .2+ transients and severely compromised ventricular contractility [Drosophila melanogaster BIN1 plays a key role in the development of TT in body muscle and is also detected at the protein level in the heart of this fruit fly [Geodia cydonium. This BIN1 homologue does not appear to be involved in the organization of subcellular structures, as it is a secreted aggregation factor involved in colony formation by this sponge [There is less information about the role of BIN1 in non-mammalian hearts. In the zebrafish heart, morpholino-knockdown of BIN1 decreased the amplitude of Caactility . Drosophruit fly . A homols sponge .BIN homologues are represented throughout Eukarya, including in yeast, green plants and animals . In the current study, phylogenetic reconstruction of the evolution of BIN1  indicatH. sapiens BIN1 exon 11 were not detectable in monotremes , a tetrapod , ray-finned fishes or invertebrates. The only amino acid conserved in mammals but not birds are a serine residue; figure 3a. Analysis of this site using the NetPhos algorithm  [The role of BIN in the evolution of cardiac dyad junctions is uncertain. The BIN homologues present in molluscs (lophotrochozoans) and arthropods (ecdysozoans) do not group with vertebrate BIN1 in phylogenetic reconstructions . It is uhos-3.1)  indicateH. sapiens BIN1 exon 13 were only detected in mammals, birds, reptiles, amphibians, lobe-finned fishes, some sharks and a limited range of ray-finned fishes. Comparison of aligned sequences revealed a motif that was conserved in mammalian BIN1 homologues, but not in those from non-mammalian species, figure 3b. Searching for this motif in the ProSite database, using the ScanProsite tool [Homologues of ite tool , revealeite tool . It is tc; electronic supplementary material, table S1. Comparison of human BIN1 with those from other taxa indicate that there is a glutamine residue (Q529 in isoform 1) encoded by exon 17 that is conserved in mammals, but not in non-mammalian animals. This residue might underpin the difference between mammals and other vertebrates in the presence of cardiac dyads: a hypothesis that is testable using mutation and heterologous expression approaches.Homologues of human BIN1 exon 17-encoded peptide are detectable in all vertebrate classes and in other chordates, but only in some echinoderms, lophotrochozoans, ecdysozoans and cnidarians, figure 3 (b)v1.2 at these CRUs [v1.2, recruiting the LTCC to the cardiac dyad [Dr.melanogaster. Heart-specific silencing or overexpression of the single JPH gene in this fruit fly significantly decreased lifespan and was associated with enlargement of the heart tube, with disorganization of the contractile apparatus [JPH are a family of proteins that link intracellular organelles, like the SR, to surface membranes such as the sarcolemma and TT . This isese CRUs . Recentliac dyad . In invepparatus . (c)Apis mellifera) CAV in a mammalian cell-line resulted in de novo formation of caveolae [Caveolae are flask-shaped infoldings of cell-surface membranes, which are nucleation sites for the production of TT in striated muscles . Caveolicaveolae . This de. 42+ signalling, such as JPH4 [v1.1/JPH1 in skeletal muscle and RyR2/Cav1.2/JPH2 in cardiac muscle) and possibly have distinct mechanisms of formation (e.g. dependent on BIN1 + 11 versus BIN1 + 13 + 17). These membrane junctions also show distinct patterns of emergence during animal evolution, e.g. ray-finned fishes possess triad junctions in their skeletal muscles but lack dyad junctions in their hearts.The current work provides insights into the evolutionary history of cardiac dyads and the roles of key proteins in their formation. A fundamental perspective is that these junctions developed independently several times during metazoan evolution, representing homoplasy (convergent evolution). Ultrastructural evidence unambiguously supports the presence of TT and dyads in the cardiomyocytes of mammals, arthropods and some molluscs. A point of conflict is whether the myocardium of reptiles, in particular turtles, possess ‘short-TT’ coupled to the SR. This is of importance and is worthy of further experimental investigation, as it places the evolution of vertebrate cardiac dyads either to a common ancestor of mammals and reptiles, or to the point of divergence of mammals from other vertebrate groups. If the former scenario is correct, it implies that birds must have subsequently lost the ability to generate cardiac dyads. This is not unreasonable, as birds are known to have lost members of protein families involved in ECC and Ca as JPH4 . It is aComparison of the phylogenetic and molecular features of proteins involved in dyad formation also supports a homoplastic mechanism of evolution. For example, BIN homologues from arthropods and molluscs do not group with vertebrate BIN1 proteins in phylogenetic analyses, nor do they share conserved exons involved in TT formation. Multiple sequence alignments of the peptides encoded by these exons revealed a glutamine residue in exon 17 (Q529) that is conserved in mammals (which form TT) but not in other vertebrates (which lack these structures). This residue potentially has a function in the formation of cardiac dyads in mammals.Platynereis dumerilii, Brunet et al. developed a framework for the evolutionary origin of smooth and striated myocytes in bilaterians [2+ influx through LTCC to drive ECC; and use the SR as a reserve mechanism in situations demanding high cardiac output [Based on the ultrastructural, transcriptomic, phylogenetic and physiological analyses of the different muscle types present in the marine annelid aterians . This moc output . The indc output . This isc output .


Background: Tuberculosis meningitis (TBM) is the most lethal form of TB. It is difficult to treat in part due to poor or uncertain drug penetration into the central nervous system (CNS). To help fill this knowledge gap, we evaluated the cerebrospinal fluid (CSF) concentrations of fluoroquinolones and carbapenems in patients being treated for TBM.Methods: Serial serum and CSF samples were collected from hospitalized patients being treated for TBM. CSF was collected from routine lumbar punctures between alternating timepoints of 2 and 6 h after drug administration to capture early and late CSF penetration. Rich serum sampling was collected after drug administration on day 28 for non-compartmental analysis.Results: Among 22 patients treated for TBM (8 with confirmed disease), there was high use of fluoroquinolones  and carbapenems . Median CSF total concentrations of levofloxacin at 2 and 6 h were 1.34 mg/L and 3.36 mg/L with adjusted CSF/serum ratios of 0.41 and 0.63, respectively. For moxifloxacin, the median CSF total concentrations at 2 and 6 h were 0.78 mg/L and 1.02 mg/L with adjusted CSF/serum ratios of 0.44 and 0.62. Serum and CSF concentrations of moxifloxacin were not affected by rifampin use. Among the 76 CSF samples measured for carbapenem concentrations, 79% were undetectable or below the limit of detection.Conclusion: Fluoroquinolones demonstrated high CSF penetration indicating their potential usefulness for the treatment of TBM. Carbapenems had lower than expected CSF concentrations. Mycobacterium tuberculosis (Mtb) and remains a major cause of mortality globally. In 2020, approximately 10 million people developed active disease and there were 1.5 million deaths, representing the first annual increase in deaths since 2005 (Tuberculosis (TB) is an infectious disease caused by nce 2005 . While tnce 2005 . TBM cannce 2005 . Mortalince 2005 .in vitro activity against Mtb. However, concentrations may be most susceptible to reduction if administered with rifampin, compromising treatment efficacy  penetration of first-line TB medications including rifampin and ethambutol can be suboptimal at standard dosages, seldom exceeding minimal inhibitory concentrations (MIC) . This isefficacy . While pCarbapenems are extended-spectrum beta-lactams with broad antimicrobial activity, including activity against Mtb . They arTo further understand the exposure of fluoroquinolones and carbapenems in TBM, our objective was to describe and compare serum and CSF concentrations of fluoroquinolones  and carbapenems (imipenem and meropenem) among persons treated for TBM.This prospective study enrolled participants at the National Center for Tuberculosis and Lung Diseases (NCTLD) in Tbilisi, Georgia. The NCTLD includes an inpatient ward for patients with TBM and directly observed therapy clinics for ambulatory care. Study participants were selected from a larger prospective cohort evaluating clinical outcomes among persons treated for TBM . Written® MTB/RIF assay  and AFB culture on a Lowenstein-Jensen solid and Mycobacterial Growth Indicator Tube liquid media. To identify drug resistance to rifampin and isoniazid, a Genotype® MTBDRplus  assay was performed on positive cultures. Serial lumbar punctures were performed at 7, 14, 28 days, and then monthly after treatment initiation per standard of care. With regards to treatment and dosing, WHO guidance was followed and treatment regimens were based on the Georgian National TB Program 2015 guidelines, as outlined previously  staining, molecular testing with Xperteviously . Both leeviously . For MDReviously . Additioeviously .Participants enrolled in the pharmacokinetic (PK) study had both serum and CSF samples collected at baseline and at approximately 7, 14, and 28 days following initiation of treatment. Monthly samples were subsequently collected during hospitalization for up to 112 days. Participants had two- and 6-h samples collected following drug administration from both serum and CSF. The CSF sampling was alternated between two- and 6-h concentrations to assess for early and delayed penetration. Rich serum sampling for non-compartmental analysis (NCA) was performed at 28 days, and the collection timepoints were 0, 2, 4, 8, 12, and 24 h after drug administration with slight variations for persons receiving bedaquiline  and delamanid . Following serum and CSF collection, samples were centrifuged then stored at −80°C at the NCTLD until shipping to the Infectious Disease Pharmacokinetic Laboratory (IDPL) at the University of Florida, with the cold chain remaining intact throughout. Total drug concentrations were quantified using validated liquid chromatography tandem mass spectrometry assays. Serum curve was used for the serum samples, while artificial CSF curve was used for CSF samples to match CSF matrix. The plasma and CSF detection ranges for levofloxacin, moxifloxacin, and ofloxacin were 0.2–15 mg/L and 2–100 mg/L for meropenem and imipenem. Intra- and inter-batch precision and accuracy were <10%. Samples below the limit of quantification (BLQ) were assigned a value of “0” for analysis.max), time to maximum concentration (Tmax), elimination rate constant (Ke), half-life (t1/2) and area under the concentration-time curve over 24 h (AUC0-24). Serum protein binding rates for levofloxacin (31%), moxifloxacin (40%), ofloxacin (32%), imipenem (20%), and meropenem (2%) were obtained from previously published literature and used to estimate free serum drug concentrations  to determine maximum serum concentration  was used for statistical analysis.Twenty-two participants were enrolled in the PK study, all of whom received a fluoroquinolone and/or carbapenem. Over half (55%) of enrolled patients were female and the median (IQR) age was 38 29.3–47.8) years. Additional information regarding baseline demographics, comorbidities, and relevant clinical measures including CSF composition are included in 9.3–47.8 max concentrations through NCA were 11.62 mg/L (8.64–13.12), with a Tmax of 4 h . Following TBM diagnosis, levofloxacin CSF concentrations increased significantly from baseline at both 2 h (p = 0.02) and 6 h (p = 0.002)  .max concentrations through NCA were 4.04 mg/L (2.78–5.34), with a Tmax of 2 h (p = 0.05). Following TBM diagnosis and treatment initiation, moxifloxacin in CSF trended towards increased concentrations, but this was not statistically significant Among the participants receiving moxifloxacin, seven received concomitant rifampin, yet no difference in moxifloxacin serum AUC0-24 were seen between patients receiving rifampin versus those not receiving rifampin . Additionally, CSF concentrations were not significantly different between patients receiving rifampin versus not receiving rifampin at 2 h  or 6 h .Ten participants received moxifloxacin during treatment, 6 (60%) following a change in therapy from levofloxacin. There were 10 and six matching CSF and serum samples at 2 and 6 h, respectively. Median (IQR) total serum concentrations at 2 and 6 h were 2.74 mg/L (1.65–4.84) and 1.91 mg/L (1.56–3.28), while median total CSF concentrations at 2 and 6 h were 0.78 mg/L (0.63–1.40) and 1.02 mg/L (0.78–1.06) . Cmax cox of 2 h . Median max concentrations through NCA were 9.47 mg/L (8.67–13.36), with a Tmax 2 h  (p = 0.04) whereas concentrations at the 6-h time-point did not (p = 0.28)  total serum concentrations at 2 and 6 h were 9.47 mg/L (8.67–13.36) and 5.40 mg/L (4.43–6.75), while total CSF concentrations at 2 and 6 h were 2.22 mg/L (0.93–3.11) and 3.20 mg/L (2.17–3.69) . Median Tmax 2 h . Median  = 0.03) . Ofloxac = 0.28) .There were 11 participants who received imipenem resulting in three matched CSF to serum samples at 2 h and two at 6 h. For meropenem, six participants with six matched CSF to serum samples at 2 h and one matched sample at 6 h were available. With the exception of 2-h serum samples, a substantial number of samples were undetectable or BLQ (<2 mg/L) for both serum and CSF . 12/15  indicating typical systemic exposure (max and AUC), with a proposed Cmax/MIC ratio target of >8–10 in TB (max/MIC or AUC0-24/MIC for this population, fluoroquinolones in our study achieved concentrations within expected ranges, suggesting potential attainment of targets. Assuming susceptible MICs of 1 mg/L for ofloxacin and 0.5 mg/L for levofloxacin and moxifloxacin, levofloxacin may have the best potential to achieve PK/PD targets given our findings (All fluoroquinolones in our study had median serum Cexposure . Our finexposure , fluoroqexposure . Our dat10 in TB . Althougfindings . In addifindings . The incfindings .in vivo animal models (, (Although carbapenems have shown efficacy in bacterial meningitis and against Mtb in vitro and l models , this stl models . CSF penl models . In the l models . Howeverl models . Techniql models , (Brunneodels (, .While increased penetration of antimicrobials into the CNS is desired for treatment of TBM, this may be associated with increased risk of CNS-related adverse effects. Previous literature has described adverse CNS effects for fluoroquinolones and carbapenems, potentially due to interferences with gamma-aminobutyric acid receptors . NotablyThere are limitations to this study, including a relatively small sample size of 22 patients. Also, due to the descriptive nature of the study, the impact of co-administered medications, renal function, and hepatic function were not controlled for in the analysis. However, expected impact is minimal. While we assumed the CSF samples with undetectable imipenem and meropenem concentrations were due to poor CSF penetration, we could not rule out other causes. The effect of carbapenem instability both before and after sample collection could not be assessed and may have contributed to the low CSF and serum concentrations, even though samples were rapidly stored at −80°C to help minimize degradation. For example, imipenem tested in conjunction with cilastatin only remained 90% stable for 2 h at 37°C or 3 h at 25°C, demonstrating its unstable nature prior to sample freezing . With stIn summary, we described fluoroquinolone and carbapenem penetration into the CSF in a cohort of patients treated for TBM. Our study is unique in that serial CSF concentrations were collected at 2 and 6 h over the first few months of TBM treatment, allowing for assessment of delayed penetration. We found that fluoroquinolones showed CSF penetration peaking after serum concentrations, potentially increasing over the duration of treatment. Carbapenems had lower than expected CSF concentrations, with the majority of samples undetectable or BLQ in the CSF while detectable in serum. These findings warrant further exploration with techniques such as cerebral microdialysis to see how carbapenem brain tissue concentrations compare to plasma and CSF concentrations.


Surface electromyography (sEMG) is commonly used to observe the motor neuronal activity within muscle fibers. However, decoding dexterous body movements from sEMG signals is still quite challenging. In this paper, we present a high-density sEMG (HD-sEMG) signal database that comprises simultaneously recorded sEMG signals of intrinsic and extrinsic hand muscles. Specifically, twenty able-bodied participants performed 12 finger movements under two paces and three arm postures. HD-sEMG signals were recorded with a 64-channel high-density grid placed on the back of hand and an 8-channel armband around the forearm. Also, a data-glove was used to record the finger joint angles. Synchronisation and reproducibility of the data collection from the HD-sEMG and glove sensors were ensured. The collected data samples were further employed for automated recognition of dexterous finger movements. The introduced dataset offers a new perspective to study the synergy between the intrinsic and extrinsic hand muscles during dynamic finger movements. As this dataset was collected from multiple participants, it also provides a resource for exploring generalized models for finger movement decoding.  Recently, high-performance myoelectric systems have been sought for accurate identification of finger movements and forces9. Such systems would enable more intuitive and streamlined human-computer interactions (HCI) in modern applications of surgical robotics10, tele-operated robotics12, or virtual reality environments14. However, as the sEMG signal is measured at the skin surface, the source signals produced by motor neurons not only undergo low-frequency filtering caused by muscle, fat and subcutaneous tissues, but they are also contaminated by elusive artefacts such as those associated with electromagnetic interference and electrode displacement15. Separating the source signals of the forearm sEMG signal is still quite challenging because of the complex spatial muscle composition16, and hence this complicates the process of decoding dexterous finger movements from sEMG signals.The motor neuronal activity within muscle fibres has been widely observed through surface electromyography (sEMG). The sEMG-based myoelectric control systems have been extensively exploited in the fields of rehabilitation engineering and robotics 28. The signals of the extrinsic hand muscles were measured by a wearable 8-channel sEMG sensor array, which is commonly adopted by most of the pertinent commercial applications27. In addition, a data-glove sensor was used to measure the finger joint angles and synchronize them with the sEMG measurements. The collected data is essentially used to for automatic decoding of human finger movements based on myoelectric signals. In particular, 12 single-finger and multi-finger flexion movements of four fingers were examined, along with the abduction (moving away from the hand center) and adduction (moving towards the hand center) of the metacarpal joints. Images of 12 gestures are shown in Fig. Recently, some studies of the myoelectric physiology suggested the presence of the sEMG sources in the intrinsic hand muscles as well as the external ones29, as shown in Fig. 30. Therefore, when fingers are moved, the forearm sEMG signals may arise from two types of source signals: signals that control fingers, and signals that maintain wrist posture. However, the majority of the sEMG databases and EMG-based hand gesture prediction approaches ignore the gravity effects. Therefore, we collected sEMG data under three typical arm postures in order to quantify the gravity effects on the wrist joints. In addition, we experimented with two finger movement speeds to better represent real myoelectric control scenarios. Moreover, the sEMG signal repeatability is commonly lower than that of the finger joint angles. Thus, we designed here three inter-session experiments, with each session containing 6 repeated trials (with three fast and three slow movements), with a total of 18 trials per gesture. This increases the abundance and diversity of the sEMG data.While the intrinsic hand muscles control only the metacarpal finger joints, the extrinsic hand muscles control the wrist as well as the proximal/distal interphalangeal finger jointsWe experimentally tested the finger movement repeatability and the synchronization of the multi-source data. Furthermore, we extracted low-dimensional components of the HD-sEMG activation map in order to visualize the relation between the single-finger and multi-finger movements. Finally, a baseline pattern recognition method was adopted to show the feasibility of dexterous finger movement classification based on the HD-sEMG signals of the intrinsic hand muscles. Our work is highly reproducible. In particular, twenty inexperienced participants were recruited for data acquisition setups that resemble real application scenarios, and enable the investigation of the robustness and generalization of EMG-based methods for identifying dexterous finger movements. Also, our dataset has 64 channels of HD-sEMG signals, and this high channel density enables the investigation of the MUAP discharge patterns of the intrinsic hand muscles. In general, our dataset paves the way of exploiting the signals of the intrinsic hand muscles for myoelectric control of dexterous finger movements.cm, and a forearm circumference range of 23.3 ± 2.8 cm). All participants were right-handed had no previous experience with myoelectric control or data-glove experiments. The demographic data of each participant is reported in the dataset31. The participants were recruited from the Southeast University in China via a mass email communication with a basic description of the experimental setup and goals. Each respondent who expressed interest was sent further details, and then scheduled to have a data acquisition session. All participants were given a full verbal description of the methods, purposes, and protocols for the experiments. Each participant read and signed an informed consent form before proceeding with the experiments. The experimental protocol was approved by the Ethics Committee of Jiangsu Province Hospital (2020-SR-362). All experiments were thus conducted according to the ethical guidelines for medical research involving human participants (according to the Declaration of Helsinki32).We collected data from 20 volunteers who had no known skeletal and/or neuromuscular disorders , respectively. Secondly, we considered the abundance of the sEMG features in the existing datasets. Indeed, Atzori et al.33 demonstrated that the EMG classification accuracy had negative correlation with the body mass index (BMI) (p = 0.026), but no significant correlation with gender (p = 1), hand dominance (p = 0.921) or age (p = 0.117). This means that a population spanning a wider range of BMI values may have more abundant sEMG features. Therefore, we selected participants with diverse BMI values ranging from 16.0 to 29.74, including “moderately thin” to “pre-obese” individuals according to the WHO standard for South-East Asia.For our database, the participant count and the selection criteria were based on two aspects. Firstly, we examined 14 publicly available sEMG databases, and found that they have mean and median numbers of participants of 20.42 and 20 . The 64-channel HD-sEMG electrode grid  used here is made from a 0.1 mm flexible printed-circuit film. The 64 electrodes were organized into 5 rows × 13 columns (with one void corner spot), where each electrode is circular with a diameter of 3 mm, and an 8 mm centre-to-centre distance. The electrode grid covers a surface area of 105 × 40 et al.34 compared the gesture classification accuracies for four types of sEMG acquisition devices (including the Delsys Trigno electrode and the MYO armband), and they reported comparable results for the Delsys Trigno electrode and the MYO armband. This indicates that the relatively low sampling rate and number of channels of the MYO armband will not have a significant impact on the gesture classification accuracy. In addition, several independent studies37 have also demonstrated the effectiveness of the MYO armband in gesture recognition applications. So, the MYO data should be reusable and valuable in sEMG-based HCI applications. The sEMG and IMU data of MYO armband are transferred via bluetooth communication protocol  by a MYO armband , which is a commercial myoelectric armband with eight bipolar dry-electrode channels. This MYO armband device uses high accuracy zero drift micropower operational amplifiers  with a CMRR of 115 dB and a GBP of 400 kHz. Recently, Pizzolato 31.The positioning of the HD-sEMG grid is shown in Fig. Finger kinematics were captured using a 22-sensor data-glove  whose data is transferred to a computer via a wired serial communication protocol  force of each movement was measured during isometric contraction (i.e. fingers being flexed to the maximum degree), and participants were asked to learn to exert 70% of the MVC for each gesture. In order to prevent muscle fatigue associated with maximal finger joint flexion during the data collecting, the participants were instructed to exert about 70% of the MVC according to their training memory. The dataset structure is described in Fig. To ensure the consistency of the finger motion speed, and the start/stop times, we designed a target trajectory guidance experiment (TGE). In particular, the joint angle curves and the target trajectories were displayed on the monitor in real-time during data collection . Each participant had a break at the middle of each session (after the first 6 movements), and also was allowed to have rest at any other time. For experiments lasting over 50 minutes, each HD-sEMG sensor would be replaced to avoid conductive paste melting and increase the crosstalk between the electrodes.rd order band-pass Butterworth filter with lower and upper cut-off frequencies of 10 and 500 Hz, respectively. Since the original 13 × 5 electrode grid had one electrode missing at one corner, the signal value at that location was estimated as the average of the values from three adjacent grids. The instantaneous signal could thus be reshaped as an image. Finally, the units for both the HD-sEMG and the MYO armband signals were set to be millivolts.The HD-sEMG data was filtered through a zero-phase 339, we set the probability of a normal sEMG as 99.7%. Then, the instantaneous amplitude of HD-sEMG outside the μ ± 3σ range were considered as outliners. Each outlier signal was replaced by the average of the remaining normal signals. The details of these processing steps are shown in Table For data validation, the sEMG signals were pre-processed and transformed into envelope signals. This pre-processing procedure was packed as an easily reusable code module for both offline and online processing. Firstly, notch filtering at 50.5 52 Hz was performed to eliminate power-line interference. Then, an outlier detection algorithm was used to screen out any abnormal signals that may result from poor electrode-skin contact. Here, we applied the Pauta criterion for outlier detection in HD-sEMG signals. Specifically, because the instantaneous sEMG amplitude can be modelled by a Gaussian distribution31. Specifically, our data is organized according to the following file structure:The data collected in our work (as well as the employed data collection methods) can be found at figshare• Info.xls• S01S01.cal– S01.png– – G01.mat– G02.mat……– G12.mat• S02……• S20In particular, a Microsoft Excel file (‘Info.xls’) contains essential pieces of information for each participant . The data files of the 20 participants are stored in ‘S01’ through ‘S20’, respectively. Each participant folder (e.g. ‘S01’) contains 14 files, among which ‘S01.cal’ is the data-glove calibration file; ‘S01.png’ is the experiment image; and the other 12 ‘Gxx.mat’ files are the collected data (where xx represents an index for each of the 12 gestures).To illustrate the data storage structure, we take ‘G01.mat’ as an example. All data is stored into a two-dimensional matrix within the struct variable ‘Data’. The 91 matrix columns represent the data features: the HD-sEMG features are those of columns 1–65; the glove features are those of columns 66–80; the MYO armband features are those of columns 81–88; and the arm posture features are those of columns 89–91. The feature index is shown in Fig. In order to quantify the gravity effects on the wrist joint, we collected sEMG data under three typical arm postures, which are denoted as P01, P02, P03 as shown in Fig. R2) between the target motion curve and the finger joint angles on a sequence of one fast trial and one slow trial , as shown at the bottom right of Fig. R2 coefficient was evaluated on more sequences associated with those movement joints. Then, we calculated the average coefficient of determination  classifier with K = 15)540. The To compare the prediction accuracy associated with the extrinsic hand muscles against that of the intrinsic hand muscles, we used the instantaneous RMS of the HD-sEMG and MYO signals separately to train two KNN models . In addition, we designed a “FUSION” group which combines the HD-sEMG and MYO features. The values of the two feature types are respectively scaled into the range of . All HD-sEMG channels share the same scale factor, while all the MYO channels have a different scale factor. Subsequently, we combined the features of the two types into a new feature vector (of 73 dimensions) and conducted KNN classification.Statistical box plots of the predicted accuracies averaged over the 20 participants are shown in Fig. The principal component analysis (PCA) of the HD-sEMG signals offers a low dimensional visual perspective on the relationship between dexterous finger gestures. We considered HD-sEMG signals of the steady-state movement, i.e. the middle 3-second segment of every 10-second trial. Then, the 65-dimensional HD-sEMG signals  were reduced into 3-dimensional signals whose components can be plot as shown in Fig. To better visualize the association among the multi-finger movements, we selectively present principal components of specific finger movements in the 2D plane. Figure kΩ. During data acquisition, the EMG artefacts were continually monitored. In offline analysis, the signal frequency spectra were checked through frequency analysis. As the sEMG intensity varies between different gestures, different signal-to-noise ratios (SNR) are obtained. Vecchio et al.27 concluded empirically that, at low EMG amplitudes, the noise power should be no more than one half of the signal power to ensure reliable decomposition. The statistical results show that the average SNR of each gesture of HD-EMG is 8.76 ± 1.26 dB, and the average SNR of each gesture of MYO is 20.60 ± 6.20 dB.Prior to data collection, the signal quality of HD-sEMG was assessed by measuring the electrode-skin impedance in the OTBioLab+ Software , with an average impedance below 40 Two types of electrodes were used in the experiments, namely ELSCH064NM3 and GR08MM1305. Some participants used both types of electrodes during the three sessions. There is indeed no difference between the two types of electrodes, except for the position of the missing electrode. The electrode type used by each participant is specified in the collected dataset. Experimental videos of all participants are available from the authors upon request.31.The A/D conversion coefficient of HD-sEMG is officially calibrated, and the unit of HD-EMG signal in this database has been converted into millivolts. However, there is no official coefficient for MYO, the coefficients in Table All the participants are able to complete the 12 types of movements, as they are commonly used in the daily interaction, but we did find some of the fingers (mainly for the ring finger) can involuntarily follow the other active fingers. Therefore, we allow this involuntary following movement happen, as long as only the fingers within the predefined gestures are correctly activated.There are also some of the limitations that should be discussed: First, in order to minimize the impedance of electrodes, the preparation for HD-sEMG signal collection is a bit tedious at present, so the electrode material optimization may help improve the ease of wearing. Second, this dataset does not measure the hand force due to the limitations on experimental equipment. Our future works include extending the current intrinsic hand muscles recording to hypothenar group muscles, and developing stretchable electrode materials to improve the robustness against electrode displacement. Furthermore, we will expand the contents of this dataset toward more hand motion scenarios, such as hand manipulation tasks, which helps to decode the hand motion intentions during the interaction between human and environment.


In combined malnourished  and at risk for malnutrition  groups (CG), compared to CTRL, all essential amino acids (EAAs) and 30% of non-EAAs were lower (p < 0.018 to 0.0001), whereas in normo-nourished ADs  the CSF levels of 10% of EAAs and 25% of NON-EAAs were decreased (p < 0.05 to 0.00021). CG compared to normo-nourished ADs, had lower CSF aspartic acid, glutamic acid and Branched-Chain AA levels . CSF/plasma AA ratios were <1 in NON-AD but even lower in the AD population. Conclusions: Compared to CTRL, ADs had decreased CSF AA Levels and CSF/plasma AA ratios, the degree of which depended on nutritional state.Cerebrospinal fluid (CSF) amino acid (AA) levels and CSF/plasma AA ratios in Alzheimer Disease (AD) in relation to nutritional state are not known. Methods: In 30 fasting patients with AD  and nine control (CTRL) matched subjects, CSF and venous blood samples were drawn for AA measurements. Patients were stratified according to nutritional state . Results: Total CSF/plasma AA ratios were lower in the AD subpopulations than in NON-AD ( There is currently no treatment able to stop the progression of Alzheimer’s disease dementia . It is d2+ homeostasis [Patients with AD (hereinafter ADs) have very complex brain metabolic alterations, including insulin and insulin growth factor 1 (IGF-1) resistances ,3, reduceostasis , all of eostasis ,23,24,25Lack of energy and oxidative stress, indeed, initiate and worsen over time the synaptic pathology in AD ,14,26,27Abnormal mitochondrial enzyme activities are associated with cognitive impairment  and clinIn the context of CGH, the brain should rely on alternative substrates to generate energy, such as Amino Acids (AAs), lactate, pyruvate, and ketone bodies ,31, giveTo the best of our knowledge, a comprehensive study on all standard individual and collective CSF AAs in their mutual interrelationships with the corresponding plasma levels, the metabolic development of AD markers and the patients’ nutritional state has not so far been carried out. We believe that the findings from such a type of study might prove useful for future investigations addressing the possibility of exogenously manipulating the brain’s amino acid metabolism.In the current prospective, observational study, we hypothesized that patients with advanced AD, compared to non-AD subjects (NON-ADs), may have lower CSF AA levels and lower CSF/plasma AA ratios, suggesting brain overutilization of AAs. The rationale of our hypothesis is based on the following two arguments: first, the CSF compartment is in free communication with the interstitial fluid (ISF) surrounding neurons and glia  and draiThe second hypothesis we formulated was that the nutritional state of ADs could influence the AA levels in the CSF compartment and the CSF/plasma ratios, as circulating AAs, which normally enter the brain in mutual competition ,44,45, mThis prospective observational study included parts of the AD dementia and CTRL patient populations investigated by our team in a previous study , in whomThe patients were cognitively impaired  and underwent diagnostic testing in the Department of General Neurology at the National Institute of Neurology IRCCS Mondino Foundation, Pavia, Italy, between 2014 and 2018.Clinical diagnosis of dementia was formulated following the current research criteria for Alzheimer’s disease . CTRL weAll procedures complied with the ethical standards of human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. The study was approved by the local Ethics Committee (Project identification code: MAIR2016). Participants or their legal representatives provided written informed consent for participation in the study. No participant received financial compensation.All enrolled patients underwent complete clinical, neurological, and neuropsychological assessment and brain magnetic resonance imaging (MRI). 2; Mid-Arm Circumference, MAC, cm), clinical evaluation and assessment of patients’ nutritional state by means of Mini Nutritional Assessment (MNA) score [In addition to physical examination, routine blood tests, anthropometric measurements  score  were carg, 15 min) in order to measure plasma AA levels. At the same time, 2 mL of CFS samples were drawn from the lumbar tract and immediately delivered to the laboratory.In both ADs and CTRL, at 8 a.m. after 12 h of overnight fasting, blood samples were drawn from an antecubital vein and immediately delivered to the laboratory, where plasma was obtained from heparinized blood using centrifugation  was calculated as follows: Among the blood variables, plasma insulin levels (µU/mL) were measured using the Cord-CT Radioimmunoassay Kit CIS (France) and Coat A Count Insulin  commercial kits. Insulin resistance was calculated using the Homeostasis Model assessment  ,51.The first objective of the study was to document possible differences between ADs and CTRL in CSF AA levels and CSF/plasma ratio.The second objective was to document whether there were differences in CSF AA levels and CSF/plasma ratio related to AD nutritional state.The normality of all variables was assessed using the Shapiro–Wilk statistical test, supported by visual inspection. Since several variables violated the assumption of normality, central tendency and dispersion of continuous variables were reported as mean ± SD, but nonparametric tests were used. Accordingly, between-groups comparisons were carried out using the Mann–Whitney U-test (for two groups) or the Kruskal–Wallis test (for three or more groups). A significant result on the Kruskal–Wallis test was followed by post-hoc analysis (Dunn–Sidak) to compare pairs of groups. Descriptive statistics for discrete variables were reported as percentage frequency. Associations between variables were assessed via both Pearson correlation coefficient and Spearman rank correlation coefficient. p < 0.05. All analyses were carried out using the SAS/STAT statistical package, release 9.4 .All statistical tests were two-tailed and statistical significance was set at It can be seen from the data in Compared to CTRL, CG had lower plasma levels of glutamate, alanine, isoleucine, leucine, total AAs (TAAs), and Essential AAs (EAAs), and a lower EAA/TAA ratio . CompareIn the CSF compartment , compareTo sum up, compared to CTRL, ADs had important reductions in numbers and types of CSF AAs. Moreover, patients’ stratification according to nutritional state indicated the existence of two CSF AA profiles: one in which reduced AAs were independent of nutritional state, the other with a further reduction in AAs associated with a deteriorated nutritional state. Moreover, in CTRL subjects the CSF/plasma AA ratios were <1 but >1 for glutamine and the BCAA/EAA ratio. In ADs, CSF/plasma ratios were even lower than in CTRL, including glutamine and the BCAA/EAA ratio.p = 0.004)  , whereasCorrelations between CSF AAs and plasma AAs were evidenced both in ADs  and CTRLp = 0.0007), BCAA/TAA ratio , and valine .In CTRL, the study found positive links for serine , and another that was found in the nutritionally deteriorated group only . The latter group of ADs therefore exhibited both nutritionally independent and nutritionally dependent CSF AA reductions.The lower CSF/plasma ratios in ADs, both as an entire group and after stratification by nutritional state, suggest that AA reductions in CSF may be due not only to insufficient supply of AAs to the brain from plasma but also to brain overutilization. The following factors may sustain the hypothesis of brain AA overutilization in ADs. First, as shown in the current study, 75% of the AAs in CSF but only 31.6% of the AAs in plasma were reduced. Second, the CSF levels of several AAs, including two EAAs, were lower in normonourished ADs despite their normal plasma levels. Third, as previously documented, AA/protein dysmetabolism is more severe in the AD brain than in extracerebral areas . AnotherFor more clarity, brain AA alterations will be discussed below as derived from two main potential mechanisms, one of which may be refereed to as “general” and the other “specific”, while being aware that these two are intimately and inextricably interconnected.In addition to the need for energy production ,55, insuThe development of insulin resistance ,3,56 migAβ itself can cause increased brain AA utilization, as demonstrated in both AD patients  and in AThe interrelationship between altered brain insulin signaling and the biosynthesis of AD markers may be another mechanism leading to brain AA overutilization. Hyperinsulinemia indeed increases tau phosphorylation ,69, favoIt is likely that low CSF AA levels were not due to the blood–brain barrier (BBB)-restricted entry of AAs into the brain from plasma, or due to Choroid Plexuses (CPs) removing AAs from CSF and/or reducing AA secretion into CSF, which are both mechanisms that, under physiological conditions, determine brain AA availability . BBB andIn AD, elevated urea cycle activity and elevated urea levels, both in the TCA cycle  and in CSome potential mechanisms underlying reduced CSF AA levels are summarized in In summary, increased AA utilization in the AD brain might over time reduce the balance between positive effects (energy production) and negative ones , resulting in abnormal neuron and glia death .The reductions in CSF asparagine, serine, glutamine, glycine, arginine, tryptophan, phenylalanine and Trp ratio were independent of patient nutritional state, suggesting that they were linked to AD per se; the brain may also use these AAs for specific metabolic tasks.Low CSF serine and glycine confirm the findings from a post-mortem study  documentGlycine is normally produced by the brain . Its redLow asparagine could be due to reduced conversion of oxalacetate  from an Low CSF glutamine levels were in line with some studies ,96 and cLow CSF arginine  may deriIn the case of AD, the only CSF amino acid that significantly correlated with insulinemia was arginine. This correlation may be explained by the hyperinsulinemia-induced upregulation of brain arginase activity, as observed in obesity . Increased arginase activity consumes arginine and reduces NO synthesis, as arginase competes with NOS for the same arginine substrate. In brief, whatever the prevalent mechanisms, the upregulation of the two enzymes (NOS and arginase) competing for the same substrate (arginine) greatly contributes to reducing brain arginine availability.Two Essential Amino Acids (EAAs), tryptophan and phenylalanine, proved to be decreased in AD CSF. Being precursors of the neurotransmitter serotonin and catecholamines dopamine, norepinephrine, epinephrine, and tyramine, their reduction suggests that brain deficits in neurochemical transmission contribute to synapse dysfunction and the loss of synapses, neurons and neuronal networks . The alterations in chemical neurotransmitters occur early in AD, before the pathological markers of AD or the loss of neurons .The decrease in tryptophan may be attributed to activation of the kynurenine pathway , which iPrevious investigations reported that phenylalanine was increased in the brain  and decrLow phenylalanine levels may result from its use for protein synthesis , increasLow Trp ratio suggests a greater prevalence of the deficit in serotoninergic transmission compared to the catecholaminergic deficit. In nutritionally deteriorated ADs, CSF levels of six EAAs , of alanine, and of TAAs and EAAs as a group were reduced. Leucine, isoleucine, alanine, TAAs, and EAAs were altered both in plasma and CSF. Thus, the brain availability of these AAs, in particular the essential leucine and isoleucine, was in part conditioned by their limited supply in plasma ,119, andChronic protein breakdown  and/or iThe differences in degree in the above correlations between ADs and controls for BCAA/TAAs ratio and valine might confirm that, in AD, an important role may be played by brain overconsumption of BCAA for both protein synthesis and  for TCA cycle activity for energy generation. Brain BCAA overutilization could occur to facilitate the synthesis of phosphorylated tau protein, the main component of neurofibrillary tangles, due to increases in the process of mTOR autophosphorylation . IncreasThe reductions in leucine and valine in CSF confirm the findings from a previous study , which aBased on the background of existing literature, it is unclear whether BCAA may potentially exert neuroprotective or toxic effects.Some experimental investigations reported unfavorable effects on the development and progression of AD ,122. In We postulate that reduced brain BCAA availability may accelerate the progression of AD for at least five main reasons. First, low brain BCAA might impair the turnover of glutamate, the main excitatory neurotransmitter in the brain ,136 as nSecond, low brain BCAA levels may increase neurotoxicity and neurodegeneration by limiting the glutamate–glutamine cycle, leading to accumulations of both glutamate and ammonia . Third, Of note, in CTRL subjects, the BCAA/EAA CSF/plasma ratio was >1, supporting, under normal conditions, the importance for the brain of maintaining high levels of BCAA among the EAAs. The decreased CSF levels of EAAs lysine and threonine, despite their normal plasma concentrations, may be secondary to increased brain oxidation and/or increased incorporation into brain proteins. These two amino acids are the most essential among EAAs, since their oxidation is irreversible, as they do not undergo transamination activities ,143. ConNeural plasticity is regulated by protein phosphorylation occurring in serine, threonine, and tyrosine residues (protein serine–threonine kinase) . The neuReduced alanine may be due to an altered glycolysis pathway, leading to decreased availability of pyruvate to be converted into alanine via the transamination process.Altered brain methionine metabolism may explain the reduced CSF levels of methionine found in the current study and may also explain the increased homocysteine (a by-product of methionine) levels in AD serum and brains  and the As, in the current study, plasma methionine could not be detected, we cannot exclude a reduced contribution of plasma methionine to decreased CSF levels. Another mechanism leading to reduced CSF methionine may be its incorporation into proteins, since it is essential for the initiation of protein and nucleic acid synthesis and its active form, S-AdenosylMethionine (SAME), is the most important methyl donor in the body. Methylation processes via SAME stabilize and protect proteins, including myelin, and are involved in the formation of creatine, carnitine, melatonine, and polyamines and in the metabolism of serotonin. Another metabolic condition leading to low methionine may be excessive amino acid conversion to cysteine (and taurine), an important precursor of the antioxidant glutathione, which may undergo a chronic decrease in AD due to high brain ROS formation .Under physiologic conditions, the brains of normal adults would seem to be spared from malnutrition because of very low cell proliferation and cell replacement in the brain , low celHowever, the findings from more recent studies seem to question the assumption that the brain could be protected from malnutrition. First, the brain protein synthesis rate in normal adults was shown to be higher than that of skeletal muscle tissue : brain pThe high rates of brain protein synthesis were documented in other previous studies ,154. SecThus, we postulate that the brain of ADs, in particular ADs with MNA < 24, might be considered an “undernourished brain”, caused by an unbalanced ratio of brain AA utilization to AA supply from plasma. Theoretically, brain AA overconsumption could lead or contribute over time to impairment of circulating EAAs, particularly in patients with poor or marginal EAA intakes and in sarcopenic patients.A problem arising from the current study is whether it is possible to reconcile two conflicting factors: the brain’s needs for EAAs/AAs to sustain metabolic processes such as repairing, circuit remodeling, and growth of the brain’s residual functioning areas and, at the same time, the need to avoid enhanced EAA utilization, which results in the synthesis of AD neuropathology markers. It has been hypothesized that a nutritional strategy could involve providing patients with a diet low in protein but with high levels of BCAAs, aromatic amino acids, glutamine, histidine, and threonine ,158. AnoRoutine determinations of AA levels, both in plasma and CSF, in ADs.Quantification of dietary protein/EAA intakes since the first diagnosis and frequent monitoring over time.Improving physical activity and exercise, aiming at the preservation of/increase in skeletal muscle mass, strength, and function, since muscle tissue is the main body store of AAs.In nutritionally deteriorated patients (MNA < 24 scores), who represented more than half of the ADs considered in this study, a supplementation of EAAs to restore their plasma levels may be needed as EAAs are also vital for maintenance of the structures and functions of extracerebral organs and tissues. Of note, supplementation of the diet with L-serine prevents synaptic loss and behavioral deficits in AD mice . Future All these clinical suggestions should be added to an antinflammatory and antioxidant diet , along wUntil investigations address these issues, a reasonable approach in clinical practice for patients with AD at present might be the following:A well-planned investigation could examine whether, in ADs with normal nutrition (MNA > 24), the prevention of nutritional deterioration and correction of brain tryptophan and phenylalanine levels should be pursued. In these patients, supplementing these two aromatic amino acids might prove useful for improving their content in the brain.The study has several limitations that require further research to be resolved.A limitation of the present investigation is the small number of subjects studied, particularly controls.Urea levels were not determined, either in AD or CTRL CSF. Elevated CSF urea in AD would have corroborated the existence of excessive brain AA consumption.Patients’ intakes of dietary proteins were not quantified. Information regarding amount and type of ingested amino acids, in particular EAAs, would have been very useful for understanding the contribution of poor diet to reduced plasma and CSF EAAs in patients with nutrition deterioration, as well as to reduced CSF tryptophan and phenylalanine in normonurished AD.Analysis of body composition with measurements of skeletal muscle tissue, the most important AA repository in the body, would have strengthened the discussion.As a strength of the study, it is important to underline that the adoption of a simple nutritional test (MNA) may be useful in clinical practice for suggesting the existence of altered AA contents both in plasma  and CSF,The study documents that AD patients, compared to CTRL, had lower CSF AA levels and lower CSF/plasma AA ratios. The reductions in CSF AAs were in part linked to the disease per se, being independent of patients’ nutritional status, and in part linked to deterioration in nutrition.Future well-planned studies are needed to understand whether and how it is possible to safely improve CSF AA levels in advanced AD patients.