We hypothesized that perioperative midazolam administration is associated with an increased incidence of myocardial injury in patients undergoing non-cardiac surgery (MINS) and that circadian biology may underlie this relationship.Animal studies have shown that midazolam can increase vulnerability to cardiac ischemia, potentially We analyzed intraoperative data from the Multicenter Perioperative Outcomes Group for the occurrence of MINS across 50 institutions from 2014 to 2019. The primary outcome was the occurrence of MINS. MINS was defined as having at least one troponin-I lab value ≥0.03 ng/ml from anesthesia start to 72 h after anesthesia end. To account for bias, propensity scores and inverse probability of treatment weighting were applied.A total of 1,773,118 cases were available for analysis. Of these subjects, 951,345 (53.7%) received midazolam perioperatively, and 16,404 (0.93%) met criteria for perioperative MINS. There was no association between perioperative midazolam administration and risk of MINS in the study population as a whole (odds ratio (OR) 0.98, confidence interval (CI) ). However, we found a strong association between midazolam administration and risk of MINS when surgery occurred overnight  or when surgery occurred in ASA 1 or 2 patients .Perioperative midazolam administration may not pose a significant risk for MINS occurrence. However, midazolam administration at night and in healthier patients could increase MINS, which warrants further clinical investigation with an emphasis on circadian biology. In the perioperative setting for non-cardiac surgery, the incidence of myocardial injury in patients undergoing non-cardiac surgery (MINS) has been reported to be as high as 16% , these eMidazolam, a benzodiazepine which binds to receptor sites in the gamma-aminobutyric acid (GABA) system, first came into use in 1976 and is on the World Health Organization’s List of Essential Medicines. It is the most commonly used pre-procedural sedative-hypnotic worldwide . BecauseRecently, our group determined that the circadian rhythm protein Period 2 (PER2) provides robust cardio-protection from myocardial ischemia (MI) in an animal model , 14. We Approval was obtained, and waiver of written informed consent was granted from the Institutional Review Board [Colorado Multiple Institutional Review Board (COMIRB)] at the University of Colorado Denver, USA (#09-0674). In keeping with the Multicenter Perioperative Outcomes Group Bylaws at the University of Michigan, this study protocol was presented to the Multicenter Perioperative Outcomes Group Perioperative Clinical Research Committee and was approved on August 12, 2019. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist in developing this manuscript.The Multicenter Perioperative Outcomes Group (MPOG) database, as well as methods for data entry, validation, and quality assurance, have been previously described  and haveData for 2,740,183 subjects undergoing non-cardiac surgical procedures between 1 January 2014, and 31 December 2019, were obtained from MPOG. Various preoperative demographic and comorbidity variables were available, as were data on the timing and dosage of midazolam administrations and the timing and magnitude of troponin-I lab values. From this sample of 2,740,183 subjects, we excluded all subjects who met one or more of the following exclusion criteria: emergent cases, outpatient procedures, patients admitted for less than 24 h, patients with ASA value 5 or 6, preoperatively intubated patients, patients with preoperative troponin elevation , pre-induction vasopressor or inotrope infusion, intraoperative transfusion >4 units of blood, estimated blood loss >2000 ml, lung-transplant surgery (CPT code between 32850 and 32856), liver-transplant surgery (CPT code between 47133 and 47147), cardiac procedures (CPT code between 33016 and 33999), and individuals under 18 years of age. 2,264,900 subjects remained after applying these exclusion criteria.Perioperative MINS was defined as having at least one troponin-I lab value ≥0.03 ng/ml in the period from anesthesia start to 72 h after anesthesia end. Perioperative midazolam administration was defined as having at least one documented midazolam administration in the period from 2 h before anesthesia start to anesthesia end. The mean dose of midazolam was 2.3 mg.As typical for large observational data extracted from electronic health records, there was some amount of missingness in our data. Of those 2,264,900 subjects who met the inclusion criteria, 9.76% had missing information regarding BMI and 0.05% regarding sex. We opted to only include those subjects without missingness in our analytic dataset. Given that our sample size was substantial, we were not concerned with efficiency loss due to a complete case analysis as opposed to an imputation approach. Recommendations from literature indicate that, as long as there is no missingness in an effect modifier variable , complete case analysis will be unbiased regardless of the missingness pattern of the covariate  . In thisComplete data from the Multicenter Perioperative Outcomes Group database included age, sex, body mass, institution, American Society of Anesthesiologists  physical status and various comorbidities .The primary outcome of our study was perioperative MINS in relation to human circadian physiology.via the “WeightIt” package (version 0.12.0). The propensity score was estimated via logistic regression using all baseline covariates as predictors, with no higher-order or interaction effects. A recounting of the baseline covariates and their stratified distribution in the analytic dataset is presented in via the “WeightIt” package, and balance was assessed as standardized mean difference <0.1 (Data are presented as coefficient estimates (adjusted odds ratios) with confidence intervals. To account for possible bias and confounders in the observational data, propensity scores were used to balance the distribution of baseline covariates between the population with and without perioperative midazolam administration , 24. Spence <0.1 .via the “survey” package (version 4.1-1). For this analysis, stabilized inverse probability of treatment weights were used. Circadian physiology analyses, including assessment of potential effect modification of time of day or ASA class on the effect of midazolam administration on perioperative MINS, were conducted using marginal structural models with an interaction effect. The time of day was divided into daytime (between 6:00 and 18:00) and a nighttime period (between 18:00 and 06:00) based on anesthesia start. Marginal structural models were also estimated via weighted logistic regression with robust variance estimation via the R package “survey.” For theses analyses via marginal structural models, weights were stabilized as per Section 12.5 of Hernán and Robins (For assessing the effect of (binary) midazolam administration on perioperative MINS, we used a weighted logistic regression model with robust variance estimation, implemented in R d Robins . This stV while adjusting for the set of baseline covariates L, the following model is used: E β0β1αβ2Vαβ3V [1]. In order to isolate the causal effect of α  on a binary outcome  while assessing potential effect modification by V , we estimated the above model in a weighted logistic regression framework via the R package survey (version 4.1-1), where weights are generated via propensity scoring. Weights are estimated as follows, per the recommendation of Hernán and Robins:Per Hernán and Robins, whether the effect of exposure α on the outcome is modified by the particular covariate V is the ASA class . For the time-of-day model, V is the time of day (either overnight or day). Thus, for each of these two paradigms, we estimated model [1] via weighted logistic regression using weights as described in [2]. For the ASA model, weights as described in [2] are equal to the ratio of the probability of midazolam administration (α) as predicted by ASA class (V) to the probability of midazolam administration as predicted by all baseline covariates including ASA class (L). For the time-of-day model, weights as described in [2] are equal to the ratio of the probability of midazolam administration (α) as predicted by time of day (V) to the probability of midazolam administration as predicted by all baseline covariates including time of day (L).We estimated two models in this framework: one to assess whether ASA class modifies the effect of midazolam administration on MINS, and one to assess whether time of day modifies the effect of midazolam administration on MINS. For the ASA model, The large sample size  and an estimated incidence of 0.4% for MINS provided a >99% power at an alpha of 0.05 to detect any differences between the groups receiving midazolam or not receiving midazolam.Of 2,264,900 cases that were eligible for analysis, 491,782 were excluded for missing data. A total of 1,773,118 cases from 50 institutions were available. Based on observations that midazolam alters the risk for myocardial ischemia in a circadian-rhythm-dependent manner, we performed two circadian physiology related analyses: (1) time-of-day dependent effects of midazolam administration on MINS risk, as circadian mechanisms are expected to cause variations in MINS risk throughout a full circadian cycle (24 h); and (2) ASA classification dependent effects of midazolam administration on MINS risk, as higher ASA classification (3+4) include an older population with more comorbidities in whom circadian rhythms are known to fade and become dysfunctional , 29.As shown in p 2 10−16), demonstrating that the effect of midazolam on MINS occurrence was modified by the time-of-day. While midazolam administration during the daytime decreased the risk for MINS , when compared to no midazolam administration. Our nighttime analysis included 161,250 individuals , demonstrating that the effect of midazolam on MINS occurrence was modified by the ASA class. Indeed, midazolam was associated with a moderately increased rate of MINS in the low ASA (1+2) class group  who had a total MINS rate of 0.23%. However, the association between midazolam and MINS was substantially attenuated, and in fact changed directions, when a subject was in the high ASA group (3+4), who had a total MINS rate of 1.44%. For a subject in the high ASA group, the administration of midazolam was associated with an odds ratio of 0.94 for MINS .Finally, we analyzed the relationship between midazolam administration and MINS risk in ASA 1+2 and ASA 3+4 patients D. We fouHowever, as shown in In this study, we examined the relationship between perioperative midazolam administration and MINS. Overall, we did not find an association between midazolam administration and the rate of MINS. However, midazolam administration was associated with an increased risk of MINS when surgeries occurred at night or in healthier patients in the ASA 1+2 class. Interestingly, we found a MINS risk reduction when midazolam was given to higher risk patients (ASA 3+4 class). When assessing the time of day, however, the increased risk of MINS at night was present in both ASA 1+2 and ASA 3+4.Per and Cryptochrome (Cry) genes, resulting in high levels of these transcripts. The resulting PER and CRY proteins translocate to the nucleus to inhibit their own transcription (Per1 and Per2 genes is mediated by the binding of phosphorylated CREB (cyclic AMP responsive element-binding protein) to a cAMP-responsive element (CRE) in the respective promoters  such as from postoperative ischemia, sepsis, neurogenic cardiomyopathy, etc., might have been missed and could therefore be possible confounders. Again, this would not fully account for the findings of our analyses regarding time-of-day or ASA-class effects.Finally, MINS was most likely underestimated in our sample as most institutions do not measure postoperative troponin routinely. As a result, our analysis treated any subject without a perioperative troponin-I as negative for perioperative MINS resulting in a MINS rate of 0.93%. However, previous studies have reported a MINS rate of 8.0% among patients that are 45 years or older . Given tThis large retrospective observational study suggests that perioperative administration of midazolam during nighttime surgeries as well as administration in healthier patients may increase the risk of MINS. The fact that we did not find an overall relationship between perioperative midazolam and MINS but found differences in risk at certain times of day suggests that chronobiology may play an important role in MINS and other perioperative outcomes. Future studies are needed that assess these time-of-day dependent effects.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Colorado Multiple Institutional Review Board (COMIRB) at the University of Colorado Denver, United States (#09-0674). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MP, JP, DD, BS, and AG: wrote the manuscript and analyzed the data. TE: designed the study, wrote the manuscript, and analyzed the data. All authors contributed to the article and approved the submitted version.


Nanoranavicina and Hazara Torrent Frog Allopaahazarensis are associated with the torrential streams and nearby clear water pools in subtropical chir pine forest and other forest types, at elevations higher than 1000 m in Pakistan. In this study, we have provided data on the extent of movement of these frog species for the first time. We installed radio transmitters on a total of 13 Murree Hills Frogs and 13 Hazara Torrent Frogs during eight consecutive days in September 2017 and 2018. Our results showed that these frogs did not move long distances along the stream or away from the stream into the forest. All the radio-tracked frogs showed movement of < 3 m. We found a significant differences only in the distance moved by Murree Hills Frogs between the two years studied. Based on our findings, we propose a movement paradigm that focuses on conservation implications for these endemic frogs.Endemic anurans are particularly vulnerable to environmental changes, and are susceptible to population declines because of their restricted distribution ranges. The Murree Hills Frog  Conservation of amphibians is becoming more vital due to the increasing global extinction rate in this group . AmphibiAllopaahazarensis and Nanoranavicina and proposed a movement paradigm that focuses on conservation implications for these endemic frogs.Studies on habitat use provide useful information for species conservation . EndemicStudy area and species.— We conducted the present study on Murree Hills Frog (Nanoranavicina) and Hazara Torrent Frog (Allopaahazarensis) at a natural freshwater stream , located in Village Parhanna, Tehsil Murree, District Rawalpindi, Punjab Province, Pakistan. This stream cascades over rapids and has a few associated ponds  and followed the attachment method by Radio tracking.—We installed radio transmitters on three Murree Hills Frogs  and five Hazara Frogs  in September 2017 and 10 Murree Hills Frogs  and eight Hazara Frogs  in September 2018 (non-breeding season) for eight consecutive days during each session. Details on specimens  and transmitters are given in Table 1. Since the two studied frog species are nocturnal, we located and observed the tracked frogs three times, every three hours from sunset to sunrise. We recorded the distance moved by each frog and calculated mean distances (m) moved for males and females of each species for the entire session.P > 0.05 for Shapiro-Wilk test) in SPSS 25, we used the Mann-Whitney test to compare distances (median) moved by males and females of each species in a given year and distances moved by radio-tracked frogs  of each species between 2017 and 2018 (α = 0.05).After testing normality of our data  during the study period and, consequently, distance data were not recorded in these cases (Table P = 0.046) in 2017 and 2018 with more distance travelled in 2017  as compared to 2018 . We did not find significant differences between any of the radio-tracked males and females of Hazara Torrent Frogs in 2017 , 2018  and of Murree Hills Frogs in 2017  and 2018  and Hazara Torrent Frogs (pooled data for the two sexes)  between 2017 and 2018.We did not observe much movement (limited to < 3 m) along the stream or away from the stream into the forest by either species. The mean distance (m) moved by radio-tracked males and females of Hazara Frogs and Murree Hills Frogs in 2017 and 2018 is given (Table Allopaahazarensis) and Murree Hills Frog (Nanoranavicina), for the first time. Our data showed that these frog species exhibit limited movement during the observed time period. A synthesis of review on movement and dispersal in amphibians by Ranaluteiventris) adults moved distances greater than 200 m. Ranasylvatica) moved distances of 2,530 m. Xenopuslaevis) made over-land movements with distances of ~ 150 m and only 91 individuals moved distances of 2.4 km. The limited extent of movement exhibited by the Murree Hills Frog and the Hazara Torrent Frog during the present study indicates that these endemic frogs depend on a specific stream to live and reproduce, which have critical implications for their conservation.We provided data on the movement pattern of two frogs endemic to Himalayan region, Hazara Torrent Frog (The Murree Hills Frogs and Hazara Torrent Frogs are facing anthropogenic threats, such as habitat degradation, urbanisation and natural threats, as well as climate change . Accordimove upstream, which would require use of energy reserves and may subject newly dispersed/immigrants to competition with individuals already inhabiting upstream areas,move downstream into unsuitable habitat in lower elevations, with more urban settlements, pollution and deforestation.The species are not expected to take the risk of dispersal into subtropical scrub streams located further south due to unfavourable habitat and unsuitable environmental conditions. Increase in the air and water temperature or water withdrawal from the streams by the local community could seriously impact populations of these species. The species might be forced to perform over-land migration through the forest to occupy nearby streams, which are situated at a distance difficult to travel by amphibians or perform upstream migration that would require considerable energy reserves and may cause stress in the individuals Fig. . VariousWe could have provided a more detailed data of movement in these frogs if we had not lost 38% of our fitted transmitters. The terrain is hilly and the stream had large boulders which were difficult to move. It was unclear whether the transmitters were lost while the frogs hid beneath the boulders, due to predation or to heavy rains which created flooding in the stream. For future research, we suggest using conventional mark-recapture study techniques or using PIT tags. It will reduce cost and more frogs could be included in the study. Likewise, we also suggest carrying out additional studies by incorporating multiple adjacent stream systems to better understand dispersal and colonisation by these frogs.


P < 1.25 × 10−10, 117 cis‐pQTLs and 59 trans‐pQTLs). Ventricular volume (measured with brain magnetic resonance imaging) was a confounder for several pQTLs. pQTLs for CSF and plasma proteins were overall correlated, but CSF‐specific pQTLs were also observed. Mendelian randomization analyses suggested causal roles for several proteins, for example, ApoE, CD33, and GRN in Alzheimer's disease, MMP‐10 in preclinical Alzheimer's disease, SIGLEC9 in amyotrophic lateral sclerosis, and CD38, GPNMB, and ADAM15 in Parkinson's disease. CSF levels of GRN, MMP‐10, and GPNMB were altered in Alzheimer's disease, preclinical Alzheimer's disease, and Parkinson's disease, respectively. These findings point to pathways to be explored for novel therapies. The novel finding that ventricular volume confounded pQTLs has implications for design of future studies of the genetic regulation of the CSF proteome.Studies of the genetic regulation of cerebrospinal fluid (CSF) proteins may reveal pathways for treatment of neurological diseases. 398 proteins in CSF were measured in 1,591 participants from the BioFINDER study. Protein quantitative trait loci (pQTL) were identified as associations between genetic variants and proteins, with 176 pQTLs for 145 CSF proteins  proteins can be explored to increase the understanding of brain disease mechanisms. This study explored protein quantitative trait loci (pQTLs) for 398 CSF proteins analyzed by highly specific protein extension assays in a large human population. While sThe overall study design is shown in Fig P < 5 × 10−8 divided by number of tested proteins, P < 1.253 × 10−10 for CSF), we found 176 significant CSF pQTLs among 145 proteins  and trans‐pQTLs (N = 59). We also found 197 plasma pQTLs (N = 141 cis‐pQTLs and N = 56 trans‐pQTLs), among 153 proteins. These main results are in Dataset P < 5 × 10−8] included to facilitate future meta‐analyses). The pQTLs were distributed across the different assay platforms used  which were non‐significant even at genome‐wide level (P > 5 × 10−8) in plasma. One example was IL‐6, with a cis‐pQTL (rs79103996) that was highly significant in CSF (P = 2.9 × 10−29) but had no association in plasma (P = 0.38), suggesting relevant tissue‐specificity of IL‐6 regulation. When testing for tissue enrichment among the genes with putative CSF‐specific pQTLs, two genes were enriched in cerebral cortex .In general, CSF and plasma pQTL were highly correlated , including six loci for CSF CCL4 (two cis‐pQTLs and four trans‐pQTLs), four for CSF RBKS , and three each for CSF CCL24 , CTSS , and LRPAP1 . Conversely, most genetic variants among the significant pQTLs were only associated with one CSF protein . This variant was in an intergenic region on chromosome 3, where other trans‐pQTLs were also nominated for some proteins . We noted that the variant rs429358 was associated with multiple CSF proteins (N = 6 trans‐pQTLs and N = 1 cis‐pQTLs). This genetic variant defines the APOE genotype, which has well‐known associations with CSF biomarkers related to Alzheimer's disease (AD), which were validated here  CSF pQTLs, there were 140 unique genetic loci and 145 unique proteins. Most CSF proteins had only one locus, predominantly acting in tein Fig . One notWe queried the EBI‐GWAS catalog (September 2022) for all Bonferroni significant CSF pQTLs identified in this study, as described in the Methods section. Novelty of the CSF pQTLs was assessed on multiple levels, depending on whether we found in the GWAS catalog any prior pQTL in the locus, a prior pQTL for the protein we observed at the locus, any prior pQTL in CSF at the locus, or a prior pQTL in CSF for the protein we observed at the locus. The result of this novelty assessment is provided in Dataset et al, cis‐acting and three were trans‐acting. Further, we identified 8 cis pQTL‐protein pairs with a proxy match (up to 10 kb) with Yang et al  but cis‐acting in the Yang et al  overlapping between the two studies, but where the direction of effect was reversed between the studies  to denote statistical significance. When comparing with the eQTL meta‐analysis study, 53 of the cis‐pQTLs identified in the current study showed previous evidence of association with gene expression in brain tissues. The effect direction was concordant for 35 and discordant for 18 of the pQTLs , where the effect direction was concordant for 5 and discordant for 2 pQTLs , indicating previous evidence of significant gene‐to‐gene links. These included CSF Aβ42, P‐tau, and T‐tau (for APOE), CCL4 (for CCR2 and CCR9), IL12A (for IL12B), and TNFSF13B  and genetic colocalization analysis. For the MR, we performed Wald ratio (WR) tests based on the independent  Dataset .cis‐pQTLs are summarized in Table cis‐pQTL proteins identified in the MR analyses and found that drug candidates were available for APOE, CD33, GRN, CD38, ADAM15, and CTSS . We found significant effects for GRN in AD, for MMP‐10 in preclinical AD, and for CD38 and GPNMB in PD, supporting the MR findings Table .et al  and AD was replicated between the studies. The other MR findings were unique to our study and to those reported by Yang et al  and the closest encoding gene upstream was OSTN (Ensembl gene ID ENSG00000188729). There were no plasma pQTLs in this region, emphasizing its specificity for the CSF proteome in this study. OSTN encodes a hormone (osteocrin) that regulates dendritic growth in cerebral cortex  is a regulator of DNA replication, with only weak expression in brain. Based on this, we suggest that OSTN is a possible causal gene for the CSF trans‐pQTLs in this region. We queried the EBI GWAS catalog (September 2022) for previous associations with variants mapped to GMNC or OSTN. There were 77 hits in total  with exact matches to variants previously described to be associated with brain volumetric measures.As noted above, intergenic variants on chromosome 3 were common among the CSF  Dataset , includiGMNC‐OSTN region for CSF trans‐pQTLs and previous links between genetic variants in this region and brain ventricle volume .Given the importance of the trans‐pQTL variant in our study (rs71635338) was associated with ventricle volume (standardized and used as outcome in linear regression), adjusting for intracranial volume (ICV), age, sex, diagnostic group, and the top 10 principal components of the genetic background. The analysis confirmed our hypothesis that this SNP (like other previously described SNPs in the GMNC‐OSTN region) was associated with ventricle volume . Note that this was a single test, based on an a priori hypothesis, whereby correction for multiple comparisons was not applicable.We first tested if the most common CSF TissueEnrich package  with lateral ventricle volume, using linear regression models with the standardized CSF protein levels as outcome variables, adjusting for ICV, age, sex, and diagnostic group. After FDR‐correction, 64 CSF proteins were associated with ventricular volume Fig . We hypoβ‐coefficients were generally reduced, while P‐values were generally increased)  were associated with ventricle volume. When adjusting for ventricle volume (and ICV), the β‐coefficients were changed by up to 14% (sed) Fig . These ret al, et al, cis‐pQTL findings, we identified CSF proteins that were implicated in different neurological diseases, including AD, PD, ALS, depression, bipolar disease, and epilepsy, as well as brain volume measures. We also demonstrate that CSF pQTLs may be susceptible to confounding effects of brain ventricular volume.In this study, we provide a comprehensive genome‐wide analysis of associations between genetic variants and a large number of CSF proteins with the aim to clarify the genetic regulation of the CSF proteome, an issue that has received only limited attention in the past , preclinical AD (MMP‐10), and PD (CD38 and GMBP) were also validated with observational differences between patients and controls. We did not find altered levels for CD33 or APOE (for AD) or ADAM15 (for PD). Hypothetically, this may be disease‐stage dependent, since causal biomarkers may not always show cross‐sectional differences during the later stages of the disease. We note that another recent CSF pQTL study also implicated CD33 for AD by MR analysis  were associated both with smaller ventricles and with increased CSF levels of the AD biomarker P‐tau  and ventricle volume  and 48 trans‐pQTLs (at Bonferroni corrected P) compared to 158 CSF cis‐pQTLs (at P < 5 × 10−8) and 59 CSF trans‐pQTLs (at Bonferroni corrected P) in our study. A direct comparison between the results showed that 15 pQTLs were exactly replicated between the studies . The overall replication rate between the studies was therefore quite low. This suggests that although some pQTLs can be robustly replicated across distinct proteomics methods, aptamer‐ and antibody‐based technologies largely provide non‐overlapping information. A recent large‐scale study directly compared the aptamer‐based SomaScan technology with antibody‐based PEA and found considerable differences in results . Mass spectrometry‐based methods, which simultaneously measure across multiple peptides per protein, may be less susceptible to this type of artifact . We cannot exclude that the MR analysis may be affected by “pseudo pQTLs” or pQTLs driven by epitope effects, i.e., that the assay detects protein with missense variant differently  from 2009 to 2014, at three neurology and memory clinics in southern Sweden. The participants included CU participants , patients with MCI, patients with dementia due to AD, FTD, DLB, PDD, VaD, or unspecified dementia. We also included patients with PD, MSA, CBS, PSP, or unspecified parkinsonism. Details on recruitment were presented before , aliquoted into 1 ml polypropylene tubes , and stored at −80°C. Before protein analyses, all samples underwent one freeze–thaw cycle and were aliquoted into 200uL LoBind tubes .CSF and plasma samples were collected and analyzed following a standardized protocol  of the panels are available from Olink Proteomics AB.Most proteins were measured using validated, highly sensitive and specific PEA developed by Olink Proteomics  as described before , Meso Scale Discovery , mass spectrometry‐based assays , Elecsys assays (for plasma Aβ40 and Aβ42) and immunoturbidimetry . The non‐Olink proteins all passed thresholds of being quantifiable and passing QC in at least 80% of measured samples.The laboratory staff analyzing samples were blinded to all data. Assay batches were randomized with respect to covariates. There was no significant variance in findings related to assay batches. Taken together, we analyzed 398 proteins in CSF and 368 in plasma .Genotyping was conducted using the Illumina platform GSA‐MDA v2. Subject‐level QC was conducted, including the removal of sexual incompatibility between chip‐inferred sex and self‐reported sex, low call rates (5% cut‐off), and extreme heterozygosity. Relatedness among the samples was eliminated by removing one participant from each pair of close relatives (first or second degree) identified as P > 5 × 10−8 and with a call rate of > 99%) were used. Imputation was carried out using the Michigan Imputation Server (link) with SHAPEIT for phasing, Positional Burrows‐Wheeler Transform (PWBT) for imputation and the entire Haplotype Reference Consortium (release 1.1) reference panel. Multi‐allelic variants and SNPs with a data imputation score < 0.2 were excluded as part of post‐imputation QC and genotype calls with a posterior likelihood < 0.9 were set to fail . SNPs with a genotyping rate > 0.9 were retained. SNPs with MAF ≥ 1% were used for the analyses.For imputation, 685,494 high‐quality variants  et al, et al, For each protein, a linear regression analysis was performed with adjustments for age, gender, diagnostic group, collection site, and time from sampling to assay execution. The residuals from the regressions were standardized and transformed using rank‐based inverse‐normal transformation  to the protein‐coding gene, and trans‐pQTLs when the genetic variant was more distant . We retained all entries that were within 250 Kbp of any of our lead SNPs. For each of our lead SNPs, we recorded (i) the total number of GWAS catalog entries, (ii) the total number of pQTLs, (iii) the total number of distinct protein biomarkers, (iv) the total number of “CNS disease” traits, and (v) the total number of distinct “CNS disease” traits within 250 Kbp of that lead SNP. A GWAS association was defined as representing a “CNS disease” trait if one or more of the Experimental Factor Ontology terms assigned by the GWAS catalog curators was a descendant of the term “central nervous system disease” in that ontology (http://www.ebi.ac.uk/efo/EFO_0009386). There are 2,717 descendants of “CNS disease” although certainly they are not all represented in the GWAS catalog.In order to assess the relevance and novelty of our pQTLs, we compared all our lead SNPs to all the lead SNPs reported in the GWAS catalog. We downloaded all the lead SNPs from the GWAS catalog . While most published pQTLs represent protein abundance from plasma or serum samples, a small number represent prior CSF pQTL studies. These were identified by the presence of the word “CEREBROSPINAL” in the “DISEASE/TRAIT” field in the GWAS catalog. Novelty of the CSF pQTLs identified in the current study was assessed on multiple levels, as described in the Results.cis‐pQTLs to model exposure and genetic case/control studies different traits as outcomes  as IVs.To test whether the CSF proteins were likely to play a causal role in neurological disease, we performed 2‐sample MR analysis using et al, et al, et al, et al, et al, P > 10−6). Imputation was performed using the European samples from the HRCr1.1.2016 reference panel (Build 37 Assembly 19). Aβ PET imaging in A4 was done using 18F‐florbetapir data, acquired 50–70 min post‐injection. Images were realigned and averaged, and then spatially aligned to a standard space template. 18F‐florbetapir, sampled in a global neocortical region for Aβ, was expressed as an SUVR with a cerebellar reference region. 18F‐florbetapir PET SUVRs were regressed on each genetic variant separately, adjusting for age, sex, and the first five principal components of the genetic background to account for unmeasured population stratification. SUVRs were modeled using ordinary least squares regression assuming a linear association with allele frequency. Statistical significance of genetic variants was determined using Wald tests and adjusted using FDR correction.The outcome data came from several sources and included GWAS summary statistics data for genetic risks for clinical AD  test. In the rare cases where there were multiple genetic variants as IV, we used the fixed effects inverse variance‐weighted (IVW) method. Estimates of the causal effect of proteins on disease were interpreted as per standard error increments in inverse‐rank normalized protein level.coloc package in R ; (ii) only trait 1 has a genetic association in the region (H1); (iii) only trait 2 has a genetic association in the region (H2); (iv) both traits are associated, but with different causal variants (H3); (v) both traits are associated and share a single causal variant (H4). It also includes specifying a prior likelihood for a SNP being correlated with disease only (p1), protein level only (p2), and with both traits (p12). With p1 and p2 set to 1 × 10−4, we applied the default P values, assuming that 1 in 10,000 SNPs is causal for either trait and p12 was set to 1 × 10−5.To estimate the prior likelihood of each genomic locus having a single variant affecting both the protein and the phenotype, we used a strict Bayesian model  using the Many brain diseases lack efficient disease‐modifying treatments. Studies of the genetic regulation of cerebrospinal fluid (CSF) proteins can give clues to novel targetable biological pathways of brain diseases. However, only a few studies have combined genotyping data and CSF data on large sets of proteins, which is needed for this type of analysis. There is especially a lack of studies using sensitive and specific antibody‐based methods, such as proximity extension assays, for a large number of proteins.We used a well‐phenotyped cohort with 1,591 study participants, who had CSF biomarker data for 398 proteins and genotyping using a comprehensive array. We identified 176 significant (after Bonferroni correction) protein quantitative trait loci (pQTL), which were associated with altered CSF protein levels. In Mendelian randomization experiments, which also leveraged genetic risk data for traits related to brain diseases, we identified proteins with shared genetic effects with several diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and others. We also identified ventricular volume as a possible confounder for CSF pQTL studies.This study provides a comprehensive overview of the genetic regulation of a large number of CSF proteins, analyzed by sensitive and specific assays. These findings can be used to better understand the unique regulation of the CSF proteome in health and disease. Key proteins that were identified with shared genetic effects with brain diseases can be explored as targets for novel treatments against neurological diseases.trans‐pQTLs, effects on CSF pQTLs from adjustment for ventricle volume, correlations between pQTLs in plasma and CSF, comparisons with eQTL findings, and summaries of Mendelian randomization results.The supplemental information includes details and summaries of pQTL findings, between‐assays comparisons for CSF biomarkers, comparisons with other CSF pQTL studies, possible gene–protein interactions for Oskar Hansson: Conceptualization; resources; supervision; funding acquisition; investigation; methodology; project administration; writing—review and editing. Atul Kumar: Data curation; software; formal analysis; visualization; methodology; writing—review and editing. Shorena Janelidze: Data curation; formal analysis; investigation; methodology; writing—review and editing. Erik Stomrud: Methodology; project administration; writing—review and editing. Philip S Insel: Formal analysis; investigation; methodology; writing—review and editing. Kaj Blennow: Resources; methodology; writing—review and editing. Henrik Zetterberg: Resources; methodology; writing—review and editing. Eric B Fauman: Data curation; investigation; methodology; writing—review and editing. Åsa K Hedman: Investigation; methodology; writing—review and editing. Michael W Nagle: Data curation; formal analysis; investigation; methodology; writing—review and editing. Christopher D Whelan: Investigation; methodology; writing—review and editing. Denis Baird: Software; formal analysis; investigation; methodology; writing—review and editing. Anders Mälarstig: Data curation; supervision; investigation; methodology; writing—review and editing. Niklas Mattsson‐Carlgren: Conceptualization; resources; data curation; formal analysis; funding acquisition; validation; investigation; visualization; methodology; writing—original draft.NMC, ES, and AK have nothing to disclose. AM, ÅKH, and EF are employees of Pfizer AB. OH has acquired research support (for the institution) from AVID Radiopharmaceuticals, Biogen, Eli Lilly, Eisai, GE Healthcare, Pfizer, and Roche. In the past 2 years, he has received consultancy/speaker fees from Roche, Genentech, Siemens, Biogen, Alzpath, and Cerveau. KB has served as a consultant, at advisory boards, or at data monitoring committees for Abcam, Axon, Biogen, JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Pharmatrophix, Prothena, Roche Diagnostics, and Siemens Healthineers, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, all unrelated to the work presented in this paper. HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Alector, Annexon, Artery Therapeutics, AZTherapies, CogRx, Denali, Eisai, Nervgen, Pinteon Therapeutics, Red Abbey Labs, Passage Bio, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure, Biogen, and Roche, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). CDW and DB are employees of Biogen Inc. MWN is an employee of Eisai, Inc.https://www.olink.com/bdnf-info/OLINK Proteomics info about CCL22 and BDNF, https://www.olink.com/resources-support/document-download-center/OLINK Proteomics info about assay performance, https://gtexportal.org/The GTEx project database, https://www.proteinatlas.orgThe Human Protein Atlas, http://www.biofinder.seThe Swedish BioFINDER study, AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Dataset EV5Click here for additional data file.Dataset EV6Click here for additional data file.Dataset EV7Click here for additional data file.Dataset EV8Click here for additional data file.Dataset EV9Click here for additional data file.Dataset EV10Click here for additional data file.Dataset EV11Click here for additional data file.Dataset EV12Click here for additional data file.Dataset EV13Click here for additional data file.Dataset EV14Click here for additional data file.Source Data for Expanded ViewClick here for additional data file.PDF+Click here for additional data file.


Testicular torsion (TT) is common surgical emergency that requires early diagnosis and immediate intervention within 6 h since its onset to salvage the testis. However, the decision was made only by the surgeon’s experience whether it has to be resected or not. Recently, indocyanine green (ICG) has become an excellent tool to identify biliary and vascular anatomy, and assess perfusion abnormalities in tissues. In this case report, we successfully salvaged the twisted testis, since the testicular blood perfusion was confirmed by means of intraoperative ICG (IICG) fluorescence imaging.A 14-year-old healthy male patient presented due to acute left testicular pain. The patient was diagnosed with TT and had immediate surgery. Macroscopically, the testis had stagnant blood flow and appeared to be dark colored. After manual detorsion, the testis remained cyanotic and with macroscopically poor blood flow. ICG angiography was performed under near-infrared light by laparoscopic camera to assess the perfusion of the affected testicle. An excellent ICG signal appeared after 45 s in the testis, and decision was made to be preserved. Therefore, left orchidopexy was performed to complete the operation. The patient had a good postoperative course and was discharged the day after surgery. Six months later, the testis did not show any shrinkage, and both sides of the testis showed the same size without any consequences.The blood flow in the testis was visually confirmed during the IICG fluorescence method. ICG fluorescence imaging may become an effective alternative to evaluate whether a testis can be preserved following TT. Testicular torsion (TT) is a common surgical emergency and requires early diagnosis and immediate intervention for testicular salvage. Most reports suggest 6 h as a ‘golden time’ for testicular viability. The viability of the salvaged testis after TT is increased if the patient presents early, however the testicular preservation rate decreases with the passage of time from onset , 2. HoweIndocyanine green (ICG) is a dye used for fluorescent-guided surgery, and widely adapted in various surgical fields. ICG fluorescence imaging is emerging as major contributions to intraoperative decision-making during surgical procedures, including identification of the biliary, and vascular anatomy, and ensure adequate vascular supply for colectomy, nephrectomy, or find lymph nodes –6.In this case report, we successfully salvaged the twisted testis, since the testicular blood perfusion was confirmed by means of intraoperative ICG fluorescence imaging.A 14-year-old healthy male patient presented with his parent at 1 pm at our pediatric department due to acute left testicular pain having suddenly started 30 min prior to presentation.At the time of this presentation he was 161 cm tall and weighed 48 kg. On physical examination, his abdomen was soft and non-tender. There was left testicular tenderness, but no swelling or erythema. The left cremasteric reflex was absent.3), with other tests being normal. An ultrasound scan showed the absence of blood flow in the left testis  light. The visualization of structures was performed by a laparoscope  with a 30º field camera of direction, which was 10 mm in diameter and equipped with a specific filter for optimal detection of the NIR fluorescence. An excellent ICG signal appeared after 45 s from intravenous injection 2 mL (5 mL/25 mg of ICG), showing sufficient blood flow of the whole testicle (Fig. On postoperative day 19, the patient came to our department. He experienced no particular problems except for occasional slight testicular pain. The testes showed symmetrical appearance and the location was at the bottom of the scrotum. Six months after surgery, duplex ultrasonography showed symmetrical testicular perfusion and testicular volumes Fig. . The sizTT occurs frequently in infancy and adolescence, and is found in 3.8/100,000 males under the age of 18 . More thIn the treatment of TT, it is important to diagnose it as soon as possible and to perform emergent surgery. At surgery, a test incision is made in the scrotum and the color tone of the testis is observed. If no problem is observed with the color tone, the testis is detorsioned and orchidopexy is performed. Even if the torsion is released, the color of the testicle may be poor, and it is often difficult to decide whether to remove or preserve it. There is also a technique that if the testicular tunica albuginea or parenchyma is incised and bleeding occurs within 10 min, it is suggestive of testicular preservation. When preserved using this technique, testicular atrophy rates have been reported to still be 17–22% , 10. TheIn this case, we evaluated testicular perfusion by means of IICG. For several years, ICG has demonstrated its application and effectiveness in tissue perfusion evaluation in a wide range of surgical procedures. In colorectal surgery, intestinal blood flow is evaluated by IICG, and it is useful for reducing the risk of anastomotic leakages .In pediatric surgery, ICG is often used for example, in the identification of metastatic lesions of hepatoblastoma , for ICGAlthough there is one case report of using IICG in a 26-year-old man with testicular torsion , there h. used ICG fluorescence imaging to assess perfusion of reconstructed gastric tubes during esophageal cancer surgery [In our pediatric case, the ICG signals appeared 45 s after the intravenous of ICG injection, and we decided to preserve the testis. There are no criteria of the ICG appearance time for evaluating the blood flow of TT. Kumagai et al surgery . After t surgery , 14. Ton surgery . ConsideThe dosage of ICG in children has not been determined. Fernández-Bautista intravenously administered 0.2 mg/kg of ICG to assess the blood flow of pediatric organs . There aThe standard operative approach for TT is scrotal approach. In our case, an inguinal incision method was selected to evaluate blood flow from the spermatic cord to the testis. The inguinal incision method requires two incisions, the inguinal region and the bottom of the scrotum. However, in the inguinal incision method, not only the testis, but also the blood flow of the spermatic cord can be observed, which may help to determine whether or not the testis is preserved.IICG may be a useful tool in the treatment of TT when macroscopic evaluation of the affected testis remains questionable after detorsion. In pediatric TT, whether the testis can be preserved is a very important issue for future tolerability, and it is expected that IICG evaluation will improve the testicular preservation rate.Blood flow can be successfully assessed after detorsion in adolescent patients with TT when using IICG. ICG could be a safe and an effective alternative that can be applied in pediatric cases of TT.


Pythium insidiosum causes pythiosis in humans and animals. Surgery is a primary treatment aiming to cure but trading off losing affected organs. Antimicrobial drugs show limited efficacy in treating pythiosis. Alternative drugs effective against the pathogen are needed. In-house drug susceptibility tests  have been established, some of which adapted the standard protocols  designed for fungi. Hyphal plug, hyphal suspension, and zoospore are inocula commonly used in the drug susceptibility assessment for P. insidiosum. A side-by-side comparison demonstrated that each method had advantages and limitations. Minimum inhibitory and cidal concentrations of a drug varied depending on the selected method. Material availability, user experience, and organism and drug quantities determined which susceptibility assay should be used. We employed the hyphal plug and a combination of broth dilution and radial growth methods to screen and validate the anti-P. insidiosum activities of several previously reported chemicals, including potassium iodide, triamcinolone acetonide, dimethyl sulfoxide, and ethanol, in which data on their anti-P. insidiosum efficacy are limited. We tested each chemical against 29 genetically diverse isolates of P. insidiosum. These chemicals possessed direct antimicrobial effects on the growth of the pathogen in a dose- and time-dependent manner, suggesting their potential application in pythiosis treatment. Future attempts should focus on standardizing these drug susceptibility methods, such as determining susceptibility/resistant breakpoints, so healthcare workers can confidently interpret a result and select an effective drug against P. insidiosum.The orphan but highly virulent pathogen  Pythium insidiosum is a member of the oomycetes that belong to the Kingdom Stramenopiles and causes a fatal infectious condition called pythiosis in humans and animals   ,128,130.P. insidiosum, were generated following the previously described methods with some modifications [2HPO4, 1 M KH2PO4, and 1 M (NH4)2HPO4), 0.1 mL solution B (0.5 M MgCl2·6H2O and 0.5 M CaCl2·2H2O), and 1000 mL sterile distilled water [3 cells/mL for broth dilution assay and 3–5 × 104 cells/mL for disc diffusion analysis (see below) [Zoospores, the asexual stage of ications ,138,139.ed water ,138,139.e below) ,140,141.P. insidiosum inoculum  in SD broth at various drug concentrations [Three in vitro susceptibility methods were used in this study: broth dilution, radial growth assay, and disc diffusion. The selection of inoculum types for each assay relied on the previously reported susceptibility assays ,123,130.trations ,130,142.P. insidiosum colony was placed face down on a drug-containing SD agar [P. insidiosum colony in a drug-containing agar compared with that in a drug-free medium (control) [The radial growth assay was conducted using a set of SD agar plates containing various drug concentrations. A test drug was added to the desired concentration in warm SD agar (~56 °C), mixed well, poured (20 mL) into a 9 cm diameter petri dish, and let the plate settle at room temperature ,125,137. SD agar ,125, inccontrol) ,122,125.TM, GE Healthcare Life Sciences, Buckinghamshire, UK) containing 20 μL of a test drug at the desired concentration [P. insidiosum inhibition zone) around the disc was measured as described elsewhere [Disc diffusion assay employed a 6 mm sterile paper disc , which indicates the lowest drug concentration that completely inhibits the P. insidiosum growth [50 and MCC50 represented the drug concentration inhibiting and killing 50% of the P. insidiosum isolates tested. MIC90 and MCC90 depicted the same but quantified the cut-off at 90% of the test isolates [Result interpretation and report for each method were performed as follows. For broth dilution and radial growth assays, anti-m growth ,130,149.m growth ,146. Minm growth ,142,150.isolates ,152,153.P. insidiosum . Disulfiram dissolved in DMSO  was 2-fold diluted to a concentration range of 2–128 μg/mL for broth dilution and radial growth assays and 1000–64,000 μg/mL for disc diffusion assay. The final DMSO concentration in each disulfiram solution, including no-drug control, was 2% (v/v) in the SD medium.Disulfiram  was used as a standard substance to compare the advantages and disadvantages of broth dilution, radial growth, and disc diffusion assays for in vitro drug susceptibility testing against 3 isolates of P. insidiosum isolates , TA , DMSO, and EtOH  were tested against all 29 isolates . Drug coP. insidiosum’s susceptibility to KI, TA, and DMSO . Regarding EtOH susceptibility testing, a P. insidiosum hyphal plug from 10 isolates was immersed in 500 µL of 25–100% EtOH for 1, 2.5, 5, and 10 min. Each EtOH-exposed hyphal plug was washed with sterile water and subcultured on a plain SD agar plate at 37 °C for 2 days [P. insidiosum isolates tested with different drug concentrations were compared using STATA 17 . The Kruskal Wallis test and quantile regression were performed with 95% confidence.Broth dilution assay and hyphal plug inoculum were employed to evaluate and DMSO . The radr 2 days  before cP. insidiosum drug used for comparing the performances of three in vitro susceptibility methods  and three inoculum types  prepared from three different isolates of the organism   . Preparadilution ,124,152.The hyphal plug was more versatile as it can be used in various susceptibility methods . Concerning the radial growth and disc diffusion assays, it is difficult to spot and keep the hyphal suspension at the inoculated location on an agar plate because the liquid nature made it scatter or splash during the assay manipulation, transportation, and incubation. Thus, the hyphal suspension was most suitable for broth dilution performed using a multi-well plate or a set of test tubes ,130,154.P. insidiosum is an oomycete whose microscopic morphology resembles filamentous fungi. A standard guideline for in vitro drug susceptibility against P. insidiosum has not been established. In vitro drug susceptibility analysis of P. insidiosum has been adapted from the standard methods of the fungi, such as CLSI M38-A2 (a procedure and interpretation guideline for broth dilution assay) [P. insidiosum drug activity [P. insidiosum activity of a drug of interest [n assay) ,155,156 n assay) ,143. Addactivity ,137,146.interest ,145,157.P. insidiosum growth and viability could be precisely assessed using mycelium dried weight measurement and MTT-based colorimetric analysis, respectively, as suggested by the other investigators [P. insidiosum. For disc diffusion, it has been commonly used for screening the anti-P. insidiosum activity of a new compound  [P. insidiosum drug screening.Broth dilution assay can be performed in test tubes or multi-well plates using any inoculum types  and a small volume of drug solution and liquid medium  ,130,150.tigators ,160. Regtigators ,162), prtigators ,125,146.tigators ,161,163. extract ,148) andf choice . The sigpotency) ,123,148.potency) ,129,162.P. insidiosum colony. This could partially prevent the organism from direct exposure to disulfiram. For the hyphal suspension, the organism was wholly immersed and exposed to the drug. Another possible explanation is that fragmented hyphae presented in the hyphal suspension (as a result of scraping the organism out of a colony) could be more vulnerable to a drug than intact organisms in the hyphal plug. When using the hyphal plug as an inoculum, broth dilution and radial growth methods demonstrated similar MICs: 32–64 μg/mL for broth dilution and 64 μg/mL for radial growth (P. insidiosum isolate tested) because the drug in the disc needed to diffuse into the plain agar and generate a drug concentration gradient. Interpretation of the disc diffusion assay relies on an organism’s inhibition zone. Suppose a markedly lower disulfiram concentration was used  as in the other methods; the disc diffusion assay could fail to generate an optimal drug concentration gradient for inhibiting the P. insidiosum growth. We found that the inhibition zone did not appear when testing a disulfiram concentration lower than 2000 μg/mL. Nevertheless, a different MIC generated by each susceptibility method is not a concern if clinical outcome data are available for determining the method-specific drug susceptible/resistant breakpoint.Different in vitro susceptibility methods or inoculum types could affect the MIC of the same drug. Some other factors, especially the medium type, could also influence the drug MIC ,162. Conl growth . In the P. insidiosum resists the conventional drugs designed to inhibit the fungi . This may be due to P. insidiosum possessing different cell wall components and sterol biosynthesis enzymes that are not proper targets of those antifungal drugs [P. insidiosum agent is one of the priorities. This study evaluated the anti-P. insidiosum activity of several alternative chemicals  against 29 clinical and environmental isolates of P. insidiosum. Because of the feasibility, robustness, and reproducibility described above, broth dilution and hyphal plug were selected as this study’s primary drug susceptibility method and inoculum type.al drugs ,165,166.50) and 90% (MIC90) of 29 P. insidiosum isolates tested were 32 mg/mL. The same concentration was also defined as MCC50 and MCC90, as KI killed at least 50% and 90% of the organism population, respectively . KI has been used to treat several fungal infections, such as sporotrichosis, basidiobolomycosis, and cryptococcosis [(1) Potassium iodide: MIC and MCC of KI ranged from ≤8 to 32 mg/mL and ≤8 to 64 mg/mL, respectively. MICs that inhibited at least 50%  killed most P. insidiosum isolates tested. Another piece of evidence supporting the direct effect of KI on a microorganism comes from Hiruma and Kagawa [Sporothrix schenckii. However, the exact mechanism of KI’s antifungal action still needs further investigation. Apart from KI, sodium iodide is another iodine salt infrequently used in treating pythiosis, providing uncertain clinical outcomes [Some investigators proposed that KI modulates the immune response  to promote the elimination of pathogenic fungi ,169,170.d Kagawa . They mioutcomes ,172,173.P. insidiosum. The broth dilution method demonstrated that the range of TA MICs and MCCs spanned from ≤32 to >512 µg/mL. MIC50, MIC90, MCC50, and MCC90 of TA were greater than 512 µg/mL  and the immunomodulatory properties of TA could contribute to the elimination of P. insidiosum.(2) Triamcinolone acetonide: TA is a synthetic glucocorticoid widely used for treating autoimmune diseases and cancers ,174,175.56 µg/mL B. This pEscherichia coli and Pseudomonas aeruginosa) and fungi  [P. insidiosum isolates. Based on the broth dilution method, DMSO MICs and MCCs ranged from 2 to 8% (v/v). MIC50, MIC90, MCC50, and MCC90 were all at 8% DMSO  and demonstrated dose-dependent growth reductions  Dimethyl sulfoxide: DMSO has been used as a solvent, antioxidant, anti-inflammatory, and antimicrobial agent ,109,181.lbicans) ,183,184.lbicans) . It was lbicans) . DMSO calbicans) ,87. In t 8% DMSO . The radsidiosum . The antactivity ,185. Varactivity , and 50%activity ,188. Foractivity . Dependiactivity ,190,191.P. insidiosum isolates at several time points . Compared with the no-drug control, the P. insidiosum growths were significantly reduced after 1 min exposure to 50% , 70% , and 100% , but not 25%  EtOH  and 10 min   Ethanol: EtOH is an antiseptic agent . Previou53) EtOH D. Like t< 0.001) . Taken t n = 24; . RegardiP. insidiosum (an orphan but highly virulent pathogen) are urgently needed. Assessing the antimicrobial activity of a drug of interest requires a standardized in vitro susceptibility test. However, no such test is available for this organism. Several in-house drug susceptibility tests  have been established, some of which adapted the standard methods  designed for fungi. Hyphal plug, hyphal suspension, and zoospores are inoculum types commonly used in the drug susceptibility assessment for P. insidiosum. We demonstrated that each method has advantages and limitations compared to the others . Other investigators have preliminarily reported these chemicals as effective agents in treating pythiosis. We augmented their findings by extensively testing these chemicals against 29 genetically diverse isolates of P. insidiosum  or locally  to treat pythiosis. The mechanism of action of these chemicals needs to be elucidated to understand how they work.In this study, we employed the hyphal plug (served as an inoculum) and a combination of broth dilution and radial growth methods to screen and validate the anti-sidiosum . The resP. insidiosum. Future attempts should emphasize standardizing the drug susceptibility methods, including determination of susceptibility and resistant breakpoints, for P. insidiosum, so healthcare workers can confidently read and interpret a result for selecting the most effective drug against the pathogen.There is no standardized method for in vitro drug susceptibility analysis of


The self-driving structure to orientate the water movement has attracted considerable attention. Inspired by the wedgelike structures of biological materials in nature, such as spider silks and cactus spines, anisotropic spreading can be realized by combining Laplace pressure gradient and hydrophilic surface. In this study, a series of groove patterns were fabricated by a combination of 3D printing and surface modification. PLA pattern was modified by the atmospheric pressure plasma, followed by grafting with hydrolyzed APTES. This work reports the anisotropic transport of water droplets on a series of designed dart-shaped groove patterns with specific angles in the main arrow and tail regions. This structure can induce capillary force to regulate droplets from the main cone to two wedgelike, whereas the droplets are hindered toward the opposite side is oat the vicinity of the groove’s tail. By means of the experiment, the mechanism of water transport in this pattern was revealed. This study can contribute a potential approach to manipulate and apply anisotropic wetting in many fields. Nepenthes alata can make use of the oriented capillarity to transfer liquid up to the peristome and create a slippery trap that catches small insects [Anisotropic wetting behavior without supplying external energy is of important interest in many research fields, such as micro-fluidic devices, bio-sensors, water/fog collection, and industrial applications ,2,3,4,5. insects ,9. Takin insects ,12,13,14 insects ,16,17. I insects . The adv insects ,20.A unidirectional wetting on a wedgelike groove is typically based on gradient surface tension , Laplace pressure gradient, wettable surface, or an anisotropic structure ,23,24,25In this study, taking the advantage of wedge-shaped features for anisotropic liquid spreading, we designed a dart-shaped structure, which consists of a main wedgelike at the main cone and two additional wedgelike at the pattern’s tail. The efficiency of directional transport with a continuous water supply would be observed through the features from the function of the transition zone under the given condition. The preparation procedure seemed to be simpler than the aforementioned methods. Instead of heterogeneous wettability, the groove surface was modified uniformly to be hydrophilic, thus liquid was easy to spread over it. The surface profiles and morphologies of the patterns were observed by laser confocal microscopy, atomic forces microscopy, and chemical composition was analyzed by electron spectroscopy. Regarding the dart-shaped groove, the gradient Laplace force at the main wedgelike induces to orientate transport direction, while the geometry of two wedgelike at the neighboring groove’s tail obstructs droplets from moving to the opposite side. We expect that these patterns could lead to the inspiration of unidirectional wetting patterns and potential practical applications.According to the study of the cacti , we concWe studied the mechanism of liquid flow in the dart-shaped groove, the five factors that manage directional movement include the length of the groove (l), the thickness of the groove (t), the width of the groove’s tip (w), the tilt angle of the main cone  was used to print the dart-shaped groove patterns. The layer resolution was 20 The quality of the modified surface was evaluated by the atomic force microscopy  under the tapping mode to observe the morphology, the electron spectroscopy for chemical analysis , and the contact angle goniometer  for wettability. The measurement of the contact angle took place at 26 °C and 60–69% relative humidity, respectively. We observed the structure of a 3D printed dart-like pattern with a laser scanning confocal microscope . Particularly, the change in the groove thickness has been conducted from 0.2 mm to 1.0 mm. We realized that when the thickness is shallow (less than 0.8 mm), resulting in droplets overflow to the borders and the spreading behavior seems to be disordered. In contrast, if the groove depth is larger than 0.8 mm, the plasma beam cannot activate the surface, leading to grafting ineffectively. In terms of the groove width, in the case of fixed other parameters, the width becomes wider, which causes a smaller Laplace pressure gradient and cannot evaluate the wetting behavior. However, to guarantee the effective unidirectional wetting, all parameters of the groove have to be changed in accordance with the width that would be difficult to design. As a result, three fixed parameters included the total length of a single groove was 6.0 mm, the width of the groove was 1.5 mm, and the thickness of the groove was 0.8 mm. The tilt angle of the main wedgelike was designed to vary from 5The contact angle of untreated polylactic acid (PLA) was measured to be approximately 73A digital camera  was used to observe and record the dynamic wetting of water droplets on flat and patterned PLA surfaces. The needle of the contact angle goniometer was placed in the middle of the pattern and continuous droplets were supplied, as illustrated in The original surface exhibited a contact angle of 73.35eactions . Consequ45.82 at 531.09 eV, and Si. A series of images show the directional movement on the dart-shaped patterns of untreated and modified surfaces.As shown in In terms of varying hin 35 s a.In summary, two major findings are the wetting performance on the modified sample is better than that of the untreated one, and the criteria of dart-shaped groove for unidirectional spreading include a) is smaller than the inward pressure of droplets (pw), which increases the attractive force within the liquid (cohesion force) and reduces the interaction with the wall. Hence, the liquid droplet is pinned at this position. For the predominant flow direction, the droplet with concave surfaces at both edges stimulates the capillary flow and allows droplets to stick on the groove wall (adhesion force) [w1 and pw2 denote droplet pressures at edge 1 and edge 2. When pw1 is larger than pw2, the droplet would move from the main wedgelike structure to the tail, in agreement with experimental observation.The anisotropic wetting mechanisms owing to hydrophilicity surface and asymmetric structure are elucidated and explained. For instance, Geng et al.  integratn force) . Let pw1Generally, the mechanism is divided into five stages to describe how the dart-shaped structure fulfills unidirectional wetting, as shown in Initially, droplets are dripped in the middle region of the sample, then fill entire grooves. The yellow arrows point in the direction of the droplet’s movement. Droplets continue wetting onto the wall of the groove to cause a force on the liquid at the edges that propel the droplet from moving along the longitudinal channel. Following the upward direction of the second stage, the end of the groove channel is the main wedge edge, which means Laplace pressure gradient force is produced to attract water droplets. In the downward direction, after escaping the channel, droplets are hindered by the geometry of the vicinity of two wedgelike from spreading and touching the groove wall. Hence, the blocking ability of the dart-shaped structure is one of the main keys to regulating the anisotropic wetting. For the third and fourth stages in the upward direction, droplets keep filling the groove’s tail, followed by Laplace pressure-driven wetting. Reaching the next groove, the process is repeated until approaching the end side of the pattern. Taking the successful cases into account, it demonstrates that if the two wedge edges are getting sharper and the proportion of their area is proper, it becomes a barrier to obstruct droplets from flowing toward the opposite side; and thus, it enhances unidirectional wetting.The cooperative effect of uniform hydrophilicity and Laplace pressure gradient enhances the unidirectional wetting of water droplets without an external energy source. In this study, we designed and fabricated a series of dart-shaped grooves by 3D printing, then proceeded with the surface modification to optimize the anisotropic spreading capacity. The major findings of this research are concluded as follows. First, the 3D printed dart-like structure achieved a high accuracy compared to the original designs. Secondly, the effectiveness of the wettable surface was verified by the observation of the long-term durability and surface analyses . Finally, the dart-shaped structure with optimized structural parameters was clarified to enhance unidirectional transport following from the main wedgelike to the tail of each groove and the wetting mechanisms were proposed.


Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the details underlying the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP.Experimental rats were employed for in vivo determinations of CSF secretion rates, ICP, blood pressure and ex vivo excised choroid plexus for morphological analysis and quantification of expression and activity of various transport proteins. CSF and blood extractions from rats, pigs, and humans were employed for osmolality determinations and a mathematical model employed to determine a contribution from potential local gradients at the surface of choroid plexus.+/K+/2Cl− cotransporter NKCC1, the Na+/HCO3− cotransporter NBCe2, and the Na+/K+-ATPase, but not on the Na+/H+ exchanger NHE1. We demonstrate that pharmacological modulation of CSF secretion directly affects the ICP.We demonstrate that CSF secretion can occur independently of conventional osmosis and that local osmotic gradients do not suffice to support CSF secretion. Instead, the CSF secretion across the luminal membrane of choroid plexus relies approximately equally on the NaCSF secretion appears to not rely on conventional osmosis, but rather occur by a concerted effort of different choroidal transporters, possibly via a molecular mode of water transport inherent in the proteins themselves. Therapeutic modulation of the rate of CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.The online version contains supplementary material available at 10.1186/s12987-022-00358-4. Our brain contains 80% water as the major constituent of the cerebrospinal fluid (CSF) that surrounds the brain tissue and fills the ventricles, the interstitial and intracellular fluid, and the blood contained in the cerebral vasculature. The CSF protects the brain from mechanical insults and serves as a route for dispersion of hormones, nutrients, and metabolites between brain structures and neighboring cells. The CSF is replenished at a rate of approximately 500 ml/day in the adult human , the maj+/K+/2Cl− cotransporter 1 (NKCC1) was recently proposed as a key contributor to CSF secretion in mice i determinations due to DIDS’ autofluoresence in these spectra. To this end, rat NBCe2 was expressed heterologously in Xenopus laevis oocytes and its activity monitored by two-electrode voltage clamp. NBCe2-mediated membrane currents were abolished with 30 µM S0859 , 28 ± 4% with NBCe2 inhibition , but unchanged with NHE1 inhibition  was 67 ± 1 mmHg, n = 21. After infusion of the inhibitor, the blood pressure was (in % of own control) 97 ± 2 for bumetanide , 94 ± 4 for ouabain , 98 ± 2 for DIDS , and 102 ± 3 for cariporide   was reduced by 39% with NKCC1 inhibition , 62% with Na+/K+-ATPase inhibition , and 35% with NBCe2 inhibition   Fig. h, i. Inh 6) Fig. h inset. + across choroid plexus i upon inhibition of NKCC1 . Notably, an earlier study showed that in Bergmann glial cells, application of bumetanide had the opposite effect, resulting in a decrease in [Na+]i [+]i also increased upon inhibition of the Na+/K+-ATPase , or NBCe2 , while the [Na+]i remained stable upon NHE1 inhibition  release  or NaCl , with no detectable delay  comparable to that obtained with the two other osmolytes  into one lateral ventricle , we observed an elevated ICP  did not significantly change the ICP over the 100 min duration of the experiment , whereas infusion of bumetanide-containing aCSF led to a pronounced decrease in the ICP . This v−1 Osm−1 ) and is −1 Osm−1 ). Accord−1 Osm−1 –74, althestioned , in partestioned , in whicestioned . An alteestioned , 40. Thiestioned , the lowestioned ). Other estioned –80. Thisestioned , 79, or estioned .from the ventricles to the blood stream was imposed across the choroid plexus. CSF secretion occurred against gradients as large as ~ 50 mOsm, which corresponds to an osmotic pressure 10 times higher than the blood pressure. Such continued fluid secretion in the face of an oppositely-directed bulk osmotic gradient has previously been observed, but not explained, in different animal species, i.e. cats and goats, and with different osmolytes, whether introduced into the vasculature or directly into the ventricular compartment  homeostasis in the excised choroid plexus. Although the absolute CSF secretion rate may differ with the ventriculo-cisternal perfusion assay (here employed) and the ‘direct’ method [+]i measurements) produce experimental read-outs at distinct time scales (minutes to hours), and are based on both in vitro and in vivo experimentation, yet still produced similar results. Such alignment may facilitate drawing conclusions regarding the direct implication of the tested transport proteins in these processes.Detection and quantification of choroidal electrolyte transport mechanisms demonstrated the Nasee e.g. , 105–114’ method , the fra’ method , 26 to o’ method , 116. Al’ method  was conf3− transporters is evident from the ability of the carbonic anhydrase inhibitor acetazolamide to reduce CSF secretion [3− transporters promoted a redistribution of other membrane transport mechanisms [With the delineation of some luminal membrane transport mechanisms supporting CSF secretion, it now remains to determine the potential contribution of the many other channels and transporters detected in the choroid plexus  and to cecretion , 119, buchanisms , 120, whElevation of ICP is generally anticipated to occur following blockage of CSF efflux routes. While such manner of ICP elevation certainly accounts for the majority of hydrocephalus cases, CSF hypersecretion, or mimicking thereof, may cause the damaging brain fluid accumulation observed in connection with a range of different pathologies (this study and ). Irresp+/K+-ATPase, and the HCO3− transporter NBCe2 as key contributors to choroidal CSF secretion. Future research will, hopefully, reveal the molecular mechanisms underlying the cotransporter-mediated water transport, determine if the Na+/K+-ATPase and NBCe2 belong to the transport proteins capable of supporting such water flux, identify other potential local osmotic gradients that contribute to the CSF secretion, and quantify the contribution from different basolateral transport proteins sustaining the fluid movement from the interstitial space into the choroid plexus epithelium. The latter identification could promote the highly desired pharmacological targets accessible from the vascular compartment and aimed at reducing CSF secretion in the pathologies comprising brain fluid accumulation and elevated ICP.In conclusion, we here propose that CSF secretion across the choroid plexus does not occur solely by conventional osmosis, whereby electrolytes are transported across the epithelium with osmotically obliged water entering via parallel routes. A trans-choroidal osmotic gradient could not be detected and the water permeability of the choroid plexus epithelium thus too low to support the well-established rate of CSF secretion. Local osmotic gradients arising in the inter-microvillar space do not appear to exist and/or suffice to support the CSF secretion. The ability of choroid plexus to continue CSF secretion even when faced with unfavorable osmotic gradients thus promotes cotransporters, and their ability to support water transport independently of the prevailing osmotic gradient in bulk solutions, as a contributing molecular mechanism supporting this pivotal fluid flow. Accordingly, we have illustrated the cotransporter NKCC1, the NaAdditional file 1. Supplementary experimental data.Additional file 2. Supplementary data for mathematical modelling.


Retinol-binding protein 4 (RBP4) is a monomeric-binding protein belonging to the lipocalin protein family, which has been reported to be dysregulated in several malignancies such as breast cancer and lung cancer. However, the expression and function of RBP4 in glioblastoma (GBM) are completely unknown. n = 73) was enrolled from our hospital to test the protein expression profile of RBP4 in GBM tissues as well as its correlation with patients' prognoses. Two human GBM cell lines, LN229 and U251, were collected to conduct overexpression and knockdown experiments targeting RBP4. The tumor-related effects of RBP4 in GBM were finally evaluated by proliferation and invasion assays.  TCGA datasets were used for analyzing the mRNA level of RBP4 in GBM and its clinical relevance. A retrospective GBM cohort ( Both the higher mRNA level and protein level of RBP4 in GBM tissues were significantly correlated with poorer patients' overall survival. Multivariate analysis identified RBP4 as a novel independent prognostic predictor in GBM patients. Overexpression of RBP4 resulted in enhanced GBM proliferation capacity, which was consistent with clinical findings on the positive correlation between RBP4 level and tumor size. Meanwhile, overexpressing RBP4 promoted GBM cell migration and invasion, while silencing RBP4 led to the opposite results.  RBP4 overexpression in tumor tissues is correlated with poorer prognosis of GBM patients, which functions by promoting GBM proliferation and invasion, thus, may serve as an invaluable predictive biomarker and therapeutic target. The most common malignancy of the central nervous system in adults is glioma, which originates in the brain or glial tissues. Pathologically, gliomas can be further classified as astrocytomas, ependymomas, oligodendrogliomas, and glioblastomas (GBM), and so on. Among them, GBM is the most aggressive type of glioma . GBM treRBP4) is a monomeric-binding protein of 21 kilo Dalton, which belongs to the lipocalin family and was initially identified as the specific carrier for retinol in plasma [Retinol-binding protein 4  was used to perform survival analysis based on RBP4 methylation using TCGA data [The RBP4-mRNA in GBM tissues was extracted from the TCGA dataset and presented as FPKM (fragments per kilobase of transcript per Million) according to the RNA-seq data. The Kaplan–Meier method was used to evaluate the prognostic effect of RBP4-mRNA. MethSurv web tool  from our hospital, including 31 females and 42 males. All cases were pathologically diagnosed as single lesion GBM without other malignancy history. All cases underwent surgical resection . None of the cases possessed distant metastasis before surgery. None of the cases underwent neoadjuvant chemotherapy or radiotherapy, but 12 cases underwent postoperative adjuvant chemotherapy while the other 61 cases rejected chemotherapy or unknown. The median age was 63 years old at the time of diagnoses. The median tumor size was 4.3 cm according to pathological records. As for the tumor location, 13 tumors located in the parietal lobe, 25 tumors in temporal lobe, 32 cases in the frontal lobe, and 3 unclear based on the surgical record. This study was approved by the Ethic Committee of Guangdong 999 Brain Hospital. Written informed consent was provided by each patient or immediate family member at the start of the study, and all procedures in this study were performed in accordance with the Declaration of Helsinki.We retrospectively enrolled a cohort of GBM cases  and cultured in DMEM medium (Invitrogen) supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO2. Knockdown and overexpression were achieved by using Lipo3000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The siRNA targeting human RBP4 was synthesized by GenePharma  as described by others . The cDNTotal protein in transfected cells was extracted with RIPA buffer , and its concentration was tested via a BCA protein assay kit (Thermo Fisher Scientific). Protein samples were then separated by SDS-PAGE and transferred to PVDF membrane . Afterwards, the membrane was blocked with 5% nonfat milk and incubated with primary antibodies  at 4°C overnight followed by secondary antibody incubation for 1 h in room temperature. The immunoreactivity was visualized by ECL reagents .μL of transfected cells were incubated in a 96-well plate  for 1–5 days. On each day, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for 4 h. The optical density was measured at 570 nm using a microplate reader. Each experiment was conducted for three independent times.The MTT assay was used to measure the proliferation of transfected cells. Briefly, 200 The colony formation assay was used to evaluate the clone-forming capacity of transfected cells. Briefly, 500 cells were inoculated into a 6-well plate and cultured for 14 days, during which period the medium was changed every five days. Afterwards, the colonies were immobilized with methanol and dyed with 0.2% crystal violet. Finally, the clone number was counted. Each experiment was conducted for three independent times.μL, containing 5,000 cells) and seeded into the upper chamber of the 24-well plate Transwell chamber , while the lower was added with 200 μl DMEM containing 10% FBS. Cells were cultured in 5% CO2 at 37°C for 48 h, and then, the chamber was taken out, cells on the upper surfaces of the chamber were wiped off with cotton swabs, and cells from the bottom surfaces were fixed and stained with crystal violet for 15 min. Migrated cells were counted under an inverted microscope. The cell invasion assay was also conducted as described above except that the membrane was precoated with diluted Matrigel . In addition, the cell seeding number for invasion experiments was 20,000 cells/well. Each experiment was conducted for three independent times.Transfected cells were resuspended in serum-free DMEM medium . Meanwhile, higher methylation of RBP4-mRNA was also correlated with poorer overall survival (P=0.029). Considering lower mRNA levels were generally correlated with higher methylation, the mRNA data and methylation data were consistent.We first evaluated the potential clinical relevance of RBP4 by retrieving its mRNA level from TCGA datasets. Accordingly, GBM patients with low RBP4-mRNA showed poorer overall survival , P=0.03.survival , P=0.029n = 73). As revealed by IHC results, GBM tissues exhibited distinct RBP4 protein expression levels. According to the immunoreactivity, 31 cases were classified as negative RBP4 expression . Consistently, the median overall survival (OS) time of younger patients was 23.6 ± 5.6 months, while it decreased to 5.8 ± 2.1 months of elder patients (P < 0.001). Although patients underwent chemotherapy showed better CSS (24.8 ± 5.9 months) than those without chemotherapy , the OS time showed no statistically significant difference . The difference between OS and CSS can be partially explained by the fact that although chemotherapy can suppress tumor recurrence, it may have systematically adverse effects and may be harmful to the human body system. Importantly, the median CSS time of patients with negative RBP4 expression was 26.6 ± 6.8 months, while it decreased to 7.3 ± 1.7 months of those with positive RBP4 expression (P=0.007). As for the OS, the median OS time of patients with negative RBP4 expression was 22.6 ± 5.9 months, while it decreased to 6.9 ± 1.6 months of those with positive RBP4 expression (P=0.013). According to our cohort, patients' sex, tumor location, tumor size, and surgery pattern had no statistically significant effect on the prognosis of GBM .Kaplan–Meier survival curves were next plotted to assess prognostic factors of GBM . The medP=0.036), highlighting its role as a novel independent prognostic predictive biomarker. Meanwhile, patients' age  and chemotherapy  were also identified as independent factors affecting the CSS of GBM patients. Similarly, we conducted multivariate analysis targeting patients' overall survival (P=0.004) and a higher RBP4  can independently help predict a worse overall survival of GBM.Since univariate data indicated that RBP4 has significant effect on the prognosis of GBM patients, its prognostic value was further validated by multivariate analysis . As a resurvival . As a reHere, we also conducted cellular experiments to validate the tumor-related role of RBP4 in GBM. The transfection efficiencies of overexpression and knockdown were tested by Western blot . CompariSince RBP4 was originally identified as a circulation protein in plasma and showed a predictive role of in several malignancies including lung cancer  and coloP=0.025). Similarly, methylation of RBP4 in esophageal squamous cell carcinoma seems more frequent than that in adjacent esophagus tissues, however, the difference was not statistically significant (P=0.08) [Besides the expression levels, online data mining revealed that the methylation of RBP4 in tumor tissues was positively correlated the overall survival of GBM patients ((P=0.08) . NeverthA recent study reported that downregulating RBP4 in colon cancer cells decreased the fraction of cancer stem cells , therefoOur study has several limitations. First, RBP4 was closely correlated with metabolism  and has In conclusion, our results suggested that RBP4-overexpression in tumor tissues is correlated with poorer prognosis of GBM patients. Therefore, RBP4 may serve as an invaluable predictive biomarker and therapeutic target for malignancies.