Breakdown of the neurovascular unit in early Alzheimer’s disease (AD) leads to leakiness of the blood-brain barrier (BBB), contributing to cognitive decline and disease pathology. Vascular stability depends on angiopoietin-1 (ANGPT1) signalling, antagonised by angiopoietin-2 (ANGPT2) upon endothelial injury. We have examined the relationship between CSF ANGPT2 and CSF markers of BBB leakiness and disease pathology, across three independent cohorts: (i) 31 AD patients and 33 healthy controls grouped according to their biomarker profile ; (ii) 121 participants in the Wisconsin Registry for Alzheimer’s Prevention or Wisconsin Alzheimer’s Disease Research study (84 participants cognitively unimpaired (CU) enriched for a parental history of AD, 19 participants with mild cognitive impairment (MCI), and 21 with AD); (iii) a neurologically normal cohort aged 23–78 years with paired CSF and serum samples. CSF ANGPT2 level was measured by sandwich ELISA. In cohort (i), CSF ANGPT2 was elevated in AD, correlating with CSF t-tau and p-tau181 but not Aβ42. ANGPT2 also correlated positively with CSF sPDGFRβ and fibrinogen – markers of pericyte injury and BBB leakiness. In cohort (ii), CSF ANGPT2 was highest in MCI. CSF ANGT2 correlated with CSF albumin in the CU and MCI cohorts but not in AD. ANGPT2 also correlated with t-tau and p-tau and with markers of neuronal injury (neurogranin and α-synuclein) and neuroinflammation (GFAP and YKL-40). In cohort (iii), CSF ANGPT2 correlated strongly with the CSF:serum albumin ratio. Increased CSF ANGPT2 and the CSF:serum albumin ratio showed non-significant associations with elevated serum ANGPT2 in this small cohort. Together, these data indicate that CSF ANGPT2 is associated with BBB leakiness in early AD and is closely related to tau pathology and neuronal injury. The utility of serum ANGPT2 as a biomarker of BBB damage in AD requires further study. Vascular pathology and dysfunction are demonstrable in most patients with Alzheimer’s disease (AD), which shares common risk factors with cerebrovascular disease (reviewed ). NeurovAngiopoietin (ANGPT) signalling via tyrosine kinase with immunoglobulin-like and EGF-like domains 1 and 2 (TIE-1 and TIE-2) receptors on endothelial cells, regulates vascular stability and BBB permeability in adult tissues , 9. ANGPWe have explored the relationships between CSF ANGPT2; markers of pericyte injury (CSF sPDGFRβ) and BBB leakiness ; established markers of AD pathology (CSF Aβ and tau); and markers of neuronal injury (neurogranin and α-synuclein) and neuroinflammation (GFAP and YKL-40), in three independent cohorts. The first cohort comprised AD and controls stratified according to CSF AD biomarkers; the second consisted predominantly of at-risk cognitively unimpaired (CU) controls, but also included individuals with mild cognitive impairment (MCI) and patients with established AD. In a third cohort, we have investigated the relationships between ANGPT2 in CSF and serum, and between ANGPT2 and the CSF:serum albumin ratio, in paired CSF and serum samples from neurologically normal adult donors.Cohort (i): CSF aliquots from clinical diagnostic CSF samples from 33 AD cases and 31 controls were kindly provided by the Clinical Neurochemistry Laboratory at Sahlgrenska University Hospital. Patients whose CSF had abnormal levels of AD biomarkers  were classified as having AD . CSF t-t® immunoassays .Cohort (ii): CSF aliquots were provided from the Wisconsin Registry for Alzheimer’s Prevention Study and Wisconsin Alzheimer’s Disease Research Center (WISC cohort). WISC participants’ cognitive performance and functional status had been adjudicated by consensus conference. Diagnoses of MCI or dementia due to suspected AD were assigned based on National Institute on Aging-Alzheimer’s Association criteria ,15, withCohort (iii): Paired serum and CSF aliquots from neurologically normal controls (n= 23) spanning a wide age-range (23–84 years) were obtained from the Blennow/Zetterberg lab. The CSF:serum albumin ratio had previously been determined by an immunoturbidimetric albumin method . The demographics of the cohort, including sex and age at LP, are presented in ANGPT2 level was measured by ELISA  according to the manufacturer’s instructions. CSF was diluted 2-fold and serum 5-fold in assay. Absorbance was read at 450 nM in a FLUOstar OPTIMA plate reader . Measurements were made in duplicate for CSF, and in a single well for serum, the concentration of ANGPT2 was determined by interpolation against a standard curve generated by serially diluting recombinant ANGPT2 (3000–23.5 pg/ml). Results are expressed in pg/ml.CSF albumin level was measured in CSF samples from the WISC cohort, by commercial sandwich ELISA (Cat no 108788)  as in our previous study . CSF samANGPT2 datasets were normally distributed. A single outlier was identified in the serum ANGPT2 measurements and was removed prior to analysis. Pearson’s test was used to assess linear correlations, which were calculated for all CSF biomarkers. Linear mixed-effects models with random intercepts, age-at-lumbar-puncture as the measure of time, and CSF vascular biomarker as the outcome were used to test associations with tau positivity (> 24.8 pg/mL), and cognitive status.CSF ANGPT2 level was significantly higher in AD patients than controls (p < 0.05) . CSF ANGCSF ANGPT2 was highest in MCI subjects and was significantly higher in MCI than cognitively unimpaired (CU) controls (p = 0.04) . AlbuminCSF ANGPT2 level correlated positively with t-tau  and p-tau , and with Aβ40  but not Aβ42 . ANGPT2 correlated with markers of neuroinflammation – neurogranin  and α-synuclein ; and markers of neuroinflammation – GFAP  and YKL-40 . A summary of the correlations between ANGPT2 and markers of AD pathology, neuronal injury and inflammation is shown in CSF ANGPT2 level correlated strongly with the CSF:serum albumin ratio in matched CSF and serum samples from neurologically normal individuals (n = 23)  . ElevateIn this study, we show that CSF ANGPT2 levels are elevated in AD and associated with BBB breakdown. ANGPT2 levels were raised in samples from individuals with CSF biomarker positivity for AD, based on established cut-off values for t-tau, p-tau and Aβ42 , and corThe ANGPT-Tie signalling pathway is a key regulator of vascular stability and is dysregulated in diseases including stroke and diabetic retinopathy, in which elevated ANGPT2 is associated with BBB leakiness and endothelial apoptosis , 22. WheCSF markers of neuroinflammation and endothelial injury  were reported to be elevated in the pre-clinical stages of AD and to be tightly associated with CSF tau, markers of cognitive decline, and cortical thinning – the relationship was strongest in individuals who were Aβ-positive on PET scan . In our CSF ANGPT2 levels do not correlate with CSF Aβ42; however, they are strongly correlated with CSF Aβ40. We and others previously reported a similar pattern of correlations for sPDGFRβ . CSF Aβ4We previously reported that serum and CSF sPDGFRβ levels correlated positively in paired serum and CSF samples healthy donors, but that serum sPDGFRβ was not related to the CSF:serum albumin ratio . In the In conclusion, CSF ANGPT2 appears to be a sensitive marker of pericyte injury and BBB breakdown in early AD. Its rise is closely related to tau pathology and neuronal degeneration, and also to neuroinflammation. The utility of serum ANGPT2 as a marker of pericyte injury and BBB breakdown merits further investigation.


The cerebrospinal fluid (CSF) is a physiological fluid that functions to protect the brain tissue and maintain intracranial pressure. Defects between the subarachnoid spaces and other spaces can cause CSF leaks. We report the case of a 37-year-old female with no known past medical history who presented to the emergency department with a history of headaches for two months, nasal drip for 1.5 months, and recurrent fevers. Idiopathic intracranial hypertension was confirmed by cranial magnetic resonance imaging (MRI) and transnasal endoscopic repair of a CSF leak defect, and an abdomen fat graft was performed followed by an Axium navigation-guided right ventriculoperitoneal shunt (VPS). The cerebrospinal fluid (CSF) is the physiological fluid produced by the choroid plexus. It functions to protect both the brain tissue and the intracranial pressure (ICP). A CSF leak may occur when there is a defect between the subarachnoid spaces and other spaces, usually resulting from meningeal damage. The most common cause of CSF leaks is head injuries, which account for more than four out of five cases . CerebroA 37-year-old female with no known previous medical illness or surgeries presented to the emergency department with a two-month history of headaches, a 1.5-month history of nasal drip, and recurrent fevers. The headache was bifrontal, pressing in quality, highest in the morning, increased when she bent forward, and was of moderate severity. Nasal discharge was almost continuous, transparent, water-like, and left a salty taste if she swallowed it. She also had recurrent subjective fevers during this period, but they were never documented. Valsalva maneuvers were not tested to examine any association between the CSF leak and increased abdominal pressure. Initial assessment in the emergency department revealed that the patient was generally well, had normal vitals, and was afebrile. Examination by the Dandy maneuver showed a positive, clear fluid leak from the left nostril upon leaning forward. Examination of the nostril showed no sign of inflammation or blockage and was unable to pinpoint the area of leakage. Endoscopic examination showed clear discharge in the nose, mostly in the left sphenoethmoid recess, and minimal septal deviation to the left side. Fundoscopy performed showed grade 1 mild papilledema indicative of increased intracranial pressure. An initial computerized tomography (CT) scan of the head showed widened empty sella, and slightly tortuous optic nerves with a prominent perioptic CSF space that can represent benign intracranial hypertension  with contrast was done, which suggested idiopathic intracranial hypertension and showed that the left olfactory gyrus was mildly sagging or protruding into the olfactory groove with scattered CSF signal intensity in the left ethmoid air cell, along the left sphenoethmoidal recess, and dependent in the left maxillary sinus Figure .The superior sagittal sinus was ruled out. There was a suspected tiny fistulous tract from the left olfactory groove into the ethmoid sinus, as seen in Figure The patient refused to do a lumbar puncture; hence, CSF was not sent for testing. Fundoscopy performed showed grade 1 mild papilledema indicative of increased intracranial pressure. The neurosurgical team, with the support of the Ear, Nose, and Throat (ENT) team, performed a transnasal endoscopic repair of the CSF leak defect and abdomen fat graft, followed by an Axium navigation-guided right ventriculoperitoneal shunt (VPS) using a Medtronic programmable strata 2 with a valve pressure of 1.5. The patient was kept in the ward for four days after the operation for observation. She was seen 11 days postoperatively in the neurosurgery clinic when the rhinorrhea had stopped, but she was still having minor mucus discharge from both nostrils and continuing to have bifrontal headaches. She was seen once more 25 days after the operation in the ENT clinic, where she showed resolution of all symptoms with only minor crusting in the nasal cavity.Electrophoresis and immunofixation showed the presence of beta-2 (B2) transferrin. This is indicative of the presence of CSF in the fluid.A defect in the skull base can result in cerebrospinal fluid (CSF) leakage as it connects the nasal cavity with the subarachnoid space. Only 5% of reported cases of CSF leakage are associated with non-traumatic causes , female Previous studies showed that persistently high intracranial pressure can lead to skull base defects leading to CSF leaks . IncreasThe first symptoms to present in spontaneous CSF leak and idiopathic intracranial hypertension are pulsating tinnitus and headache . Our patThe investigation of choice to differentiate rhinorrhea due to CSF leak from other rhinorrhea causes is beta-2 (B2) transferrin, which carries a specificity of 95% and a sensitivity of 100%. Our patient had positive B2 transferrin, and the physician's clinical judgment as well as radiological findings confirmed the diagnosis ,12.In contrast, the most accurate methods of differentiating non-spontaneous rhinorrhea from spontaneous CSF rhinorrhea are computerized tomography (CT) scans and high-resolution magnetic resonance imaging (MRI). Although these two methods do not demonstrate the leak, they help in the localization of leaks when the leak is related to traumatic fractures or masses.The gold standard for determining the amount as well as the origin of the CSF leak is the use of magnetic resonance/computerized tomography (MR/CT) cisternography. The downside to this technique is its redundant invasiveness, which is not required to establish a diagnosis of CSF leak . ImagingIn non-operative management of spontaneous CSF leaks, acetazolamide can be given; alternatively, we can pursue surgical options in case of failure.  SurgicalIntracranial pressure tends to increase after surgical closure of the leakage due to diminished CSF drainage . CSF shuOur neurosurgical team, with the support of the ENT team, performed a transnasal endoscopic repair of the CSF leak defect, followed by an Axium navigation-guided right VP shunt. This was done to avoid the recurrence of spontaneous CSF leakage after the repair of the defect.Spontaneous cerebrospinal fluid (CSF) rhinorrhea is an uncommon finding. It is associated with a high body mass index (BMI), increased intracranial pressure (ICH), and female gender. It commonly presents with headaches and tinnitus. Magnetic resonance/computerized tomography (MR/CT) cisternography is the gold standard for detecting the tract of the CSF leak. Management can be conservative (acetazolamide) or surgical .


Effective communication is imperative for successful interprofessional collaborative interactions that augment both patient-centred and evidence based care. Inquiry into the prevalence of chiropractic-related terminology on South African chiropractor’s webpages has not been explored to date. The implications of such analysis could indicate the professions’ ability to effectively communicate in interdisciplinary settings.From 1 to 15 June 2020, Google search was used to identify the webpages  of South African private practice chiropractors registered with the Allied Health Professions Council of South Africa (AHPCSA). Webpages were word-searched for eight chiropractic terms with context: subluxation; manipulate(-ion); adjust(-ing/-ment); holism(-tic); alignment; vital(-ism/-istic); wellness; and innate intelligence. Data collected was transferred to an Excel spreadsheet. Accuracy of information was verified by the researchers through a process of double checking. The number of instances each term was used, and certain socio-demographic data were recorded. Descriptive statistics and bivariate analyses were used to summarise and analyse the data.p = 0.015). The longer a chiropractor was in practice the more likely they were to use profession-specific terms (p = 0.025). The most frequently occurring combination of terms were adjust(-ing/-ment) and manipulate(-ion), found in 38 out of 336 webpages .Among 884 AHPCSA-registered South African chiropractors, 336 webpages were identified and analysed. From 1 to 15 June 2020, the most commonly found terms on 336 South African chiropractic webpages were 'adjust(-ing/-ment)', 'manipulate/manipulation', and 'wellness', with prevalence estimates of 64.1% , 51.8% , and 33.0% , respectively. The least commonly found terms were 'innate intelligence' and 'vital(-ism/-istic)', with prevalence estimates of 0.60%  and 0.30% , respectively. Manipulate(-ion) was used more by male chiropractors (The use of chiropractic-related terminology on South African chiropractic webpages was common, with the prevalence of term use varying by type of terms, by gender of the chiropractor, and by clinical practice experience. Better understanding of the effects of chiropractic terminology use on interprofessional and patient interactions and communication is warranted.The online version contains supplementary material available at 10.1186/s12998-023-00483-3. Patient or person-centred care (PCC) is an ethical approach  that aimOften the individual members of a medical interprofessional collaborative team emerge from differing education routes, have specialized skills that use different professional languages that align them to diverse aspects of healthcare , 20. FolChiropractors in South Africa have been shown to have poor interprofessional-referral relationships with other musculoskeletal professionals, with 60.2% of chiropractors almost never receiving a referral from an orthopaedic specialist . HoweverSimilar international studies undertaken in Australia and the United Kingdom (UK) indicate that majority of the chiropractor websites screened in those countries included more than one chiropractic-related term. Interestingly, the statistical comparison between these two studies showed that while the chiropractic terms were used in the same order of preference (adjust (-ing/-ment) mostly used followed by wellness, holism (-tic), subluxation and finally innate intelligence) the frequency of usage was to a lesser extent in the UK , 30.There is currently no literature on the prevalence of chiropractic-related words used on South African chiropractors’ websites or webpages, and whether the educational collaborative statement has had any impact on the use of chiropractic-related terminology post 2014. These insights are relevant in documenting the trends of South African chiropractor communication skills. Chiropractic-centric terminology primarily includes the words subluxation, manipulate (-ion), adjust (-ing/-ment), holism (-tic), alignment, vital (-ism/-istic), wellness, and innate intelligence, detailed in Table n = 884 chiropractors in South Africa at the time of the study [The Allied Health Professions Council of South Africa (AHPCSA) documented the registration of he study . Inclusihe study . Each elThe use of chiropractic-related terminologies was documented based on the context and value used. Thus, any sentence that devalued the meaning of a chiropractic-related word by rejecting its chiropractic-related meaning, whilst still using the word in the sentence, was not tallied. A conjectural example of this is: “We treat vertebral subluxations” was included, but “We believe the vertebral subluxation model is only a historical model and has no clinical value” was excluded . FurtherNumber of citations per page was converted into a binary categorical variable where 0 was no citation and 1 was at least one citation. Additional variables were computed, including a variable showing how many different terms occurred on a webpage and a variable that showed the combination of terms used on a webpage. The frequency procedure was used to establish how many webpages featured each term, to show the distribution of number of citations for each term, to show how many different terms occurred on a webpage, and the main combinations of terms on the webpages. The findings were reported using descriptive statistics such as frequencies with percentages, means with standard deviations where appropriate. The 95% confidence intervals for proportions was calculated using the Wilson method to report on where the population parameter was most likely located between. Bivariate analysis in the form of Pearson's correlation coefficient and chi-square test was used to assess for relationships between chiropractic terms and socio-demographic variables.p value of less than 0.05 was be considered significant. IBM SPSS version 28 was used for the analysis.Of the 884 chiropractors in the country, 336 webpages were found through census sampling where all the webpages found were then analysed for relevant terms. Fourteen webpages were excluded as 3 of the chiropractor’s webpages were in fact not located in South Africa, 5 webpages were unable to load, and 6 webpages only mentioned the chiropractor’s name with a link to their social media accounts. A n = 336 webpages were appraised in this study. Gender was recorded for 288 chiropractors, where n = 169 (58.7%) were male and n = 119 (41.3%) were female. Just over 90 percent of chiropractors were located in three provinces: Gauteng ; KwaZulu-Natal (KZN) ; and Western Cape (WC) . The remaining chiropractors were located in Eastern Cape , Free State , North West , Limpopo , Mpumulanga  and Northern Cape  provinces. It was possible to record number of years the chiropractor was in practice for ninety-eight chiropractors. The minimum and maximum number of years in practice were one year and 47 years respectively. The average number of years in practice was 13.83 (SD = 8.97). Place of study could be identified from 248 of the 336 webpages. Most chiropractors had obtained their qualification from a South African institution, either the University of Johannesburg (UJ)  or the Durban Institute of Technology (DUT) . Smaller numbers had studied at an institution either in the United States of America (USA)  or in the United Kingdom (UK) .A total sample of Analysis of use of terms per webpage is presented in Table From the sum of number of times a term was used, adjust(-ing/-ment), manipulate(-ion) and wellness were also the most frequently used terms. Adjust(-ing/-ment), was used 1106 times, manipulate(-ion) 611 times and wellness 324 times. Innate intelligence and vital(-ism/-istic) were used twice and once respectively. Subluxation, which occurred in just 17.6% of webpages (ranked 6) was the fourth most frequently used term (used 231 times). Analysis of mean number of times a term was used follows a similar pattern to the sum of use. Adjust(-ing/-ment), manipulate(-ion) and wellness were the three top ranked terms based on average use . Subluxation was the fourth most used term based on mean (0.69 citations per webpage on average) and innate intelligence and vital (-ism/-istic) were used the least , manipulate manipulate(-ion) and wellness were used in more webpages and were used more often and innate intelligence and vital(-ism/-istic) were very rarely used. There was some variation in ranking for alignment, holism(-tic) and subluxation depending on what measure was used. Subluxation was used in fewer webpages than alignment and holism(-tic) but when it was used it was used more often than the other two terms.n = 70;20.8%) or three  of the eight terms. No webpages cited all eight or seven of the eight terms. Sixty webpages (17.9%) used none of the eight terms.It was also of interest to examine the combination of terms used by webpages. Number of different terms used per webpage is shown in Table Bivariate analysis was used to establish if there was a significant linear relationship between number of times different terms were used. A significant positive linear relationship would demonstrate when use for one term was low or high use of another term would be similarly low or high. Significant positive correlations were observed Table  for the The bivariate analysis provides further evidence that some pairs of terms were more likely to be related than other pairs.Number of webpages that cited a term at least once was used as the indicator to test if sociodemographic characteristics were associated with use of terms. For this analysis only the three most frequently reported provinces—Gauteng, KwaZulu-Natal and Western Cape—and the two most frequently reported universities—UJ and DUT—were included. Years in practice was recoded into categories 1–5 years, 6–10 years, 11–15 years, 16–20 years and 21 or more years. Only statistically significant results are reported. There were no significant associations between place of study and proportion of webpages using different terms.p = 0.015). For other terms similar proportions of male and female chiropractors cited the term on their webpage.Male chiropractors were more likely to use the term manipulate(-ion) on their webpages. Of the male chiropractors, 104 (61.5%) used manipulate(-ion) at least once on their webpage compared with 56 (47.1%) of female chiropractors .Chiropractors with fewer than six years and those with over 20 years in practice were less and more likely to use the term alignment on their webpage respectively. While between 31.3% and 33.3% of chiropractors with 6–10 and 16–20 years of practice used the term alignment at least once on their webpage, just two (10.0%) of chiropractors with 1–5 years practice and ten (62.5%) with 21 years or more in practice featured the term on their webpage. This association was statistically significant . Wellness was more likely to be used by chiropractors in the WC where 44 (43.6%) of chiropractors in WC included at least one instance of wellness on their webpage compared to 32 (31.7%) and 28 (27.7%) in Gauteng and KZN, respectively. This association was statistically significant . Holism(-tic) was more likely to be used by chiropractors in the WC where 41 (40.6%) of chiropractors in WC include at least one instance of holism(-tic) on their webpage compared to 28 (27.7%) and 19 (18.8%) in KZN and Gauteng, respectively. This association was statistically significant .Frequency of use of three terms show an association with province. Adjust(-ing/-ment), was more likely to be used by chiropractors in WC and Gauteng than those in KZN. Seventy-four (73.3%) and seventy-one (70.3%) of chiropractors in WC and Gauteng included at least one citation of adjust (-ing/-ment), on their webpages compared to just 49 (48.5%) of chiropractors in KZN. This association was statistically significant  chiropractor webpages which showed an 85% and 75% prevalence of chiropractic-specific words, respectively. This suggests that South African chiropractors may be similarly inclined to promote the more traditional unsubstantiated chiropractic historical beliefs , 44, 45.This preference within South African chiropractors of the word ‘adjustment’ rather than the word ‘manipulation’ may arise from the notion that an adjustment is more specific in its intention having broader mechanical and neurological effects that are perhaps absent in the type of manipulation provided by other health-care professionals , 46, 47.The moderate to strong correlational relationship between the prevalence of the words alignment, adjustment and wellness to the word subluxation found in the South African sample is somewhat predictable considering these terms are used to convey similar concepts. Early alignment-subluxation based chiropractors used radiographic imaging to study spinal alignment to identify the location of a ‘subluxated’ or misaligned vertebrae, a practice that is strongly discouraged in allopathic medicine and evidence-based chiropractic today . Likely Another relevant association was observed between the number of years a South African chiropractor was in practice to both the term ‘alignment’, where the longer the chiropractor was in practice (the older he/she is) the more likely they were to use chiropractic-related terminology (and those practicing for a very short time were much less likely to use the term than those practicing for a moderate time). It is possible that these results could be attributed to the adoption of the International Chiropractic Education Collaboration, Clinical and Professional Chiropractic Education Position Statement , post-20This study showed that male South African chiropractors were more likely to use the word ‘manipulation’ on their webpage compared to their female counterparts. Most studies investigating chiropractic sex differences focus on the number of working hours and number of patients seen weekly. One study shows that female chiropractors spend more time with their patients on direct patient care. They also found that female chiropractors treated more patients with acute conditions, whereas male chiropractors treated more chronic conditions . No studChiropractors’ webpages in the Western Cape province showed the highest overall prevalence for the use of chiropractic-related terminology—specifically terms adjustment, wellness and holistic (but not the other terms)—when compared to the other two predominant provinces (KZN and Gauteng). The average age of chiropractors in the Western Cape Province is 37 years . AccordiThis study advances on previous studies as it adds similar data based on a South African perspective while including gender and number of years in practice as variables to be considered, something not previously reported before.Google search is not impartial to webpages that have not been skilfully marketed so this may have had an unaccountable effect on the results. The prevalence of chiropractic-related terms on a chiropractor’s website is not an automatic representation of their beliefs. The results found in this study are specific to the educational and professional contexts of chiropractic in South Africa that may not be directly generalised to other parts of the world. Chiropractors that work in group settings or do not have webpages remain unaccounted for. Counting words and determining the context of the words used on a website opens up the possibility to human error or oversight. This study did not investigate any differences found between provinces or regions.Two-thirds of South African chiropractors use at least two chiropractic-related words on their webpages with a partiality to the words adjustment, manipulate and wellness. While South African chiropractors seem attached to historical underpinnings of chiropractic to a lesser extent than Australian and UK chiropractors, chiropractic-related terminology does seem to be used by the chiropractors webpages in this study to distinguish their professional identity. The vitalistic chiropractic opinion, albeit in the minority, has survived to some extent. Evidence-based education has had a positive influence on the way chiropractors communicate and on the prevalence of chiropractic-related terminology. Certain demographics such as older age, the male sex and regional location in the Western Cape Province in South Africa seem to show increased usage of chiropractic-related words. Chiropractors in South Africa may have difficulty integrating and communicating effectively with other medical professionals in interdisciplinary settings as their interprofessional communication skills seem to be more informed by chiropractic philosophies rather than medical perspectives. Further investigations are warranted on the perceptions of chiropractic communication competency and interpretation by other health professionals.Additional file 1. References for Supplementary Material.Additional file 2. Further descriptions of chiropractic-related terms.


Capsicum annuum L.) with different particle sizes (255–901.7 nm) were prepared using three industrialized pulverization-inducing techniques . Subsequently, the PO emulsion was microencapsulated via spray drying. The effects of the different induction methods on the physicochemical properties, digestive behaviour, antioxidant activity, and storage stability of PO microencapsulated powder were investigated. The results showed that ultrasound and high-pressure homogenization induction could improve the encapsulation efficiency, solubility, and rehydration capacity of the microcapsules. In vitro digestion studies showed that ultrasound and high-pressure homogenization induction significantly increased the apparent solubility and dissolution of the microcapsules. High-pressure homogenization induction significantly improved the antioxidant capacity of the microcapsules, while high-intensity ultrasound (600 W) induction slowed down the degradation of the microcapsule fats and oils under short-term UV and long-term natural light exposure. Our study showed that ultrasound and high-pressure homogenization equipment could successfully be used to prepare emulsions containing nanoscale capsicum oil resin particles, improve their functional properties, and enhance the oral bioavailability of this bioactive product.As an indispensable process in the microencapsulation of active substances, emulsion preparation has a significant impact on microencapsulated products. In this study, five primary emulsions of paprika oleoresin (PO, the natural colourant extracted from the fruit peel of  Paprika oleoresin (PO) is a tetraterpenoid natural pigment extracted from chilli peppers as a deep red viscous oily liquid. PO contains a large number of carotenoids, such as capsanthin, capsorubin, β-carotene, zeaxanthin, cryptoxanthin, and lutein, as well as phenolic compounds, which have a high antioxidant capacity ,2. StudiMicroencapsulation utilizes certain stabilized materials to microencapsulate a core ingredient . This is an effective way to enhance the stability of an unstable bioactive ingredient by providing a protective outer wall . Among tThe spray-drying microencapsulation preparation process is typically divided into two steps: (1) the encapsulated and microencapsulated core materials are dissolved or dispersed in water via auxiliary drive measures  to prepare a primary emulsion or suspension; and (2) the emulsion or suspension is atomized and spray-dried to form the final microencapsulated product . There aCommonly used industrial techniques, including high-pressure homogenization and ultrasonic induction methods, are widely used for emulsion production in the food and pharmaceutical industries . The useThe particle sizes and polydispersity index (PDI) of PO emulsions prepared via ultrasonication and high-pressure homogenization were determined, as shown in p > 0.05).In addition, as can be seen from One of the purposes of microencapsulation is to promote the uniform stabilization of fat-soluble substances in water. Therefore, good solubility is a key factor in promoting the application of microencapsulated products in the food industry . It was Wettability reflects the rehydration capacity of the microcapsules. From p < 0.05), indicating that these three powder products contained a greater amount of PO. The CV can be used to assess the pigment content and examine the PO residue of microencapsulated products.The colour value (CV) can reflect the pigment content to some extent. Observation of the colour value column showed that the CV values of U-600, H-50, and H-100 were significantly larger   images of microcapsules consisting of the encapsulated materials, inulin and sodium starch octenyl succinate are shown in −1. The stretching vibration peak of –CH was located at 2925 cm−1, the strong absorption peak of C=C was located at 1640 cm−1, and the strong absorption peak was located at 1025 cm−1. Moreover, the spectra of pure PO appeared near 3009 cm−1, 2925 cm−1, 2850 cm−1, 1740 cm−1, 1458 cm−1, and 722 cm−1, indicating the presence of aromatic rings, polar groups , and hydrophobic groups [−1, the aromatic moiety was the most important band for this oleoresin. Meanwhile, the stretching vibrations of C–H groups in the regions of 2850 cm−1 and 722 cm−1 represented saturated aliphatic compounds [FTIR can detect the introduction of new functional groups or the formation of new chemical bonds in PO microcapsules, and can characterize their molecular structure. ompounds .−1 to 2925 cm−1 was associated with capsaicin, particularly paprika oleoresin stimulating compounds. The peaks at 1740 cm−1 and 1640 cm−1 correlated to the stretching vibration of C=O, and the peak at 1458 cm−1 corresponded to the stretching vibration of the N–H group. In addition, the band at 722 cm−1 characterized the regional vibrations of C–H and C–C, which were considered to be the aromatic bonds of the capsaicin benzene ring. The characteristic absorption peaks of the encapsulated paprika oleoresin powder corresponded to the EMs’ characteristic absorption peaks. Finally, loading chromatography of the particles showed band deformation at the carbonyl and hydroxyl groups, suggesting that PO may bind to the chemical groups of the encapsulated material.It was found that the signal peak at 2850 cmH-100 > TgH-50; TgU-600 > TgU-200). Among them, U-600 had the highest absorption peak temperature and the highest EE  curves were analysed and compared using TA software (Version: 4.1.1.33073) to study the thermal properties of PO microcapsules, which was beneficial for evaluating product quality and stability. The DSC curves and glass transition temperatures (Tg) of the five PO microcapsules were shown in ghest EE , and thep < 0.05), while there was no significant difference in the release rate between microcapsules prepared by ultrasound and high-pressure homogenization (p > 0.05). The results showed that using ultrasound and high-pressure homogenization for the preparation of emulsions could improve the release characteristics of PO microcapsules and enhance bioavailability.PO release analysis was performed after 2 h of digestion in simulated intestinal fluid (SIF), and the results are shown in This may be because ultrasonication and high-pressure homogenization induced the formation of a more homogeneous solution, which facilitated the entry of PO into the cavity formed by the EMs and introduced more water-soluble hydroxyl groups to the particles, thus reducing the hydrophobicity of the PO microcapsules. Ultrasound and high-pressure homogenization induction improved the degradation of PO microcapsules in intestinal digestion by increasing the dispersibility and wettability of the PO microcapsule particles, thus accelerating the release of PO. Therefore, particles prepared by ultrasound and high-pressure homogenization induction showed better release characteristics in SIF.The antioxidant capacity of the five types of microcapsules was investigated by analysing and comparing their free radical scavenging capacity. As shown in The retention of PO in the five types of microcapsules was investigated under UV irradiation, as shown in The thermal stability of PO is displayed in The effect of the preparation method on the stability of PO particles in terms of shape, colour, and PO content was evaluated by storing them for 30 days under two different treatments: natural light at room temperature and darkness at room temperature. Colour is an important parameter that influences consumers when purchasing food products. Carotenoids in PO were crucial in determining the colour of the particles . The carp < 0.05). The CK and U-200 particles exhibited a significant decrease in colour parameters  under the influence of natural light (p < 0.05). Unexpectedly, U-600 particles exhibited a slight increase in colour parameters and less loss of carotenoids compared to the initial state, followed by H-50 and H-100 microcapsules, similar to the results in Measurements of PO particles stored in different environments were evaluated using the CIELab system . The results for storage stability of PO microcapsules in the presence or absence of natural light are shown in PO was provided by Luck Huaguagn Graphics Co., Ltd. Sodium starch octenylsuccinate and inulin were purchased from Thousand Taste Food Technology Co., Ltd. , and Baiyun Aroma Chemical Co., Ltd. , respectively. Organic reagents such as ethanol (99.7%) and hexane were of analytical grade and purchased from Shanghai Titan Technology Co., Ltd. . α-amylase was from Yuanye, Shanghai, China, trypsin, pancreatic lipase, and potassium persulfate from Aladdin, Shanghai, China, porcine bile salts from Solarbio, Beijing, China, and DPPH and ABTS were from Macklin, Shanghai, China.The process of preparing the emulsion as well as the microcapsules is shown in Spray-dried emulsions of paprika oleoresin were separately prepared by ultrasound induction and high-pressure homogenization. (1) Ultrasonic induction method: the mixed solution was ultrasonicated for 10 min  using a 6 mm probe ultrasonic cell crusher  with output power of 200 W or 600 W, abbreviated as U-200 and U-600, respectively. (2) High-pressure homogenization method: the final emulsion was processed with an ultra-high-pressure homogenizer  for 10 cycles at pressures of 50 MPa or 100 MPa. The treated samples were abbreviated as H-50 and H-100, respectively. The temperature of the emulsion was controlled at <35 °C during the preparation.3/min and 0.5 MPa, respectively. The inlet temperature was set at 155 °C, and the outlet temperature varied between 75 and 85 °C. The spray-dried powder was collected from the bottom of the cyclone and transferred to a ziplock bag, and then stored at −18 °C.PO particles were prepared using an experimental spray dryer . The PO suspension was continuously stirred during the spraying process and fed into the atomization chamber at a feed flow rate of 450 mL/h. The suction air rate and spray pressure were set at 0.55 mThe particle sizes and polydispersity index (PDI) of the biopolymer dispersions and the zeta potential of the emulsions were measured using a Zetasizer Nano ZS . The samples were diluted to 1 mg/mL with ultrapure water, and the measurements were repeated five times.The five prepared emulsions were left in a room temperature environment for seven days to observe the suspension of the emulsions and to compare the colour, clarity, and floating of oils and fats via sensory comparison.Determination of moisture: the moisture of the particles was evaporated in an oven at 105 °C until a constant weight was reached. Then, the moisture content was determined based on the weight difference method.Determination of EE was based on the literature . First, g rpm/min for 10 min at 20 °C using a centrifuge . A total of 5 mL of the supernatant was transferred to a pre-weighed petri dish and then dried in an oven at 105 °C for 5 h. Solubility was calculated based on the weight difference.Pellets (0.2 g dry basis) were added to 20 mL of distilled water and stirred for 5 min. The pellets were centrifuged at 10,000× An accurately weighed amount of microencapsulated powder (0.1 g) was sprinkled into 50 mL of deionized water at room temperature and then stirred with a magnetic stirrer at 450 rpm/min until completely submerged. We recorded the time at which the powder was fully immersed .To determine the colour of the particles, the powders were evaluated using a Colour Reader , D65 light source, and 10° angle, using the colour parameters  of the CIELab scale. a* represented the change in colour between red (+) and green (−), b* indicated the change in colour between yellow (+) and blue (−), and the a* and b* values were used as colour parameters after the samples were stored for 30 days under light and dark room temperature storage conditions.The powder obtained after spray drying was used to determine the CV according to the method of the American Spice Trade Association . A certaFollowing the method of Ding et al. , the theThe morphology of the spray-dried powder samples was observed using a scanning electron microscope  at 10 kV. To obtain images , dried samples were taken and directly glued to a conductive adhesive sprayed with gold , observed, and photographed.−1 on a background of KBr slices, the test wave numbers ranged from 400 to 4000 cm−1 [A Fourier transform infrared spectrometer  was utilized for determination and analysis. The samples and dried KBr powder were mixed, ground, and pressed into flakes. Scanning at 4 cm000 cm−1 .g for 15 min to obtain the supernatant.A static in vitro intestinal digestion model was used to determine and analyse the bioavailability of PO in microcapsules obtained by spray drying. Simulated in vitro intestinal digestion was performed as described in the literature . To prepThe digested supernatant was collected. Absorbance was measured at 460 nm using a UV–Vis spectrophotometer, and CV was calculated using the equation in 1) was measured at 517 nm. The absorbances of blank (D2) and control (D0) samples were determined using the same method. In the blank, ethanol was used instead of the DPPH solution; in the control, ethanol was used instead of sample solution. The DPPH free radical scavenging capacity was calculated using the equation:The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity of the samples was determined according to a minor modification of the method described by Meng et al. . Two milv/v) ratio to generate ABTS radicals. The mixture was then placed in the dark at 25 °C for 16 h. The absorbance measured at 734 nm was 0.7 after dilution with PBS  to obtain the ABTS working solution. Then, 0.6 mL of sample (30 mg/mL), 0.6 mL of ABTS working solution, and 2.8 mL of PBS were mixed, reacted for 5 min, and absorbance (As) was measured at 734 nm. The control (Ac) used 10 mM PBS instead of the sample solution. We used the following formula to determine the removal capacity of the samples:The ABTS free radical scavenging capacity was assessed, with some modifications, according to Meng et al. . ABTS .Data were presented as means with standard deviations. One-way ANOVA was performed using Prism 8  and In this study, oil-in-water microcapsules were prepared by the spray drying method using paprika oleoresin as the core substance and inulin and sodium starch octenylsuccinate as the wall materials. The objective of this study was to improve the stability and water solubility of the active ingredients in the microencapsulated core material by induction of ultrasound and high-pressure homogenization. The results showed that the particles in the PO emulsion were nanoscale, and after the emulsion was left to stand for one week, PO in the emulsion prepared by high-pressure homogenization was less likely to float to the surface, and the emulsion was stable. The five types of microcapsules were structurally intact, with no obvious pores or ruptures on the outer surface, with smooth surfaces, while concave wrinkles were more obvious with increases in mechanical shear strength. Rehydration was continuously enhanced. The EE of the U-600 and H-100 microcapsule systems, with high mechanical strength, increased solubility, and the highest heat absorption capacity, showed good thermal and photochemical stability. Microcapsules produced by ultrasound and high-pressure homogenization preparations had comparable intestinal digestibility, which was significantly higher than that of the control, and microcapsules prepared by high-pressure homogenization showed significant antioxidant activity. The PO microcapsules produced by 600 W ultrasonication showed a positive effect on the retention of lipid content in both short-term UV irradiation and long-term natural light storage tests.These results indicate that the spray drying method is effective for the preparation of PO using ultrasound and high-pressure homogenization as induction means. PO microcapsules are characterized by uniformity and stability, high digestibility, good water solubility, and high content, and they can be added to feeds as dietary supplements to improve the absorption of carotenoids in poultry. Our findings provide a basis for the use of ultrasonic and high-pressure homogenization equipment for microcapsule production to prepare emulsions containing PO microcapsules, prolonging the shelf-life of this bioactive product and improving its oral bioavailability.


A unique feature of normothermic ex vivo perfusion on the TransMedics® Organ Care System (OCS™) is that the donor heart is maintained in a beating state throughout the preservation period. We applied a video algorithm for an in vivo assessment of cardiac kinematics, video kinematic evaluation (Vi.Ki.E.), to the donor hearts undergoing ex vivo perfusion on the OCS™ to assess the feasibility of applying this algorithm in this setting.Reliable biomarkers for assessing the viability of the donor hearts undergoing n = 6) were procured from Yucatan pigs and underwent 2 h of normothermic ex vivo perfusion on the OCS™ device. During the preservation period, serial high-resolution videos were captured at 30 frames per second. Using Vi.Ki.E., we assessed the force, energy, contractility, and trajectory parameters of each heart.Healthy donor porcine hearts . Similarly, there were no significant changes in the force, energy, or trajectory parameters. Post-transplantation echocardiograms demonstrated robust contractility of each allograft.ex vivo perfusion is feasible on the TransMedics OCS™, and we observed that the donor hearts maintain steady kinematic measurements throughout the duration.Vi.Ki.E. assessment of the donor hearts undergoing  Ex vivo machine perfusion has transformed organ transplantation outcomes by minimizing ischemic injury and reconditioning the organs prior to transplantation (ex vivo perfusion (NEVP) has allowed for the expansion of the donor pool through the utilization of hearts from donors after circulatory death  (ex vivo cardiac kinematics while a porcine donor heart is undergoing NEVP on the TransMedics® Organ Care System (OCS™). A successful measurement of cardiac kinematics while the donor hearts are beating on the OCS™ could allow for this technology to be applied as a biomarker to predict cardiac fitness.We applied a well-characterized video method for i.Ki.E.) –10 and an = 6) were procured in a standard fashion through a sternotomy. The hearts were then prepared on a back table and subsequently mounted on an OCS™ and underwent 2 h of NEVP at 34°C–35°C as described by Mendiola Pla et al. (2), perfusate lactate, and perfusate hematocrit (Hct) were obtained.This study was approved by the Duke University Institutional Animal Care and Use Committee. Female Yucatan pigs  aged 7–9 months were utilized for this study. Baseline cardiac troponin I values and cardiac magnetic resonance imaging (cMRI) were obtained prior to surgery. In preparation for surgery, the animals were anesthetized and intubated for mechanical ventilation. The donor hearts  or a Canon EOS Rebel T8i equipped with a Canon EFS 18–55 mm lens . Once perfusion of the donor heart was established on the OCS™ and the heart was beating, the camera was positioned approximately 30–40 cm perpendicularly in front of the heart . The disex vivo perfusion and video acquisition, the hearts were cooled to 14°C–16°C, then arrested and removed from the OCS™. The heart was then prepared for transplantation in a standard fashion and transplanted into the recipient pig in an intra-abdominal position with the graft aorta anastomosed to the recipient aorta and the graft pulmonary artery anastomosed to the recipient inferior vena cava  capable of returning the XY coordinates of the marker movement for every cardiac beat. The selected VST kernel followed the heart movement and created the trajectory of contraction and relaxation  and returns the kinematic parameters such as contractility , cardiac force, energy expenditure, and trajectory perimeter (tissue compliance), as previously described by Rozzi et al. .To investigate whether the kinematic parameters such as contractility , contraction force (indicating cardiac fatigue), energy (expenditure of energy during contraction/relaxation), and trajectory perimeter (indication of cardiac compliance) were modified over time, data acquisition was divided into four temporal windows  .post-hoc test for multiple comparisons. Statistical analyses were performed using GraphPad Prism version 9.5.1 . The details of the specific test used for each experiment are reported in the figure legends. P-values <0.05 were considered statistically significant.Data are expressed as mean ± SD. Normality was assessed by the Kolmogorov–Smirnov test. Comparisons were performed using one-way ANOVA with Bonferroni The baseline cardiac MRI demonstrated no evidence of compromised function in any of the donor hearts with the ejection fraction (EF) measuring >50%. The representative MRI data are shown in 2, 88.4%–96.0%; and Hct, 18.7%–28.4%. The average total perfusion time was 140 min with a standard deviation of 18 min. The composition of OCS™ perfusate is shown in 2)/beat at the start of perfusion to 3,350 ± 897.4 (px/s2)/beat at the end of perfusion 2/beat at the end of perfusion . This is of great value since moderate PGD is associated with a 12% risk of mortality or re-transplantation and severe PGD with a 40%–50% risk  platform that can predict PGD outcomes in patients based on the beating activity of the donor heart on the OCS™. The application of Vi.Ki.E. to guide AI assessments of the heart has been previously described . Potentiex vivo beating hearts on the OCS™ system using the Vi.Ki.E. system. The results suggest that the cardiac function and performance remain stable on the OCS™, which is an encouraging finding for the expansion of the utility of normothermic ex vivo perfusion for donor heart preservation during transplantation.In conclusion, our study provides valuable insights into the performance of


Journal of Clinical Medicine.We are pleased to see that Peter Wostyn has contributed a Comment: “Could Young Cerebrospinal Fluid Combat Glaucoma?”  in respoThe author proposed a treatment strategy to control intracranial pressure (ICP) based on our findings that lower cerebrospinal fluid (CSF) pressure, which can be expected with a small optic nerve sheath diameter (ONSD), may be involved in the pathogenesis of normal-tension glaucoma (NTG) . In partRecent studies have suggested that changes in the composition of CSF may be involved in the pathogenesis of NTG. Wostyn et al. proposed an alternative pathogenesis of NTG in which CSF circulatory failure results in reduced neurotoxin clearance along the optic nerve . AdditioOur study only indirectly speculated that a small ONSD was associated with a low CSF pressure  and did In conclusion, modifying the composition of CSF to treat glaucoma is an innovative and intriguing idea that warrants further investigation. However, it is questionable whether the small ONSD in NTG patients can justify such treatment. Further research is required to enhance insight into the role of CSF pressure and composition in the pathogenesis of NTG.


We performed group comparisons, association studies with diagnosis, age, and APOE ε4 status, overrepresentation analysis (ORA), and gene set enrichment analysis (GSEA) to determine differentially expressed proteins and dysregulated pathways. (3) Results: GSEA results demonstrated an enrichment of granulocyte-related and chemotactic pathways . Moreover, some of the well-replicated, differentially expressed proteins in CSF included: ITGAM, ITGB2, C1QA, TREM2, GFAP, NEFL, MMP-10, and a novel tau-related marker, SCRN1. (4) Conclusion: Our results highlight the upregulation of neuroinflammatory pathways, especially chemotactic and granulocyte recruitment in CSF of early AD patients.(1) Background: Despite the existence of well-established, CSF-based biomarkers such as amyloid-β and phosphorylated-tau, the pathways involved in the pathophysiology of Alzheimer’s disease (AD) remain an active area of research. (2) Methods: We measured 3072 proteins in CSF samples of AD-biomarker positive mild cognitive impairment (MCI) participants ( Numerou1–40 , and Aβ140 [1–42  in cerebMild cognitive impairment (MCI) refers to the transitional stage between healthy aging and dementia where the earliest symptoms of cognitive decline occur while daily functions are still preserved . ProteinTo investigate upstream mechanisms of AD pathophysiology and the associated protein profile, systems-based approaches, such as proteomics, have been employed ,8. Among1–42/Aβ1–40 and high CSF p-tau181) individuals with MCI compared to controls.In this pilot study, we used the Explore Olink PEA assay to quantify the differential expression of approximately 3000 proteins in CSF of AD biomarker-confirmed . The intra-assay coefficients of variation for this assay were 3.4% for Aβ1–42, 2.7% for Aβ1–40, and 1.8% for p-tau181. The ratio of CSF Aβ1–42/Aβ1–40 and p-tau181 were used in the current analyses. The participants with an Aβ1–42/Aβ1–40 ratio below 0.068 and p-tau181 levels above 50.6 pg/mL were identified as AD-biomarker positive.CSF was collected from 48 cognitively normal and 38 MCI participants. Participants underwent a lumbar puncture and blood collection in the fasted state. Twenty ml of CSF was directly collected in a 50 mL polypropylene tube and transported on ice to the lab along with 20 mL of whole blood in EDTA tubes, where they underwent centrifugation at 2500× All MCI participants selected for this study were required to be AD-biomarker positive based on their CSF values, whereas all controls were required to be AD-biomarker negative based on the cut-offs established by Greenberg et al. .The Olink Explore panel, a highly sensitive and specific technique, measures the expression of 3072 proteins in 10 µL CSF . CSF protein measurements were conducted using PEA technology, following the manufacturer’s protocol .Pre-processing handling of data included plate-based normalization and QC checks based on appropriate Olink protocols . Outliert-test analysis and Benjamini–Hochberg (BH) post hoc analysis. Differentially expressed proteins were defined as assays with false discovery rates (FDR) below 0.05 .Statistical analyses were performed with R software (R-project.org) using the publicly available OlinkAnalyze package. Groups were compared using Welch’s two-sample, two-sided Multivariate linear regression models were used to test for the association of CSF mean NPX values of each protein with each covariate  and for interactions between the covariates, specifically age with diagnosis See . All staAfter performing differential abundance analysis on our proteomics dataset, we conducted overrepresentation analysis (ORA) to identify differentially expressed pathways. This bioinformatics analysis helped us identify pathways that were enriched with significant changes in protein abundance. The goal of this analysis was to gain a better understanding of the functional implications of the differentially expressed proteins and to identify potential biological processes involved in the observed phenotypes.We performed overrepresentation analysis on differentially expressed proteins  using Gene Ontology (GO) biological process and the Wikipathway database . ORA wasGene Set Enrichment Analysis (GSEA) is a computational method that determines whether the expression of a set of genes is significantly different between two phenotypes . GSEA waIn addition to ORA, we also performed GSEA in our proteomics study of CSF from MCI patients. We chose to use both methods because they provide complementary information and can help to validate each other’s results.ORA allowed us to identify pathways that are overrepresented in our list of differentially expressed proteins, whereas GSEA enabled us to assess the enrichment of pre-defined gene sets in the list of differentially expressed proteins ranked by normalized enrichment scores, which can provide a more comprehensive understanding of the biological processes affected by the disease.Using both methods also helped to overcome the limitations of each method, such as the dependence on pre-defined gene sets in ORA and the need for a large reference gene set in GSEA. Overall, by using both ORA and GSEA, we were able to identify novel and previously known pathways that are dysregulated in MCI and gain a better understanding of the underlying mechanisms contributing to the pathogenesis of this disease.We used PEA technology with the Olink Explore panel to determine the differential expression of approximately 3072 proteins in AD-biomarker positive MCI patients compared to the healthy controls . At leasp-value < 0.05; After the pre-processing steps, 2936 proteins were assayed in CSF. Among these, 117 proteins were found to be differentially expressed in CSF based on group comparisons (FDR-adjusted The results of the group comparison conducted between participants younger and older than the median age (71 years old), demonstrated that no proteins were differentially expressed in CSF of younger participants compared to the older participants .Even though, the overrepresentation analyses of the differentially expressed proteins using GO databases did not show any pathways to be significantly enriched among these differentially expressed proteins, it is worth noting that “microtubule polymerization or depolymerization” and “leukocyte activation involved in inflammatory response” were among the top two biological processes that were found to be enriched in the CSF proteins in MCI. The proteins contributing to these terms included: ITGAM, ITGB2, TGFB2, MAPT, HSPA1A, MAP2, and SNCA. ORA performed using the Wikipathways database did not yield FDR-significant results; however, the “Microglia Pathogen Phagocytosis Pathway” was the top enriched biological process in CSF. The proteins that contributed to the enrichment of these pathways included: ITGB2, ITGAM, C1QA, TREM2, MAPT, CRKL, and MAP2K1.Multivariate linear regression analysis of individual proteins with covariates of age, diagnosis, sex, and APOE genotype revealed the assays that were associated with each of these variables and a full model including all covariates in CSF .p-value < 0.05; The multivariate analysis identified 147 proteins in CSF that were significantly associated with diagnosis  was the most enriched term among age-associated proteins. Enrichment ratio is the ratio of the number of observed, differentially expressed genes of a certain pathway divided by the expected value if the pathway was neither enriched nor downregulated. Many chemokine motifs were among assays that contributed to this result. Moreover, C1QA, ITGAM, and ITGB2 are prominent complement-related biomarkers seen in this list.The most enriched biological processes among diagnosis-associated assays were “inclusion body assembly”  and “apoptotic mitochondrial changes” (ER = 4.4). Nonetheless, they were not found to be FDR-significant.Interestingly, the APOE genotype  was significantly correlated with APOE and FGFBP1 in CSF .t-test and the association analysis of diagnosis in the multivariate association study, with and without interactions  = 0.75; The only pathway found to be significant among all canonical pathway databases was the granulocytes pathway in BioCarta , which was associated with diagnosis but not age. Secernin-1 is a novel and specific phosphorylated tau binding protein that has been recently shown to be abundantly present in amyloid plaques and NFTs . Little This study also identified pathways that were enriched in MCI participants compared to controls . Most imDysregulation of the mitogen-activated protein kinase (MAPK) pathway has been demonstrated in AD for several decades ,31. MAPT1–42 oligomers and inducing mitochondria-dependent cell death [The third mechanism prominent in our results is the cellular chaperone network. Chaperones  have been shown to be upregulated in AD animal models as a neuroprotective mechanism inhibiting the aggregation of tau and Aβ fibril formation ,36,37. IOligodendrocyte myelin glycoprotein (OMG) is among the three proteins found to be downregulated in CSF in participants with MCI compared to controls. As evident from its name, OMG is suggested to have a role in regulation of collateral sprouting of intact axons in response to injury and is restricted to oligodendrocytes in the brain . Zhang eMultiple studies have assessed the proteomics of CSF in AD dementia patients using Olink platforms ,30,41,42Apart from other Olink studies, many CSF proteins determined through our experiments have been previously reported in other proteomics studies of AD  ,11,26,42t-test, we cannot exclude the effect of age on our differential analysis by diagnosis. Multivariate analysis, including age as a covariate, identified proteins that showed a statistically significant effect with age for this sample. Moreover, we acknowledge the limitation of the relatively small sample size of our study. We conducted a power calculation for our study, which can be found in the A major limitation of our study is that the control and MCI groups differed by age. While we did not see any differential expression of proteins in CSF on the basis of age alone in the group comparison performed with Welch’s In conclusion, we identified both well-established and novel markers, such as the tau-binding protein secernin-1, in CSF of MCI participants compared to controls. The utilization of pathway analysis methods further highlighted the role of neuroinflammation and especially opsonization of complement-marked synapses by macrophages in early AD biology. Other pathways, including protein misfolding and microtubule assembly pathways, were also implicated based on our results. Taken together, these findings emphasize the involvement of several pathways in early AD pathogenesis and protein markers associated with it, which require further validation.


Patients with subarachnoid hemorrhage (SAH) may develop posthemorrhagic hydrocephalus (PHH), which is treated with surgical cerebrospinal fluid (CSF) diversion. This diversion is associated with risk of infection and shunt failure. Biomarkers for PHH etiology, CSF dynamics disturbances, and potentially subsequent shunt dependency are therefore in demand. With the recent demonstration of lipid-mediated CSF hypersecretion contributing to PHH, exploration of the CSF lipid signature in relation to brain pathology is of interest. Despite being a relatively new addition to the omic’s landscape, lipidomics are increasingly recognized as a tool for biomarker identification, as they provide a comprehensive overview of lipid profiles in biological systems. We here employ an untargeted mass spectroscopy-based platform and reveal the complete lipid profile of cisternal CSF from healthy control subjects and demonstrate its bimodal fluctuation with age. Various classes of lipids, in addition to select individual lipids, were elevated in the ventricular CSF obtained from patients with SAH during placement of an external ventricular drain. The lipidomic signature of the CSF in the patients with SAH suggests dysregulation of the lipids in the CSF in this patient group. Our data thereby reveal possible biomarkers present in a brain pathology with a hemorrhagic event, some of which could be potential future biomarkers for hypersecretion contributing to ventriculomegaly and thus pharmacological targets for pathologies involving disturbed CSF dynamics. Non-traumatic subarachnoid hemorrhage (SAH) accounts for 5% of all strokes ,2,3 and During the last decade there has been a sustained effort to identify various types of acute brain injury-associated biomarkers for indication of cerebral damage ,14,15, aThe lipid profile of ventricular CSF in the group of patients with SAH remains unresolved and, therefore, per extension, also the potential presence of other lipids that may serve modulatory roles in the CSF secretion apparatus. The development of lipidomics,  has allowed insights into the complexity of lipid profiles and their dynamics. Such analysis provides a complete lipid profile of a given compartment and is thus suitable for exploring lipid homeostasis in brain diseases. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), as employed in this study, is an effective analytic platform for untargeted lipidomics with high accuracy and resolution. Here, we employed this technique to determine the lipid profile in CSF obtained from healthy individuals undergoing prophylactic vascular clipping of an unruptured aneurysm (henceforth referred to as controls) and patients with SAH who subsequently developed chronic hydrocephalus in order to identify a specific lipid signature in patients with SAH-associated hydrocephalus that may modulate the CSF dynamics and potentially contribute to the ventriculomegaly. We successfully demonstrate dysregulated CSF lipid levels in patients with SAH-induced PHH.n = 8) or as soon as possible hereafter (n = 5) (range: 3–74 h). Only patients with no signs of neuroinfection at admission or during their treatment were selected to ensure that measured CSF parameters were not affected by neuroinfections requiring antibiotic treatment. Patient samples were randomly chosen from our biobank to fit the following criterion: all enrolled patients with SAH received a permanent ventriculo-peritoneal shunt because of continued need for CSF diversion (development of PHH). Patients who had milder PHH, and thus did not need a shunt, were therefore not included in this study. As control subjects, 11 patients undergoing preventive surgery for unruptured aneurysms (vascular clipping) were enrolled, and CSF was collected from the basal cisterns during surgery prior to clipping of the aneurysm . CSF samples obtained from patients within the same cohort had previously been analyzed for inflammatory markers [Ventricular CSF samples were collected between June 2019 and September 2021 from 13 patients with SAH admitted and treated at the Department of Neurosurgery at Rigshospitalet, Copenhagen, Denmark . All pataneurysm . There w markers ,32. ® filter with extraction solvent, and centrifuged at 1400 rpm at 5 °C for 2 min. Following this, the sample was transferred into a high recovery HPLC vial with eluent mix and capped. Liquid chromatography-mass spectroscopy  coupled to Thermo Q Exactive HF mass spectrometers) was used as untargeted analysis of the CSF samples and conducted by MS-Omics, Denmark. Ionization was performed in positive and negative ionization mode using an electrospray ionization interface. The chromatographic separation of lipids was carried out on a Waters®  ACQUITY Charged Surface Hybrid (CSH™) C18 column . The column was thermostated at 55 °C. The mobile phases consisted of (A) acetonitrile/water (60:40) and (B) isopropanol/acetonitrile (90:10), both with 10 mM ammonium formate and 0.1% formic acid. Lipids were eluted in a two-step gradient by increasing B in A from 40 to 99% over 18 min. Flow rate was 0.4 mL/min. The MS was operated with the following settings: m/z range: 200–1500; AGC target: 1 × 106; resolution: 120,000; collision energy: 30. Compounds were extracted based on features (a peak characterized by one mass and one retention time), and additional information . The retention time of compounds within the same lipid class is estimated using the relation between retention time and the chain length and number of double bonds. Three levels of annotation were used (level 1–3) with level 1 representing the most confident identifications  and estimated retention time), level 2, based on two of these sets of information, is divided into two sublevels; accurate mass and estimated retention, and level 3 is based on library searches using the accurate mass and elemental composition alone. In this study, level 3 annotations were not included. As a quality control, a mixed pool sample was created by taking a small aliquot from each sample to be analyzed with regular intervals through the sequence. Splash lipidomix (Avanti 330707), a single-vial prepared lipidomic analytical standard for human plasma lipids, was diluted 1:50 as prescribed by the manufacturer and used as internal standards for QC check  to ensure that all samples were analyzed correctly. The mixture components were 15:0–18:1(d7)PC (150.6 μg/mL), 15:0–18:1(d7)PE (5.3 μg/mL), 15:0–18:1(d7)PS (3.9 μg/mL), 15:0–18:1(d7)PG (26.7 μg/mL), 15:0–18:1(d7)PI (8.5 μg/mL), 15:0–18:1(d7)PA (6.9 μg/mL), 18:1(d7)LPC (23.8 μg/mL), 18:1(d7)LPE (4.9 μg/mL), 18:1(d7)cholesterol (329.1 μg/mL), 18:1(d7)MG (1.8 μg/mL), 15:0–18:1(d7)DG (8.8 μg/mL), 15:0–18:1(d7)-15:0 TG (52.8 μg/mL), 18:1(d9)SM (29.6 μg/mL), cholesterol(d7) (98.4 μg/mL). Data were processed using Compound Discoverer 3.0 . CSF was transferred into a Spin-Xn = 11; SAH, n = 13) and the number of outliers was counted for each sample. If a patient sample presented more than 20% outliers, this patient sample was excluded from the rest of the analysis . All 358 compounds were manually curated into 35 main classes of lipids, non-biological lipid compounds, and non-classified (‘others’), of which the latter two groups were excluded from the analyses. Fourteen groups, all containing less than four lipids , were assembled into one group . The resulting 17 groups with 244 compounds were employed for the analyses. An enrichment plot of the number of lipids detected within each group was generated for the 17 groups counting all compounds assigned to each group. To obtain the total lipid abundance for each group, the lipids were normalized based on the geomean of the quality control samples (n = 15). An enrichment plot was generated for normalized mean abundance by summing the normalized values of lipids in each group upon which the mean of all controls or patients with SAH was calculated. Principal component analysis (PCA) [t-test followed by the Benjamini−Hochberg method  [https://github.com/Sorennorge/MacAulayLab-SAH-Metabolomics (accessed on 8 August 2023).A Smirnov−Grubbs test  was performed for all detected compounds 358) within each patient group   plots we of 10%) ,35. All 8 within To delineate the lipid composition in the cisternal CSF obtained from control subjects  undergoiTo reveal a potential sex-dependent CSF lipid distribution in control individuals, we employed a principal component analysis (PCA). The comparison of overall lipid profiles of each sample revealed a similar lipid distribution in CSF obtained in males and females with no distinct grouping of sexes A, which To map the lipid composition in patients with SAH , we emplp = 0.49). To identify the individual lipid classes that drive the diversion of the overall SAH lipid profile from the control subjects, we compared the total abundance of each lipid class between the two groups. On a lipid class basis, the abundance of fatty acids, phosphatidic acids, plasmenylphosphatidylethanolamines, and monoglycerols was significantly elevated in the CSF from patients with SAH  to associate with the female sex , althougPatients with SAH demonstrated a distinct CSF lipid signature with upregulation of several classes of lipids, i.e., phosphatidylcholines, plasmenylphosphatidylethanolamines, phosphatidic acids, fatty acids, and monoacrylglycerols. In addition, a cluster of lipids were detected as highly elevated in patients with SAH, but only significantly so without correction for multiple testing . With analysis of each lipid class in isolation, a range of individual lipids stood out as significantly upregulated in the patients with SAH and could potentially serve as biomarkers for SAH progression and PHH formation. Phosphatidylcholines, several of which ) were upregulated in the CSF from patients with SAH, play critical roles in signaling cascades in cell apoptosis and various inflammatory responses contributing to SAH-induced early brain injury in animal models . In addiWe identified five members of the phosphatidic acid lipid class being upregulated: PA22:0, PA24:0, PA26:0, PA28:0, and PA45:4. Phosphatidic acids are the simplest (diacyl)glycerophospholipids present in cells, a second messenger, and the common intermediate in synthesis of all phospholipids. The levels of phosphatidic acids are often seen to be elevated following activation of inflammatory cells . LysophoThe SAH-induced lipid abundance could arise with the entry of blood into the ventricular compartment or as a biological response to the hemorrhagic event. Irrespectively thereof, the CSF lipid composition could contribute to a coupling between a brain hemorrhagic event and the ensuing ventricular enlargement signifying PHH. Removal of a choline group from the lysophosphatidylcholine, by the action of a lysophospholipase, yields LPA, which has earlier been detected in patient CSF following TBI  and SAH In the current study we employed an untargeted mass spectroscopy-based platform to quantify the lipid content in ventricular and cisternal CSF, which is the first of its kind, contrasting with earlier targeted studies based on CSF obtained from the lumbar CSF compartment . LimitatBiomarkers which reliably reflect CSF homeostasis following SAH would be extremely useful for clinical assessment and decision making. If such biomarkers can be used to distinguish between individuals with only a temporary need for CSF diversion and those with a long-term or permanent need, EVD discontinuation in the former group vs. shunt insertion in the latter group could be decided earlier, making it possible for patients to leave intensive care unit environments and begin rehabilitation. Biomarkers reflecting the extent of brain tissue damage would be useful to assess functional prognosis and rehabilitation potential and might also assist in distinguishing exhausted rehabilitation potential due to the primary brain damage from shunt dysfunction.In conclusion, we demonstrate that, of the 357 detected compounds, 244 lipids assigned to 17 different classes made up the lipid profile of the control subjects. The lipid distribution is not sex dependent but does fluctuate with age. Our results revealed a CSF lipid signature for patients with SAH, which encourages future studies aimed at elucidating if any of the identified dysregulated lipids induces pathophysiological changes in the brain fluid dynamics occurring with SAH. Such studies could aim to identify novel agonists of the molecular machinery underlying lipid-induced CSF hypersecretion, and thus novel lipid biomarkers with the potential to be employed in predicting the shunt dependency and/or outcome of SAH.


Pelvic obliquity (PO) has not been extensively investigated, and there is no gold standard for measurement. The PO is essential for surgeons in planning hip arthroplasty, which includes the restoration of leg length discrepancy (LLD). We aimed to establish a normative range of PO angles by measuring healthy individuals without musculoskeletal disorders.Our study included 134 consecutive cases  referred to our institution between April 2020 and September 2021 for non-orthopedic problems. Patients were screened for normal gait and posture using the visual observation method (VOM) and the Modified Gait Abnormality Rating Scale (GARS-M). In standing standard radiographs, the PO angle was measured as the angle between the horizontal plane and the inter-teardrop line.P < 0.001). The PO angle did not differ significantly between males and females , nor did it correlate significantly with age (P = 0.24). Considering the 95% percentile of PO angles was 5.6°, this range (0°—5.6°) was regarded as a normative value.Patients' mean age and Body Mass Index (BMI) were 39.7 ± 16.8 and 22.3 ± 3.1, respectively. PO angles did not follow a normal distribution, with a median (IQR) of 2.0° (0.9°—3.1°). According to the Wilcoxon one-sample test, the median PO angle differed significantly from zero (Normative values for PO in the normal healthy population range from 0° to 5.6°, with a median value of 2.0°. The PO angle was independent of age and sex and differed significantly from 0°. Slight pelvic obliquity may be normal, and physicians should not always assume that it is caused by pain, scoliosis, or weakness of the abductors.III.The online version contains supplementary material available at 10.1186/s40634-023-00613-z. Pelvic obligity (PO) has not been extensively studied, and there is no gold standard for measuring it . A balanPO can be defined as a deformity between the spine and the pelvic axis . Several. reported that the mean PO angle in asymptomatic patients is – 0.1 ± 1.6 [PO can result from various etiologies, including spinal deformity, hip contracture, leg length discrepancy (LLD), antalgic positions, muscle weakness, and idiopathic reasons . The pre.1 ± 1.6 . For sco.1 ± 1.6 , 20.The present study aimed to define a normative range of PO angles by measuring healthy people without musculoskeletal disorders.N = 15 excluded). Also, the postural state was assessed by the visual observation method (VOM) [N = 10 excluded). Further, all patients were evaluated by standard 3-joint views  referred to our institution for non-orthopedic problems from April 2020 to September 2021. Inclusion criteria included patients without spinal, pelvic, or lower extremity abnormalities or a history of orthopedic surgery. Any gait or posture abnormality, significant LLD (> 1 cm), and significant varus/valgus lower limb mal-alignment were considered exclusion criteria  , 13. AftFor AP X-rays of the pelvis, all patients underwent the same protocol in a similar radiology department. The patients were standing, both legs in 15° internal rotation. Film and tube distances were standardized at 1.15 m. The beam was centered on the pubis symphysis. The coccyx should be aligned with the pubic symphysis with 2.5 cm intervals between them in the female sex and 1.5 cm in the male sex, as described by Tannast et al. . PO anglP-values of less than 0.05 were considered significant.Finally, data were analyzed with IBM SPSS software . Data are presented as mean and standard deviation (SD) for normal continuous variables and median, interquartile range (IQR) for non-normal continuous variables, while categorical data are presented as numbers and percentages. Kurtosis, skewness, Kolmogorov–Smirnov, and Shapiro–Wilk tests were used to determine whether the samples had a normal distribution. One-sample Wilcoxon test was used to compare the PO angle and zero. Mann–Whitney U and Spearman's correlation tests were used to analyze the association between PO angle and sex and age. The Institutional Review Board (IRB) reviewed and approved the study design and confirmed no ethical concerns in the present study. Furthermore, we obtained informed consent from all patients in this study.P < 0.05).There was a mean age of 39.7 ± 16.8 and a mean Body Mass Index (BMI) of 22.3 ± 3.1. Kurtosis and skewness tests indicated that PO angles were not normally distributed (kurtosis and skewness to standard error ratio = -0.32 and 3.4). The Kolmogorov–Smirnov and Shapiro–Wilk tests were also in agreement (P < 0.001). According to the Mann–Whitney U test, there were no significant differences in PO angle between males and females (2.0° (0.7 – 2.8) vs. 2.0° (0.9 – 3.3), P = 0.46). There is no correlation between age and PO angle   PO angle was 2.0° 0.9 – 3.1) Fig.  . PO angles of 95% of the patients were less than 5.6°, suggesting that 0°—5.6° is the normal range for PO in a healthy normal population .This study found that the median (IQR) of PO angle in the normal healthy population without musculoskeletal problems was 2.0 (0.9—3.1), significantly different from zero , which have different treatments such as fixing the spine deformity and balancing both legs.There were limitations to this study. First, we did not assess PO angle with other measurement methods but only by evaluating the angle between the horizontal plane and the Inter-teardrop line. The second limitation was that we only studied the normal PO angle in the small sample size, which could limit generalizations to the entire population. Moreover, the cohort consisted of patients without musculoskeletal problems; however, these were not healthy adults as they were referred to a clinic for non-orthopedic problems. Thus, healthy asymptomatic volunteers should be recruited to identify PO in the completely healthy normal population.The normal range of PO angle was 0° to 5.6° degrees, with 95% of the population having a PO angle within this range. A physiologic PO angle was not associated with age and sex and differed significantly from 0.Additional file 1: Supplementary Table 1.Modified Gait Abnormality Rating Scale (GARS-M) described by Wolfson et al in 1990. Supplementary Table 2.The postural examination from head-to-toe with visual observation method (VOM).


Serum troponin levels correlate with the extent of myocyte necrosis in acute myocardial infarction (AMI) and predict adverse outcomes. However, thresholds of cardiac troponin elevation that could portend to poor outcomes have not been established.In this cohort study, we characterized all cardiac troponin elevations > 0.04 ng/mL  from patients hospitalized with an ICD-9/10 diagnosis of AMI across our health system from 2012–2019. We grouped events into exponential categories of peak cardiac troponin and evaluated the association of these troponin categories with all-cause mortality, heart transplants, or durable left ventricular assist devices (LVAD). Patients with cardiac troponin > 10,000 × ULN were manually chart reviewed and described.There were 18,194 AMI hospitalizations with elevated cardiac troponin. Peak troponin was 1–10 × ULN in 21.1%, 10–100 × ULN in 34.8%, 100–1,000 × ULN in 30.1%, 1,000–10,000 × ULN in 13.1%, and > 10,000 × ULN in 0.9% of patients. One-year mortality was 17–21% across groups, except in > 10,000 × ULN group where it was 33% (adjusted hazard ratio (99%CI) for > 10,000 × ULN group compared to all others: 1.86 ). Hazards of one-year transplant and MCS were also significantly elevated in the > 10,000 × ULN group.Elevation in cardiac troponin levels post AMI that are > 10,000 × ULN was rare but identified patients at particularly high risk of adverse events. These patients may benefit from clarification of goals of care and early referral for advanced heart failure therapies. These data have implications for conversion to newer high-sensitivity cardiac troponin assays whose maximum assay limit is often lower than traditional assays.The online version contains supplementary material available at 10.1186/s12872-023-03168-0. Cardiac specific troponins, including Troponin T and Troponin I, are regulatory proteins unique to the myocardium. The serologic concentration of these proteins rise and fall in the setting of myocardial injury . The utiSeveral studies have shown a correlation between the magnitude of cardiac troponin elevation and the extent of myocyte necrosis, the development of heart failure, and the risk of mortality –7. HowevPatients with massive myocardial injury are at high risk for adverse outcomes, including death. However, the clinical course can be variable and difficult to predict. These patients may be eligible for durable left ventricular assist device (LVAD) or cardiac transplantation, but getting patients to such therapies usually requires timely engagement of an advanced heart failure program, sometimes with early initiation of temporary circulatory support. Late referral after massive myocardial infarction when patients have progressed to multi-organ failure can cause patients to “miss the window” for advanced therapies .Given the known relationship of cardiac troponin levels to degree of cardiomyocyte injury and based on anecdotal cases of patients whose cardiac troponin level was greater than assay maximum, we hypothesized that markedly elevated peak cardiac troponin is associated with particularly high rates of adverse outcomes. Better characterizing this relationship of very high cardiac troponin levels to outcomes could improve rapid triage of in-hospital care, guide post-discharge planning, and encourage completion of advance care directives for this high-risk population. In addition, if an absolute threshold of cardiac troponin elevation could be identified beyond which outcomes were worse than that seen with LVAD and transplant, a simple rule of thumb could be created to prompt immediate consideration for engagement of advanced heart failure or palliative care consultation.Therefore, we aimed to systematically characterize all cardiac troponin elevations across a diverse health system and compare absolute cardiac troponin levels to 1-year outcomes , and heart transplant) in these populations.https://www.healthdatacompass.org/). All procedures were approved by the Colorado Institutional Review Board and were conducted in accordance with the principles outlined in the Declaration of Helsinki.UCHealth includes 12 hospitals and over 30 clinics within Colorado that cover 19% of the 5.8 million Coloradans, as well as referrals from relatively rural areas in surrounding states. Major efforts were made in 2011 to convert the entire system to a single instance of the Epic electronic health record for all inpatient and outpatient activities. Since 2011, all laboratory test results, as well as diagnoses, vital signs, procedures, encounters, and deaths are reported in the UCHealth Epic record and then downloaded into the Health Data Compass virtual data warehouse  based on International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9)  and ICD-10  codes. Based on this cohort, additional data collected were demographics , labs obtained during AMI encounters , and primary outcomes in the following year after the hospitalization event .th percentile of the target population. However, troponin data stored in the electronic health record did not include assay details. Within this context, we used a singular cutoff of 0.04 ng/mL for this analysis. This approach ensured patients met criteria for the Fourth Universal definition of myocardial injury (i.e. a serum cardiac troponin test result > 99th percentile of the ULN). This approximation also reflected chest pain-myocardial infarction treatment pathways at all health system sites which used a cutoff of 0.04 ng/mL regardless of assay. We limited our patient selection to age ≥ 18 years. For patients with multiple events, all events and associated cardiac troponin were considered.All hospitals used a standardized “Chest Pain Protocol” pathway, which recommended cardiac troponin testing at presentation, 3 h, and 6 h. During the study period, the cardiac troponin assay most commonly used for clinical diagnosis across hospitals was the ADIVA Centaur cardiac troponin I assay. The reference range for the assay was 0.0–0.04 ng/mL. A minority of troponin values reflected the use of alternative assays, primarily the Ortho Clinical Diagnostics VITROS 5600 (reference range 0.00–0.03 ng/mL). Upper limit of normal (ULN) was determined as above the 99For the subgroup of patients with cardiac troponin > 10,000 times the ULN (> 400 ng/dL), the clinical course was further explored via manual chart review . Data obtained included MI type, procedures, and types of temporary MCS utilized, as well as confirmation of death, durable MCS, and heart transplant.Patients and/or the public were not involved in the design, conduct, reporting, or dissemination plans of this research.Patients were described using counts and percentages, both overall and by cardiac troponin level. cardiac troponin was grouped into exponential categories: 1–10 × ULN . Initial survival comparisons between groups were performed using Kaplan–Meier plots, and Kaplan–Meier estimates were used to obtain outcome rates at 1 year; death estimates account for censoring at the time of data collection for patients with recent events, while other estimates account for censoring both at the time of data collection and due to death. Hazard Ratios (HRs) and associated confidence intervals (CIs) for each troponin category compared to the reference category were obtained via Cox models, with robust sandwich covariance matrix estimates to account for the correlation among multiple events for some subjects. Analyses were repeated with the addition of several potential confounders–age, race, ethnicity, diabetes, hypertension, smoking status, and creatinine level– added to the model to obtain adjusted hazard ratios. Finally, all analyses were repeated with lowest 4 categories combined, compared with the > 10,000 × ULN (> 400 ng/mL) group, to provide hazard for this group compared to all other subjects. CIs presented are 99% confidence intervals, corresponding to an alpha of 0.01. Analyses were conducted in SAS version 9.4.Between 2012 and 2019, 27,570 hospitalizations occurred where a primary diagnosis of AMI was assigned. Of these, 8,210 (30%) did not have a cardiac troponin value recorded, and 1,166 (4%) had a peak cardiac troponin value ≤ 0.04, resulting in an analytic sample of 18,194 events. Some patients had multiple events in the dataset, resulting in a patient-level sample of 15,800 unique patients . Baselinn = 3,833) had a peak cardiac troponin 1–10 × ULN  had a peak cardiac troponin 10–100 × ULN  had a peak cardiac troponin 100–1,000 × ULN  had peak a cardiac troponin 1,000–10,000 × ULN  had peak troponin > 10,000 × ULN (> 400 ng/mL),  received durable LVAD placement and 2.5% (n = 3) received a heart transplant within one year. Fewer than 1% of patients in the other groups were noted to have received a LVAD or heart transplant within one year. For LVAD this difference was not statistically significant; for transplant the AHR (99% CI) for the cardiac troponin > 10,000 ULN group compared to all others was 17.04 .At 1 year, mortality rates were highest with highest levels of cardiac troponin Fig. . Of the n = 158). Most patients received a PCI within 7 days of cardiac troponin peak  and only 5 patients received CABG within 7 days (3%). In our manual chart review performed in this group, it was noted that 42 MCS procedures performed prior to transfer between hospitals were not captured by the automatic data extraction leading to a difference between patients requiring temporary MCS in the data extraction at 16% vs 41% when manual chart review was performed in this group. For those n = 69 (41%) who received temporary MCS during the hospitalization, n = 40 (24%) received intra-arterial balloon placement, n = 27(16%) received Impella support, and n = 10 (6%) needed extracorporeal membrane oxygenation. Five deaths were picked up in the > 400 ng/mL group through manual chart review which were missed during automated data extraction.For all 168 patients with cardiac troponin > 10,000 ULN (> 400 ng/dL), the clinical course was further explored via chart review Table . Almost This retrospective observational study aimed to identify whether a threshold peak cardiac troponin level at the time of AMI could quickly identify patients at particularly high risk of subsequent adverse outcomes, and thus rapid consideration of MCS, LVAD placement, heart transplant, or involvement of palliative care. We identified that an inflection point indicating much worse outcomes exist in patients with cardiac troponin levels elevated > 10,000 times the ULN (> 400 ng/mL). Though this is an infrequent occurrence, accounting for only 1% of all our cases, this group had a statistically higher incidence of death at 33% vs 17–21% in all other groups (AHR (99% CI): 1.86) and captured all use of advanced therapies (transplant and LVAD) in the following year.n = 168), one third died, more than double the rate of death after transplant or durable LVAD implantation. We therefore conclude that early consideration and referral for advanced therapies in this small sub-set of patients would be reasonable.Although there is a well understood relationship between the magnitude of troponin elevation and the extent of myocyte necrosis , the quan = 132). This group was noted to have significantly worse outcomes, including mortality, than the more modest troponin elevations. This is a noteworthy observation as there exists uncertainty as to the clinical significance of periprocedural cardiac biomarker elevation [Peak troponin was noted in the post-PCI period for most patients in the highest troponin group . Third, low event numbers for some outcomes, particularly LVAD and transplant, resulted in wide confidence intervals and limited precision in our estimates and between-group comparisons. Fourth, our estimation of hazards of MCS, transplant, and LVAD in the presence of the competing risk of death assumes that events are independent, and our hazard ratios may be altered to the extent that this assumption is not met. Fifth, we only assessed cardiac troponin I and not other cardiac troponins. The goal of our study was not to assess the relative performance of multiple assays, but rather to assess the prognostic information conferred by orders of magnitude serum troponin elevation above ULN. Last, HsTrop are currently being increasingly adopted and traditional troponin assays will likely be phased out in the future.Overall, cardiac troponin > 10,000 times the ULN (> 400 ng/mL), though rare, has particularly poor outcomes in regard to requiring mechanical circulatory support and death. This serves to identify a group where clarification of goals of care and consideration for early referral for advanced therapies may prove valuable. Here we also highlight the relevance of measuring cardiac troponin until peak even if patients undergo PCI. Current HsTrop assays that are being increasingly adopted, often have lower upper limits of quantification, which may lead to a loss of prognostic information after AMI.Additional file 1: Supplemental Table 1. Patient-level and event level demographics.