Mitochondrial OCRs were similar between the FGR and control groups for palmitoyl-carnitine (fatty acid-driven) or pyruvate plus palmitoyl-carnitine metabolic substrates. A total of 2284 genes were differentially expressed in the semitendinosus muscle from growth restricted fetuses  ≤ 0.05). A pathway analysis showed that the upregulated genes (FGR compared to control) were overrepresented for autophagy, HIF-1, AMPK, and FOXO signaling pathways . In addition, the expression of genes modulating pyruvate’s entry into the TCA cycle was downregulated, whereas the genes encoding key fatty acid oxidation enzymes were upregulated in the FGR muscle. These findings show that FGR skeletal muscle had attenuated mitochondrial pyruvate oxidation, possibly associated with the inability of pyruvate to enter into the TCA cycle, and that fatty acid oxidation might compensate for the attenuated energy metabolism. The current study provided phenotypic and molecular evidence for adaptive deficiencies in FGR skeletal muscle.Fetuses with intrauterine growth restriction (FGR) have impaired oxidative and energy metabolism, with persistent consequences on their postnatal development. In this study, we test the hypothesis that FGR skeletal muscle has lower mitochondrial respiration rate and alters the transcriptomic profiles associated with energy metabolism in an ovine model. At late gestation, mitochondrial oxygen consumption rates (OCRs) and transcriptome profiles were evaluated in the skeletal muscle collected from FGR and control fetuses. The ex vivo mitochondrial OCRs were reduced  Neonates born with fetal growth restriction (FGR) have lower birth weights and increased risks for developing metabolic diseases in adulthood, such as type 2 diabetes, obesity, and cardiovascular diseases ,2,3. FetIn response to placental insufficiency, FGR fetuses slow protein accretion rates for new tissues to preserve energy for basal metabolic requirements . DespiteAlthough there is evidence that growth restricted fetuses have impaired global oxidative metabolism, the metabolic adaptation in skeletal muscle mitochondria function and the underlying molecular mechanisms are less understood. Therefore, we proposed to test the hypothesis that FGR fetal sheep had impaired skeletal muscle ex vivo mitochondrial oxygen consumption rates. In addition, a comparative transcriptomic analysis (RNA-seq) was conducted to identify the molecular mechanisms governing the metabolic adaptation in FGR skeletal muscle.n = 9) compared to those of the control  . Placentae from the FGR group weighted 59% less than those from the CON group . The FGR fetuses had lower semitendinosus  and biceps femoris  muscle weights. In addition, the FGR fetuses tended to have lower semitendinosus muscle weights normalized to fetal weights . However, the relative biceps femoris weights normalized to fetal weights were not different between the CON and FGR groups .At late gestation , fetal weights were lower in the FGR group (p = 0.027) in the FGR compared to the CON group  were reduced  between the FGR and CON fetal skeletal muscle . Among tAn gene ontology overrepresentation analysis was performed for the up- and downregulated genes separately. For the upregulated genes (FGR compared to CON), the HIF-1 signaling pathway (oas04066), the AMPK signaling pathway (oas04152), autophagy (oas04140), mitophagy (oas04137), the FoxO signaling pathway (oas04068), and insulin resistance (oas04931) were among the KEGG pathways that were enriched  A. In addp < 0.001) for the log2 fold changes in candidate genes between these two experiments, showing the validation of the RNA-seq findings.Twenty candidate DEGs that were among the top fold changes and were associated with the FoxO signaling pathway, the AMPK signaling pathway, and fatty acid oxidation were chosen for our qPCR experimental validation. The log2 fold changes identified between the RNA-seq and qPCR experiments are plotted in In the present study, we used an ovine model of FGR to demonstrate that FGR fetuses had lower ex vivo mitochondrial oxygen consumption rates in permeabilized soleus muscle when glucose-derived pyruvate was the metabolic substrate. FGR muscle had a normal mitochondrial OCR when exposed to fatty acids  or pyruvate plus fatty acid substrate pairs. Our further exploration of the skeletal muscle molecular adaptation showed that the lower pyruvate oxidation was partially explained by lower pyruvate carboxylase (PC) and citrate synthase (CS) mRNA expression, and higher pyruvate dehydrogenase kinase (PDK) mRNA expression which may prevent pyruvate’s entry into the TCA cycle for subsequent oxidative respiration. We also showed lower expression of genes encoding TCA cycle enzymes  in FGR muscle , which iPermeabilized soleus muscle fibers from FGR fetuses have lower mitochondrial OCRs with pyruvate as the substrate but were not different from those with fatty acids as the substrate. These data indicate that deficiencies reside in pyruvate’s entry into the mitochondria rather than TCA cycle deficiencies because acetyl-CoA derived from fatty acids had a normal OCR in FGR muscle. Upon its transport into the mitochondria, pyruvate enters into the TCA cycle via its conversion to either acetyl-CoA or oxaloacetate, catalyzed by pyruvate dehydrogenase (PDH) or PC, respectively. Both acetyl-CoA and oxaloacetate are further catalyzed by CS, the first enzyme in the TCA cycle. Supporting the current data on lower pyruvate oxidative respiration, we observed lower expression of genes encoding PC and CS. In addition, there was higher expression of genes encoding PDK1 and PDK4, which phosphorylate and inactivate PDH. The current data are also supported by our previous proteomic data showing the downregulation of mitochondrial pyruvate carrier 2 (MPC2) and PC which governs pyruvate’s flux into mitochondria and the TCA cycle . AnotherIn FGR muscle, a lower OCR with pyruvate but not with pyruvate plus fatty acid substrates indicates that fatty acid oxidation was increased when pyruvate oxidation was attenuated under the condition that both substrates are supplemented ex vivo. Although fetal fatty acid oxidation rates are low and are not deemed to be a major energy source for the fetus under normal circumstances , the expAn interesting finding is that the pathways associated with autophagy and mitophagy were upregulated in FGR muscle, suggesting increased proteolysis. Skeletal muscle proteolysis is largely driven by the autophagy-lysosomal pathway and the ubiquitin proteasome pathway . We obseA limitation of this study is that evidence from the transcriptomic level was explored while changes in protein abundance were beyond its scope. Future studies are warranted to validate whether these gene expression changes can be translated to functional and protein changes, although the parts of gene markers identified in the current study have been validated at the protein and functional level in recent studies from our lab and others ,21.All experiments on animals were approved by the Institutional Animal Care and Use Committee at The University of Arizona (Protocol #08-132), which is accredited by the American Association for Accreditation of Laboratory Animal Care International (AAALAC).−1) and phenytoin sodium . Fetal and placental gross morphometry was conducted postmortem following dissection. Placental and fetal weights were measured and recorded. Fetal semitendinosus muscle was removed, weighed, and immediately frozen in liquid nitrogen. The soleus was dissected from the hindlimb and transferred into cold phosphate buffered saline for fiber dissection and further analysis.Fetal muscle samples were obtained from a subset cohort of animals used in previously published studies ,35. Pregn = 12 ; FGR, n = 9 ). The muscle fibers were then permeabilized with saponin (50 μg/mL) in the BIOPS solution . The permeabilized muscle fibers were thoroughly rinsed in the respiration buffer containing 0.5 mM EGTA, 20 mM HEPES, 3 mM MgCl2·6H2O, 10 mM KH2PO4, 20 mM taurine, and 110 mM mannitol. Mitochondrial respiration for the permeabilized soleus muscle fibers was measured in a Fluorescence Lifetime Micro Oxygen Monitoring System  with the following substrates added: the pyruvate substrate , the fatty acid substrate , or the pyruvate + fatty acid substrate . Mitochondrial state 3 OCRs were determined from the slope of pO2 disappearance over time in a linear range and calculated using the solubility of oxygen at 37 °C [2 consumed per minute per gram of tissue. Cytochrome C and cyanide controls were used to verify that the procedure of permeabilization did not impact mitochondrial function and are commonly used procedures in metabolic studies.Oxygen consumption rates were measured in the permeabilized soleus muscle as per the protocol previously described with modifications . Brieflyat 37 °C . Values n = 4 ), FGR n = 4 ) was extracted from the semitendinosus muscle using the RNeasy Mini Kit  as per the manufacturer’s instructions. RNA concentration was determined by measuring absorbance at 260 and 280 nm with the NanoDrop ND-1000 Spectrophotometer . RNA integrity was evaluated with an Experion Automated Electrophoresis System , and samples that had RNA Quality Index values of 9.6 to 10 were used for downstream analyses. The RNA samples were then submitted to University of Arizona Genomics Core for RNA-sequencing (RNA-seq). Messenger RNA (mRNA) was selected and double-stranded cDNA libraries with ligated sequencing adapters were constructed with the Illumina TruSeq RNA Sample Prep Kit . Cluster generation was conducted with Illumina TruSeq 100bp PE (paired end) cluster kit before running it on the Illumina HiSeq2500 platform .RNA  ≤ 0.05 were considered as differentially expressed. Candidate differentially expressed genes were then subjected to gene ontology and pathway enrichment analyses  in KOBAS dataset version 3.0 [p value (FDR) ≤ 0.05 were considered enriched or overrepresented.Quality control of sequencing reads was performed using FastQC . The paired-end read numbers ranged from 33 to 48 million and were aligned to the sheep reference genome n 2.7.3a . The aven 2.7.3a . Differen 2.7.3a  in R vern 2.7.3a . Log folsion 3.0 , annotatn = 11) and FGR (n = 11) ST muscle samples. Twenty candidate DEGs that were among the top fold changes and were associated with FOXO signaling, AMPK signaling, and fatty acid oxidation pathways were chosen for qPCR experimental validation. The qPCR assays were conducted in accordance with the guideline [Validation of RNA-seq data was performed via quantitative PCR (qPCR) on a larger cohort of CON (uideline . The oli−ΔΔCT method [t test. Means are presented ± SEM. A p value < 0.05 was considered significant, and 0.5 ≤ p < 0.1 was considered a trend.Skeletal muscle from male and female fetuses were included in all groups but effects of sex were not evaluated due to low numbers and because fetal sex was not a factor in previous studies ,11. CorrT method . MitochoThe findings from the current study demonstrated that the skeletal muscle from FGR fetuses near term had lower ex vivo mitochondrial oxygen consumption rates with pyruvate substrates. The lower respiration rates were potentially caused by the transcriptional regulation of enzymes that inhibited pyruvate’s entry into the TCA cycle . However


The prognosis of upper urinary tract urothelial carcinoma (UTUC) is associated with tumour grade (G) and stage. Despite preoperative risk stratification and radical treatment, recurrence and progression are common. Thus, prognostic and monitoring biomarkers are needed. This feasibility study aimed to investigate if targeted analyses on circulating tumour DNA (ctDNA) in plasma could identify tumour-specific gene variants, and thus have potential for further evaluation as a biomarker in UTUC.Nine UTUC patients with genetically characterised tumours were included in this prospective pilot study. Two tumour-specific variants were chosen for targeted analyses with multiplex droplet digital PCR on cell-free DNA (cfDNA) from plasma at diagnosis or from recurrence.2 in size. Three of these patients progressed in their disease and the fourth had the largest G3 tumour at sampling. In contrast, the two patients with undetectable ctDNA in diagnostic plasma had a G1 tumour and G3 carcinoma in situ (CIS), respectively. The patient with G3 CIS had detectable ctDNA later during follow-up and progressed thereafter with aggressive intravesical recurrence and CT-scan-verified CIS progression in the upper urinary tract. In three patients with small recurrent G1 or G2 tumours, none had detectable ctDNA in plasma and all were progression free.Of six patients with diagnostic plasma samples, ctDNA was detected in four with G2 or G3 tumours and tumours > 300mOur early findings demonstrate that ctDNA in plasma can be detected by targeted analysis in patients with UTUC. However, further studies are needed to determine its role as a potential biomarker.The online version contains supplementary material available at 10.1007/s00345-023-04583-w. Although upper urinary tract urothelial carcinoma (UTUC) is rare, the incidence has been increasing during the last decades . The proWhen cell death occurs by apoptosis or necrosis, small fragments of cell-free DNA (cfDNA) are released into body fluids such as plasma . In cancThis prospective study included nine patients diagnosed with UTUC at ureterorenoscopy (URS). Six patients underwent organ-sparing surgery; one patient (PP-6) received radiotherapy as palliative treatment, one patient received BCG-instillation in a solitary kidney with CIS (PP-7) and one patient (PP-8) underwent nephroureterectomy. Patients were diagnosed between 2018 and 2020 and were followed-up routinely  to censor date (February 2023) or death. A next-generation sequencing (NGS) gene panel consisting of 387 genes was performed on barbotage specimens obtained at URS at diagnosis or local recurrence for genetic characterisation of all tumours prior to inclusion . The NGSExtraction of cfDNA from plasma and droplet digital PCR (ddPCR) was performed as previously published and described in Supplementary methods . TargetsAll patients had verified UTUC at the time of first plasma sample collection, 6/9 at initial diagnosis of UTUC and 3/6 at recurrence. The follow-up time after UTUC diagnosis was 4–55 months and no patient was lost to follow-up. Clinical characteristics including age at diagnosis, sex, tumour grade (G) and follow-up data of each patient are summarised in Table Two SNVs or indels were selected as targets for each patient based on NGS gene panel data on barbotage tumour cells. Multiplex ddPCR assays were performed on same tumour cell DNA and also cfDNA from plasma samples . VAF, false positive rate, concentration of mutant and wild type allele and total cfDNA are listed in Table n = 2) or G2 (n = 1) tumours and were progression free at follow-up. None of these three patients had detectable ctDNA in plasma.Tumour-specific variants in cfDNA from plasma were analysed by multiplex ddPCR in 6/9 patients at initial UTUC diagnosis and at recurrent disease for the remaining 3/9 patients. In addition, more than one plasma sample was analysed for three patients. The results from ddPCR in relation to clinical status and outcome are presented in Fig. 2. This specific plasma sample was taken at diagnosis in a patient with G1 tumour. In contrast, in patients where ctDNA was detected, the smallest tumour size at sampling was 300 mm2. The only exception was the third sampling time point that was positive for ctDNA in the patient with G3 CIS where the tumour was macroscopically undetectable. Amongst the patients with detectable ctDNA at diagnosis, the largest tumour size at sampling was 2491 mm2. This specific patient did not suffer disease progression, but had a large inoperable G3 tumour at the time of sampling and later received palliative treatment with radiotherapy.Tumour size was estimated by URS at the time point of each plasma sampling Table . We obseAlthough a number of studies have been performed assessing the utility of ctDNA as a prognostic and monitoring biomarker in urothelial carcinoma with promising results –17, onlyIn our pilot cohort, where all had detectable tumour-specific gene variants in tumour cells received from focal barbotages, 5/9 patients had detectable ctDNA in plasma during their clinical course. Four of them, including one patient with CIS, later progressed in their disease with either distant metastasis, progression to higher tumour grade or death from UTUC 4–12 months after the sampling time point, which might suggest that ctDNA could have potential as a biomarker for disease progression. These results are consistent with previous findings where ctDNA was found in patients with metastatic UTUC and was shown to predict UTUC patients with poor prognosis , 8. The TERT promoter mutation C228T, which highlights the need to perform genetic testing on tumour material to select patient-specific genetic variants for monitoring tests. NGS analysis of tumour cells will likely become part of future clinical routine as the cost of sequencing comes down which will simplify the selection of tumour-specific gene targets. Tumour genetic heterogeneity and subclonal genetic evolution during progression affect the genetic landscape and level of ctDNA [In this study, targeted multiplex ddPCR was used to detect known patient-specific gene variants in plasma. This method offers a fast, high-sensitivity, minimally invasive and easy-to-interpret approach to detect a few patient-specific variants at once and could, thus, serve as a promising method for surveillance of UTUC if deemed clinically relevant. The only recurrent mutation analysed in the cohort was the of ctDNA  and are Detection of tumour-specific gene variants in ctDNA from plasma by targeted multiplex ddPCR is a feasible method in patients with UTUC. Our early observations suggest that detection of ctDNA by this minimally invasive approach could have potential as a biomarker in UTUC. However, no clinical conclusions can be drawn and further studies are warranted to determine its role as a biomarker.Supplementary file1 (PDF 5674 KB)Supplementary file2 (PPTX 81 KB)Supplementary file3 (DOCX 15 KB)Supplementary file4 (DOCX 14 KB)Below is the link to the electronic supplementary material.


Head kinematics are altered in individuals with vestibular schwannoma (VS) during short duration gait tasks , both before and after surgery, yet whether these differences extend to longer duration gait exercises is currently unknown. Here we examined the effects of vestibular loss and subsequent compensation on head kinematics in individuals with VS during gait exercises of relatively extended versus short duration (< 10 versus 30 s), compared to age-matched controls. Six-dimensional head movements were recorded during extended and short duration gait exercises before and then 6 weeks after sectioning of the involved vestibular nerve (vestibular neurectomy). Standard functional, physiological, and subjective clinical assessments were also performed at each time point. Kinematics were differentially altered in individuals with vestibular loss at both time points during extended versus short duration exercises. Range of motion was significantly reduced in extended tasks. In contrast, movement variability predominately differed for the short duration exercises. Overall, our results indicate that quantifying head kinematics during longer duration gait tasks can provide novel information about how VS individuals compensate for vestibular loss, and suggest that measurements of range of motion versus variability can provide information regarding the different strategies deployed to maintain functional locomotion. In healthy individuals, coordinated vestibular-motor pathways make essential contributions to the multisensory mechanisms required to maintain stable balance during our everyday activities (reviewed in Ref.2). For instance, to transition from a stable standing posture to locomotion, the brain switches between reflex and voluntary vestibular motor pathways3, in a coordinated, goal directed, manner consistent with optimal feedback theory5. The reduction or absence of peripheral vestibular input to these pathways increases the brain’s reliance on information from other sensory modalities , which in turn contribute to long-term vestibular compensation (reviewed in Refs.8). However, such compensation is incomplete, resulting in individuals with vestibular loss requiring significantly more time to perform daily life activities compared to controls9.The vestibular system detects the head’s six-dimensional motion and orientation relative to space to provide essential sensory information to the brainstem, cerebellum, and vestibular cortex during our everyday activities12 as well as free overground walking19). Overall, these studies have led to the proposal that head stabilization, particularly in the pitch axis, is a vital feature of inertial guidance and postural control during locomotion14. To understand how loss of vestibular sensory input alters locomotion, several research studies have measured and quantified head kinematics in individuals with peripheral vestibular loss. Measurements have been made using magnetic search coils12, 3D video motion capture21, and most recently inertial measurement units (IMUs)25. Compared to controls, individuals with peripheral vestibular loss display marked changes in head movement variability, principally along the vertical axis, as well as time for task completion. Overall, these studies have contributed to the proposal that head stabilization is important for navigation and postural control. The use of IMUs to quantify kinematics in more than 2 dimensions is relatively novel and unexplored. Indeed to date, only two studies have measured 6-dimensional head kinematics in individuals with peripheral loss24. Both of these prior studies focused on performance during a traditional clinical gait measure, the Functional Gait Assessment (FGA), which comprises only short duration gait tasks . Thus, it remains unknown how 6-dimensional head kinematics are altered in such individuals during longer duration bouts of locomotion that are common in daily living.Locomotion, the ability to walk from place to place, is an essential aspect of our daily living. To date, head kinematics have been studied in healthy individuals during locomotion, in conditions including treadmill walking26, as well as during the 10 standard tasks of the FGA (< 10 s in duration). Notably, 4 of these former tasks were extended versions of FGA tasks. We first tested whether head kinematics distinguished VS from healthy control participants in either the pre- and postoperative state during extended gait tasks. We then assessed whether there were significant differences in kinematics of these individuals during extended versus short duration tasks, relative to healthy controls. Finally, we investigated whether and how the quantification of head kinematic data during both extended and short duration gait tasks can be utilized to predict standard clinical, functional, or physiological outcomes in the VS pre and/or postoperative state relative to healthy controls.Accordingly, via this exploratory study we measured and compared head kinematics during extended versus short duration gait tasks in individuals with vestibular loss pre and post vestibular deafferentation. Six-dimensional head kinematics were recorded in vestibular schwannoma participants, at pre-operative and 6 weeks post-operative time points, relative to healthy controls. Measurements were made using light weight IMU sensors during 7 30-s vestibular gait exercises detailed by the Clinical Practice Guidelines for vestibular rehabilitation in individuals with peripheral vestibular hypofunctionSubjects: 18 participants with vestibular schwannoma (VS) tumor were recruited and scheduled for surgical resection between February 2019 and January 2020. 9 VS participants  were assessed before (mean = 8 ± 13 days) and 6 weeks after (35–45 days) surgery, . We chose this 6-week post-operative time point to correspond when participants returned to Johns Hopkins for their scheduled post-operative follow up otology appointment. 9 aged matched healthy controls were also recruited . Details of the VS participant demographic information as well as tumor size, laterality, and Koos score are provided in Supplementary Table 27. Participants performed the DVA test while seated, 2 m from the portable tablet. 10 optotypes (C D H K N O S R V Z) were randomly presented during 3 different conditions: (1) head stationary  (2) sinusoidal head rotations with the optotype presented during active right head rotation and (3) active left head rotation. The scores were calculated in the logarithm of the minimal angle resolution (LogMAR). Possible LogMAR scores ranged from − 0.3 to 1.7 . Corrected DVA accounted for static visual acuity scores by subtracting SVA from DVA.DVA measures visual acuity during active head rotation as a proxy of the vestibular ocular reflex using a portable test developed and validated at our institution. DVA is a behavioral measure of the vestibular ocular reflex with active head rotation with visual fixation of an optotype. The DVA test comprises of a Samsung Galaxy Pro tablet  and a head mounted motion sensor . We used the DVA protocol as previously described by Millar et al.28. Higher TUG scores, > 11.1 s, correlate with reports of falls in individuals with vestibular dysfunction29.Participants were asked to stand from a seated position, walk 3 m, turn 180° and then return to a seated position. The TUG task was performed for 2 trials, with an ispilesional turn and contralesional turn, respectively. For healthy controls, the two trials equivalent to ipsilesional and contralesional turns were carried with a right turn and left turn, respectively. The TUG has excellent inter and intra-rater reliability in community dwelling elderly (ICC = 0.99)The Ten Meter Walk Test (10MWT) was performed, measuring participants’ self-selected walking pace. The walking duration was timed over a 10-m distance. Participants started and stopped their walking distance both 2 m before and after the 10-m timed portion of the walk to ensure the acceleration and deceleration phases of locomotion were not included in the timed portion of the test. The average gait velocity (meters/second) was computed based on two consecutive trials.31: (1) Gait on a level surface, (2) Change in gait speed, (3) Gait with horizontal head turns, (4) Gait with vertical head turns, (5) Gait with a single 180° pivot turn, (6) Gait while stepping over an obstacle, (7) Gait with narrow base of support , (8) Gait with eyes closed, (9) Backwards gait, and (10) Steps. Figure The FGA is a measure of dynamic postural stability and includes 10 walking tasks33. We did identify corrective saccade metrics . Yet, given we found no significant correlations in our analyses, we opted not to include the corrective saccade metric data in our methods and results.Vestibular ocular reflex (VOR) gain (eye velocity/head velocity) was measured using the ICS Otometrics system . Participants were seated 1 m from a stationary visual target, in room light. Right eye velocity and head velocity were sampled at 220 Hz during 12 passive head rotations among both directions of each of the three semi-circular canal planes: yaw, left anterior/right posterior (LARP), right anterior/left posterior (RALP). vHIT traces were deleted if the eye velocity trace preceded head velocity, or if the passive head rotation trace did not match the acceleration profile suggested by the manufacturer. VOR mean gain values were calculated based on the area under the curve of eye/head velocity. VOR gains within 0.8–1.2 with standard deviation < 0.12 were considered normal35, Activities Balance Confidence Scale (ABC)37, as well the Headache Impact Test (HIT)38, and Beck Anxiety Inventory (BAI)39.Participants rated self-perceived severity of symptoms of dizziness, imbalance, headache, and anxiety using the Dizziness Handicap Inventory (DHI)In addition, to the short-duration FGA exercises detailed above, we also asked participants to perform 7 traditional vestibular rehabilitation gait exercises, for a single trial, to assess how these tasks can distinguish between VS patients and healthy controls Fig. A. These ).Participants completed the seven 30-s vestibular rehabilitation gait exercises as well as the 10 short-duration FGA tasks while wearing 6 (51 mm × 34 mm × 14 mm) micro-electromechanical (MEMS) sensors   in 6 dimensions from the 4 shared tasks that were performed both as part of the extended duration gait exercises (30 s) and the standard FGA tasks (< 10 s). The global kinematic score ranged between 0 (most severely impaired) to 100  and was computed as follows: (i) each selected kinematic measure  during each task was normalized using a linear transformation of mean ± 2SD to a number between 0 and 100 ; (ii) in order to keep the range of scores between 0 and 100, the outliers  were projected to the closest number within this range ; and (iii) the normalized numbers across all three kinematic measures, 6 dimensions, and 4 tasks were then averaged. We compared head kinematic measures at the preoperative and 6-week postoperative time points and then contrasted these with head kinematic measures obtained from age-matched healthy control participants.For the comparison between the kinematic measures of the vestibular participants (pre- and postoperative) and age-matched healthy controls we used a non-parametric paired sample permutation (re-randomization) test. Specifically, we generated 2000 randomized rearrangements of the observed data points and then computed p-values of the actual observed measures. Pearson correlations were used to compute the correlation coefficients and p-values of the correlations between kinematic and clinical measures. To find the consistent trends across several exercises we assessed whether the correlation for the majority of exercises (i) were significant (p < 0.05) and (ii) had the same sign . Correction for multiple comparisons were not performed since the goal of this exploratory study was to investigate unilateral vestibular participants already known to be different from healthy controls based on clinical assessment; performing correction would have exaggerated Type II errors. Throughout the text, values are expressed as mean ± 1 SD and significance is reported at p < 0.05. All data processing and statistical tests were performed using MATLAB .Figure , which summarizes the kinematic measures computed for each of the 7 gait exercises (top panels) and 10 standard FGA tasks (bottom panels) for all participants. These measures include the variability [coefficient of variation (CV) and standard deviation (SD)] and range of head motion for all 6 axes, average and variability of the step cycle duration, and time and length asymmetry (see “Methods”). The four 30-s vestibular gait exercises that were extended-duration versions of standard FGA tasks are highlighted in color. We found that the range of motion and variability of head motion were both altered for the extended duration gait exercises and standard FGA tasks in both pre- and post-operative VS participants as compared to healthy controls . To address this question, we evaluated the correlations between clinical measures and head kinematic measures computed for the 7 extended gait exercises as well as 10 standard FGA tasks (see “Methods”). Supplementary Tables Finally, we completed a complementary analysis of the relationships between head kinematic measures during the 7 gait exercises and 10 FGA tasks, and clinical measures after surgery Fig. . The ploTo our knowledge, this is the first study to have compared head kinematics using IMUs in six dimensions during gait tasks performed over an extended, 30-s period (gait rehabilitation exercises), versus gait tasks performed for a short, 10-s duration (via the standard FGA). Additionally, this study is the first to have utilized kinematic measures recorded during either extended duration exercises or short duration gait tasks to predict clinical measures in individuals with vestibular loss (pre and post vestibular deafferentation).41 and was not paired with significant lengthening of gait cycle duration, which otherwise would have been considered a more conservative, protective gait strategy . We speculate that this behavior likely puts VS participants at a greater risk of falls during extended gait tasks, where balance control could be compromised by greater gait cycle instability41 uncoupled from compensatory temporal modifications in cycle duration, leading to slower gait speed.We found that: (i) head motion variability and range of motion is altered in VS participants relative to controls during several extended duration gait exercises and short duration FGA tasks, both pre and postoperatively. (ii) Only minor differences were observed when the same comparisons were made between pre and postoperative groups. (iii) Head kinematics and step cycle duration were predictive of clinical measures  for both gait task groups. (iv) A global kinematic score computed from variability measures, among the 4 shared short duration FGA gait tasks was more informative than that computed for the corresponding 4 extended duration gait exercises. (v) In contrast, a global kinematic score computed from range of motion measures was more informative in distinguishing VS (both pre and postoperative) from healthy controls when computed across the 4 shared extended gait exercises as compared to the corresponding shorter FGA tasks. This change in range of motion is suggestive of increased dynamic instability44. The dynamic upweighting of these extra-vestibular sensory  signals, as well as inputs from motor pathways is evident even at the first central stage of sensory motor processing48. The strategy of implementing such available adaptive central mechanisms likely evolves over the course of tumor growth in response to the progressive impairment. Thus, an individual’s adaptative strategy may not be optimal when considered over longer durations and/or for compensating for additional perturbations such as vestibular deafferentation. Indeed, recent reports describing head movement kinematic in pre versus postoperative VS patients during gaze and balance exercises are consistent with this view49.In the context of VS, the slow growth of the tumor allows for the putative development of motor strategies that would enable patients to remain functional through most activities of daily living. Individuals with vestibular loss rely on adaptive or compensatory mechanisms including visual and somatosensory substitution as well as changes in feedforward motor control50, followed by a second adaptive phase characterized by a task-dependent kinematic strategy wherein range of motion increase with the extent of challenge imposed by the task . Our present results, focused on extended duration exercises, suggest that while gait cycle variability was present in this latter phase, the outputted gait cycle amplitude was also significantly enhanced in what could be considered a second, additional compensation strategy. Our results thus suggest that maintaining stability during extended gait recruits this latter feature of an evolving kinematic strategy, which like the initial phase of gait, is more challenging for VS participants. Interestingly, our present results align with previous reports of dynamic regulation of sensorimotor integration in human postural control, in which the repetition of a motor task (here the extended gait exercises) is taken as means towards reducing movement-to- movement motor variability, yet can potentially decrease final performance when there is inadequate regulation of corrective responses51. This abnormal motor response was likely aggravated in our study population by the biased influence of the tumor over what had been, prior to the presence of the VS tumor, a normal state of the neural circuitry that together with the locomotor apparatus modulate the feed-forward information process warranted in effective, healthy locomotor adaptation52. One could speculate that the kinematic changes observed overtime were unrelated to changes in motor control adaptative strategies, but rather were the result of biomechanical entrainment to the task.We speculate that relative increase in variability of VS participants for short versus extended duration gait tasks reflects that their postural maintenance follows a kinematic sequence that is initially characterized by greater movement to movement variability 54. However, few studies have focused on head kinematics during gait in VS participants. Paul et al. studied 1D head kinematics (yaw axis) and reported reduced purposeful movement in postoperative VS participants during gait including the FGA22 and community ambulation19, compared to controls. To date, only a single study24 has quantified 6D head kinematics in VS participants during the standard FGA. Importantly, several of the main findings of our present study are consistent with those of this prior study, which focused on a separate population of VS participants. Notably, here as well as this prior report, VS participant head kinematics differed from those of controls, both before and after surgery. As reviewed above, here we have further established that while measures of variability are more informative among the short duration FGA tasks, measures of range of motion are more informative for extended duration exercises.In the present study we chose to focus on head kinematics because head movements not only activate the vestibular sensors, but they are also modulated by vestibular-motor pathways. Furthermore, it has been proposed that humans strongly rely on head-based orientation for inertial guidance56 and balance49). Moreover, we found that kinematic measures from both short and extended duration conditions better predicted physiological  measures preoperatively than postoperatively. Thus, although the duration of the presence of the vestibular schwannoma tumor and the VS participants’ level of central adaptation, prior to participation, was unknown, our exploratory findings suggest that the quantification of 6D kinematics across multiple time points can be utilized to predict clinical outcomes in VS pre and/or postoperative state, as well as for informed exercise prescription in the future.Finally, here we have further shown that head kinematics and step cycle duration—both during short duration FGA and extended duration gait rehabilitation exercises—are predictive of several clinical measures, but not of subjective quality of life assessments. In this context, our findings add to a growing body of work demonstrating comparable conclusions based on head movements during other classes of vestibular exercises (i.e., gazeTaken together, our findings reveal vestibular schwannoma patients alter their head variability and range of motion during short and extended duration gait tasks pre and postoperatively in contrast to healthy controls. Variability and range of motion kinematic measures provide different information regarding the strategy individuals deploy to maintain functional locomotion. We also found that kinematic measures are predictive of functional gait clinical measures in individuals with vestibular deafferentation. The global head kinematic score may prove useful among future studies in individuals with vestibular loss across multiple time points and levels of rehabilitative progression.Supplementary Information.


Despite the effectiveness of the various targeted therapies currently approved for solid tumors, acquired resistance remains a persistent problem that limits the ultimate effectiveness of these treatments. Polyclonal resistance to targeted therapy has been described in multiple solid tumors through high-throughput analysis of multiple tumor tissue samples from a single patient. However, biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient.We analyzed circulating tumor DNA (ctDNA) with a large next-generation sequencing panel to characterize the landscape of secondary resistance mechanisms in two independent prospective cohorts of patients  with solid tumors who were treated with various types of targeted therapies: tyrosine kinase inhibitors, monoclonal antibodies and hormonal therapies.Emerging alterations involved in secondary resistance were observed in the plasma of up 34% of patients regardless of the type of targeted therapy. Alterations were polyclonal in up to 14% of patients. Emerging ctDNA alterations were associated with significantly shorter overall survival for patients with some tumor types.This comprehensive landscape of genomic aberrations indicates that genetic alterations involved in secondary resistance to targeted therapy occur frequently and suggests that the detection of such alterations before disease progression may guide personalized treatment and improve patient outcome.The online version contains supplementary material available at 10.1186/s12943-023-01878-9. The development of targeted therapies has revolutionized the systemic approach to the treatment of cancer. Indubitably, hormone receptor targeting is one of the earliest examples of targeted therapy and has significantly affected the survival of patients with breast and prostate cancer, which are ‘lineage-addicted’ to estrogen receptor and androgen receptor, respectively. Therapies targeting mutated or overexpressed kinases were developed more recently and showed improvement in patient outcome in a variety of tumor types, including non-small cell lung cancer (NSCLC), melanoma, colorectal cancer, and gastrointestinal stromal tumors.Responses to these generally well-tolerated therapies often last several months and sometimes several years. However, almost all patients treated with targeted therapies eventually demonstrate progression of their disease, a clinical setting described as acquired resistance.Secondary alterations of the oncogene drug target and mutation of alternate components of oncogene-induced signaling pathways have been described as crucial mechanisms of acquired resistance to targeted therapies. However, their true incidence in the real-life setting and their impact on patient outcome are still unknown. Indeed, although genetic analysis of tumor tissue is the standard technique for identifying DNA alterations in malignancies, this approach has several shortcomings, including invasiveness and an inability to capture intratumor spatial and temporal heterogeneity. In contrast, next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) is a method that is increasingly being used for genomic profiling of cancer and has several advantages in comparison with NGS of tissue biopsies: it is a noninvasive method, is easily achievable and repeatable, and may provide a more holistic view of actionable mutation targets in a patient’s disease, particularly in the case of treatment resistance Patients were included in two independent ongoing precision medicine studies . Patients were eligible if they (i) presented a locally advanced, unresectable, or metastatic solid tumor and (ii) were being treated with targeted therapy as per standard of care at the time of plasma sampling for ctDNA profiling. All patients provided written consent for the genomic analyses. This study was approved by the Institutional Review Board.Genomic analysis was performed for each patient by using the Foundation One Liquid CDx Assay  Descriptive statistics were used to describe population characteristics. Qualitative data are expressed as numbers and percentages. Quantitative data are expressed as the mean and standard deviation or median, 1st and 3rd quartiles and range when applicable. Data were analyzed using the software R version 4.0.3.Here, we performed ctDNA molecular profiling to assess the incidence of genomic aberrations involved in acquired resistance and their impact on the outcome of patients treated with targeted therapies in the advanced setting. To identify candidate mechanisms of acquired resistance, in each case, cell-free DNA (cfDNA) sequencing data were compared to sequencing data from pre-treatment tumor tissue to identify emergent alterations Therefore, we evaluated a prospective cohort of 626 patients with advanced cancer who were treated with a targeted therapy and enrolled in an institutional molecular profiling program  to obtain a molecular profile based on ctDNA profiling Fig. A. Their KRAS/BRAF wild-type (WT) colorectal cancer patients treated with anti-EGFR monoclonal antibodies, prostate cancer patients treated with antiandrogen therapy, hormone receptor-positive breast cancer patients treated with an aromatase inhibitor-based regimen and EGFR-mutated NSCLC patients treated with an anti-EGFR tyrosine kinase inhibitor. To confirm the findings observed in the STING study, we analyzed the same subgroups of patients enrolled in an independent precision medicine study . Their characteristics are described in Table We then analyzed the patterns and prognostic impact of ctDNA alterations in 4 specific subgroups of patients enrolled in the STING study: EGFR mutations , MET amplification/mutation (n = 4), PI3KCA mutation (n = 3), HER3 mutation (n = 1), and BRAF fusion (n = 1). Overall, 9 patients (20%) exhibited polyclonal resistance. The median OS time was significantly lower in patients with at least one identified emerging ctDNA alteration: 6.7 (95% CI 4.2–9.1) vs. 10.8 months (95% CI 7.8–13.8), p = 0.04   out of 45 with colorectal cancer  included in the STING cohort . Among them, 28 patients (14.9%) harbored ≥ 2 variants (up to five), and 25 harbored an AR amplification (13.3%). Hotspots are essentially located in the ligand binding domain and are involved in resistance to androgen receptor signaling inhibitors. The most frequent mutations included W742C/F  (n = 4), H875Y (n = 9), F877L (n = 3) and T878A (n = 25) , and L702H  (n = 25). Less common AR resistance mutations were V716M (n = 3), G689A (n = 1) and D891H (n = 2); such mutations have been previously described in ctDNA from patients with metastatic castration-resistant prostate cancer (mCRPC) who progressed on bicalutamide or abiraterone. Additional AR gene rearrangements that consisted of deletions or inversions impacting the genomic segment containing AR exons 5–7 and have already been reported in patient-derived xenograft models were identified in 5 patients. Overall, polyclonal resistance was observed in 40 patients (21%). The median OS time was significantly lower in patients with at least one identified emerging ctDNA alteration than in those with no identified alterations: 7.9 months vs. not reached (95% CI 5.5–10.3), p < 0.001  harbored ≥ 2 variants (up to five) and 2 harbored an AR amplification (1.9%). Polyclonal mechanisms of resistance were identified in 10 patients (9%). Median OS was not reached in patients with no emerging mutation or patients with at least one emerging ctDNA alteration, but the survival time was significantly lower in patients with at least one emerging ctDNA alteration (p < 0.001)  out of 188 included in the STING study. ESR1 mutations, which represent a key driver of endocrine therapy resistance, were identified in 31 patients (32%) out of 97 with advanced breast cancer who were treated with aromatase inhibitors in the STING study , BRAF (n = 1) and NTRK1 (n = 1). Overall, the mechanisms of resistance were polyclonal in 8 patients (14%). In the BIP study, emerging alterations involved in resistance were observed in 4 patients (25%) out of 16 and included EGFR mutations, PI3KCA mutation and AKT amplifications  out of 57 with EGFR-addicted NSCLC . In our study, which included patients with KRAS WT colorectal cancer receiving anti-EGFR monoclonal antibodies, the detection of emerging resistant ctDNA aberrations was associated with significantly worse OS in two independent cohorts. This suggests that switching from an anti-EGFR-based regimen to another line of treatment upon early identification of the resistance mutation in plasma—before disease progression—may improve patient outcome and deserves further investigation in a randomized setting.We report here a comprehensive study investigating the incidence of genetic aberrations involved in resistance to targeted therapies detected through ctDNA sequencing of patients with advanced cancer. By analyzing two independent cohorts of patients, we were able to demonstrate that such aberrations are present prior to disease progression and are polyclonal in up to 14% of patients. This polyclonality illustrates the utility of ctDNA sequencing compared to tumor tissue sequencing in characterizing the tumor molecular profile. The clinical impact of the detection of genetic aberrations involved in resistance through ctDNA profiling remains to be demonstrated through randomized clinical trials. Our study suggests that further exploration into using ctDNA sequencing results to potentially guide therapeutic decisions might be of value. In colorectal cancer, the results of the CRICKET study presented the first evidence that RAS WT status, as assessed by liquid biopsy, could represent an appropriate tool to select patients who might benefit from anti-EGFR rechallenge therapies AR alterations also advocates for the implementation of serial ctDNA sampling to identify mutational mechanisms of drug resistance to antiandrogen therapy. Indeed, this can result in timelier and more appropriate therapeutic decisions that can improve patient outcomes. For instance, responses to withdrawal of enzalutamide, bicalutamide, nilutamide, and flutamide are well described Androgen deprivation therapy has been a cornerstone of treatment for patients with prostate cancer since early 1940. Several new potent androgen axis inhibitors such as enzalutamide, apalutamide and darolutamide abiraterone acetate are now being approved for castration-sensitive metastatic disease ESR1 mutations that have been associated with resistance to aromatase inhibitors ESR1 mutations in the ctDNA of patients treated with a combination of aromatase inhibitor and CDK4 inhibitor, as illustrated by the recently reported results of the PADA-1 study ESR1 mutations in patients being treated with an aromatase inhibitor plus palbociclib. Patients found to have a mutation prior to disease progression were randomly assigned to continue treatment or switch to fulvestrant plus palbociclib. Interestingly, switching therapy resulted in a doubling of median PFS time, from 5.7 months with standard treatment to 11.9 months . Data regarding OS are not yet available and are eagerly awaited.A significant proportion of patients with advanced breast cancer and treated with aromatase inhibitors displayed EGFR T790M mutation EGFR T790M mutation occurring before progression as per RECIST. However, the median OS was not significantly different between the two arms , suggesting that further studies are needed to clarify the role of ctDNA monitoring in this setting.We found that up to 33% of patients with EGFR-mutated NSCLC harbored newly acquired mutations involved in resistance to EGFR TKIs at the time of ctDNA sequencing, and the emergence of such mutations was not associated with OS. This lack of prognostic impact may be related to the availability of highly efficient treatment in patients with acquired mutations such as osimertinib for patients harboring the One main limitation of our study is that ctDNA profiling cannot capture all the complexity of the processes involved in resistance to targeted therapies. For instance, this approach does not allow the detection of epigenetic alterations or functional activation of redundant kinases involved in bypass pathways. However, thanks to a robust methodology , our findings demonstrate the complex and heterogeneous molecular mechanisms of resistance in cancer patients and illustrate the great potential of ctDNA profiling for guiding treatment decision-making and improving prognosis in clinical practice.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3


Cassiopea andromeda jellyfish:a unique benthic jellyfish known to favor (sub-)tropical coastal regionswhich are potentially exposed to plastic waste from land-based sources.Juvenile medusae were exposed to fluorescent polyand polypropylene microplastics (<300 μm), resin embedded,and sectioned before analysis with confocal laser scanning microscopyas well as transmission electron microscopy and Raman spectroscopy.Results show that the fluorescent microplastics were stable enoughto be detected with the optimized analytical protocol presented andthat their observed interaction with medusae occurs in a manner whichis likely driven by the microplastic properties .Many researchershave turned their attention to understanding microplasticinteraction with marine fauna. Efforts are being made to monitor exposurepathways and concentrations and to assess the impact such interactionsmay have. To answer these questions, it is important to select appropriateexperimental parameters and analytical protocols. This study focuseson medusae of  Whileplastic particles have been an increasingly popularresearch topic within the past decade, much remains uncertain abouttheir fate within marine environments and food chains. This studypresents optimized analytical protocols that allow assessment of interactionsbetween microplastics and jellyfish to provide insight into how theproperties of the plastics may influence their fate. Within their study, Sucharitakulet al. found that there was limited ingestion and no hazardous effectsafter exposure to polystyrene (PS) MPs, while Costa et al. reportedingestion and negative physiological impacts after exposure to polyethylene(PE) MPs.24 Still fewer studies have focused on theinteraction of MPs with Cassiopea sp. jellyfish specifically.Iliff et al. demonstrated that some MPs were present in their wild-caught Cassiopea sp. samples, but as they were pre-processed withchemical digestion of the jellyfish tissue to make detection easier,all potential spatial information for the MPs within the tissue waslost.11Despite such promising qualities, few studies existto date whichfocus on the interaction of MPs with jellyfish, and the reported resultsoften differ.25 Thus, it is clear that there is a need inthe field for optimization of analytical techniques that will allowfor the detection of MPs within marine organisms. To that end, juvenilemedusae of the jellyfish Cassiopea sp., specifically, Cassiopea andromeda (C. andromeda), were exposed to thermostable, fluorescently labeled poly (PET) and polypropylene (PP) MPs (<300 μm).Samples of C. andromeda medusae withMPs were then resin embedded, sectioned, and analyzed with confocallaser scanning microscopy (cLSM), transmission electron microscopy(TEM), and Raman spectroscopy to detect and study any interactionsoccurring (Scheme S1).This highlights the compromiseswhich must be made due to the multipleanalytical challenges presented by MPs. Such challenges are the resultof limitations for common techniques that are influenced by factorssuch as the MP’s small size, low estimated environmental concentration,and organic composition, as well as the high variability in bioticand abiotic factors within the surrounding natural environments ofinterest.27 Additionally, it isoften not possible to image the fluorescence of a sample after thenecessary pre-processing steps for TEM due to the degradation of themolecules which give fluorescence  or tocollect Raman spectra from highly fluorescent bulk samples withoutsacrificing signal intensity through the use of lower energy nearinfrared lasers.31 Thus, our work focused on overcoming these analytical concerns forthe first time for samples of whole marine organisms exposed to MPs.A protocol was developed which allowed for an initial insight intohow interactions with C. andromeda medusamay differ depending on the MP type, providing a platform for futureresearch which can further probe such differences in these and othermarine organisms.While correlativemicroscopy with TEM providing a high-resolutionvalidation of initially observed fluorescence signals has been highlyutilized in other fields, very little work has been conducted to optimizethis work flow for MP detection in tandem with Raman spectroscopyin whole marine organisms.33 Briefly, stock pellets of PETwere purchased from Goodfellow Cambridge Ltd., and pellets of isotacticPP were purchased from Sigma-Aldrich. These pellets underwent a sequentialmelt processing and milling protocol to create the final MP stocksused for the study. First, thin films of the as-received pellets wereprepared by compression molding 2.5 g of each polymer between Kaptonsheets in a hot press (Carver) at 170 °C for PP or 255 °Cfor PET at 2 tons for 1 min and then 5 tons for an additional 1 min.The films were removed from the press and cooled to room temperature.To create fluorescently labeled plastics, the dye 1,4-bis(α-cyano-4-methoxystyryl)-2,5-dimethoxybenzene(C1RG) was added onto the films in concentrations of 0.01% (by weight)for PP (PPC1RG) and 0.1% for PET (PETC1RG). The films with dye werefolded to seal in the dye and compression-molded again using the parameterspreviously described. The films of labeled or unlabeled plastic werefed into a Haake Mini Lab II twin screw extruder (Thermo Fisher) fortwo 5 min mixing cycles under recycling conditions at 170 °C(PP) or 255 °C (PET) to ensure even mixing and obtain extrudedfilaments of plastic which could be cut into pellets. These pelletswere placed in a polycarbonate chamber with a steel milling rod andsteel chamber plugs to be milled under cryogenic conditions in a 6770Freezer Mill (SPEX) with a 15 min pre-cooling followed by 2 cyclesof 3 min milling at 12 cycles per second (cps) with a 2 min coolingperiod in between. Finally, the cryo-milled MPs were sieved with a0.3 mm stainless-steel mesh  to select for thesize range of interest.Theexact protocol for creation and characterizationof the MPs utilized in this study has been previously described byCaldwell et al.2; Tescan).To assess theirsize and shape, MPs were placed onto aluminum SEM holders (Agar Scientific)and 10 μL of ethanol  was dropped onto the sampleto disperse them across the surface of the holder. Samples were driedovernight prior to sputter coating a 2.5 nm layer of gold using a208 HR sputter coater (Cressington Scientific Instruments). All imagingwas carried out with a Mira3 LM FE scanning electron microscope . To maintain clean tank conditions, UV, mesh (200 μm),and skimmer filtration were utilized, and the tank was cleaned twicea month with a 30 L water renewal.C. andromeda tank wascollected and passed through a 0.22 μm syringe filter (Macherey-Nagel)to remove large organic matter. Subsequently, 1 mL per well of filteredtank water was pipetted into 16 wells in two different 12-well plates.A transfer pipette (Merck) was cut and used to transfer a single C. andromeda medusa at a time into the wells of 12-wellplates. MPs, which had been pre-massed using an AG204 Delta Rangebalance (Mettler-Toledo) into labeled Eppendorf tubes (Sigma-Aldrich)to ensure a final exposure concentration of 120 μg/mL, weresuspended in 200 μL of filtered tank water, mixed thoroughly,and then pipetted into the appropriately labeled well with the medusa(Table S2). Each tube was rinsed with additional300 μL of filtered tank water to ensure that all MPs were removed.The rinse water was added to the respective well so that the finalvolume of water introduced with plastic MPs was 500 μL. Forthe blank control samples, 500 μL of filtered tank water withoutplastic particles was used. An additional 1 mL of filtered tank waterwas introduced per well to help facilitate mixing. A small aliquotof water was taken from an Artemia hatcher and filtered,and then 20 μL of filtered Artemia hatcherwater was dropped into each well to facilitate feeding behavior. The12-well plates were placed in an incubator to ensure a constant temperatureof 25 °C and light exposure of 150 μmol photons m–2 s–1 per organism during a 6 h exposure period.An aliquot of waterfrom the main At the end of the 6 h exposure,transfer pipettes were used to move each juvenile medusa to a cleanEppendorf tube and 1.5 mL of a fixative solution (pH 8) containing9% (by volume) sucrose (Sigma-Aldrich), 4% paraformaldehyde (ElectronMicroscopy Sciences), and 2.5% glutaraldehyde (Electron MicroscopySciences) was added. All water was removed, and samples were maintainedin fixative at room temperature 2 h before storing at 4 °C untilfurther work was conducted.Sample dehydration with a gradedseries of ethanol solutions  was conducted prior to an infiltration step witha graded series of EPON resin:100% ethanol mixtures , two times in pure resin 1 h each, and then a finalresin bath with catalyzer for 12 h. Samples were then left to polymerizefor 3 days at 60 °C.Fully polymerized resin blocks couldbe trimmed, and micron-sizedstep sections could be made until a viable start position for analysiswas reached using a Leica UC6 Ultramicrotome (Leica). Once the blockwas adequately trimmed, serial ultrathin sections of 400 nm thicknesswere cut and placed onto glass microscopy slides (Thermo Fisher Scientific).Subsequent ultrathin sections of 70 nm thickness were then cut andplaced onto carbon film on copper 300 square mesh grids (ElectronMicroscopy Sciences).To improve contrastfor bright fieldimaging, all sample sections on glass microscopy slides were stainedwith a toluidine blue solution (Merck). All slides containing samplesections were placed, samples facing up, on a hot plate at 50 °Cfor 2 min prior to coating the full slide surface with a 0.5% toluidineblue solution. After 3 min, the slides were removed from the hot plateand rinsed copiously with Milli-Q water to remove excess stainingsolution. Slides were returned to the hot plate until dry.Toimprove contrast for TEM imaging, sample sections on TEM grids werestained with commercially available UranyLess (Electron MicroscopySciences) and lead citrate (Electron Microscopy Sciences) solutionsin a protocol adapted from the manufacturer recommended use. Per samplegrid, a single drop of UranyLess was prepared in addition to threewash droplets of 100 μL of Milli-Q water followed by a singledrop of lead citrate and three final 100 μL droplets of Milli-Qwater. Grids were placed for 1 min into the UranyLess, removed, blottedwith a Kim wipe, and then placed onto the first Milli-Q droplet for5 min. This wash process was repeated twice for a total of three Milli-Qwash steps. After the third wash, samples were blotted and transferredto a drop of a lead citrate for 5 min. The grids were removed fromlead citrate, blotted, and then placed onto the first 100 μLMilli-Q wash droplet for 5 min. This wash process was repeated twicefor a total of three Milli-Q wash steps. Finally, the grids were removedfrom the last Milli-Q wash, blotted, and left to dry overnight atroom temperature.All fixedsamples were imaged with a Zeiss LSM 710 META confocal microscope(Carl-Zeiss AG). Excitation laser wavelengths of 488 nm (C1RG) and440 nm (Chlorophyll) were utilized for fixed samples prior to resinembedding. Resin embedded samples were imaged with bright field microscopyparameters and 488 nm (C1RG) excitation.All TEM sampleswere imaged with a Tecnai Spirit transmission electron microscope(FEI) operating at 120 kV with a wide angle Veleta CCD camera .Table S3.All Raman measurementswere conductedwith a WITec Alpha300 Access confocal Raman microscope using a 633nm laser with a power of 1–5 mW, 20× or 50× magnificationair objectives, and a built-in CCD camera for obtaining the brightfield images (WITec). TEM grids and loose MP powders were supportedon clean glass coverslips to ensure that the samples were not lost.Raw spectral data were extracted from the accompanying WITec Control5 software so that cosmic ray removal (zap) and baseline corrections(multi-point baseline subtraction) could be conducted using GramsAI software . The final presentedspectra were obtained by accumulating multiple (300–1000) 0.5s measurements and averaging them. A full table of the exact measurementparameters for each of the presented spectra can be seen in Tile scan stitching was conducted automaticallythrough the accompanying Zen 2010 software of the Zeiss cLSM. Alladditional image processing for SEM, cLSM, and TEM, including scalebar inclusion, channel merging, look up table application, contrastadjustments, and noise reduction, was conducted using Fiji (ImageJversion 1.53).During exposureexperiments, covered sample plates were used to limit the risk ofatmospheric deposition of contaminants. Multiple control exposureswere conducted, ensuring a control was present in each sample plateused, and the blank samples were processed for imaging in an identicalmanner to true samples. Images were acquired for “empty”control regions . Furthermore, slides were stored in closedcontainers during all procedural steps which did not directly involvetheir handling and during the time between sample creation and analysis.When samples were handled, cotton lab coats and latex gloves wereworn.Figure S1) showed high heterogeneity in the shape, size, and surfaceroughness of the particles present. Measurements obtained from theseimages indicated that all MPs were, at their largest point, 300 μmor less in size (Table S1) and thus smallerthan the average size of newly hatched Artemia nauplii C. andromeda were fed, but within a size range comparable to that of MPs reportedto be found in wild-caught jellyfish.35 Proceduralblank images  obtained underthe same conditions showed no dust or other particulate matter contaminationwithin the relevant size range.Representativesub-samples foreach of the MP stocks imaged with SEM , the concentrations it was possibleto prepare were higher than anticipated environmental contaminationlevels of a few micrograms to nanograms per liter.25As an initial method validationand control, 1,4-bis(α-cyano-4-methoxystyryl)-2,5-dimethoxybenzene(C1RG) labeled MPs were embedded into EPON epoxy resin and sectionedfor cLSM imaging  for further processing.Representative cLSM Z-projections shown in Upon chemicalfixation of the juvenile Figures S4–S8).cLSM imagingof the final resin embedded sample sections on glass microscopy slides3 confirmFigure S9).For PETC1RG samples, the C1RG fluorescence could be clearly observedeven with sample sections of only 70 nm thickness. However, it isapparent that gaps are present in some MPs observed either as theresult of uneven surface topography in the MPs themselves or of slightsample damage during sectioning. Despite this, correlative imagingwith TEM was possible and the exact ROIs imaged in cLSM could be observedat significantly higher magnification (S10). Correlationwith electron microscopy allows for further validation of the MP presence,which is of particular importance for the unlabeled MPs, and servesto confirm that the MPs are located inside the gastric cavities.Tile scan imaging of the full sample sections placed on TEMgridsallows for identification of specific landmarks  which were important for location triangulationin subsequent TEM imaging , and showed none of the peaks observed in the MP control measurement than water and PET (1.35g/cm3). Furthermore, PP has been previously shown to bemore hydrophobic than PET (Table S1).32 These properties directly impact the behaviorof the MPs during the exposure experiments, with PET and PETC1RG MPssedimenting over time while PP and PPC1RG MPs were buoyant and onlymixed into lower parts of the water column as a result of currentsin the water created by the movement of the C. andromeda medusa. As C. andromeda are benthicby nature, they remain at the bottom of the well (or tank) which theyare in. Thus, imaging revealed that the differences in propertiesof the MPs could already be observed to impact their interactionswith C. andromeda. PET and PETC1RGexposed medusae were shown to grab the MPs with their oral arms andingest them, while no such interaction was observed for PP and PPC1RGexposed medusae. Findings reported by Iliff et al. and Sun et al.also showed that polyesters  were among the most commonMP type observed in wild-caught jellyfish samples.35 This highlights an interesting potential for future studies whichfocus on further probing such differences through altering experimentalconditions such as exposure time, probing whether these differencesin interaction remain after the MPs are biofouled , and conducting MPexposures to Cassiopea sp. at different stages inthe life cycle. Furthermore, work could be conducted to expose the Artemia sp. prey to MPs prior to Cassiopea sp. feeding in order to gain insight into potential biomagnificationof MPs across multiple trophic levels as was recently proposed forPP MPs by Jeyavani et al. or observed for PS MPs with Aurelia coerulea by Sucharitakul et al.40 As a final consideration, researchers could attempt to enact a morecomplex exposure setup which better mimics the anticipated physicalenvironment . Such studies would allow researchers to gain furtherinsight into why certain MP types appear to be preferentially ingestedand what factors influence the interactions which occur.Initial characterization of the MPs used in thisstudy revealedthat the top-down mechanical degradation protocol used to create themresulted in a high heterogeneity in the size, shape, and surface roughnessof the MP stocks. This heterogeneity makes the MP stocks a good representationof MP fragments commonly reported to be collected in environmentalsampling campaigns.Figure S9),such a property would be of greater importance for smaller plasticparticles such as submicron (100 nm–1 μm) or nanoplastics whichcannot be detected in regular bright field microscopy due to resolutionlimits and contrast issues.41 This provides the potentialfor future studies to sequentially mill plastic particles to smallersizes and study their interactions with organisms when their movementin water would be driven by Brownian motion as opposed to buoyancyor sedimentation caused by the plastic’s density.42Inaddition to confirming the relevant physical properties wereas desired, it was possible to confirm that the C1RG fluorophore utilizedto create the labeled MP stocks was not degraded by the resin embeddingprocess and could still be observed even after ultrathin sectioningand heavy metal staining (UranyLess and lead citrate). While thiswas already beneficial for observing the MP locations on the TEM gridsto facilitate ROI selection  would be furthersupported by the high resolution one can obtain with a TEM.C. andromeda medusae, while the PP and PPC1RG MPs were not.Furtherinvestigation into the exact properties of the particlesbeing imaged through the use of Raman spectroscopy, a technique whichyields spectra based on material-specific inelastic scattering oflight, serves as the final validation that of MP presence or absencewithin the sample of interest. These measurements could be conductedwith a 633 nm excitation laser and multiple accumulations as the thinsample section could be photobleached more readily than a bulk sample.However, this prolonged photobleaching is not necessary for sampleswhich are not fluorescently labeled. Regardless of if the MPs werefluorescently labeled, Raman spectra could be obtained for all sectionsobserved to contain PET and PETC1RG MPs, indicating that the protocolutilized is reliably reproducible. The lack of Raman peaks for thePPC1RG MP exposed medusa and the medusa not exposed to MPs is in goodagreement with the findings from the cLSM and TEM, where no MPs couldbe observed. Thus, Raman spectra serve to confirm that in this experiment,the PET and PETC1RG MPs were ingested by the Scheme S1), one would be ableto analyze not just samples with labeled reference MPs, but also trueenvironmental samples as well. Thus, the protocol could be employedas a complimentary analysis for studies such as the one conductedby Iliff et al. where Cassiopea sp. jellyfish werecollected from their natural habitat to check for interaction withMPs. In such an analysis, one could select a few representative jellyfishfor sectioning, imaging, and spectroscopy, then digest the remainingjellyfish, and present average MP per organism count data alongsiderepresentative images of where MPs are located within the organisms.This would allow for a more holistic understanding of MP interactionwith Cassiopea sp. in their native environment andset the foundation for utilizing studies of MP presence in Cassiopea sp. jellyfish to monitor the overall pollutionlevels in the region they are collected from.Such findingsdemonstrate that with the work flow presented inthis study (


TREM2 expression and its association with brain pathologies have been limited by sample size. This study sought to correlate region-specific TREM2 mRNA expression with diverse neuropathological measures at autopsy using a large sample size (N = 945) of bulk RNA sequencing data from the Religious Orders Study and Rush Memory and Aging Project (ROS/MAP). TREM2 gene expression of the dorsolateral prefrontal cortex, posterior cingulate cortex, and caudate nucleus was assessed with respect to core pathology of Alzheimer’s disease , cerebrovascular pathology , microglial activation (proportion of activated microglia), and cognitive performance. We found that cortical TREM2 levels were positively related to AD diagnosis, cognitive decline, and amyloid-β neuropathology but were not related to the proportion of activated microglia. In contrast, caudate TREM2 levels were not related to AD pathology, cognition, or diagnosis, but were positively related to the proportion of activated microglia in the same region. Diagnosis-stratified results revealed caudate TREM2 levels were inversely related to AD neuropathology and positively related to microglial activation and longitudinal cognitive performance in AD cases. These results highlight the notable changes in TREM2 transcript abundance in AD and suggest that its pathological associations are brain-region-dependent.Previous post-mortem assessments of The online version contains supplementary material available at 10.1007/s00401-023-02564-2. TREM2) mutations from genome-wide association studies [TREM2 expression has also been documented in AD and other neuroinflammatory diseases. TREM2 signaling has been tied to mechanisms of phenotypic switching of microglia, regulation of neuropathology, and cognition [TREM2 gene expression with all three of these critical components of AD in the human brain. The following analyses expand upon our understanding of how post-mortem TREM2 mRNA levels in brain relate to disease by investigating regional, neuropathological, and retrospective cognitive functioning associations.The discovery of increased Alzheimer’s disease (AD) risk in carriers of rare Triggering Receptor Expressed on Myeloid Cells-2  risk, dysregulation of TREM2 expression is found in the AD brain. TREM2 mRNA and protein expression are upregulated in severe AD frontal cortex versus aged control [TREM2 has been found to track with both protein levels in the cortex as well as with clinical progression of the disease [TREM2 expression changes in AD may be regionally dependent as contrasting reports of TREM2 protein expression changes in the hippocampus—a region vulnerable early-on to the accumulation of AD-related pathology—yielded differential findings depending on the study and form of TREM2 measured [TREM2 expression is a late event which reflects a response to the accumulation of disease pathology [In addition to rare mutations in  control . Further disease . Howevermeasured , 42, 61.TREM2 with respect to disease pathology. For example, TREM2 protein is enriched in brain regions associated with high levels of amyloid-β accumulation in mice similar to humans [Additionally, from transgenic mouse models of AD, several important findings were gleaned concerning expression changes of o humans . Specifio humans , 64, 65.o humans .TREM2 transcription may relate to PAM in post-mortem tissue and whether this association is dependent on the presence of classical AD neuropathology. Additionally, we investigate whether PAM corresponds to TREM2 levels in subcortical areas and if this may reflect age- or disease-related cerebrovascular changes as these areas contain smaller blood vessels residing in deep brain areas which are particularly vulnerable to injury from inflammation, arteriosclerosis, and ischemic lesions [Accompanying the response of TREM2 protein to plaque development is a transcriptional and phenotypic transition from a maintenance to an activated microglial cell. The proportion of activated microglia (PAM) has been found to be elevated in AD compared to age-matched non-AD subjects , 20. Thi lesions .TREM2 expression changes are associated with several neurodegenerative and inflammatory diseases [TREM2 dysregulation may be associated with post-mortem amyloid-β levels as well as other concomitant pathology related to underlying immune dysregulation. Strong evidence links chronic inflammation to the development of small vessel disease and dementia. Effects of immune activation on blood vessels, such as proinflammatory cytokine, complement, or reactive oxygen species release by myeloid cells , may manifest as localized infarcts, diffuse white matter hyperintensities, or microbleeds, increasing blood–brain barrier permeability in old age and disease [TREM2 variant rs6918289 has been associated with increased risk of atherosclerosis [diseases , 44, 59. disease . This ox disease , 58, 59. disease , 43. Furclerosis .TREM2 bulk transcript levels with cognitive functioning, microglial activation, and AD-related neuropathology, including measures of cerebrovascular pathology. We evaluated expression in the dorsolateral prefrontal cortex (dlPFC), the posterior cingulate cortex (PCC), and the head of the caudate nucleus (CN) to investigate the neuropathological correlates of TREM2 levels across multiple brain regions. We hypothesized that higher expression of TREM2 mRNA at autopsy will correspond to increases in AD neuropathology, microglial activation, cerebrovascular injury, and worse cognitive performance.The present analyses investigated the relationship between Autopsy and cognitive data from the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP), or ROS/MAP, were leveraged to conduct this study . Data coAPOE genotyping was performed by investigators blinded to cohort data at Polymorphic DNA Technologies. The APOE gene was sequenced defining differential isoforms of APOE- ε2, APOE- ε3, and APOE- ε4 by codons 112 and 158 on exon 4.Whole blood lymphocytes or frozen brain tissue was used to extract DNA and previously defined quality control (QC) measures were employed . APOE geNeuropsychological testing details are previously published , 8, 9. TA clinical diagnosis was made at each participant visit based on the combination of cognitive tests scores, clinical judgement by a neuropsychologist, and diagnostic classification by a clinician  as previously described , 8, 9. C1-42 and abnormally phosphorylated tau, AT8 epitope, respectively, based off the average of eight regions . Braak staging of neurofibrillary tangle distribution and severity along with complimentary measures of neuritic plaque abundance by Consortium to Establish a Registry for Alzheimer’s disease (CERAD) scoring and spatial–temporal Aβ deposition capturing both neuritic and diffuse plaque progression by Thal phasing were also included as neuropathological outcomes and described elsewhere [All neuropathological marker quantifications have been previously described , 8, 9. Blsewhere , 12, 39.Rating of large vessel cerebral atherosclerosis was performed by visual inspection of the vertebral, basilar, posterior cerebral, middle cerebral, and anterior cerebral arteries of the circle of Willis, as well as their proximal branches and graded based on severity including the number of arteries involved and extent of artery involvement . Additionally, given visual identification of occlusion, an artery was bisected to assess the degree of occlusion which would then be incorporated into the final score . ArterioA standard post-mortem biological specimen protocol was utilized across centers where autopsies were performed and has been previously described . RNA extSequencing was carried-out in multiple phases; phase one included the dorsolateral prefrontal cortex (dlPFC) while phase two added additional dlPFC samples as well as samples from the posterior cingulate cortex (PCC) and head of the caudate nucleus (CN). Phase three ran additional participant samples from the dlPFC. Detailed information on RNA processing and sequencing can be found on synapse (syn3388564). In summary, phase one utilized poly-A selection, strand-specific dUTP library preparation, and Illumina HiSeq with 101 bp paired-end reads achieving a coverage of 150 million reads of the first 12 reference samples. The deep sequencing of these 12 reference samples included 2 males and 2 females of non-impaired, mild cognitive impaired, and Alzheimer’s disease cases. The remaining samples underwent sequencing with a coverage of 50 million reads. Phase two, library preparation utilized KAPA Stranded RNA-Seq Kit with RiboErase (kapabiosystems) with which ribosomal depletion and fragmentation was performed. Sequencing of this phase was performed on an Illumina NovaSeq6000 using 2 × 100 bp cycles targeting 30 million reads per sample. Phase three, RNA was extracted with a Chemagic RNA tissue kit  using a Chemagic 360 instrument and ribosomal RNA was depleted using RiboGold . Sequencing of phase three was performed on an Illumina NovaSeq6000 with 40-50 M 2 × 150 bp paired-end reads.N = 631, N = 278, and N = 104, 1–3, respectively) and independently processing the other brain regions . In brief, all samples with a RIN score below 4 and/or a post-mortem interval greater than 24 h were removed. Alignment was made to the hg38 reference genome using STAR. Read counts per million (CPM) values were quantile normalized to adjust for global variability between samples using the R package, cqn (version 1.30.0), controlling for GC-content and gene length. The R package, limma, was used to adjust for batch effects. In addition, principal component outliers outside five standard deviations as well as genes missing covariates of interest or those necessary for normalization were removed. Statistical outliers of gene expression outside four standard deviations from the mean were removed.Data processing and QC of RNA sequencing runs was performed by the Vanderbilt Memory and Alzheimer’s Center Computational Neurogenomics Team using an automated pipeline . This inN = 48 individuals and 80,660 single-nucleus transcriptomes) which was used to find the best predictors of each cellular component  using all of the genes in the RNAseq data to build models with the most optimized set of genes. The isolation and extraction of nuclei from frozen tissue has been described previously [A deconvolution technique was previously employed to obtain cellular fraction data on a subset of ROS/MAP participants . This meeviously . Brieflyeviously  with modQuality control for cell inclusion has been previously described in detail . The fin6 yielding a composite average density.Microglial density measurements were previously obtained from brain samples using immunohistochemistry performed by an Automated Leica Bond immunostainer (Leica Microsystems Inc.) and anti-human HLA-DP, DQ, and DR antibodies  . A blindhttps://www.rstudio.com/). To evaluate the data, a multiple linear regression model  as well as a linear mixed-effects model  were used. Models were run separately by regional TREM2 expression. For binomial and multinomial cerebrovascular outcome variables, generalized linear and proportional odds models were substituted, respectively. Linear regression models covaried for age at death, sex, post-mortem interval, education, and the interval from the last visit to death. In mixed-effects regression models, time was modeled as years from final visit with an additional covariate for the time between final visit and death. Both time and intercept were entered as fixed and random effects in the model. Immunohistochemistry and silver staining measurements of AD pathology were square root transformed to better approximate a normal distribution. Secondary analyses were conducted to account for possible variation in model predictions due to microglial cell-type fraction by including this estimate as a covariate as well as analyses assessing differences in results between AD cases and controls by stratifying the data.Statistical analyses were performed in R v3.6.1 using R Studio IDE (N = 30). Among the 945 participants with TREM2 mRNA measurement from the dlPFC, 505 participants also had TREM2 measurement from the PCC and 670 participants also had measurement from the CN. There were 430 participants with TREM2 measurement from all three brain regions.All primary models were corrected for multiple comparisons using the Benjamini and Hochberg (1995) false discovery rate based on the total number of tests completed, accounting for all gene-tissue combinations across main effect models assessing neuropathological outcome measures   but no different across APOE-ε4 carrier status . Levels of TREM2 mRNA in the CN neither differed across clinical diagnosis nor APOE-ε4 carrier status  as compared to NCI individuals without or with low likelihood of AD as classified by NIA-Reagan criteria . In contrast, caudate TREM2 levels did not differ across clinical or neuropathologically confirmed AD . As expected, TREM2 mRNA was found to be significantly correlated with the microglial cell-type fraction compared to other fractions .TREM2 mRNA levels were associated with higher amyloid burden in both cortical regions and across both amyloid measures . Main effect results per regional measurement of amyloid yielded similar outcomes and are provided in Supplementary Table 2. PCC and dlPFC but not CN TREM2 expression was higher in participants with higher amyloid burden as determined by CERAD scoring . In contrast, when assessed across Thal phases considering spatial–temporal distribution, there are significant increases in PCC TREM2 only .As expected, TREM2 mRNA expression and tau neuropathology were weak. TREM2 mRNA was negatively associated with tau burden in the CN, albeit nominally , and positively associated with the two cortical regions  and AD cases but not controls . Tau results were similar in sensitivity analyses adjusting for microglial fraction (Supplementary Table 3). Main effect results per regional measurement of phosphorylated tau are provided in Supplementary Table 2. Subtle increases in cortical TREM2 levels were observed across Braak staging supporting the linear regression results with tau . Interestingly, decreases in CN TREM2 mRNA are shown in later Braak stages which may help explain the negative model estimates in this region .In post-hoc models adjusting for Aβnt Table . When plTREM2 mRNA at autopsy may reflect concomitant cerebrovascular pathology, this was largely unsupported by results using cross-sectional data from ROS/MAP. Supplemental Table 4 summarizes the main effects analyses. Cell-type sensitivity analyses using dlPFC data yielded insignificant results and are reported in Supplementary Table 5.Despite the initial hypothesis that upregulation of TREM2 and AD neuropathology described above were accompanied by TREM2 associations with microglial density and/or activation in the same regions. A subset of participants  had transcript and microglial activation data available for analysis (see Supplementary Table 6 for participant characteristics). Interestingly, TREM2 measurements from cortical regions were not correlated with microglial outcome measures as we anticipated. However, TREM2 levels were significantly correlated with the activated component of microglial density cis-regionally in the caudate .Next, it was investigated whether the associations between TREM2 mRNA expression associations with composite global cognition scores. Cross-sectional results revealed TREM2 mRNA levels were not significantly associated with cognition prior to death in any brain region (p > 0.15). However, TREM2 mRNA levels related to faster decline in longitudinal analysis from the dlPFC . TREM2 mRNA levels in the PCC and CN were not significantly associated with longitudinal global cognition . Interestingly, diagnosis-stratified graphs revealed positive relationship between TREM2 and cognition in AD cases only when TREM2 was measured from the CN .Next, we explored potential n p > 0.1. Howevern p > 0.1. HoweverTREM2 levels on longitudinal cognition scores. TREM2 levels did not significantly interact with amyloid positivity on cognitive trajectory (p > 0.08), plots stratified by amyloid status are presented in Supplementary Figure 12.Finally, we tested whether amyloid status interacted with TREM2 gene expression on AD neuropathology, cognition, and concomitant pathways of injury in the human brain. We found striking evidence that TREM2 transcript associations are region-specific, with high cortical TREM2 relating to both amyloid pathology and clinical AD, while high caudate TREM2 was associated with microglial activation, less AD neuropathology, and a slower rate of global cognitive decline in AD cases. These findings highlight the regional complexity of TREM2 and suggest that changes in cortical TREM2 signaling may be particularly relevant to amyloid deposition while changes in caudate TREM2 signaling may be relevant at the onset of AD diagnosis.This work evaluated the effects of TREM2 and Aβ burden. This is consistent with the fact that functional responses of microglia to plaque have been shown to reflect increases in transcription of TREM2 which, in turn, is vital to microglial barrier formation and subsequent compaction of the plaque residue. These associations were consistent in cortical brain tissues and supported by previous work showing post-mortem immunoreactivity of cortical TREM2 in microglia, particularly those surrounding plaque [TREM2 corresponding with the progression of amyloid pathology [TREM2 mRNA levels from the CN did not relate to Aβ burden  and across CERAD scores .Amongst neuropathology outcome measures, perhaps the most robust was the association between g plaque . Furtherathology . The CN athology . Yet, TRden Fig. . This ise cortex , 11, 54.TREM2 expression also related to cognitive decline, although this association was weak and limited to the dlPFC in primary analyses. We expand upon this result by showing that the association with cognition is likely related to neuropathological tissue status, as differences between AD cases and controls were most pronounced when using a neuropathologically confirmed diagnosis , it should be noted that we did not observe a statistically significant interaction between amyloid burden and TREM2 abundance on cognitive decline. Ultimately further work is needed, perhaps focusing on cell-specific expression, to better understand how and when TREM2 changes with disease.Cortical CD44, CD45, CD11B, SPP1, and LGALS3) and observed that their expression levels, like those of TREM2, differ regionally; more pronounced positive associations were found in the dlPFC (with the exception of LGALS3), while none of these markers were significantly associated with amyloid when measured from the CN (Supplementary Table 8). TREM2 and other microglial transcripts were positively correlated amongst each other regardless of region . These results highlight the need to further investigate differences in microglial activation and subpopulation heterogeneity using multiple markers between cortical and subcortical brain regions and their potential differing roles in neurodegeneration in the presence of amyloid.To better understand how other microglial inflammatory markers relate to amyloid, we employed a post-hoc analysis of a small panel of transcripts  as well as caudate TREM2 and cognition , when the associations are limited to AD cases only, suggesting the negative relationship between caudate TREM2 and neuropathology may be relevant in disease. However, these stratified results need replication in an independent cohort. Previous work has found a similar positive association between TREM2 expression in human temporal cortex and phosphorylated tau [TREM2 transcriptional upregulation and Aβ deposition is thought to be deleterious to microglia and the immune landscape, whereas the development of tau pathology downstream has been shown to be dependent to some extent on a priori amyloid accumulation [TREM2 mRNA expression may be closer coupled to levels of amyloid rather than tau pathology in earlier affected cortical regions; this is despite concurrent elevations in its soluble protein fragment and phosphorylated tau in CSF described in MCI and early AD [TREM2 and low tau pathology in AD-stratified data  lending the possibility that subpopulations of microglia residing in this subcortical region may have a beneficial role staving off neurodegeneration as disease progresses. Our results highlight the possibility that TREM2 associations with tau may vary by brain region and disease state, though more confirmatory work is needed.In contrast to amyloid, there was weaker evidence of cortical sue Fig. C. This rated tau . This maated tau , there iated tau . Furthermulation . Therefoearly AD , 51. In TREM2 expression relates to amyloid while caudate TREM2 expression, instead, relates to microglial reactivity. This is interesting given that it is not explicitly clear whether there are disparate functions of TREM2 in microglia derived from these distinct brain regions. Evidence from transcriptomic- and other omic-profiling has revealed distinctive spatial and molecular patterns of microglia across the brain [TREM2 across Braak stages Supplementary Figure 8C, phospho-tau levels by amyloid status Supplementary Figure 6, and annual changes in global cognition stratified by diagnosis Supplementary Figure 11C). Due to the finding that increased TREM2 in caudate relates to the microglial state cis-regionally, we are left to speculate as to why this association does not extend to the cortex Below is the link to the electronic supplementary material.