Hgd−/− mice . Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry . Raw data were processed using a targeted feature extraction algorithm and an established in-house accurate mass retention time database. Matched entities  were filtered based on their frequency and variability. Experimental groups were compared using a moderated t-test with Benjamini–Hochberg false-discovery rate adjustment. Results: L-Tyrosine, N-acetyl-L-tyrosine, γ-glutamyl-L-tyrosine, p-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance  in the mice that received nitisinone. Several other metabolites of interest were matched, but no significant differences were observed, including the aromatic amino acids phenylalanine and tryptophan, and monoamine metabolites adrenaline, 3-methoxy-4-hydroxyphenylglycol, and octopamine. Conclusions: Evaluation of the CSF metabolome of a murine model of AKU revealed a significant increase in the abundance of a limited number of metabolites following treatment with nitisinone. Further work is required to understand the significance of these findings and the mechanisms by which the altered metabolite abundances occur.Background: Nitisinone-induced hypertyrosinaemia is well documented in Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a decline in cognitive function and/or mood by altering neurotransmitter metabolism. The aim of this work was to evaluate the impact of nitisinone on the cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to providing additional insight into metabolic changes that occur following treatment with nitisinone. Methods: 17 CSF samples were collected from BALB/c  The widespread metabolic complication of nitisinone therapy in Alkaptonuria (AKU) ,5,6,7,8 Much of the literature available on hypertyrosinaemia relates to the clinical consequences that have been observed, for example corneal keratopathy and skin rashes ,13,14,15Several mechanisms relating hypertyrosinaemia to altered neurotransmitter metabolism have been proposed, including: (i) increased transport of L-tyrosine into the brain, leading to increased DP in the central nervous system (CNS); (ii) decreased transport of other large neutral aromatic amino acids via the large neutral amino acid transporter 1 (LAT-1) into the brain , leading to decreased serotonin in the CNS, and (iii) altered serotonin metabolism due to the direct inhibition of tryptophan hydroxylase  activity (rate-limiting step in serotonin biosynthesis) by L-tyrosine ,18,19.Escherichia coli (BL21), exhibited prominent substrate inhibition kinetics, and that enzyme activity decreased at elevated L-tyrosine levels. This supports the earlier supposition, but again in this study no downstream metabolites were measured .Thimm et al. , in a stThere is no direct evidence of altered cognition or central neurotransmitter metabolism in AKU patients with hypertyrosinaemia. In order to assess central neurotransmitter metabolism, either CSF or a brain biopsy are required, as they directly reflect neurotransmitter metabolism in the CNS. This is an accepted practice in the assessment of neurotransmitter disorders , but to A study in a murine model of AKU has also reassuringly demonstrated that monoamine metabolite patterns in brain tissue did not change following treatment with nitisinone, with the exception of the trace amine tyramine, which increased 25-fold ,26,27. TIn the absence of biological group QCs, only system QC samples were run. https://www.metabolomicsworkbench.org/, Study ID: adavison_20220505_020022_mwtab.txt and data track ID: 3248, accessed 5 May 2022), respectively, using an in-house AMRT database. After filtering entities based on their frequency  and variability across the experimental groups (CV < 25%), 82 and 51 entities were retained from positive and negative polarity profiling experiments, respectively. Of these, five were shown to have a log2 fold change (FC) > 2.0, 3 of these entities were shown to be significantly different (p < 0.05), respectively  between the BALB/c Hgd−/− mice that did and did not receive nitisinone therapy.Clear separation was observed between the CSF metabolite profiles from mice in the two treatment groups . These, however, had raw signal abundance CVs of 53 and 64%, 100 and 61% in the mice that were and were not treated with nitisinone, respectively. These metabolites were therefore excluded from further statistical analysis. Interestingly, homogentisic acid (HGA) was not observed in any of the CSF samples analysed, including those that had not received nitisinone.m/z of each metabolite in positive and negative polarity, respectively.In this study, the monoamine metabolites HVA, DP, VMA, tyramine, and kynurenine were not matched using the targeted feature extraction in Profinder or through manual extraction of chromatograms in MassHunter Qualitative software (Build 07.00) using the Hypertyrosinaemia is a universal biochemical consequence of nitisinone treatment in AKU ,5,6,7,8 The study presented herein is the first of its kind to report on the assessment of the CSF metabolome in a murine model of AKU. The reason for the paucity of the literature likely reflects the rarity of AKU, but also the small volume of CSF that can be collected from a mouse (~3 µL). The latter point presents a significant analytical challenge in terms of sample handling  and also in that the metabolites of interest are present at picomolar to nanomolar concentrations. Moreover, the technical expertise required to collect non-blood-stained CSF from a mouse means the procedure is not frequently performed without some sample contamination.p < 0.05) following treatment with nitisinone. These changes have previously been reported following treatment with nitisinone in serum [Three metabolites were shown to have an increased abundance  tryptophan and phenylalanine were observed in this study, suggesting that the excess L-tyrosine observed following nitisinone treatment does not compete for uptake into the CNS via the LAT-1 transporter. This contrasts with what has been proposed in PKU or HT1, where the excess of phenylalanine and L-tyrosine, respectively, have been proposed to alter neurotransmitter metabolism ,45,46,47Encouragingly, MHPG  and adrenaline, 5-HIAA (serotonin metabolite) and DOPAC (DP metabolite) were also not significantly altered in nitisinone-treated mice. These metabolites did, however, show large variation in their respective treatment groups, so it is important to not over-interpret this trend. In contrast, in a recent study by Pilotto et al. , patientIt is uncertain whether the high variation in metabolite abundance was a reflection of ion suppression due to the CSF matrix, or indeed a limitation of the instrumentation utilised herein. Previous studies have utilised quantitative high pressure liquid chromatography with fluorescence or electrochemical detection ,49 for tIt should be borne in mind that a major limiting factor in this unique study was the very limited sample volume available for analysis. This precluded sample clean up or concentration, or indeed the application of a quantitative LC-MS/MS methodology with the use of an internal standard and matrix matched calibrators. One approach adopted by previous authors  has beenDisappointingly, DP, and its major metabolites 3-MT and HVA, and the major metabolite of adrenaline, VMA, were not detected in the samples analysed. The reasons for this are not known, as these metabolites were present in the AMRT database used in this study and are likely to be present at similar concentrations to 5-HIAA, as they are in humans and have a short half-life . The predata not shown), in which pooled CSF samples from respective treatment groups were analysed. This meant a larger sample volume was available for analysis and samples were not diluted. Disappointingly, this strategy did not improve the detection of 3-MT, HVA, tyramine, or VMA, meaning that the impact of hypertyrosinaemia on DP metabolism in CSF could not be fully evaluated.To investigate the impact of diluting CSF samples on the detection of metabolites, additional experiments were performed . Methanol, acetonitrile, and isopropanol were purchased from Sigma Aldrich . Formic acid and ammonium formate were obtained from Biosolve, . All reagents were LC-MS grade. The 2 mL amber screw vial and 150 µL glass inserts with polymer feet  were purchased from Agilent  and artificial CSF from Harvard apparatus . All analytical standards were obtained from Sigma (UK) as 1 mg/mL Cerilliant standards.Hgd−/− mice . A total of seventeen BALB/c All mice were culled by carbon dioxide asphyxiation and CSF was removed from the cisterna magna following the puncture technique of Liu et al. , with soMurine CSF samples were prepared by diluting 2 µL CSF with 2 µL deionized water (DIRECT-Q 3UV Millipore water purification system). CSF samples were pipetted directly into a 150 µL glass insert with polymer feet, which sat in a 2 mL amber screw vial . These vials were used to ensure maximal recovery of samples. Deionised water was also added directly to the glass insert, and mixed with the CSF sample using a pipette. The glass vials were centrifuged at 600 rpm for 10 min to ensure the diluted sample was at the bottom of the vial for sampling. Then, 1 µL of diluted CSF was analysed in negative and positive polarities, respectively.As the CSF sample volume was very limited (~3 µL per mouse), biological group QCs were not prepared. The performance of the analytical run was assessed using system quality control samples that were prepared in artificial CSF . These contained phenylalanine (100 µmol/L), L-tyrosine (100 µmol/L), nitisinone (10 µmol/L), and succinylacetone (30 µmol/L). Phenylalanine, L-tyrosine, and nitisinone were assessed in positive polarity and succinylacetone in negative polarity. Each analytical run  commenced with 20 replicate injections of the system quality control sample to condition the system.The order of individual samples was randomised computationally. System QC samples were interspersed throughout the analytical sequence every sixth injection, to assess the analytical system performance. Binary pump pressures and reference ions were also monitored to assess the performance of the LC-QTOF-MS .LC-QTOF-MS conditions used for the analysis of murine CSF samples were the same as those previously reported for the analysis of urine  and seruData were acquired using Acquisition . Quality checks and processing of raw data files (Agilent ‘.d’ files) were performed with Qualitative Analysis software .Extracted ion chromatograms of reference masses were performed to check mass accuracy remained <5 ppm throughout the run, and that the reference ion signal did not drop out during the chromatographic run. In addition, to check chromatographic reproducibility, binary pump pressure curves for injections across each analytical sequence were overlaid. Mass accuracy and chromatographic reproducibility were acceptable for all experiments performed.+, Na+ and NH4+ (positive polarity) and H- and CHO2− (negative polarity). Dimers were allowed for both polarities. The charge state range was 1–2. Data files were then exported from Profinder as ‘.CEF’ files as a whole batch for each profiling experiment, and imported to Mass Profiler Professional (MPP) software for statistical analysis .The acquired profiling sample data were mined for signals against an established in-house AMRT database of compounds  ,26,27 usIn the absence of biological group QC samples (due to low CSF sample volume), analytical performance was assessed using spiked artificial CSF samples. Criteria for deeming the analytical run as acceptable were: CV of raw area abundance for target compounds <15%; mean error from target mass <5 ppm; and mean difference from target retention time <0.3 min.p value was <0.05 and a log2 FC >2.0. Principal component analysis (PCA) employing a 4-component model was performed on each filtered dataset using MPP.In MPP, sample data files were scaled to the median of all samples, and chemical entities were filtered based on their frequency  and variability across the experimental groups (CV < 25%). Statistical analyses performed in MPP were based on compound signal intensity, expressed as total peak area. Profiles from the experimental group that received nitisinone were compared to the profiles that did not receive nitisinone using a moderated t-test with Benjamini–Hochberg false-discovery rate adjustment. Log2 fold change was calculated between each experimental group, and this was based on raw peak area data. Changes were deemed significant if the asymptotic This is the first study to report the impact of treatment with nitisinone on the CSF metabolome, and specifically on monoamine neurotransmitter metabolites. This study showed that L-tyrosine and a small number of related metabolites had a greater abundance in mice that were treated with nitisinone. Of particular interest is the observation that the abundance of HPA increased following treatment with nitisinone, suggesting that there may be an increase in the turnover of the trace amine tyramine. Further work is required to understand the significance of these findings and the mechanisms by which the altered metabolite abundances occur. This in turn will help inform the implications these changes may have in patients with hypertyrosinaemia.


Periodontitis (PO), a chronic microbially-induced inflammation of the supporting tissues of the tooth, is linked to various systemic diseases. We analyze its bidirectional relationship to chronic kidney disease (CKD), a major health-care problem with impressive excess mortality. Overwhelming associative relationship between CKD and PO are analyzed. Major pathophysiologic mechanisms that link CKD to PO are then presented: systemic inflammation, endothelial dysfunction, and imbalance of oxidative stress characteristic of CKD have a role in PO development and might influence escape mechanisms of oral microbiota. Subclinical local and systemic inflammation induced by PO might influence in turn CKD outcomes. Homeostatic changes induced by CKD such as mineral bone disorders, acidosis, uremic milieu, or poor salivary flow are also relevant for the occurrence of PO. There is insufficient evidence to recommend a standardized diagnostic and therapeutic approach regarding association of PO to CKD. Chronic kidney disease (CKD) is a major socio-economic and health-care problem, with a global prevalence of almost 7 billion people worldwide . GloballThe leading cause of CKD is diabetes, followed by hypertension and glomerular diseases , but irrTraditional cardiovascular risk factors, such as those identified in the Framingham hearth study , fail toPO is a highly prevalent disease, affecting around 50% of the population . InvestiPO is a chronic microbially-induced inflammation of the supporting tissues of the tooth that results in loss of periodontal attachment, alveolar bone, and finally of the tooth. The homeostasis of the periodontal tissues is ensured between the hosts’ immune-inflammatory reactivity and the subgingival microbiome . FactorsPOs’ severity is influenced by various pathological conditions and, in turn, PO may also impact systemic health, and it has been independently associated with many chronic noncommunicable diseases. The evidence supporting these associations mainly focused on diabetes, pregnancy, and cardiovascular disease, but other conditions such as obesity, auto-immune conditions, certain cancers, respiratory diseases, and cognitive disorders including Alzheimer’s disease must be considered ,17,18. LThis review aims to present associative relationships between these two conditions and report concepts on the major pathophysiologic mechanisms that link CKD to PO, such as particularities of oral microbiota, inflammation driven by innate and adaptative immunity, endothelial dysfunction, and imbalance of oxidative stress. Lastly, we will discuss the treatment of periodontal disease in CKD patients.Prevalence of PO in CKD: PO is more common in pre-dialysis CKD patients than in the general population, and this has been consistently described in various cohorts ,22,23,24Prevalence of CKD in PO patients: Conversely, kidney disease, defined as decreased glomerular filtration rate (GFR), hematuria, or albuminuria, was more frequently reported in patients with PO in different cohorts [ cohorts ,34. Thes cohorts ,37,38,39 cohorts .Link of severity of PO and CKD: There seems to be a link between severity of PO and that of CKD: PO is more severe in CKD than in the general population [pulation , subjectpulation ,40, and pulation ,42 and ppulation ,43.From cross-sectional studies, causality cannot be inferred; indeed, some overlap of CKD and PO might be expected, as they are both very common conditions, and prevalence increases with age. However, there is mounting evidence that CKD and PO might have common pathophysiologic mechanisms, based on increased inflammatory state, impaired immune clearance, and other factors such as xerostomia, gingival hypertrophy, or uremic milieu. PO was predictive for occurrence of CKD in large epidemiologic studies such as NHANES III   or otherComplex statistical analysis revealed that the association of PO with CKD has most likely bidirectional determinism. As such, in a recent analysis data from a prospective study (RIISC)  with longitudinal data on PO, kidney function, and oxidative stress available, structural equation modelling was used to test causal assumptions between PO and kidney function. This study was positive in that 10% increase in periodontal inflammation resulted in a 3.0% decrease in renal function and a 10% decrease in renal function resulted in a 25% increase in PO, and the common denominator is thought to be oxidative stress .Genetic predisposition does not seem to be an important pathogenic factor between PO and CKD: using genome-wide association studies, single nucleotide polymorphisms (SNP) identified as genetic causes of PO were not related to kidney function and vice versa, and multiple SNP identified as genetic causes of CKD were not associated with PO, using 2 large genetic databases of PO  and of CKD (Chronic Kidney Disease Genetics—CKDGen Consortium) .The main cause for the increase in mortality of patients with both CKD and PO is the renal condition. As a result, PO can be studied as a modifier of this risk. Not only is there an epidemiologic link between CKD and PO, but the presence of periodontal disease was found to exacerbate cardiovascular morbidity  and the However, negative studies are also reported: a recent large database study from Taiwan does not support the idea that PO increases mortality in CKD patients or that it influences progression of kidney disease , and in 2 or the presence of one of more markers of kidney damage, such as albuminuria, urine sediment abnormalities, electrolyte, and other abnormalities due to tubular disorders, abnormalities detected by histology, structural abnormalities detected by imaging, and history of kidney transplantation. The staging of CKD relies on a decrease of estimated GFR and includes 5 categories: G1 (GFR > 90 mL/min/1.73 m2), G2 (GFR 60–89 mL/min/1.73 m2), G3a (GFR 45–59 mL/min/1.73 m2), G3b (GFR 30–44 mL/min/1.73 m2), G4 (GFR 15–29 mL/min/1.73 m2), and G5 (GFR < 15 mL/min/1.73 m2). Three categories for albuminuria are also defined based on urinary albumin to creatinine ratio or 24 h urinary albumin excretion: A1 (<30 mg/24 or mg/g creatinine), A2 (<30–300 mg/24 or mg/g creatinine), and A3 (>300 mg/24 or mg/g creatinine). The combination of the G and A categories defines severity of CKD, risk of progression, risk of cardiovascular morbidity and mortality, and need for intervention. This definition, dating back to 2012, is globally implemented. Nevertheless, across studies that addressed the link of PO to CKD GFR, estimation equations used are not uniform and GFR thresholds used for inclusion of patients are widely different, with many studies including only dialysis patients.Controversial findings in different studies are at least in part due to disparate sets of criteria that have been used to diagnose PO and CKD. Currently, according to KDIGO  , CKD is Periodontal diseases represent a wide range of inflammatory conditions that affect the supporting structures of the teeth , which could lead to tooth loss and contribute to systemic inflammation . The curThe human microbiome consists of the totality of microorganisms in the human body, which are distributed in various organs, with the oral cavity being an important site. Changes in the composition of microbiome or in its metabolites can trigger and aggravate several systemic diseases . The humAlterations in systemic or local homeostasis triggered by disease in distant organs could be a major factor in altering oral microbiota. As such, the interrelation of CKD and gut microbiome is well-documented, but changes of oral microflora in CKD are less studied . Oral keAntibodies to selected periodontal pathogens, including Porphyromonas gingivalis, are associated with increased odds of having an impaired renal function .Alterations in oral microflora can influence distant organs in various ways, such as translocation and bacteremia or toxic effects of bacterial compounds , but theLPS-mediated activation of the innate immune system might also have systemic consequences relevant for distant organs, such as the kidney. In fact, endothelial cells express TLR2 and TLR4 in diabetic environment, including in renal microcirculation and translocated Porphyromonas gingivalis from the subgingival biofilm, bind to these cell surface receptors, activating endothelial cells and causing overexpression of adhesion molecules like VCAM1 and E-selectines. This, in turn, leads to leukocyte margination and glomerular, as well as tubulointerstitial inflammation .On the other hand, CKD-induced alterations of microenvironment also play a permissive role: both innate and adaptive immunity are impaired. In CKD, antigen presentation capabilities of dendritic cells and macrophages are diminished , leadingPathogenic mechanisms linking PO to CKD are depicted in CKD is a proinflammatory state characterized by an increase of inflammatory markers, including cytokines. Multiple lines of evidence have supported a direct pathogenic role for inflammation in progression of CKD, but also in development of various complications such as malnutrition, coronary artery calcification, atherosclerosis, cardiovascular disease, and enhanced CKD mortality. Relevant proinflammatory molecules that are increased in CKD are interleukin (IL) 6, TNFα, and adhesion molecules and adipokines ,82. A stPO is also a cause of subclinical inflammation and associated with an increase of markers of systemic inflammation . In theiIn CKD patients, the association of PO with increased inflammatory markers, such as C- reactive protein (CRP), has repeatedly been reported; in a recent systematic review this issue was studied. With 2 exceptions out of 8 reported studies, CRP was significantly elevated in CKD patients with PO . Low albOne special mention is needed regarding the contribution of matrix metalloproteinases (MMP) in pathogenesis of PO- and CKD-induced lesions. MMPs are a group of enzymes involved in tissue repair and apoptosis, and some of them are upregulated during periodontal inflammation . In the Endothelium is much more than a mechanical barrier separating blood from tissues; it exerts active regulatory functions on the balance of oxidative stress, vasoconstriction/vasodilation, platelet aggregation, and leukocyte adhesion. PO can influence all these functions and, given the fact that kidneys receive up to 25% of cardiac output to assure glomerular filtration, the effects of PO on glomerular endothelial function are significant ,95.Porphyromonas gingivalis LPS has been detected in various endothelial cells; it triggers induction of increased reactive oxygen species (ROS), followed by NF-kB-induced inflammation, polynuclear adhesion, and cell apoptosis. ROS produced by leukocytes during inflammation are an important defense mechanism in PO targeting bacterial DNA , howeverAs mentioned before, an analysis of a large longitudinal cohort seems to indicate that CKD and PO are reciprocal risk factors, and that one important mechanism for their interaction is oxidative stress .PO also influences vasodilation/vasoconstriction balance; endothelial dysfunction measured by flow-mediated vasodilation of the brachial artery is associated with PO and various markers of inflammation .glomerulosclerosis, and interstitial fibrosis, exacerbating the severe inflammatory processes already underway. At a systemic level, CKD-related oxidative stress is also responsible for several pathological conditions contributing to morbidity and mortality such as atherosclerosis and cardiovascular disease. Last, but not least, ROS are able to further enhance the inflammatory response by triggering pro-inflammatory mediators  [Oxidative stress is frequently observed in CKD and dialysis patients and is nowadays considered a non-traditional risk factor for all-cause mortality in CK ,101. Elecascade) .Alterations to internal homeostasis of CKD that might be relevant for pathogenesis of PO are depicted in The term ‘mineral bone disorders’ depicts the whole spectrum of abnormalities of biochemical parameters, bone structure changes, and vascular calcifications secondary to altered mineral metabolism of CKD. Among biochemical parameters, hypocalcemia, hyperphosphatemia, increased fibroblast growth factor 23 (FGF23), parathormone (PTH), and alkaline phosphatase, as well as 1,25-dihydroxy vitamin D3 deficiency, are the most common findings. It is not clear to what extent mineral bone disorders of CKD might be linked to PO. One study identified positive correlation of serum calcium and phosphate to the severity of PO reflected by CAL  . HoweverIn a small case-control study, vitamin D deficiency was more severe in patients with CKD and PO than in controls without PO ; clinicaIn a mouse model of diabetic nephropathy Porphyromonas gingivalis, LPS exposure induced accumulation of FGF 23 in kidneys . FGF 23 Metabolic acidosis is yet another homeostatic imbalance associated to renal disease: usually high anion gap acidosis, due to accumulation of phosphate and sulphate containing acids, is found, but hyperchloremic acidosis due to tubular dysfunction might also contribute; significant acidosis becomes prevalent in stage 4 CKD and is constant in dialysis patients. Long-standing acidosis is known to cause bone loss and might contribute via decreased support of alveolar bone to PO .Saliva is a major controlling factor of intraoral biofilm; decreased salivary flow and decreased oral hydration can cause both gingivitis and PO. Xerostomia is an important symptom of CKD; it might be present in pre-dialysis phases in certain conditions, e.g., rheumatic disease such as Sjogren syndrome. Singh et al. demonstrated that, in Sjogren syndrome patients, the number of sites with CAL ≤ 4 mm was significantly higher compared to the healthy controls . DecreasIncreased salivary urea, whose hydrolysis engenders an alkaline salivary pH , can infFor unclear reasons, in end-stage renal disease patients, dialysis modality might play a role with HD, having a negative impact on oral health and on indices like visible plaque index, CAL, and candida colonization. The effect is reversible when switching to PD .Association of PO with other comorbidities of CKD should be considered when analyzing a possible pathophysiologic link between these conditions. Extensive evidence suggests that diabetes is more prevalent in PO patients, with these facts confirmed in a recent systematic review . DiabetiAs noted above, cardiovascular disease and associated mortality are major problems in CKD, both in predialysis and in dialysis stages. PO is recognized as a major contributor to atherosclerosis via endothelial disfunction, inflammation, and progression of atherosclerotic plaques ,126. It Immunosuppressive treatment  can also contribute to propagation of PO. The bacterial species associated with PO in immunosuppressed patients depends on the immunosuppression regimen, with certain species (Pavimonas micra and Capnocytophaga species) favored by corticoid and mycophenolic acid salt therapy. Worse periodontal condition is described in patients under cyclosporine when compared to other regimens . HoweverA special mention needs gingival overgrowth, a common side-effect of calcineurin inhibitors used for treatment of various glomerular conditions and for kidney transplantation. In the latter setting, up to 10% of patients have gingival hypertrophy . TacroliTreatment of CKD is standardized; a part of the etiologic treatment, it is addressed for halting progression of CKD, treating complications and comorbidities, and replacing renal function by dialysis or transplantation. We have found no prospective studies or trials on the impact of a specific CKD treatment on PO as an outcome.Standard therapy in PO is outlined in the current treatment guidelines  and consAmong prophylactic measures, oral hygiene is important, from the perspective of it being correlated to gingival overgrowth . ImproveRegarding treatment of PO, there is indirect evidence of the potential benefit of non-surgical treatment on inflammatory markers: intensive treatment of PO was found to be associated to decrease of markers of inflammation and endothelial dysfunction  91,98,1,198,137 There are few prospective studies with endpoints defined according to renal or PO outcomes in these patients at risk. Non-surgical treatment of PO lead to improved renal function in small prospective studies ,140,141,Treatment of PO in CKD patients may be more difficult, as pathogenic subgingival microbiota tends to persist after periodontal treatment .Given the vast evidence of association of PO to CKD, one should expect increased awareness of this matter in both renal physicians and dentists in order to generate evaluation protocols and treatment guidelines. However in this matter there is room for improvement: most renal physician and nurses, despite having correct knowledge on PO, do not routinely inspect oral cavities of patients, and they refer them to dental care in less than 30% of cases . AdditioPO is not listed as a complication for CKD and not addressed by current KDIGO guidelines. Additionally, current PO guidelines  do not rOur review aims to present data on the interplay of PO and CKD, a necessary step given the overwhelming evidence of epidemiologic association between the two conditions and their respective impact on general health. The strength of our review is the complex perspective from both nephrologists’ and dentists’ point of view regarding pathogenetic pathways that can play a role in the bidirectional relationship between the two conditions. Understanding mechanisms of oral and systemic inflammation and homeostatic imbalances in PO patients with CKD is key to efficient intervention. The limitations of our work are the inability to draw conclusions on best clinical practice in PO patients with CKD due to insufficient data derived from clinical trials or prospective studies, regarding cost-effectiveness and outcomes of specific therapeutic measures. This holds true when referring to the lack of data on optimal evaluation of these patients.There is overwhelming evidence of the association of CKD and PO, however a few studies also support directional relation between the two pathologies. PO and CKD share common pathogenic mechanisms such as protracted, inefficient inflammation, oxidative stress, and endothelial dysfunction; PO may be facilitated by homeostatic changes found in CKD, comorbidities, or immunosuppressive treatment. Further prospective studies as well as randomized controlled trails, especially clarifying effects of PO treatment on renal outcomes, are needed for better insight into the relationship between CKD and PO.


The choroid plexus (CP) is a delicate and highly vascularized structure in the brain comprised of a dense network of fenestrated capillary loops that help in the synthesis, secretion and circulation of cerebrospinal fluid (CSF). This unique neuroanatomical structure is comprised of arachnoid villi stemming from frond-like surface projections—that protrude into the lumen of the four cerebral ventricles—providing a key source of nutrients to the brain parenchyma in addition to serving as a ‘sink’ for central nervous system metabolic waste. In fact, the functions of the CP are often described as being analogous to those of the liver and kidney. Beyond forming a barrier/interface between the blood and CSF compartments, the CP has been identified as a modulator of leukocyte trafficking, inflammation, cognition, circadian rhythm and the gut brain-axis. In recent years, advances in molecular biology techniques and neuroimaging along with the use of sophisticated animal models have played an integral role in shaping our understanding of how the CP–CSF system changes in relation to the maturation of neural circuits during critical periods of brain development. In this article we provide an ontogenetic perspective of the CP and review the experimental evidence implicating this structure in the pathophysiology of neurodevelopmental and neuropsychiatric disorders. The strA diversity of ion channels, pumps and transporters are expressed at the basolateral and apical membranes of the CPE cells that modulate the net flux of sodium, chloride, and bicarbonate ions across the CPE from the blood-to-CSF side . This geChoroid plexus development begins at week 7 of gestation with mesenchymal cells invaginating into the neural tube at the sites of cerebral ventricles formation, starting with the fourth, lateral, and then third ventricles , 29. TheAlthough little is known about the molecular mechanisms directing fenestrated vessel formation in the CP, a recent study using zebrafish larval CP and CPEs demonstrated ultrastructural conservation of the CPs across vertebrate species . SpecifiThe CP serves a diversity of physiological functions in the CNS ranging from CSF production, neural stem cell behaviour and immune cell trafficking to modulating the microbiome gut-brain axis, circadian rhythms and cognition  can access the brain parenchyma via the CP and circulate within the CSF . Specifind Cxc10 . Notablynd Cxc10 . In anotnd Cxc10 . This stnd Cxc10 . The autnd Cxc10 . These fnd Cxc10 , 67.The CP also plays a role in gut microbiota-immune interactions . NotablyHelicobacter suis (H. suis) infection [H. suis-evoked gastrointestinal permeability and subsequent peripheral inflammation induces changes in brain homeostasis via changes in blood-CSF barrier integrity [A growing stream of evidence has linked alterations in gut microbiome composition to the anomalies in CSF observed in neuropsychiatric disorders . In thisnfection . These cntegrity . Interesntegrity . The samntegrity .Per1, Per2, Cry1, and Cry2 genes whose protein products PER and CRY repress their own transcription [Bmal1, Per1, Per2 and Cry2 fluctuating throughout the day [Per1, Per2, and Bmal1 genes in the lateral and fourth ventricles was modulated in a circadian manner and may control CSF secretion [Circadian rhythms are a fundamental biological phenomena in almost all organisms, and control not only rest/activity rhythms but also a variety of other physiological functions such as hormone secretion, blood pressure, and body temperature regulation . The molcription . Animal cription , with ex the day . Recentl the day , a proce the day . In a stecretion . Other eecretion .The structural and functional implications of the CP in neuropsychiatric and neurodevelopmental disorders have been neglected for many years . HoweverCP abnormalities in schizophrenia are described as early as 1921 demonstrating morphological changes to the CPE, vascular endothelium, as well as hypersecretion , 88. SevOur group previously reported CP volume enlargement in a large sample of patients with schizophrenia, schizoaffective, and bipolar disorder with psychosis compared to both their first-degree relatives and healthy controls . GreaterConsistent with the neuroimaging findings above a recent study reported a larger CP volume in schizophrenia patients with orofacial tardive dyskinesia (TD) . This stThe CP has gained significant attention since the advent of the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), commonly referred to as the COVID-19 pandemic. While the virus primarily impacts the respiratory system, neurological complications, including cerebrovascular injury, encephalopathy, neuropsychiatric and neurocognitive disorders have also been described . Viral gSimilar to schizophrenia, there is a growing interest in understanding CP morphology in autism. The expansion of cranial ultrasounds led to the identification of minor abnormalities in neonates, such as subependymal pseudocysts, frontal horn cysts, or CP cysts. In a large sample of neonates receiving cranial ultrasound testing one week after birth, these patients were prospectively followed and received repeated neurodevelopmental assessments between the ages of 5 and 15. The authors found that while CP and frontal horn cysts were not associated with autism risk, subependymal cysts were associated with developmental delay in 5.5% of children and the odds ratios for developing autistic spectrum disorder was 28.54 . In a laCerebral folate deficiency (CFD) syndrome is a neuropsychiatric condition associated with low CSF 5-methyltetrahydrofolate (MTHF) and normal folate in the blood . This syThe high metabolic activity  and well developed extracellular matrix (ECM) of the CP provide important neuropathological implications for when the CP is altered. Notably, the CP can be induced to elicit neuroinflammatory responses in the presence of peripheral insults . FurtherUnderstanding the mechanisms by which homeostatic disturbance at the CP–CSF interface is involved in neurodevelopmental and neuropsychiatric disorders can give new insights into therapeutic strategies. Delivery of therapeutic agents to the CNS is challenged by the barriers in place to regulate brain homeostasis. This is especially true for protein therapeutics. Targeting the CP barrier may be a surrogate brain delivery strategy to circumvent the BBB. However, as noted above for CFD the treatment would involve providing additional folate to overcome the FRα autoantibodies or FLOR-1 mutations . HeterogMore recently, exosomes—a type of extracellular vesicle that contain constituents  of the cells that secrete them—have been recognized as potential therapeutic conveyors for brain-related diseases given their known role in the modulation of transcription, neurogenesis, plasticity, and neuroinflammation, each of which are affected in neuropsychiatric and neurodevelopmental disorders . In thisThe unique anatomical localization of the CP directly influences brain function through CSF mediated transport of secreted proteins such as cytokines and hormones that in turn regulate neuronal signalling, brain plasticity and cognition. The recent discovery of CSF clearance routes, namely glymphatic efflux and the meningeal lymphatic network are of particular significance given the high metabolic rate and exquisite sensitivity of neurons and glia to alterations in their extracellular environment. It follows that understanding how the CP modulates CSF homeostasis including the entrance, maintenance, and exit of immune populations, extracellular proteins and micronutrients will provide an important framework to understand how perturbations in these key steps have an impact on brain development and are disrupted in neurodevelopmental and neuropsychiatric disease. In this context, the use of recently developed technologies such as iDISCO that enable high resolution imaging of structures deep within the brain—including the CP , 134—wil


To investigate the association between serum total testosterone (TT) levels and metabolic syndrome (MetS) or its components among adult women.2,678 women from NHANES 2011-2016 were included in this cross-sectional study. MetS was determined according to the National Cholesterol Education Program Adult Treatment Panel III guidelines. The association between serum TT levels and MetS was evaluated by two logistics regression models and the adjusted restricted cubic spline (RCS). Stratified analysis and sensitive analysis were also conducted.p for trend < 0.001). Similar trend was observed in Model B. Multivariate-adjusted logistic regression with RCS exhibited that TT had a L-shaped dose–response association with MetS or its components. Interaction analyses revealed that women who were less than 50 years old , with depression  or being smokers  showed lower ORs than those who were over 50 years old , without depression  or non-smokers  when measure the association between ln TT and the occurrence of MetS.Continuous TT levels were negatively associated with the occurrence of MetS, and the ORs associated with per SD increase in ln TT were 0.70 (95%CI: 0.58-0.85) in 2011-2014 and 0.56 (95%CI: 0.39-0.79) in 2015-2016 in Model A. High TT group were less likely to have MetS  when compared to the low TT group. When TT levels were divided into quartiles, TT levels were negatively correlated with the incidence of MetS (Our study indicated that TT levels are negatively correlated with the occurrence of MetS, with interaction effects of age, smoke behaviors, and depressive status. These abnormalities would increase the risk of cardiovascular disease and all-cause mortality –3. The pAlthough previous studies have reported that sex hormones are related to an increased cardiometabolic risk and mortality, including MetS, type 2 diabetes mellitus (T2DM), and hypertension –8, the rThus, this cross-sectional study aims to investigate the association between TT levels and MetS or its components among adult women by using the large and nationally representative survey of the US population from NHANES 2011-2016. This work may provide insightful suggestions on the impact of TT levels on women, which may further be derived to clinical evidence on the controversy of hormonal therapies for women in preventing and managing MetS.22.1NHANES  is a majhttps://www.cdc.gov/nchs/nhanes).All NHANES data collection protocols were approved by the National Center for Health Statistics Institutional Review Board and all participants signed an informed consent. NHANES survey data, detailed survey operation manuals, consent documents, and brochures of each period are publicly available on the NHANES website , women who were less than 20 years , pregnant (n=155) and lactating women (n=108) from our analysis. Those with missing information on TT, MetS or covariates were further excluded. Therefore, the final sample size of our analysis was 2,678 women Figure 12.3The diagnosis of MetS was determined according to the National Cholesterol Education Program Adult Treatment Panel III guidelines . The stu2.4The serum samples were processed, stored, and transported to the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health for analysis. The concentration of TT was performed by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method, which was developed by the CDC. Detailed quality control and quality assurance instructions were discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM).2.53. The neutrophil-to-lymphocyte ratio (NLR) was measured as the ratio of neutrophil count-to-lymphocyte count.Based on previous studies and clinical plausibility, the following covariates were included in our analysis to reduce potential confounding bias. Information on age, race , education , marital status , and ratio of family income to poverty  were obtained through self-reports of demographic questionnaires. The body mass index  was categorized into 4 groups: underweight (<18.5 kg/m2), normal weight (18.5-24.9 kg/m2), overweight (25.0-29.9 kg/m2), obesity (≥30 kg/m2). Alcohol use referred to participants who had at least 12 drinks of any type of alcoholic beverage in any one year. Alcoholic beverages included liquor, beer, wine, wine coolers, and any other type of alcoholic beverage. Information about smokers was that participants who had smoked ≥100 cigarettes in their lifetimes. NHANES has incorporated the Patient Health Questionnaire (PHQ-9) since 2005, which is a self-reported assessment based on nine signs and symptoms of depression over the past two weeks. The score for each item ranged from 0 to 3, and the total score for each participant ranged from 0 to 27. A total score of ≥5 was used as the cut-off for depression. Participants were asked “Ever told doctor had trouble sleeping?”, and those who answered “yes” were considered to have sleep disorders. The total caloric intake per day was estimated based on the types and amounts of foods and beverages  consumed during the 24-hour period prior to the interview (midnight to midnight). Lymphocyte and neutrophil counts were assessed using automated hematology analysis devices and expressed as ×1,000 cells/mm2.6p values were 2-sided, and the significance level was set at 0.05. R software (Version 4.1.2) was used for statistical analysis.Categorical variables were expressed as percentage (%) and compared using the chi-square test or Fisher’s exact test when appropriate. Continuous data with normally distributions were expressed as mean (± standard deviation [SD]) and compared by independent samples t-test. Variables with skewed distributions were expressed as median (interquartile range [IQR]) and compared using the nonparametric Wilcoxon rank sum test. In the data analysis of NHANES 2011-2014, we used two logistics regression models to investigate the association between serum TT levels and MetS among women. Model A only adjusted for age, race, and BMI. Model B further adjusted for other demographic characteristics, lifestyle variables, and some related health indicators: education level, marital status, ratio of family income to poverty, alcohol use, smoking-cigarette use, depression, sleep disorders, total caloric intake per day, and NLR. We also evaluated the presence of nonlinear dose-response relationships between ln-transformed TT (ln TT) and MetS or its components by the adjusted restricted cubic spline (RCS) with three knots . We measured the interaction of TT with various clinical parameters with product interaction terms. To investigate the modified effect of age, depression, smoking status and TT on the incidence of MetS, we stratified the study population by age (less than 50 years or above), depression status (No or Yes) and smoke (No or Yes). We also performed sensitive analysis after excluding participants who were taking antidiabetic drugs, hypertension medication or lipid-lowering medication or women with ovary removed, and comparing results across samples over different time periods. For the female population from NHANES 2015-2016, we also used two logistics regression models with increasing degrees of adjustment to evaluate the association between TT levels and the odds ratio of MetS. Meanwhile, we also stratified women by age (less than 50 years or above) to determine the relationship between TT levels and MetS at different age stages. All reported 33.1p<0.05). The median (IQR) of TT levels in MetS group were 17.80  ng/dL, which were lower than that in non-MetS group . In general, TT levels were negative correlated with age (The baseline characteristics of the study population with weighted estimates were presented in 3.2p for trend < 0.001). Women in Quartile 4 (30.10-575.00 ng/dL) in 2011-2014, Quartile 3 (20.40-28.10 ng/dL) and Quartile 4 (28.30-444.00 ng/dL) in 2015-2016 showed statistically significant lower odds of MetS. Similar trend that a statistically lower occurrence of MetS was observed among women with high TT levels were obtained in Model B: OR=0.70 (95%CI: 0.56-0.87) in 2011-2014 and OR=0.57 (95%CI: 0.33-0.99) in 2015-2016 per SD increase in ln TT; OR=0.61 (95%CI: 0.45-0.81) in 2011-2014 and OR=0.47  in 2015-2016 in high TT group. Multivariate-adjusted logistic regression with RCS exhibited that TT levels had a L-shaped dose–response association with MetS or its components, when analyzed the association between TT levels and the incidence of MetS or its components (3.3p for interaction < 0.05). As shown in p > 0.05). In addition, when the TT levels were divided into quartiles, there was no statistical difference in the occurrence of MetS between the groups, except for women in Quartile 4 who had a decreased risk of MetS than that in Quartile 1.To investigate the potential modified effect of TT levels and the other factors on the occurrence of MetS, we stratified the study population with the mentioned modifiers. Interaction analyses revealed that TT levels were significantly associated with MetS for the following factors: age, depression, and smoking behaviors (all 3.4Given the observation that the concentration of TT was prognostic for women with MetS, a sensitivity analysis was investigated. We conducted sensitivity analyses after excluding the participants who were taking antidiabetic drugs, hypertension medication or lipid-lowering medication or those women with ovary removed, and comparing results across samples over different time periods, and similar results were obtained (4We used the datasets from NHANES 2011-2016 for multi-dimensional analysis in order to obtain reliable results of the relationship between TT and Mets in the adult female population. In this large cross-sectional study of nationally representative US adult women, we found that TT levels negatively correlated with the occurrence of MetS, which was in accordance with previous observational studies in the male population , 23, 24.In our study, median testosterone in women was 20.50  ng/dL, and TT levels were negative correlated with age. Our study suggested that TT had a L-shaped dose–response association with MetS and its components, and the relationships between TT levels and the outcome were not exactly the same when the concentration of TT was in different quartile range. Although these results are consistent with some previous studies, the discrepancies in different studies may be associated with different genders and ages. TT levels vary greatly in different genders, ages, or conditions. The normal physiologic range of TT in male population is 450-600 ng/dL , 26, andAndrogen excess is one of the main characteristics of PCOS and is present in 70% of diagnosed women , 31. PCOIn terms of the MetS components, the relationships showed a L-shaped dose–response between TT levels and all components in our study, which was consistent with most previous results in male population , 35. An Given that previous studies on the relationship between TT and MetS in certain conditions or different age groups had obtained different results, we expanded our study by stratified analyses to demonstrate that TT had different impacts on MetS at different ages. Globally, the mean age of natural menopause is around 50 years, with remarkably little geographic variation , 37. TheHowever, the research has several limitations too. In this research, we did not include evaluation of albumin and sex hormone binding globulin levels to calculate free androgen index or bioavailable TT, and did not consider other androgens, such as dehydroepiandrosterone and androstenedione. Besides, the cross-sectional nature of the survey could not determine causal inference. In addition, although we controlled for wide ranges of major potential confounders including demographics, self-reported diseases, medications, lifestyles, and dietary risk factors in the multivariable logistic models, there might be residual confounding from unmeasured factors which could have impacted the effect size estimates. Unfortunately, women with PCOS cannot be excluded from the datasets in this study, which may cause bias.To our knowledge, this is the first large scale epidemiological study that used nationally representative US population data and examined the association between TT levels and the incidence of Mets or its components among adult women of all ages by multi-dimensional analysis which includes stratified analysis and sensitive analysis. In addition, our results are robust after adjustment for a wide spectrum of potential confounders.5The present cross-sectional study indicated that TT levels were negatively correlated with the occurrence of MetS or its components, and TT had a L-shaped dose-response association with MetS or its components. In addition, the trend of negative correlation between TT levels and the occurrence of MetS was more obvious among women who were less than 50 years old, with depression or being smokers than those who were over 50 years old, without depression or non-smokers. However, different trends were observed when we stratified women according to the average menopausal age. There was no statistically significant association between serum TT levels and the occurrence of MetS among women aged 50 or above. Future studies are necessary to determine the cut-off value for abnormal TT levels in women and the impacts of TT on Mets in different ranges.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by National Center for Health Statistics Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.YH contributed to the conception and design of this study. CL and MZ organized the database and CL wrote the first draft of the manuscript. MZ performed the statistical analysis. YZ put forward academic suggestions and revised the draft. All authors contributed to the article and approved the submitted version.


To compare the anaerobic work capacity  assessed using four different approaches/models applied to time-trial (TT) cycle-ergometry exercise.AO). Linear relationships between power output (PO) and submaximal metabolic rate were constructed to estimate TT-specific gross efficiency (GE) and AnWC, using either a measured resting metabolic rate as a Y-intercept (7 + YLIN) or no measured Y-intercept (7-YLIN). In addition, GE of the last submaximal bout (GELAST) was used to estimate AnWC, and critical power (CP) from TTAO (CP3´AO) was used to estimate mechanical work above CP .Fifteen male cyclists completed a 7 × 4-min submaximal protocol and a 3-min all-out TT . The AnWC were 269 ± 60, 272 ± 55, 299 ± 61, and 196 ± 52 J∙kg−1 for the 7 + YLIN, 7-YLIN, GELAST, and CP3´AO models, respectively . For the three pair-wise comparisons between 7 + YLIN, 7-YLIN, and GELAST, typical errors in AnWC values ranged from 7 to 11 J∙kg−1, whereas 7 + YLIN, 7-YLIN, and GELAST versus CP3´AO revealed typical errors of 55–59 J∙kg−1.Average PO during TT3´AO and the other models. The 7 + YLIN and 7-YLIN generated 10% lower AnWC values than the GELAST model, whereas 7 + YLIN and 7-YLIN generated similar values of AnWC.These findings demonstrate a substantial disagreement in AnWC between CP However, due to the different energy equivalents for fat and carbohydrate oxidation, and the effect of submaximal exercise intensity on substrate utilization  or anaerobic work capacity  during whole-body exercise  is calculated as PO above CP integrated over time and is often referred to as a marker of AnWC  method. With the CP concept, the CP threshold equals the maximal PO that is generated by primarily aerobic energy sources and can, at least in theory, be maintained indefinitely, whereas the exercise duration above CP is finite . Although, Hill and Smith  and “MAOD”/”GE” methods used in previous studies  against the AnWC estimated with the MAOD and/or GE methods  using four different models: the 7 × 4-min linear regression method based on PO and MR with, and without, the inclusion of a resting baseline MR value as a Y-intercept ; the GE method using the last exercise intensity (GELAST); and the CP method based on the 30-s average end power as CP (CP3´AO).Given the limited number of methodological studies, the primary aim of the current study was to compare estimates of AnWC and/or AnC generated during a 3-min all-out time trial  the GEW’/AnWC between the CP3´AO versus the 7 + YLIN, 7-YLIN, and GELAST models.2) there would be disagreement in The hypotheses of the current study were as follows: −1 min−1; no previous experience of laboratory cycle-ergometry tests; and injury or illness.Fifteen highly trained male cyclists  were recruited for this study  and carbon dioxide (CO2) sensors were calibrated using a two-point calibration procedure with ambient air conditions and the anticipated expiratory gas percentages using a known calibration gas containing 15% O2 and 5% CO2 . The flow volume was calibrated using a 3-L syringe . Blood lactate concentration was measured via ear-lobe capillary samples (20 μL) that were subsequently analyzed using a Biosen S-line . The Biosen S-line was calibrated with a known standard solution of 12 mmol L−1. The blood samples were collected at the final minute of the 6-min warm-up, directly after completion of the submaximal protocol, 1 min prior to the start of the TT, and 2 min after the TT  equipped with road race handlebars and standard shifting mechanics . Cycling PO was logged continuously as second-by-second data. The bike was fully adjustable to the rider’s preferences . The participant’s stature and body mass were measured before the first test using an electronic scale . Respiratory variables were measured using a Cosmed Quark CPET mixing chamber ergospirometry system  with raw data as 10-s values. This set-up was used to provide valid and reliable metabolic measurements, especially at high ventilation rates in highly trained athletes , raw 10-s respiratory data were transferred to second-by-second data  and smoothed using a 9-s counterbalanced moving average , which was conducted twice. For the start-point of the TTAO, a gradual increase in the smoothing function time window was used up to the fifth second, whereafter the 9-s counterbalanced moving average was used. For the endpoint of TTAO, the same principle was employed, but with a gradual decrease in the smoothing time window over the last four seconds. The highest 20-s moving average during the TTAO was used to calculate Respiratory and heart rate data were collected continuously during the submaximal exercise and the TT−1 and RER values (≤ 1.00) during the final minute of each stage of the submaximal exercise protocol.Energy expenditure was calculated from  by Weir  and thenGE was calculated as:Net efficiency was calculated as:BL is the baseline MR calculated from an average 3-min baseline where MRLIN) or excluded (7-YLIN) from the model. In the latter case, the Y-intercept was based on all data points in the regression but excluding the baseline resting value of MR. The 7 + YLIN and 7-YLIN regression equations were used to estimate the required instantaneous MR during the 3-min TT (MRTT_req) at each 1-s time point. Submaximal GE from the last submaximal stage (GELAST) was also used to estimate the MRTT_req at each 1-s time point of the TT. MRTT_req was calculated by dividing instantaneous PO with GELAST. For illustrative purposes, GELAST was converted to a PO-MR linear equation, where the slope was calculated as the reciprocal value of GELAST, with a Y-intercept of zero due to the constant GE assumption for the GELAST model. The instantaneous second-by-second GE values during the TT were also calculated for the 7 + YLIN and 7-YLIN models (GEREG) as instantaneous PO divided by the instantaneous required MR calculated from the regression equation.A linear relationship between PO (W) and MR (W) during the final minute of each of the 7 × 4-min submaximal stages was derived for each participant with the baseline MR  included  at each 1-s time-point (t) of the TT could then be expressed as:For the 7 + YAE is the aerobic MR calculated according to Eq. where MR−1]) was calculated by integrating MRan over the 3-min TT.For all three models, the total anaerobic energy production   at each 1-s time point (t) of the TT was calculated for the 7 + YLIN and 7-YLIN models as:Anaerobic PO contribution  over the TT duration (s).The AnWC in joules (J) was calculated for the 7 + Y3´AO model, the total mechanical work above CP  was calculated as the PO above CP integrated over time. The CP was determined as the average PO of the last 30 s of TTAO  and expressed as an average TT value.To calculate the average supramaximal GE during the TT for both the 7 + Yα = 0.05. Normality of data was confirmed by visual inspection of Q–Q plots and histograms together with the Shapiro–Wilk analysis. Accordingly, data are presented as mean ± standard deviation, except in the case of peak heart rate and rating of perceived exertion where data are presented as median and interquartile range. In addition, the different AnWC estimates are presented as mean and 95% confidence interval. The PO-MR relationships for the 7 + YLIN and 7-YLIN models were assessed using linear regression analyses. One-way repeated measures ANOVA tests were used to compare GE and net efficiency between the seven submaximal stages as well as the GE, required MR, and AnC associated with the TT. A paired t test was used to compare the linear regression coefficients for the 7 + YLIN and 7-YLIN models. The precision of the two linear regression equations was assessed with the standard error of the estimate. For the ANOVA tests, the assumption of sphericity was assessed using Mauchly’s test. For violated sphericity, a Greenhouse–Geisser correction of the degrees of freedom was used (epsilon ≤ 0.75). Eta-squared effect size was reported for the one-way repeated measures ANOVA tests. Bonferroni α corrections were applied to all ANOVA tests.All statistical tests were processed using Office Excel 2016  and the Statistical package for the social sciences . The level of statistical significance was set at t-test and the standardized mean difference  was reported according to the equations presented by Lakens . The GE increased from the first to the last submaximal stage prior to the TT , whereas the net efficiency remained unchanged during all the submaximal stages prior to the TT .The PO, cadence, physiological responses, and two types of efficiency  at the seven submaximal stages are shown in Table AO was 5.43 ± 0.30 W kg−1 at an average cadence of 92 ± 9 rev min−1. The peak PO was 9.3 ± 1.2 W kg−1 and the peak cadence was 127 ± 19 rev∙min−1. The average cadence during the last 30 s of the TTAO was 90 ± 14 rev min−1. The −1 min−1 (5.0 ± 0.6 L min−1) at an RER of 1.10 ± 0.10. The average −1 min−1, which resulted in an average fractional utilization of 80 ± 3% of −1. The blood lactate concentrations measured 1 min prior to the TT and 2 min after the TT were 2.1 ± 0.5 and 11.5 ± 2.6 mmol L−1. The median rating of perceived exertion value immediately after the TT was 20 (interquartile range = 19–20).The average PO during TTLIN and 7-YLIN models and calculated from GE based on the last submaximal stage (GELAST) with extrapolation up to the TT are displayed in Fig. LIN and 7-YLIN models are displayed in Fig. LAST), and CP are presented in Fig. W’  estimated with the CP3´AO model. The CP was 4.5 ± 0.2 W kg−1, which was equivalent to 93 ± 10%, 93 ± 9%, and 96 ± 10% of the peak aerobic MR  are presented in Fig. 3´AO model compared to 7 + YLIN, 7-YLIN, and GELAST models, while the GELAST generated the highest values of AnWC.The mean PO-MR regression lines that were based on the seven submaximal stages for the 7 + YLIN versus the 7-YLIN regression model. The regression slope and Y-intercept values were similar for the 7 + YLIN and 7-YLIN models, which resulted in similar average GE values during the TT. The average GE during the TT was ~ 0.9 percentage points lower for the GELAST than the 7 + YLIN and 7-YLIN models.The data presented in Table LIN and 7-YLIN models together with GE calculated from the two linear regression equations for the submaximal stages and the TT, and measured GE, as well as the estimated AnWC for the 7 + YLIN, 7-YLIN, and GELAST models are shown in Fig. AO using the different models are presented in Table LIN and 7-YLIN generated similar mean values of AnWC and the typical errors between the models were relatively low. The GELAST model generated significantly higher values (~ 12% higher) of AnWC than the 7 + YLIN and 7-YLIN models, due to the lower GE  than the 7 + YLIN, 7-YLIN, and GELAST models and the typical errors were high (23–26%) for all the respective comparisons  the typical errors in AnWC between the 7 + YLIN, 7-YLIN and GELAST models were low; 3) the AnWC estimated with the CP3´AO model was substantially lower and demonstrated considerably higher typical errors versus the 7 + YLIN, 7-YLIN, and GELAST models.The main findings were as follows: 1) the GELAST model generated significantly higher values of AnWC compared to the 7 + YLIN and 7-YLIN models (~ 12% higher), as well as to the CP3´AO model (~ 63% higher). The higher AnWC estimates for GELAST, when compared to 7 + YLIN and 7-YLIN, could be explained by the positive Y-intercept in the 7 + YLIN and 7-YLIN regressions  in the linear PO-MR regression could only be justified if it is reasonably aligned with the submaximal stages of exercise. It appears that this is the case for the current study, because the slopes, delta efficiencies, and Y-intercept values were similar for the 7 + YLIN and 7-YLIN models. This finding is contrary to previous findings for other exercise modalities, such as diagonal-stride treadmill roller-skiing and treadmill running, where the inclusion of a Y-intercept lowered the slope of the regression line significantly and resulted in significantly lower estimates of AnC in highly trained participants agreement between four different models of estimating AnWC during cycle ergometry exercise. As hypothesized, the GEodels ~ 1% higher,odels ~ 1% higher,LIN, 7-YLIN, and GELAST models were relatively low (2–4%). This could be partly explained by the relatively similar within-athlete regressions for the 7 + YLIN and 7-YLIN models , it is probably still wise not to use different models interchangeably when testing athletes regularly.In the current study, the typical errors for the AnWC estimates for the 7 + Yls Figs. A, B. Forvely low –4%. Thissee Fig. A. Since see Fig. , the typ3´AO model was lower compared to the 7 + YLIN, 7-YLIN, and GELAST models and showed substantial typical errors in AnWC versus the 7 + YLIN, 7-YLIN, and GELAST models  aerobic contribution to PO and the similar aerobically attributable PO and CP during approximately the second half of TTAO  based on a conventional CP concept. Some previous studies have shown W′, based on any of the CP concepts, to be correlated with other method-specific estimates of AnWC/AnC, which suggests that W′ is likely to represent an AnWC  based on CP derived from the 30-s end power of a 3-min TTAO. The results showed that even if the AnC and W′ measures were correlated (r = 0.654), there was still a poor agreement between the two measures, suggesting that one, or both, of the methods, was/were unable to accurately measure AnWC/AnC. Thus, both the study of Muniz-Pumares et al.  steady-state. However, based on previous research findings , entails quite different methodological concepts. An important factor that is likely to explain the (dis)agreement between W’  based on the CP3´AO model and the other three models is rooted in the aerobic component of W’, which arises when aerobically attributable PO is in the intensity domain between CP and the maximal aerobically attributable PO  were approximately the same after 90 s of TTAO, which indicates that the AnWC/W′ was depleted after ~ 90 s with a following plateau of PO during the later stages of the TT, similar to previous studies  , which means that torque increases linearly with cadence and that PO increases quadratically with cadence. The use of a linear factor could, potentially, trigger more of a true all-out maximal effort as PO increases quadratically with cadence and may result in a higher initial PO along with higher exhaustion in the second half of the TT and, thus, a more realistic 30-s end test CP. However, the self-selected cadence approach that was used in the current study resulted in a reasonable cadence for supramaximal exercise and it was only slightly higher than the cadence of 80–90 rev·min−1 that was used in the study by Burnley et al.  during the second half of the TT. Although the decline in GE during supramaximal exercise is problematic to measure and includes several assumptions agreement between different models of estimating AnWC/AnC. Exercise physiologists should be aware that the use of these models is not interchangeable. The optimal model is likely to be, at least to some extent, case-by-case dependent, however, this study suggests that particular caution should be used when interpreting LIN and 7-YLIN models generated 10% lower AnWC values than the GELAST model. This result was caused by the ~ 0.9 percentage points higher supramaximal GE for the 7 + YLIN and 7-YLIN . When expressed as an AnC, the same comparison generated 17% lower values for the 7 + YLIN and 7-YLIN models versus the GELAST model. Due to the similar slopes and Y-intercepts for the 7 + YLIN and 7-YLIN models, supramaximal GE and AnWC/AnC were the same for both models. The within-participants variation in AnWC estimates between the 7 + YLIN, 7-YLIN, and GELAST models was low, as indicated by the low typical errors that ranged between 7 and 11 J∙kg−1 (or 2–4% of the respective pair-wise grand mean). The CP3´AO model generated the least valid and reliable estimate of AnWC, as revealed by the 30% lower AnWC values (on average) compared to the other models, and the substantial between-models typical errors .In conclusion, the 7 + Y


The sperm reserves per testis (SR), sperm reserves per gram testis (SRG), daily sperm production per testis (DSP), and daily sperm production per gram testis (DSPG) were  million sperm cells, respectively. The cauda epididymal sperm reserves were 1,386 ± 456 million sperm cells, and the sperm motility (M%), morphologically normal viable cells (N%), dead sperm cells (D%), and morphologically abnormal sperm cells (A%) were 89 ± 3.2, 70.13 ± 8.0, 13.55 ± 5.05, and 16.33 ± 7.9, respectively. In conclusion, testicular morphometric parameters in Ghanaian West African Dwarf rams are highly and positively correlated (r = 0.7487–0.9354) with sperm reserves and production.The current study was conducted to investigate the association between testicular morphometric parameters and sperm reserves in Ghanaian West African Dwarf rams. The intact scrotum along with testes and epididymides from twelve rams were collected immediately after slaughtering and transported to the Regional Veterinary Laboratory. The left testes and epididymides from all rams were dissected and the testes were individually weighed. The individual length, width, and volume of the testis and cauda epididymis were determined. Tissue samples were taken from testicular parenchyma, homogenized, and used to determine gonadal sperm concentration using a hemocytometer. Spermatozoa were recovered from the cauda epididymis by the incision method and used to determine the spermiogram. The weight, length, width, and volume of the testis were 63 ± 11 gram, 6.7 ± 0.44 cm, and 4.6 ± 0.40 and 74 ± 19 cm Small ruminant production plays an important role in the economies of developing countries. The major breeds of sheep reared in Ghana include the West African Draft (WAD) or Djallonke, long-legged Sahelian, and crossbreeds. The sheep population is estimated to be 4,335,000 . IndigenEvaluation of sperm reserves includes the assessment of gonadal and extragonadal sperm reserves, which are crucial in the selection of potential good breeders . ExtragoSperm reserves have been evaluated from slaughtered domestic animals , while sThe study was conducted from May to June 2021, at the Akwatia Line slaughter slab, located in the centre of the Kumasi Metropolis. Kumasi is the regional capital of the Ashanti region and is located geographically within the forest zone of Ghana. It is a commercial city, where most animals brought for slaughtering, originate from Northern Ghana.2, where L represents the length and W is the width of the testis.The scrotal sacs from twelve clinically healthy WAD rams, aged 18–24 months, were collected immediately after slaughtering. The testes were collected after slaughtering because this procedure was less expensive. Moreover, the rams were humanely slaughtered at an approved slaughter facility where slaughtering processes adhered to laid down animal welfare principles. The procedures used in this study were approved by the animal ethics committee of the university. The body weight and age of the selected rams were estimated before slaughtering. The collected samples were packaged aseptically in polythene bags and transported on an ice chest to the Regional Veterinary Laboratory for analysis, within one hour after slaughtering. The left testes and epididymides were dissected, freed from all connective tissue and fats, and the testes were weighed individually with an electronic weighing scale . The length and width of the left testis and cauda epididymis in each sample were measured using Vernier calipers, while their respective volumes were estimated using the modified formula of a prolate spheroid  as folloSegments of testicular parenchyma were taken from the proximal and distal regions, weighed, and then homogenized with a high-speed blender for 2 min with 20 ml normal saline solution containing antibiotics  to prevent bacterial growth. The homogenate was stored overnight at 5°C to allow sperm cells to ooze out of the tissues. An aliquot of this homogenate was filtered and used to determine the gonadal sperm concentration, using an improved Neubauer hemocytometer after appropriate dilution in 0.05% formal saline as previously described . The speSpermatozoa were collected from the cauda epididymis by multiple longitudinal incisions into its parenchyma, followed by immersion into a Petri dish containing 2.9% sodium citrate buffer solution and placed in a water bath at 37°C for 30 minutes . Cauda er).The general linear model was used to analyse the results. The results obtained were expressed in the form of mean ± SD, using the GraphPad Prism software . The ass3, respectively, while the length, width, and volume of the cauda epididymides were 3.2 ± 0.35 cm, 1.8 ± 0.16 cm, and 5.48 ± 1.5 cm3, respectively.The data for morphometric parameters of the testes and cauda epididymides of WAD rams are presented in The mean values of sperm reserve of whole testis (SR), sperm reserve per gram testis (SRG), daily sperm production per testis (DSP), and daily sperm production per gram testis (DSPG) were 11,740 ± 386, 250 ± 6, 2350 ± 7.7, and 50 ± 1.0 million sperm cells, respectively .r = 0.8262–0.9676) with the weight, length, width, and volume of testes and also with sperm reserves and sperm production. Testicular morphometric parameters correlated highly and positively with each other (r = 0.7487–0.9354) and also with sperm reserves and sperm production. Sperm reserves correlated positively and highly (r = 0.9804–1.000) with sperm reserves per gram testis, daily sperm production, and daily sperm production per gram testis.The correlations between testicular morphometric parameters and sperm production are illustrated in The cauda epididymal sperm reserves were 1,386 ± 456 million sperm cells, while the sperm motility (M%), morphologically normal viable cells (N%), dead sperm cells (D%), and morphologically abnormal sperm cells (A%) were 89 ± 3.2, 70.13 ± 8.0, 13.55 ± 5.05, and 16.33 ± 7.9, respectively .Our results in terms of length and width of the testis were similar to the values of 6.3 ± 0.16 cm and 4.4 ± 0.11 cm, respectively, as previously documented in WAD rams in North Benin . However3, respectively, reported for WAD rams in Akwa Ibom State, Nigeria [The mean values of weight and volume of the testis of rams in the current study were higher than 51.20 gm and 53.7 cm Nigeria , 20. The Nigeria .However, the mean values of testes' weight, length of testes, and cauda epididymides were lower than 99.8 ± 2.6 gram, 8.7 ± 0.2 cm, and 4.4 ± 0.7 cm, respectively, as reported for Yankasa rams reared in Zaria, Nigeria . MoreoveThe mean values of SR, DSP, and DSPG in this study were higher than 2,150 ± 110, 600 ± 10, and 9.4 ± 2 million sperm cells, respectively, reported for WAD rams in Nigeria . These dThe testicular morphometric parameters highly correlated with each other and with sperm reserves and daily sperm production. Our findings agree with previous reports that high correlations existed between testicular weight, sperm reserves, and daily sperm production in WAD rams . MoreoveThe mean values of cauda epididymal sperm reserves in this study were higher than reported for WAD rams in Ibadan, Nigeria , and YanThe mean values of percent sperm motility, sperm viability, and morphological abnormal sperms in this study were within the ranges of 66.25–85.66, 84.17–87.66, and 11.84–12.96, respectively, as documented for WAD rams in Ibadan, Nigeria . On the r = 0.7487–0.9354) with sperm reserves and production. Future research should be conducted to investigate the fertilizing ability of spermatozoa recovered from recently slaughtered animals.In conclusion, testicular morphometric parameters in Ghanaian West African Dwarf rams were highly and positively correlated (


Diagnosis and longitudinal monitoring of neurological diseases are limited by the poor specificity and limited resolution of currently available techniques. Analysis of circulating cells in cerebrospinal fluid (CSF) has emerged as a promising strategy for the diagnosis, molecular characterization, and monitoring of neurological disease. In comparison to bulk sequencing analysis, single-cell sequencing studies can provide novel insights into rare cell populations and uncover heterogeneity in gene expression at a single-cell resolution, which has several implications for understanding disease pathology and treatment. Parallel development of standardized biofluid collection protocols, pre-processing strategies, reliable single-cell isolation strategies, downstream genomic analysis, and robust computational analysis is paramount for comprehensive single-cell sequencing analysis. Here we perform a comprehensive review of studies focusing on single-cell sequencing of cells in the CSF of patients with oncological or non-oncological diseases of the central nervous system. The current approach to diagnosing and monitoring neurological diseases is largely reliant on imaging and tissue biopsy. Imaging does not provide molecular or biological information regarding diagnosis, response to therapy, or progression of the disease. Furthermore, obtaining tissue samples in the central nervous system (CNS) is not always feasible owing to the location of the lesion, disease type, and potential complications. Longitudinal tissue biopsies in the context of the brain are very challenging, again due to the inherent risks of neurosurgical biopsy .A liquid biopsy involves the study of analytes  in biofluids  to allow for the diagnosis and monitoring of disease. Analysis of circulating cells has evolved as a promising strategy to study neurological diseases. Combining circulating cell analysis with advanced sequencing methodologies such as single-cell RNA sequencing (scRNA-seq) can provide valuable insights into disease biology and immunology, diagnosis, and response to therapy. CSF, already considered a biofluid of high significance in the diagnosis of CNS pathologies due to its proximity, accessibility, and role in immunosurveillance, holds unique cell populations which can be analyzed to improve our understanding of the pathology, diagnosis, and treatment of CNS disease. In this review, we discuss emerging studies exploring scRNA-seq of circulating cells in CSF to investigate oncological and non-oncological diseases of the CNS.CSF surrounds the brain and the spinal cord and plays a vital role in CNS homeostasis . Τhere aCSF is considered a biofluid of high significance in the diagnosis of CNS pathologies due to its proximity, accessibility, and role in immunosurveillance. In several neurological diseases, the CSF composition, opacity, and opening pressure can be altered. By measuring various CSF components using methodologies such as cell counts, cytology, staining, culture, immunoglobulins, and lactate levels, the diagnosis, severity and prognostication of conditions including infection, subarachnoid hemorrhage (SAH), and autoimmune demyelinating conditions can be made . AdditioMycobacterium tuberculosis, Listeria monocytogenes, Treponema Pallidum, or Borrelia burgdorferi also results in mononuclear pleocytosis [CSF pleocytosis is a hallmark of neuroinflammation commonly seen in meningitis, autoimmune diseases, and leptomeningeal disease . For insocytosis . Autoimmocytosis .Recent studies in the neuro-oncology space have pioneered and demonstrated the feasibility of studying various liquid biopsy analytes in primary and metastatic brain tumors ,17,18,19While traditional bulk transcriptomic sequencing provides an average expression signal for an ensemble of cells, there is increasing evidence in tissue analysis that heterogeneity in gene expression exists even among very similar cell types, driving different cell fates . PioneerResearchers have applied such advances in single-cell analysis to the study of circulating cells in the CSF . ParalleFor all liquid biopsy-based assays, including single-cell assays, reliable sample collection and preprocessing is necessary. The volume of CSF required for adequate single-cell isolation may vary depending on disease-specific quantities of cells present in the CSF. For instance, circulating tumor cells (CTCs) are extremely rare and thus may require larger volumes of CSF to be collected for adequate analysis in comparison to the smaller volumes needed to study infectious diseases . ImmediaAlthough CSF is an ideal biofluid from an anatomic proximity standpoint, emerging studies have shown that other easily obtainable biofluids such as blood/plasma also have circulating cells of interest. However, blood contains an abundance of normal cells, and isolating disease-specific circulating cells in blood has several inherent limitations . ParalleIsolating single cells from CSF is the next step in the single-cell transcriptomic analysis pathway. Arguably, this is the greatest source of technical variation, contamination, and batch effect in any single cell study . Unlike A common method of single-cell isolation within CSF is antibody-mediated capture (positive selection) using Flow-Activated Cell Sorting (FACS). In this method, cells are tagged with fluorescent monoclonal antibodies via targeting extracellular membrane proteins for cell sorting . AnotherThe CellSearch single-cell capture system, approved by the FDA in 2004 for detecting CTCs in peripheral blood, uses antibodies conjugated to magnets to enumerate CTCs from patient blood samples . PlatforLibrary preparation protocols depend on downstream sequencing platforms. Usually, the extracted nucleic acid from each cell is labeled with barcodes then pooled together for sequencing. Several next-generation sequencing methodologies have been developed for single-cell sequencing . The advDownstream data processing primarily involves the conversion of raw reads into standardized FASTQ files (text-based files used to store nucleotide sequences), demultiplexing, quality control, filtering, alignment and finally data visualization and interpretation. Several scRNA-seq analysis pipelines have been developed to assess gene expression and interpret single-cell transcriptomics . As discCTCs are cancer cells that leave the primary tumor, enter biofluids such as blood or CSF, and play a vital role in tumor dissemination and distant metastasis. Several groups have reported the presence of CTCs in the peripheral blood of patients with GBM ,37,38,39Ruan and colleagues studied CSF CTCs from patients with leptomeningeal metastasis of adenocarcinoma of the lung . SignifiRuan and colleagues also used scRNA-seq analysis to explore the possibility of studying CSF to determine the origin of the primary tumor in carcinoma of unknown primary (CUP) based on the gene expression profile of CTCs. Although results were inconclusive, this highlights a future diagnostic avenue using transcriptome analysis of CSF CTCs in patients with leptomeningeal dissemination of CUP to guide diagnosis and therapy .Studying CSF immune cells and correlating them with immune cells in the primary tumor site, metastatic site or peripheral blood can provide unique insights into the immune interactions and response in each respective compartment. There is mounting evidence of the ability of cancers to modulate tumor microenvironments into an immune suppressing and tumor supporting system, and scRNA-seq can provide minimally invasive insights into this immune microenvironment. Smalley et al. determined that the CSF immune microenvironment in patients with leptomeningeal metastasis secondary to melanoma was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases . This stChi and colleagues used scRNA-seq to understand how CSF CTCs adapt to microenvironmental challenges including inflammation and sparse micronutrients: breast cancer and non-small cell lung cancer cells in CSF express and use iron-binding protein lipocalin-2 (LCN2) and its receptor SCL22A17 to outcompete macrophages for iron. This may permit tumor cells to evade the immune system by reducing the efficacy of macrophages .Ruan and colleagues studied over a thousand diffuse large B cells in the CSF (CSF-DLBCs) in 6 patients with CNS lymphoma . When coscRNA-seq of both CTCs and immune cells in the CSF has provided insights into the immune response to cancer treatment. This may aid in understanding differential responses, diagnosing treatment failure, and providing molecular insights into the adaptations that could be contributing to such failure. A recent study by Mochizuki and colleagues used scRNA-seq of the CSF to evaluate patient response to chimeric antigen receptor (CAR) T-cell therapy for the treatment of H3K27M-mutant diffuse intrinsic pontine glioma (DIPG) and spinal cord diffuse midline glioma (DMG). Patients who demonstrated clinical/radiographic benefit from CAR-T therapy exhibited fewer S100A8 + S100A9+ myeloid suppressor-cells and CD25 + FOXP3+ regulatory T-cells in the CSF pre-infusion when compared to the patient in which a therapeutic response was not achieved. In a subject with DIPG who demonstrated improvement, polyclonal CAR T-cells detectable in CSF at Day +14 demonstrated enrichment of CD8A, GZMA, GNLY and PDCD1 compared to the pre-infusion CAR T-cells by trajectory analysis, suggesting differentiation toward a cytotoxic phenotype. The same subject exhibited increased numbers of S100A8 + S100A9+ myeloid cells and CX3CR1 + P2RY12+ microglia over time. These results provide evidence that the presence of immunosuppressive myeloid populations, detectable in CSF, may correlate with clinical response in CAR T cell therapy for DIPG/DMG .Prakadan and colleagues studied malignant cells and immune cells in patients with leptomeningeal metastasis of solid tumors who underwent treatment with immune checkpoint inhibitors (ICI). An immediate increase in the number of CD8+ T Cells in the CSF following intravenous ICI administration was noted, in addition to increased IFN-γ signaling and cytotoxicity post-treatment, suggesting immunomodulation by ICIs. A decline in immune response to ICIs over time was also noted, emphasizing the value of studying tumor behavior in response to therapy at multiple time points . SimilarCSF is composed of multiple analytes including circulating free DNA (cfDNA), circulating free RNA (cfRNA), and extracellular vesicles (EVs) in addition to CTCs. cfDNA is released by dying tumor cells, while EVs are actively secreted by tumor cells as molecular messengers ,47. UsinStudying the cellular landscape of CSF has been an avenue of interest in several non-oncologic diseases where routine biopsy is not obtained or feasible. Emerging studies have used scRNA-seq to gain insights into disease progression and neuroinflammation .Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment without a definitive cure. There has been significant interest in understanding the immune response and its contribution to AD progression. scRNA-seq of CSF in patients with AD revealed that T-cell receptor (TCR) signaling was upregulated in circulating CSF CD8+ T- effector memory CD45RA+ (TEMRA) cells, which was negatively associated with patient cognition . Gate anscRNA-seq has also been employed to investigate the mechanism by which some patients develop neurological symptoms such as anosmia, dysgeusia, headache, impaired consciousness, and seizures as a result of COVID-19 infection. Song and colleagues studied the CNS immune response in individuals with neurological sequelae of COVID-19 using scRNA-seq and cytokine analyses of CSF and blood, finding evidence of CNS-specific T cell activation and B cell responses distinct from peripheral responses during infection. In addition to uncovering the transcriptional profiles of CSF immune cells in patients with neurological symptoms of COVID-19 infection, it was found that typical interactions between CD4+ T cells and monocytes were diminished, suggesting a dysregulated innate-to-adaptive immune interface .Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS with ongoing research using scRNA-seq to elucidate the etiology and potential treatments of aberrant immune activation. scRNA-seq of immune cells in the CSF has provided evidence that in MS, CSF B cells are driven to inflammatory and clonally expanded memory and plasmablast/plasma cell phenotypes . scRNA-sResearchers have been studying the role of CNS immune activation in patients with neurocognitive impairment in the setting of HIV, even during sustained long term suppressive antiretroviral therapy. Farhadien and colleagues used scRNA-seq of CSF and blood from adults with and without HIV to identify a rare subset of myeloid cells with a genetic signature that overlaps significantly with neurodegenerative disease-associated microglia. CNS myeloid cells and microglia in particular have been proposed to play several important roles in HIV infection, including acting as a potential site of viral replication .Cryptococcus cell culture was negative, while the Cr-Ag agglutination test was weakly positive (1:1). Thus, scRNA-seq of the CSF was employed to identify the etiological agent: C. gattii sensu stricto. This case highlights the applicability of scRNA-seq in establishing the etiology of rarer causes of meningitis when initial testing is inconclusive.Researchers have begun using scRNA-seq to study the CSF cellular landscape in patients with bacterial meningitis that is unresponsive to antibiotic treatment . Chen etCirculating cells in CSF are relatively difficult to isolate due to low concentration in the CSF, necessitating larger volumes of CSF to obtain adequate cellular yield. However, ex vivo culture of circulating cells can generate patient-specific disease models to allow individualized treatment planning. Another limitation of scRNA-seq of the CSF that has been touched upon throughout the manuscript is inherent to the novelty of this technology: because the field is still relatively new, definitive standards for quality control, data processing, and interpretation have yet to be established and implemented. Such standardization will be necessary to ensure results are reproducible and translatable from benchtop cell cultures to bedside diagnostics. Available studies have limitations including small sample sizes and variability in processing, sequencing, and downstream analysis . Thus, lRapid technological development and improvement in high-throughput single-cell sequencing, together with the methodological advances in single-cell isolation and downstream processing of CSF in patients with neurological diseases, have advanced the field of liquid biopsy. In this review, we presented studies that leverage scRNA-seq of CTCs and immune cells in the CSF which identify unique cell populations and characterize the immune microenvironment to diagnose and treat both neoplastic and non-neoplastic diseases of the CNS. While standardized protocols must be developed and implemented for scRNA-seq technology to be consistently translatable to clinical practice, this field holds tremendous promise for new diagnostic methods and targeted individualized treatments.