The workshop’s goal was not to address EHE per se but to discuss larger questions around Hippo signaling and ways of controlling or regulating YAP/TAZ-TEAD activity. The role of this workshop in advancing EHE research is best understood in the context of the TSRC itself.Funding the research needed to advance our understanding of rare cancers is very challenging. Given the limited potential for financial reward and lack of public awareness, inspiring government, industry, or society to fund research for individual diseases is extremely difficult. Nevertheless, patients and families affected by rare cancers will often find novel ways to support and pay for the science that is necessary to promote knowledge of underlying diseases and develop more effective treatments. This is the case for epithelioid hemangioendothelioma (EHE), a one-in-a-million vascular sarcoma caused by oncogenic fusion proteins involving YAP or TAZ, two downstream, negative effectors of the Hippo Pathway. In June of 2023, scientists active in Hippo-YAP/TAZ research assembled at a workshop hosted by the Telluride Science Research Center  and partially remotely (2021&2022). So far, it has limited participation each year to 20 people to stay true to the characteristics that have defined the TSRC for nearly 40 years. Namely, a small, topic-focused workshop seeking productive collaboration through interactive presentations both in the meeting room and during walks in the woods. It has had participants from every continent (excluding Antarctica) and at every stage of their career, ranging from MacArthur Grant awardees, Howard Hughes Investigators, and department chairs to graduate students and post-docs. This includes prominent scientists from both academia and industry, many of whom regularly speak at international conferences on Hippo signaling. The workshop has achieved its scientific goal of increasing collaboration and interaction. The initial example was a collection of Hippo-related papers that included manuscripts from several of the workshop participants ,11,12,13The ‘real-world’ impact of the TSRC model is well demonstrated by many substantive advances in EHE research since the inaugural “YAP/TAZ and TEAD: at the crossroads of cancer” workshop. These include the first transgenic mouse models of EHE ,15, the Notably, these collaborations have also resulted in improved EHE awareness and funding for EHE-focused basic research. This also results from mutually beneficial interactions among workshop participants and supportive foundations and EHE advocacy groups, including the CRAVAT Foundation, the Richard Eaton Foundation, and EHE foundations located in the United States, United Kingdom (England), and Australia. In addition to funding many of the EHE projects mentioned above, these organizations have helped defray workshop costs, allowing promising young researchers to attend. In turn, the foundations have also benefitted. They have drawn on workshop participants to present research at foundation-sponsored conferences, patient outreach events, and fundraising efforts. This is yet another example of how the TSRC model promotes fruitful collaborations that help advance basic science.However, the purpose of this workshop is not solely to promote interaction and collaboration to advance EHE research; it is to do this across the entire Hippo-YAP/TAZ-TEAD field. This pathway has strong therapeutic promise in cancer and other diseases, and despite substantial progress over the past 3 decades, there is still a great deal we do not understand about the pathway and how it contributes to human disease. This is reflected in the diversity of topics presented at the workshop throughout its first 7 years, including talks focused on: Hippo Pathway signaling; mechano-transduction; structure and targeting of TEADs; transcriptional regulation by YAP/TAZ-TEADs; regulation of YAP and TAZ nuclear/cytoplasmic shuttling; novel YAP/TAZ-TEAD regulators; epigenetics; and crosstalk between the Hippo Pathway and other critical signaling cascades.Cancer is a major focus of the workshop, and numerous cancers, in addition to EHE have been discussed. As has virtually every aspect of cancer biology. Other diseases, such as fibrosis of the kidney and lung, pulmonary hypertension, and even COVID-19, have also been featured. Stem cells, cell fate decisions, development, and tissue homeostasis are other frequent topics. The model systems and research tools presented are equally diverse and innovative and include in vitro work in cell lines as well as in vivo work in flies, zebrafish, mice, rats, and humans. Many important discoveries in the Hippo-YAP/TAZ-TEAD field were previewed by scientists sitting in a classroom at the Telluride Middle School, and discussed with enthusiasm, not unlike that of a middle schooler learning about the wonder of science and discovery for the first time. While the contribution of the workshop to the field is difficult to quantify, the true indication of its value to researchers in this field is the fact that many return and actively participate each year.Cancers were authored by researchers who have participated in many of the previous workshops. The recipe in 2024 will remain unchanged. High-level scientists will present novel, unpublished data on the biology of the Hippo Pathway and the potential for targeting YAP, TAZ, and their binding partners, the TEADs. The workshop will continue to promote active collaboration that leads to advances in our understanding of the Hippo-YAP/TAZ-TEAD pathway and, ultimately, to improved outcomes for all cancer patients, including those with EHE.Given the therapeutic relevance of the Hippo-YAP/TAZ pathway, there is potential for competition amongst researchers and companies active in this field. Despite this, the TSRC “YAP/TAZ and TEAD: At the crossroads of cancer” workshop has remained an open and highly interactive oasis that has fostered extensive collaboration, not competition. The character of its core participants, the unique 40-year-old TSRC recipe, and the inspiring setting of Telluride all play big roles in this. In 2024, the workshop will mark its eighth year, and it is fitting that several of the manuscripts in this Special Edition of


Despite recent literature supporting non-inferiority of oral therapy, many patients are treated with traditional extended intravenous (IV) antimicrobial courses. The objective of this study was to evaluate the attitudes of infectious disease (ID) specialists on utilizing oral antimicrobials (PO-A) for the treatment of bone and joint infections (BJI).This was a multicenter clinician survey study of ID specialists from 1/10/23 – 1/31/23, regarding their attitude towards using PO-A for the treatment of BJIs. Survey respondents were presented with statements and rank order questions and asked to indicate their level of agreeance using a 5-point Likert scale. The primary outcome was agreement with the phrase ‘I am willing to prescribe PO-A for treatment of BJIs’. Secondary outcomes included patient case and ID specialist characteristics associated with willingness to prescribe PO-A. Study utilized descriptive statistics and univariate tests.A total of 216 surveys were distributed to ID specialists of which 65 recorded responses with 50 complete responses . Of the 50 complete surveys, the majority of respondents (60%) indicated they prescribe PO-A in ≤25% of their patient-case load as definitive treatment of BJIs. A total of 41/50 respondents (82%) indicated strong agreement with comfortability prescribing and dosing PO-A for BJIs (Table 1) and were most willing to prescribe PO-A ( >70%) in patients with diabetic foot infection (Table 2). ID specialists’ willingness to prescribe PO-A for treatment of BJIs significantly correlated with their current clinical practice. Respondents who disagreed with the statement ‘I am willing to prescribe PO-A’ currently prescribe PO-A in less than 25% of their BJI patients compared to respondents who agreed (37% prescribe PO-A 26-50% of their patients with 35% of those who strongly agree prescribe PO-A it in >50% of their BJI patients (p=0.004). The most common concern (62%) when prescribing PO-A for BJIs is the identified pathogen may not be susceptible to the preferred antibiotic (Table 3).The findings of this survey suggest that most ID specialists in this sample are comfortable prescribing PO-A for treatment of BJIs and was significantly related to current practice.Jessica Seidelman, MD, MPH, Uptodate: content editor for pelvic osteomyelitis page Kristen Dicks, MD, UpToDate: Advisor/Consultant


Embolus shedding is one of the important complications in carotid artery stenting (CAS). Carotid high-resolution magnetic resonance imaging (HR-MRI) is often used to directly reflect important biological characteristics, such as plaque size and composition, as well as the structure of the carotid artery wall. The aim of this study was to investigate the predictive values of carotid HR-MRI for large embolus shedding in CAS.In total, 195 patients with carotid stenosis were enrolled. Preoperative carotid HR-MRI was performed to define the nature of the carotid plaques. CAS was performed in all patients, and intraoperative embolic protection devices were used to collect the shed emboli. According to the diameter and number of shed emboli, the patients were divided into the small-embolus group (group X) and large-embolus group (group Y). Logistic regression analysis was used to analyze the risk factors of large embolus shedding.Group Y included 58 patients, and group X included 137 patients. Age, stenosis length, smoking, and ≥3 transient cerebral ischemic attacks were risk factors for large embolus shedding. Two cases of shed large emboli developed from stable plaques, and 56 cases of large emboli developed from vulnerable plaques. When vulnerable plaques were associated with more risk factors, the incidences of large embolus shedding in cases with vulnerable plaques combined with 0, 1, 2, 3, and 4 risk factors were 44 % (4/9), 68.1% (15/22), 72.2% (13/18), 76.5% (13/17), and 84.6% (11/13), respectively.Carotid HR-MRI can predict the incidence of large embolus shedding in CAS. Cerebral infarction (CI) is one of the main causes for the death and disability in elderly individuals, and carotid stenosis is an important risk factor for CI. It has been reported that about 26%–28% of patients with a carotid stenosis degree of 70%–90% as well as cerebral ischemic symptoms will develop CI within 1 year , and CEACarotid high-resolution magnetic resonance imaging (HR-MRI) can directly reflect important biological characteristics such as plaque size and composition, as well as the structure of the carotid artery wall. Carotid HR-MRI has unique advantages in evaluating different types of atherosclerotic plaques, especially in evaluating the stability of vulnerable plaques, compared with those of other methods . Whether9/L; leucopenia, <4×109/L; neutropenia; cerebral hemorrhage within nearly 3 months; allergic to aspirin or clopidogrel; severe heart or lung disease; and bead-string-like CAS. If the patient newly developed CI, the surgery was routinely delayed for 3 weeks. If the patient did not present with symptom aggravation within 3 weeks and diffusion-weighted imaging (DWI) or computed tomography (CT) showed good absorption of the new lesion or the lesion was significantly reduced than before, the patients could undergo CAS. The study was supervised by the Medical Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University, and all the patients or their legal guardians signed the informed consent.A total of 195 patients who underwent CAS in the Affiliated Provincial Hospital between October 2012 and December 2015 were enrolled. The inclusion criteria were as follows: carotid stenosis ≥70%, symptomatic or asymptomatic poor collateral circulation, and age >45 years. The exclusion criteria were as follows: thrombocytopenia, <100×10All the patients underwent digital subtraction angiography using one angiography instrument . After local anesthesia was administered, the femoral artery was punctured using the Seldinger method, and one 5F vascular sheath was implanted. The carotid and vertebral arteries underwent arteriography in turn using the catheter. Carotid stenosis was measured according to the criteria of the North American Symptomatic Carotid Endarterectomy Trial . Before Stable plaques are type VII  and type VIII (fibrous plaque) in the classification of AS plaques. The MRI signals of type VII plaques showed low signals in T1WI, T2WI, and PDWI; type VIII plaques showed iso-signal or high signal on T1WI, slightly high or iso-signal on PDWI and slightly high signal or iso-signal on T2WI . As a noWhen performing CAS, the right femoral artery was first punctured using the Seldinger method, and 4000–5000 units of heparin saline (70 units/kg) was then injected together with an intraoperative intravenous push of 2000 units of heparin saline every other hour to ensure the heparinized state of the whole body (even if the clotting time was maintained between 250 and 300 s). One 8F catheter was then inserted into the stenotic vessel with its tip placed 20 mm below the stenosis. In order to ensure that the maximum diameter of the cerebral protection device  could safely pass through the stenosis, if the residual luminal segment at the stenosis was <2 mm, a 2-mm balloon system  was delivered via a micro-wire to preexpand the stenosis.After EPDs were successfully placed, the appropriate balloons  were used to preexpand the stenosis, and after the expansion, the appropriate stents  were selected for CAS. After the stent was released, if the residual vascular lumen was ≥30%, the balloon system was reused for the post-stenting expansion to achieve the purpose of reducing the residual vascular lumen to <30%.−1·min−1) could be administered to increase the blood pressure and to ensure that the systolic blood pressure was not less than 90 mmHg. Meanwhile, the systolic blood pressure could not exceed 120 mmHg to prevent hypertension-induced high perfusion syndrome. After releasing the stent, EPDs were carefully recycled, marked, and recorded correspondingly for the statistical analysis later.During stent release, the patient’s heart rate may decrease rapidly, so the patient was asked to cough immediately after the stent was released; if the heart rate still increased slowly 10 s later, atropine  was intravenously injected immediately. If the intraoperative blood pressure dropped, there was no need to increase the blood pressure immediately, and observation alone could be performed if no symptoms appeared. If the blood pressure dropped to 90/60 mmHg or less, dopamine  was used for the statistical analysis. Logistic regression analysis was used to compare the characteristics and disease history of the patients to determine the risk factors that might result in the occurrence of large emboli during CAS. The p value <0.05 was defined as statistically significant.Before surgery, carotid HR-MRI showed that, of 195 patients, 116 patients had stable plaques and 79 patients had vulnerable plaques .A total of 195 patients successfully underwent CAS. The procedure of CAS is shown in After surgery, 58 cases of large emboli and 137 cases of small emboli were collected using EPDs. Among 58 cases of large emboli, 56 cases involved vulnerable plaques and 2 cases involved stable plaques.On the day of the surgery, hyperperfusion symptoms occurred in 6 patients. Two patients had intracranial hemorrhage on the day and on the second day of surgery, respectively.Logistic regression analysis of the risk factors of large emboli is shown in The incidences of large embolus shedding for vulnerable plaques combined with risk factors are shown in Presently, there exist controversies about the two main surgical methods of carotid stenosis, namely CAS and CEA. However, the Carotid Revascularization Endarterectomy versus Stenting Trial (CREST) shows that complications such as stroke, myocardial infarction, or mortality occurred in the two methods are not significantly different, but CAS has greater advantages for intra-carotid stenosis in high-cervical and intracranial segments .Esposito-Bauer et al.  performeIn this study, 26 patients had difficulties recovering with the use of an EPD, which was finally solved after allowing these patients to turn their necks, perform a swallowing action, or pressing the neck. Müller-Hülsbeck et al. , 30  are important indications for the application of EPDs. Therefore, we recommend that the patients with vulnerable plaques should receive EPDs during CAS. Carotid HR-MRI also showed that 116 of the 195 patients had stable plaques, and finally, 2 large emboli were observed in cases with stable plaques, with an incidence of large emboli of 1.7%. The incidence of large emboli in cases with stable plaques is low, so not applying EPDs can be considered. However, small emboli have clinical uncertainty in patients with stable plaques, so whether patient with stable plaques need EPDs still needs further experimental studies.There still exist several shortcomings in this study. First, no cranial DWI scanning was performed after CAS, so no preoperative and postoperative DWI comparison can be performed, and no timely diagnosis can be made regarding the existence of asymptomatic infarction. Second, statistical analysis was not performed for the micro-plaques in EPDs, thus resulting in statistical errors. Finally, the MRI scanning time was too long and the number of scanning sequences were too many to be easily accepted by elderly patients.In conclusion, carotid HR-MRI provides a new imaging method for clinically studying the rupture mechanisms of vulnerable plaques and detecting vulnerable plaques early, which has confirmed its high detection rate and strong specificity toward both vulnerable and stable plaques. Therefore, in combination with patients’ own risk factors, it can be used to predict the incidence of intraoperative embolus shedding.


SHANK3 gene or heterozygous pathogenic variants in SHANK3. Pathophysiologically, this syndrome has been regarded as a synaptopathy, but current data suggest an additional concept, since axonal functions of neurons are also impaired, thus, the specific pathophysiological processes in this disease are not yet fully understood. Since symptoms of the autism spectrum, regression, and stagnation in development occur, we investigated whether neuroinflammatory and neurodegenerative processes may also play a role. To this end, we analysed biomarkers in cerebrospinal fluid (CSF) and parameters from magnetic resonance imaging with high-resolution structural T1w volumetry and diffusion tensor imaging analysis in 19 Phelan-McDermid syndrome patients.Phelan-McDermid syndrome (PMS) is a rare multisystem disease with global developmental delay and autistic features. Genetically, the disease is based on a heterozygous deletion of chromosome 22q13.3 with involvement of at least part of the CSF showed no inflammation but abnormalities in tau protein and amyloid-ß concentrations, however, with no typical biomarker pattern as in Alzheimer’s disease. It could be demonstrated that these CSF changes were correlated with integrity losses of the fibres in the corticospinal tract as well as in the splenium and dorsal part of the cingulum. High CSF levels of tau protein were associated with loss of integrity of fibres in the corticospinal tract; lower levels of amyloid-ß were associated with decreasing integrity of fibre tracts of the splenium and posterior cingulate gyrus. Volumetric investigations showed global atrophy of the white matter, but not the grey matter, and particularly not in temporal or mesiotemporal regions, as is typical in later stages of Alzheimer’s disease.In summary, alterations of neurodegenerative CSF markers in PMS individuals could be demonstrated which were correlated with structural connectivity losses of the corticospinal tract, the splenium, and the dorsal part of the cingulum, which can also be associated with typical clinical symptoms in these patients. These findings might represent a state of dysfunctional processes with ongoing degenerative and regenerative processes or a kind of accelerated aging. This study should foster further clinical diagnostics like tau- and amyloid-PET imaging as well as novel scientific approaches especially in basic research for further mechanistic proof. SHANK3 on chromosome 22q13.3 [SHANK3 involvement have been recognized who have a similar clinical phenotype so that the nomenclature of the syndrome has recently been adjusted in PMS-SHANK3 related or PMS-SHANK3 unrelated [The Phelan-McDermid syndrome (PMS) is a disease OMIM#606,232) characterized by a global developmental delay with motor deficits (muscle hypotonia), expressive and receptive language development delay, cognitive impairment, epilepsy, and other neuropsychiatric comorbidities such as mood disorders, regression, and autism spectrum disorders . The cli32 characSHANK3 encodes structural proteins of the postsynapse of excitatory neurons [SHANK3 is not only expressed within the brain in the hippocampus, amygdala, cingulate gyrus, and cerebellum [ neurons . The com neurons . SHANK3 rebellum , but alsrebellum  and neurrebellum .Meanwhile, there are also data that the PMS is not only a disease of the synapses, but also of the white matter with impairment of the directionality of certain fibre tracts , 10. ThuThe current study is based on multimodal data from diagnostic processes and addresses the following questions:Can cerebrospinal fluid be used to demonstrate inflammatory or neurodegenerative processes in patients with PMS?Is there a morphological correlate in the brain for changes in the CSF biomarkers that can be associated by means of advanced volumetric or microstructural MRI data?SHANK3 related and PMS-SHANK3 unrelated?Are there differences in the results related to the classification of patients into PMS-In the CSF routine parameters white cell count/µl, total protein in mg/dl, lactate in mmol/l and oligoclonal IgG, none of the patients showed any pathological findings. Results were displayed in a standard Reibergram and measured automatically in our hospital CSF laboratory according to established formulas of Prof. Reiber . Here, wNormal values were also shown for the cytokine CXCL13 as an inflammation marker . In a next step, markers were examined that reflect neurodegeneration. For pNF-H , none of the patients showed abnormal values either. The degeneration markers tau protein (reference range < 400 pg/mL) and amyloid-ß (reference range > 600 pg/mL) revealed pathological values for one of the two parameters in 63% of all patients . It is noteworthy that only one patient had abnormal values for both biomarkers. The amyloid-ß quotient 1–42/1–40 (reference range > 0.07) was not abnormal in any of the patients, p-Tau 181 (reference range < 60 pg/ml) also showed normal values in all patients. Therefore, there seemed to be no typical Alzheimer’s constellation of these biomarkers in laboratory CSF tests (Table SHANK3 related or SHANK3 unrelated), language, motor function, ASD, epilepsy, cognition, and ADHD, respectively (data not shown).Since neurodegenerative processes in PMS have not yet been described, the question arose whether the current results could be dependent on different variables. No associations were observed between the biomarkers tau protein / amyloid-ß and the variables age, gender, genetic findings , indicative of globally altered cerebral white matter microstructure.A significant association cluster for amyloid-ß was observed in the splenium and dorsal part of the cingulum Fig. . For tauThe investigation of ICV-corrected GM and WM volumes of PMS patients compared to controls showed significantly reduced WM substructure volumes , whereas almost no significant differences for GM substructure volumes were observed in the hemispheres Table . The onlSHANK3 involvement or not, according to the current classification into PMS-SHANK3 related and PMS-SHANK3 unrelated. However, only descriptive statements can be made here, as the group of PMS-SHANK3 unrelated with one patient does not allow to make a statistical statement. Alterations were usually detected in just one of these biomarkers. As the typical pattern like in Alzheimer’s disease  [PMS is a genetic multisystem disease whose genotype and phenotype have been characterized in increasing detail , 14, but0 ratio)  could, tNext, we analysed whether changes in tau and amyloid-ß biomarkers had associations with structural or microstructural measures of the brain, as assessed by MRI. To this end, we observed significant volume reductions in the patients of this study localized in the white matter of supratentorial structures, but not in the grey matter and especially not temporo-parietally or mesiotemporally as one would expect in AD-like pathology. These results led to the assumption that not gross volume changes, but microstructural processes are apparently impaired in PMS patients, as suggested by the very widespread white matter alterations in an unbiased whole brain DTI analysis. Thus, using whole-brain-based spatial statistics of DTI, a voxel-based correlation to tau and amyloid-ß showed a clear pattern for each of the biomarkers: Specifically, the direction of the fibres of the corticospinal tract was negatively correlated with tau values, meaning that pathologically high tau values were associated with a greater reduction in the integrity of the fibres of this motor tract. Possibly, plastic changes in the sense of degenerative and regenerative processes of the microtubules may represent a pathophysiological correlate of these alterations. There is a possible clinical connection between these results and motor functions in the affected patients who have a motor developmental delay with global muscular hypotonia, gait ataxia, and fine motor dysfunction.The association analysis with amyloid-ß showed a pattern with impairment of the integrity of fibre connections of the splenium and the dorsal cingulum, i.e., the pathologically lower the amyloid-ß values, the lower was the directionality of the nerve fibres. The posterior part of the cingulum receives connections from the posterior parietal cortex, but other cortical afferents to this part of the cingulate gyrus include fibres from the visual and auditory association areas . The funIn summary, changes in the neurodegeneration markers tau protein and amyloid-ß in the CSF of patients with PMS seem not to show the typical biomarker pattern of Alzheimer’s pathology. In line with this, there are neither morphological indications of atrophy nor clinically assessable indications of the development of a dementia syndrome. Unexpected was the regional association for both biomarkers with tau/corticospinal tract and amyloid-ß/structures of the limbic system. Based on our microstructural MRI results, one could argue that these results may not reflect degenerative processes sensu stricto, but rather dysfunctional processes which may also be an expression of accelerated aging, as described by Braak and coworkers in autopsies of children and adolescents . AlternaSHANK3 animal models to further characterize the (patho-)physiological assignment of our results.It will be exciting to address these questions with further diagnostic tools like tau/amyloid-PET imaging as well as basic research using iPS cell models and translational More than 60% of the Phelan McDermid patients investigated here have alterations in the CSF degeneration markers tau protein and amyloid-ß without exhibiting the typical pattern seen in patients with Alzheimer disease. The tau and amyloid-ß changes are associated with both functional changes in specific tract systems and clinical symptoms. It remains exciting for further investigations whether these results represent adaptation processes to external stimuli or premature aging processes.SHANK3 , one deletion at 22q13.3 without involvement of SHANK3 (PMS-SHANK3 unrelated), three cases with disruption of SHANK3 due to structural abnormalities  and five intragenic SHANK3 variants  had a genetically proven diagnosis of PMS. Genetic diagnoses revealed ten deletions at chromosome 22q13 including ts Table .Developmental stages and clinical characteristics of patients were classified for motor skills, speech, cognition, autism and autism spectrum symptoms, attention deficit hyperactivity syndrome, epilepsy, and regressive symptoms Table . CognitiAll subjects or their caregivers gave written informed consent for the study protocol according to institutional guidelines which have been approved by the Ethics Committee of Ulm University, Germany (reference # 321/16) and which were consistent with the declaration of Helsinki.After informed consent of the patient/caregiver, an anaesthesiologist performed sedation with propofol while continuously monitoring of the vital parameters during MRI acquisition. Directly afterwards, the lumbar puncture was performed while the patient was still sedated. The CSF was immediately taken to the laboratory for further routine diagnostics. The CSF was then frozen at -80 °C, and the biomarkers for the scientific questions were measured at a later point in time.Measurement of total-tau (t-tau), pTau181 and Aβ42, Aß40 in CSF: the neurodegenerative biomarkers t-tau, pTau181 and Aβ42, Aß40 in CSF were measured after routine diagnostic work-up of patients. The following assay was used: Fujirebio, Gent, Belgium. Measurement of the inflammatory biomarker CXCL13 was performed using an ELISA . Measurement of the axonal biomarker pNF-H was performed using an ELISA  (Table 2) and four scans with b = 0, TE and TR were 95 ms and 8000 ms. Further scanning included a T2-weighted data set (Fluid Attenuated Inversion Recovery/FLAIR) with 40 coronal slices of 3.0 mm thickness, 0.45 mm x 0.45 mm in-plane resolution and 512 × 448 voxels matrix dimension, TR/TE 6180ms/112ms).MRI scanning was performed on a 1.5 Tesla Magnetom Symphony ; the T1-weighted imaging (MPRAGE) consisted of 144 sagittal slices of 1.2 mm thickness, 1.0 mm x 1.0 mm in-plane resolution in a 256 × 248 matrix, echo time (TE) was 4.2 ms, repetition time (TR) was 1640 ms; the diffusion tensor imaging (DTI) study protocol consisted of 52 volumes , representing 48 gradient directions (b = 1000 s/mmTensor Imaging and Fiber Tracking (TIFT) [The postprocessing and statistical analyses were performed by the software platform g (TIFT) . In ordeg (TIFT) . From thg (TIFT) . In a coUnbiased WBSS was performed by comparing voxelwise FA values of PMS patients and controls at the group level (non-parametric Mann-Whitney-U-test).3) [Associations of CSF tau protein and amyloid-ß with FA values were voxelwise calculated by non-parametric Spearman correlation. Both WBSS and the correlation analyses were thresholded at p < 0.05, corrected for multiple comparisons and followed by a clustering procedure discarding isolated clusters (< 256 mm3) .http://www.fil.ion.ucl.ac.uk/spm) and masks predefined in the reference space by means of different brain atlases. For the purpose of the current study, the volumes of main intracranial compartments as well as gray matter (GM) and white matter (WM) structures were determined according to the LONI Probabilistic Brain Atlas and corrected for intracranial volume (ICV) [As described previously , 28, theme (ICV)  (Table 3Associations were calculated by Spearman’s correlation coefficient between the CSF markers tau protein and amyloid-ß and the variables age, gender, genetic findings, language, motor function, ASD, epilepsy, cognition, and ADHD as well as the volumetric results and voxelwise the FA data.


The prognosis of hepatocellular carcinoma (HCC) with vascular invasion is extremely poor, especially in patients with tumor thrombus (TT) of the inferior vena cava (IVC), which is an oncological emergency with a high risk of sudden death due to TT extension or migration. Herein, we describe a case of HCC with TT of the IVC that rapidly extended into the right atrium (RA), in which right hepatectomy was performed under cardiopulmonary bypass.A 64-year-old man was diagnosed with HCC with IVC TT, and right hepatic lobectomy was scheduled. While awaiting surgery, he complained of respiratory distress and rushed to the emergency room. The TT had reached the RA, and the patient was in a state of oncologic emergency. We requested the cooperation of the cardiovascular surgery department, and under artificial cardiopulmonary support, the right atrium was incised, and a part of the TT was removed. The IVC was clamped to prevent tumor dispersal, and right hepatic lobectomy was performed. The remaining thrombus was excised along with the right lobe of the liver by incising the IVC. There were no serious postoperative complications, and the patient is alive 1 year and 5 months postoperatively.Hepatic resection with cardiopulmonary bypass could be an option for HCC with TT reaching the RA. The effectiveness of liver resection for hepatocellular carcinoma (HCC) with hepatic vein tumor thrombus (HVTT) has been previously reported . HoweverA 64-year-old man was referred to our hospital for further examination of a tumor in the right lobe of the liver, which was found on computed tomography (CT) by his previous physician. He had a history of stroke and diabetes mellitus but was generally in good condition, and his liver function was rated as Child–Pugh A and Liver damage A. Abdominal CT showed a tumor on the right side of the liver with TT extending to the IVC Fig. . The patOn the second postoperative day, the total bilirubin level increased to 4.5 mg/dl, but then plateaued and subsequently normalized. Postoperative complications included SSI corresponding to Clavien Dindo-II. Prophylactic antibiotic administration caused drug eruption, generalized skin rashes, and fever, which improved gradually. The patient was discharged from the hospital on postoperative day 74 because of the time required for rehabilitation due to the decline in ADL caused by these complications.Eight months after surgery, the patient was diagnosed with hepatic and adrenal recurrence, and bevacizumab and immune checkpoint inhibitors were initiated.He is still alive 1 year and 10 months after the surgery.In previous studies, the frequency of HCC with IVC-TT was approximately 1.4% , 6. AlthIn general, hepatic resection with IVC-TT is a high-risk procedure, with a mortality rate of 15% , 8.Recent improvements in the surgical techniques for total hepatic vascular exclusion (THVE), CPB, and the Cell-Saver technique have improved the safety of the procedure , 9–11. WThe prognosis for HCC patients with IVC or RA is very poor, with MST within 5 months in untreated patients , 8. CompOur case demonstrates that hepatic resection with CPB is an option for HCC with TT reaching the RA. The removal of the TT before hepatectomy to prevent tumor dispersal was useful approach.


We measured the intracranial vascular volume (arteriovenous stroke volume: SVvasc) and CSF  volume variations during the cardiac cycle. Results: In the whole population, we observed a significant correlation between SVvasc and SVCSF . In the population unlikelyCH, this correlation was significant . In the population probableCH, this correlation was not significant . Conclusions: These results show that the link between the compliance of the oscillating CSF and the abrupt arterial inflow seems to be altered in CH. CSF oscillations between intracranial and cervical fluid spaces limit the impact of the abrupt arterial inflow.Background: During a cardiac cycle, intracranial pressure is related to arterial entry into the cranium and its interaction with intracranial compliance. The arterial inflow is compensated by intracranial compliance and, initially, the flushing of cerebrospinal fluid (CSF) into the cervical subarachnoid spaces. Our objective is to analyze the interactions between intracranial arteriovenous exchange and cerebrospinal fluid oscillations. Method: A total of 23 patients (73 ± 8 years) with suspected chronic hydrocephalus (CH) underwent an infusion test and phase-contrast MRI. Rout is an important factor in the diagnosis of CH. Patients were divided into 2 populations:  Diagnosing chronic hydrocephalus (CH) is a challenging task. Currently, the most widely accepted definition involves the presence of ventricular dilation in association with neurological disorders that are either completely or partially reversible after cerebrospinal fluid (CSF) diversion surgery . In CH, Intracranial pressure refers to the pressure of cerebrospinal fluid (CSF), and its generation is a result of the interplay among CSF, arterial, and venous flows. This interaction is traditionally governed by the Monro–Kellie doctrine . It statThere is both a pulsatile and slow circulation of CSF, with each type governed by different physiological and physical laws. During a long period, the Monro–Kellie doctrine is crucial. Indeed, a disorder in the volume of a compartment needs to be compensated by the others to preserve the mean intracranial pressure. During the cardiac cycle, the application of the Monro–Kellie doctrine is not appropriate, suggesting its inadequacy in certain scenarios. The intracranial pressure pulse waveform is not constant, changing with both the respiratory and cardiac cycles. The pulsatility of intracranial pressure is well documented ,9,10. WiPhase-contrast MRI (PCMRI) is a unique tool to non-invasively evaluate this pulsatile circulation of blood and CSF during a cardiac cycle ,16,17,18Our objective is to analyze arterial blood, venous blood, and CSF oscillations and interactions using PCMRI in CH patients and patients that present a passive ventricular dilation. We included 23 prospective patients (73 ± 8 years) from the REVERT project (revertproject.org). All of these patients presented suspected CH. They underwent morphological MRI with PCMRI acquisitions. The hydrocephalus of all patients was communicating after neuroradiological morphologic analysis.After MRI, patients underwent a lumbar infusion test. The delay between PCMRI and infusion test was 2 months or less. A senior neurosurgeon performed an infusion test in patients with suspected CH in the lateral decubitus. An 18-gauge needle was inserted into the lumbar intrathecal space and connected to a pressure transducer and syringe pump via a 3-way tap.® software (version 9.1.0.17) (icmplus.neurosurg.cam.ac.uk (accessed on 16 march 2023)). Baseline intracranial pressure (Pbaseline) was measured for 15 to 20 min. Saline was then injected at a rate of 1.5 mL/min until plateau pressure (Pplateau) was reached. Intracranial pressure was analyzed using ICM+® (icmplus.neurosurg.cam.ac.uk). Rout was calculated via this by using the formula Intracranial pressure was analyzed using ICM+probableCH if Rout > 12 mmHg/mL/min (13 patients) and unlikelyCH if Rout < 12 mmHg/mL/min (10 patients). Patients were divided in two groups: All included patients underwent morphological analysis through MRI and the dynamic study of blood and CSF flows through PCMRI. Planes of slices were placed perpendicularly to the presumed flow direction of the studying compartment. Vascular analysis was based on a plane upstream of the Willis circle . CSF floSequence parameters are presented in Data were analyzed using in-house software (Flow 2.0—march 2021) for the semi-automatic delineation of the blood vessels and CSF regions . VelocitFor CSF analysis, the curve of flow evolution over the cardiac cycle was determined. We secondarily integrated this curve. One part was negative and the other was positive. We averaged the absolute value to obtain the stroke volume of the CSF measured at the cervical level. This is the volume displaced through this cross-sectional plane, characterizing the variation in intracranial CSF volume during a cardiac cycle ,14.a) was defined as the sum of the flow through the internal carotid and basilar artery. Cerebral venous blood flow (CBFv) was measured as the sum of the flow of the superior sagittal sinus and straight sinus. As CBFv does not drain all the CBF itself , a venous correction factor (α factor) was calculated as equal to the mean(CBFa)/mean(CBFv). It was thus possible to provide a theorical correctedCBFv flow curve = α factor * CBFv flow. When this venous factor is equal to 1, the correctedCBFv is equal to the CBFv. This corresponds to exclusive jugular venous drainage. When the correction factor is >1, it shows the magnitude of accessory drainage paths.Cerebral arterial blood flow . This project has been approved by our ethics committee (ID-RCB: 2021-A00240-41).AV and SVCSF were not different between unlikelyCH and probableCH populations. All results are presented in We found no difference in CSF and arteriovenous stroke volumes in our two populations. SVAV and SVCSF     A. In the= 0.001) B. In the = 0.16) C.unlikelyCH was 1.55 ± 0.25 and that in probableCH was 2.13 ± 1.71. This index was not different between our two populations (p = 0.03).The hemohydrodynamic ratio in Arteriovenous and CSF interactions can be analyzed using PCMRI. However, the response of CSF to arteriovenous changes during the cardiac cycle differs between CH and non-CH patients. This reflects an inappropriate response of CSF to changes in intracranial vascular volume, thus indicating a disturbance in intracranial pressure.AV was not different between unlikelyCH and probableCH populations. Balédent et al. [SVt et al.  studied t et al. . Arteriot et al. . Temporat et al. ,16. A veCSF was not significantly different between unlikelyCH and probableCH populations. This result is in line with previous studies [SV studies . They ha studies , possibl studies ,29,30. I studies . However, analyzing CSF alone may not provide diagnostics or pathophysiological insights. CSF has a passive circulation and responds to variations in intracranial pressure, which is influenced by vascular volume changes and the biomechanical properties of the brain. The intracranial pressure waveform is primarily determined by intracranial vascular and CSF volume variations , highligAV and SVCSF in the overall population. This causes a response of CSF to the intracranial vascular volume variations, which generate intracranial pressure [We found a correlation between SVpressure ,31. Intrpressure . Other cpressure . AV and SVCSF is present in the unlikelyCH population but is not present in the probableCH population. For similar changes in intracranial vascular volume, the hydrodynamic response is different in our two populations. This could be linked to other compensatory mechanisms of intracranial pressure. But these remain unknown.These assumptions can explain the existence of different responses of the CSF function of these alternative compensatory mechanisms to preserve intracranial pressure. A correlation between SVCSF is mainly reduced in front of SVAV. In a few cases, we observed a very high SVCSF in front of SVAV. The hemohydrodynamic ratio is not useful for CH diagnostics. There is an important heterogeneity in the hydrodynamics in CH. An increase in SVCSF relative to SVAV suggests an alteration in intracranial compliance, whereas a decrease indicates a spinal compliance alteration. Both of these hydrodynamic patterns are associated with a clinical history of CH, reflecting different underlying pathophysiologies. The classification of chronic hydrocephalus may need to be revisited based on these distinct hydrodynamic behaviors and suspected alterations.The analysis of the hemohydrodynamic ratio shows that SVCraniospinal compliance cannot be considered as a single entity. In fact, intracranial and spinal compliance separately exist on the scale of the cardiac cycle. Intracranial compliance  is the rprobableCH group. Indeed, a larger number of patients would enable us to better observe and understand the heterogeneity of results concerning alterations in CSF pulsatility. This would enable us to approach disorders associated with altered intracranial or spinal compliance.Our study is limited in terms of the number of patients. It seems necessary to increase our populations in order to better discriminate the different hydrodynamic alterations predicted in the Our groups are based on the analysis of intracranial pressure and CSF flow resistance. However, this parameter has a low negative predictive value . A futurThe hydrodynamic response to the intracranial hemodynamic constraints is disturbed in CH. There are multiple hydrodynamic responses with a hyperdynamic CSF in some cases and an hypodynamic CSF in others. These alterations are different but link to CH development. Hemohydrodynamic analysis seems to be essential to understand neurofluid flows in CH, which is not an entity but an association of multiple pathologies. These different alterations are probably linked to different alterations in compliance. Compliance is an emerging concept in CSF research. This concept needs to be precisely defined. Indeed, there are many anatomic sites that regulate craniospinal compliance. In addition, it is necessary to determine the behavior of the different temporal scales of analysis: the supracardiac scale, where Monro–Kellie’s law can be applied, or the cardiac cycle scale, where Monro–Kellie’s law cannot be applied.


We aimed to estimate the prevalence of trachomatous inflammation–follicular (TF) in 1–9-year-olds and trachomatous trichiasis (TT) unknown to the health system in ≥15-year-olds in Benishangul Gumuz (BGZ) region, Ethiopia. This will help to assess progress towards the elimination of trachoma as a public health problem and determine the need for future interventions against trachoma in the region.Cross-sectional population-based trachoma prevalence surveys were conducted in four evaluation units (EUs) of BGZ using World Health Organization-recommended survey methodologies. Individuals were examined for clinical signs of trachoma. Household access to water, sanitation and hygiene facilities (WaSH) was assessed.A total of 11,778 people aged ≥1 year were examined. The prevalence of TF in 1–9-year-olds was <5% in three EUs and ≥5% in one EU. The prevalence of TT unknown to the health system in people aged ≥15-years was ≥0.2% in all four EUs. The proportion of households with an improved drinking water source within a 30-minute round-trip ranged from 27−60%. The proportion of households with an improved latrine ranged from <1−6%.Surgical interventions for TT are required in all EUs in BGZ. One annual round of mass drug administration (MDA) of azithromycin is required in one EU before resurvey to reassess progress in lowering TF prevalence below the WHO elimination threshold of 5% in 1–9-year-olds. MDA should be stopped in the other three EUs and trachoma surveillance surveys should be conducted at least 24 months after the surveys described here. Ongoing strengthening of WaSH infrastructure may help sustain the low prevalence of trachoma. Chlamydia trachomatis.1 Repeated infection2 and the associated inflammatory response lead to scarring of the tarsal conjunctivae, trachomatous trichiasis (TT), corneal opacity and visual impairment.1 Infection spreads from person-to-person on hands, fomites and flies that have been in contact with discharge from the eyes or nose of an infected person.3–5 Trachoma is more common in people who live in poor and overcrowded communities with lack of access to water and sanitation.6–9 In May 2020, globally, a total of 137 million people were living in endemic areas and of these, 50% (68.5 million) were in Ethiopia.10Trachoma is an infectious disease of the eye caused by the bacterium 12 Elimination is achieved when the prevalence of TT unknown to the health system is <0.2% among people aged ≥15 years, prevalence of trachomatous inflammation–follicular (TF) is <5% in children aged 1–9 years, and there is written evidence of a system to identify and manage incident cases of TT, in all formerly endemic districts.13Trachoma can be eliminated as a public health problem through the use of a package of interventions known as the Surgery, Antibiotics, Facial cleanliness and Environmental improvement (SAFE) strategy.14 Four EUs (covering 11 woredas) were identified in which the prevalence of TF was ≥5% and therefore qualified for implementation of the A, F and E components of SAFE. Among these four EUs, TF prevalence was ≥10% in two EUs covering four woredas  which, therefore, required three rounds of annual antibiotic mass drug administration (MDA). The other two EUs in which interventions were required, covering seven woredas , had TF prevalences between 5 and 9.9% and were eligible to receive one round of MDA.14Between December 2013 and January 2014, the Benishangul Gumuz (BGZ) Regional Health Bureau participated in the Global Trachoma Mapping Project (GTMP), undertaking baseline surveys to determine the need for implementation of the SAFE strategy in the region. The 20 woredas (districts) in the region were grouped into seven evaluation units (EUs), based on common borders and similar socioeconomic characteristics. The prevalence of TT in people aged ≥15 years was ≥0.2% in all seven surveyed EUs.16As a result of the GTMP survey data, MDA was conducted in all four EUs with TF prevalence ≥5%. The two EUs (four woredas) requiring three rounds started MDA in 2014 just after the baseline surveys finished; one round of MDA was delivered to the remaining two EUs (seven woredas) in 2017. Active TT case finding and corrective surgery were launched throughout the region in 2019 as well as implementation of water, sanitation and hygiene (WaSH) interventions and coordinated neglected tropical disease activities targeting trachoma.This survey series aimed to determine how far BGZ remains from elimination of trachoma as a public health problem. We used population-based trachoma surveys to measure prevalence of TT unknown to the health system and TF after the interventions undertaken so far, to provide evidence with which to plan whether future interventions are needed.https://www.tropicaldata.org/). All individuals noted to have active trachoma were provided with 1% tetracycline eye ointment, and those with TT were referred to the nearest appropriate healthcare facility for surgical correction.Ethical approval was obtained from the ethics committees of the BGZ Regional Health Bureau and the London School of Hygiene & Tropical Medicine (16105). Regional, zonal and woreda officials were engaged and provided letters of support. The purpose of the survey was explained to the members of each participating household before enrollment and informed verbal consent was obtained from all adult participants before examination. For participants aged <18 years, informed verbal consent was obtained from the head of household, a parent or guardian. Consent was documented electronically using the Tropical Data app . For these trachoma impact surveys, four EUs were created with boundaries that matched those from the pre-MDA baseline surveys.14BGZ is one of the eleven regional states of Ethiopia , map. It18 The sample size was calculated to estimate a prevalence of TF in 1−9-year-olds of 4% with an absolute precision of ±2% at the 95% confidence level, assuming a design effect of 2.6318,19 and accounting for non-response with an inflation factor of 1.2. TT was determined using the number of people aged ≥15 years living in selected households.20A two-stage cluster sampling design was developed that complied with World Health Organization (WHO)-recommended technical parameters for trachoma surveys.20In the first stage, villages (clusters) were sampled. In each EU, a complete list of villages was obtained from the BGZ Regional Health Bureau, and 26 were selected systematically with a probability of selection proportional to population size. In the second stage, 30 households were selected per cluster using compact segment sampling. All residents of selected households aged ≥1 year were eligible for inclusion.21 For trainee graders to be certified for deployment to the field for data collection, they had to pass two inter-grader agreement assessments, comparing their grades to expert consensus on a set of photographs, followed by a comparison to a certified grader trainer on 50 live participants. Only graders who achieved a kappa of ≥0.7 for TF on both assessments participated in the survey. Survey recorders were trained to recognize water, sanitation and hygiene facility types, and to utilize the Tropical Data electronic data capture system. The recorder had to attain an agreement of 90% on a set of test data to be certified. Qualified grader and recorder pairs participated in practical team training before beginning field work.The training for both graders and recorders was conducted over four days using version 2 of the Tropical Data standardized training programme.22 For individuals who had TT, additional questions were asked about whether they had been offered and/or taken up management for TT, and the conjunctiva was examined for trachomatous scarring (TS). TT “unknown to the health system” was defined as an eye with TT for which the participant could not remember being offered surgery or epilation.Both eyes of all consenting residents aged ≥1 year were examined using adequate light (torch or sunlight) and a 2.5× magnifying loupe. Participants were examined for the presence of TT, TF and trachomatous inflammation–intense (TI) using the WHO simplified trachoma grading system.20Information on WaSH access was collected by recorders through interviews with heads of households and observation of sanitation and handwashing facilities. Variables related to water access and source type for both drinking and washing, and sanitation types were collected using a version of the WHO/United Nations Children’s Fund (UNICEF) Joint Monitoring Programme (JMP) household questionnaire adapted for the context of trachoma.24 Descriptive data and prevalence results were analyzed using open source R-based analysis scripts , as described elsewhere.20 TF prevalence in children aged 1–9 years was adjusted for age in 1-year age-bands, and TT prevalence in people aged ≥15 years was adjusted for age and gender in 5-year age-bands, using 2007 census data.25 Confidence intervals (CIs) were produced by bootstrap resampling of adjusted cluster-level outcome proportions.26 WaSH facilities were categorized according to the WHO/UNICEF JMP definitions.27Associations between TF and individual- and household-level variables were analyzed using mixed-effects binomial regression, in a similar approach to that of previous studies.28 As there were only four EUs surveyed in this series, the number of levels was insufficient to warrant inclusion of EU as a random effect. Similarly, the number of TF cases was insufficient to enable convergence of complex multivariable models with household included as a random effect. Cluster of residence was, therefore, the only random-effect variable included. Age and gender were included a priori as fixed-effect variables due to their known relationship with trachoma. Household-level fixed-effect variables were first tested for correlation and removed or amalgamated where correlation was found based on a Spearman’s rho >0.8. Fixed-effect variables were then tested in univariable analysis; those for which evidence of association with TF was strong (p < .05) were carried into the multivariable analysis. Finally, stepwise addition and removal of further fixed-effect variables was undertaken to finalize model structure. Comparison between models was achieved through likelihood ratio testing (LRT), with and without the variable in question .Data quality measures were implemented as previously described.Associations with TT were analyzed using the same approach. All TT cases, regardless of management status, were included in this analysis.A total of four EUs (comprising 11 woredas) were surveyed from March 2018− March 2019. A total of 3,081 households were visited in 103 clusters, from which 13,100 people were enumerated. Of these individuals, 11,778 (89.9%) consented to participate and were examined, 1287 (9.8%) were absent at the time of the team’s visit and 0.3% refused to take part. Of those examined, 6,329 (53.7%) were female. A total of 4,590 children aged 1–9 years and 5,825 people aged ≥15 years were examined .Table 1.TF was present in 259 (5.6%) of examined 1–9-year-olds. EU-level age-adjusted TF prevalence ranged from 2.2%  in Sherkole, Menge, Kurmuke and Homosha woreda of Assosa zone to 9.1% (95% CI: 5.8 − 12.8%) in Dibate and Bullen of Metekel Zone. There was only one EU which had a TF prevalence ≥5% , Table 2The change in prevalence from baseline to these impact surveys is shown in Overall, 78 cases of TT were identified across all EUs, of whom 73 (94%) reported not having been offered surgery or epilation and were classified as “unknown to the health system”. The age- and gender-adjusted prevalence of TT unknown to the health system in people aged ≥15 years ranged from 0.4% (95% CI: 0.1 − 0.8%) in Wombera, Guba and Dangure woredas to 0.9% (95% CI: 0.4 − 1.6%) in Sherkole, Menge, Kurmuke and Homosha woredas. The TT prevalence unknown to the health system in people aged ≥15 years was, therefore, ≥0.2% in all four EUs .The proportion of households with an improved drinking water source within a 30-minute round-trip ranged from 27 − 60%. The proportion of households with an improved latrine ranged from <1%−6%. The proportion of households with a latrine (status not specified) that had a hand wash station with water and soap was <1%−16% .Table 4.Compared to 1–3-year-olds, TF was less common in 4–6-year-olds  and still less common in 7–9-year-olds . TF tended towards being more common in female than male children; however, there was weak evidence to reject the null hypothesis of no gender difference . None of the household-level WaSH variables measured were associated with TF . The ageTT was more common in older individuals and in females. Compared to the reference age group of 15–35 years, the aOR for TT in 36–55-year-olds was 6.6 (95% CI: 3.8–11.5), for 56–75-year-olds was 8.5 (95% CI: 4.20–17.9) and for >75-year-olds was 18.0 . The aOR for TT in females compared to males was 3.9 . The age-distribution of TT in five-year age brackets is shown in 29 In the fourth EU surveyed, a substantial decrease in TF prevalence was observed, but one additional round of MDA is still required before another impact survey can be conducted, as per WHO recommendations.30In BGZ, the prevalence of TF in 1–9-year-olds was lower following implementation of MDA than at baseline. In three of four EUs surveyed, this reduction allowed MDA to be discontinued. This is encouraging, given the importance of Ethiopia to the global trachoma program overall.This survey series was not designed to measure changes in TT prevalence between the pre-MDA and post-MDA surveys: the sample did now allow generation of sufficient precision in TT prevalence estimates. However, it is notable that the prevalence of TT remained above the elimination threshold  in all four EUs, just as it was during the GTMP surveys several years previously. With 94% of individuals with TT reporting not having been offered management surgery or epilation, increased TT surgery provision and uptake are urgently needed. Active TT case finding should take place and surgical services should continue to be strengthened. The age and gender distribution of TT suggest programs should ensure older women, in particular, have equitable access to TT management.C. trachomatis.31 Guidelines for sustainable elimination of NTDs, including trachoma, suggest that prolonged investment in WaSH infrastructure beyond meeting disease elimination thresholds would be needed to maintain the benefits of MDA programmes.32 This is perhaps beyond the scope of the trachoma programme alone. Instead, these data could add leverage to the case for ongoing, general improvements to WaSH infrastructure.WaSH access in BGZ also requires improvement. Between one- and two-thirds of all households in the areas we surveyed did not have access to a water source within a 30-minute round trip. This could lead to prioritization of water for drinking and cooking and, thus, limited water use for hygiene purposes, particularly (from a trachoma perspective) facial cleanliness. An overwhelming majority of households did not have an improved sanitation facility. Unimproved latrines and open defecation could serve as breeding sites for the flies which transmit ocular 8One limitation of this study was the modelling approach used to identify factors potentially associated with TF. Pre-filtering variables for inclusion in a multivariable model risks falsely reducing variance and over-fitting the model. In this case, we elected to pre-filter the variables on the basis that the variables we had collected data on would not cumulatively predict trachoma risk. Instead, our aim was to estimate the magnitude of any relationship between household WaSH facility access and TF, for which an explanatory model would be preferable, and inclusion of unrelated variables could confound the estimate. Despite the risk of overfitting, no significant relationships were identified between TF and WaSH variables. The lack of association between TF and household-level WaSH variables should not deter regional health authorities from greater focus on the F and E components of SAFE. Because of the relatively low current prevalence of TF in these EUs, analysis of the small number of TF cases was probably underpowered to detect associations of TF and WaSH variables. Larger, multi-country analyses provide compelling evidence of association.These data represent a significant step forwards towards elimination of trachoma in BGZ. We have demonstrated that, in addition to the woredas found to have a TF prevalence <5% at the time of the GTMP, a further nine woredas no longer require MDA. Program momentum should be maintained to ensure the gains made by MDA are sustained long-term.


Atomic layer deposition (ALD) allows excellent conformal growth, which can be exploited on 3D nanoelectrode arrays. In this work, we disclose the growth of nanocrystalline rutile IrO2 at T = 150 °C adopting a new plasma-assisted ALD (PA-ALD) process. The morphological, structural, physical, chemical, and electrochemical properties of the IrO2 thin films are reported. To the best of our knowledge, the electrochemical characterization of the electrode/electrolyte interface in terms of charge injection capacity, charge storage capacity, and double-layer capacitance for IrO2 grown by PA-ALD was not reported yet. IrO2 grown on PtSi reveals a double-layer capacitance (dlC) above 300 µF∙cm−2, and a charge injection capacity of 0.22 ± 0.01 mC∙cm−2 for an electrode of 1.0 cm2, confirming IrO2 grown by PA-ALD as an excellent material for neuroelectronic applications.In vitro and in vivo stimulation and recording of neuron action potential is currently achieved with microelectrode arrays, either in planar or 3D geometries, adopting different materials and strategies. IrO The investigation of neural networks in vitro is fundamental for the understanding of the mechanisms involved in neurological diseases such as Alzheimer’s. In this context, the study of the short- and long-distance interactions between neurons is possible thanks to the development of microelectrode arrays (MEAs), which can be used for the stimulation of neurons and the recording of neuronal signals . MEAs arThe electrical interaction between MEAs and neurons can take place according to two main different charge transfer mechanisms that are determined by the electrical properties of the electrode’s material. The so-called capacitive charge transfer takes place when a dielectric material, which forms a capacitor at the electrode/electrolyte interface, is polarized, and it transfers the polarization to the electrolyte. The other mechanism, commonly occurring for metallic electrodes, consists of a direct, Faradaic transfer of charges between the electrode and the electrolyte . Among t2 has been widely applied in the neuroelectronic field because of its pseudocapacitive behavior, along with good stability and non-toxicity  dissolved in ethyl cyclo-hexane as a precursor, and a mix of O2 and H2 plasma as co-reactant. The same combination of precursor and co-reactant was applied in 2014 by Kim et al. [2 nanodots. It is worth mentioning that (EtCp)Ir(CHD) was originally designed and synthesized by Kawano et al. [2 (on silicon with its native oxide) using O2 plasma as a reactant and (Methylcyclopentadienyl)  Iridium(I) [(MeCp)Ir(COD)].There are few reports, to the best of our knowledge, using a plasma-assisted ALD (PA-ALD) process for the preparation of IrOi et al.  reportedm et al.  for PA-Ao et al.  in 2004 o et al.  have rep2. The process is based on the application of a mix of Ar/O2 plasma as a reactant in combination with (EtCp)Ir(CHD) as a precursor. Compared to previous works on PA-ALD of IrO2 using (EtCp)Ir(CHD), we did not have to dissolve the precursor in cyclo-hexadiene or feed the plasma source with H2 gas. The ALD process is characterized in situ by spectroscopic ellipsometry (SE). In situ SE is a key tool for the characterization of ALD processes, since it is able to monitor thickness changes in the films deposited down to the atomic scale [2, a Drude–Lorentz oscillator was selected, generally applied for modeling the absorption of light of conductive materials [In this work, we report on a novel PA-ALD approach for the growth of IrOic scale ,29,30,31aterials ,29,30,312 thin films have been fully characterized to assess key functionalities relevant to neuroelectronics. Specifically, an extensive electrochemical characterization has been performed, providing key parameters for neuroelectronics, such as the double-layer capacitance, the charge storage capacity, and the charge injection limit. Furthermore, the interpretation of the impedance spectroscopy measurements has been corroborated by the cyclic voltammetry measurements. This last aspect is not very common in the literature on neuroelectronics and can be of broader interest in the electrochemical field.The chemical, physical, and electrochemical properties of the IrO2. The choice of the substrate is motivated by the planned integration of IrO2 onto vertical nanopillar arrays to be used as scalable nanoelectrodes fabricated starting from silicon nanopillars [PtSi has been selected as a conductive substrate for the deposition of IrOopillars .2 deposition was performed using a PICOSUN R-200 Advanced ALD system, equipped with a remote inductively coupled plasma source. The base pressure of the ALD reaction chamber is within the range of 0.2–4.0 hPa. The plasma source operates in the range of 1.9–3.2 MHz, with the plasma power adjustable from minimum of 300 W to maximum of 3000 W. The distance between the plasma source and the sample holder is about 75 cm, in order to reduce any possible damage to the substrate from highly energetic ions. (EtCp)Ir(CHD) (99%), from Strem Chemicals, was kept in a stainless steel cylinder heated to 100 °C, while the line from the cylinder to the deposition chamber was heated to 120 °C. (EtCp)Ir(CHD) precursor should be handled with care, since it causes skin and eye irritation and may cause respiratory irritation. N2 gas (99.9999%), used as carrier for the precursor, was flowed at 200 sccm. A mix of Ar gas (99.9999%) and O2 gas (99.9999%), flowing at 40 sccm and 190 sccm, respectively, was used to feed the plasma. During the plasma step, the power of the plasma source was set to 2500 W.IrO2 is flowed through the plasma source for 1 s in order to stabilize the flow before igniting the plasma for 40 s. Afterward, a purge step of 4 s closes the ALD cycle.The ALD recipe starts with the dosing of (EtCp)Ir(CHD) for 6 s, followed by 15 s of purge. Then, the mixture of Ar/O2 layer. The fitting of thickness and optical constants of IrO2 thin films was performed using a Drude–Lorentz oscillator.Film Sense FS-1™ ellipsometer system was used for in situ and ex situ characterization of thickness and optical constants of the deposited IrO2 thin films were characterized by X-ray diffraction in grazing incidence mode (GI-XRD), transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM).ALD-prepared IrO2. The elemental maps were acquired and elaborated by Velox software.Transmission electron microscopy (TEM) techniques were conducted on electron-transparent lamellae obtained by focused ion beam (FIB). The alloy microstructure was observed by bright-field TEM and high-angle annular dark-field STEM (HAADF-STEM), while high spatial resolution chemical analyses were carried out by energy dispersive spectroscopy (EDS). The lamellae were obtained using a Thermofischer Helios G5UX FIB. Low energy milling was used during the final thinning steps to limit heating and ballistic effects of ion irradiation on alloy film. The TEM images were performed with a Thermofischer Themis Z G3 aberration-corrected transmission electron microscope equipped with an FEG electron gun operating at 200 kV acceleration voltage. To minimize the electron beam damage, all the TEM/STEM images and EDS maps were acquired with a low beam current (0.5 nA). The EDS measures were carried out using a Dual-X sensor made of two detectors of 100 mmGI-XRD was performed on a Rigaku Smartlab SE equipped with a Cu Ka source (Ka1 = 1.540598) operating at 40 KV and 30 mA. Data were collected under parallel beam conditions, at the angle of incidence of 0.2°, in the range of 20°–50°.+ ions at 1 keV (113 nA) and rastering over a 300 × 300 mm2 area. Analysis was performed by means of Ga+ ions at 25 keV (1.2 pA) rastering over a 50 × 50 mm2 area.ToF-SIMS profiles were performed using a dual beam IONTOF IV system operating in negative polarity. Sputtering was accomplished using Css signal at 284.5 eV was used to correct the binding energy scale.XPS analysis was performed on a PHI5000 Versaprobe III system equipped with a monochromatic Al Ka X-ray source (1486.6 eV) and a concentric hemispherical analyzer with a take-off angle of 45°. Survey spectrum was acquired with a band-pass energy of 280 eV. High-resolution spectra were acquired with a band-pass energy of 55 eV. C 1http://gwyddion.net/; accessed on 7 March 2023) to derive the root mean square (RMS) roughness and the correlation length (L). RMS accounts for the height fluctuations of the surface features, whereas the correlation length is the measure of the length beyond which surface heights are not significantly correlated and it was estimated by the gaussian fitting of the height–height correlation function (see Equations (S1)–(S3), Film surface morphology was analyzed by atomic force microscopy, using a commercial system (Bruker Dimension Edge). Measurements were carried out in non-contact mode using sharp silicon probes with typical tip radius of 10 nm and resonance frequency of approximately ~320 kHz. Several square scans (1- to 5-micron side) were taken at various surface locations. The acquired data were analyzed by Gwyddion  was performed using a Zurich Instruments MFIA impedance analyzer, applying a test signal of 100 mV in the frequency range from 0.1 Hz to 100 kHz. EIS characterization of the interface IrO2/electrolyte was performed in a two-electrode configuration, with two IrO2 samples facing each other in electrical contact through the electrolyte. The phosphate buffer (PBS) used as electrolyte for all the electrochemical characterizations was prepared by diluting 10 mL of PBS 10× purchased from Sigma-Aldrich with 90 mL of deionized water.Electrochemical tests were performed using a double-sided-magnetic mount photo-electrochemical cell from Redoxme. Unless differently specified, the electrode area is 1.0 cmCyclic voltammetry (CV) and voltage transient measurements (VT) were performed using a BioLogic VMP3 multi-channel potentiostat in a three electrodes configuration, i.e., the sample under study as working electrode (WE), an Ag|AgCl wire as reference electrode (RE) and a Pt wire as counter electrode (CE). The Ag|AgCl was selected as reference electrode since it is commonly applied in the field of neuroelectronics [2 (on PtSi) via spectroscopic ellipsometry is shown in 2. Nonetheless, the different GPC could be explained by the use of different precursors, i.e., (EtCp)Ir(CHD) in this work and (MeCp)Ir(COD) in the work of Simon et al. [2 focus on the deposition of nanodots; therefore, the GPC value of the process is not specified. From The in situ characterization of ALD of IrOn et al. . On the n et al.  and Kim n et al.  on PA-ALThe saturation of each ALD dosing step was tested by independently changing the dosing times of precursor and co-reactant. 2 thin films was investigated by grazing incidence X-ray diffraction (GI-XRD). 2 prepared by ALD at 150 °C reveals the characteristic pattern of IrO2 in the rutile phase, with the three peaks corresponding to the <110>, <101>, and the <200> planes, as reported in the literature [2 in the thin film [2 thin film was determined to be 5.3 nm ± 0.4 nm.The crystallinity of the ALD-prepared IrOterature . The fulhin film ,35. Assu2 grown on PtSi/Poly-Si/SiO2/Si. 2 layer, clearly visible because of the difference in Z-contrast with respect to the PtSi substrate. The layer is homogenous with a thickness of about 13 nm, in line with SE measurements. 2 layer on PtSi. Considering the surface roughness of PtSi, the interface between IrO2 and PtSi is sharp, with no clear indication of interdiffusion between the two layers. The surface roughness of the underlying substrate has a strong influence on the surface morphology of the IrO2 deposited, as suggested by AFM measurements performed on IrO2 grown onto Al2O3 on c-Si . The EDS line profile in 2 is due to the Pt deposited as a contrast layer during the specimen preparation. No further contamination from other elements was detected in the IrO2 film within the sensitivity limit of the EDS technique.2 thin film, shown in 2 layer on top of the PtSi substrate. 193IrO− secondary ion signal and 195Pt− and 30Si− secondary ion signals are reported as markers of the IrO2 film and PtSi substrate, respectively. These signals clearly indicate no diffusion of Pt and Si from the substrate into the IrO2 film during the ALD growth. The broadening of the 193IrO−, 195Pt−, and 30Si− secondary ion signals at the IrO2/PtSi interface is fully consistent with the roughness of the PtSi surface that was highlighted by the TEM analysis shown above in 2 film, within the sensitivity limit of the technique, confirming the good quality in terms of impurities for the IrO2 thin film prepared by ALD.ToF-SIMS depth profile of the ALD-prepared IrO2 film deposited on top of the PtSi substrate. The different core lines in the spectrum correspond to signals from the Ir and O atoms in the IrO2 film. No signals from the underlying PtSi substrate were detected, further supporting the idea of a homogeneous IrO2 film, perfectly covering the PtSi substrate. f and O 1s core lines, respectively. Since rutile-type IrO2 is a metallic conductor, an asymmetric line shape is expected for the Ir 4f core lines. The high-resolution Ir 4f spectrum was fitted using a doublet of asymmetric functions with spin–orbit splitting of 3 eV to capture the main 4f lines and a doublet of Gaussians at 1 eV higher binding energy to capture the primary shake-up satellites. A secondary satellite of the Ir 4f5/2 peak at ~3 eV above the main line was introduced to obtain a satisfactory fitting of the experimental data, in agreement with previous results reported in the literature [f7/2 core line is determined to be 61.7 ± 0.1 eV. The high-resolution O 1s spectrum was fitted using asymmetric functions. The position of the O 1s core line is found to be 529.9 ± 0.1 eV. Two additional Gaussian functions were introduced to correctly fit the experimental data. These functions account for hydroxyl groups (BE ~ 531.3 eV) and adsorbed water (BE ~ 532.4 eV) on the IrO2 surface [f and O 1s core lines are perfectly consistent with data available in the literature for rutile IrO2 [The results of the chemical characterization performed by XPS are reported in terature . The pos surface . The binile IrO2 ,38.2 films is strictly related to the surface morphology of the supporting substrate. In 2 surface and of the bare PtSi surface are shown. IrO2  used to model the bulk resistivity of the solution. The data analysis was performed using EIS Spectrum Analyser software [2 behaves in solution as an ideally polarized blocking electrode, i.e., no DC is flowing at the electrode/electrolyte interface. The parameters of the CPE, as reported in Q and n are the parameters defining the impedance of the CPE (see Equation (S4) in supporting information). For the results shown in −2. This value obtained for ALD-prepared IrO2 is very promising, considering that the electrochemically active surface area influences the dlC, and how in this work, IrO2 was deposited onto a relatively flat substrate. As a comparison, a dlC value of 270 µF∙cm−2 has been reported for IrO2 electrodeposited onto Ti felt [Electrochemical impedance spectroscopy (EIS) was used to characterize the charge transfer properties of the interface between the ALD-prepared IrOsoftware . The modtion (1) ,41:(1)Cd Ti felt .dlC value extracted from EIS data, the double-layer capacitance was determined via cyclic voltammetry (CV) as well. A series of CV measurements were carried out, varying the scan rate from 5 mV∙s−1 to 10 mV∙s−1 in the voltage range between 0.35 V and 0.45 V, where no faradaic processes are supposed to occur [c) and the capacitive anodic current (Ia) as a function of the voltage of the working electrode (Ewe) for different scan rates. The double-layer capacitance can be calculated from the CV measurements via Equation (2) [c/ai is the capacitive current, cathodic or anodic, and dE/dt is the scan rate. As reported in −2 which is in good agreement with the value obtained by EIS, considering that two different methods, based on different working principles, were used. The high values of dlC obtained for ALD-prepared IrO2 can be explained by considering the good pseudocapacitive behavior of IrO2 due to the oxidation/reduction of the Ir(III)/Ir(IV) species at the surface of the electrode [In order to corroborate the to occur . The restion (2) :(2)CdlCVlectrode ,44. It i2 in the range of −0.6–0.8 V vs. Ag|AgCl. 2 thin film deposited onto PtSi (blue line) compared to the bare PtSi substrate (black line). The current density measured for the IrO2/PtSi electrode is much larger than the current density recorded for the PtSi substrate. Since the Faradaic contribution due to the oxidation and reduction of water is negligible below 0.8 V and above −0.6 V, the higher current density of IrO2 over the PtSi substrate can be attributed to the pseudocapacitive behavior of IrO2, which allows accumulating a larger amount of charge.Cyclic voltammetry was then performed in order to determine the charge storage capacity (CSC) of ALD-prepared IrOCSC by mean of Equation (3) [aE and cE are the anodic and cathodic limits of the potential, i is the measured current, v is the scan rate, and A is the surface area of the electrode [2, the value of CSC was calculated to be 1.9 ± 0.1 mC∙cm−2, while the bare PtSi substrate exhibited a CSC of (2.4 ± 0.2)·10−2 mC∙cm−2. The value of CSC obtained for ALD-prepared IrO2 is slightly lower compared to the non-activated sputtered IrO2 reported in the literature (2.8 mC∙cm−2) [2 deposited by reactive sputtering is generally characterized by higher surface roughness, while ALD-prepared thin films are generally smoother since they replicate the surface features of the underneath substrate conformally, as mentioned above in the AFM measurement paragraph.The CV curves reported in tion 3) ,9:(3)CSClectrode . For ALD ,9:(3)CS2 electrode can deliver without overcoming the cathodic limit for the water reduction, voltage transient (VT) measurements were performed. A current squared wave of different amplitudes was forwarded to the sample, recording the voltage (Ewe) over time. The results reported in we as a function of time for values of current amplitude between 0.5 mA and 2.5 mA (mc) was then calculated by the difference between the voltage peak (Ep) and the access voltage (Ea) [mc as a function of the charge injected. By linear regression of the data, it was possible to calculate that for Emc = −0.6, the charge injected is 0.22 ± 0.01 mC∙cm−2. Taking into account that the CIC is inversely proportional to the electrode’s surface area [2, in view of its application onto microelectrode arrays. We shall also note that CIC values higher than 1.0 mC∙cm−2 can be detrimental to neural stimulation because of possible neural damage [To determine the maximum charge that the IrOd 2.5 mA a. The maage (Ea) . Figure ace area , the vall damage .2 using (EtCp)Ir(CHD) as the precursor and a mix of Ar/O2 plasma as the reactant. The growth characteristics, the physicochemical as well as the electrochemical properties of IrO2 grown by PA-ALD were determined in view of the application in neuroelectronics. The use of the plasma allowed deposition at a relatively low temperature (150 °C), which can be helpful for the integration of the layer on existing devices as well as on flexible substrates. XRD results show the characteristic peaks of the metallic rutile-phase IrO2, indicating that the layer so prepared is nanocrystalline. TEM/EDS and ToF-SIMS analysis of ALD-prepared IrO2 indicates that the layer is compact and continuous with low carbon contamination. TEM and AFM results highlight the conformality of the layer to the PtSi substrate. XPS results further confirm the presence of the metallic rutile phase of IrO2.In this work, we disclosed a novel PA-ALD process for the deposition of IrO2 grown by PA-ALD has competitive performances for application in neuroelectronics. Specifically, EIS results revealed a strong capacitive coupling between the IrO2 electrode and the electrolyte, with a dlC of 301 ± 4 µF∙cm−2, higher than the value reported in the literature for IrO2 electrodeposited on Ti felt [dlC obtained from CV was about 341 ± 1 µF∙cm−2, in general agreement with the value determined with EIS, considering the different working principles of the two methods. To the best of our knowledge, the electrochemical characterization of the electrode/electrolyte interface in terms of charge injection capacity, charge storage capacity, and double-layer capacitance for IrO2 grown by PA-ALD was not reported yet. Furthermore, the comparison between EIS and CV results for the determination of the double-layer capacitance is also an element of novelty in the field of neuroelectronics.Electrochemical characterization showed that IrO Ti felt . The valCSC of IrO2 was calculated in the range of −0.6–0.8 V vs. Ag|AgCl, where the oxidation and reduction reactions of water are negligible. The CSC value of 1.9 ± 0.1 mC∙cm2 is slightly lower than what is reported for non-activated electrodeposited IrO2 (2.8 mC∙cm2), although it should be considered that surface roughness has a big role in the electrochemical results. Finally, voltage transient measurements were performed in order to determine the maximum charge that the IrO2 electrode can deliver without overcoming the cathodic limit for the water reduction (−0.6 V). The charge injection capacity found for ALD-prepared IrO2 was 0.22 ± 0.01 mC∙cm−2, in line with the literature [2 onto MEAs, since the charge injection capacity is inversely proportional to the surface area (1.0 cm2 in this work) of the electrodes.The terature . The val


Metabolomics technology facilitates studying associations between small molecules and disease processes. Correlating metabolites in cerebrospinal fluid (CSF) with Alzheimer’s disease (AD) CSF biomarkers may elucidate additional changes that are associated with early AD pathology and enhance our knowledge of the disease.The relative abundance of untargeted metabolites was assessed in 161 individuals from the Wisconsin Registry for Alzheimer’s Prevention. A metabolome-wide association study (MWAS) was conducted between 269 CSF metabolites and protein biomarkers reflecting brain amyloidosis, tau pathology, neuronal and synaptic degeneration, and astrocyte or microglial activation and neuroinflammation. Linear mixed-effects regression analyses were performed with random intercepts for sample relatedness and repeated measurements and fixed effects for age, sex, and years of education. The metabolome-wide significance was determined by a false discovery rate threshold of 0.05. The significant metabolites were replicated in 154 independent individuals from then Wisconsin Alzheimer’s Disease Research Center. Mendelian randomization was performed using genome-wide significant single nucleotide polymorphisms from a CSF metabolites genome-wide association study.Metabolome-wide association study results showed several significantly associated metabolites for all the biomarkers except Aβ42/40 and IL-6. Genetic variants associated with metabolites and Mendelian randomization analysis provided evidence for a causal association of metabolites for soluble triggering receptor expressed on myeloid cells 2 (sTREM2), amyloid β (Aβ40), α-synuclein, total tau, phosphorylated tau, and neurogranin, for example, palmitoyl sphingomyelin (d18:1/16:0) for sTREM2, and erythritol for Aβ40 and α-synuclein.This study provides evidence that CSF metabolites are associated with AD-related pathology, and many of these associations may be causal. The neuropathological changes of Alzheimer’s disease (AD) consist of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles of hyperphosphorylated tau proteins in the brain . Well-esUntargeted metabolomics technology is a promising approach that can simultaneously identify and quantify a large number of small molecules  in a biological sample . Previoun < 10) to include in the analyses.The Wisconsin Registry for Alzheimer’s Prevention (WRAP) began recruitment in 2001 as a prospective cohort study, with initial follow-up 4 years after baseline and subsequent ongoing follow-up every 2 years. WRAP is comprised of initially cognitively-unimpaired, asymptomatic, middle-aged (between 40 and 65) adults enriched for parental history of clinical AD . At eachInvestigating Memory in Preclinical AD-Causes and Treatments (IMPACT) cohort] (n < 10) to include in the analyses.The Wisconsin Alzheimer’s Disease Research Center’s (ADRC) clinical core cohorts started in 2009 and are comprised of well-characterized participants who undergo cognitive testing and physical exams every 2 years . The Wis cohort] . From thThis study was conducted with the approval of the University of Wisconsin Institutional Review Board, and all participants provided signed informed consent before participation.Fasting CSF samples were collected via lumbar puncture using a Sprotte 25- or 24-gauge spinal needle at the L3/4 or L4/5 interspace with gentle extraction into polypropylene syringes. More details can be found in the previous study . The CSF® Aβ(1–42), P-tau(181P), and T-tau, as well as S100b and IL-6, were performed on a cobas e 601 analyzer  using an untargeted approach, based on Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry platform (UPLC-MS/MS) . DetailsA total of 412 CSF metabolites were identified and quality control procedures were performed. First, 46 metabolites missing for at least 80% of the individuals were excluded. Then the values for each of the remaining metabolites were scaled so that the median equaled 1. Two metabolites with an interquartile range (IQR) of zero were excluded and no metabolites had zero variability between individuals. Log10 transformation was applied to normalize the data. After quality control, 269 metabolites with known biochemical names remained for this investigation. The missing percentage of each metabolite in WRAP and Wisconsin ADRC is available in ® DNA Isolation Kit, and the concentrations were quantified using the Invitrogen™ Quant-iT™ PicoGreen™ dsDNA Assay Kit. More details can be found in the previous study (R2 < 0.8), with a low minor allele frequency (MAF) (<0.001), or out of HWE were removed. The genetic ancestry was assessed by using Principal Components Analysis in Related Samples (PC-AiR) because of the sibling relationships present in the WRAP cohort.In the WRAP participants, DNA was extracted from whole blood using the PUREGENEus study . Genotypus study . Brieflyp < 1e-6 due to differences in sample sizes and the number of SNPs between the two cohorts.Genetic data in the Wisconsin ADRC were generated from DNA extracted from blood samples at baseline and genotyped with either the Infinium OmniExpressExome-8 Kit or the Infinium Global Screening Array-24 Kit. Genetic data for the Wisconsin ADRC underwent the same quality control (QC) and imputation as the WRAP data except samples and SNPs missing in >2% were excluded and HWE threshold was p-values for multiple testing; the FDR corrected q value was used to determine statistical significance in each analysis. Potential functional pathways of the replicated significant metabolites were identified by pathway analyses using the web-based software Metabo-analyst 5.0 (A metabolome-wide association study (MWAS) was conducted in the WRAP cohort between 269 individual CSF metabolites and 13 CSF NTK biomarkers using linear mixed-effects regression models with random intercepts to account for repeated measures and family relationships (10 families with two or more siblings) and fixed effects for age at CSF collection, sex, and years of education. Replication of each CSF metabolite significantly associated with one or more biomarkers in WRAP was then conducted in the Wisconsin ADRC cohort using linear regression adjusting for the same covariates. Both Bonferroni and false discovery rate (FDR) methods were used to correct the lyst 5.0  based on2 in the combined and imputed cohorts of WRAP and the Wisconsin ADRC. For this analysis, we only included the first available measures of independent participants from WRAP. Since the number of significant metabolites for each biomarker was large and some of the metabolites were highly correlated, elastic net regression  base model, which only included the demographics of age, sex, years of education, and indicator of cohort, (2) metabolite model, which included the demographics in the base model plus all the replicated significant metabolites, and (3) elastic net-selected metabolite model, which contained the demographics and elastic-net-selected metabolites.The variance for each biomarker explained by its corresponding significant metabolites was evaluated using rgression  was emplp < 5 × 10–8) from a previous genome-wide meta-analysis of CSF metabolites  for correlated IVs that were relatively weak   which usabolites  were extabolites . Next, tely weak . The conely weak , were coivmodel” .The genetic data have been uploaded to the National Institute on Aging Genetics of Alzheimer’s Disease Data Storage Site (NIAGADS) and metabolomic data are being uploaded to Accelerating Medicines Partnership Program for Alzheimer’s Disease (AMP-AD).Characteristics of the WRAP and Wisconsin ADRC participants can be found in The significant MWAS results in WRAP and the Wisconsin ADRC are summarized in The functional pathways for replicated significant metabolites with known human metabolome database (HMDB) IDs for each CSF NTK biomarker are shown in 2 and presented in 2 of the base models, which only included the demographic variables, ranged from 0.01 to 0.25. Adding the replicated significant metabolites increased the r2 substantially for each biomarker, ranging from 0.23 to 0.74. The elastic net regression further prioritized candidate metabolites associated with each biomarker. For example, 14 of the original 40 significant metabolites were selected by the elastic net as important independent metabolites for P-tau. Initially, 40 significant metabolites explained about 72% of the variance in P-tau; the 22 elastic net-selected metabolites still explained 67% of the variance.The prediction performance of replicated significant metabolites was measured by rAccording to the F statistics, we employed the LIML method for MR. The full results of the test statistics are provided in In this analysis, we tested the associations between CSF metabolites and CSF NTK biomarkers representing different pathologies of AD in initially cognitively-unimpaired individuals. Significant metabolites were identified in the WRAP cohort using linear mixed effects regression and most of the metabolites were replicated in the Wisconsin ADRC cohort. The elastic net regression method reduced the number of CSF metabolites by selecting the important and independent metabolites for each CSF biomarker. This provides a smaller, more practical set of metabolites to focus on in future research. The results of the MR analyses suggested several metabolites that may play a causal role in AD pathology. A detailed look into these associations, such as the contributing genes and their corresponding functions, is worth exploring.We have identified and replicated multiple CSF metabolites that were associated with CSF NTK biomarkers for AD pathology; most of these CSF metabolites were lipids, particularly sphingolipids, phosphatidylcholines, and lysophospholipids, which are all types of phospholipids. Phospholipids are a class of lipids that construct the cellular membranes and are involved in many complex activities of membrane proteins, receptors, enzymes, and ion channels in the cell or at the cell surface . In the r = 0.55), as it was in our study. N-acetylneuraminate is an acetyl derivative of the amino sugar neuraminic acid, which occurs in many glycoproteins, glycolipids, and polysaccharides. Specifically, it is a functional and structural component of gangliosides, which are found predominantly in the nervous system and are abundant in the brain, especially in the grey matter , 1-stearoyl-2-arachidonoyl-GPC (18:0/20:4), sphingomyelin  and sphingomyelin  were only associated with α-synuclein. These metabolites may be helpful to study synaptic dysfunction and could potentially be used as biomarkers to differentiate AD pathologies.The significant associations between a number of metabolites and both Aβ42 and Aβ40, but not with Aβ42/40 may indicate that the metabolites associated with Aβ42 and Aβ40 only influence the production of amyloid in general versus clearance of the pathological form, Aβ42. Our analysis also suggested that no metabolites were associated with IL-6, consistent with two other studies in the WRAP and Wisconsin ADRC cohorts that found no associations between twelve SM metabolites and IL-6 , and no By utilizing Mendelian randomization, we found causal evidence for several of the associations between CSF metabolites and CSF NTK biomarkers. Among these metabolites, most of them were lipids, with some amino acids and cofactors/vitamins, and a xenobiotic metabolite, erythritol. Another metabolite of interest, homocarnosine, is an inhibitory neuromodulator synthesized in the neuron from gamma-aminobutyric acid (GABA) and histidine . The levin vitro are necessary for these results to be clinically relevant. Our analysis also excluded several metabolites with unknown biochemical names, but due to the development of Metabolon’s library and rapidly increasing studies in this area, it would be worthwhile to re-examine these metabolites when we have more knowledge. Finally, the MR conclusion can be nullified if the underlying assumptions are violated. For example, our conclusions may be sensitive to the presence of invalid IVs due to potential pleiotropy of metabolites. In general, the research confirmed that several novel metabolites changed along with AD CSF biomarkers and extended several developing and understudied AD pathologies, e.g., synaptic dysfunction, based on untargeted CSF metabolomics and will expand our knowledge of the biological mechanisms behind AD.This study has some limitations. First, the analysis only included non-Hispanic white individuals, so the results may not extrapolate to other racial/ethnic groups. Second, the sample sizes of both the WRAP and Wisconsin ADRC cohorts were relatively small and will need to be replicated in a larger independent sample. Although we excluded the metabolites with very high missingness (>80%), there were 10 metabolites with missing values in over 50% of individuals in WRAP and/or the Wisconsin ADRC . The mishttps://www.niagads.org/home), accession number: NG00067. The metabolomic data presented in the study are deposited in Synapse (https://www.synapse.org/), project SynID: syn52222896.The genetic data presented in the study are deposited in the National Institute on Aging Genetics of Alzheimer’s Disease Data Storage Site (NIAGADS, The studies involving human participants were reviewed and approved by the University of Wisconsin Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.RD conceived and designed the analysis, contributed data or analysis tools, performed the analysis, and wrote the manuscript. QL, HK, YD, and CV contributed data or analysis tools, modified the manuscript, and provided suggestion. IS, GK, NW, HZ, and KB collected the data, modified the manuscript, and provided suggestion. RA modified the manuscript and provided suggestion. CC, SA, and SJ collected the data. CE conceived and designed the analysis, collected the data, wrote the manuscript, and provided suggestion. All authors contributed to the article and approved the submitted version.