Pan troglodytes verus and Pan troglodytes schweinfurthii) subspecies and communities living in their natural environments. We conclude that the three most predominant perspectives on gestural acquisition—Phylogenetic Ritualization, Social Transmission via Imitation, and Ontogenetic Ritualization—do not satisfactorily explain our current findings on gestural interactions in chimpanzees in the wild. In contrast, we argue that the role of interactional experience and social exposure on gestural acquisition and communicative development has been strongly underestimated. We introduce the revised Social Negotiation Hypothesis and conclude with a brief set of empirical desiderata for instigating more research into this intriguing research domain.Scientific interest in the acquisition of gestural signalling dates back to the heroic figure of Charles Darwin. More than a hundred years later, we still know relatively little about the underlying evolutionary and developmental pathways involved. Here, we shed new light on this topic by providing the first systematic, quantitative comparison of gestural development in two different chimpanzee ( Displacement, which refers to the ability to encode and understand meanings about past, future, or distant referents.Productivity/openness, which denotes the skill to invent and understand new utterances/signal combinations.Duality of patterning, which refers to the ability to combine meaningless units (phonemes) into meaningful ones (morphemes), which can then be combined into larger meaningful units (e.g. sentences).Traditional  transmission, which denotes the fact that languages (or communication systems) are learned and not genetically encoded Fitch , 1963.Language, more than anything else, distinguishes us from the rest of the animal kingdom , to enable the most efficient and least costly communication transfer, and is open to subsequent adaptation  community (Hobaiter and Byrne Pan paniscus) groups  and a chimpanzee community  had relatively low levels of concordance between individuals . leaf clipping consists in some communities of ripping parts of the leaf with the mouth .However, despite these research efforts, a detailed understanding of the impact of social transmission on gestural acquisition has been hampered by the lack of systematic comparative data across field sites and species. Although group-specific gestures have been reported in chimpanzees .lowers her back to allow the infant to climb up).The recipient, individual B, reacts in a predictable way .On some subsequent occasion, individual B anticipates this action sequence on the basis of its first step .The initiator, individual A, learns over repetitions of this sequence to shorten its behaviour to just that initial step  over two different field periods  per community and during two consecutive years. The age of the offspring ranged from nine to 69 months. At Taï South, one mother gave birth to another infant in the second field period, resulting in observations of twelve chimpanzee mothers and 13 infants. We applied a focal behaviour sampling approach  design, enable researchers to gain detailed insight into nonhuman primates’ communicative development, but are still rare outside of captive settings . Although we applied a different observational data design and did not aim to assess the community repertoires, we ensured to maximize observation periods and field observation time. We observed all 13 dyads for a minimum of 150 days  of field observation time, resulting in a total of 169 h of video footage. Furthermore, Byrne and colleagues . The coding of the data set resulted in a total (number of cases) of 442 actions , 599 gestures , 51 bimodal , and 80 vocalisations   Dice , which r see Box  gestures see Box  gestures see Box . Indeed, see Box .The findings of the joint travel study led us to conclude that meaningful and thus functional gestural signals do not represent simple innate, fully formed constant means of communication  contact of the palm and/or fingers of signallers on the body of the recipient, each gestural performance of a touch gesture, by a given signaller, is a highly variable online adjustment . The coding of the videos resulted in a total (number of cases) of 1174 gestures , including 109 audible , 646 tactile , and 419 visual gestures  and 74 cases of self-handicapping gestures .In the play study, we investigated communicative exchanges during play interactions and examined the influence of demographic factors, namely the interactants’ age, sex, and kin relationship on gestural signalling  and self-handicapping (e.g. poke while lying in a supine position) gestures were of crucial importance for initiating play with partners of the same age and younger, respectively. The play study demonstrated that play interactions with peers and other non-maternal individuals may serve as an essential training ground to provide an opportunity for experiencing the production and usage of communicative signals. Here, individuals can test the effectiveness of, and practice, gestural signalling that might be of vital importance for negotiating and maintaining social relationships in later life.We analysed the form of signals in relation to developmental phase, context, and interactional partner and found a strong effect of age on the production of different gesture categories: visual and audible gestures were produced at the expense of tactile signals with increasing age. In addition, there was a sex difference in gestural usage, with males employing more tactile signals than females. Tactile gestures differ from visual gestures in both physical effectiveness and potential demonstration of physical strength, suggesting a sex difference in signal directness in terms of the level of physical contact involved. In line with these results, a recent study by Lonsdorf et al.  reportedN = 260; joint travel: N = 392; social play: N = 468). We identified a total of 1066 gestures, with 301 infant gesture cases produced during the context of food sharing, 77 to initiate joint travel, and 688 gesture cases produced in the play context. Overall, we described a total of 55 different gesture types , with seven types being exclusively produced in the food sharing context and 37 in the play context. Five gesture types were utilized in all three contexts, four in both the food sharing and the play contexts. Two gesture types were utilized in both the joint travel context and the play context.In the context study, we compared gestural signalling across three different communicative contexts—food sharing, joint travel, and social play  and spatial independence  of immature apes, which optimally complement the fine-grained analysis of gestural production via video recordings. Therefore, by using this method multiple domains of development can be assessed simultaneously.chest beat or slap ground—while other gesture types incorporate highly variable movements under the same umbrella term (e.g. touch). Originally intended to cluster the behaviour of animal species into recognizable and reliable units , resulting in overlooking individual differences, function, online adjustment, and downplaying the impact of social learning on gestural acquisition.With this article, we make the case that the most predominant perspectives of gestural acquisition—Phylogenetic Ritualization, Social Transmission via Imitation, and Ontogenetic Ritualization—do not satisfactorily explain the variability and distribution observed in chimpanzees’  natural communicative gesturing. In contrast, our results suggest that the role of interactional experience and social exposure on gestural acquisition and communicative development of chimpanzees has been severely underestimated. We propose that the production and usage of communicative gestures highly depend on social negotiation between interactants and are open to online adaptation and refinement. This results in a shared understanding that specific behaviours can be used to achieve communicative goals and carry distinct meanings linked to particular social contexts. Chimpanzees' gesture acquisition thus highly matches the learning process suggested by Acredolo and Goodwyn  for humaBy applying a comparative, developmental approach to understand the role of learning in communicative signalling of our closest living relatives, we hope to have also stimulated future research shedding additional light on learning mechanisms involved in early gesturing of human children (Acredolo and Goodwyn


Changes in drug absorption and first-pass metabolism have been reported throughout the pediatric age range. Our aim is to characterize both intestinal and hepatic CYP3A-mediated metabolism of midazolam in children in order to predict first-pass and systemic metabolism of CYP3A substrates.Pharmacokinetic (PK) data of midazolam and 1-OH-midazolam from 264 post-operative children 1–18 years of age after oral administration were analyzed using a physiological population PK modelling approach. In the model, consisting of physiological compartments representing the gastro-intestinal tract and liver,intrinsic intestinal and hepatic clearances were estimated to derive values for bioavailability and plasma clearance.versus 527 L/h (RSE 7%) for a 16 kg individual, respectively). When expressed per gram of organ, intrinsic clearance increases with increasing body weight in the gut wall, but decreases in the liver, indicating that CYP3A-mediated intrinsic clearance and local bioavailability in the gut wall and liver do not change with age in parallel. The resulting total bioavailability was found to be age-independent with a median of 20.8% in children (95%CI: 3.8–50.0%).The whole-organ intrinsic clearance in the gut wall and liver were found to increase with body weight, with a 105 : 5–405) times lower intrinsic gut wall clearance than the intrinsic hepatic clearance (i.e. 5.08 L/h (relative standard error (RSE) 10%) In conclusion, the intrinsic CYP3A-mediated gut wall clearance is substantially lower than the intrinsic hepatic CYP3A-mediated clearance in children from 1 to 18 years of age, and contributes less to the overall first-pass metabolism compared to adults.The online version of this article (10.1007/s11095-018-2458-6) contains, which is available to authorized users. Differences in drug absorption and first-pass metabolism have been reported in children compared to adults –3. Thesein vitro and in vivo studies on the ontogeny of CYP3A in children (With cytochrome P450 (CYP) being an enzyme family involved in metabolism of many drugs ,7. From children –10, therchildren . Howeverchildren . In ordechildren ,14. Thischildren .In this study, the aim is to characterize both intestinal and hepatic CYP3A-mediated metabolism of midazolam in children between 1 and 18 years of age. We will adopt the above described physiological population PK modelling approach, in which we account for known changes in the physiology of the gastrointestinal tract, and use the available PK data from the population, to estimate the whole-organ intrinsic intestinal and hepatic CYP3A-mediated midazolam clearance in children. For this analysis, we had access to data from a clinical study in which the CYP3A substrate midazolam which is considered a probe drug for CYP3A , was admIn 873 plasma samples from 266 patients of the Children’s Hospital of Philadelphia, PA, midazolam and 1-OH-midazolam concentrations were measured . The chiB) were estimated based on the measured plasma concentrations (P) using eq. Blood concentrations (u is the fraction unbound in plasma and Kp is the unbound partition coefficient of the red blood cells to plasma (assumed to be constant between adults and children)  . The frahildren) :2\documeterature ,22. The mentclasspt{miniman = 4 (0.5%) and n = 5 (0.6%) of midazolam and 1-OH-midazolam measurements respectively).Measurements below the lower limit of quantification were discarded according to the M6 method   based on first-order conditional estimation with interaction, and for visualization of data Pirana 2.9.0, R (version 3.3.1), and R-studio (version 0.98.1078) were used. A physiological population pharmacokinetic (PK) model, earlier developed and applied by Yang et al. , Frechen  was calculated based on body surface area   :4\documeVolume of the portal vein was assumed to be equal to the reported value of 5.2 mL in adults . To calcTo calculate organ weight, the organ volumes are multiplied by the organ density of 1040 g/L  and an oh) was assumed to be a fixed percentage of the cardiac output (CO), which was calculated based on BSA  for midazolam could not be estimated, and was therefore fixed at 4.16 h−1, yielding peak concentrations to be reached round 30 min (tmax), which is in agreement with the observed tmax and other reported literature values  was calculated using:a is the fraction absorbed, which is assumed 1 for midazolam , and Fh is the hepatic bioavailability, which is equal to 1 minus the hepatic extraction ratio (Eh). The hepatic extraction ratio of midazolam (EH) and 1-OH-midazolam  were defined by the well-stirred model:H,int is the estimated intrinsic hepatic clearance (whole-organ), fu,B is the fraction unbound in the blood and Qh is the hepatic blood flow. The gut wall extraction ratio is described using the Qgut model , fu,G is the fraction unbound in the gut, which was assumed to be 1, and Qgut is the effective blood flow at the gut wall  for midazolam was included as well.In which CLjth observed concentration Y of the ith individual was modelled according to:pred,ij is the jth predicted midazolam concentration of the ith individual, ε1ij and ε2ij are random variables from a normal distribution with a mean of zero and variance of σ2.Residual unexplained variability was modelled using a combined proportional and additive error model for both midazolam and 1-OH-midazolam. The G,int, CLH,int, CLH,int,M) and intercompartmental clearance ) was performed in which the following covariates were tested for significance: age, body weight, height, body surface area, and sex. For sex, the typical value (θTV) for girls was estimated relative to the value for boys. The remaining continuous covariates were tested using a power  of the covariate (COV) and θCOV the estimated exponent or slope for a power or linear function respectively.A covariate analysis for the estimated parameters (whole-organ intrinsic clearance (CLower Eq.  or lineaower Eq.  functionp < 0.05) was considered statistically significant. In addition, goodness-of-fit plots of midazolam and 1-OH-midazolam were assessed, in which observed versus individual- and population-predicted concentrations and conditional weighted residuals (CWRES) versus time and population predicted concentrations are visualized. Moreover, the condition number, the confidence interval of the parameter estimates, and visual improvement of the individual plots were used to evaluate the models.Structural models were evaluated by comparison of the objective function values . A decrease of 3.84 in the OFV between nested models (p < 0.01) was considered statistically significant, while for the backward deletion a more stringent p value  was used. Moreover, to retain a covariate in the model, the inter-individual variability in the PK parameter should decrease.For inclusion of covariates, a drop in OFV by at least 6.64 points (n = 250) was performed to evaluate model stability and parameter precision  of 1  of 1 , on the p1 = 55.2 L), body weight was found as a covariate, while no covariate was identified for intercompartmental clearance to the second peripheral compartment (with Vp2 = 79.1 L). Lastly, a covariate was included in the model correlating weight to intrinsic hepatic clearance of the metabolite with an exponent of 0.651 (RSE 9%). Age, height, body surface area, and sex were not identified as covariates for the estimated parameters. The gut wall metabolism of the metabolite could not be estimated independently due to model instability, and was therefore estimated as a fraction of the gut wall metabolism of midazolam. The bootstrap results confirmed the model stability and the precision of parameter estimates of the model  decrease in hepatic intrinsic CYP3A activity per gram of liver with age, while no change with age is observed for gut wall intrinsic CYP3A activity per gram of small intestine except for a small drop around the age of 4–5 years.Figure Using eq. g and Eh  were derived. Figure p < 0.001, Wilcoxon-Mann-Whitney U-test) .Using the estimated whole-organ intrinsic clearance values, together with blood flow and fraction unbound, the extraction ratios E Eh eqs.  and 9 anst) Fig. . With res Fh eq. , Fig. 4 versus the observed concentrations  is smaller than 100%, the median total bioavailability does not change considerably. While the hepatic bioavailability is not impacted, the gut wall bioavailability however increases by 4.1 and 18.7% for an Fa of 0.90 and 0.80, respectively. A change in the volume of distribution of the primary metabolite leads to considerable changes in all clearance parameters. All results of the sensitivity analysis are summarized in the supplemental material . The fraction escaping gut wall metabolism was found to be smaller  than the fraction escaping hepatic metabolism . These values for hepatic bioavailability are in agreement with previously reported values in adults (gut) increases less with age than the whole-organ intrinsic intestinal clearance.The extraction ratios Eund Eqs.  and 9, an adults ,37. The g and Fh around 0.2 and 0.7, respectively  across the pediatric age range is similar to the reported median value of 13.4%  in adultversus predicted concentrations and versus time after first dose, which demonstrates accurate prediction of the midazolam and 1-OH-midazolam concentrations and variability, with only a small over-prediction of the variability of midazolam.All parameters in the model could be estimated with good precision


One of the greatest challenges in neuro-oncology is diagnosis and therapy (theranostics) of leptomeningeal metastasis (LM), brain metastasis (BM) and brain tumors (BT), which are associated with poor prognosis in patients. Retrospective analyses suggest that cerebrospinal fluid (CSF) is one of the promising diagnostic targets because CSF passes through central nervous system, harvests tumor-related markers from brain tissue and, then, delivers them into peripheral parts of the human body where CSF can be sampled using minimally invasive and routine clinical procedure. However, limited sensitivity of the established clinical diagnostic cytology in vitro and MRI in vivo together with minimal therapeutic options do not provide patient care at early, potentially treatable, stages of LM, BM and BT. Novel technologies are in demand. This review outlines the advantages, limitations and clinical utility of emerging liquid biopsy in vitro and photoacoustic flow cytometry (PAFC) in vivo for assessment of CSF markers including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA (miRNA), proteins, exosomes and emboli. The integration of in vitro and in vivo methods, PAFC-guided theranostics of single CTCs and targeted drug delivery are discussed as future perspectives. Leptomeningeal and brain metastasis (LM and BM) as a result of metastatic dissemination of solid tumors  and hematological neoplasms as well as primary brain tumors  are commonly fatal with minimum treatment options ,9,10,11.Among other parts of central nervous system (CNS), cerebrospinal fluid (CSF) is the easiest accessible medium that can directly uptake tumor markers from different parts of CNS ,15,16,17In tumor patients with CNS involvement, CSF contains various markers associated with disease progression and responses to therapy ,28,29,30Based on the physiology of CNS and mechanisms of tumor development , CTCs, Thus, testing CSF might predict deadly LM, BM and BT; and advanced methods to assess CSF tumor markers in CSF are urgently needed to prolong life of patients suffering from CNS tumor lesions.The gold standard for routine clinical examination of CSF is cytology after lumbar puncture ,24,42,43The limitations of cytology and deadly nature of LM, BM and BT encouraged researchers and clinicians to develop more sensitive and accurate markers using modern technologies. During the past decade, substantial efforts have been made to assess CSF samples using new concept of liquid biopsy  2,15,16,46,47,48Several years ago, Patel et al. showed that FDA-approved CellSearch method can be used to identify CTCs in 7.5 mL CSF samples of breast cancer patients . CompareUsing real-time polymerase chain reaction (RT-PCR) for examination of patients with BM and LM has demonstrated higher sensitivity than conventional cytology . HoweverAnother promising emerging data of CSF liquid biopsy have been obtained using immunofluorescence in situ hybridization (FISH) technology ,58,59,60Integration of array comparative genomic hybridization (ACGH) analysis and whole genome amplification provided achieving the genomic characterization of rare CSF CTCs ,62. The Analysis of CSF samples with conventional flow cytometry in vitro has been reported to diagnose CTCs in CSF ,64,65. FIn the past few years, the clinical potential of some other technological platforms including microfluidic technology, immunomagnetic platform, high performance liquid chromatography-mass spectrometry, next generation sequencing (NGS) and proteolytic activity matrix assay (PrAMA) has also been demonstrated ,67,68,69Based on this, liquid biopsy in vitro can provide advanced molecular and genetic analysis of tumor associated markers in CSF but it cannot detect rare CTCs at early stage of LM and BM and possibly, before LM and BM initiation . The rarDespite significant progress in neuroimaging in vivo  ,72,73,74The most promising method for detecting CTCs in CSF is photoacoustic (PA) flow cytometry (PAFC), which compared to other in vivo diagnostic techniques, demonstrated ultra-sensitive molecular detection and counting of single cells in different body fluids  ,81,82,83The first successful demonstration of PAFC’s capability to diagnose CSF tumor markers was reported using preclinical models of breast metastatic cancer b–d 71]..71]. It Another interesting finding is the existence of CTC-containing emboli in CSF in vivo d. Identi3 –fold increased sensitivity compared to existing CTC assays) [Overall, the success of preclinical studies together with the simplicity and safety of PAFC give confidence to rapidly translate this method into clinical practice. PAFC diagnosis of CSF in human subarachnoid space and spinal canal at a depth of 1–3 cm seems possible and was supported by the reports on high sensitivity and resolution of PA methods in deeper tissues. Recently, the clinical relevance of PAFC was successfully demonstrated in clinical trials with melanoma patients by detecting blood CTCs in 1–2 mm hand vessels at depth of 1-3 mm with a detection limit of 1 CTC/1000 mL  .To date, crucial steps in increasing the survival of patients with LM and BM are (1) early diagnosis; (2) initiating preventive therapy such as targeted therapy of single CSF CTCs and their emboli and (3) assessing therapeutic efficacy in order to optimize an individual course of therapy.Although many promising technologies to detect various CSF tumor markers during liquid biopsy have been reported, there is no standardized and validated assay that is currently ready to introduce for daily clinical practice as an advanced alternative or supplement of conventional cytology.Novel approaches integrating unprecedented high sensitivity of in vivo flow cytometry and comprehensive molecular and genetic characterization of tumor markers in CSF in vitro are highly desired for clinical needs.In addition, one of the possible future alternatives is CSF diagnosis in vivo using updated GILUPI CellCollector. This method was introduced in 2016 for EpCam-based detection of CTCs in blood by introduction of EpCAM-coated wire into a vein of the patient . HoweverThe new looks are also suggesting continuous cell exchange between CSF, blood, lymph and brain tissue ,91,92 thMinimal treatment options in current management of LM and BM lead to poor prognosis for patients due to low efficacy, late therapy initiation, use of common  therapeutic schematics and high toxicity. From this, one of the top future priorities is development of novel targeted and immune therapies. The molecular-targeted nanotechnology platform is highly promising for targeted drug delivery. For this purpose, nanoparticles should have high sensitivity, specificity and selectivity as well as safety, multifunctionality, multimodality, ability to penetrate BBB and high efficiency of drug delivery to tumor. Among existing nanoparticle-based drug cargoes, the most promising candidates include low toxic individual nanoparticles, high-contrast spasers, liposomes, polymer micelles, lipid micelles packaged with semiconducting polymer dots as simultaneous MRI and PA imaging and photodynamic and photothermal dual-modal therapeutic agents, layer by layer based composite structures (core-shells) and microcapsules (shells) and biocompatible natural magnetic nanoparticles ,96,97,98There is a high therapeutic potential of modern technologies for creating synthetic truncated antibodies  and scafd~10−14 M. The strategy is applicable to any proteins or nanoparticles that can be functionally attached to the barstar and barnase, especially for production of heterooligomeric constructs because the extremely specific barnase barstar interaction eliminates reliably the mispairing problems. This universal platform is a promising alternative to commonly used chemical conjugation techniques in nanobiotechnology, theranostics and clinical applications. It provides a straightforward technology to design wide range of multifunctional nanoheterostructures for the highly efficient delivery of active agents to tumor cells for theranostics [The novel targeted CSF therapy may also use the advanced design of heterostructures based on the barnase:barstar pair . The ribanostics ,116,117.A very exciting future direction is the possibility of integration of in vivo molecular diagnosis, targeted therapy and estimation of therapeutic efficacy in one technological platform of PAFC ,79,118. It is expected that technological innovations and ongoing clinical trials would contribute to the finding of novel approaches to provide advances in BM and LM theranostics at the earliest possible stages before development of overt deadly lesions, to select patients with high risk of BM and LM for personalized therapy, to identify early disease progression and thereby improve survival rates of cancer patients.


Serum retinol-binding protein 4 (RBP4) plays a critical role in insulin resistance. The mechanism behind the impact of laparoscopic sleeve gastrectomy (LSG) on glucose metabolism is unclear. Hence, we aimed to investigate the triangle relationship between the RBP4, glucose metabolism, and LSG in patients of Chinese ethnicity.The study enrolled eighty-two obese patients. Glucose-lipid metabolic index, uric acid (UA), superoxide dismutase (SOD), free triiodothyronine (FT3), free thyroxin (FT4) and thyrotropin (TSH) were measured. RBP4 levels were detected by enzyme-link immunosorbent assay. 30 obese patients underwent LSG were studied. All these markers were measured again at a time interval of 3 and 6 months after surgery.P < 0.05). RBP4 levels in the patients with impaired fasting glucose (IFG), insulin resistance or hyperinsulinemia were significantly higher than the patients without IFG, insulin resistance or hyperinsulinemia . (2) LSG resulted in significantly decreased FBG, FINS, FCP and HOMA-IR at 3, 6 months after surgery . The RBP4 levels were significantly decreased after surgery  with no gender difference. (3) The change in RBP4 levels was significantly associated with the change in FINS, FCP, HOMA-IR, and HOMA-β at 6 months and the change in TSH at 3 months after surgery in males . The change in RBP4 levels were significantly associated with the change in FINS, FCP, HOMA-IR, HOMA-β, and TCH at 3 months after surgery in females .(1) Circulating RBP4 levels were positively associated with body mass index(BMI), blood glucose in 0 min (BG0), BG30, BG120, BG180, fasting inulin(FINS), fasting C peptide(FCP), homeostasis model of assessment for insulin resistance index (HOMA-IR), SOD, TSH and negatively associated with Matsuda index in obesity with a significant difference (Overall, our results interpret the significant correlations between RBP4, glucose-lipid metabolism, oxidative stress and thyroid function in obese patients. Further, the LSG brings a decline in RBP4 levels and that may contribute partly to the improved insulin resistance in obese Chinese patients. Lifestyle changes contribute significantly to the increased prevalence of obesity worldwide. Obesity causes type 2 diabetes and associated chronic complications –3. ThereP < 0.001) , uric acid (UA) and superoxide dismutase (SOD) were measured by Roche Cobas c701 fully automatic biochemical analyzer. Free triiodothyronine (FT3), free thyroxin (FT4) and thyroid-stimulating hormone (TSH) were measured by ADVIA Centaur XP Immunoassay System. A 75 g-oral glucose tolerance test (OGTT) was performed to estimate glucose metabolism and insulin clearance according to the methods demonstrated by World Health Organization . The gluThe HOMA of insulin resistance (HOMA-IR) was calculated to assess insulin resistance by the following formula: fasting serum insulin concentration (uIU/mL) × fasting blood glucose concentration (mmol/L)/22.5 . HOMA-β ×MeanIns . Serum rImpaired fasting glucose (IFG) was defined by fasting glucose levels ≥ 5.6 mmol/L . HyperinP value of less than 0.05.SPSS20.0 statistical software was used to analyze the data. Descriptive statistics were expressed as mean ± standard deviation (X ± SD). Count data were expressed as number (n). An independent sample-test was used to compare the data between two independent groups. Statistical significance before and after surgery was evaluated using the paired two-tailed t-test. Nonparametric tests were used for non-normal distributed data. Pearson’s and Spearman’s correlation coefficient was used for correlations between RBP4 levels and other markers. The difference was considered significant at a 2. 10 healthy persons aged 32.08 ± 9.84 years old  with average BMI 20.22 ± 1.15 kg/m2 were enrolled as a control group. RBP4 levels were significantly higher in obese patients than healthy weight controls  as presented in Fig. P = 0.416). Additionally, the average BMI of 30 obese subjects  who underwent LSG was 38.97 ± 4.83 kg/m2 and the average age of them was 33.16 ± 11.03 years old. Markers of glucose-lipid metabolism of the 30 obesity subjects were presented in Table There were a total of 82 obese patients enrolled  with average BMI 35.20 ± 6.69 kg/mP < 0.05). RBP4 levels were significantly and positively associated with UA, BG0, BG30, BG120, BG180, INS0, CP0, HOMA-IR, HGB, SOD, TCH and TG in females with obesity . RBP4 levels were only significantly and positively associated with HOMA-IR, SOD and negatively with the Matsuda index in males with obesity . All the results were showed in Table P = 0.035; 55.18 ± 16.90 mg/L vs. 39.57 ± 12.45 mg/L, P = 0.001; 55.58 ± 17.81 mg/L vs. 44.33 ± 13.67 mg/L, P = 0.007). Additionally, RBP4 levels of the patients with hypertriglyceridemia or hypercholesterolemia were significantly higher than patients with normal TG or TCH . RBP4 levels of the patients with higher LDL-C or lower HDL-C were slighter higher than corresponding control groups . All the data were presented in Fig. RBP4 levels were significantly and positively associated with BMI, BG0, BG30, BG120, BG180, INS0, CP0, HOMA-IR, SOD, TSH and negatively associated with Matsuda index in all obesity . TG at 3 and 6 months, UA at 6 months, SOD at 3 months after surgery were significantly decreased and HDL-C at 3, 6 months after surgery were significantly increased in females . SOD and HDL-C were significantly increased at 6 months after surgery in males . All the results were showed in Table P < 0.05) as shown in Fig. P < 0.05) as presented in Fig. P = 0.002). RBP4 levels were significantly decreased from 43.50 ± 12.11 to 31.6 ± 4.74 mg/L at 3 months, and to 28.83 ± 13.74 mg/L at 6 months after surgery in males (P = 0.020 and P = 0.017). RBP4 levels were significantly decreased from 43.07 ± 15.48 to 34.79 ± 11.76 mg/L at 3 months, and to 32.00 ± 11.94 mg/L at 6 months after surgery in females (P = 0.037 and P = 0.027). It need be mentioned that RBP4 levels were slightly decreased from 3 months to 6 months after surgery without statistic difference in females, males and all subjects with obesity . All these results were showed in Fig. Weight, BMI, WC, HC and, TG were significantly decreased in males and females at 3, 6 months after surgery . In females, the change in serum RBP4 levels were significantly related to change in FINS, FCP, HOMA-, HOMA-β and TCH at 3 months after surgery . The change in serum RBP4 levels were not significantly associated with the change in lipid profile while were significantly associated with FCP at 6 months in all obesity . All results are presented in Table In males, the change in serum RBP4 levels were significantly associated with a change in FINS, FCP, HOMA-IR and HOMA-β at 6 months and were associated with a change in TSH at 3 months after surgery  [As a metabolic risk factor in obesity, RBP4 has been reported to be associated with insulin resistance and adipose accumulation. RBP4 levels are elevated in obesity and are positively associated with BMI and WHR –27. In o < 0.05) . Another < 0.05) . By takiRBP4 was mostly secreted by the human liver and adipose tissue as an adipokine . RBP4 coIn addition to the influence of RBP4 on glucose metabolism, it also has a specific role in lipid metabolism. A previous study proved that elevated serum RBP4 is associated with increased serum TG levels and decreased HDL-C levels . In thisAs a novel adipokine secreted by adipocyte, RBP4 is significantly higher in obese subjects than adults with normal weight , 30. RBPTSH levels present a positive association with BMI and are increased in obese patients when compared to the control group with normal weight . Obese pMetabolic surgery is more and more popular worldwide for reducing body weight and improving metabolism. Insulin resistance is significantly improved earlier than weight loss after surgery . In our There also some limitations to our study. The sample size is relatively smaller, and the follow-up time is relatively short. We will further expand the sample size and extend the follow-up time to observe the change in RBP4 for the long-term.Overall, we relate the association between RBP4 with glucose metabolism, oxidative stress and thyroid function in patients of Chinese ethnicity. After the LSG surgery, the patients have shown improved glucose metabolism and decreased RBP4 levels in the 3rd and 6th months. Our study also suggests, decreased RBP4 levels may partly account for the improved glucose metabolism in obese patients after LSG. Future in-depth analyses on this area could project LSG as the most reliable treatment method for obesity and RBP4 as a trustable biomarker.


The results indicated the risk of accelerating the pathogenesis of AD by midazolam and suggested that TFEB might be a candidate target for reduction of midazolam-dependent neurotoxicity.Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases, and β-amyloid (Aβ) plays a leading role in the pathogenesis of AD. The transcription factor EB (TFEB), a main regulating factor of autophagy and lysosome biosynthesis, is involved in the pathogenesis of AD by regulating autophagy-lysosomal pathways. To date, the choice of anesthetics during surgery in patients with neurodegenerative diseases and evaluation of the effects and underlying mechanisms in these patients have rarely been reported. In this study, the HEK293-APP cells overexpressing APP and Hela cells were used. The cells were treated with midazolam at different concentrations and at different times, then lysosomes were stained by lysotracker and their morphology was observed under a fluorescence microscope. The number and size of lysosomes were analyzed using the ImageJ software. The levels of TFEB in the nucleus and APP-cleaved intracellular proteins were detected by nuclear separation and Western Blot. Finally, ELISA was used to detect the levels of Aβ40 and Aβ42 in the cells after drug treatment. We found that 30 μM midazolam decreased the number of lysosomes and increased its size in HEK293 and HeLa cells. However, 15 μM midazolam transiently disturbed lysosomal homeostasis at 24 h and recovered it at 36 h. Notably, there was no significant difference in the extent to which lysosomal homeostasis was disturbed between treatments of different concentrations of midazolam at 24 h. In addition, 30 μM midazolam prevents the transport of TFEB to the nucleus in either normal or starved cells. Finally, the intracellular C-terminal fragment β (CTFβ), CTFα, Aβ40 and Aβ42 levels were all significantly elevated in 30 μM midazolam-treated HKE293-APP cells. Collectively, the inhibition of TFEB transport to the nucleus may be involved in midazolam-disturbed lysosomal homeostasis and its induced Aβ accumulation  HEK293 cells are primary embryonic human kidney cells, HEK293-APP are APP-overexpressed HEK293B cells, and these cells were kindly provided by WJ in Shanghai Jiao Tong University. HeLa cells are human cervical cancer cell lines, and they were kindly provided by Professor Longping Wen in the University of Science and Technology of China. Nutrient starvation assays were performed in the presence of Earle’s Balanced Salt Solution (EBSS).Cells were cultured at 37°C with 5% CO4 cells per well, incubated in 37°C, 5% CO2 for 24 h and then treated with midazolam for different times and washed twice in PBS. Next, the cells were incubated in medium containing 1 μM LysoTracker Red DND-99  dye for 10 min. Cells were washed twice in PBS and observed under an LSM 710 confocal microscope . The size and number of lysosomes were measured by ImageJ software using its “analyze particle” analysis tool with default image/adjust/threshold settings. For lysosomal morphology and size, as well as the effect size between the two groups were calculated using Ellis  for 10 min, the cellular fluorescence was observed by confocal microscopy . The HeLa EGFP-TFEB cell lines were kindly provided by professor Wen Longping, which expressed strong green fluorescence under a fluorescence microscope.2, 1 m magnesium acetate, 250 × 10−3 M ethylenediaminetetraacetic acid (EDTA), 100 × 10−3 M dithiothreitol (DTT), and 100 × 10−3 M phenylmethylsulfonyl fluoride (PMSF)] was used to separate nuclear and cytoplasm fraction. Briefly, cells were collected from a 24-well cell culture plate after treatment and washed by PBS, and then 100–200 μL sucrose NP-40 (0.5% NP-40 in sucrose buffer) buffer was added. Those samples were put on ice for 15 min and centrifuged for 10 min at 1,000× g. The supernatant was the cytoplasm fraction. In contrast, the precipitation was the nuclear fraction and washed by sucrose buffer before adding cell lysis buffer and boiling.The sucrose buffer [1 M sucrose, 0.1 M CaClCells were lysed with sample buffer and boiled for 10 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred onto nitrocellulose membranes. The membranes were incubated with the primary antibody at 4°C overnight and the second antibody for 1 h at 37°C. Membranes were incubated with the ECL kit and visualized using a chemiluminescence instrument .The cells were homogenized in PBS followed by RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1× protease inhibitor  For lysosomal homeostasis detection, HEK293 and Hela cells were divided into two groups: 15 μM and 30 μM midazolam treatment groups. At 0, 24 h and 36 h, the size and morphology of the lysosomes were observed; (2) for TFEB levels detection, HEK293 and Hela cells were divided into two groups: 15 μM and 30 μM midazolam treatment groups. Detect the levels of TFEB at different times  in the nucleus; (3) for TFEB levels detection in the case of starvation with or without 30 μM midazolam, HEK293 and Hela cells were divided into four groups: control, midazolam, starvation and midazolam+starvation. Detect the level of TFEB in the nucleus; and (4) for Aβ levels detection, HEK293 cells were divided into control and 30 μM midazolam treatment group. Detect the C83, C99 levels.t-test for detecting significant differences between two groups. For lysosomal homeostasis detection, the lysosomal size and number were analyzed by one-way ANOVA followed by post hoc Tukey’s test. The Western Blot results were analyzed by one-way ANOVA, followed by Tukey’s Post hoc test or two-tailed Student’s t-test. For Aβ40 and Aβ42 detection using ELISA, the results were analyzed by two-tailed Student’s t-test. Differences were considered statistically significant at *p < 0.05 and **p < 0.01, ***p < 0.001.All data are shown as Mean ± SEM. Data were analyzed by two-tailed Student’s p < 0.001, effect sizes: 2.820) and size  of lysosomes were transiently decreased and became larger at 24 h compared with 0 h, respectively. The above changes were counteracted at 36 h  and swelled lysosomes  until 36 h  and size  of lysosomes temporarily decreased and became larger at 24 h, and these changes were offset at 36 h , but 30 μM midazolam continued to reduce the number of lysosomes  and lysosomes expand  until 36 h. Notably, the size and number of lysosomes had no significant difference under different concentrations of midazolam treatment at 24 h, indicating the similar extent of damage to the lysosomal homeostasis by midazolam (To evaluate the effect of midazolam on lysosomes, HEK293 and HeLa cells were treated with different concentrations of midazolam for 24 h or 36 h. After 15 μM midazolam treatment, the number (til 36 h . What’s til 36 h , at 15 μidazolam .As TFEB is the major regulator of lysosome biosynthesis, the levels of TFEB in the nucleus was detected in midazolam-treated cells. The results showed that nuclear TFEB levels were reduced at 24 h in both HKK293 and HeLa cells treated with 15 μM midazolam compared with 0 h, but it was recovered at 36 h . HoweverIn starvation condition, TFEB will transport from cytoplasm to nucleus to upregulate lysosomal function and autophagy, and then provide enough nutrients for the cells to survive (Settembre et al., Aβ has been shown to be neurotoxic and plays a leading role in the pathogenesis of AD. The lysosome is an important organ required for Aβ degradation (Xiao et al., In this study, we found that the clinically used anesthetic sedative midazolam increased intracellular Aβ levels, suggesting that midazolam may promote the pathological process of AD. Additionally, our previous studies have also shown that midazolam increases intracellular mutant huntingtin protein, revealing its risk of accelerating the pathogenesis of polyglutamine diseases (Zhang et al., Notably, midazolam-induced accumulation of neurotoxic protein aggregates was always accompanied by impaired lysosomes/autolysosomes function. In this article, we demonstrate that midazolam destroys lysosomal homeostasis primarily in HEK293 cells which is commonly used instrumental cells with high transfection efficiency and convenient operation in the study of molecular mechanism. They are also often used to study the pathogenesis of Alzheimer’s disease. HT22 is hippocampal neuronal cell lines of mouse. And we further test the inhibitory effect of midazolam on lysosomal homeostasis in HT22 cells, the result is the same as HEK293 cells . Some otThe TFEB, a major regulator of lysosomal biosynthesis, is coordinated by driving autophagy and expression of lysosomal genes (Settembre et al., in vitro since in vitro cell experiments are unilateral and the pathological process cannot be completely simulated in vivo. Thus, the effect and mechanism of action of midazolam in AD should be further investigated in vivo. Studies have shown that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aβ generation. Activation of TFEB in neurons is an effective strategy to attenuate Aβ generation and attenuate amyloid plaque deposition in AD (Xiao et al., The impaired autolysosomal function has been reported in sevoflurane-exposed AD mice, and it may be involved in the acceleration of the AD pathological process by sevoflurane (Geng et al., DC and QT designed and conducted the study, analyzed and interpreted the data and wrote the manuscript. QZ participated in the work of additional figures 1 and 2 in the later period and participated in the revision of the article. JZ, XH and PF helped conduct the study. WJ provided us with a strain of cells. XL conceived and designed the study, analyzed and interpreted the data, and drafted and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since there are many manufacturers of cardiac troponin I assays, the true incidence of a false positive result is unknown. The authors report a case of an 18-year-old patient with previous history of recurrent myopericarditis and admitted to hospital twice again with a suspicious of myopericarditis.Troponin I was found to be a false positive and alkaline phosphatase interference was proved to have been the responsible for this. However®  is used while in the routine laboratory - that executes tests for inpatients - the Abbott Architect STAT high sensitive TnI®  is performed.In our institution, for practical reasons, two different troponin I assays are employed. In the emergency laboratory - which executes tests for patients in the emergency department - the Beckman Coulter Access AccuTnI+32We report a case of a 18-year-old female patient with previous history of recurrent myopericarditis in 2015 and 2016. In both episodes, troponin I was elevated  and slowly declined. In the last episode she took ibuprofen 600 mg every 8h for 2 weeks and colchicine 0.5 mg twice a day for 6 months. Physical activity was restricted for 6 months. Transthoracic echocardiography was normal but cardiac Magnetic Resonance Imaging (MRI) performed latter demonstrated residual fibrosis in the lateral wall of left ventricle.The patient remained asymptomatic until June 2017 when she complained of palpitations and mild chest pain. Physical examination, Electrocardiogram (ECG) and transthoracic echocardiography were normal but troponin I was positive  so myopericarditis was assumed and the patient was admittedto the hospital and ibuprofen and colchicines were initiated. The next day and until discharge, troponin I remained negative and the patient was free from symptoms. Serology for virus, interferon gamma release assay and clinical or laboratory tests for autoimmune diseases were all negative. The possibility of a false positive test on admission was thought due to the immediate fall in troponin level. However, owing to the relief of symptoms with medication, myopericarditis could not be excluded and the patient was discharged on ibuprofen and colchicine at the same dose as before.1). Once more, a false positive result was a possibility since the first measurements were performed by the emergency laboratory (Beckman Coulter Access AccuTnI+3®) and the next ones by the routine laboratory (Abbott Architect STAT high sensitive TnI®). To rule out a false positive result in the first measurement, performed by emergency laboratory, two samples of blood were collected at the same time and sent to both laboratories. The result was positive in the emergency laboratory (1.03 ng/mL) and negative in the routine laboratory (0.00 ng/mL). Although alkaline phosphatase activity was within the normal range , alkaline phosphatase interference was proved in the Beckman Coulter central laboratory. To demonstrate that, beta human chorionic gonadotropin (βhCG) was measured using the Beckman Coulter Dxl 800 Total β-hCG®  assay [Three weeks later she returned to the hospital complaining of similar symptoms. Once again, initial measurement of troponin I was positive (1.31 ng/mL) and incessant myopericarditis was assumed. Physical exam remained unremarkable as well as the ECG and transthoracic echocardiography. During hospitalization, measurements of troponin I remained always negative. She had a cardiac MRI once again but no signals of active myocarditis were detected and residual fibrosis remained as previously described  so myopericarditis indeed existed. In order to avoid future hospital admissions, information regarding alkaline phosphatase interference with troponin I and other assays was provided to the patient and added to the patient´s files.3Currently, cardiac troponin is the most widely used cardiac biomarker in emergency departments. In the correct clinical context, troponin elevation may confirm the diagnosis of acute coronary syndromes, stratify the risk of further adverse cardiac events or result from a variety of non-coronary causes of cardiac myocyte damage or necrosis, such as myocarditis. Several immunoassays are commercially available for determination of cardiac troponin I concentrations, while only one assay for troponin T is obtainable.® uses alkaline phosphatase for signal amplification while the Abbott Architect STAT high sensitive TnI® uses acridinium, the first suspicion was interference of alkaline phosphatase. In fact, elevated alkaline phosphatase can interfere with contemporary alkaline phosphatase-dependent immunoassays as recently demonstrated [The Access AccuTnI assay is a two-site immunoenzymatic “Sandwich” assay. A sample is added to a reaction vessel along with monoclonal anti-troponin I antibody conjugated to alkaline phosphatase and paramagnetic particles coated with monoclonal anti-troponin I antibody. Human troponin I binds to the anti-troponin I antibody on the solid phase, while the anti-troponin I antibody- alkaline phosphatase conjugate reacts with different antigenic sites on the troponin I molecules. After incubation, the chemiluminescent substrate Lumi-Phos* 530 is added and light generated by the reaction is measured with a luminometer. The light production is directly proportional to the concentration of troponin I in the sample -8. The Anstrated  but thisnstrated , it is sUnfortunately, since there are the many manufacturers of troponin I assays, the prevalence of clinically significant inaccurate quantification of troponin I is unknown . Given iThe authors demonstrate that a false positive troponin I test may lead to inaccurate diagnosis with important clinical implications, if clinical judgment is not used. In this case, two hospital admissions were not necessary; in other situations, as acute coronary syndrome suspicion, a false positive result lead in an unnecessary angiography , 13. As


Testicular trauma is relatively uncommon. However, severe injuries can result in many complications and should be carefully diagnosed and managed. We present a case of testicular fracture diagnosis made by ultrasonography. The surgical exploration revealed the fracture as well as complete rupture of the tunica albuginea. Testicular rupture is the disruption of the tunica albuginea, while testicular fracture is a “break” in the testicular parenchyma. Management could be conservative in mild fracture cases without rupture while suspected or confirmed fracture should be treated by surgical exploration. Testicular trauma is relatively uncommon as the organs are protected by the dependent and mobile nature of the scrotum. However, severe testicular injury can result in organ loss which can affect fertility contribute to hypogonadism and affect social confidence. Rupture or fracture of the testis might result in the release of spermatic antigens and the subsequent exposure to the immune system resulting in the production of antisperm antibodies. This might lead to infertility by affecting sperm function , and earA 32-year-old motorcyclist was admitted following a collision with a car travelling at 30 mph. His scrotum impacted the motorbike handlebars and he was thrown over the car bonnet. He complained of right-sided testicular pain. The past medical history was uneventful. Clinical examination revealed normal lie of the right testicle with a small contained haematoma. The left testicle was entirely normal. Abdominal examination was unremarkable. The patient was cleared of all nonurological injuries.Scrotal ultrasonography showed irregular hypoechoic regions in the lower pole of the right testis with no significant Doppler flow evident. The tunica albuginea appeared intact and there was slightly increased Doppler flow in the rest of the testis. The left testis was normal. Figures  and 2. TA decision for surgical exploration was made and through a midline scrotal incision, the right haematocoele was evacuated. The lower pole of the right testis was seen to be fractured in a bi-valve pattern. The tunica albuginea was circumferentially dismembered (Ruptured) and the testicle was seen as nearly two separate lobes. As both halves appeared largely viable, it was decided to spare the testis and reconstruct. An area of necrotic tissue was excised, after which the tunica albuginea was closed with 4/0 vicryl continuous suture. There injury to the tail of the epididymis was also repaired. Histopathology of the excised testicular tissue confirmed haemorrhage and focal necrosis.A follow-up ultrasound scan testes at one month revealed an entirely normal testicle Figures  and 4. TBlunt injury accounts for most of testicular trauma cases and it occurs most commonly during sporting injuries, straddle injuries, motor vehicle accidents, and assaults, mostly affecting males aged between 10 and 30 years . A detaiThe tunica albuginea is a tough, fibrous outer covering of the testis. Testicular rupture is defined as interruption of the tunica albuginea (or change in contour) or extrusion of seminiferous tubules . TesticuA retrospective study by Dalton et al found that the discontinuity of the echogenic tunica albuginea is the main sign of testicular rupture which indicates the necessity of surgical intervention . SonograAnother paper used the absence of normal vascularity within the testis as an indicator that may help characterize a rupture .An abnormality of the contour of the testis is considered indirect evidence of testicular rupture in the case of a large extratesticular haematocoele or a large scrotal wall hematoma that may obscure the site of tunical disruption . Due to Testicular fracture refers to a break or discontinuity in the normal testicular parenchyma. It is diagnosed using ultrasonography and appears as a linear hypoechoic and avascular area within the testis. This finding can be associated with rupture of the tunica albuginea. Only 17% of cases demonstrate a fracture line –15.In such cases, color Doppler imaging plays an important role in directing management . The preIn our case, the patient was reported to have a fracture of the testis on ultrasonography; however on exploration the testis was completely ruptured. In these cases, magnetic resonance imaging does have higher sensitivity and a diagnostic accuracy of 100% . HoweverIn patients with testicular trauma, ultrasonography performed by an experienced operator can yield useful information to guide management. Accurate terminology should be used when reporting the findings—testicular rupture is the disruption of the tunica albuginea, while testicular fracture is a “break” in the testicular parenchyma. The two findings can occur in synchrony. Management should always be tailored to the specific injuries; however urgent scrotal exploration should be the gold standard in investigating and treating suspected testicular rupture.


Neuronal and glial cell interaction is essential for synaptic homeostasis and may be affected in Alzheimer’s disease (AD). We measured cerebrospinal fluid (CSF) neuronal and glia markers along the AD continuum, to reveal putative protective or harmful stage-dependent patterns of activation.n = 36) and Aβ-positive (Aβ+) cases ) with either subjective cognitive decline , mild cognitive impairment , or AD dementia (n = 27). The following CSF markers were measured: a microglial activation marker—soluble triggering receptor expressed on myeloid cells 2 (sTREM2), a marker of microglial inflammatory reaction—monocyte chemoattractant protein-1 (MCP-1), two astroglial activation markers—chitinase-3-like protein 1 (YKL-40) and clusterin, a neuron-microglia communication marker—fractalkine, and the CSF AD biomarkers , total tau (T-tau)). Using ANOVA with planned comparisons, or Kruskal-Wallis tests with Dunn’s pairwise comparisons, CSF levels were compared between clinical groups and between stages of biomarker severity using CSF biomarkers for classification based on amyloid pathology (A), tau pathology (T), and neurodegeneration (N) giving rise to the A/T/N score.We included healthy controls (p < .01), MCI (p < .05), and AD dementia cases (p < .001) and increased in AD dementia compared to MCI cases (p < .05). MCP-1 was increased in MCI (p < .05) and AD dementia compared to both healthy controls (p < .001) and SCD cases (p < .01). YKL-40 was increased in dementia compared to healthy controls (p < .01) and MCI (p < .05). All of the CSF activation markers were increased in subjects with pathological CSF T-tau (A+T−N+ and A+T+N+), compared to subjects without neurodegeneration (A−T−N− and A+T−N−).Compared to healthy controls, sTREM2 was increased in SCD  were noted only at the MCI and AD dementia stages, respectively, and in Aβ+ cases (A+) with pathological T-tau (N+). Possible different effects of early and later glial activation need to be explored.The online version of this article (10.1186/s12974-019-1399-2) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) may be described as a biological continuum that includes the hallmark pathological processes of amyloid-beta (Aβ) dysmetabolism, formation of amyloid deposits (A), neurofibrillary tangles (T), neurodegeneration (N), determined by measuring cerebrospinal fluid (CSF) levels of Aβ42, phosphorylated tau (P-tau), and total tau (T-tau) respectively. The presence or absence of pathological markers can be summarized as an A/T/N score, an unbiased classification of pathology and severity along the AD continuum , 2. In cWhile genetic evidence indicates that Aβ dysmetabolism is causal in familial AD, the initial sequence of events and causality in sporadic AD is still not determined. However, reduced Aβ clearance and deficient innate immune activity related to the triggering receptor expressed on myeloid cells 2 (TREM2) and clusterin (Apo J) function may play a role –11. WhilGlial activation occurs as part of altered immune cytokine activities, which also change towards increased inflammatory activity during AD progression. However, micro- and astroglial activation are interlinked, and genetic evidence suggests that innate immunity could be a prime mover in the AD cascade . Based oSoluble TREM2 is released upon microglial activation, leading to increased levels of CSF sTREM2 in AD , 30. ThiThese individual markers have been studied in predementia and in AD dementia stages with variable reported findings , participants with SCD (n = 18), and MCI (n = 20) patients, both with CSF Aβ42 confirmed amyloid pathology, were selected from the Norwegian multicenter study, “Dementia Disease Initiation” (DDI) [n = 27) and an additional 19 MCI patients with CSF Aβ42 confirmed amyloid pathology were included from the Norwegian part of the Gothenburg-Oslo MCI (MCI-GO) cohort [18F]-Flutemetamol uptake study [For the purposes of the present study, we selected 121 participants from two Norwegian cohorts. Healthy controls with normal CSF (n” (DDI) . A patie) cohort . Classifke study . Cases wke study . For P-tFurther criteria for inclusion were age between 40 and 80 years and a native language of Norwegian, Swedish, or Danish. Exclusion criteria were brain trauma or disorders, including clinical stroke, dementia, severe psychiatric disorder, and severe somatic disease that might influence the cognitive functions, intellectual disability, or other developmental disorders.Both DDI and MCI-GO employ a standardized protocol for participant selection, assessment, and disease stage classification according to published criteria , 4, 6. AParticipants were classified as SCD according to the SCD-I framework, which requires normal objective cognitive performance on neuropsychological tests while experiencing a subjective decline in any cognitive domain . MCI wasg for 10 min at room temperature. The supernatant was subsequently transferred to new defined tubes, directly frozen at − 80 °C on site and kept at − 80 °C until thawed for analysis. All CSF samples were analyzed either at the Department of Interdisciplinary Laboratory Medicine or Section of Clinical Molecular Biology (EpiGen) at Akershus University Hospital. The exception was the sTREM2 analysis, which was assayed at the Department of Pharmacology at the University of Oslo.Lumbar punctures were performed similarly on four sites all following a detailed BIOMARKAPD SOPs as described previously . BrieflyCommercial enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies were used to measure CSF levels of the following protein biomarkers: Aβ42, T-tau, and P-tau. They were determined using Innotest β-Amyloid (1-42), Innotest T-tau Ag, and Innotest P-tau (181P), respectively.CSF sTREM2 was also analyzed using a sandwich ELISA as described earlier  with somThe QuickPlex SQ 120 system from Meso Scale Discovery  was used to measure YKL-40, MCP-1, and fractalkine in a U-plex format and clusterin in an R-plex format, where YKL-40 and clusterin were in a singleplex setup and MCP-1 and fractalkine were in the same multiplex setup. The MSD analyses were carried out according to the manufacturers’ procedures, with the adjustments that CSF samples were diluted 200 times prior to YKL-40 and clusterin analyses, and the multiplex setup was used with 100 μl neat CSF and 25 μl buffer.All the lower limits of quantifications (LLOQs) were defined as the lowest concentration at which the coefficient of variation (CV) of the calculated concentration was < 20% in > 75% of the analyses or the mean CV was < 20% in our test set. All biomarker values in all samples were well above LLOQ. All samples were analyzed in duplicates and reanalyzed if relative deviations (RDs) exceeded 20%. In addition, quality control samples with RD threshold of 15% assured inter-plate and inter-day variation.Normality was assessed through the inspection of Q-Q plots, histograms, and the Shapiro-Wilks test of normality.In order to explore and adjust for age and sex, and APOE-ɛ4 allelic effects on CSF inflammatory markers in healthy aging, simple and multiple regression analyses  were performed between these variables and CSF immune markers within the healthy control group. If a significant relationship was observed between these covariates and an inflammatory marker in the healthy control group, the standardized residuals from the pertinent regression model was obtained for the entire sample and used in further analysis in order to adjust for these covariates in between-group comparisons. To assess differences in biomarker levels between groups, we performed one-way ANOVAs with planned comparisons for variables with normal distributions. We performed Kruskal-Wallis test with Dunn’s non-parametric pairwise post hoc test with Bonferroni corrections to assess group differences in variables with non-normal distributions . Non-parametric pairwise comparisons and ANOVA contrasts were performed in a hierarchical manner. We compared Aβ + SCD, MCI, and AD dementia groups to healthy controls, and finally we compared the SCD with the MCI group and both SCD and MCI to the AD dementia group. The dichotomous variable “sex” was assessed using a chi-square test. For the A/T/N groups, A−T−N− and A+T−N− were compared to all other groups. Only one patient had A+T+N− classification, and this patient was excluded from both statistical analysis and figure.r within the pertinent symptomatic groups .To assess clinical stage dependent relationships between the innate immune response to AD pathology, correlational analyses between the inflammatory markers  and CSF AD biomarkers  were performed using Pearson’s p < .05.All analyses were performed in the Statistical Package for Social Sciences (SPSS) version 25, and the significance level was defined as The regional medical research ethics committee approved this study. Participants gave their written informed consent before taking part in the study. All further study conduct was in line with the guidelines provided by the Declaration of Helsinki  and the Norwegian Health and Research Act (2009).See Table β = .477, p < .001) and T-tau  were shown to predict CSF YKL-40  , when controlling for the effects of T-tau  in a multiple regression analysis . No relationships were found between age and CSF MCP-1, clusterin, or fractalkine within the healthy control group in any regression models. Thus, only YKL-40 was adjusted for age-effect prior to analysis of between-group differences in clinical staging.Neither APOE-ɛ4 status nor sex was associated with any of the CSF inflammatory markers in healthy controls when using simple regression analysis. When age and CSF T-tau were included in multiple regression models, both age  = 3.282, p < .01), Aβ + MCI subjects (t(116) = 2.364, p < .05), and subjects with AD dementia (t(116) = 4.213, p < .001), compared to healthy controls. A higher CSF sTREM2 level was also found in subjects with AD dementia compared to Aβ + MCI subjects (t(116) = 2.135, p < .05).CSF sTREM2 values were higher in Aβ + SCD subjects (t(117) = 2.370, p < .05) and healthy controls (t(117) = 3.096, p < .01).No difference in the CSF YKL-40 level was found between healthy controls, and Aβ + SCD or Aβ + MCI subjects. However, an increased level of CSF YKL-40 was found in subjects with AD dementia compared to both Aβ + MCI subjects (t(117) = 2.480, p < .05) and (t(117) = 3.704, p < .001) respectively).No differences in CSF MCP-1 levels were demonstrated between Aβ + SCD and healthy controls. However, CSF MCP-1 levels were equally increased in Aβ + MCI subjects and subjects with AD dementia, and levels where higher in these groups compared to healthy controls  = 2.574, p = .057), with markedly higher CSF clusterin levels in the dementia group compared to the control group  compared to participants with cognitive symptoms with A+T−N− (n = 23). However, the levels of these markers were all equally increased in A+T−N+ (n = 30) and A+T+N+ (n = 31), p < .01–p < .001 and p < .05–p < 01 respectively, please see Table n = 9) and those with normal CSF levels, for either CSF sTREM2, YKL-40, MCP-1, clusterin, or fractalkine (independent samples T-test), nor no significant correlations between CSF MCP-1 or clusterin and T-tau or P-tau. However, there were significant positive correlations between CSF sTREM2, YKL-40, and fractalkine with both T-tau and P-tau within the SCD group  and A+T+N+ (n = 31) compared to patients with pathological CSF Aβ without tau pathology or neurodegeneration markers A+N−T− (n = 23) (p < .001 and p < .01 respectively), in accordance with the association between fractalkine and T-tau (Table p = .057). CSF clusterin levels were equally increased in both A+T−N+ (n = 30) and A+T+N+ (n = 31) compared to healthy controls with normal CSF  (p < .01 and p < .05 respectively) and also in accordance with the association with T-tau  in the SCD group. CSF T-tau correlated strongest with all of the before mentioned activation markers when all symptomatic subjects were combined, in addition to several of the subgroups. For details, please see Table There were no significant correlations between sTREM2, YKL-40, MCP-1, or fractalkine and CSF Aβ42 in our cohort, neither in subgroups controlled separately nor when all symptomatic subjects were combined. There was, however, a correlation between CSF clusterin and CSF Aβ42 , indicating neurodegeneration, compared to those subjects without neurodegeneration (A−T−N− and A+T−N−). Interestingly, neither glial activation markers nor inflammatory markers were significantly increased in cases with only Aβ+ pathology (A+T−N−) compared to healthy controls (A−T−N−), though CSF fractalkine showed a non-significant reduction. For both CSF fractalkine and CSF clusterin, the increase with increasing neurodegeneration may be masked when comparing the clinical groups, as these contain subjects with and without neurodegeneration, but it became evident when we employed the A/T/N classification. For CSF fractalkine, we found no between group differences between the control group/A−T−N− compared to the other clinical groups. However, when comparing A/T/N groups as a measure of AD biomarker severity, the Aβ42-positive group without neurodegeneration  showed significantly lower CSF fractalkine levels compared to the Aβ42-positive groups with neurodegeneration . CSF fractalkine showed a clear association to T-tau in all clinical subgroups, see Table Our findings in the clinical stages are in accordance with imaging data suggestive of a biphasic microglial response, though the sTREM2 level in our material at the SCD stage was only nominally higher but not significantly different from that at the MCI stage , 26. AgiOur findings may help interpret studies of immunomodulating therapies towards reducing the Aβ deposition by increasing the microglial clearance . A too aA limitation of these findings is that these markers are expressed or secreted by multiple cell types and are not completely specific to neuronal, microglial, or astrocyte expression. Fractalkine is mainly expressed on neurons in the CNS , but expIn addition to a relatively small sample size, a further limitation in this work is the cross-sectional design, as longitudinal sampling is necessary for a better interpretation of the sequence of events along the AD continuum. Furthermore, a more extensive mapping of inflammatory cytokine markers will be needed.We here demonstrate increased microglial activation at a preclinical AD stage, with increased CSF sTREM2 levels in Aβ + SCD. The MCP-1 level was significantly enhanced at the Aβ + MCI stage, and CSF YKL-40 in AD dementia, suggesting a shift towards a more harmful stage of immune activation as AD progresses. Furthermore, our findings suggest that inflammation is associated with neurodegeneration, but not with amyloid pathology alone.Additional file 1:Table S1. Literature overview CSF immune markers in AD clinical groups. (DOCX 72 kb)Additional file 2:Figure S1. Correlation between CSF YKL-40 and age. (PDF 805 kb)Additional file 3:Figure S2. Between-group comparisons of microglial- and astroglial activation with inflammation based on clinical staging. (PDF 277 kb)


Trachomatous trichiasis (TT) is a painful and blinding condition in which the eyelashes turn inwards and damage the cornea. TT surgery is the remedy.more than 230,000 operations across 35 countries to treat trachomatous trichiasis (TT), the late blinding stage of trachoma.In 2017, the global trachoma elimination programme conducted The World Health Organization recognised the challenge and convened a meeting in 2015 to discuss the development of a system to track TT patients through the steps of surgical intervention.Surgical teams using the TT Tracker enter patient information into electronic forms at each stage in the patient's journey, which reduces the need to collect and file paper-based forms. The information collected includes:Registration and evaluation. Demographic information, TT diagnosis, recommended interventionSurgery. Type of operation/sutures, name of surgeon, related complicationsFollow-up . Surgical outcome assessment and actions required to address complicationsOrganizing trichiasis surgical outreach’ preferred practice manual.Common details related to surgical provision, such as the type of suture used, are included in the predefined list of selections (adapted for each country); these are based on the International Coalition for Trachoma Control (ICTC) ‘All data are owned by the government and collected on password-protected devices. Surgeons are granted access to patient information within their ministry-designated working area only.TT Tracker uses the information collected by surgical teams to generate the following:Patient follow-up lists. After surgery, all patients are automatically placed on follow-up lists based on the relevant follow-up time-period; lists can be accessed by programme staff and officials approved by the Ministry of Health. Reports are electronically sent to supervisors detailing where and when follow-up must take place. Surgical outcomes are assessed during follow-up visits and are added to patient records to ensure each surgical record is complete.Surgical performance evaluation. The TT Tracker creates performance assessments for individual surgeons so supervisors know when enhanced supervision or additional training is needed. To provide encouragement, surgeons also receive personalised emailed reports including their individual outcomes and contribution to the national elimination effort.Timely, easy-to-access reports. The TT Tracker includes a reporting tool that analyses data automatically. Reports are updated daily and are easy to understand, allowing programme staff to use the data for decision making and to improve the programme, which in turn encourages regular use.All national trachoma programmes are eligible to use the TT Tracker to support elimination activities; however, programmes must cover implementation costs, including training, phones and outreach. The tool has been successfully piloted in Sightsavers programmes in Nigeria and Tanzania in 2017/2018 as part of the DFID SAFE programme, followed by a country-wide roll-out in Uganda in July 2018 as part of The Queen Elizabeth Diamond Jubilee Trust's Trachoma Initiative. Full expansion is planned in 2019 in Benin, Guinea, Nigeria, Senegal, Tanzania, and Zimbabwe in the new Accelerate Trachoma Elimination Programme.As more countries show positive results using the TT Tracker, there is potential to apply it to other surgical eye interventions or other neglected tropical diseases, further contributing to global targets for universal eye health coverage and the UN sustainable development goals.www.tttracker.org/homeFor more information about the TT Tracker or to get in touch with the team, please visit:


A novel framework based on the fast orthogonal search (FOS) method coupled with factorization algorithms was proposed and implemented to realize high-accuracy muscle force estimation via surface electromyogram (SEMG). During static isometric elbow flexion, high-density SEMG (HD-SEMG) signals were recorded from upper arm muscles, and the generated elbow force was measured at the wrist. HD-SEMG signals were decomposed into time-invariant activation patterns and time-varying activation curves using three typical factorization algorithms including principal component analysis (PCA), independent component analysis (ICA), and nonnegative matrix factorization (NMF). The activation signal of the target muscle was obtained by summing the activation curves, and the FOS algorithm was used to create basis functions with activation signals and establish the force estimation model. Static isometric elbow flexion experiments at three target levels were performed on seven male subjects, and the force estimation performances were compared among three typical factorization algorithms as well as a conventional method for extracting the average signal envelope of all HD-SEMG channels (AVG-ENVLP method). The overall root mean square difference (RMSD) values between the measured forces and the estimated forces obtained by different methods were 11.79 ± 4.29% for AVG-ENVLP, 9.74 ± 3.77% for PCA, 9.59 ± 3.81% for ICA, and 9.51 ± 4.82% for NMF. The results demonstrated that, compared to the conventional AVG-ENVLP method, factorization algorithms could substantially improve the performance of force estimation. The FOS method coupled with factorization algorithms provides an effective way to estimate the combined force of multiple muscles and has potential value in the fields of sports biomechanics, gait analysis, prosthesis control strategy, and exoskeleton devices for assisted rehabilitation. Muscle force estimation refers to the prediction of contraction force produced by a piece of muscle or multiple muscles during human movement. Precise and reliable muscle force estimation is important to many research fields such as sports biomechanics ,2, gait The surface electromyogram (SEMG) is one such noninvasive method to detect muscle activities at the skin surface. It can reflect the compound neuromuscular activity and has been widely used in the field of force estimation. SEMG signals used for muscle force estimation were recorded by one or several separated unipolar or bipolar electrodes in a number of previous related studies ,14,15,16High-density SEMG (HD-SEMG) grids, which consist of tens or even hundreds of electrodes and cover a large portion of target muscles, have shown good performances in some SEMG-based applications including force estimation ,23,24,25In HD-SEMG-based force estimation, researchers found that HD-SEMG signals recorded from target muscles were high dimensional and some of them might be redundant for force estimation. In many previous studies, a common electrode dimension reduction method was to calculate the average rectified signal of all channels or a part of them ,25,26,27In one example, Staudenmann et al. compared the performances of several HD-SEMG processing methods in the estimation of elbow extension force and found that PCA coupled with HD-SEMG could markedly reduce the root mean square difference (RMSD) . Huang eAlso, a set of physiological and phenomenological models has been proposed to establish the relationship between SEMG and the muscle force. For instance, Bai et al. used a third-order polynomial model to estimate the force exerted by lower limbs with the help of SEMG from the quadriceps femoris and hamstring muscles . MobasseTo extract more useful muscle activation information from HD-SEMG signals and improve the performance of force prediction, a SEMG-force estimation framework based on the FOS method coupled with factorization algorithms  was proposed in this study. More specifically, factorization algorithms were used to decompose HD-SEMG signals from a target muscle into several time-invariant activation patterns and corresponding time-varying activation curves. The activation signal of the target muscle was obtained by summing the activation curves. The FOS method was then utilized to create basis functions constructed by activation signals and establish the force estimation model. The feasibility and superiority of the proposed SEMG-force estimation framework were evaluated under the static isometric elbow flexion task where the generated elbow force was estimated using HD-SEMG signals from the upper arm muscles.The proposed SEMG-force estimation framework based on factorization algorithms and the FOS method can be summarized into four steps , with no known neuromuscular deficits of the right arm, were recruited. Prior to the experiment, all of them signed the informed consent approved by the Ethics Committee on Clinical Research of the First Affiliated Hospital of Anhui Medical University (No. PJ 2017-04-06).The experiments were performed on a homemade apparatus a. A one-During the experiment, subjects were asked to sit upright with the right forearm positioned vertically and the upper arm positioned horizontally. The pads were used to fix the elbows of subjects into the proper position. The force sensor was regulated at the same height as the wrist, and the wrist was tightly tied using an inelastic strap to the board to immobilize the wrist joint decreasing the influence of the wrist force. The subjects were then instructed to keep their left arms and hands in an absolutely relaxed state; the elbow flexion task was performed only on the right upper arm muscles, but this was done as far as possible. Also, their right hand, wrist, and shoulder were all kept static to limit the contributions of forearm and shoulder muscles to the elbow force. The sum of the force moments and the forces acting on the arm could be zero because the isometric elbow flexion task was static—this is obvious in Here, In the first session, each subject was asked to perform the maximum voluntary contraction (MVC) three times, and the largest value was adopted as the final MVC. In each trial, subjects were asked to generate the measured force increasing linearly from zero to a target value within three seconds while keeping this target value state for another three seconds by performing elbow flexion. The three targeted values were 20%, 40%, and 60% of MVC in the experiment. At each level, the motion task was repeated 7 times. To avoid muscle fatigue, a one-minute rest was enforced between two motion cycles. Before data collection, subjects were asked to practice elbow flexion task using their upper arm muscles until they could successfully track the force profiles.m = 32 channels, t = 6000 samples), each column of n = 2 or 3 activation patterns) that describes the spatially correlated activations of m channels by m weighting factors, and each row of Factorization algorithms including PCA, ICA, and NMF were used to process SEMG signals. In simple terms, factorization algorithms refer to problems where both the sources and the mixing methodology are unknown, and only mixture signals are available for further separation. As shown in Equation (2), factorization algorithm can decompose a matrix More specifically, PCA is an orthogonal feature projection method, which aimed to find orthogonal eigenvalues with greatest variance among the data . PCA proICA is a widely used multivariate data analysis technique that can transform the original vector data into mutually statistically independent components (ICs) . Based oNMF was proposed by Lee and Seung  and is aConsidering the anatomical structure of the mainly contained muscles (the biceps brachii has two heads and the triceps brachii muscle has three heads) , two actFor comparison, a force estimation framework based on the conventional averaging method (hereafter referred to as AVG-ENVLP method) was also implemented ,25,26,27The FOS algorithm was first developed by Korenberg  and is aUtilizing the Gram-Schmidt orthogonal method, the candidate basis functions In this case, it becomes easier to calculate the coefficients of the orthogonal basis functions found in ,41:(5)amOverall, the FOS algorithm searches among a set of candidate functions and selects the function that leads to the highest mean square error (MSE) reduction between the estimated and measured system outputs to generate mathematical model. The model iteratively adds one selected function at a time until some criteria are met. For example, the overall MSE of the FOS estimation model has been small enough; a certain number of functions have been selected for the model; or the remaining candidate functions are unable to significantly reduce the MSE.M) are two crucial procedures to establish a force estimation model using the FOS method. In related studies [Establishing a candidate basis function pool and selecting a function number in the model ):To quantify the estimation performance of the trained FOS-based force model, two frequently-used criteria were evaluated: RMSD ) and In Equations (7) and (8), R2 to better reflect the superiority of the proposed method compared with the conventional AVG-ENVLP method and to examine the significant differences among them. The design included one independent within-subjects factor consisting of four signal processing methods . The significance level in the statistical tests was set to p < 0.05. All statistical analyses were carried out using SPSS software .A one-way repeated-measure ANOVA was applied to RMSD and The PCA, ICA, and NMF factorization algorithms were used to extract input signals of the FOS-based force model. Data from Subject 1 were used as an example to determine the number of basis functions and to establish the most suitable force estimation model. R2 for all seven subjects are presented in R2 values. The RMSD values decrease and R2 values increase conversely with force levels rising from 20% MVC to 60% MVC for all subjects.Next, cross-validation force estimation experiments were conducted to verify the proposed force estimation framework. For each subject, one repetition from the seven repetitions was iteratively selected as the training set, and the remaining 6 repetitions were test sets. The overall RMSD and 2 values, and there were significant differences between different methods. p < 0.0001) for both RMSD and R2; no such significant difference exists among the three factorization algorithms except for PCA and ICA with p < 0.05. Thus, we concluded that the proposed muscle force estimation framework that integrates factorization algorithms could significantly reduce the estimation error compared to the conventional AVG-ENVLP method.A one-way repeated-measure ANOVA analysis was performed on RMSD and RWe also estimated the measured force using signals from only one piece of HD-SEMG grid via the NMF + FOS framework. This better reflects the performance of the proposed framework and proves that FOS is especially suitable for estimating the combined force of multiple muscles. Here, the number of FOS input signals changed from 2 to 1, and the number of basis functions was reduced to 5 (including 2 values are higher when using SEMG signals from two muscles to estimate the measured force.Previous studies have demonstrated that SEMG signals can offer a comparatively accurate estimation of relevant force. In most HD-SEMG based force estimation studies ,23,24,25R2 versus AVG-ENVLP.The present study deeply explored the activation of target muscles via factorization algorithms such as PCA, ICA, and NMF. The factorization algorithms could decompose HD-SEMG signals into different activation patterns and corresponding activation curves . The actIn addition, because it contained multitudinous nonlinear basis functions, FOS offered better estimation performance than simple linear model used in previous studies . Taking In summary, this study validated the feasibility of a novel muscle force estimation framework based on factorization algorithms and the FOS algorithm. Only the SEMG signals from the upper arm muscles were collected to estimate the generated elbow force. While the subjects were asked to avoid activation of forearm muscles during elbow flexion task, forearm muscles inevitably have some contribution to the force measured at the wrist. This is the source of some errors between the estimated and measured forces. Consequently, contributions from the forearm muscles including brachioradialis, flexor carpi radialis, and extensor digitorum should be considered in future work. In addition, apart from the elbow-induced force at the wrist, other combined forces of multiple muscles could be studied to verify the feasibility of the proposed force estimation framework. Further research will also evaluate dynamic contraction tasks.A HD-SEMG-force estimation framework based on FOS and factorization algorithms was proposed and implemented, and its feasibility and superiority were verified under static isometric elbow flexion task. The results demonstrated that factorization algorithms could extract more detailed muscle activation information at a deeper level than the conventional method. Coupled with factorization algorithms, fast orthogonal search method provided an effective way to realize the combined force estimation of multiple muscles. The framework proposed here can be employed in many significant applications, including sports biomechanics, gait analysis, myoelectric prosthesis, and control of exoskeleton devices.


MYD88 and CD79b. The MYD88 p.L265P mutation occurs at high frequency in CNS lymphoma and is extremely rare in non-hematologic malignancies. Currently, brain biopsy is considered the gold standard for CNS lymphoma diagnosis. However, brain biopsy is invasive, carries a risk of complications, and can delay initiation of systemic therapy. Circulating tumor DNA (ctDNA) in the cerebrospinal fluid (CSF) can be utilized to detect tumor-derived mutations. Testing of CSF-ctDNA is a minimally-invasive methodology that can be used to assess the genomic alterations present in CNS malignancies. We present a case of an 82-year-old man with a history of testicular lymphoma who presented with speech difficulty and a multifocal enhancing left inferior frontal mass. Analysis for both CSF-cytology and flow cytometry did not show evidence of neoplastic cells. A brain biopsy was performed and microscopic examination showed DLBCL. We isolated CSF-ctDNA and used droplet digital PCR (ddPCR) to detect the most common lymphoma-associated mutations in MYD88, L265P, and V217F. In conjunction, we evaluated the patient-matched CNS lymphoma tissue for MYD88 mutations. We detected the MYD88 p.L265P mutation in formalin fixed paraffin embedded (FFPE) tissue from the brain biopsy and the CSF-ctDNA. In contrast, both the tumor tissue and the CSF ctDNA were negative for the MYD88 p.V217F mutation. This study shows that testing CSF ctDNA for MYD88 mutations is a potentially minimally-invasive approach to diagnosing patients with suspected CNS lymphomas.Primary Central Nervous System Lymphoma (PCNSL) and Metastatic (or Secondary) Central Nervous System Lymphoma (MCNSL) are rare central nervous system (CNS) malignancies that exhibit aggressive clinical behavior and have a poor prognosis. The majority of CNS lymphomas are histologically classified as diffuse large-B cell lymphoma (DLBCL). DLBCL harbors a high frequency of mutations in  Lymphoma of the central nervous system (CNS) includes two major subtypes: primary central nervous system lymphoma (PCNSL) arisen and confined to the CNS and metastatic or secondary CNS lymphoma (MCNSL), which originates at sites outside the CNS , 2. AccoMYD88) gene. Specifically, the p.L265P amino acid substitution is the most commonly occurring MYD88 mutation in CNS lymphoma  and activates interleukin-1 (IL-1) and IL-18 receptor signaling pathways. The MYD88 p.L265P mutation is also present in virtually all cases of Waldenstrom macroglobulinemia  as wells as discuss the potential application of this assay for minimally-invasive diagnosis of CNS lymphomas.Studies on the genomic alterations of CNS lymphoma have reported a high frequency of mutations in the myeloid differentiation factor 88  tissue was used to extract DNA using QIAamp DNA FFPE Tissue Kit . A lumbar puncture was performed (L3-L4) ~4 weeks after surgery and 13 ml of CSF were collected. One milliliter of CSF was used for ctDNA isolation . The ddPCR mutation detection assay is a 20X concentrated, ready-to-use primer-probe mix optimized for use with ddPCR supermix for probes. The droplet generation process was performed using the Bio-Rad QX-100 emulsification device following the manufacturer's instructions. The droplets were subjected to amplification followed by transferring to a Droplet reader (Bio-Rad) and were analyzed with the Quanta Soft analytical software package (Bio-Rad) (MYD88 p.L265P mutation was detected in DNA extracted from tumor tissue and ctDNA obtained from the CSF (CSF-ctDNA). In contrast, the p.V217F mutation was not detected either in the tissue or in the CSF-ctDNA, highlighting the specificity of the assay.Droplet digital polymerase chain reaction (ddPCR) mutation detection assay for Bio-Rad) . As showMYD88 p.L265P mutation as a promising complementary method to assist in establishing the diagnosis of CNS lymphoma.Currently, establishing a diagnosis of CNS lymphomas often requires invasive procedures such as craniotomy or stereotactic biopsy. Minimally invasive procedures such as MRI, CSF-cytology, and flow cytometry, have limited value due to low specificity and sensitivity , 16. In MYD88 mutations in cancers mostly occur as a single base substitution at c.794T > C resulting a leucine to proline (L265P) amino acid change in the Toll/IL-1 receptor domain. This genetic alteration causes recruitment of the MYD88 protein to the cytoplasmic tail of the TLRs to form an active complex and subsequent activation of the NF-kB signaling pathway , peritoneum (n = 1), large intestine (n = 1), and prostate tumors (n = 1) (MYD88 mutation (p.V217F) has been reported in lymphomas  . A less ymphomas . Therefoutations . To our lymphoma .MYD88 mutations in patients with CNS lymphoma. This could potentially be used as an alternative and minimally-invasive method for diagnosis. However, further investigation in a larger cohort of patients is required to establish this assay as a routine clinical test.In summary, our study demonstrates that ddPCR analysis of CSF-ctDNA is a promising methodology to detect This project was approved by the institutional review board of the University of Texas Health Science Center at Houston and Memorial Hermann Hospital, Houston, TX. Residual tumor tissue and CSF were analyzed with written consent from the patient. In addition, the written informed consent was obtained from the patient for the publication of this case report.LB designed and supervised the study and data analysis. SZ, JW, and DD designed and performed the experiments. GL and J-JZ are responsible for clinical information gathering, sample collection, and patient consent. SZ and YE prepared the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.