TroponinT levels are frequently elevated after subarachnoid hemorrhage (SAH). However, their clinical impact on long term outcomes still remains unclear. This study evaluates the association of TroponinT and functional outcomes 3 months after SAH. Data were obtained in the frame of a randomized controlled trial exploring the association of Goal-directed hemodynamic therapy and outcomes after SAH (NCT01832389). TroponinT was measured daily for the first 14 days after admission or until discharge from the ICU. Outcome was assessed using Glasgow Outcome Scale (GOS) 3 months after discharge. Logistic regression was used to explore the association between initial TroponinT values stratified by tertiles and admission as well as outcome parameters. TroponinT measurements were analyzed in 105 patients. TroponinT values at admission were associated with outcome assessed by GOS in a univariate analysis. TroponinT was not predictive of vasospasm or delayed cerebral ischemia, but an association with pulmonary and cardiac complications was observed. After adjustment for age, history of arterial hypertension and World Federation of Neurosurgical Societies (WFNS) grade, TroponinT levels at admission were not independently associated with worse outcome (GOS 1–3) or death at 3 months. In summary, TroponinT levels at admission are associated with 3 months-GOS but have limited ability to independently predict outcome after SAH. A majority of SAH patients develops electrocardiogram (ECG)—changes and more than 50% have increased Troponin levels3. Besides, some patients develop a severe form of stress-induced cardiomyopathy, neurogenic stunned myocardium (NSM) characterized by depressed ventricular function and regional wall motion abnormality (RWMA)4.Cardiac irregularities after subarachnoid hemorrhage (SAH) are common and include a wide range of manifestations like hyper- and hypotension, arrhythmias, myocardial injury or cardiac failure6. RWMAs, elevated NT-proBNP levels as well as some ECG changes, for example ST-segment depression, were significantly associated with an increased risk of death7. Although the elevation of cardiac troponins has been the topic of several investigations, its impact on outcome remains unclear. While there are studies linking cardiac troponin elevation with long-term outcome8, the association was not present after discharge in other trials3. Therefore, the aim of this study was to evaluate whether hsTroponinT values after SAH are associated with functional outcome at 3 months.Numerous studies have explored the association of these findings with complications and outcome9. The study was approved by the local ethics committee  and was first registered at 16/04/2013 at clinicaltrials.gov; Identifier: NCT01832389. All research was performed in accordance with the Declaration of Helsinki.Troponin T measurements were obtained prospectively in the frame of a randomized, controlled clinical trial exploring the association of goal-directed hemodynamic therapy and outcomes after SAH at a University Hospital in Germany 10.Patients older than 18 with an aneurysmal SAH diagnosed by CT and confirmed by angiography were eligible for enrollment. Exclusion criteria were: traumatic SAH, congestive heart failure, severe diseases of aorta or aortic valve, pregnancy, calcium antagonist intolerance. Written consent was obtained from each patient or their legal representative. Clinical management was performed according to current guidelines and was described previously in detail11. The participants were usually seen in the neurosurgical outpatient department by a neurosurgical consultant unaware of measurement results, otherwise they were contacted by telephone.Outcome was assessed 3 months after discharge using Glasgow Outcome Scale (GOS): GOS 1 = death, GOS 2 = persistent vegetative state, GOS 3 = severe disability, GOS 4 = moderate disability and GOS 5 = good recovery14.To describe medical history at admission the following factors were considered: arterial hypertension, history of coronary artery disease, arrhythmia, history of cerebrovascular disease, tobacco use, history of asthma/chronic obstructive pulmonary disease (COPD), diabetes mellitus and chronic kidney disease. Further, the severity of SAH was assessed using Hunt–Hess grade, World Federation of Neurologic Surgeons (WFNS) grade and modified Fisher scale, taking into consideration the amount of blood in the initial CT scan15. Vasospasm was diagnosed by daily transcranial Doppler measurements and defined as peak-value increase by > 50 cm/sec/24 h compared to the previous result or a mean value > 120 cm/sec in one of the main supply branches16. DCI was defined as a new focal neurological deficit or a cerebral infarction in the presence of vasospasm revealed by radiologic imaging, or both. Other causes of neurological aggravation had to be excluded18.Complications were assessed as follows: cardiac complications , pulmonary complications , cerebrovascular complications , need for intra-arterial vasodilator therapy). Cardiac and pulmonary complications were assessed based on the current clinical practice involving symptoms, laboratory and radiological parameters, as well as current guidelinesTroponin T was measured daily starting from admission for consecutive 14 days or, if shorter, until discharge from ICU. For troponin T measurements the Roche high-sensitive troponin T Elecsys-assay was used in our laboratory.19. All statistical tests were done using SPSS statistics 27 . A p value < 0.05 was considered statistically significant.Variables are presented as median and interquartile ranges or as mean and standard deviation, as appropriate. Continuous, normally distributed variables were compared using a two-sided Student’s t test, and a Mann Whitney-U-test was used for variables with skewed distributions. Spearman rank test was used to test correlations in non-normally distributed variables. Troponin T values at admission were divided into tertiles. Logistic regression models were applied to assess the relation between initial troponin T values and admission as well as outcome parameters. In order to test whether troponin T is an independent risk factor for worse outcome, a multivariate regression was performed considering age, history of arterial hypertension and WFNS score according to SAHIT core criteria9. Both, outcome and troponin t data, were available in 106 patients. From the initially included 108 patients two were excluded: one due to missing outcome data, the other due to missing troponin T data.Patients were enrolled between March 2013 and December 2015The median age was 55 years and 81% of the patients were female. Almost half of the patients had a history of arterial hypertension and 8% had a history of coronary artery disease. An overview of baseline characteristics is presented in Table Troponin T measurements were available in 104 patients (98%) at admission (ICU day 1). The median interval between admission and troponin T peak was 2 days [1–5 days]. In 36% of the patients, the first measurement with a value of 0.011 µg/L [0.005–0.031 µg/L] was the highest. There was a close correlation between the initial troponin T measurement and the peak troponin T value . Mean troponin T values were higher (p < 0.001) on days 1–3  compared to days 4–7 (0.027 ± 0.053 µg/L) and days 8–14 (0.016 ± 0.021 µg/L). An overview of troponin T distribution over time is shown in Fig. GOS was assessed 3 months after discharge: GOS5: 58 patients (55%), GOS4: 10 (9%), GOS3: 12 (11%), GOS2: 12 (11%), GOS1: 14 (13%). There was a significant association between GOS and troponin T values at all 14 days (p < 0.05). Patients with good recovery (GOS4–5) 3 months after admission had lower initial troponin T levels than patients with GOS 1–3   had a therapy-relevant arrhythmia and one case of congestive heart failure was registered (1%). None of the patients developed a myocardial infarction. Troponin T values at admission were associated with cardiac complications: patients with cardiac complications had higher troponin T levels than those without . 23% of the patients developed pulmonary complications and showed higher troponin T levels at admission compared to those who did not . Vasospasm was observed in 55% of the patients and occurred on day 4 [3–6]. There was no significant difference in initial troponin T levels between those who developed vasospasm and those who did not . Intra-arterial vasodilator therapy was performed in 10% of the cases with no significant differences in initial troponin T levels between patients requiring the therapy and those who did not . Further, 23% of the patients developed DCI on day 5 [4–8]. Similarly, no significant difference was observed in patients developing DCI (23%) and those who did not .In order to assess the relationship between troponin T levels and admission as well as outcome parameters, troponin T values at admission were stratified by tertiles . An overview is shown in Table In this prospective study, we were able to show that troponin T levels measured for the first 14 days are associated with 3 months outcomes assessed by GOS. However, when adjusted for age and Hunt–Hess scores, troponin T levels at admission were not independently predictive of death (GOS 1) or worse outcomes (GOS 1–3) at 3 months.23. While there is widely agreement about the mechanism behind these findings involving abnormal sympathetic innervation with an excessive norepinephrine release, their impact on short and long term outcome remains controversial24. There are only very few studies who have prospectively evaluated the association between cardiac troponins and outcome after SAH, and only two of them used a high sensitive troponin essay similar to the one in the present study25. To the best of our knowledge, the present study is the first one prospectively conducting serial hs troponin T measurements for 14 days after admission.Cardiac impairment and its impact on outcomes after SAH has been an object of great interest in recent years26. Neither vasospasm nor DCI nor the need for intra-arterial vasodilator therapy showed an association with initial troponin T levels in our study. Neither did the modified Fisher scale which is closely related to vasospasm14. Due to the disparity of definitions of vasospasm and DCI in previous trials, the results in this field are poorly comparable. While no association between cardiac troponins and vasospasm was found by some investigators27, other reported a relation between cardiac troponins and DCI8. For further research, standardized definitions and approaches are needed in this field and, according to our data, considerably larger sample sizes are warranted in order to draw robust conclusions considering vasospasm and DCI. Further, our work confirms an association between troponin T levels and cardiac as well as pulmonary complications28. Finally, while there was an association in the univariate analysis between troponin T levels and 3 months outcomes at all 14 days of measurement, the relation between troponin T tertiles at admission and death as well as worse outcome was no longer present in the multivariate analysis. This finding is in line with previously published retrospective data and even expands those due to the prospective approach5. Moreover, in a multicenter cohort study, regional wall motion abnormalities (RWMA) after SAH were found to be an independent predictor of outcome after SAH while troponin T elevation was not7. Our results confirm the latter observation, but there are also trials suggesting a causal relationship between the early biomarker release such as troponin T and outcome after SAH25. Further research in a preferably multicenter setting is warranted to clarify the impact of the elevated biomarker levels on the outcome after SAH.We were able to show that initial troponin T levels are strongly correlated to peak troponin T levels. Additionally, there was an association in the univariate analysis between troponin T levels at admission and severity scores like Hunt-Hess and WFNS grade. Both findings confirm previously published results and support the hypothesis that troponin T elevation is likely to reflect an initial SAH severity with its influence on myocardial dysfunctionOur study has several limitations. First, this study does not investigate the mechanisms behind the biomarker elevation nor does it consider other cardiac parameters. Second, it is a single center trial with a limited number of patients. Nevertheless, the data were obtained prospectively and continuously. Besides, troponin T as well as 3 months outcome data were collected in all included patients. Third, our follow-up period ended at 3 months. We cannot preclude that a longer follow-up might have led to different results.29. We were able to show that troponin T measurements are associated with outcome after SAH as well as cardiovascular and pulmonary complications and might be useful in risk stratification of SAH patients. However, our results suggest that troponin T has limited ability to independently predict outcome after SAH. Further studies are needed to test the utility of hsTroponin T measurements in the early phase after SAH.Nevertheless, our results add to previous literature as the high sensitive troponin T essay was used as suggested by the current guidelines


E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five  Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), also known as colony-stimulating factor 2 (CSF-2), enhances the growth and differentiation of hematopoietic progenitor cells to various lineages, including granulocytes, macrophages, eosinophils, megakaryocyte, and red blood cells , althougSaccharomyces cerevisiae [E. coli [Escherichia coli (E. coli) is a reducing environment however so that expressed recombinant hGM-CSF cannot be properly folded, and it thus driven into the aggregated structures known as inclusion bodies [E. coli including periplasmic expression [E. coli strains [hGM-CSF is a 127-residue monomer with six glycosylation sites  and two revisiae , and the[E. coli . The gly[E. coli  by decren bodies ,14,15,16pression , differe strains , several strains , thiored strains , and int strains .E. coli strains, and temperatures. We also established a simple purification protocol for solubly overexpressed hGM-CSF fused to the protein disulfide isomerase (PDI) b′a′ domain. The hGM-CSF produced with this method showed a robust stimulatory effect on the proliferation of TF-1 cells.In our present study, we tested additional approaches to addressing the solubilization and refolding problems with hGM-CSF by screening various combinations of holdase tags, E. coli and then chemically synthesized  and Lobster strains grew fastest, the SHuffle strain was in the middle, and the ClearColi BL21 (DE3) and Origami2 (DE3) strains showed the slowest growth rates , SHuffle, and Origami2 (DE3) strains.The growth rate of the host bacteria is another important factor for the overall production level of recombinant proteins. This was measured in our current analyses in a standard manner using optical density readings at a 600 nm wavelength OD600; . Withoutth rates A. In the reduced B. HoweveE. Coli, we chose the PDIb′a′-hGM-CSF from the ClearColi BL21 (DE3) strain, inoculated at 30 °C with IPTG induction. The final hGM-CSF preparation was purified using a 2-step chromatography approach  and ion exchange chromatography (IEC)) with TEV protease cleavage after the IMAC step  protease, as the cleavage site for this enzyme had been placed downstream of the PDIb′a′-tag. The dialyzed fractions of this fusion protein obtained with IMAC were cleaved with the TEV protease, and then verified using SDS-PAGE B, Lane 5Both the target protein and PDIb′a′-tag were next purified by anion exchange chromatography based on the isoelectric point of hGM-CSF. The purity of the eluted hGM-CSF (14.6 kDa) B, Lane 650 and Hill coefficient of the purified hGM-CSF were 16.4 ± 2 pM and 0.49 ± 0.08 (n = 3), respectively. For the commercial hGM-CSF, the EC50 and Hill coefficient were 10.3 ± 3.4 pM and 0.63 ± 0.22 (n = 3), respectively.The biological effects of the purified hGM-CSF proteins were tested on the TF-1 human leukemia cell line using the CCK8 assay, which measures cell viability. Exposure of these cells to our purified hGM-CSF stimulated their growth in a dose-dependent manner similar to that of the commercial hGM-CSF . The HilE. coli and aggregate to form inclusion bodies. Recombinant GM-CSF also accumulates in this insoluble fraction when expressed in E. coli and requires manual solubilization and refolding [E. coli strain, expression temperature, and protein tag, were varied to determine the best protocol for obtaining a soluble and substantial hGM-CSF preparation from a bacterial expression system. Among these three variables, the tag was found to be the most influential  were developed to promote better cytoplasmic disulfide bond formation by engineering them to express trxB, gor, and ahpC* genes. In addition to these genes, SHuffle T7 expresses DsbC, which further alters the redox state of the cytoplasm [The hGM-CSF protein product has two intramolecular disulfide bonds and the naturally reducing environment in the cytoplasm of ytoplasm . In our ytoplasm ,38. Not E. coli is not mediated enzymatically. Another possibility in this regard is that a higher temperature increases the production of the protein but with a more limited quality control, leading to the generation of more misfolded protein. However, low temperature cultivation can decrease the growth rate of E. coli [As indicated in  E. coli  and consPichia pastoris can produce a yield of up to 760 mg/L of mouse GM-CSF when the culture is batch-fed [The lower mobility of hGM-CSF in a reducing SDS-PAGE gel suggests that the purified protein contains disulfide bonds that make it compact. The single band that was obtained under both the reducing and non-reducing condition also suggested that the purified GM-CSF was a monomer. We successfully obtained 0.65 mg of hGM-CSF with a 97.5% purity from a 0.5 L flask culture , which watch-fed . The lowE. coli. Several methods have been applied to ameliorate this such ultrafiltration, activated carbon, surfactants, anion-exchange chromatography, histamine- and histidine-immobilized Sepharose, and polymyxin B-immobilized Sepharose [E. coli strain, ClearColi BL21 (DE3), as it produces a modified and non-immunogenic LPS [E. Coli, we anticipate that more optimal protocols can be developed in the future to give a higher yield and better biological activity.Endotoxin has been always a problem when producing human proteins in Gram-negative bacteria such as epharose ,46,47,48enic LPS . This elE. Coli showed a robust stimulatory effect on the proliferation of TF-1 cells, a human leukemia cell line that responds to multiple cytokines, such as interleukin-3 and GM-CSF. The EC50 of this stimulation was 16.4 ± 2 pM, which was equivalent to 0.23 ± 0.03 ng/mL ethanesulfonic acid (MES monohydrate) were obtained from Duchefa Biochemie . Dithiothreitol (DTT) and 1-thio-β-d-galactopyranoside (IPTG) were acquired from Anaspec . Tris-Cl and Coomassie brilliant blue R-250 were purchased from Amresco . Other chemicals such as sodium hydroxide, sodium chloride, and glycerol were acquired from Samchun Chemical  and imidazole was purchased from Daejung Chemicals . Protein Marker (EBM-1014) were sourced from Elpis . Silver Staining kit Plus was obtained from Bio-Rad Laboratories .E. coli strain was obtained from Invitrogen . ClearColi BL21 (DE3) E. coli strain was purchased from Lucigen . The LOBSTR E. coli strain was modified from the BL21(DE3) strain and was obtained from Kerafast . The Origami2 (DE3) E. coli strain was purchased from Novagen . The SHuffle T7 E. coli strain was obtained from New England Biolabs . The TF-1 cell line was obtained from ATCC .The BL21 (DE3) The cell disruptor JY99-IIDN was purchased from Ningbo Scientz Biotechnology  and the Smart R17 micro refrigerated centrifuge was sourced from Hanil Science Industrial . The polyethersulfone (PES) membrane  and syringe filter  were purchased from Hyundai Micron . Acrodisc syringe filters were acquired from Pall Korea . Dialysis membranes were purchased from Viskase  and Amicon Ultra was purchased from Merck Millipore . The IMAC column, Anion exchange column, and ÄKTA Start system were purchased from GE Healthcare .Cell culture materials and assay kits were purchased from different suppliers, including RPMI-1640 growth medium powder from GIBCO , fetal bovine serum (FBS) from Avantor , recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) from Peprotech , penicillin-streptomycin from Biological Industries , and Cell Counting Kit-8 from Dojindo .E. coli expression and synthesized by Cosmogenetech . The tobacco etch virus recognition site (TEVrs: ENLYFQ/G) sequence was inserted at the N-terminus of the hGM-CSF (18–144) gene. The entry vector pENTRY-hGM-CSF was created via a BP reaction through the homogenous recombination of the attB and attP sites on the synthesized gene with the pDONR207 vector, in accordance with the manufacturer’s instructions. This entry vector was then recombined with other destination vectors  to generate expression constructs with different tag proteins. These expression vectors were validated by sequencing analysis prior to use .All bacterial expression vectors used in the current study were constructed using the BP and LR reactions of the Gateway cloning system. The lambda integrase and excisionase which were used in the BP and LR reactions were supplied by Elpis Biotech . The hGM-CSF gene region encoding amino acids 18–144 of the protein product was codon-optimized for E. coli strains BL21 (DE3), ClearColi BL21(DE3), LOBSTR, SHuffle T7, and Origami2 (DE3) using the heat shock method to test their expression efficiency of recombinant hGM-CSF. To screen for colonies expressing high levels of protein, transformed E. coli were grown on LB agar plates containing 100 μg/mL ampicillin and selected colonies were inoculated in LB media also containing 100 μg/mL ampicillin and cultured overnight at 37 °C, with shaking at 250 rpm. The overnight suspension was inoculated the following day at a 1:100 ratio into 10 mL fresh LB medium supplemented with 100 μg/mL ampicillin and then cultured at 37 °C, with shaking at 250 rpm until the OD600 reached 0.6–0.8. Next, the E. coli suspension was separated into three different round-bottom tubes and induced with 0.5 mM IPTG. These tubes were cultured under distinct conditions including 37 °C for 4 h, 30 °C for 6 h, and 18 °C overnight with shaking at 200 rpm. The bacterial pellets were collected from 1 mL of each culture by centrifugation at 13,000 rpm for 5 min at 4 °C. These pellets were then lysed on ice using an ultrasonic cell disruptor with a 35% amplitude, and 5 sec ON/5 sec OFF pulse for 10 cycles. The lysed bacterial suspensions were then centrifuged at 13,000 rpm for 5 min at 4 °C to separate the soluble and insoluble fractions. The expression and solubility level of the hGM-CSF protein under each set of experiment conditions was evaluated using 10% tricine SDS-PAGE and Gel Analyzer software .The recombinant plasmids His-hGM-CSF, PDIb′a′-hGM-CSF, and MBP-hGM-CSF were transformed into the E. coli cultured was inoculated the following day at a 1:100 ratio into 1 L of fresh LB medium containing 100 μg/mL ampicillin and cultured at 37 °C with shaking at 250 rpm until the OD600 was approximately 0.6–0.8. IPTG was then added to a final concentration of 0.5 mM and the culture was shifted to growth at 30 °C for a further 6 h. The bacteria were then harvested by centrifugation at 3500× g for 45 min at 4 °C. After centrifugation, the supernatant was removed, and the bacterial pellets were stored at −20 °C.For purification of the recombinant hGM-CSF protein, a single bacterial colony was selected for seeding into a large-scale culture. The single colony was first inoculated in 10 mL of LB supplemented with 100 μg/mL ampicillin at 37 °C, with shaking at 180 rpm, overnight. This overnight v/v), pH 10.0 with 1× cocktail protease inhibitor) and disrupted by sonication in an ice-bath with an ultrasonic cell disruptor. The lysed cells were then centrifuged at 11,000× g for 25 min at 4 °C and the supernatant was filtered through a 0.45 μm pore membrane. The filtered supernatant was added to buffer at a final concentration of 500 mM NaCl and 25 mM imidazole and applied to an IMAC column (HiTrap FF 5 mL) which had been pre-equilibrated with a buffer containing 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole 5% glycerol (v/v), and a pH of 8.0. After loading of the protein sample, the IMAC column was washed with at least 5 column volumes (CV) of the equilibration buffer to remove non-specifically bound proteins. To increase the purity of the protein, the column continued to be washed with 10 CV of 7% elution buffer , pH 8.0). The protein was eluted with 400 mM imidazole. The fractions containing the fusion protein were dialyzed in Tris buffer  at a 1:10,000 ratio to remove the NaCl and imidazole. The fusion protein was then treated with TEV protease (Addgene: 8827) to remove the PDIb′a′ tag (at a 10:1 (w/w) ratio of the fusion protein and TEV protease) at 18 °C for 16–20 h.To extract the recombinant protein, the stored bacterial pellets were first resuspended in 50 mL of Tris buffer , the hGM-CSF protein was eluted in 5 CVs of 20% elution buffer (corresponding to 200 mM NaCl) and then exchanged in PBS, pH 7.4, using a desalting column. The purified hGM-CSF was concentrated by centrifugation in a 3 K Amicon Ultra column at 3000× Proteins were separated on 10% Tris-Tricine gels and visualized by staining with Coomassie Brilliant Blue R-250. The expression, solubility and purity of these products were quantified using Gel Analyzer software. Silver staining was performed using the Silver Staining kit Plus. Briefly, following electrophoresis, the polyacrylamide gel was placed in fixative solution  for 20 min and then washed twice in distilled water for 10 min to increase the sensitivity and contrast of the staining. The staining and development steps required approximately 20-min incubations with subsequently mixed fresh solutions (prepared before use) of silver complex solution, reduction moderator solution, image development reagent and development accelerator solution. The stained gel was placed in 5% acetic acid and rinsed in high purity water to stop the reaction.F is the amount of fusion protein; T is the total amount of cellular protein after induction with IPTG; S is the amount of fusion protein in the supernatant (S) and P is the amount of fusion protein in the pellet (P).Expression testing of the hGM-CSF fusion proteins and all protein sample testing during the purification steps was conducted using 10% Tricine SDS-PAGE. Briefly, the protein fractions were boiled for 10 min at 100 °C in 5× sample buffer  to denature the proteins. Under non-reducing conditions, the protein sample was applied in the 5X sample buffer without DTT. To detect protein bands, SDS PAGE gels were stained with Coomassie brilliant blue R-250 solution. The expression and solubility levels of the fusion proteins and the purity of the target proteins were analyzed using Gel Analyzer software and calculated using a Microsoft Excel spreadsheet using the equations:5.The purified hGM-CSF protein band in the SDS-PAGE gels was collected and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) which can both accurately identify and quantify the substance in question. LC-MS/MS is a combination of mass spectrometry (MS), that provides a structural identity of individual components with high specificity and detection sensitivity, and high-performance liquid chromatography (HPLC) that can separate mixtures with multiple components. Briefly, the gel was washed and digested into peptides by trypsin, which were harvested and desalted if necessary. The protein was verified with GelDoc (Protein A280) and analyzed by LC-MS/MS. In the LC phase, the sample was separated using a PepMap RSLC 75 × 50 column containing solution A , and solution B (0.1% formic acid in Acetonitrile) over 200 min with a flow rate 200–280 nL/min. Fractions were gradient-eluted from 5% solution B to 90% solution B in 180 min. The MS analysis was separated in two phases: MS1 with a resolution of 70,000 and maximum fill time of 50 msec in which the results were analyzed by data-dependent acquisition (DDA) methods; and MS2 with a resolution of 17,500 and maximum fill time of 150 msec, whereby the auto gain control for the peaks was 1 × 102 incubator. The cells were washed with PBS to remove all cytokines, plated at a density of 7500 cells per well in a 96-well plate, and then maintained without hGM-CSF or FBS in the growth medium for 24 h before the activity test. The TF-1 cells were then treated with different concentrations of hGM-CSF from 0.01 pM to 10 nM  for 48 h at 37 °C in a humidified atmosphere of 5% CO2. The negative control was TF-1 cells treated with PBS and the blank control was the culture media containing the highest concentration of hGM-CSF without cells. All of these experiments were repeated three times. After a 48 h incubation, 10 μL CCK-8 reagent was added to each well and the plate was incubated for 4 h at 37 °C in the dark. The absorbance of the sample was measured at 450 nm with a microplate reader. The data were processed with Microsoft Excel software using the following Hill equation:Re is the response of the cells, Bl is the baseline at the low concentration of exposure, Max is the maximum response, conc. is the concentration of the protein, and Hs is the Hill coefficient of stimulation.The in vitro biological activity of the purified hGM-CSF was measured by its ability to enhance the proliferation of the human erythrocytic cell line, TF-1. TF-1 cells were cultured in RPMI 1640 medium, 10% fetal bovine serum (FBS), 2 ng/mL hGM-CSF , and 1% penicillin and streptomycin in a 37 °C, 5% COt-test or one-way analysis of variance (ANOVA). p < 0.05 was considered significant.All data are presented as the mean ± standard error. Group means were compared using the Student’s


In conclusion, the head-nodding has obvious effects on CSF circulation and head movement is one of the important drivers of cerebrospinal fluid circulation. We propose that the suboccipital muscles, participating in various head movements, might pull the dura sac via the myodural bridge, and thus, head movement provides power for the CSF circulation.The myodural bridge (MDB) is a dense connective tissue bridge connecting the suboccipital muscles to the spinal dura mater, and it has been proven to be a normal common existing structure in humans and mammals. Some scholars believe that the suboccipital muscles can serve as a dynamic cerebrospinal fluid (CSF) pump via the MDB, and they found head rotations promote the CSF flow in human body, which provided evidence for this hypothesis. Head movement is a complex motion, but the effects of other forms of head movement on CSF circulation are less known. The present study explored the effects of head-nodding on CSF circulation. The CSF flow of 60 healthy volunteers was analyzed via cine phase-contrast magnetic resonance imaging at the level of the occipitocervical junction before and after one-minute-head-nodding period. Furthermore, the CSF pressures of 100 volunteers were measured via lumbar puncture before and after 5 times head-nodding during their anesthetizing for surgical preparation. As a result, it was found that the maximum and average CSF flow rates at the level of the upper border of atlas during ventricular diastole were significantly decreased from 1.965 ± 0.531 to 1.839 ± 0.460 ml/s and from 0.702 ± 0.253 to 0.606 ± 0.228 ml/s respectively. In the meantime, the changes in the ratio of cranial and caudal orientation of the net flow volume were found differed significantly after the one-minute-head-nodding period (p = 0.017). And on the other hand, the CSF pressures at the L3–L4 level were markedly increased 116.03 ± 26.13 to 124.64 ± 26.18 mmH Recently, the existence of the MDB has been revealed in five mammalian species and it is presumed that the MDB commonly exists in mammals9. The MDB-like structures were also found in the common rock pigeon (Columba livia)10, Gallus domesticus11 and Crocodile (Crocodylus siamensis)12. This shows that the MDB is a highly conserved evolutionary structure. In general, a universal and highly conserved structure always has its own unique functions.The MDB is a kind of dense connective tissue bridge which connects spinal dura mater to suboccipital muscles and ligaments17 speculated that the MDB could prevent dura mater infolding and maintain normal CSF circulation. Other scholars18 believed that changes of dural tension could be transmitted through the sensation, triggering contraction or relaxation of the suboccipital muscles. Recent studies19 proposed that the MDB may act as a dynamic pump for CSF circulation. A study confirmed that head rotations could affect the CSF flow rates20. During the head-rotation, the suboccipital muscles could pull the upper cervical dura sac via their MDBs acting as a pump to provide dynamics for the circulation of the CSF20.Some scholarsHead movements are complicated, and varied, while except for head rotation, the effects of other forms of head movement on CSF circulation are less well known. In order to verify relationships between the CSF circulation dynamics and head movements, and to provide physiological data for revealing the role of MDB, the present study explored the effects of head-nodding movement on CSF circulations.Approval from the Ethics Committee for Research at the Basic Medical College of Dalian Medical University was obtained for this study.The experimental protocol of this study was carried out in accordance with the approved guidelines. In this study, a group of 60 adult volunteers  was subjected to MR imaging . Informed consent was obtained from all of them, and none of them had any history of cardiovascular, neurological, endocrine and cervical disorders.21 was used to measure the cardiac-gated CSF flow through the transverse plane at the level of the upper border of atlas with a peripheral pulse trigger . Imaging parameters were collected as follows: TR, 33 ms; 10 TE, 10 ms; flip angle, 20°; imaging matrix, 256 × 192; FOV, 240 cm2; section thickness, 5 mm; and 2 signal intensity average. The encoding direction was in the head-to-foot orientation for all volunteers. PC-images were obtained at each time point, for a total of 25 measurements equally distributed over the cardiac cycle. The imaging time duration varied between 2 to 5 min, depending on the volunteer’s heart rate.A cine-PC MR imaging methodEach volunteer was scanned twice. The first scanning was performed after 10-min resting period. The second scanning was performed after one-minute-head-nodding. Before the cine-PC scan, sagittal T2 weighted images of head and neck were obtained by a quick MR scan to provide anatomic details. Based on the median sagittal images, a transverse plane for the cine-PC scan was designated at the level of the upper border of atlas  for CSF flow analysis using the analyzing (Report Card 4.0) software. Using the region-of-interest function of the analyzing software, an irregular contour was drawn manually to encompass the entire cross sectional area of the subarachnoid space at the level of the upper border of atlas Fig. b. The re100 patients  without nervous system diseases or other diseases that can influence the CSF circulation and pressure, and no adverse reaction was found after the operation.A disposable puncture anesthesia kit  and cerebral pressure measuring tube  were used in this study.2O), blood pressure (mmHg), and heart rate (times/min) were recorded. Subsequently patients remained in the lateral position with a cushion pillow and gently performed nodding movement 5 times. When the fluid level in the piezometer tube got stabilized again after the head-nodding movement, the above parameters were recorded again.The patients were prepared for a routine lumbar puncture, and blood pressure were monitored. Patients maintained lying on the side with their elbow, hip and knee joints flexed. The surface of back maintained vertical perpendicular, and head cushion is prepared to make sure the head, neck, and spine horizontal. The L3–L4 posterior intervertebral space was selected as a puncture point. A 5# (1.6 × 80 mm) epidural needle and a pen tip type 16# (0.5 × 110 mm) spinal needle were used. When CSF flows out, the piezometer tube was connected. When the fluid level in the piezometer tube slowly rose and stabilized, the CSF pressure . The respiratory rate values before and after the head-nodding period were of 15.75 ± 3.37 and 15.56 ± 3.932 times per minute, respectively, showing no significant change in respiratory rate after the head-nodding period (p = 0.509). The correlation between heart rate and MDFR was not significant , but its correlation with ADFR was significant .2O. Significant differences were found in CSF pressure (p = 0.000), heart rate (p = 0.000) and systolic blood pressure (p = 0.000), but there was no significant difference in diastolic blood pressure (p = 0.293).The CSF pressures and heart rates and blood pressures before and after head-nodding period were presented in the Tables Further analysis of the correlations showed no statistically significant difference among the CSF pressures, heart rates and blood pressures before and after the head-nodding period. The p value between CSF pressures and heart rates is 0.804, and their correlation coefficient is 0.029. The p value between CSF pressures and systolic blood pressures is 0.162, their correlation coefficient is 0.160. The p value between CSF pressures and diastolic blood pressures is 0.456, their correlation coefficient is − 0.087.6. Furthermore, studies have shown that the suboccipital musculatures including RCPmi, RCPma, OCI, and the nuchal ligament (NL) participated in forming the MDB complex22. The MDB fibers from different origins could associate with each other and synergistically exert a pulling effect on the dura mater23.Head movements are complicated movements involving activities of many muscles. Amongst these muscles, the rectus capitis posterior minor (RCPmi), rectus capitis posterior major (RCPma), and obliquus capitis inferior (OCI) are located deep and posterior to the axis (C1) and atlas (C2). It was well known that they were connected to the upper cervical spinal dura mater via the MDBs through the posterior atlanto-occipital and atlanto-axial interspaces19 and Zheng et al.6 proposed that the MDB may act as a pump for CSF circulation dynamics, and Xu et al.20 research demonstrated that CSF diastolic flow was significantly affected by the one-minute-head-rotation period, indirectly supporting the MDB's CSF circulation dynamics hypothesis.Sui et al.As a further investigation upon the function of head movements, the present study found that the CSF flow was significantly affected in the one-minute-head-nodding period and the MDFR and ADFR were significantly decreased. That means nodding-head has a strong tendency to slow down the cerebrospinal fluid pulsation velocity toward the cranial cavity, although there was no obvious impact on the volume parameters  of CSF flow.Furthermore, the present research found that the effect of head-nodding on CSF circulation was based on the initial NV flow direction. In 70.4% of the subjects with an initial caudal NV flow direction, the NV values were strengthened in the caudal orientation following the one-minute-head-nodding period, while in 60.6% of the subjects with an initial cranial NV flow direction, the NV values were strengthened in the cranial orientation. This indicates that the effect of head-nodding on CSF circulation may have individual differences and depend on its initial direction. For most subjects, the effect of nodding on CSF circulation may increase net flow in the same direction.20 found that the MDFR and ADFR were significantly increased following the one-minute-head-rotation period. That means head-rotating has a strong tendency to speed up the cerebrospinal fluid pulsation velocity toward the cranial cavity. That is contrary to the effects of head-nodding. According to the anatomy and physiology of head movements, nodding motion happens at the atlanto-occipital joint along the coronal axis, while head rotating happens at the atlantoaxial joint along the vertical axis. They are different head movements. During nodding head, the RCPma and RCPmi assist in this movement. Their MDB may pull the upper cervical spinal dural sac along the sagittal plane, and cause the posterior wall of the dural sac to move backwards. And then, that may result in the decline of the MDFR and ADFR with some kind of kinetic mechanism. However, during head shaking, the OCI works with other muscles, such as the sternocleidomastoid, to move the atlantoaxial joint. In this process, the OCI’s MDB may pull the posterior wall of upper cervical spinal dural sac laterally and cause the dural sac to twist following the head-rotating. And then, that may lead to the increase of the MDFR and ADFR with some kind of kinetic mechanism. For the conflict between CSF flow rate and volume, we purposed it may be related to changes of the cross-sectional area at the measurement location. However, we still need to explore this conflicting finding in future study.Compared with the previous studies, Xu et al.Accordingly, the present study also found that the CSF pressure in the lumbar region was significantly increased during 5 times head-nodding movements. Based on the results of Cine-PC MRI measurements, the cranial flow rate of CSF was slowed down during head-nodding. it means that more and more cerebrospinal fluid might be transported into the spinal canal during head-nodding and as a result, the CSF pressure would be increased of in the spinal canal. The results of CSF pressure measurement proved the speculation. Consequently, the present study suggests that head-nodding may provide driving force for the diffusion of cerebrospinal fluid from the cerebellomedullary cistern into the spinal canal.Due to the subjects keeping stationary during the MRI scan, the CSF flow parameters were obtained not in sync with head-nodding but 1.17–4.00 min after head-nodding period. The immediate effects of head-nodding movement might be more pronounced than the measured effect because of the time lap between the head nodding occurring and the CSF measuring. In this way, the results of this study might be follow-up effects of nodding motion and motion mechanisms such as inertia might provide explanation for this effect.In addition, at the level of the occipital cervical junction, the cerebellar tonsils are suspended in the cerebellomedullary cistern. During the head flexion and extension, the size of the space between the tonsils and the atlas (C1) will be changed. In this way, we suggest that the relative movement of the cerebellar tonsils might also be a factor affecting CSF circulation during head-nodding movement. That will be studied in the future.25 and respiration29 are well known to affect CSF circulation. In this study, it was found that the heart rate decreased significantly after head-nodding period, which might be related with volunteers’ adaptation of the MR working environment. According to the dynamic mechanism of CSF circulation30, the rhythmic beating of the arteries directly transmits the pulse pressure to the CSF in the brain and promotes the flow of CSF. This study showed that there was no significant correlation between heart rate changes and MDFR, but the heart rate was significantly correlated with ADFR. The lower heart rate could prolong the DDF, thus ADFR may be affected by the lower heart rate. In addition, some authors have shown that when the heart rate is less than 90 beats per minute, the change in heart stroke volume is very weak31. According to the way that heart rate influences the CSF, we may conclude that the heart rate changes in the study may not lead to CSF flow changes. In our current study, the respiratory rate remains statistically unchanged before and after one-minute-head-nodding movement. Therefore, it was reasonable to eliminate the interference of respiratory changes in the studies.Moreover, there are many factors that affect CSF circulation. The heart rate34 and syringomyelia37. Anatomy and pathologic changes of the head movements, the MDB, or the suboccipital muscles might be related with them. Additionally, Clinical studies have shown that the dysfunction of the suboccipital muscles under various kinds of pathologic status might be correlated with chronic cervical headache40. The findings of this study may be a clue to its potential pathogenesis.The results of this study also provide a basis for etiology and diagnosis of CSF circulation-related diseases. Several studies have found that changes of CSF circulation are tightly associated with some clinical disorders, such as type I Chiari malformationIn conclusion, head-nodding can significantly affect the CSF circulation and head movement is one of the important drivers of cerebrospinal fluid circulation.


For fecal microbiota transplantation (FMT) to be successful in immune diseases like inflammatory bowel disease, it is assumed that therapeutic microbes and their beneficial functions and immune interactions must colonize a recipient patient and persist in sufficient quantity and for a sufficient period of time to produce a clinical benefit. Few studies, however, have comprehensively profiled the colonization and persistence of transferred microbes along with the transfer of their microbial functions and interactions with the host immune system. Using 16S, metagenomic, and immunoglobulin A (IgA) sequencing, we analyzed hundreds of longitudinal microbiome samples from a randomized controlled trial of 12 patients with ulcerative colitis who received fecal transplant or placebo for 12 weeks. We uncovered diverse competitive dynamics among donor and patient strains, showing that persistence of transferred microbes is far from static. Indeed, one patient experienced a dramatic loss of donor bacteria 10 weeks into the trial, coinciding with a bloom of pathogenic bacteria and worsening symptoms. We evaluated the transfer of microbial functions, including desired ones, such as butyrate production, and unintended ones, such as antibiotic resistance. By profiling bacteria coated with IgA, we identified bacteria associated with inflammation and found that microbial interactions with the host immune system can be transferred across people, which could play a role in gut microbiome therapeutics for immune-related diseases. Our findings shed light on the colonization dynamics of gut microbes and their functions in the context of FMT to treat a complex disease—information that may provide a foundation for developing more-targeted therapeutics. Clostridium difficile infections  which microbes transfer and persist across hosts, (ii) the microbial functions that accompany them, or (iii) whether immune functions of gut bacteria also transfer from donor to recipient. Previous reports of fecal transplants in IBD patients observed variable colonization by bacterial taxa from donors to recipients but did not categorize the functions and immune interactions that were also transferred –23. Furt–We comprehensively profiled the colonization dynamics of microbes and functions from a small randomized controlled clinical trial of 12 patients who had mild to moderate ulcerative colitis and were treated with FMT . By brinn = 4) received capsule fecal transplant material from an alternate donor for 4 weeks in the middle of the clinical trial, after which they returned to taking material from their original donor .We obtained samples from a small clinical cohort recruited at the University of Vermont Medical Center . In brieal donor , providi10.1128/mBio.00975-21.1FIG S1FIG S1, PDF file, 0.1 MB.Detailed clinical trial treatments for each patient. The unbalanced clinical trial design resulted from insufficient patient recruitment. (a) Five placebo patients received antibiotics followed by colonoscopic and capsule sham FMT. (b) Three FMT patients received antibiotics followed by colonoscopic and capsule FMT from the stool of Donor am. For 4 weeks in the middle of the capsule FMT period, the donor material was switched to stool from Donor az. (c) One FMT patient received antibiotics, followed by colonoscopic and capsule FMT from the stool of Donor az. For four weeks in the middle of the capsule FMT period, the donor material was switched to stool from Donor am. Because of an error in FMT processing, two FMT patients (d and e) received FMT material from only a single donor. Stool collection method (mailed samples stored in RNAlater or fresh samples stored in glycerol buffer) did not systematically alter microbiome diversity metrics. (f) We compared paired microbiome samples—instances when a fresh stool sample (stored in glycerol) and a mailed stool sample (stored in RNA later) were collected within a week of each other (excluding samples taken before and after antibiotics) or on the same day—to test whether stool collection method systematically altered alpha diversity (Shannon index). Lines connect paired samples and show no systematic bias. Download Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00975-21.2FIG S2FIG S2, TIF file, 2.1 MB.Related to Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00975-21.3FIG S3FIG S3, PDF file, 2.1 MB.Individual patient trajectories through PCoA space. (a) PCoA of Bray-Curtis distances based on 16S, same as Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://github.com/nathanieldchu/uc_fmt/tree/master/supplemental_materials.) To identify the abundance of different amplicon sequence variants , we processed the 16S rDNA sequences using QIIME 2 .We sequenced DNA from these stool samples at the Broad Institute  using 16S ribosomal DNA (rDNA) sequencing and shotgun metagenomic sequencing, producing data sets comprising an average of ∼250,000 16S rDNA sequences and ∼30.5 million metagenomic DNA sequences per sample (see Table S1). , samples from both donors clustered together , and patient-to-patient differences drove most of the variance in both 16S and metagenomic analyses . We categorized transferred ASVs in each patient as persistent or temporary colonizers on the basis of whether an ASV was present in follow-up samples  from the donor, patient, and environment compete and coexist in treated patients. Previous reports have demonstrated that recipient and donor strains of the same bacterial species can coexist within fecal transplant recipients. Our analysis of frequent longitudinal samples allowed us to ask how the dynamics of this coexistence unfold over time. In particular, we sought to determine whether donor strains could dominate over patient strains—which, because they are endogenous, may have a competitive advantage—and whether the competitive balance of strains changes over time.We used two complementary strategies to evaluate the contributions of different strains to the resulting bacterial community in each patient: a flexible genome approach  and a single-nucleotide-polymorphism (SNP) approach using StrainFinder  . We focu1Persistent colonization and dominance. The donor strain colonized after a fecal transplant, came to dominate the strain community, and persisted as the dominant strain throughout the trial. In 2Temporary colonization and dominance. The same as pattern 1, but all strains were subsequently lost or undetected. In 3Persistent colonization and temporary dominance. The same as pattern 1, but at later time points, another strain from the patient or from an unknown source became equally or more abundant than the donor strain. In 4Ambiguous persistent colonization. Strains of this bacteria persistently colonized the patient, but it is unknown if those strains were from the donor, patient, or environment. In 5Ambiguous temporary colonization. Strains of this bacteria temporarily colonized the patient, but it is unknown if those strains were from the donor, patient, or environment. In 6No colonization. In Using this flexible genome approach, we first confirmed that our methodology provided intuitively reasonable results by testing a number of control comparisons  and two samples from different donors (b), with red circles indicating genome segments that were present in one sample, but absent in the other. (c) Flexible genome strain matches against donor samples for Bacteroides ovatus, B. caccae, B. fragilis, and P. merdae indicated further instances of transferred donor strains in fecal transplant recipients. To evaluate our flexible genome method, we confirmed that samples from placebo-treated patients did not match any of the donor samples (d), and samples from fecal transplant recipients who received an alternate donor did not match samples from that alternate donor except for isolated cases after the alternate donor period (e). (f to i) StrainFinder analyses of B. fragilis (f), B. thetaiotaomicron (g), Bifidobacterium longum, (h) and Eubacterium eligens (i). Download FIG S5, PDF file, 1.3 MB.Related to Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bacteroides species were successful in persistently and dominantly colonizing (pattern 1), including B. vulgatus , B. dorei (patients E and W), and B. uniformis (patient E)  appeared to be more resistant to colonization by donor Bacteroides species, nearly always showing a mix of strains (pattern 4 or 6). We also observed temporary colonization and dominance for B. ovatus and B. caccae in fewer patients   to d. Thents see . With rewith IBD —some patwith IBD .B. dorei temporarily dominated the community of patient F, but later strain matching gave ambiguous results, which could mean that the donor strain disappeared from the community or that it coexisted with another strain (n = 1 to 5 samples) into five time points  were shared between the donor and patient   , 35. Likable S5) , and theable S5) .10.1128/mBio.00975-21.6FIG S610 cumulative abundance of all virulence factors in each patient across the trial period. (g) Cumulative abundance of virulence factors for all patients across the trial period. (h) Cumulative abundance of Proteobacteria in patient V during the trial period. Download FIG S6, PDF file, 0.7 MB.Related to Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Overall, fecal transplant recipients showed greater richness in glycoside hydrolase genes than did placebo-treated patients, suggesting increased capacity to digest dietary polysaccharides . On the Not all microbial functions transferring to fecal transplant recipients are beneficial. Although, clinically, fecal transplants can effectively clear patients of antibiotic-resistant infections, debate continues as to whether the therapy could introduce novel resistance genes, with negative clinical effects . We obseIn addition, we found that antibiotics triggered a short-lived increase in specific antibiotic resistance genes across all patients Methods, , regardlE. coli, and patient placebo V  Source plots as in Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In the context of IBD, interaction with the host immune system is perhaps the most important microbial function. Although it is reasonable to expect that transferring microbes would also transfer their endogenous metabolic capacities, it is much less certain whether transferred bacteria will elicit similar immune interactions—particularly adaptive immune responses—in a new host with a different immune system.in vivo and at high throughput for bacteria in stool samples. To identify IgA-coated and -uncoated bacteria, we used fluorescence-activated cell sorting (FACS) to separate these fractions of gut microbiome samples, and we 16S-sequenced the fractions  to a median depth of 165,000 reads. We calculated IgA coating scores for each bacterium as the log-fold change in abundance of that bacterium in the IgA-coated and IgA-uncoated fractions, such that a positive value indicated high IgA coating and a negative value indicated low IgA coating. To quantify similarity in IgA coating between samples, we calculated the Pearson correlation of IgA coating scores across all bacterial ASVs.To begin understanding host response to transferred gut bacteria, we used immunoglobulin A sequencing (IgA-seq) to profile bacteria coated with IgA antibodies . Secretor = 0.7, P < 1e−25) (r > 0.7), although for a couple of samples this pattern did not hold, potentially suggesting that IgA coating of bacteria in apparently healthy subjects may vary occasionally over time. We then compared the mean IgA coating in samples from Donor am with the mean IgA coating scores across all patients’ baseline samples and found moderate correlation  . To furt< 1e−25) . We foun10.1128/mBio.00975-21.9FIG S9r values for all time-point comparisons within each donor. Some time points did demonstrate variation, with correlation coefficients under 0.6. (c to h) Some patients’ baseline samples had much greater correlation of IgA coating with a healthy donor than others’ samples. (a and b) Line plots (a) and corresponding Pearson r correlation values (b) for each patient at baseline. (c to e) IgA coating scores (a.k.a. IgA enrichment scores) did not correlate with a bacterium’s abundance (c), variance (d), or bimodality (e). (f) IgA coating scores from each sample largely followed a normal distribution. Download FIG S9, TIF file, 1.9 MB.Related to Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.0001 by a permutation test) that were reliably and strongly IgA coated across patients and within patients through time, including before and after FMT . These bacteria were a phylogenetically diverse group comprising organisms from all major phyla (Bacteroides) and many taxa that transferred from donors to fecal transplant recipients . We also identified some Proteobacteria (including known opportunistic pathogens) and Ruminococcus gnavus—as well as the closely related R. torques—as reliably IgA coated in patients and donors  in our patient cohort; the species was essentially absent in our donors . Download Copyright © 2021 Chu et al.2021Chu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the n = 14, P < 0.0001 by a permutation test) were less phylogenetically diverse and tended to represent known commensals. All of these bacteria were Firmicutes, including known butyrate producers and immuno-modulatory taxa like Faecalibacterium, Alistipes, Roseburia, Oscillibacter, Butyricicoccus, and other Lachnospiraceae species  divergent host immune responses or (ii) strain specificity of IgA coating. If the second process were responsible, a given bacterium would be IgA coated or uncoated because each patient would be responding differently to different strains of bacteria with identical 16S sequences, the genetic marker for our IgA-seq data. But each of these patients would nevertheless respond in the same way to the exact same strain, and thus transfer of the same strain across patients would transfer a similar IgA response.To establish the role of strain specificity in explaining variably IgA-coated bacteria, we examined two bacterial subsets in fecal transplant recipients: bacteria that were shared by donor and patient and bacteria that transferred from the donor to the patient after a fecal transplant . Thus, for each bacterial 16S sequence, the first subset included a potential mix of donor and patient strains, while the second subset likely contained only one strain from the donor that then transferred to the patient. We found that the subset of strains transferred from the donor (exact strain matches) had higher correlations of IgA coating scores than bacteria shared between the donor and recipient (mixed strains) ; this par = 0.3), this finding further established that immune functions of transferred microbes may be broadly replicated in fecal transplant recipients, bolstering the prospects of engineering the gut microbiome to modulate host immunity and disease.These results suggest that exactly matching strains may trigger more-similar immune responses than do mixed strains with identical 16S sequences, indicating that variable IgA coating of bacteria could in part be explained by strain specificity. Although in one patient the correlation of IgA coating scores of exactly matching strains was still weak , suggesting that essentially all bacteria can be transferred between people. This finding reaffirms that the gut microbiome can be clinically engineered by transplanting whole gut microbial communities. It remains unknown, however, whether more-targeted therapeutics using synthetic communities will show the same ability to colonize recipient hosts.For fecal transplants or other targeted microbial therapeutics to have a clinical effect, colonized microbes must persist at sufficient abundance. We observed a variety of fates of colonizing microbes in each patient over time—from wholesale colonization and persistence of new taxa to fleeting passage of individual strains. A significant fraction of microbes that colonized recipients for multiple weeks later disappeared, suggesting that even though many bacteria can colonize a patient temporarily, competition, nutritional requirements, or immune system interactions may hamper persistence. This problem might be addressed by administering fecal transplants along with other treatments aimed at maintaining colonized bacteria  , 45, 46.Coexisting conspecific strains also showed a range of competitive dynamics: in some patients donor strains dominated endogenous strains, while in others, endogenous strains remained more abundant. Competitive dynamics like these may contribute to variable clinical responses to whole-gut microbiota transplantation and are likely to play an even greater role in more-targeted microbial therapeutics, whose efficacy hinges on the dynamics of a small number of strains.In addition, patient FMT A offered an example of how patient health can affect the colonization of donor bacteria. After more than a month of stable colonization, this patient lost a large portion of transferred strains in a short period, which coincided with—or potentially preceded—a bloom in pathogenic bacteria and severe worsening of symptoms. This dramatic decline warns us that continued patient monitoring may be needed to maintain treatment efficacy, particularly with chronic diseases like IBD. Patients who lose colonized donor bacteria could be retreated, restarting the clock on donor-strain persistence and intended clinical effect. Furthermore, although it remains unknown which of these shifts came first—loss of donor bacteria, bloom of pathogens, or worsening of symptoms—the progressive unfolding of these events raises the possibility that real-time tracking of patient microbiomes may enable early intervention and prevention of IBD flares.We found that specific beneficial functions transferred from donors to patients and could also persist. Many gut microbiome studies have focused on the benefits of butyrate production, for example, and we were able to track the transfer of butyrate production genes from donor to patient. But even after receiving new butyrate genes from a donor, fecal transplant recipients did not show higher butyrate gene diversity compared to placebo-treated patients. This observation suggests that it may be difficult to increase overall genetic capacity for butyrate production via fecal transplants. Of course, the diversity of genes related to a function does not necessarily reflect the activity of those biochemical pathways or the resulting amount of butyrate in the colon. Indeed, best-practice methods to measure clinically relevant butyrate production are not well established, since butyrate is continually produced by the microbiome and absorbed by the epithelium. Instead, it may be more fruitful to focus on which butyrate-producing organisms are present (Are some microbes more productive than others?) and which nutrients  are available to those bacteria. Examining how colonization, persistence, and environmental context alter the activity of transferred gut bacteria is likely to bring us closer to understanding the pharmacokinetics of gut microbiome engineering.We found that fecal transplants can also transfer unintended functions . To date, no evidence suggests that such unintended transfers have appreciable clinical effects , 47, butR. gnavus and E. coli), as well as known commensals (Bacteroides and Blautia)—a finding that complicates the frameworks of research suggesting that IgA-coated bacteria are largely pathogenic and inflammatory  and stored at −80°C  in PBS/1% bovine serum albumin (BSA) and incubated the samples for 20 min on ice. After incubation, we added 25 μl of 1:12.5 α-mouse-IgA-PE  to each sample and incubated samples on ice for 30 min. Finally, we washed samples three times in 1 ml PBS/1% BSA, resuspended them in PBS/1% BSA, and transferred them to blue filter-cap tubes (VWR 21008-948) for flow sorting. We sorted an average of 50,000 cells from the IgA-positive and IgA-negative bacteria into sterile microcentrifuge tubes on the BD FACSAria II at the MIT Koch Institute Flow Cytometry Core . We then centrifuged the samples, removed the supernatants, and resuspended the pellets in a final volume of 10 μl of sheath fluid. Samples were stored at −80°C until DNA library prep, in which 2 μl  was used directly as the template for PCR.Samples were processed as described previously . We centWe analyzed 16S data using Qiime2 , DADA2 , and cusTo track the sources of various bacteria, we defined all bacteria observed in any of a patient’s baseline samples and in the donor sample as “Shared,” all other bacteria present in baseline samples as “Patient,” all bacteria absent from baseline but shared with the donor as “Donor,” and finally all others as “Unknown.” We defined “persistent” colonization as a bacterium (ASV or metagenomic species) that transferred exclusively from a donor and appeared in at least three samples after the initiation of FMT and remained present in at least one follow-up sample at ∼18 weeks after initial transplant. We defined “temporary” colonization similarly, except that such bacteria did not appear in any follow-up samples.E. coli, all transplant and placebo-treated patients and the donors shared a single E. coli ASV, but it is highly unlikely that every patient in fact shared the same E. coli strain. Thus, our strategy may occasionally falsely identify the source of a given ASV. Consequently, we focused on the overall frequency (occurrences) of ASVs from different sources, instead of the abundance of each ASV. This approach minimizes the signal from highly abundant but potentially incorrectly identified ASVs, since it weights all ASVs equally.Assigning sources using this strategy has its limitations. Because many strains within common bacterial taxa have identical 16S sequences, 16S-based techniques may register many unique strains as a single ASV. For example, in the case of To quantify the transfer of bacterial functions, we used ShortBRED  to deterTo quantify the transfer of strains, we used two strategies: one based on flexible genome content and the other on single-nucleotide variants. For the first, we used a strategy similar to that described previously . Briefly2-fold change between the IgA-coated and -uncoated fractions. We observed that IgA coating scores somewhat followed a normal distribution . Metadata are included as supplementary tables which can be found at the accompanying GitHub repository . Additional 16S ASV tables and metagenomic species tables, as well as the code used to generate the figures and analyses, can be found at https://github.com/nathanieldchu/uc_fmt.We deposited raw sequence files of 16S, metagenomic, and IgA-sequencing results in NCBI’s SRA database (BioProject


Polyphenols are important bioactive compounds that are affected by processing. The consumer’s demand for minimally processed products contributes to the increase in non-thermal technologies such as high-pressure processing (HPP) in the food industry. This review is aimed at critically discussing the positive and negative effects of thermal treatment (TT) and HPP on the stability of different polyphenol families in agro-food products obtained from strawberry and apple, two of the most used fruits in food processing. Our findings show that the phenolic content was affected by processing, fruit type, polyphenol family, and storage conditions (time and temperature) of the final product. To increase shelf life, manufacturers aiming to preserve the natural content of polyphenols need to find the sweet spot between polyphenol stability and product shelf-life since the residual enzyme activity from HPP can affect polyphenols negatively. Strawberries and apples are fruits consumed worldwide, both fresh and processed , due to their desirable sensory characteristics, nutritional value, and bioactive compounds. The main bioactive compounds found are polyphenols, the most significant dietary antioxidants present in fruit and vegetables, with a wide variety of biological activities linked to the associated health benefits . These iIn particular, in strawberries, flesh and achenes have a different polyphenol profile. In the flesh, the major phenols are flavan-3-ols/proanthocyanidins (F3OLs/PACs) and anthocyanins (ATs), while in achenes, the highest concentrations are for ellagitannins (ETs) and ellagic acid (EA) ,6. SimilThere is growing consumer awareness of the importance of consuming food products that preserve nutritional and functional properties . ConsumeMeanwhile, non-thermal treatments include high-pressure processing, dehydration, freezing, pulsed electric fields, cold plasma, ultrasound processing, magnetic field, and ozone ,17. TherFurthermore, one of the most studied non-thermal treatments is HPP. It works with high hydrostatic pressure and is commonly used at the industrial level between 300 and 600 MPa for 3–5 min ,22. As aBriefly, given the above facts and considerations summarized as follows: (1) polyphenols are important bioactive compounds that are affected by processing, (2) consumers are increasingly demanding “minimally processed” products, and (3) several non-thermal processing technologies such as HPP are now prevalent on the market, here, the objective of this article is to conduct a review of the literature about the differences between TT and HPP on the impact in polyphenol degradation in strawberry and apple products, two of the most used fruits in food processing.We systematically searched, evaluated, and synthesized research articles on the effects of traditional TT and HPP and their subsequent storage on the main polyphenols found in strawberries and apples. In strawberries, we analyzed F3OLs/PACs, ATs, ETs, and EA. Whereas for apple, F3OLs/PACs, FOLs, DHCs, and HCAs were reviewed. The major databases for the topic were used . The search terms used were: polyphenols or proanthocyanidins or anthocyanins or ellagic acid or flavonols or dihydrochalcones or hydroxycinnamic acids and thermal treatment or high-pressure processing or high hydrostatic pressure and strawberry or apple.The search was focused on studies published over the last 10 years. Articles were included: (i) if the phenols were quantified by high-performance liquid chromatography (HPLC); (ii) when at least three studies were found for strawberry and apple identifying the effect of the related processing technology; (iii) when there was a control of fresh strawberry and apple or their unprocessed products. Articles were excluded: (i) when the analyzed sample was a mix of more than one fruit; (ii) when the HPP processing was combined with any thermal treatment higher than 50 °C; (iii) when the TT led to a concentration or reduction in the final product, e.g., jams.For comparison purposes, the quantification of the different strawberry and apple polyphenols in the literature were converted to mg/100 g FW (fresh weight) to provide values closer to the fruit servings used in nutritional studies. The conversion of values based on DW (dry weight) to FW assumed that a fruit retains 10% of the original fresh weight after drying.Overall, 26 articles were reviewed to address the study question: what are the effects of TT and HPP on the stability of polyphenols?Strawberry was the most studied fruit with 15 articles, whereas 11 were examined for apple products;A total of 19 articles reported the effects of thermal treatment and 12 for high-pressure processing. The reviewed studies examined 199 different trials at different processing and storage conditions .The concentrations reported in these studies for phenols of the same family were added. Each group’s total polyphenols were compared before and after processing, obtaining a percentage of increase or decrease attributed to the processing effect. A total of 4 of the 26 articles reviewed did not provide quantitative information to calculate these percentages, and therefore they were not included in the figures. The phenolic concentrations recorded after processing were considered control to determine the storage effect, and the results obtained during storage were compared against the control.This section will discuss the positive and negative effects of TT and HPP treatments and their subsequent storage on the stability of the main polyphenols in strawberries  and applProanthocyanidins, or condensed tannins, are plant secondary metabolites that could be found as oligomers or polymers of flavan-3-ol monomeric units. The most common monomeric building blocks are catechin, (epi)-catechin, (epi)-afzelechin, and (epi)-gallocatechin . F3OLs/PIn the case of strawberry, Nowicka et al.  recordedFor TT, a total of 8 studies with 30 trials including heat treatments from 70 to 98 °C during 0.4 to 15 min provided concentration data to calculate percentages of change of total F3OLs/PACs. Increments  from 4% to 1800% were observed on total F3OLs/PACs in apple puree and juice, pasteurized strawberry and strawberry pulp, and puree ,44,46,48Overall, HPP maintained the content of F3OLs/PACs closer to the fresh control in both fruits. In contrast, the impact of TT on the degree of change of F3OLs/PACs was positive or negative depending on the study, influenced by the matrix, the TT conditions, and the response of the fruit cultivar to the processing technology ,7 Figur. In addiFor TT, Stübler et al.  found a As mentioned before, in general, HPP showed minor variation in F3OLs/PACs levels compared to control. In the case of strawberry puree pressurized at 600 MPa for 1 min, Stübler et al.  detectedAnthocyanins (ATs) are water-soluble compounds found in different tissues of the plant, such as leaves, roots, flowers, and fruits . These pFor TT, 7 studies with 23 trials comprising temperatures from 70 to 100 °C for 1 to 15 min provided concentration data to calculate percentages of change of total ATs. Reductions  from 5% to 44% were observed in pasteurized strawberry, strawberry purees and pulp ,41,42,44Regarding HPP, a total of 6 studies, including 40 trials with pressurization conditions from 100 to 600 MPa for 1 to 20 min at 0–50 °C, were analyzed. Degradations  from 7% to 28% were reported in strawberry pulp and puree ,39,41,44In general, just after pressurization, the levels of ATs were preserved close to those found in fresh strawberries. In contrast, a more significant impact was observed in the degree of change of ATs due to TT, with a downward trend in both cases A.Since ATs are very chemically sensitive compounds, most of the studies registered the degradation of ATs after processing and storage. For instance, pasteurization of entire strawberries at 90 °C for 5 min resulted in a reduction of 5–35% of cyanidin-3-glucoside, pelargonidin-3-rutinoside and pelargonidin-3-glucoside ,32. HighInterestingly, many authors have reported different degradation percentages depending on the strawberry variety, concluding that the effect of TT and subsequent storage on ATs also depends on the response of the fruit variety to the treatment ,37,42. TAlthough overall TT led to degradation on ATs, some studies reported a positive impact after processing. Garzoli et al.  reportedThe effect of refrigerated storage (4–6 °C), from 14 to 180 days, on total ATs after different TT and HPP conditions is presented in Ellagitannins (ETs) constitute complex molecules with variable water solubility structured by one or more hexahydroxydiphenoyl (HHDP) moieties, which could be hydrolyzed to release ellagic acid (EA) . ETs andFor TT, 5 studies with 10 trials including temperatures from 55 to 90 °C for 1 to 15 min provided concentration data to calculate percentages of change of EA. Increases  from 8% to 66% were observed in pasteurized strawberries, strawberry pulp, and strawberry purees ,40,41,44In general, TT and HPP influenced the level of EA both positively and negatively depending on the conditions. Still, the most significant increases were observed as a result of heat treatment A.For TT, Cao et al.  reportedFor HPP, Cao et al.  reportedFlavonols (FOLs) are plant secondary metabolites that could be found in foods as aglycones or much more frequently as glycosidic conjugates . The mosFor TT, 3 studies with 17 trials including temperatures from 71 to 98 °C during 0.4 to 15 min provided concentration data to calculate percentages of change of total FOLs. Increments ranging from 4% to 69% were observed in five trials with apple juice and applesauce . In contOverall, HPP had positive effects on FOLs concentration. With TT, the results depended on the conditions and the apple varieties with a general trend to decrease their content .For instance, FOLs contents increased 49% and 69% after mild (71 °C/0.4 min) and intense (90 °C/14.8 min) pasteurization of apple juice, respectively, which indicates that thermal treatment favored the release of phenolic compounds and reduced PPO activity . AdditioAbout HPP, no significant differences were reported in quercetin levels after apple juice pressurization at 300 and 450 MPa for 5 min and in multi-pulsed HPP at 300 MPa x 3 pulses, each 5 min. However, after HPP treatment at 600 MPa for 5 min, a minor but significant increase from 0.73 to 0.91 mg/L (25%) was quantified for quercetin . These rThe dihydrochalcones (DHCs), phloretin and phloridzin, are characteristic compounds of apple and apple products. Since these compounds are exclusive of apples, these have been used to detect adulterations . DHCs leFor TT, 3 studies with 16 trials comprising thermal treatments from 71 to 98 °C during 0.4 to 15 min provided concentration data to calculate percentages of change of total DHCs. Increments 12 trials) ranged from 8% to 767% in applesauce and apple juice . Convers trials rOverall, TT and HPP changed the content of DHCs of apple products  positiveFor TT, Le Bourvellec et al.  reportedFor HPP, Szczepańska et al.  reportedp-coumaric, caffeic, ferulic, and sinapic acids [Hydroxycinnamic acids (HCAs) are the major subgroup of phenolic acids and may occur either in their free or conjugated forms, including amides, esters, and glycosides . The maiic acids . More fric acids . In applic acids . Ranges ic acids ,12. Wheric acids ,10. Someic acids ,90,91.For TT, four studies with 19 trials comprising heat treatments from 71 to 98 °C for 0.4 to 30 min provided concentrations data to calculate percentages of change of total HCAs. Increments  ranged from 8% to 925% in apple sauce and apple juice . On the In general, both TT and HPP caused degradation of HCAs after processing. Nevertheless, although a higher degree of degradation was observed after heat treatment, it also caused the most significant increase in two trials with apple juice  .p-coumaroylquinic acid. A total of 205% and 925% increments on total HCAs were reported after the mild (71 °C/0.4 min) and intense (90 °C/15 min) pasteurization of apple juice, respectively. The increase in HCAs could result from the favoring effect of thermal treatment in enzyme inactivation and the release of chlorogenic acid from the cell walls to the food matrix [p-coumaroylquinic acid after TT were due to apple variety. After cooking (95 °C/2 min) and pasteurization (90 °C/5 min), nine varieties experienced reductions from 1% to 48% in 5’-caffeoylquinic acid, whereas increases of 8%, 4%, and 30% were observed in Golden Delicious, Freiberg, and Granny Smith varieties, respectively.For TT, Alongi et al.  identifid matrix . These rd matrix , who cond matrix  reportedd matrix , suggestd matrix ,50. In aFor HPP, Marszalek et al.  found noThis review analyzed the effects of TT and HPP treatments on the phenolic compounds in strawberry  and apple  products. Our findings show that the effect on polyphenols content (positive or negative) was contingent upon the type of processing, type of fruit, polyphenol family, and the shelf-life conditions (time and temperature during storage) of the final product. The impact of TT relied mainly on the food matrix and the thermal stability of the different phenolic compounds.TT had positive effects in strawberry products, as was observed for F3OLs/PACs and EA. However, it had negative effects on the ATs content. These were due to the thermal instability of ATs and the enhanced extraction of EA and condensed tannins from achenes with thermal treatments. It is well known that ATs are heat-labile compounds, susceptible to oxidation and condensation reactions, which was confirmed in most of the studies where degradation of ATs was observed after TT. On the contrary, TT increased EA content due to the hydrolysis from ETs and release from the lignified matrix of the achenes.In apple products, TT had positive effects, significantly promoting the release of FOLs and DHCs from peel and seeds. However, most of the studies observed a negative effect of F·OL/PACs, principally due to the extraction processes that quantified flavan-3-ols oligomers rather than monomers. Nevertheless, the final concentrations were variable depending on the variety, and the TT conditions applied. HPP treatments maintained the concentrations of phenolic compounds closer to those of the fresh or unprocessed samples regardless of the food matrix.The impact of storage after TT and HPP had only been described for ATs in strawberry products. In general, TT has positive effects preserving better the ATs during storage. However, in the only study that examined the effect of HPP on ATs during storage, a negative effect was described showing a higher degradation than TT. Negative effects were also observed as the fast and pronounced degradation of FOLs, DHCs, and HCAs in apple products after HPP and storage.The phenolic compounds’ degradation after storage of HPP products could be due to the limitations in oxidative enzyme inactivation when thermal treatments were insufficient. From the industrial perspective, manufacturers aiming to preserve the natural content of polyphenols need to find the sweet spot between polyphenol stability and product shelf life. Further studies are recommended to compare how both technologies influence the content of the different families of polyphenols and their bioavailability and bioactivity.


Purpose125I and 103Pd Collaborative Ocular Melanoma Study (COMS) plaques in ocular brachytherapy.The purpose of this study is to compare optic disc dose (ODD) between Methods125I (model: IAI-125A) and 103Pd (model: IAPd-103A) seeds. ODD calculations were repeated for prescription depths from 2 mm to 10 mm in 1 mm intervals. A prescribed dose of 85 Gy (irradiation time: 120 hours) was normalized to each prescription depth. Dose conversion factors (DCFs) for each prescription depth were calculated by taking a ratio of [total reference air kerma (TRAK) per seed]prescription depth to [TRAK per seed]5 mm. ODD reduction by notched COMS plaques was calculated for each prescription depth by subtracting ODD for notched COMS plaques from ODD for standard COMS plaques.A previously validated in-house brachytherapy dose calculation program was used for ODD calculations. ODD was calculated as a function of tumor margin-to-optic disc distance (DT) up to 5 mm for various tumor basal dimensions (BDs), for a prescription depth of 5 mm, and for standard and notched COMS plaques loaded with Resultst) for each BD in each plaque at which ODD for 125I COMS plaques equals that for 103Pd COMS plaques. For small BDs, at DT <dt, ODD for 103Pd COMS plaques is higher than that for 125I COMS plaques while at DT >dt, the opposite is observed. For the largest 1-3 BD(s), contrarily, dt occurs within the tumor, and thus, ODD for 125I COMS plaquesis always higher. Trends of ODD reduction by notched COMS plaques as a function of DT for various BDs are the same for the two seed types except that maximum ODD reduction by 103Pd COMS notched plaques is larger. DCF increases with increasing prescription depth for both seed types.Trends of ODD as a function of DT for various BDs are similar between the two seed types in both standard and notched COMS plaques. However, due to the energy difference, there exists a transition distance (dConclusions125I and 103Pd COMS plaques and the differences depend on DT, BD, plaque size, and prescription depth.There exist ODD differences between  Likewise, DCFs for 103Pd standard COMS plaques were obtained. DCFs will allow for estimating ODD at any prescription depth by multiplying ODD at 5 mm by DCF at a depth of interest.Dose conversion factors (DCFs) for different prescription depths were generated for 125I notched COMS plaques and 103Pd notched COMS plaques. ODD calculations for 125I notched COMS plaques were repeated for various prescription depths from 2 mm to 10 mm in 1 mm intervals. For each prescription depth, calculations were performed for one seed removal (plaques from 12 mm to 22 mm) and two seeds removal (plaques from 14 mm to 22 mm). The same dose (85 Gy) for the same irradiation time (120 hours) was prescribed to each depth after seed(s) were removed. TRAK per seed for each depth and each plaque was calculated and then DCF for each depth and each plaque was obtained in the same way as for 125I standard COMS plaques. Likewise, DCFs for 103Pd notched COMS plaques were obtained.In addition, DCFs for different prescription depths were generated for 125I and 103Pd notched COMS plaquesODD reduction by 125I standard COMS plaques and 125I notched COMS plaques, ODD reduction by 125I notched COMS plaques was calculated as a function of DT for various BDs. Absolute ODD reduction, From ODDs for  103Pd notched COMS plaques was also calculated as a function of DT for various BDs. ODD reduction by 125I and 103Pd standard COMS plaques: prescription depth of 5 mmODD comparison between 125I versus 103Pd standard COMS plaques as a function of DT for various BDs when 85 Gy was prescribed to a central-axis depth of 5 mm. Trends of ODD for 125I standard COMS plaques are similar to those for 103Pd standard COMS plaques. ODD for both seed types usually decreases as DT and BD increase. On the other hand, there are differences in ODD between 125I and 103Pd standard COMS plaques. Compared with that for 125I plaques, ODD for 103Pd plaques is higher, becomes the same, and then is lower as DT increases. Therefore, for each BD (excluding the largest 1-2 BDs) in each plaque, there exists a transition distance (dt) at which ODD for 103Pd standard plaques is equivalent to ODD for 125I standard plaques. For each plaque, dt becomes closer to the optic disc by about 1 mm with increasing BD by 2 mm. For the largest 1-2 BDs in plaques ≥18 mm, however, dt occurs within the tumor, and as a result, ODD for 125I standard plaques is always higher than ODD for 103Pd standard plaques.Figure 125I and 103Pd notched COMS plaques: prescription depth of 5 mmODD comparison between 125I versus 103Pd notched COMS plaques with one seed removed as a function of DT for various BDs when 85 Gy was prescribed to a central-axis depth of 5 mm. As for standard COMS plaques, trends of ODD for 125I notched COMS plaques are similar to those for 103Pd notched COMS plaques: ODD for both seed types usually decreases as DT and BD increase. Also, trends of an ODD comparison between the two seed types for notched plaques are similar to those for standard plaques: dt, at which ODD for 103Pd notched plaques equals ODD for 125I notched plaques, exists for each BD (excluding the largest 1-3 BDs) in each plaque and ODD for 103Pd notched plaques is higher than that for 125I notched plaques at DT <dt but the opposite is observed at DT >dt. For the largest 1-3 BDs, ODD for 125I notched plaques is always higher.Figure 125I versus 103Pd notched COMS plaques with two seeds removed as a function of DT for various BDs are plotted in Figure 125I and 103Pd notched plaques with two seeds removed are similar to those with one seed removed but ODD for two seeds removal is lower.ODD for 125I and 103Pd notched COMS plaques: prescription depth of 5 mmODD reduction comparison between 125I versus 103Pd notched COMS plaques with one seed removed as a function of DT for various BDs when 85 Gy was prescribed to a central-axis depth of 5 mm. These plots were obtained by subtracting plots in Figure 125I notched plaques are the same as those for 103Pd notched plaques except that ODD reduction is larger with 103Pd notched plaques. ODD reduction increases with increasing DT, reaches the maximum value  at a prescription depth of 10 mm.Table 125I and 103Pd notched COMS plaques with one seed removed. Trends of DCFs for notched plaques are the same as those for standard plaques: DCF increases with increasing depth in each plaque for both seed types. DCFs for 103Pd notched plaques are smaller than those for 125I plaques at a depth of <5 mm, whereas the opposite is observed at a depth of >5 mm.Table 125I and 103Pd notched COMS plaques with two seeds removed is shown in Table A comparison of DCFs for prescription depths from 2 mm to 10 mm between Estimation of ODD: clinical application of this study125I versus 103Pd), COMS plaque type (standard versus notched), and prescription depth (COMS protocols versus ABS guidelines), 12 scenarios are possible, and ODD can be estimated for each scenario (Table 103Pd standard plaque is higher than that (190.43 Gy) for the 125I standard plaque. On the other hand, ODD (131.89 Gy) for the 103Pd notched plaque with one seed removed is similar to that (131.52 Gy) for the 125I notched plaque with one seed removed and ODD (93.27 Gy) for the 103Pd notched plaque with two seeds removed is lower than that (96.71 Gy) for the 125I notched plaque with two seeds removed. Fourth, when 85Gy was prescribed at the tumor apex , ODD (156.38 Gy) for the 103Pd standard plaque is higher than that (154.25 Gy) for the 125I standard plaque. The opposite trend is observed for notched plaques . Therefore, among the 12 scenarios, the 103Pd standard plaque prescribed at 5 mm and the 103Pd notched plaque with two seeds removed prescribed at 4 mm give the highest ODD (195.48 Gy for scenario #4) and the lowest ODD (73.68 Gy for scenario #12), respectively, and the dose difference between these two scenarios is 121.80 Gy (143.29% of the prescribed dose).For a clinical example , a practical application of the results (figures and tables) from this study is shown in Table 125I standard and notched COMS plaques are similar to those for 103Pd standard and notched COMS plaques, respectively  which leads to a more drastic decrease of relative dose with distance for 103Pd seeds than for 125I seeds [103Pd plaques is higher at relatively short DT and becomes lower at longer DT than ODD for 125I plaques. As a result, there exists the transition point  at which ODD for 103Pd plaques is the same as that for 125I plaques. For small BDs in each plaque, dt becomes closer to the optic disc with increasing BD because the region of drastic dose fall-off within the tumor increases as BD increases.Despite the aforementioned similarities, there are differences in ODD between 5I seeds . Therefo125I and 103Pd notched COMS plaques , COMS plaque type (standard or notched), and prescription depth (5 mm or tumor apex) affect ODD. ODD for 103Pd seeds is not always lower than that for 125I seeds, contradicting the study by Finger et al. [125I seeds can be lower when the tumor is closer to the optic disc than dt (scenarios #1 versus #4). Notched plaques are usually beneficial in reducing ODD. Prescribing to a shallower depth is also beneficial in reducing ODD. For tumors with apical height ≥5 mm, however, a prescription depth is always the tumor apex and thus, a prescription depth would not be a parameter to reduce ODD.The clinical example comparing ODDs among 12 different scenarios would help the clinician choose the best option to minimize ODD. As shown in Table r et al. . ODD forOne limitation of this study is that heterogeneity corrections were not taken into account. It has been reported that dose attenuated in the silastic insert and gold plaque is significant (≥10%) . Hence, 125I and 103Pd seeds for both standard and notched COMS plaques. However, due to the energy difference between the two seed types resulting in different relative dose variations with distance, there exists a transition distance (dt) for each BD in each plaque at which ODD for 125I plaques equals that for 103Pd plaques. For small BDs, at DT <dt, ODD for 103Pd plaques is higher than that for 125I plaques while at DT >dt, the opposite is observed. For the largest 1-3 BDs, contrarily, dt exists within the tumor and thus, ODD for 125I plaques is always higher than that for 103Pd plaques. Trends of ODD reduction by notched plaques as a function of DT for various BDs are the same for both seed types except that 103Pd notched plaques is larger than that by 125I notched plaques. ODD and DCF increase with increasing prescription depth for both seed types but DCF for 103Pd plaques increases more drastically than for 125I plaques.This study has demonstrated that ODD has dependence on DT, BD, plaque size, plaque type, prescription depth, and seed type. Trends of ODD as a function of DT for various BDs are similar between