TT consisted of 90% δ-TT and 10% γ-TT. In this metabolomic study, we evaluated the placebo and 600mgTT at baseline and 12 weeks. As expected, TT and its metabolite levels were higher in the supplemented group after 12 weeks. At baseline, there were no differences in demographic parameters or comprehensive metabolic panels (CMP). Metabolomics analysis of serum samples revealed that 48 biochemicals were higher and 65 were lower in the 600mg TT group at 12 weeks, compared to baseline. The results confirmed higher serum levels of tocotrienols and lysophospholipids, but lower acylcarnitines and catabolites of tryptophan and steroids in subjects given 600mg TT. In summary, 12-week TT supplementation altered many serum metabolite levels in postmenopausal women. The present study supports our previous findings that TT supplementation helps reduce bone loss in postmenopausal osteopenic women by suppressing inflammation and oxidative stress. Furthermore, the body incorporates TT which restructures biomembranes and modifies phospholipid metabolism, a response potentially linked to reduced inflammation and oxidative stress.Osteoporosis is a major health problem in postmenopausal women. Herein we evaluated the effects of 12-week tocotrienols (TT) supplementation on serum metabolites in postmenopausal, osteopenic women. Eighty-nine participants (59.7 ± 6.8 yr, BMI 28.7 ± 5.7 kg/m Vitamin E contains two subgroups, namely tocopherols and tocotrienols (TT) and their corresponding isomers , with a common chromanol ring and a saturated or unsaturated side chain . Most plRecent studies provide strong evidence for age-associated mitochondrial dysfunction, dysregulated intracellular lipid metabolism, and insulin resistance , all of Increased oxidative stress and inflammation in skeletal muscle contributes to structural and functional changes of mitochondria, the important organelle for energy supply, redox regulation, and apoptosis . The mitDue to the unsaturated isoprenoid side chain and the ability to easily penetrate tissues, some of the biological effects of TT are superior to those of alpha-tocopherol (the most common type of vitamin E with a saturated side chain). Previous studies have shown the osteoprotective effects of tocotrienols on animals with estrogen-deficient bone loss –33 as weMetabolomics profiling technologies provide a systematic approach for evaluating the metabolic response to nutritional interventions by simultaneously measuring and identifying a large number of small molecules. Metabolomics is particularly useful in interpreting human responses to dietary manipulation, including dietary supplement, and improves the capacity to capture both complex and subtle influences on whole body metabolism and physiology. The primary goal of metabolomics is to correlate changes in the biochemical profile of a sample with a corresponding shift in physiology due to the dietary intervention. We recently showed that dietary supplementation with annatto-extracted TT lowered the levels of serum metabolites, dicarboxylic fatty acids, and inflammatory/oxidative stress markers  in obese mice, suggesting TT supplementation reduces inflammation and oxidative stress (oxidized glutathione and GSH/GSSH) and improves macronutrient metabolism (carbohydrates) .In the current study, we have translated animal findings to focus research on a clinical trial using postmenopausal women (PMW). In PMW, the rate of bone loss increases dramatically due to estrogen deficiency and contributes to greater bone fractures. Estrogen is a phenolic compound that shares similarities with the structure of well-known lipophilic antioxidants such as α-tocopherol . Thus, eWe previously reported that 12-week annatto-extracted TT supplementation decreased bone resorption and improved bone turnover rate via suppressing bone remodeling regulators, namely serum soluble receptor activators of nuclear factor-kappaB ligand and serum osteoprotegerin, in PMW with estrogen deficiency . Therefo2) with no known bone metabolic disease or serious health issues  were recruited for this study. Participants were randomly assigned into three groups: Placebo, 300mg TT, and 600mg TT for 12 weeks. All participants were provided with dietary calcium  and vitamin D  daily during the study period. Serum samples were collected at baseline, 6 weeks, and 12 weeks (end) for outcome measures. Eighty-seven subjects completed the study, with two dropouts due to loss of interest. Pill compliance rates for placebo, 300mg TT, and 600mg TT were 93, 92, and 91%, respectively. We have previously reported the flow of subject recruitment, exclusion, dropout, and the final number of subjects in the main study  have been previously described . In brieg for 20 min), and stored in −80°C freezer before analysis.Demographics and medical history were obtained at the time of enrollment. Food intake was determined by a semi-quantitative Harvard Willett Food Frequency Questionnaire, physical activity level via Godin Leisure-Time Exercise Questionnaire, and body composition by SC-331S Body Composition Analyzer  were recorded at baseline and after 12 weeks. Overnight fasting blood was drawn from a superficial arm vein, and serum samples were collected after centrifugation . Sample preparation was carried out in a manner similar to a previous study . In brieMetabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon. Identification of known chemical entities was based on comparison to metabolomic library entries of purified standards . The idehttp://cran.r-project.org/) was used. For statistical analyses and data presentation, biochemicals were rescaled to set the median to one, based on all samples. Following log transformation and imputation of missing values, any missing values were assumed to be below the limits of detection and were imputed with the compound minimum . As the minimum observed value for each compound, ANOVA contrasts were used to identify biochemicals that differed significantly between experimental groups.Two types of statistical analyses for metabolites were performed: (1) significance tests and (2) classification analysis. Standard statistical analyses were performed in ArrayStudio on log-transformed data. For analyses not standard in ArrayStudio, the R program (http://cran.r-project.org/) was used to compare between the placebo- and High TT-treated groups at baseline and after 12 weeks. P-values <0.05 were considered significant. We further performed Welch's t-test, a standard statistical test completed with R program (http://cran.r-project.org/), for ratio High-TT (12 weeks vs. 0 week) to the ratio Placebo (12 weeks vs. 0 week) to identify biochemicals that differed significantly (p < 0.05) between experimental groups. An estimate of the false discovery rate  was calculated to account for the multiple comparisons that normally occur in metabolomic-based studies. Classification analyses used included principal components analysis (PCA), hierarchical clustering, and random forest. Analyses were graphed using Array Studio software, version 7.2 . The Random forest is a supervised classification technique based on an ensemble of decision trees  a standard statistical test completed with R program (on trees . For then = 234), decline to participate (n = 80), and other reasons (n = 12). The remaining 89 subjects were randomized into three groups  and 87 subjects completed the study (attrition rate: 2%) with a >90% compliance rate by a pill count. In this metabolic profiling study, only serum samples from Placebo and 600mg TT groups at baseline and after 12 weeks were performed. Participants in the Placebo and 600mg TT groups showing no significant differences in age (60.8 ± 5.2 yr for Placebo vs. 59.2 ± 6.6 yr for 600mg TT), weight (71.8 ± 12.6 kg for Placebo vs. 71.9 ± 14.8 kg for 600mg TT), height (158.8 ± 6.1 cm for Placebo vs. 159.2 ± 5.9 cm for 600mg TT), and BMI (28.5 ± 4.9 kg/m2 for Placebo vs. 28.2 ± 5.5 kg/m2 for 600mg TT). We did not find significant changes in (a) macro- and micronutrients intakes, (b) physical activity level, and (c) body composition between the Placebo and the 600mg TT groups at both baseline and at the end of study (p > 0.05). The detailed report of medical history, macro- and micro-nutrients intake, physical activity, and body composition of study participants has been published previously  were detected and categorized into six major categories, namely: amino acids, cofactors and vitamins, lipids, nucleotides, peptides, and xenobiotics.p < 0.05). Principal component analysis (PCA) for serum revealed no separation between the Placebo and the 600mg TT groups  and significantly lower in the 600mg TT group vs. Placebo at 12 weeks of treatment.The levels of serum TT and their metabolites are listed in The changes in metabolites associated with bone matrix are listed in p = 0.081) and proline-hydroxyproline (pro-hydroxy-pro) (p = 0.0003) were lower after 12 weeks of TT supplementation. Furthermore, there was a trend toward a lower level in N-acetylglucosamine/N-acetylgalactosamine  and higher serum glucuronate levels (p < 0.05) in the 600mg TT groups from 0 week to 12 weeks.Compared to the 600mg TT group at 0 week, both of C-glycosyltryptophan (p < 005). In this case TT supplementation may conserve met and cys amino acids, sulfur derivatives or lead to a lower catabolism of these amino acids. Furthermore, the sulfur containing amino acids , when comparing serum levels after 12 weeks of TT administration (600mg TT after 12 weeks vs. 600mg TT baseline) as shown in no acids  such as no acids .Tyrosine and tryptophan metabolites were altered after TT supplementation . ChangesIn terms of tryptophan metabolites, the subjects given 600 TT for 12 weeks had lower levels of n-acetyltryptophan and kynurenine, while a higher level of 3-indoxyl sulfate . SimilarSteroid hormone metabolism was affected in the Placebo group and to a greater extent in the 600mg TT group after 12 weeks of the study . When coCompared to baseline, the levels of several serum lysophospholipids were higher in individuals consuming TT for 12 weeks, e.g., 2-palmitoyl-glycerophosphocholine (GPC), 1-palmitoleoyl-GPC, 2-palmitoleoyl-GPC, 1-stearoyl-GPC, 1-oleoyl-GPC, 1-linoleoyl-GPC, and 1-arachidonoyl-GPC, 1 palmitoyl- glycosylphosphatidylinositol (GPI), 1-stearoyl-GPI, 1-oleoyl-GPI, 1-linoleoyl-GPI, and 1-archidonoyl-GPI . In contThe levels of several acyl carnitine metabolites  were lower only in the serum samples of subjects consuming TT group at 12 weeks compared to baseline as shown in This study is the first double-blinded placebo-controlled randomized study to evaluate the effect of dietary annatto-extracted TT supplement on serum metabolites in postmenopausal women using a non-targeted metabolomics analysis. Tocopherols can be metabolized to carboxyethyl-hydroxychroman (CEHC) derivatives and excreted in the urine . CEHC arThe findings that TT supplementation resulted in the higher levels of gamma-TT, gamma-CEHC , and gamma-CEHC glucuronide (a gamma-CEHC metabolite) in the serum of subjects corroborates with findings of previous studies , 55. Frep = 0.0003) and C-glycosyltryptophan (p = 0.081) were decreased in the serum after 12 weeks of TT supplementation suggests that TT favors bone remodeling by a decreased bone resorption/collagen degradation in collagen turnover.Bone and skin contain large amounts of proline/hydroxyproline, and bone is a mixture of hydroxylapatite and collagen . Bone mip = 0.09) and a 25% higher serum glucuronate levels further support that dietary TT favors bone metabolism in postmenopausal women, in part, by reducing the release of bone matrix (Proteoglycans are composed of glycosaminoglycans (GAGs) covalently linked to the core protein, with each GAG, such as chondroitin sulfate or heparin sulfate, consisting of repeating disaccharide units that contain a sulfated amino sugar, e.g., N-acetylglucosamine or N-acetylgalactosamine, linked to glucuronic acid or iduronic acid . In the e matrix . All fine matrix .P < 0.001) in the 600mg TT group relative to the placebo group  after 12 weeks of TT supplementation. Methionine can be used to synthesize cysteine, a limiting factor in glutathione synthesis, which is utilized in antioxidant defense . In thisbo group . FurtherTyrosine and tryptophan metabolite levels were lower after TT supplementation . ChangesKnudsen et al. reported that butyrate has been shown to be a potent metabolic and inflammatory modulator in the development of non-alcoholic fatty liver disease. Phenylacetic acid, imidazole propionate, and HPLA have been identified as potential inducers of steatosis and hepatic inflammation, whereas indolic compounds (indole and indole-3-acetate) seem to preserve liver integrity . Caussy Steroid hormones are synthesized from cholesterol in the adrenal glands and can undergo esterification with sulfate in the liver and brain . In addi1) and Sn-2 (PLA2) position of phospholipids  were postulated to promote inflammatory effects until recent opposing evidence . Specifiholipids . The difone mass .Carnitine (levocarnitine) is a naturally occurring compound found in all mammalian species. An important biological function of L-carnitine is in the transport of fatty acids into the mitochondria for subsequent β-oxidation, a process which results in the esterification of L-carnitine to form acylcarnitine derivatives. As such, the endogenous carnitine pool is comprised of L-carnitine and various short-, medium- and long-chain acylcarnitines. The physiological importance of L-carnitine and its obligatory role in the mitochondrial metabolism of fatty acids have been clearly established . It has The effects of TT on fatty acid synthesis/metabolism are intriguing, and have been observed previously. Allen et al. reported that TT increased carnitine palmitoyltransferase (CPT) 1A and CPT2 . Gamma-TFurther, it appears that TT resulted in higher levels of PUFA, which might be related to greater elongation . Moreover, TT led to higher levels of lysophospholipids. Lysophospholipids are bioactive signaling lipids that are generated from phospholipase-mediated hydrolyzation of membrane lipids. Other findings show that a placebo group (860 mg of olive oil per day) had lower saturated and monosaturated metabolite levels, and PUFA metabolism consistent with obese mice .In conclusion, our global metabolomic study of postmenopausal women with low bone mass found that the 12-week TT supplementation altered a number of biochemicals representing metabolism related to redox homeostasis, bioactive lipids, amino acid metabolism, steroid hormones synthesis, and bone degradation/collagen turnover. Changes in redox homeostasis manifested in transmethylation and transsulfuration pathway biochemicals could stem from the antioxidant activity of TT. Differences in compounds associated with bone degradation/collagen turnover indicate that, consistent with our previous findings, TT affects bone metabolism and collagen synthesis/degradation. Further, TT supplementation may conserve methionine and cysteine amino acids, sulfur derivatives or lead to a lower catabolism of these amino acids. Changes in tyrosine and tryptophan metabolites would suggest changes in the gut microbiome activity. Finally, differences in steroid hormones may point toward yet unreported effects of TT on the expression of steroid biosynthetic enzymes. As the members of the vitamin E family gaining more recent interests, tocotrienols have demonstrated broad biological effects on oxidative stress, inflammation, cell proliferation, adipogenesis, osteoclastogenesis, and osteoblastogenesis, and lipid metabolism. Our findings provide evidence to pursue future studies that would afford a mechanistic understanding of the nutritional values and health benefits of TT on bone remodeling and osteoblast functions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Texas Tech University Health Sciences Center Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.CLS, HM, DMD, and BAW: conceptualization and writing—review and editing. CLS and BAW: methodology and writing—original draft preparation. CLS: supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.Department of Pathology, Texas Tech University Health Sciences Center, Lubbock, TX, provided fund for metabolomics analysis. American River Nutrition, LLC., Hadley, MA supplied the tocotrienol supplement and funded this clinical trial. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of American River Nutrition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There were no differences between the groups in the Lunge Test  or in pressure algometry  . No significant short-term significant differences were found in the rest of the variables. (4) Conclusions: Diathermy can induce changes in the absolute temperature of the medial gastrocnemius muscle.(1) Background: Myofascial pain syndrome (MPS) is a clinical condition characterized by localized non-inflammatory musculoskeletal pain caused by myofascial trigger points. Diathermy or Tecar therapy (TT) is a form of noninvasive electro-thermal therapy classified as deep thermotherapy based on the application of electric currents. This technique is characterized by immediate effects, and its being used by high performance athletes. (2) Methods: A total of thirty-two participants were included in the study who were professional basketball players. There was a 15-person Control Group and a 17-person Intervention Group. TT was applied in the Intervention Group, while TT with the device switched off (SHAM) was applied in the Control Group. The effects were evaluated through the Lunge test, infrared thermography, and pressure threshold algometry at baseline, 15, and 30 min after the intervention. (3) Results: the Intervention Group exhibited a greater increase in absolute temperature (F Electrical or electromagnetic stimulation-based physical therapy has been applied in rehabilitation with successful results. Specifically, resistive capacitive electrical transfer therapy has been used in physical rehabilitation and sports medicine to treat muscle, bone, ligament, and tendon injuries ,2. RadioTecar therapy (TT) is a form of non-invasive electrothermal therapy classified as deep thermotherapy based on the administration of electric currents within the radiofrequency range, constituting a monopolar capacitive resistive radiofrequency of 448 KHz ,3,4,5. TMyofascial pain syndrome (MPS) is a clinical condition characterized by localized non-articular musculoskeletal pain  as a conMPS is caused by trauma or muscle overuse in certain sports or activities. It can also appear in a weak muscle when demands exceed capacities in terms of the muscle activity not being able to withstand the strain exerted . In addiTravell and Simons used the term myofascial trigger points (MTrPs) , charactTriceps sural injuries are common in a wide number of sports but are mainly prominent in soccer ,22. TherHowever, the acute impact of the TT therapeutic tool on the treatment of medial gastrocnemius related to MTP has not yet been investigated. Thus, the main aim of the present study was to analyze the acute effect of TT on latent MTPs on skin temperature, ankle ROM, and pain in professional basketball players. We hypothesized that the use of TT would produce an acute increase of skin temperature and ankle ROM and, at the same time, cause a decrease of hyperalgesia associated with MTPs.This is a randomized clinical trial involving thirty-two participants. Once the participants completed the preliminary stages, they were randomized using a simple Excel procedure in two groups: The Diathermy Group and Control Group. The process described above was managed by a staff member who did not participate in the study. All research staff were blinded to this allocation process. Single-blinding was used to reduce bias in the interpretation of the results. The patients were not informed of the treatment being performed in the immediate post-treatment and successive measurements.n = 32) amateurs basketball players were involved in the present study coming from the Picanya national category basketball team . The mean ± SD of age, height, body mass, and body mass index were 22.84 ± 5.86 years; 179.33 ± 7.98 cm; 75.73 ± 11.51 kg; and 23.51 ± 2.81 kg/m2. All of the participants were previously informed about the procedure and signed the informed consent prior to their participation. A parental or guardian’s authorization and consent form was attached for the participants who below 18 years of age. The research was previously registered in ClinicalTrials.gov .A total of thirty-two (The following exclusion criteria were applied: (a) connective tissue pathology; (b) lymphatic disorders (lymphadenopathy); (c) skin injuries ; (d) peripheral neuropathies; (e) previous fractures; (f) previous lower limb surgeries (in the past 12 months). There were three adverse events during the investigation process (post-treatment skin reactions) resulting in a total of three dropouts who were excluded from the initial sample.The inclusion criteria were: (a) be an active national-level male basketball player; (b) be aged between 16–39 years of age; (c) not having suffered leg injuries in the past 6 months; (d) not having suffered a triceps sural rupture in the past two years; (e) show a difference of 1.5 cm between limbs in ankle dorsiflexion restriction measured through the lunge test; (g) be diagnosed with latent MTPs in the gastric–soleus complex through manual therapy in the dominant side.Latent TrPs were identified as follows: (1) palpable taut band within the muscle; (2) presence of a hypersensitive spot in the taut band; (3) presence of a local twitch response of the taut band with palpation .® therapy machine   see . The thealy) see . The tecThe head of the device was applied over the treated area for 25 min (15 min of capacitive head and 10 min of resistive head), maintaining the same intensity and at all times. It was applied along and in circles over the medial gastrocnemius, generating light pressure above the muscle belly. The base plate was located below the region of the tibia to close the current circuit. The head of the device was applied over the entire medial gastrocnemius and not just over the latent MTP to improve the blood flow speed .At baseline, data were collected through thermography (temperature), algometry (pain), and the lunge test (ankle ROM) before the intervention. All of the measurements were repeated immediately, 15′ after and 30′ after treatment. To eliminate any cross-effect that may interfere with the results, immediately after the post-treatment measurements, the participants were seated by research staff in a chair with 90° hip and knee flexion in a controlled room at a preset temperature see .The same protocol as the Diathermy Group was performed but with the device in off mode (SHAM).The thermography protocols were performed according to the International Academy of Clinical Thermology . The equFor the thermographic assessment, the ideal temperature values (18–25 °C), relative humidity (39.8%), and atmospheric pressure  ,30,31 s.The participants were placed barefoot on a 35-cm-high platform (Step) facing the wall with their hands on their waists with their bare legs 40 cm apart against a neutral black background so as to not interfere with body temperatures .Images of both triceps surals were taken and interpreted with FLIR Tools software. Through the analysis, maximum (TMAX), minimum (TMIN), and mean (TMED) absolute temperatures were obtained from the medial gastrocnemius at the selected times.2  = 9.64, p = 0.001, eta = 0.24), showing no significant differences in the comparison Group x Time . In the within-group comparison, the Diathermy Group showed differences in the comparison of post-immediate vs. pre-  and post-15 vs. pretreatment . In relation to the Control Group, Bonferroni’s post hoc showed statistically significant differences in the comparison of post immediate vs. pre-captures . No statistically significant differences were found between any of the possible between-group comparisons (p > 0.05).Considering the pain values , the of the mixed factorial ANOVA showed statistically significant differences in the main effect of Time  in the main effect of time . After transforming the scores of the time variable regarding to pre-intervention values, the model was corrected to F = 2.91, p = 0.072, η2p = 0.08, without ever obtaining statistically significant differences. In regard to ankle mobility, RM ANCOVA showed no statistically significant differences (Fp = 0.213). However, statistically significant differences were found in the comparison of the main effect of the time variable ) and post-15 min vs. 30 min .The Bonferroni post hoc analysis showed that there were no statistically significant differences in the comparison of the variables between the groups . Bonferroni’s post hoc analysis showed no statistically significant differences for any of the comparisons , measurements post-15 min , and measurements post-30 min ). On the other hand, no statistically significant differences were found in the effect of Group  and in the interaction between Group x Capture . Bonferroni’s post hoc analysis showed statistically significant differences between the Diathermy Group vs. the Control Group . Regarding muscle temperature, the mixed factorial ANOVA showed significant differences in the main effect for The main objective of the study was to analyze the acute effect of TT on latent MTPs on skin temperature, ankle ROM, and pain in professional male basketball players. Our results showed a significant increase in the absolute temperature in the diathermy group, with no significant differences within and between groups in the rest of the parameters. An increase in pain (VAS) was shown in the TT group, which describes an increased sensitivity in the MTP following the application of this technique. Currently, only few studies have analyzed the effects of TT applied on medial gastrocnemius MTPs in basketball players on pain. However, to our knowledge, this is the first study to evaluate the effects of TT on temperature, hyperalgesia, and ankle ROM.TT produces a thermal effect, assumed to be a biological effect related to hyperthermia. Our results are similar to previous studies regarding the temperature due to its increase after applying the technique . For insBy contrast, other investigations found no significant changes when TT was applied over other anatomical regions. For instance, TT did not alter the circulation in the peritendinous region of the Achilles tendon .Furthermore, the present study noted that after 15 min of the application, there is a 1.5° drop from the absolute temperature peak. According to Kumara and Watson’s study on the thermophysiological effects of the skin after applying TT, this technique was compared to pulsed short-wave therapy, which obtained better results than those in the TT group . These fRegarding ankle mobility, we did not find any significant improvement, which was in contrast with previous investigations. MTPs may adversely affect clinical effects on restricted ROM . AccordiCurrent evidence describes an increase in tissue elasticity after TT . Such inVarious treatments for MTPs have been shown to improve clinical outcomes, including improved muscle strength, ROM, and pain reduction . For exaApplying TT alone on a MG latent MTP does not produce changes in ROM when it is measured with the lunge test. On the other hand, Draper et al. reported that the combination of TT and joint mobilization is considered to be an effective treatment to recover joint ROM in elbow extension in post-surgical patients .Despite our results, other studies that have used the same treatment technique have found an increase in ROM in other joints, such as in the right rotation of the cervical spine . HoweverRegarding pain, we did not find any changes in pain assessed by VAS scale. This result is in line with the study of Aguacil I et al. , which dThe assessment of the sensitivity of the palpable nodule in the latent MTP through algometry justifies the VAS measurement evaluation of this study. An advantage of TT was that pain scarcely increased compared to other techniques commonly used in physiotherapy, although an increase in hyperalgesia was noted in the post-immediately measurement.Some possible factors that can explain the reduction of hyperalgesia on VAS thirty minutes after treatment that we can find are the natural evolution of the disease ; the speContrary to our results, other similar treatments were effective in reducing mechanical hyperalgesia compared to control groups. The main difference was that the technique was applied on active MTPs rather than on latent MTPs . DespiteFurthermore, previous studies have shown that pressure inhibition treatment reduced pain in MTPs in the upper trapezius . These sIf we extrapolate the effects of TT in patients diagnosed with different pathologies, the literature shows that TT is a useful therapy for reducing pain in patients with osteoarthritis . In otheAlthough no promising changes in the studied variables were obtained, it has been observed that currently, there are a wide range of standardized protocols for the application of this technique . This meThe theoretical consequences of this work entail the need to evaluate this technique using other methods or assessment tests and to verify or contrast our results. The importance of this lies in the possibility of a new hypothesis and new experimental studies. Possible practical applications of this study are related to the treatment of muscle stiffness in athletes in a non-invasive, painless, and effective way of using TT.The present study is not without limitations. Although thermography is an innovate technique for measuring skin temperature, we extrapolate that the external temperature reflects the internal or muscle temperature. In addition, the lack of reliability analysis of the algometer assessment could be also considered to be a limitation.Future research with larger sample sizes should examine further does–response relationships between TT and other physiological parameters in latent and active MTPs. In addition, further studies are needed to compare the effects of TT against other treatment modalities.In conclusion and according to our results, TT can induce temperature changes in the medial gastrocnemius in professional male basketball players, generating an increase of local temperature and a decrease of local pain (VAS) at the MTP after treatment. In addition, TT does not affect the ankle dorsiflexion ROM.These results suggest that use of TT could be useful to improve muscle recovery. Further studies with similar characteristics and implemented during an experimental period to assess chronic adaptations would be needed to gain in-depth knowledge of the advantages of this technique.


P = 0.017). Compared with prophylactic G-CSF alone, prophylactic G-CSF and antibiotics significantly suppressed the occurrence of FN.The aim was to investigate the efficacy of prophylactic antibiotics for the prevention of febrile neutropenia (FN) during cabazitaxel therapy for castration-resistant prostate cancer (CRPC) with G-CSF. We retrospectively studied 443 cycles of cabazitaxel therapy given to 56 patients with CRPC at Keio University Hospital between May 2012 and August 2018. Statistical analysis was conducted to determine whether the combination of prophylactic G-CSF and antibiotics was more effective in preventing FN, compared with prophylactic G-CSF alone. Prophylactic PEG-G-CSF or G-CSF was administered in all 443 cycles. Only fluoroquinolones were used as prophylactic antibiotics and were administered in 328 cycles (74.0%). FN occurred in 5 cycles (1.1%). Prophylactic antibiotics were administered in 327 cycles (74.6%) in the FN-negative group and in only 1 cycle (20.0%) in the FN-positive group. Chi-square test indicated the incidence of FN was significantly lower in the group that received prophylactic antibiotics compared with the group that did not receive prophylactic antibiotics ( However, cabazitaxel therapy often has a high risk for hematologic toxicity, such as neutropenia. According to the TROPIC study, World Health Organization (WHO) grade 3 or higher neutropenia occurred in 82% and febrile neutropenia (FN) occurred in 8% during cabazitaxel therapy1. On the other hand, a phase I study in Japan reported high rates of WHO grade 3 or higher neutropenia in 100% and FN in 54.5%2. The American Society of Clinical Oncology (ASCO) guideline recommended prophylactic administration of granulocyte-colony-stimulating factor (G-CSF) for regimens that have a reported FN incidence of ≥ 20%3. In Japan, a phase IV trial  revealed a 9.5% rate of FN among all patients (N = 21) who were administered prophylactic polyethylene glycol (PEG)-G-CSF4. These results indicated that prophylactic G-CSF was effective for prevention of FN.Cabazitaxel for metastatic castration-resistant prostate cancer (mCRPC) has been recommended as second-line chemotherapy after docetaxel. A phase III trial  demonstrated improved overall survival in patients with CRPC after or during docetaxel-containing therapy8. Our previous research on the same patient population (N = 41) reported a 7.3% incidence rate for FN9; this rate was slightly lower, compared with the rates in the other reports in Japan. On hindsight, we deemed that the reason for this low rate was the administration of prophylactic antibiotics, with informed consent, for patients in whom the risk for FN was considered high. Our previous study revealed that patients aged ≥ 75 years and those with a history of radiation therapy were at high risk for FN when treated with docetaxel for CRPC10.To date, in Japan, the reported incidence of WHO grade 3/4 FN ranged from 16.3 to 30.6% and prophylactic G-CSF administration was reported in 65.9% to 95%11.On the other hand, the ASCO guideline recommended administration of prophylactic antibiotics only for patients who were expected to have < 100 neutrophils/μL for > 7 daysIn cabazitaxel therapy, the utility of prophylactic administration of antibiotics for the prevention of FN is unclear. The aim of this study was to reveal the efficacy of prophylactic antibiotics for the prevention of FN during cabazitaxel therapy with G-CSF.2 (median 22.5 mg/ m2) with prednisone 10 mg/ day every 3–4 weeks. All patients were administered prophylactic PEG-G-CSF or G-CSF. Only fluoroquinolones were used as prophylactic antibiotics. Subcutaneous injection of pegfilgrastim (3.6 mg) was administered as PEG-G-CSF for 411 cycles. Subcutaneous injection of lenograstim (100 μg) daily was administered as G-CSF for 32 cycles. At the beginning of the enrollment period, pegfilgrastim was not approved in Japan, so we administered lenograstim daily. But after approval, we have unified lenograstim with pegfilgrastim. Lenograstim was administered to 8 patients for 32 cycles. Regarding the administration of prophylactic antibiotics, we administered ciprofloxacin 200 mg orally twice daily or levofloxacin 500 mg orally once daily or sitafloxacin 50 mg orally twice daily for 7 days. In Japan, a phase IV trial 4 determined that the median time to nadir in the Japanese population was 8 days (range 7–9 days), so it is considered necessary to cover the median time to nadir on the administration start date. We administered prophylactic G-CSF on day 2–4 and prophylactic antibiotics on day 4–5 to cover the nadir period.We retrospectively researched a total of 443 cycles of cabazitaxel therapy given to 56 patients who had progressive castration-resistant prostate adenocarcinoma during docetaxel therapy at Keio University Hospital between May 2012 and August 2018. The work described has been carried out in accordance with the Declaration of Helsinki for experiments involving humans. Obtaining informed consent was exempted because our study design was retrospective and all identifying information was removed. For each cycle, we retrospectively investigated the cabazitaxel dose, prophylactic antibiotics, prophylactic administration of G-CSF, and the development of FN. Almost all patients were given intravenous cabazitaxel at 20–25 mg/ m12.According to the Infectious Diseases Society of America (IDSA), we defined neutropenia as an absolute neutrophil count of < 1000/ μL and severe neutropenia as an absolute neutrophil count of < 500/ μL. We defined FN as a single oral temperature of ≥ 38.3 °C or a sustained temperature of ≥ 38.0 °C for more than 1 hourP < 0.05 was considered to indicate statistical significance. All statistical analyses were performed using the Statistical Package of the Social Sciences, version 25.0 .The continuous variables of the FN-positive and FN-negative groups were compared using the chi-square test. For multivariate analysis, logistic regression was used. For all statistical analyses, tests were one-sided and All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the Declaration of Helsinki (1964) and its later amendments or comparable ethical standards. The study was approved by the Institutional Ethics Board of Keio University Hospital (No. 20150159).This study was approved by the Institutional Ethics Board of Keio University Hospital (No. 20150159), which exempted obtaining informed consent because our study design was retrospective and all identifying information was removed.1 and the TED 11,576 study in Japan13. The dose of cabazitaxel was ≤ 20 mg/m2 in 37.9%, 22.5 mg/m2 in 29.6%, and 25 mg/m2 in 32.4%. The reasons for dose reduction to ≤ 20 mg/m2 were old age, reduction of PS during the cabazitaxel administration cycle, and grade 3/4 adverse effect (AE) in the previous cycle. Prophylactic PEG-G-CSF or G-CSF was administered in all cycles, whereas prophylactic antibiotics were administered in 328 cycles (74.0%). No AE was observed after prophylactic antibiotics. FN occurred in 5 cycles (1.1%) in 4 patients. Cabazitaxel in combination with prednisone was well tolerated. Table All patient characteristics are shown in Table Table Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter freundii complex were detected in sputum cultures. Blood cultures were negative. Ciprofloxacin-resistant bacteria were not detected. Meropenem (1 g) was administered intravenously thrice a day. He was discharged on the 10th day of illness after the recovering from pneumonia.Prophylactic antibiotics were administered in 327 cycles (74.6%) in the FN-negative group and in only 1 cycle (20.0%) in the FN-positive group. The patient in whom FN occurred with prophylactic antibiotics was 71 years old. FN occurred on day 6 of the 7th cycle of cabazitaxel. PEG-G-CSF was administered on day 3, and oral ciprofloxacin administration was started on day 3 for 7 days. He had cough and sputum. Chest X-ray film revealed a consolidation in the left lower lung zone, suggesting pneumonia. P = 0.017).Dose reduction was classified as < 25 mg, but no significant difference was observed between the two groups. The patients were grouped by age (≥ 75 years or < 75 years) and by the ECOG PS at the time of introduction . No significant difference was observed. Results of the analysis revealed that prophylactic antibiotics significantly reduced the FN rate (P = 0.017), but the dose, age, and ECOG PS were not. Prophylactic G-CSF has been shown to reduce the incidence of FN, which can be burdensome for patients, especially those who go through a serious course. Therefore, keeping the incidence of FN as low as possible is necessary. Although the ASCO guideline recommended prophylactic antibiotics only for patients expected to have < 100 neutrophils/ μL for > 7 days3, we considered that concomitant administration of antibiotics and G-CSF would be a superior method.Our results showed a significantly lower FN incidence in the group that received prophylactic antibiotics than in those that did not  reported that prophylactic antibiotics helped reduce overall mortality  and infection-related mortality 14. Most of these reports were on chemotherapy for hematologic malignancies. Only few reports have compared prophylactic G-CSF with antibiotics and prophylactic G-CSF alone for other solid carcinomas; we summarized these reports in Table 419. Many of these studies reported a reduced incidence of FN by concomitant use of antibiotics. One report stated that there was no significant difference in the incidence of FN between the G-CSF plus prophylactic antibiotic and the G-CSF alone groups; however, G-CSF alone had very low FN incidence in 1 of 23 patients17. One study reported no significant difference when the neutropenia grade was 3 or less, but in the group with grade 4 neutropenia, infections were significantly suppressed 19. In Japan, the rate of WHO grade 3/4 neutropenia due to cabazitaxel was high2, and prophylactic antibiotics are expected to be efficacious. For solid carcinoma, one study on 161 patients who received cyclophosphamide, doxorubicin, and etoposide regimen for small cell lung carcinoma showed 55% reduction of FN incidence with the administration of G-CSF plus prophylactic antibiotics than of G-CSF alone18. There were very few reports with similar comparisons for each solid carcinoma or regimen.The use of prophylactic antibiotics has been most commonly reported in the hematologic oncology field. A systematic review of 109 trials  Gram-negative coliforms, and methicillin-resistant Staphylococcus aureus (MRSA) between the placebo group and the levofloxacin group21. It would be necessary to keep in mind the possible emergence of resistant bacteria and to refrain from unnecessary long-term use of antibiotics.Fourth, a possibility of the emergence of resistant bacteria with the administration of prophylactic antibiotics is concerning. In our study, none of the patients developed resistant bacterial infection, and none of the reports listed in Table Compared with G-CSF alone, prophylactic administration of G-CSF preparations plus antibiotics significantly reduced the occurrence of FN during cabazitaxel therapy for CRPC.Supplementary Information


Background: Non-small cell lung cancer (NSCLC) accounts for the largest pathological type of lung cancers, and it is characterized by high incidence and poor prognosis. However, the molecular mechanisms involved in development and progression of NSCLC are not well elucidated. In this study, we aimed to explore the role and regulatory mechanism of RPS9 in NSCLC.Materials and methods: The RPS9 expression in NSCLC tissues and cell lines was assessed by qRT-PCR and western blot. Knockdown of RPS9 induced by RNA interference (RNAi) method in PC9, A549 and H1299 cells. Overexpression of RPS9 induced by transient transfection in H292 cells. Cell proliferation, colony formation, metastasis and apoptosis abilities were determined by CCK-8 assay, colony formation assay, transwell assay and flow cytometry, respectively. The host signaling pathways affected by RPS9 were screened by antibody library and proved by western blot.Results: RPS9 was significantly upregulated in NSCLC tissues and cell lines. Overexpression of RPS9 predicted poor prognosis of NSCLC patients. Knockdown of RPS9 obviously repressed cell proliferation, metastasis, and induced apoptosis. Mechanistically, suppression of RPS9 inhibited the expression level of phosphorylation of Stat3 and Erk.Conclusion: Our study clarified that knockdown of RPS9 inhibits the progression of NSCLC via inactivation Stat3 and Erk signaling pathways. Lung cancer is one of the most frequently diagnosed malignant cancer and the first cause of cancer-related death in the world RPS9 is ribosomal protein gene and located at chromosome 19ql3.4 Previous studies have suggested that RPS9 dysregulation was involved in tumorigenesis, such as pancreatic cancer, head, and neck cancer (such as head and neck squamous cell carcinoma) and so on In this research, we provide evidence for the first time that RPS9 is upregulated in NSCLC patients and cell lines and overexpression of RPS9 indicates poor prognosis. Strikingly, RPS9 knockdown inhibits NSCLC cell proliferation, colony formation and metastasis. In addition, RPS9 suppressed apoptosis in NSCLC cells. More interestingly, we found that knockdown of RPS9 could inhibit the progression of NSCLC via regulating Stat3 and Erk signaling pathways. This study highlights the function of RPS9 in NSCLC growth and metastasis and may facilitate development of new diagnostic or therapeutic biomarker to improve the outcome of NSCLC patients.A total of 143 pairs of primary NSCLC and matched adjacent normal lung tissues were collected from patients who underwent surgery between 2017 and 2019 in Huashan Hospital, Fudan University. Written informed consents were signed before the research started. And this research was authorized by the Ethics Committee of Huashan Hospital, Fudan University. All procedures were carried out in accordance with the ethical guidelines of the Declaration of Helsinki. Three NSCLC cell lines  and normal human bronchial epithelial cells (16HBE) were obtained from the Cell Bank of the Chinese Academy of Sciences . All the cells were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37 ºC. Cells were used when they were in the logarithmic growth phase.The total RNA from NSCLC tissues and cell lines was purified using RNAiso Plus reagent . Then, RNA was converted to complementary DNA (cDNA) by the PrimeScript RT Reagent Kits  according to the manufacture's protocol. The qRT-PCR assays were carried out using SYBR Premix Ex Taq II Kit . The following primer sequences were used: RPS9: Forward: 5'-GAAGCGGAGCCAACATGC-3', Reverse: 5'-ATACTCGCCGATCAGCTTCAG-3'. GAPDH served as an internal control.The siRNAs targeting RPS9, and corresponding negative control siRNAs (si-NC) were designed and synthesized by Genechem . Plasmid used for transient transfection were designed and synthesized by Ribobio . Cells were plated at 60-70% confluence in a 6-well plate. Cell transfection was performed using Lipofectamine 2000 reagents . After transfection for 48 hours, the knockdown and overexpression efficiency were validated by both qRT-PCR and western blot.For transwell migration assays, cells were resuspended in DMEM without FBS and 4 × 10^5 cells were seeded into the upper chamber of a 24-well insert  precoated with Matrigel. 800-µL DMEM supplemented with 10% FBS was placed in the lower chamber. After incubation for 24 hours, the invaded cells on the lower surface were fixed with methanol, stained with crystal violet and the number of invaded cells from five random fields was counted under a light microscope. For the invasion assay, 1 × 10^6 cells were seeded into the upper Matrigel invasion chambers , and the following procedures were carried out similarly as what was described in the transwell migration assays.Cells were seeded into 96-well plates in triplicate and cell viability was detected by the Cell Counting Kit-8 (CCK-8) assay  according to the manufacturer's instructions. For colony formation assay, cells were plated in 6-well plates (1000 cells/well) and cultured for 14 days. After washed with phosphate-buffered saline (PBS) for three times, the colonies were fixed in methanol and subsequently stained with crystal violet for 30 min.For cell cycle analysis, after washed with PBS for three times, cells were collected and fixed with 70% ethanol. Following PBS washing, the cells were stained with 500μl propidium iodide (PI)  contained RNaseI  according to the manufacturer's instructions. Subsequently, the cell cycle was analyzed by a FACSCalibur flow cytometer . For apoptotic assay, after washed with PBS for three times, cells were stained with Annexin V-FITC and PI  at room temperature for 30 min. Then, the apoptotic cells were assessed using flow cytometer.Protein lysate were extracted using RIPA lysis buffer  contained protease and phosphatase inhibitors . Subsequently, proteins were electrophoresed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes . After blocked with 5% bovine serum albumin for 1 hour, the membranes were incubated with following primary antibodies at 4℃ overnight: RPS9 ; P- mTOR, mTOR , P-FAK, FAK, P-FoxO3a, FoxO3a, P-FoxO1, FoxO1, P-Stat3, Stat3, NF-κB, P-AMPK, AMPK, P-Smad2, Smad2, P-AKT, AKT, TGF-β, P-Erk and Erk ; β-actin . Next day, after washed in PBST for three times, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit antibodies  at room temperature for 1 hour. Finally, the bands were visualized and analyzed by SuperSignal West Femto Maximun Sensitivity Substrate .The results were presented as mean ± SEM (Standard error of mean). The unpaired two-tailed Student's t-test was used to compare the differences between two groups and the data were visualized by Graphpad prism version 8.0 software. All statistical analysis was conducted with SPSS version 22.0 software. A p-value less than 0.05 was considered statistically significant.To examine the potential role of RPS9 in NSCLC tissue, we firstly analyzed RPS9 mRNA levels in NSCLC data from TCGA database via UALCAN, the results confirmed that RPS9 was highly expressed in primary adenocarcinoma samples compared with normal samples Figure A. KaplanTo identify the biological function of RPS9 in NSCLC, we assessed the RPS9 expression levels in three NSCLC cell lines  and the normal human bronchial epithelial cells (16HBE) by qRT-PCR and immunoblotting analysis. As shown in Figure in vitro.To investigate the impact of RPS9 in NSCLC, we next silenced RPS9 expression in PC9, A549 and H1299 cell lines using si-RNA. The effective knockdown efficiency was validated by both qRT-PCR and immunoblotting analysis, demonstrating that RPS9 was significantly inhibited in PC9, A549 and H1299 cell lines after transfection with si-RPS9 Figure A-C. The To further examine whether RPS9 affects cell apoptosis capacities of NSCLC, flow cytometry was also utilized to determine the percentage of cell apoptosis. As shown in Figure Seeking to define the potential mechanism of RPS9 promoting NSCLC progression, the common phosphorylated protein related antibodies and their total protein antibodies including P- mTOR, mTOR , P-FAK, FAK, P-FoxO3a, FoxO3a, P-FoxO1, FoxO1, P-Stat3, Stat3, NF-κB, P-AMPK, AMPK, P-Smad2, Smad2, P-AKT, AKT, TGF-β, P-Erk and Erk from antibody library were utilized. Subsequently, the above phosphorylated protein and total protein levels were examined by western blot. As shown in Figure Ribosomal proteins, as indispensable components of living cells, are long considered as highly conserved and exert diverse roles in different cells Previously, RPS9 could act as a tumor suppressor in breast cancer, pancreatic cancer, and glioma Reversely, we revealed that RPS9, as a member of ribosomal proteins, was significantly up regulated in NSCLC tissue samples compared to the corresponding non-cancerous tissues. In addition, high levels of RPS9 in NSCLC tissues are related to poor prognosis. In the present study, RPS9 knockdown suppressed tumor growth, metastasis and promoted cell apoptosis, which is consistent with the previous researches in osteosarcoma, glioma and cervical carcinoma In NSCLC, it has been reported that RPS9 was involved in the activation of JAK-STAT and calcium signaling pathways. In the previous study, knockdown of RPS9 offset the promoting effects of si-BRCAT54 on cell proliferation and silencing of RPS9 activated JAK-STAT pathway and suppressed calcium signaling pathway gene expressions In conclusion, our findings exposed that RPS9 acted an oncogene in NSCLC via regulating the phosphorylation expression of Stat3 and Erk, promoting cell proliferation, migration, invasion, and inhibiting apoptosis in NSCLC. The role of RPS9 on the occurrence and metastasis of lung cancer needs more research. With the emergence of more studies, RPS9 may serve as an important marker for clinical prediction and diagnosis.Yiru Kong involved in data curation, methodology, writing—review and editing. Shuangshuang Deng involved in formal analysis, investigation, writing—review and editing. Xiaohua Liang involved in resources, writing—review and editing. Xinli Zhou involved in conceptualization, resources, writing—review and editing.


Central nervous system (CNS) infections caused by Gram-negative bacteria (GNB) are sometimes hard to treat due to antibiotic resistance and difficulty with penetration into cerebrospinal fluid (CSF). Cefiderocol (CFDC) which was approved by the FDA and the EMA in 2019 to 2020 is a siderophore cephalosporin with potent activity against various GNB including carbapenem-resistant strains. In this study, we evaluated the penetration of CFDC into CSF using a rat meningitis model. 1 CFU of E. coli SR200138. 200 mg/kg or 50 mg/kg of CFDC was administered via tail vein bolus injection to uninfected rats (n=4/sampling point) and rats with meningitis (n=4/sampling point) 24 hours after infection. CSF was collected by cisternal puncture and blood was collected from heart. The samplings were performed 0.25, 0.5, 1, 3, and 5 hours after dosing. The concentrations of CFDC in plasma and CSF for individuals were determined by LC/MS/MS. PK parameters for the average values in plasma and CSF were calculated. To induce meningitis, the anesthetized immunocompetent rats were infected by intracisternal inoculation of a bacterial suspension of 8.7×10CSF/AUCplasma: 0.149-0.183) compared with the uninfected rats (AUCCSF/AUCplasma: 0.0508-0.0588). The penetration rates of CFDC in the meningitis were comparable to those of piperacillin, cefepime, and meropenem in human  [1]. In both groups, elimination of CFDC from CSF was slower compared with that from plasma as seen with other β-lactam antibiotics such as meropenem, suggesting that T> MIC, an indicator that correlates with the efficacy of β-lactams, may be higher in CSF [2].CFDC concentration and the PK parameters are shown in Figure and Table, respectively. The penetration of CFDC from plasma to CSF was observed in both uninfected and meningitis groups, and the penetration rates increased in the rats withs meningitis :858–883.2. Nau, R. et al. Antimicrob Agents Chemother. 1998 Aug;42(8):2012–2016.Miki Takemura, MS, SHIONOGI & CO., LTD. (Employee) Sachi Kanazawa, PhD, Shionogi & Co., Ltd. (Employee) Naoki Kohira, PhD, Shionogi & Co., Ltd. (Employee) Yuki Aoe, BS, Shionogi TechnoAdvance Research Co., Ltd. (Employee) Atsushi Morimoto, n/a, Shionogi TechnoAdvance Research Co., Ltd. (Employee) Kana Horiuchi, MPharm, Shionogi & Co., Ltd. (Employee) Yuji Inoue, MPharm, Shionogi & Co., Ltd. (Employee) Yoshinori Yamano, PhD, Shionogi (Employee)


Idiopathic intracranial hypertension (IIH) is a rare disease of unknown aetiology related possibly to disturbed cerebrospinal fluid (CSF) dynamics and characterised by elevated intracranial pressure (ICP) causing optic nerve atrophy if not timely treated. We studied CSF dynamics of the IIH patients based on the available literature and our well-defined cohort.A literature review was performed from PubMed between 1980 and 2020 in compliance with the PRISMA guideline. Our study includes 59 patients with clinical, demographical, neuro-ophthalmological, radiological, outcome data, and lumbar CSF pressure measurements for suspicion of IIH; 39 patients had verified IIH while 20 patients did not according to Friedman’s criteria, hence referred to as symptomatic controls.0) were higher in IIH than symptomatic controls (p < 0.01). The mean CSF pressure was higher in IIH patients with psychiatric comorbidity than without (p < 0.05). In IIH patients without acetazolamide treatment, the RAP index and power of slow waves were also higher (p < 0.05). IIH patients with excess CSF around the optic nerves had lower relative pulse pressure coefficient (RPPC) and RESP than those without (p < 0.05).The literature review yielded 19 suitable studies; 452 IIH patients and 264 controls had undergone intraventricular or lumbar CSF pressure measurements. In our study, the mean CSF pressure, pulse amplitudes, power of respiratory waves (RESP), and the pressure constant  as key findings in IIH. Our study confirmed significantly higher lumbar CSF pressure and increased CSF pressure waves and RAP index in IIH when excluding patients with acetazolamide treatment. In overall, the findings reflect decreased craniospinal compliance and potentially depleted cerebral autoregulation resulting from the increased CSF pressure in IIH. The increased slow waves in patients without acetazolamide may indicate issues in autoregulation, while increased P Idiopathic intracranial hypertension (IIH) is a disease with an incidence rate of 0.5–2.0/100,000/year , 31. An . Deranged CSF dynamics and aquaporins could play a role in dysregulation of CSF production and in turn play a role in developing cerebral edema in IIH ;Neuro-ophthalmological findings ;Magnetic resonance imaging (MRI) findings evaluated by a neuroradiologist;Lumbar CSF pressure parameters determined with a CELDA CSF infusion apparatus.Baseline at diagnosis of IIH:Treatment types: (a) conservative , (b) surgical treatments , (c) combined;Treatment outcomes after (a) medical, (b) both medical and surgical treatments combined;Neuro-ophthalmological outcome: (a) no papilledema, (b) partial resolution, (c) no improvement;Symptomatic outcome: (a) symptomless, (b) partial recovery, i.e. ongoing symptoms such as headache, tinnitus, fatigue, dizziness, balance problems and need for continuing medical treatment, (c) no improvement.Follow-up during treatment of IIH:0, a constant in the mathematical model describing CSF dynamics  (p = 0.001). Verified IIH patients had more often severe optic nerve findings as compared with the symptomatic controls. Twenty (51.3%) IIH patients had visual field defects at diagnosis whereas no patient from the symptomatic control group had a visual field defect (p = 0.001). However, there was no difference in visual acuity at presentation (Table The mean of first opening CSF pressure was significantly higher [29.5 (SD 5.5) mmHg] for the verified IIH patients, as compared with the symptomatic controls [18.5 (SD 9.7) mmHg] (p < 0.01) except the RAP index, SLOW and RPPC. Fifty percent of the IIH cases and 25% of the controls were taking acetazolamide at the time of the pressure measurement (p = 0.068). To ensure that this did not skew the comparison between the groups, a corresponding analysis was also performed for only the cases without acetazolamide treatment. For these 33 subjects (18 with verified IIH), the identified differences between the groups were similar to the original analysis, except that the RAP index and SLOW were also significantly higher in the IIH cases (RAP index: median (IQR) 0.86(0.13) vs 0.59(0.45), p = 0.017; SLOW: median (IQR) 1.1(1.2) mmHg vs 0.7(1.0) mmHg, p = 0.025).The lumbar CSF pressure parameters from the CELDA measurements are presented in Table p = 0.033). None of the pressure parameters showed significant rank correlation to levels of CSF protein (p > 0.2). When analysing only the IIH subjects that were not taking acetazolamide at the time of measurement, the correlation between RESP and BMI was stronger , but no other trends were identified. In subjects with excess CSF around the optic nerve the RPPC  and RESP  were significantly lower than in those without, respectively. RPPC was also lower in IIH subjects with empty sella compared to those without  0.45(0.14) vs 0.63(0.28), p = 0.041). Comparisons between IIH subjects with or without psychiatric comorbidity (N = 21 vs 17) showed higher lumbar CSF pressure (median (IQR) 22.7(7.0) mmHg vs 17.6(8.3) mmHg, p = 0.038), as well as trends toward higher RESP (median (IQR) 0.9(0.6) mmHg vs 0.7(0.6) mmHg, p = 0.083), and lower RPPC  0.45(0.20) vs 0.61(0.26), p = 0.061) for subjects with comorbidity than those without .Only the power of respiratory waves showed significant positive correlation to BMI   IIH patients were prescribed acetazolamide medication during lumbar CSF pressure measurement due to debilitating symptoms and assessment for possible CSF diversion. A total of 18 46.2%) patients had no medication or it had been started within two weeks prior to CELDA investigation. The acetazolamide treatment was reported beneficial, i.e. complete resolution of all symptoms was observed in 17 (48.6%) of all patients, while partial recovery only or no improvement was noted in 18 (51.4%) patients at the end of follow-up (Table 6.2% patip = 0.018 right eye and p = 0.001 left eye). Neuro-ophthalmological outcome was assessed as a degree of resolution of papilledema, and up to 65.7% of all patients had physiological papillae at the end of follow-up, and partial resolution was noted in 28.6% and 5.7% had papilla atrophy at the end of follow-up , but none of the pressure parameters differed significantly between these groups (p > 0.2). There were no differences in lumbar CSF pressure parameters between IIH patients with complete resolution of papilledema and those with partial or no resolution . When comparing results from IIH subjects who were not taking acetazolamide at the CSF pressure measurement to those who were (N = 18 vs 17), there were trends towards higher lumbar CSF pressure , increased RAP index  and increased power of slow waves  in patients without acetazolamide treatment.For the IIH cases, values of the lumbar CSF pressure parameters grouped according to outcome (favourable versus unfavourable) are presented in Table In this study, we conducted a literature review of CSF dynamics in IIH and investigated different lumbar CSF pressure parameters in a well-described retrospective cohort. The systematic review of the literature mainly revealed consistently increased CSF pressure and increased resistance to CSF outflow. Our analysis of the retrospective cohort showed that mean lumbar CSF pressure and BMI were higher in verified IIH patients compared to symptomatic controls, and that both fast and slow dynamic changes in lumbar CSF pressure were increased. We confirmed that psychiatric comorbidity is common in IIH and found that lumbar CSF pressure was more often higher in cases with such comorbidity. We observed that the use of acetazolamide may have obscured an increase in RAP and SLOW measures in true IIH patients.ClinicalTrials.gov ID: NCT03948971). CSF pressure and SSP appears to be coupled in IIH and when CSF pressure increases during CSF infusion, it produces an increase in SSP [In our literature review of CSF dynamic findings in IIH, we found that lumbar CSF pressure or ICP was elevated in IIH patients , 48, 51,e in SSP  and its e in SSP . During e in SSP , 38. Opee in SSP . The crae in SSP , 24, 42,e in SSP . One pree in SSP , which ce in SSP . AdditioIncreased venous sinus pressure has been regarded as one of the possible causes of IIH. In addition, changes of the cerebral cortical capillaries and blood–brain barrier dysfunction are associated with evolvement of IIH , 18. ClaThe mean age and CSF protein count at diagnosis was similar in both groups. The presenting symptoms were similar and most commonly these patients presented with headache and visual disturbances. Neuroradiological findings did not differ between verified IIH patients and symptomatic controls, most commonly these patients presented with empty sella or increased CSF around the optic nerve. However, the mean measured opening pressure and BMI were significantly higher for the verified IIH patients, as compared with the symptomatic controls, as expected. In addition, the true IIH patients more often had severe optic nerve findings and visual field defects at diagnosis, but there was no difference in visual acuity at presentation.0 in IIH patients compared to symptomatic controls could support the previous finding of increased sagittal sinus pressure in IIH [0 sets the “baseline” for the lumbar CSF pressure level, i.e. a change in P0 results in an equal change in (mean) lumbar CSF pressure. In terms of the classical exponential pressure/volume curve for CSF pressure, a change in P0 corresponds to moving the entire curve along the pressure axis, rather than moving the state of the patient along the curve. Accordingly, craniospinal compliance decreases as the difference between lumbar CSF pressure and P0 increases, i.e. an increased lumbar CSF pressure would not result in reduced craniospinal compliance if P0 increased in parallel. Based on our results, the increase in P0 in IIH was less than half of the increase in lumbar CSF pressure  as well as increased RAP index likely reflect reduced craniospinal compliance in IIH, as would be expected at the increased lumbar CSF pressure level. These results are not entirely unexpected but have not been previously established in the literature. Amplitudes of CSF pressure waves, specifically pulse amplitudes, measured via the lumbar route, with the methodology used in this study, are typically somewhat smaller than if measured intracranially, though there is a very strong correlation. This ise in IIH , 29, 38,e in IIH  — and ate in IIH . AccordiThe increased value of SLOW we found in the IIH cases without acetazolamide treatment could stem from a decreased craniospinal compliance, resulting in higher amplitudes, but it could also mean that SLOW waves appear more often in IIH. The power of slow waves reflects both the degree of occurrence and the amplitude of the waves, i.e. the power can increase if waves occur during a higher proportion of time or if the amplitude of the waves increase. Since slow waves are expected to appear in response to variations in intracranial blood volume or blood pressure, this could potentially indicate an issue with cerebral autoregulation in IIH. However, without simultaneous measurement of arterial blood pressure, the origin of the waves cannot be confirmed, and thus, autoregulation was not specifically assessed. Also, with the relatively short duration of measurement of this study, i.e. < 1 h, it was not possible to reliably determine if there is more slow wave activity in IIH in general, though such results would be in line with Gjerris et al . These rIn IIH patients, there was significant correlation between power of respiratory waves and BMI, but not between any other lumbar CSF pressure parameters and BMI or between lumbar CSF pressure parameters and CSF protein levels. When analysing only the IIH subjects that were not using acetazolamide at the measurement occasion, the correlation between RESP and BMI increased further. The respiratory waves reflect variations in venous cerebral blood volume and/or CVP, so this observation could relate to increased venous pressure or increased respiratory variation in venous pressure in subjects with high BMI.Comparing lumbar CSF pressure parameters in IIH cases with and without empty sella turcica did not reveal any differences. In IIH subjects with excess CSF around the optic nerve, RPPC and power of respiratory waves were significantly lower than for subjects without this radiological finding. The implications of these results are unclear; though a reduced RPPC may be indicative of a worsened pressure-independent component of craniospinal compliance, i.e. the aspect of compliance that defines the steepness of the pressure/volume curve, which is often described by the PVI .Only about half of the IIH patients benefitted from acetazolamide treatment, and further surgical intervention was required for eleven patients. After conservative and operative treatments, the overt outcome improved slightly as 60% of patients reported themselves to be symptomless, hence having favourable outcome. The visual acuity at diagnosis was better for the IIH patients with favourable outcome as compared with IIH patients with unfavourable outcome. This finding might suggest that if the IIH is diagnosed early and the treatment has been started promptly, the IIH-related symptoms have progressed less leading to better outcome. No lumbar CSF pressure parameters differed significantly between these groups. Neuro-ophthalmological outcome was assessed as a degree of resolution of papilledema, and up to 65.7% of all patients had physiological papillae at the end of follow-up, there were no differences in lumbar CSF pressure parameters when stratified by neuro-ophthalmological outcome.When comparing results from IIH subjects who were taking acetazolamide to those who were not, there were trends toward higher lumbar CSF pressure, power of slow waves (SLOW) and RAP index in subjects without acetazolamide. While not statistically significant, the trends for RAP index and SLOW motivate the post hoc analysis comparing only verified IIH cases and symptomatic controls without acetazolamide treatment, where these two parameters showed significant differences between the groups that were not revealed in the original comparison. Our findings suggest that in some cases the lumbar CSF pressure was reduced by acetazolamide, as is expected , and thaPreviously, we have reported that psychiatric disorders are very common in IIH patients compared to general population . In thisOur retrospective analysis is limited by factors inherent to a secondary analysis of retrospectively collected data as well as the small sample size. Nevertheless, this issue is addressed in limited amount of previous data, as our systematic literature review shows. This study represents a detailed investigation, including continuous lumbar CSF pressure measurements, based on a well-defined cohort, which ensures a clinically comprehensive analysis and long follow-up times for these patients, which we regard as strengths of this study.2O. The cutoff point for lumbar CSF pressure might not unambiguously separate IIH patients from symptomatic controls, especially in patients with increased AMP and decreased craniospinal compliance.The major concern of this study is the risk for circular reasoning with the definition of symptomatic controls. Somewhat surprisingly some of these patients had also slight papilledema without visual field defects. Perhaps, this could represent early stage of IIH patients who have part of the time lumbar CSF pressure above 25 cm HHenceforth, the definition of symptomatic control is somewhat controversial. To overcome this problem, in the future, patients with suspected IIH and borderline lumbar CSF pressure, might benefit from control lumbar CSF pressure measurement.www.iih.fi). In this study, we prospectively perform continuous lumbar CSF pressure measurement, collect biological samples for further metabolomics and genetic studies to advance understanding of the complex pathophysiology of the IIH disease, in addition to gathering all clinical variables and administrating validated questionnaires to screen and follow-up on mental well-being of patients with IIH, quality of life, and impact of their possible residual symptoms.Further studies, focusing on the role of CSF dynamics in patients with IIH in larger patient cohorts are needed. In 2017, we initiated a prospective collaborative multicentre study on IIH (This study confirmed that mean lumbar CSF pressure is increased in verified IIH patients compared to symptomatic controls, and also revealed that both fast and slow dynamic changes in lumbar CSF pressure were increased in IIH cases, indicating a reduced craniospinal compliance. Furthermore, we confirmed that psychiatric comorbidity is common in IIH, and found that lumbar CSF pressure was higher in cases with comorbidity than those without. Changes in some dynamic lumbar CSF pressure parameters in true IIH, such as RAP and SLOW, may be obscured by prescribed acetazolamide, which may advocate CELDA-measures before medication in feasible cases.


Agitation is common in subarachnoid hemorrhage (SAH), and sedation with midazolam, propofol and dexmedetomidine is essential in agitation management. Previous research shows the tendency of dexmedetomidine and propofol in improving long-term outcome of SAH patients, whereas midazolam might be detrimental. Brain metabolism derangement after SAH might be interfered by sedatives. However, how sedatives work and whether the drugs interfere with patient outcome by altering cerebral metabolism is unclear, and the comprehensive view of how sedatives regulate brain metabolism remains to be elucidated.For cerebrospinal fluid (CSF) and extracellular space of the brain exchange instantly, we performed a cohort study, applying CSF of SAH patients utilizing different sedatives or no sedation to metabolomics. Baseline CSF metabolome was corrected by selecting patients of the same SAH and agitation severity. CSF components were analyzed to identify the most affected metabolic pathways and sensitive biomarkers of each sedative. Markers might represent the outcome of the patients were also investigated.p = 0.0000107, impact = 0.35348) and propofol  groups. On the contrary, dexmedetomidine decreased levels of sedoheptulose 7-phosphate (p = 0.002) and NADP (p = 0.024), and NADP is the key metabolite and regulator in pentose phosphate pathway. Midazolam additionally augmented purine synthesis  and propofol enhanced pyrimidine synthesis , whereas dexmedetomidine weakened pyrimidine synthesis . Reduced guanosine diphosphate  was the significant CSF biomarker for midazolam, and uridine diphosphate glucose  for propofol, and succinyl-CoA  plus adenosine triphosphate  for dexmedetomidine. Down-regulated CSF succinyl-CoA was also associated with favorable outcome .Pentose phosphate pathway was the most significantly interfered (upregulated) pathway in midazolam (Pentose phosphate pathway was a crucial target for sedatives which alter brain metabolism. Midazolam and propofol enhanced the pentose phosphate pathway and nucleotide synthesis in poor-grade SAH patients, as presented in the CSF. The situation of dexmedetomidine was the opposite. The divergent modulation of cerebral metabolism might further explain sedative pharmacology and how sedatives affect the outcome of SAH patients. Subarachnoid Hemorrhage (SAH) is a neurological emergency, and the mortality of poor-grade SAH patients is relatively high . AgitatiSedation is the essential and major treatment of agitation in SAH , 7 and iIt is still unclear whether and how agitation management affect the neurological outcome of the SAH patients . NotablyPrevious studies have proved that these three sedatives present metabolic suppression in generally anesthetized patients by decreasing cerebral blood flow and regulating cerebral metabolic rate –26. MeanMeanwhile, the three drugs display significant disparity in cerebral metabolic regulation, and even controversy exists. Clinical study has revealed that propofol and other sedatives differ in the alterations of plasma and cerebral metabolomic signature , 30. In 1HMR) spectroscopic approaches demonstrate how brain metabolism is changed in situ, they still have drawbacks. The identified compounds are limited, which is inadequate to provide sufficient metabolic information [In clinical settings, although non-invasive PET and proton magnetic resonance  as the partial representative of cerebral metabolism, based on its exchange with extracellular space of the brain (ECSB), which is the interstitial fluid of the brain . CSF couIn SAH patients, it would be difficult to distinguish where the CSF alterations come from— the sedatives or the disease itself— for SAH brings blood to subarachnoid space and involves CSF composition alterations. To solve this problem, we have demonstrated in our previous research that CSF composition patterns could be divided by clinical SAH severity , and is not associated with hematoma volume . Namely,For these reasons, we hypothesized that the sedatives differentially modulate brain metabolism in the acute phase of SAH, and therefore influence their long-term outcome. We therefore undertake this observational study to portray the CSF metabolic profile of SAH patients treated with different sedatives , to compare their metabolome, and to identify possible biomarkers of satisfying sedation as well as outcome of the patients.As an observational study, we collected CSF samples from SAH patients admitted to the Neurosurgical Critical Care Unit in Nanjing Drum Tower Hospital Department of Neurosurgery between September 2017 to December 2018. The CSF was sampled within 7 days after SAH onset. The study adhered to ethical guidelines, and was approved by the Ethical Committee of Nanjing Drum Tower Hospital. Written informed consent was obtained from all individual participants or their legal guardians included in the study. Hunt-Hess scale , Fisher The CSF samples were centrifuged at 3000 rpm and the supernatants were transferred to fresh conical tubes and stored at − 80 °C before use. The samples were applied to high performance liquid chromatography coupled with mass spectrometer as previously described . Compounp value less than 0.05 was considered significant.Statistical analyses were performed using MetaboAnalyst   and SPSSFrom September 2017 to December 2018, we included 42 SAH patients with Hunt-Hess Scale above III . Those patients utilized single or none sedative agent. Among the patients, 11 were sedated merely with midazolam, 13 with propofol and 13 with dexmedetomidine, while 5 underwent no sedation. The patient characteristics were shown in Table To acquire a global view of if sedatives change CSF metabolome, we compared the metabolite data of the four groups. In Fig. p = 0.0000107, impact = 0.35348, Fig. p = 0.00000787).To clarify if sedatives alter brain metabolism and CSF composition, we firstly compared CSF metabolome between SAH patients treated with midazolam or no sedation. As shown in Fig. To further ensure whether midazolam inhibited or upregulated pentose phosphate pathway, we compared the peak intensity of metabolites identified by sparse PLS-DA and one-way ANOVA. As listed in Fig. p = 0.00175, Fig. Meanwhile, pathway analysis showed that purine metabolism was also influenced by midazolam treatment, with the impact at 0.13481 (p = 0.00506). Multivariate biomarker analysis did not show significant results.Univariate biomarker analysis demonstrated that reduced guanosine diphosphate (GDP) was the significant CSF indicators of midazolam treatment ose/xylulose-5-phosphate, D-glyceraldehyde 3-phosphate, D-erythrose 4-phosphate, fructose 6-phosphate levels significantly increased. .We also applied the metabolic data to biomarker analysis to search for appropriate indicators of propofol sedation. Multivariate analysis did not reach a significant outcome. Univariate analysis showed that decreased uridine diphosphate glucose (UDP-Glc) level represented satisfying propofol sedation , and the impact was as low as 0.02108, as shown in Fig. p = 0.000112, Fig. In term of pentose phosphate pathway, pathway analysis reveals that pentose phosphate pathway metabolites in CSF were affected by dexmedetomidine treatment, however, it did not meet the statistical significance (p = 0.000000000594), with the highest impact at 0.24922 in pathway analysis  was also significantly interfered  and ATP  concentrations were the significant CSF indicators of dexmedetomidine treatment. Multivariate biomarker analysis did not show significant results.Moreover, as shown in Fig. Therefore, dexmedetomidine affected CSF metabolome in SAH patients by inhibiting pyrimidine metabolism, and meanwhile pentose phosphate pathway was also slightly down-regulated. The suppressed energy supply, as represented by diminished succinyl-CoA and adenosine triphosphate concentrations, was the biomarker of dexmedetomidine sedation. The modulation of pentose phosphate pathway was divergent in CSF of patients treated with midazolam, propofol and dexmedetomidine.p = 0.029333, sensitivity 0.7, specificity 0.7) in poor-grade SAH patients. Interestingly, down-regulated CSF succinyl-CoA level was also one of the biomarkers of dexmedetomidine sedation. Dexmedetomidine, although failed to bring a better prognosis to patients in the present study  was weakened, and the production of ribose-5-phosphate through PPP, which supports nucleotide biosynthesis, was thereby insufficient. Importantly, nucleotide synthesis is essential in maintaining normal brain function .The CSF metabolome, in dexmedetomidine-treated group, was diverged from that of the control group, and the diminished pyrimidine synthesis was the most influenced metabolic pathway. Given the pentose phosphate pathway was mildly inhibited, its assistance of pyrimidine synthesis was thereby weakened. Thus, the decreased concentration of the CSF metabolites in pyrimidine metabolism could at least partly explained. In pyrimidine metabolism, ATP and PRPP activate carbamoyl phosphate synthase (CPS) II and UTP inhibits the enzyme, and CTP inhibit CTP synthase . For ATPIn addition, decreased succinyl-CoA and ATP levels are sensitive and specific biomarkers of dexmedetomidine sedation. Meanwhile, alteration in CSF succinyl-CoA concentration was also associated with patient outcome, which might be the key compound linking dexmedetomidine pharmacology and attenuation of neurological deficit in poor-grade SAH patients. Moreover, as crucial indicators of energy supply, the two reduced biomarkers suggested a relatively low activity of citrate cycle. However, the alteration of this mitochondrial process, which could not be completely presented in the ECSB/CSF, should be carefully studied in the future.There were limitations of this study. The mechanisms underlying the observations in the present study require more research, focusing on how sedatives alter metabolic pattern of the brain on molecular level and whether drug action on neuron and glia differs. Secondly, the metabolomic approach has its own defect that it is not able to tell regional or local data . For insBy analyzing CSF metabolome of poor-grade SAH patients sedated with midazolam, propofol and dexmedetomidine, we discovered that these drugs changed CSF metabolic profiles in divergent ways. The influence of CSF composition by SAH severity was excluded by baseline calibration. Midazolam and propofol up-regulated pentose phosphate pathway metabolites and enhanced purine and pyrimidine synthesis respectively. On the contrary, dexmedetomidine down-regulated pentose phosphate pathway metabolites and attenuated pyrimidine synthesis. We also linked satisfying sedation with their CSF metabolites as biomarkers. Down-regulated GDP and UDP-Glc levels were biological indicators of desirable midazolam and propofol sedation, whereas diminished succinyl-CoA and ATP concentrations represented that of dexmedetomidine treatment. Succinyl-CoA level down-regulation was also associated with better clinical outcome of these poor-grade sedated SAH patients. How sedatives alter brain metabolism and how these metabolic profile alterations associate with the prognosis of the SAH patients remained to be further elucidated.


To understand inter-individual variability of fecal microbe transplantation (FMT) to enhance anti-PD-1 immunotherapy (IT) for melanoma, we analyzed the data sets from two recent publications with a microbial strain-tracking tool to determine if donor strains were dominant in the recipient feces following FMT.Analysis of the Baruch et al. data set found that the presence of commensal donor microbes in recipient feces post-FMT did not correlate with the patient response to IT. From the Davar et al., data set, we found 4 patients that responded to IT had donor’s related strain post-FMT, while 2 patients that did not respond to the IT also had donor’s strain post-FMT. Importantly, we identified no donor microbes in the feces in one recipient post-FMT that responded to IT. Furthermore, in depth analysis from two patients who responded to IT revealed both donor and recipient strains at different times post-FMT. Colonization of the gastrointestinal tract niches is important for the interaction with the host immune system. Using a separate data set, we show that mucosa from the cecum, transverse colon, and sigmoid colon share strains, providing a large reservoir of niches containing recipient microbes.We demonstrated using strain-tracking analysis individual variation with the respect to the presence of fecal dominant donor microbes in the recipient following FMT that did not correlate with the response to anti-PD-1 immunotherapy. The inter-individual differences of FMT to enhance IT might be explained by the variability of the donor microbes to occupy and outcompete recipient microbes for the gastrointestinal niches. The result from our study supports the use of new approaches to clear the niches in the gastrointestinal tract to promote donor colonization to reduce inter-individual variability of IT for melanoma and potentially other cancers.The online version contains supplementary material available at 10.1186/s12866-021-02312-0. The composition of the gut microbial community has been shown to be a regulator of the response to anti-PD-1 immunotherapy (IT) for a variety of cancers . RecentlClostridium difficile [C. difficile results in the presence of donor microbes in the feces of recipients for up to two years with no evidence of residual recipient fecal stains [Most of the clinical success for FMT to improve or alleviate symptoms has been found when given to relieve patient’s chronic recurrent ifficile –10. Subsifficile . To furtifficile –13. In aifficile . Using pifficile , 14, 15.l stains .In the current study, we have used the WSS strain-tracking method to analyze the FMT from two studies that have used FMT to enhance IT , 6. BothAlistipes putredinis, Bacteroides vulgatus, Bifidobacterium adolescentis, Collinsella aerofaciens, Coprococcus eutactus, Eubacterium eligens, Eubacterium rectale, Faecalibacterium prausnitzii A2, Faecalibacterium prausnitzii L2, Prevotella copri, and Roseburia intestinalis was related to the recipients’ post-FMT strain at certain time points  were showing that 36 cases had donor’s related strain in recipient’s post-FMT regardless of whether there was a clinical response or no response to IT  used his/her fecal sample for the FMT on a single recipient while the second group of donors (n = 4) used his/her fecal sample in multiple recipients  shahii, (B) uniformis, B. sp. 1-1-6, B. sp. 2-1-16, and B. cellulosilyticus were found in the recipient (19 − 0013) even though this recipient did not respond to the IT  vs. 19–0026 (recipient), and 18 − 0014 (donor) vs. 19 − 0007 (recipient)) that did not respond to IT had donor’s related strain in (A) putredinis, (B) uniformis, and B. vulgatus (B. vulgatus is specific for 18 − 0014 vs. 19 − 0007 pair)  shahii, (B) vulgatus or B. ovatus in the recipients at times up to 187 days post-FMT  in recipients post-FMT. For the first donor (18 − 0014), we analyzed 4 different recipients: 19–0024 and 18–0032 that responded to IT and 19–0026 and 19 − 0007 that did not respond to IT  shahii, and P. merdae were dominant in the recipient at all times up to 535 days post-transplant. However, in the case of (B) ovatus, we found that the donor strain was present in only the sample collected at 90 days. Interestingly, we found that there were pre-FMT-related strains present, including B. uniformis, B. vulgatus, and B. stercoris for the 18 − 0002 vs. 18–0018 pair  in which IT was enhanced after FMT. To do this, we compared each recipient’s pre-FMT sample with available longitudinal post-FMT samples. Here we found that for the 18 − 0002 transplant into 18–0018 that donor strains of air Fig. A. In theair Fig. B. The reair Fig.  or recipair Fig. . Finallyair Fig. .Fig. 4LBacteroides spp. (no unrelated strains) and Parabacteroides spp. (one unrelated strain)  Fig. . From ouIn this study, we have utilized strain-tracking analysis to investigate the inter-individual variability seen for FMT to respond to IT. Strain-tracking analysis using two recent data sets demonstrates the presence of donor microbes in the recipient does not correlate with the response to IT. Further support that the link between donor microbes in the feces of recipients and response to IT is incongruent comes from the analysis of longitudinal fecal samples from recipient’s post-FMT that revealed an ongoing competition over time between the donor and recipient microbial strains for fecal dominance. Using a separate data set, we also determined that the microbial strains in the intestinal mucosa of the cecum, transverse, and sigmoid colon are shared, indicating an extensive reservoir for the recipient microbial strains to interact with the immune system. The results of our strain-tracking analysis provide evidence that the conditions used for transplant of donor microbes into existing microbial communities of the recipient do not ensure comprehensive replacement of the recipient microbes with donor and new insights to explain the inter-individual variability of FMT to enhance IT.8 CFU per gram [12 CFU per gram [The number of microbes in the cecum is estimated to be approximately 10per gram . Both Baper gram , 6. Althper gram . Thus, tper gram . Collectper gram .One of the striking results of our strain-tracking analysis was that we identified one patient from Davar et al. following FMT 19 − 0010) that responded to IT without having donor’s (18 − 0008) strain in the feces in the recipient post-FMT . This re − 0010 tC. difficile resulted in the donor microbe colonization that remained stable for up to two years, while in another study Smillie et al. found donor microbes in the recipients post-FMT for months after FMT [We acknowledge a limitation with our analysis that we did not directly analyze the patient’s mucosal tissue in our study for microbial colonization of the intestinal mucus following FMT. Given the invasiveness of the procedure to obtain intestinal mucus from patients at varying times post- FMT, it would be unrealistic to accomplish these studies in patients undergoing cancer therapy. An alternative might be to enhance the possibility that donor microbes from an FMT access the intestinal mucosa might be to modify the FMT procedure. In our previous study, we found FMT in patients with recurrent fter FMT . In bothfter FMT , 27, 28.fter FMT . FollowiIn this study, we demonstrated using strain-tracking analysis individual variation with the respect to the presence of fecal dominant donor microbes in the recipient following FMT that did not correlate with the response to anti-PD-1 immunotherapy. To help to explain this result, we show conserved microbial strains throughout the colon that could limit the opportunities for colonization by the donor microbes following FMT. Our study then supports that to reduce the inter-individual variation in the colonization of recipient’s post-FMT it might be necessary to first clear the recipient microbe niches to provide opportunities for interactions with the host immune system. Finally, additional studies will be needed to delineate the immune system function variation between individuals to fully understand the differences in the effectiveness of IT for melanoma and potentially other cancers.+IgA+ or IgG−IgA+, sequenced, and then deposited by James et al. Further detailed sample information has been reported by James et al. [We used publicly available data sets, James et al. , Davar es et al. . For Davs et al. . We seleA total of 3,350,868,513 metagenomic sequencing reads were downloaded from all data sets; 357,236,853 from James et al., 2,256,240,779 from Davar et al., and 737,390,881 from Baruch et al.  cecum vs. transverse colon, 2) cecum vs. sigmoid colon and 3) transverse colon vs. sigmoid colon), a WSS score for each species was compared against each species’ cut-off value . For Davar et al., each donor sample was compared with the paired recipient’s pre- and post-FMT samples to determine donor’s related strain in the recipient’s samples. The paired relationships between donors and recipients are included in Table SAdditional file 1: Figure S1. Other species’ summarized WSS scores from Baruch et al. WSS analysis of the sample pairs used for this figure is provided in Fig. Additional file 2: Figure S2. Other species’ summarized WSS scores from donors with having a single recipient. WSS analysis of the sample pairs used for this figure is provided in Fig. Additional file 3: Figure S3. Other species’ summarized WSS scores from donors with having multiple recipients. WSS analysis of the sample pairs used for this figure is provided in Fig. Additional file 4: Figure S4. Other species’ summarized WSS scores from the longitudinal analysis. WSS analysis of the sample pairs used for this figure is provided in Fig. dditional file 5: Figure S5. WSS scores between colon segments from IgG data set. Samples that were able to provide WSS scores for each species were selected to compare the WSS scores between (A) cecum vs. transverse colon; (B) cecum vs. sigmoid colon; and (C) transverse colon vs. sigmoid colon from IgG data sets. The summarized WSS scores were grouped into different color boxes (see the figure key). Each column in the table matches the label shown in Table SAAdditional file 6: Table S1. Sample and sequence read information. The original sequence files were sequenced and deposited by (A) James et al., (B) Davar et al., and (C) Baruch et al. The table represents the sample information along with sequence read information.Additional file 7: Table S2. WSS scores for Baruch et al. All pairwise comparisons were conducted between a donor-recipient paired sample from Baruch et al. The resultant scores (%) are shown as a numerical value. *CO indicates the cut-off value for each speciesAdditional file 8: Table S3. WSS scores for Davar et al. All pairwise comparisons were conducted between donor-recipient paired samples. The resultant scores (%) are shown as a numerical value. *CO indicates the cut-off value for each species.Additional file 9: Table S4. WSS scores for James et al. All pairwise comparisons were conducted among colon segment samples. The resultant WSS scores (%) are shown as a numerical value under the ‘WSS’ column.Additional file 10: Table S5. Boundary cut-off values. A list of WSS cut-off values is provided for each species. These values were previously established based on the Human Microbiome Project (HMP) using our classifier and can be used to assess the relatedness of the strains in two separate samples in the same individual .