Lung microbiome dysbiosis affects the immune system balance and promotes lung inflammation. We aimed to characterize and compare the lung bacteriome composition and the cytokine profile in women with normal lung function exposed to risk factors for chronic lung diseases (tobacco smoking and biomass‐burning smoke exposure).n = 11) and current smokers women . The bacteriome composition was performed in induced sputum, sequencing the 16 rRNA gene. Cytokine levels were measured using enzyme‐linked immunosorbent assay multiplex assay in the supernatant of induced sputum. For quantitative variables, we used medians and minimum and maxim values. For the amplicon sequence variants (ASV) differential abundance testing between groups.We included women with biomass‐burning smoke exposure ; however, after the false discovery rate adjustment, this difference was not retained (p = .288). We found a higher concentration of IL‐1β in the TS group than in the BE group . Women with high biomass‐burning smoke exposure in an hour per day had a positive correlation with the abundance of Bacteroidota  and Fusobacteriota . FEV1/FVC had a positive correlation with an abundance of Bacteroidota, Proteobacteria, and Fusobacteria . In tobacco smoking, women had a positive correlation  between cigarettes per day and Firmicutes' abundance.At the taxa level, the phylum Proteobacteria was found in a higher proportion in the TS group concerning BE (Compared to biomass‐burning smoke‐exposed women, current smokers have poor lung function and high levels of IL‐1β in sputum. Women with biomass‐burning smoke exposure present an increased abundance of Bacteroidota and Fusobacteriota. This is the first study that evaluates the bacterial composition in exposed women to known risk factors for lung diseases with normal lung function. The pro‐inflammatory mediators play an essential role in the initiation and maintenance of inflammation. The compounds and particles in these noxious gases cause changes in pH, oxygen tension, oxidative stress, pro‐inflammatory conditions, and even dysbiosis of the microbiome in the pulmonary microenvironment.Veillonella and Megasphaera) and the genus Prevotella. At the same time, this variable is inversely associated with Proteobacteria (genera Eikenella and Haemophilus).Haemophilus and elevated levels of interleukin (IL)‐1β and tumor necrosis factor (TNF) in the sputum.Cigarette smoking influences the composition of the lung microbiome,3). The same behavior is observed in 24 h CO concentrations (13.4 vs. 2.0 ppm).Reports regarding the involvement of the lung microbiome due to biomass‐burning exposure are very scarce; this smoke contains thousands of harmful substances, including particulate matter at most 10 (PM10) or 2.5 (PM2.5) microns in diameter.Streptococcus and Neisseria).In the COPD Risk Cohort, the authors evaluated the influence of the environmental PM2.5 concentration on the lung microbiome for 14 days. The microbiome showed significant changes from the sixth day of exposure, especially in some Operational Taxonomic Units (OTUs). This change was maintained until the last monitored day.22.1n = 11), recruited in rural and suburban populations by the Department of Research in Smoking and COPD (DITABE) of the Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas (INER) in Mexico City and current smoking women . The general inclusion criteria were women, age ≥40 years, with lung function test  values of ≥70%. The TS group had a history of ≥3 packs per year, and the BE group had a biomass smoke exposure index (BSEI) of ≥100 h per year. Both groups had exposure to corresponding environmental risk factors for ≥10 years. In addition, all participants had a physical examination by pulmonology specialists. The exclusion criteria were a record of respiratory diseases , lung transplant, cerebrovascular disease, psychiatric disorders, infectious disease, antibiotics, or immunosuppressive treatments in the previous 3 months, or evidence of acute upper respiratory symptoms for the preceding 4 weeks.We designed an observational, descriptive, and retrospective study, including women with biomass‐burning smoke exposure , or BSEI.The lung function tests were performed by spirometry technicians considering the criteria established by the American Thoracic Society and the values calculated in the Mexican population.2.2The women who agreed to participate voluntarily and signed an informed consent document were provided with a personal data protection certificate. The INER committee reviewed and approved the study on bioethics and research (protocol number B01‐17).The women were required to stop smoking or leave biomass‐smoke exposure for at least 12 h before sample collection. Then, for sputum induction, the participant rinsed their mouth with water. Next, we administered sterile saline solution (7%) by nebulization for 5 min, followed by a 5‐min break. Participants performed this technique a maximum of three times.In a biosafety cabinet, using a transfer pipette, saliva was eliminated. In a 1:1 relation, sterile saline solution (0.9%) was added to sputum using a sterile syringe and 20‐gauge needle to disaggregate sputum (up and down a minimum of 10 times); subsequently, we used a 22‐gauge needle until complete disintegration. The resulting sample was filtered  and centrifuged at 3000 rpm at 20°C for 10 min. The supernatant was stored at −80°C. The cellular pellet was employed for bacterial DNA extraction. Bacterial DNA was purified using the ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research Corp.), following the instructions for liquid samples (200 µL) as the supplier recommends. The DNA was quantitated utilizing the Qubit dsDNA High Sensitivity (HS) Assay Kit (Invitrogen) and assessed for integrity by electrophoresis on agarose gel. We included two saline samples to identify bacteria procedures of sources of contamination; these controls were treated in the same conditions that the sputum samples.2.3The libraries were prepared for targeted amplicon sequencing following the “16 Sr DNA Metagenomic Sequencing Library Preparation” guide . We used the primer pair sequence for the V3 and V4 regions of the 16 S ribosomal RNA gene (16 Sr DNA), Nextera XT indices, and PhiX control. The sequencing was performed on the MiSeq platform using paired 300 bp reads and MiSeq v3 reagents. We employed MiSeq Control Software v 3.1.1.13 (Illumina) to generate the FASTQ files.2.4We analyzed FASTQ files using RStudio,2.5According to the manufacturer's instructions, sputum supernatant cytokine levels were determined using a commercially available multiplex Human Cytokine Magnetic 10‐Plex Panel (Thermo Fisher Scientific). Proteins evaluated include IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, tumor necrosis factor (TNF)‐α, Interferon (IFN)‐γ, and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF).The multiplex assay was performed according to the manufacturer's instructions. Samples were homogenized and adjusted for further quantification in the Luminex LAB‐Scan 100 (Luminex Corp.) system. The cytokine concentrations were calculated using the standard curve. The xPONENT 3.1 software (Luminex Corp.) was used for data acquisition.2.6U test. For the ASV differential abundance testing between groups, we employed the negative binomial generalized linear model (GLM) and obtained maximum likelihood estimates for an ASV log‐fold change between two study groups; the p‐value was adjusted by the false discovery rate (FDR) method.U test with a Bonferroni posthoc correction. A value of p < .05 was considered statistically significant. For the quantitative variables and abundance of bacteria, Spearman's correlation was made. The description of the results was carried out using the ggplot2The analysis for anthropometric characteristics, TI or BSEI, spirometry data, and cytokine levels was performed using SPSS version 15 . For quantitative variables, we used a mean and standard deviation or medians and minimum and maxim values according to the distribution of the variables and used parametric or non‐parametric tests. Alpha diversity was also assessed using the Mann–Whitney 33.1The groups included in the study did not show statistically significant differences in age, body mass index, forced vital capacity (FVC), or expiratory volume in the first second FEV1), as shown in Table , as showp = .001).Regarding the BE group, 45.5% of the women were born in Oaxaca state, the rest in the State of Mexico's rural and suburban areas, and reported having >210 h per year of exposure to smoke from biomass burning. The participants did not present any lung affection. The post‐bronchodilator FEV1/FVC was >70% in the study groups; however, the TS group had a lower FEV1/FVC ratio than the BE group . The most abundant phyla were Actinobacteriota, Firmicutes, Bacteroidota, Proteobacteria, and Fusobacteriota. Proteobacteria was found in a higher proportion in the TS group concerning BE (p = .045); however, after the adjustment by FDR, this difference was not maintained (p = .288)  Figure . The topa Figure . Howeverρ = 0.71, p = .014) and Fusobacteriota . FEV1/FVC had a positive correlation with an abundance of Bacteroidota, Proteobacteria, and Fusobacteria . In the bacterial abundance analysis, we found that Firmicutes and Actinobacteria had a positive correlation . Bacteroidota had a positive correlation with Proteobacteria and Fusobacteriota  and Proteobacteria had a positive correlation with Fusobacteriota   and abundance of Bacteroidota  Figure .p = .009, ρ = 0.77). Firmicutes versus Proteobacteria and Actinobacteriota showed a positive correlation , and Proteobacteria and Actinobacteriota    as well as GM‐CSF  and IL‐2 ; however, with a Bonferroni posthoc correction, only IL‐1β maintained a significant association (p = .010).Regarding evaluated cytokines Table , we foun4The current study was carried out in women with exposure to known risk factors for the development of chronic lung diseases and without the presence of airflow limitation (FEV1/FVC ≥ 70).Pneumocystis pneumonia.We used sputum specimens to evaluate the microbiome in the women in the present study. The presence of oropharyngeal microbiota does not confuse the meaningful microbial signal in sputum, which is correlated with established indices of lung health.p = .288). The genera most abundant were Streptococcus, Veillonella, Haemophilus, Rothia, and Prevotella. We found no statistically significant difference between the BE and TS groups at the genus level.The pulmonary bacterioma showed that the phylum Proteobacteria was statistically higher in the TS group concerning the BE group; however, this difference was not maintained after the Bonferroni correction  in both groups compared to the lung microbiome of healthy subjects (27.8%–46.0%).Streptococcus and Neisseria are pathogenic bacteria.Exposure to smoke from biomass burning has been less explored; however, a biomass study on healthy people showed a higher abundance of In this study, in women with biomass‐burning smoke exposure, no significant correlation between IL‐1β levels and the abundance of OTUs; however, high levels of BiEx increased the abundance of Bacteroidota and Fusobacteriota; Bacteroidota in the gut stimulate TLR2 but not TLR4; in the lung, this mechanism is unknownIn this study, we reported that the abundance of Proteobacteria has been correlated with FEV1/FVC in women exposed to biomass burning; the same observation exists in the abundance of Bacteroidota and Fusobacteriota. The phylum Proteobacteria has been associated with asthmaIn smoking women, the cpd was positively correlated whit Firmicutes abundance; previous studies showed that current smoking was strongly associated with the rate of FEV1 decline, and the most abundant phyla was Firmicutes.Alterations in the microbiota composition of the respiratory tract have been reported in the development of many chronic lung diseases  and are associated with more advanced conditions. Our pilot study is not exempt from limitations, such as the small sample size or the lack of a group of women without exposure to risk factors . However, we included other reports in the literature to compare our results.This is the first study that evaluates the bacterial composition in exposed women to known risk factors for lung diseases with normal lung function. We identified high levels of IL‐1β in the sputum of tobacco‐smoking women. In women with biomass‐burning smoke exposure exist a positive correlation between BiEx and abundance of Bacteroidota and Fusobacteriota. These changes in women exposed to environmental risk factors, despite not having lung function impairment, alter bacterial composition that contributes to inflammatory diseases such as COPD or asthma.5Compared to biomass‐burning smoke‐exposed women, current smokers have poor lung function and high levels of IL‐1β in sputum. Women with biomass‐burning smoke exposure present an increased abundance of Bacteroidota and Fusobacteriota.Fernando Morales‐González: Data curation; formal analysis; methodology; software. Juan Alberto Lira‐Lucio: Data curation; formal analysis; methodology; software. Ramcés Falfán‐Valencia: Conceptualization; funding acquisition; investigation; writing—original draft. José Eduardo Márquez‐García: Resources; validation. Edgar Abarca‐Rojano: Resources; validation; writing—review & editing. Alejandra Ramírez‐Venegas: Conceptualization; investigation; writing—original draft. Raúl H Sansores: Methodology; visualization. Leonor García‐Gómez: Resources; validation. Andrea Hernández‐Pérez: Resources; validation. Gloria Pérez‐Rubio: Conceptualization; formal analysis; project administration; resources; software; writing—original draft.The authors declare no conflict of interest.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the INER committee on bioethics and research (protocol number B01‐17).Informed consent was obtained from all subjects involved in the study.Supporting information.Click here for additional data file.


Frailty develops as a result of age-related decline in many physiological systems and is associated with increased vulnerability to adverse outcomes following thoracic surgery. We prospectively tested our hypothesis that pre-operative pulmonary rehabilitation (Prehab) improves frailty, as suggested by a frailty index > 3 (FI > 3) and fitness, and thereby reduces the risk of post-surgical complications and death in vulnerable elderly lung cancer patients.221 surgical patients, 80 with FI > 3 vs. 141 patients with FI < 3, following Prehab proceeded to surgery. Their Frailty index (FI), dyspnoea scores, performance status (PS), level of activity (LOA) and six-minute walk test (6MWT) prior to and following Prehab were determined. The post-operative length of hospital stay (LOHS), complications, mortality and mid-term survival at 1100 days were compared. Similarly, outcomes for elderly patient ≥ 70 years with FI > 3  were compared with younger patients < 70 years with FI ≤ 3 .Patients with FI > 3 were significantly older, had lower 6MWT and higher thoracoscores hence, 82.5% of patients with FI > 3 vs. 33.3% (p = 0.02) with FI ≤ 3 were considered high risk for surgery and postoperative adverse events. With Prehab there was significant improvement in the FI, dyspnoea scores, PS, LOA and 6MWT. Following surgery, there were no differences in major complication rates (8.8% vs. 9.2% p = ns); LOHS median (IQR) [7 (6.8) vs. 8 (5.5) days]; mortality at 30-days ; 90-days  and 1-year survival (81.1% vs. 83.7% p = ns). Survival at 1100 days was . Likewise, 87.7% elderly ≥ 70,FI > 3 patients were considered high-risk for surgery and postoperative adverse events vs. 35.1% younger patients < 70,FI ≤ 3 (p = 0.0001). Following Prehab and surgery, there were no significant differences in complications, LOHS, mortality at 365 days between the two groups. Survival at 1100 days for ≥ 70,FI > 3 was 55.2% vs. 79.96% for < 70,FI ≤ 3; .Our study suggests that Prehab optimises vulnerable high-risk elderly lung cancer patients with frailty allowing them to undergo surgery with outcomes of post-surgical complications, LOHS and mortality at 365 days no different to patients with no frailty. However, mid-term survival was lower for elderly patients with frailty.The online version contains supplementary material available at 10.1186/s13019-023-02433-9. Over the past two decades, the number of elderly patients undergoing major surgical procedures have increased substantially. Adverse postoperative outcomes, particularly medical complications, are more frequent in this elderly group of patients when compared with their younger counterparts , 2. WhilFrailty develops with aging as a result of age-related decline in many physiological systems . CollectThe benefits of Prehab for patients undergoing lung resection for NSCLC is being increasingly recognized –18. At oThe aim of the study was to review our prospectively collected data and determine whether Prehab, by improving frailty along with the subjective fitness criteria, makes elderly surgical patients with frailty, as suggested by a frailty index > 3, suitable for surgery and thereby allows them to safely undergo curative lung resection with outcomes, namely post-operative complications, LOHS, mortality and survival following surgery no different to patients with no frailty, as suggested by a frailty index of ≤ 3.Our pragmatic study was carried out between January 2017 and December 2019 at Morriston Hospital, Swansea, United Kingdom . The ser10.4% (n = 45) of patients diagnosed with other cancers or benign diseases were excluded from the study as were 17.2% (n = 75) of patients who either declined, did not attend, had high cardiovascular risk for Prehab, died prior to Prehab or proceeded straight to surgery.The diagnosis and stage of lung cancer of the remaining 314 patients were validated. For clinical lung cancer staging the 8th edition of the International Association for the Study of Lung Cancer staging system was used . AssessmFollowing surgery, outcomes measured were major complications, LOHS, mortality and survival at 365 and 1100 days. The extended Clavien-Dindo classification for grading complications was used and grades 3a to 5 considered major complications . AdverseTo test our hypothesis and determine the impact of Prehab on patients with frailty we initially determined the impact of Prehab in the total cohort. Patients with FI > 3 were first compared with patients with FI ≤ 3. Next, to test the hypothesis and determine the impact of Prehab specifically on elderly patients with frailty for surgery, patients with stage III and IV disease requiring radiotherapy, chemotherapy and palliative care were excluded from the analysis. Elderly patients ≥ 70 years with FI > 3  being considered for surgery, were compared with younger patients < 70 years with FI ≤ 3  for surgery. The percentage of patients unfit to proceed with surgery at first assessment and the percentage of patients suitable to proceed with surgery following Prehab were determined.Our Prehab program, delivered by trained cardiothoracic physiotherapists, are describe in supplementary table and previously, based on standard guidelines , 24, 25.Descriptive statistics are expressed as percentage (numbers), and means and 95% confidence interval (± 95% CI) or median and interquartile range (IQR). Intergroup differences were analysed by 2-sided unpaired t-test, Mann–Whitney test, chi-square test or the Fisher’s exact test as appropriate. Due to missing data, as some patients had to proceed to surgery quickly or patients exercising at home who could not travel back for reassessment, we were unable to use the paired samples t-test or ANOVA. The Kaplan Meier log rank test was used to estimate survival at 365 days and 1100 days following surgery. Statistical analysis of the data was performed using IBM SPSS Statistics, 2018 software. We used the Strengthening the Reporting of Observational Studies in Epidemiology guidelines for reporting our pragmatic study.The baseline characteristics of the total cohort of 314 patients are described in Table Impact of Prehab on the total patient cohort: Over an average of 44.6 ± 6.9 days there was a significant improvement in the overall fitness of patients with Prehab as demonstrated graphically in Fig. At the time of assessment, 58.9% of patients were considered high-risk for any radical treatment (surgery or radiotherapy) Table . FollowiPost-surgical outcomes are described in Table (i) Entire surgical cohort of 221 patients: The mean age of patients with FI > 3 was significantly higher at 72.9 ± 1.6 years vs. 70.9 ± 1.3 years (p < 0.034) for patients with FI ≤ 3  Elderly patients ≥ 70 years with frailty vs. younger patients < 70 years with no frailty: The mean age of patients ≥ 70,FI > 3 was significantly higher than patients < 70,FI ≤ 3 (Table 1 and DLCO, 6-MWT distance and high thoracoscores compared with younger patients < 70,FI ≤ 3, and required a longer period of optimisation with Prehab (Table  3 Table . However 3 Table . A signiab Table .With Prehab there was clinical and statistically significant improvement in PS, dyspnoea, LOA, and FI hence, fitness of patients ≥ 70,FI > 3 Table . Hence, Our study is unique in that we have used the 9-point Clinical Frailty Scale as an index to assess patients for levels of frailty , 26. TheOur study takes into account that 90% of patients with FI > 3 were ever-smokers hence, likely to have underlying smoking related cardiopulmonary disease. Loss of lung tissue with surgical resection in such patients may grossly impair their post-operative ventilatory function or diffusion capability, predisposing them to dyspnoea, complications and death 19,26,28]. Hence, a comprehensive assessment of elderly patients for co-morbidity and modifiable factors is important. Our study demonstrates that with a standardised Prehab program, it is possible to improve their modifiable factors of dyspnoea, PS, LOA, FI and achieve optimisation for surgical resection with good outcomes , 14, 28.. Hence, The five-year survival of patient with lung cancer amongst other cancers is poor  and a muInterestingly our study, in keeping with the findings of a randomized trial by Wolfram Karenovics et al. , shows tOur study has certain limitations. This is a single-institution, pragmatic, real life provision of a standardized Prehab program to optimise lung cancer patients with frailty for surgery and may not fully address confounding factors. Objectively counting a patient’s clinical deficits for frailty, which although reproducible and correlates highly with mortality, is unwieldy . NeverthIn conclusion, our pragmatic study demonstrates that with a standardised Prehab program, vulnerable high-risk elderly patients with high frailty index for lung resection, can be suitably optimised to proceed to safe surgical resection and have outcomes similar to low-risk patients with low frailty index. Therefore, at lung cancer MDTs the management plan for high-risk elderly patients with frailty, who are otherwise deemed vulnerable to adverse events and death, should include consideration of referral for a period of structured Prehab. Bearing in mind that optimisation with Prehab takes longer in this elderly group of patients, the duration of Prehab is guided by their stage of disease and risk of progression. Those suitably optimised with Prehab can be expected to safely proceed with curative lung resection. Nevertheless, a suitably powered randomized control trial is required to confirm our observations and establish whether a structured Prehab program helps improve the long-term survival of elderly lung cancer patients with frailty.Below is the link to the electronic supplementary material.Supplementary Material 1


Embryonal tumor with multilayered rosettes (ETMR) is a deadly grade IV pediatric brain tumor. Despite an intensive multimodal treatment approach that includes surgical resection, high‐dose chemotherapy, and radiotherapy, the progression‐free survival at 5 years is less than 30%.We report a case of long‐term survival in a 5‐month old female with a large mass in the posterior fossa, diagnosed as ETMR, which subsequently underwent treatment‐induced maturation. Prior to chemotherapy, histopathology revealed an abundance of highly proliferative, undifferentiated cells and multilayered rosette structures. Conversely, post‐treatment histopathology revealed cell populations that differentiated into neuronal and ganglionic phenotypes. At 5‐year follow‐up, the patient remains progression‐free.This finding contributes to the few reports to date of post‐treatment differentiation/maturation of ETMR cell populations, with an implication for less cytotoxic therapeutic interventions aimed at differentiation. Jessa et al.Herein, we report a case of long‐term survival (greater than 5 years) in a patient with ETMR, whose post‐treatment histopathology revealed maturation of undifferentiated embryonal cells into mature neuronal and ganglionic phenotypes. This case is important, because it builds upon the postulation made by Jessa et al. and others supporting differentiation as a mechanism for treating malignant, embryonal tumor cell populations, demonstrating the feasibility of treatment‐induced differentiation for prolonging patient survival. Furthermore, we review the literature on post‐treatment differentiation of ETMR, of which there are only three other publications, as well as other pediatric brain tumors. These reports on treatment‐induced differentiation support the notion of differentiation as a promising therapeutic aim for novel drug development for the treatment of deadly pediatric brain tumors.22016 WHO Classification of Tumors of the Central Nervous System had just defined ETMR as a single entity, and diagnosis required detection of the amplification of the C19MC miRNA cluster on Chr19q13.42. IHC staining for LIN28 was also characteristic of an ETMR diagnosis. Patient samples were sent to NYU Langone Health  and the Hospital for Sick Children  for confirmatory diagnostics. Methylation profiling and positive LIN28 IHC staining confirmed the diagnosis of ETMR.In 2016, a 5‐month‐old female presented to the Emergency Department of the Joseph M. Sanzari Children's Hospital at Hackensack University Medical Center with vomiting and somnolence. After ruling out infectious and metabolic differentials with diagnostic laboratory tests, an MRI of the brain was obtained and showed a 6.5 × 6.5 × 4.4 cm calcified, heterogeneously enhancing mass in the right posterior fossa Figure . SubsequFollowing diagnosis, the patient underwent 4 cycles of induction chemotherapy, according to the DFCI‐IRS‐III regimen.2.12 i.v.; Cisplatin 90 mg/m2 i.v.; Doxorubicin 30 mg/m2/day with continuous infusion over the course of 48 h; Cyclophosphamide 300 mg/m2/day with continuous infusion over the course of 72 h and Cyclophosphamide 600 mg/m2 i.v.; Etoposide 100 mg/m2 i.v.; Actinomycin D 1.2 mg/m2 i.v.; and Topotecan 0.2 mg/m2 intrathecally. Regarding the timepoints of administration, Vincristine was administered on day 1 of weeks 1–13 and week 16 of therapy.2/day was administered on days 2, 3, and 4 of weeks 1, 13, and 16.2 was administered on day 2 of weeks 7 and 10.The chemotherapy induction regimen based upon the DFCI‐IRS‐III approach, as detailed by Hanson et al.2.2An adverse effect of this patient's chemotherapeutic course was sinusoidal obstructive syndrome, which was rapidly responsive to treatment with defibrotide. Defibrotide is a polydisperse polydeoxyribonucleotide that has thrombolytic effects that are beneficial for the hypercoagulable sequelae of cancer states, and it also demonstrated anti‐tumor activity when used as a single agent against multiple myleoma.2.3Following the first resection of tumor tissue, and prior to chemotherapy, pathological examination of the collected specimen revealed a plethora of characteristic multilayered rosettes in a background of primitive, undifferentiated cells with high mitotic activity, atypia, and overabundant cellular density Figure . The tum2.4Final treatment included high‐dose chemotherapy and stem cell rescue (HDCT/SCR) with a carboplatin and thiotepa conditioning regimen, with maintenance chemotherapy for 18 months with DFMO, everolimus, and intrathecal topotecan. The patient is now 6 years old (5 ½ years from diagnosis) and attends school. The patient requires hearing aids secondary to cisplatin‐related ototoxicity, which further highlights the need for less cytotoxic therapies in the pediatric population.3The case presented here is the fourth known case in the literature describing a patient with ETMR, whose post‐treatment pathology showed differentiation of tumor cells along their programmed lineage; this is in comparison to the pre‐treatment pathology of rapidly dividing undifferentiated cells. Importantly, to the best of our knowledge, this is the only report of an ETMR patient with post‐treatment differentiation of tumor cells who has exceeded the 5‐year survival mark following initial diagnosis.Of the three post‐treatment differentiation cases published to date for ETMR, the study by Antonelli et al.Moreover, our case contributes to the growing number of reports of post‐treatment differentiation in embryonal tumors in general. In addition to this case, Levine et al.Furthermore, Lafay‐Cousin et al.Interestingly, both Levine et al. and Lafay‐Cousin et al. present cases of patients who did not have impressive post‐treatment reduction in tumor bulk, both of whom had recurrence that eventually led to patient demise. Conversely, in the case that we present and in Antonelli et al., there were more impressive reductions in tumor bulk following chemotherapy treatment, and the patients had longer‐term survival. However, in all four cases, it is apparent that the molecular footprint of ETMR destines it for neuronal maturation, and the maintenance and extent of that maturation may dictate stability and survival.Other instances of pediatric embryonal tumors undergoing post‐treatment maturation include: medulloblastoma, neuroblastoma, and high‐grade neoplasm with PNET‐like features. Wu et al.Additionally, Bidgoli et al.Our case contributes to the relatively few pieces of literature that elucidate the clinical association of post‐treatment differentiation of tumor tissue on pathology, reduced tumor burden on imaging, and prolonged survival of the patient. A pre‐clinical correlate can be seen with the findings of Jessa et al.,In summary, we have presented a case with prolonged survival (>5 years) after post‐treatment differentiation of the patient's pathology and reviewed similar accounts reported in the literature. These collective findings support the notion for the development of novel therapeutics that are less cytotoxic than chemotherapy, but that achieve the objective of inducing maturation of embryonal tumor tissue in order to potentiate cancer remission.Francesca M. Gualano contributed to conceptualization, validation, writing , and visualization. Patrice Hassoun contributed to visualization and review. Claire L. Carter contributed to conceptualization, validation, writing (review and editing), visualization, supervision, and project administration. Derek Hanson contributed to conceptualization, validation, writing (review and editing), visualization, supervision, and project administration.The authors have stated explicitly that there are no conflicts of interest in connection with this article.We obtained a written statement of informed consent from the patient's parents for the publication of case details and use of images. The case discussed in this manuscript does not include patient‐identifying information, nor does it report a new study that required IRB approval.


FGFR1 (Fibroblast Growth Factor Receptor 1) and RET (Ret Proto‐Oncogene) and, (c) later in the course of the disease, PARP (Poly(ADP‐Ribose) Polymerase) inhibition with olaparib prompted by signs of defective homologous recombination DNA repair. In addition, our data provided new insights into the molecular landscape of PC with respect to the genome‐wide footprints of specific mutational processes and pathogenic germline alterations. These data underscore the potential of comprehensive molecular analyses to improve care for patients with ultra‐rare cancers based on insight into disease biology.Parathyroid carcinoma (PC) is an ultra‐rare malignancy with a high risk of recurrence after surgery. Tumour‐directed systemic treatments for PC are not established. We used whole‐genome and RNA sequencing in four patients with advanced PC to identify molecular alterations that could guide clinical management. In two cases, the genomic and transcriptomic profiles provided targets for experimental therapies that resulted in biochemical response and prolonged disease stabilization: (a) immune checkpoint inhibition with pembrolizumab based on high tumour mutational burden and a single‐base substitution signature associated with APOBEC  overactivation; (b) multi‐receptor tyrosine kinase inhibition with lenvatinib due to overexpression of  MUTHY heterozygous germline mutation as a potential predisposing factor. Therapeutically, transcriptome sequencing enabled the detection of recurrent FGFR1 and RET overexpression and guided targeted therapy.Our case study on advanced parathyroid carcinoma using whole genome and transcriptome sequencing improved our understanding of the molecular pathogenesis of the disease by identifying a relevant contribution of the APOBEC signatures SBS2 and SBS13 to the mutational process, as well as a  APOBECapolipoprotein B mRNA editing enzyme catalytic polypeptide‐likeCCcell cycleCNVcopy number variationDDRDNA damage repairDEVdevelopmental regulationDKFZGerman Cancer Research CenterDKTKGerman Cancer ConsortiumFGFR1fibroblast growth factor receptor 1IEimmune evasionindelsmall insertions and deletionLNlymph nodeLOH‐HRDloss of heterozygosity‐homologous recombination deficiencyLSTlarge‐scale state transitionMASTERMolecularly Aided Stratification for Tumor Eradication ResearchMSImicrosatelite instabilityMTBmolecular tumour boardNCTNational Center for Tumor DiseasesOTHotherPAMPI3K‐AKT‐mTORPARPpoly(ADP‐ribose) polymerasePCparathyroid carcinomaRETRet proto‐oncogeneRMERAF‐MEK‐ERKRNA‐seqRNA sequencingSBS3single‐base substitution signature 3SNVsingle‐nucleotide variantSVstructural variationTKtyrosine kinaseTMBtumour mutational burdenTPMtranscript per millionWGS/WESwhole‐genome/exome sequencing1Parathyroid carcinoma (PC) is an ultra‐rare malignancy with an incidence of approximately 0.03 per 100 000 persons per year . In the In a retrospective study of 83 patients with PC, disease‐specific overall survival was favourable, but nearly 40% of patients experienced recurrence. Factors associated with longer recurrence‐free survival were low tumour stage, a Ki67 index < 10%, normalization of calcium levels after surgery and absence of lymph node (LN) invasion . RecurreCDC73 and MEN1. Furthermore, activation of the PI3K‐AKT‐mTOR (PAM) pathway and amplification of CCND1 have been observed in up to one‐third of patients [Known molecular drivers of PC include germline and somatic mutations in patients , 13, 14.The National Center for Tumor Diseases (NCT), the German Cancer Research Center (DKFZ) and the German Cancer Consortium (DKTK) conduct MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a prospective observational study that applies whole‐genome/exome sequencing (WGS/WES) and RNA sequencing (RNA‐seq) to inform the care of young adults with advanced malignancies and patients with incurable rare cancers , 16. Her22.1Metastases of four patients aged 39–82 years with advanced PC and imminent need for systemic treatment, followed between 2017 and 2020 by two tertiary referral centres, were studied by WGS and RNA‐seq as part of NCT/DKFZ/DKTK MASTER. In all cases, peripheral blood was used as a control. The trial was approved by the Ethics Committee of Heidelberg University (protocol no. S‐206/2011), and all patients provided written informed consent. Clinical data were collected as part of clinical routine. The study was conducted in accordance with the Declaration of Helsinki.2.2Sample processing was done as previously published : A freshyapsa  on cosmic v3 signatures [msisensor algorithm [platypus (version 0.8.1.1) and further annotated with gnomad (version 2.1) and the local controls database using vcfanno (version 0.3.2), and variants with a minor allele frequency > 0.005 in gnomad and a frequency > 0.05 in the local control database were removed. vep (version 97) was used to annotate the rare variants in detail. Rare germline variants were evaluated according to American College of Medical Genetics and Genomics and Association for Molecular Pathology criteria and further specifications [MEN1 and CDC73 were manually inspected and did not indicate structural variants. Immune infiltration analysis was performed using transcript per million (TPM) values with seven different tools via the unified interface of immunedeconv (version 2.0.2) [Mapping and alignment of sequencing reads, detection of somatic single‐nucleotide variants (SNVs), small insertions and deletions (indels), DNA structural variations (SVs) and copy number variations (CNVs), and assessment of gene expression were performed using previously reported bioinformatics workflows , 20, 21.gnatures . Genomicgnatures , 24, 25.lgorithm . Clusterications , 28. Mapn 2.0.2) .progeny algorithm (R package progeny version 1.12.0) [m to consider only high‐affinity binding. A kinase gene was considered overexpressed if the expression was higher than that of 75% of all kinase genes across all samples (Tables The  1.12.0)  was used 1.12.0)  using ans Tables  and S2.Biological curation and clinical annotation of genomic and transcriptomic alterations were performed as previously described , and tre33.1Key clinical characteristics are provided in Table CDC73, MEN1, MSH6, CCND1, DICER1 and HRAS) were altered in more than one case . PC‐B had biallelic CDC73 inactivation due to a CDC73 K34Rfs*3 frameshift deletion and LOH. PC‐C showed a deletion with LOH at chromosome 11q13.1, including MEN1. PC‐D harboured a MEN1 p.Q547_S561delinsH in‐frame deletion. MSH6 was affected by two somatic missense mutations (p.D135N and p.S574L) in PC‐A, which were classified as variants of unknown significance. Immunohistochemistry showed no loss of mismatch‐repair protein expression in this case, suggesting benign mutations. CCND1 was amplified in PC‐B and PC‐C. Outlier overexpression of CCND1 was observed in PC‐C and PC‐A, but we observed no somatic or germline alterations affecting CCDN1 in PC‐A  was considered high [In one case each, we found a p.E2181K missense mutation in the phosphatidylinositol 3‐ and 4‐kinase domain of red high , with 50Single‐base substitution signature 3 (SBS3), which is highly prevalent in HRD‐related cancers , was detA total of 821 CNVs were identified in the four patients Fig. , with coMUTHY p.G393D variant in PC‐D, was classified as pathogenic. MUTYH encodes the DNA glycosylase mutY, which removes adenine residues mispaired with 8‐oxo‐deoxyguanosine or ‐guanine. PC‐D also had somatic LOH at MUTYH, resulting in biallelic inactivation of MUTYH, a constellation conferring a heritable predisposition to colorectal carcinoma and possibly other malignancies termed MUTYH‐associated polyposis. Interestingly, this sample also displayed SBS18, most likely caused by the MUTYH variant and somatic LOH [The four patients harboured 13 rare germline alterations affecting eight known cancer predisposition genes  Table S. One of atic LOH , 34. Imm3.2RET and FGFR1 in PC‐A and PC‐B compared with PC‐C and PC‐D . We noticed striking overexpression of C‐D Fig. .BRAF‐specific inhibitor approved for melanoma and non‐small‐cell lung cancer. Nintedanib has activity against VEGFR, FGFR and PDGFR and is approved for non‐small cell lung cancer in combination with docetaxel [Considering the top 25% expressed kinase genes across the four samples, seven kinase inhibitors with at least five targets in at least one sample were identified as potential therapies Fig. . Dasatinocetaxel . Ibrutinocetaxel . Lenvatiocetaxel , 42.To explore changes in pathway activity that cannot be inferred from the expression of individual kinase genes, we further used the PROGENy algorithm. An enrichment of PIK3CA, MAPK and WNT signalling was observed in PC‐A, whereas PC‐D showed upregulation of the NFkB, TNFa, JAK‐STAT, VEGF and Trail pathways Fig. .3.3In the MASTER program, biomarkers and treatment recommendations are categorized into eight molecular intervention baskets based on the cellular pathways or processes involved: tyrosine kinases (TKs), PI3K‐AKT‐mTOR (PAM), RAF‐MEK‐ERK (RME), cell cycle (CC), developmental regulation (DEV), DNA damage repair (DDR), immune evasion (IE) and other (OTH).At least two targeted therapies were recommended for each patient. These were assigned to the IE (three patients), TK (three patients), PAM (two patients), DDR (two patients), DEV (one patient) and CC (one patient) baskets. Treatment combinations were recommended in two cases Table . In two PC‐A, a 43‐year‐old woman with locally advanced PC, remained in remission for 12 years after primary treatment when she developed abdominal LN metastases. Based on a high TMB  and the APOBEC mutational signatures SBS2 and SBS13 in a metastasis sample, the PD‐1 inhibitor pembrolizumab was recommended. Interestingly, compared with the other three cases, the CIBERSORT deconvolution algorithm, an immune infiltration analysis tool in immunedeconv, indicated high fractions of activated CD8‐positive T cells, T‐follicular helper cells, inflammatory M1 macrophages and a small fraction of regulatory T cells (Tregs), suggesting a responder phenotype for immune checkpoint inhibitor treatment .FGFR1 and RET, which prompted treatment with lenvatinib. The patient experienced complete biochemical remission and remained on therapy for 3 months. Due to adverse events, treatment was discontinued, and radiologic and biochemical disease progression occurred 5 months later. The second molecularly informed treatment with the PARP inhibitor olaparib, informed by somatic loss of BRCA2 and detection of SBS3 [Patient PC‐B, a 51‐year‐old man with localized PC, relapsed 4 years after primary resection with lung and skull base metastases, for which he underwent repeat surgical interventions. Rapid progression required radiation to the skull base. Molecular analysis of a skull base metastasis revealed overexpression of  of SBS3 , resultePatient PC‐C was a 39‐year‐old man who underwent left parathyroidectomy and hemithyroidectomy followed by remission for 13 years. He subsequently developed local relapse with LN metastases, for which he underwent multiple surgical procedures before enrolling in the MASTER program. He was lost to follow up, and no information is available on treatment or response.Finally, patient PC‐D was an 82‐year‐old woman with localized PC who developed lung metastases 1 year after the initial surgery. High expression of somatostatin receptor 5 detected by immunohistochemistry prompted two courses of peptide receptor‐mediated radionuclide therapy with Lu‐177 DOTATOC, but this did not halt the progression of LN metastases. A surgical biopsy of one LN was subjected to molecular analysis, but the patient died of progressive disease before targeted therapy could be considered. Also in this patient, immune checkpoint inhibition was recommended based on high TMB, although immune cell deconvolution suggested a protumoral tumour microenvironment characterized by higher proportions of Tregs and immunosuppressive M2 macrophages and fewer M1 macrophages, CD8‐positive T cells and T‐follicular helper cells Fig. .4This is the first study to perform genome‐ and transcriptome‐wide characterization of advanced PC to guide clinical management of patients with this ultra‐rare cancer after failure of standard therapy. Analyses were performed using the standardized workflow established in the NCT/DKFZ/DKTK MASTER program for young adults with advanced cancers and patients with refractory rare malignancies . MoleculBoth DNA and RNA sequencing provided clinically valuable information. Because cryopreserved tumour tissue is required for whole‐genome and RNA sequencing, as used in our study, a direct comparison with archival tissue samples from primary and metastatic lesions to shed light on spatial and temporal heterogeneity in PC was not possible. However, previous targeted RNA expression studies on formalin‐fixed, paraffin‐embedded samples have revealed marked differences in gene expression between primary tumours and metastases from different patients .CDC73 mutations in two of four patients, which have been linked to a more aggressive disease course and increased TMB [CCND1 as a recurrent alteration in PC [TP53 mutations are rare in PC and associated with a more aggressive clinical course [TP53‐mutant PC in our cohort relapsed after 1 year with rapid clinical deterioration. The pathogenetic role and potential clinical significance of additional tumour suppressor genes in PC are largely unknown. Our observation of deletion of chromosome 3q in three of four samples suggests that one or more such genes may be located in this region.In line with previous data, we detected somatic ased TMB , featureon in PC , suggestl course . ConsistMUTYH was observed in the tumour and associated with the characteristic SBS18, supported by evidence of increased oxidative DNA damage on immunohistochemistry. While biallelic germline mutations in MUTYH are associated with adenomatous polyposis and increased risk of colorectal cancer, the relative cancer risk for heterozygous carriers is less clear. Monoallelic MUTYH variants contribute to tumorigenesis through loss of the remaining functional MUTYH allele via LOH. The characteristic C‐to‐A base substitutions in MUTYH‐deficient tumours increase the formation of stop codons, contributing to the inactivation of tumour suppressor genes [MUTYH carriers [MUTYH in the pathogenesis of this PC case.The increased TMB observed in two cases could be attributed to two different genetic mechanisms. In PC‐D, LOH for a heterozygous germline mutation in or genes , 48. Thecarriers , 49 and In addition, PC‐D, the second patient with elevated TMB, exhibited SBS2 and SBS13, which are attributed to APOBEC cytidine deaminase activity and account for more than one‐third of human cancers signatures , 22. As Thus, the clinical courses of PC‐A and PC‐D underscore the clinical utility of integrating composite biomarkers – in these cases, TMB and mutational signatures – in the workup of advanced PC. This general strategy, which requires comprehensive molecular profiling, is also supported by patient PC‐C, in whom evidence of HRD and HRD‐LOH provided a rationale for PARP inhibition, which was implemented after treatment with lenvatinib and led to prolonged disease stabilization.FGFR1 and RET prompted the recommendation of treatment with lenvatinib by the MTB. This led to a dramatic biochemical response in PC‐B, even entailing hypocalcemia as an indicator of response, and morphologic disease stabilization for 8 months. In this exceptional responder, only RET but not FGFR1 was among the targets of lenvatinib when only the top 25% expressed kinases with a high binding affinity  were considered. Together with the strong overexpression of RET in PC‐A, these observations suggest a role for RET signalling in the development and/or progression of a subset of PC, a hypothesis that requires further validation in preclinical models and the clinical setting. Notably, a previous study comparing parathyroid adenoma, non‐metastatic and metastatic PC found that FGFR1 is one of the genes overexpressed in metastatic versus non‐metastatic PC, also emphasizing FGFR1 inhibition as a potential therapeutic strategy [Recent studies have demonstrated that transcriptome analysis can substantially increase the number of patients treated in molecularly informed clinical trials , 52. Accstrategy .5MUTYH as potential predisposing factors, and recurrent overexpression of FGFR1 and RET. Notably, some of these features provide novel therapeutic targets. Our work thus underscores the scientific and clinical value of multi‐omics‐based and entity‐spanning precision oncology programs, which is particularly apparent in very rare and poorly understood cancers for which there are few standards of care, such as PC.In summary, our findings reveal novel aspects of the molecular pathogenesis of PC, particularly the importance of mutational processes reflected in the APOBEC signatures SBS2 and SBS13, heterozygous germline mutations in C. Terkamp: Consulting or advisory board membership: AstraZeneca, Lilly, NovoNordisk; honoraria: AstraZeneca, Lilly, Merck Sharp and Dohme, NovoNordisk, Takeda; travel or accommodation expenses: AstraZeneca, Ipsen, Lilly, NovoNordisk, Pfizer. S. Fröhling: Consulting or advisory board membership: Bayer, Illumina, Roche; honoraria: Amgen, Eli Lilly, PharmaMar, Roche; research funding: AstraZeneca, Pfizer, PharmaMar, Roche; travel or accommodation expenses: Amgen, Eli Lilly, Illumina, PharmaMar, Roche. M. Kroiss: Consulting or advisory board membership: Advanz, Bayer, Eisai, Eli Lilly; research funding: Eli Lilly, Ipsen, Loxo; honoraria: Eli Lilly, Ipsen, Merck Sharp and Dohme, Roche, Sanofi; travel or accommodation expenses: Eli Lilly. The other authors declare no competing interests.SF and MK designed the study. M‐VT, CTF, NP, SP, AM, SK, L‐SL, AJ, and DH performed experiments and analysed data. AM, CT, CL, SK, NS, AS, MF, HG, SF, and MK contributed to collection and interpretation of data. M‐VT, CTF, SF, and MK drafted the manuscript. All authors critically revised and contributed to the manuscript and approved its final version.Fig. S1. Genomic landscape of PC.Fig. S2. Expression of CCND1, RET, FGFR1 in samples PC‐A, B, C, and D compared to 149 background samples from the NCT/DKFZ/DKTK MASTER cohort.Fig. S3. RNA‐based pathway activity inference using the PROGENy algorithm.Fig. S4. Immune cell infiltration.Table S1. Kinase gene expression.Table S2. Kinase gene outlier expression.Table S3. Raw and processed patient‐individual sequencing results.Click here for additional data file.


To analyze the clinical characteristics and bronchoalveolar lavage fluid pathogens in elderly patients with community‐acquired pneumonia (CAP).This was a retrospective observational epidemiological study using that elderly cases diagnosed with community‐acquired pneumonia receiving treatment at the Affiliated Hospital of North China University of Technology, Tangshan Hongci Hospital and Tangshan Fengnan District Hospital of Traditional Chinese Medicine. A total of 92 cases were divided into two groups according to age. There were 44 patients over 75‐year‐old and 48 patients between 65 and 74‐year‐old.p = 0.007) and are more likely to have mixed infections  or larger lesions . Their hospital stays will also be extended , and the albumin level , the neutrophils level (9.09(6.26–10.63) vs. 7.18(5.35–9.17),p = 0.026) is significantly lower and the d‐dimer , PCT  levels are significantly higher.Compared with the elderly 65 to 74‐year‐old, the elderly over 75‐year‐old with diabetes are more likely to suffer from CAP (35.42% vs. 63.64%, d‐dimer can predict the prognosis of patients.The clinical symptoms and signs of elderly CAP patients are not so typical, and the infection is more serious. Attention should therefore be paid to elderly patients. Hypoalbuminemia and high  The clinical signs of elderly community‐acquired pneumonia patients are not so typical. Attention should be paid to elderly patients. Compared with patients 65 to 74‐year‐old, patients over the age of 75 were more likely to have infections with mixed infection. They have higher PCT、D‐D and lower ALB, which indicated severe infection. Developing countries bear a significant brunt of the disease, impacting healthcare.Population aging is a problem for countries all over the world. People aged 60 and older make up 12.3% of the global population. The number will rise to almost 22% by 2050.Bronchoalveolar lavage fluid (BALF) is a noninvasive, well‐tolerated procedure that is an important diagnostic tool for the diagnosis of various lung diseases.According to these data and background, in this study, we evaluated the clinical characteristics and pathogen analysis results of the disease in the elderly patients by screening elderly patients with CAP being treated at the Affiliated Hospital of North China University of Technology, Tangshan Hongci Hospital and Tangshan Fengnan District Hospital of Traditional Chinese Medicine, providing some ideas for the management in CAP elderly patients.22.1This retrospective study selected 92 elderly patients with community‐acquired pneumonia. Most patients live with their spouses or children, while a few patients live alone. All the patients were admitted to the Department of Respiratory Medicine, Affiliated Hospital of North China University of Science and Technology, Tangshan Hongci Hospital and Tangshan Fengnan District Hospital of Traditional Chinese Medicine from September 2020 to September 2021, and were divided into two groups according to age. There were 48 patients between 65 and 74‐year‐old (69.21 ± 2.52 years) and 44 patients over 75‐year‐old (78.23 ± 2.93 years). The selected patients all met the diagnostic criteria, inclusion criteria, and exclusion criteria of the Guidelines for the Diagnosis and Treatment of Community‐Acquired Pneumonia.2.2d‐dimer (D‐D), neutrophils (NEU), high‐sensitivity C‐reactive protein (hsCRP), serumamyloid A (SAA), fibrinogen (FIB), the erythrocyte sedimentation rate (ESR), procalcitonin (PCT), and albumin (ALB). Pathogenic detection:collect and culture of BALF.General information collected includes: gender, age, body mass index (BMI), combined underlying diseases  Table . ClinicaChinese Expert Consensus on Pathogen Detection of Bronchoalveolar Lavage in Pulmonary Infectious Diseases,Mycoplasma pneumoniae antibodies were detected by passive agglutination method.According to the 2017 2.34 CFU/mL (semiquantitative culture +~++) or sterile antipollution BALF ≥ 103 CFU (semiquantitative culture+) is positive, and the dominant strains have moderate growth (+++) which is clinically significant; two serum M. pneumoniae antibody titers collected at intervals of 2 to 4 weeks showed a ≥4‐fold increase or decrease with clinical significance.According to the expert consensus,2.4t‐test or Mann–Whitney U test. For categorical variables, we used the χ2 test. p < 0.05 (two‐tailed) was considered statistically significant.Categorical variables are shown as proportions, and continuous variables are shown as means with standard deviation (SD). The statistical tests conducted for continuous variables were the 33.1p = 0.007) than patients 65 to 74‐year‐old. There was a statistically significant difference in the number of days in hospital between the two groups. There were no differences in age, BMI, coronary heart disease, hypertension, COPD, hypokalemia, hyponatremia, and pleural effusion between the two groups (p > 0.05).Patients older than 75‐year‐old had a higher incidence of diabetes (3.2p = 0.003), dyspnea  and anorexia (p = 0.049) were identified between the two groups (p < 0.05), but no significant difference in other clinical manifestations was detected (p > 0.05). There was no significant difference in pulmonary signs between the two groups (p > 0.05), but pulmonary infection in the elderly over 75‐year‐old was more likely to involve more than three lung segments  and anorexia (58.33% vs. 84.09%) were common clinical manifestations in both groups. Fever (62.50% vs. 31.82%) was also more common in patients aged 65 to 74‐year‐old than in patients over 75‐year‐old. There were differences in fever (3.3p = 0.023). The number of infected strains in the two groups of patients is compared in Table Among the 92 patients in this study, 57 patients had pathogens detected bronchoalveolar lavage fluid cultures or through passive agglutination tests in serum infection. The detection rate was 61.96%. The bronchial lavage fluid of all patients was stained with gram. A total of 32 patients detected Gram‐negative bacilli (13 in the younger group and 19 in the other group), and 24 patients detected Gram‐positive cocci (10 in the younger group and 14 in the other group). A total of 107 strains of 11 categories were detected. The composition ratio is shown in Table 3.4p = 0.026, p = 0.000, p = 0.011, p = 0.001), as well as other indicators. The mean levels of NEU (9.09（6.26–10.63） vs. 7.18（5.35–9.17）) and ALB (30.93 ± 6.58 vs. 37.51 ± 8.92) in patients over 75‐year‐old were lower than those 65–74 years old. The mean levels of D‐D (611.82 ± 195.85 vs. 505.42 ± 197.12) and PCT (0.12 ± 0.07 vs. 0.08 ± 0.04) were higher in patients over age of 75‐year‐old than those aged 65–74‐year‐old. There was no significant difference between the two groups in terms of levels of PLT, FIB, hsCRP, SAA, ESR, and other indicators , M. pneumoniae (13.08%), and H. influenzae (12.15%). At the same time, the number of patients with mixed infection in the two groups is large. People who regularly take antibiotics have immune disorders. They are more easily to meaning that they produce drug‐resistant strains, thus increasing the chance of infection with multiple pathogens.M. pneumoniae infection is also more common. Therefore, it is necessary to cover this pathogen when taking medicine. The literature shows that drug‐resistant Gram‐negative bacilli mainly include Acinetobacter baumannii, K. pneumoniae, ESBL‐producing Enterobacterales and Pseudomonas aeruginosa.According to several literatures, the most common pathogen of CAP is the It has beend‐dimer in patients with severe CAP was significantly higher than that in patients with nonsevere CAP, and D‐D was considered to have extremely high clinical value, which could be used as a means of judging the prognosis of patients degree indicator. This is consistent with the analysis results of our study.When the body is in a state of infection, platelets will decrease. In the American CAP guidelines, thrombocytopenia is also listed as one of the diagnostic criteria for severe CAP.This study also has shortcomings. Due to limited equipment and conditions, virus isolation and detection in patient lavage fluid was not performed; and the sample size needs to be further expanded.5In conclusion, clinical manifestations are not typical in patients with CAP over 75‐year‐old. They are usually serious, and mostly have a mixed infection. They usually have longer hospital stays and are more difficult to treat, and the prognosis may not be ideal. Therefore, attention should be paid to the elderly group and prevention should be strengthened. It is an important measure to improve the knowledge of such people and their families about related diseases. ALB and D‐D can predict the prognosis of patients to a certain extent.Conception and design of the research: Yan Huang. Acquisition of data: Cai‐Hong Li. Analysis and interpretation of the data: Bai‐Yi Liu. Statistical analysis: Dan Zhang, Zhu Fan, and Jing‐Jing Jin. Obtaining financing: Xue‐Juan Li. Writing of the manuscript: Rui‐Nan Guo. Critical revision of the manuscript for intellectual content: Yan Huang. All authors read and approved the final draft.The authors declare no conflict of interest.This study was conducted with the approval of the Affiliated Hospital of North China University of Science and Technology Ethic Committee. This study was conducted in accordance with the declaration of Helsinki. Written informed consent was obtained from all participants.


The effects of FGF21 on Parkinson's disease (PD) and its relationship with gut microbiota have not been elucidated. This study aimed to investigate whether FGF21 would attenuate behavioral impairment through microbiota–gut–brain metabolic axis in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) induced PD mice model.Male C57BL/6 mice were rendomized into 3 groups: vehicle (CON); MPTP 30 mg/kg/day i.p. injection (MPTP); FGF21 1.5 mg/kg/d i.p. injection plus MPTP 30 mg/kg/day i.p. injection (FGF21 + MPTP). The behavioral features, metabolimics profiling, and 16 s rRNA sequencing were performed after FGF21 treatment for 7 days.Clostridiales, Ruminococcaceae, and Lachnospiraceae, thereby rescuing the PD‐induced metabolic disorders in the colon.MPTP‐induced PD mice showed motor and cognitive deficits accompanied by gut microbiota dysbiosis and brain‐region‐specific metabolic abnormalities. FGF21 treatment dramatically attenuated motor and cognitive dysfunction in PD mice. FGF21 produced a region‐specific alteration in the metabolic profile in the brain in ways indicative of greater ability in neurotransmitter metabolism and choline production. In addition, FGF21 also re‐structured the gut microbiota profile and increased the relative abundance of These findings indicate that FGF21 could affect behavior and brain metabolic homeostasis in ways that promote a favorable colonic microbiota composition and through effects on the microbiota–gut–brain metabolic axis. FGF21 attenuated the motor and non‐motor dysfunction in PD mice. FGF21 regulated choline metabolism, glutamate‐glutamine cycle, and energy metabolism in the brain. FGF21 treatment restores PD‐induced shifts of colonic microbiota and modulates colon metabolism. The patient's quality of life is significantly impacted by both motor symptoms like rigidity and resting tremor as well as non‐motor symptoms like depression and cognitive dysfunction.1H NMR) based metabolomics was applied to evaluate the metabolic signatures in different stages of PD, and results suggest that the overactive glutamate–glutamine metabolic cycle in the striatum was linked to the dopaminergic neuronal loss and motor dysfunction in PD mice.Recently, metabolic disorders have been considered to contribute to the development of neurodegenerative diseases including PDFibroblast growth factor 21 (FGF21), a new member of the fibroblast growth factor family, is a recently discovered endocrine hormone primarily expressed in the liver and exerts diverse effects on metabolic health.More importantly, as a classic metabolic regulator, the effects of FGF21 on PD and its relationship with gut microbiota have not been elucidated. The current study addressed this issue by investigating the potential effect of FGF21 on metabolic homeostasis in the brain and colon in PD mice. Moreover, the molecular insights connected with the microbiota–gut–brain axis in the beneficial role of FGF21 in PD were discussed as well.22.1n = 10 in each group): CON, MPTP, and FGF21 + MPTP groups. Mice in the MPTP and FGF21 + MPTP groups were injected intraperitoneally (i.p.) 30 mg/kg MPTP–HCl  daily for 5 consecutive days,Male C57BL/6 mice (20–26 g), were purchased from the SLAC Laboratory Animal Co. Ltd. Shanghai, China, and then maintained in the laboratory animal center of the Wenzhou Medical University under regular laboratory conditions. A previous study showed that males of at least 8 weeks of age weighing at least 22 g are the ones that produced the most consistent results in MPTP induced PD mice model.2.2Behavior performance was assessed using open field and Y maze tests before the sacrifice. The open‐field device is designed to measure rodents' spontaneous activity. Mice were tested in the open field for 5 min. Gross movement  and time spent in the different zone were measured using video‐tracking software. Additionally, the Y maze was adapted to measure cognitive function. Mice were subjected to the Y maze by placing them in the start arm and were allowed to explore freely all three arms of the maze for 5 min. The number and the sequence of arms entered were recorded and used to calculate spontaneous alteration.2.3Brain samples were rapidly dissected and then sectioned at a thickness of 5 μm. The paraffin‐embedded sections were then deparaffinized and rehydrated, followed by incubation with primary antibody to tyrosine hydroxylase  at 4 °C for 24 h. And then incubated with a second Goat Anti‐Rabbit IgG H&L antibody, the immunoreactivity was visualized with a Nikon ECLIPSE 80i (Nikon) and then analyzed using Image J software.2.4Microbiota sequencing was conducted to determine the effects of FGF21 on PD‐induced microbiota alteration Method . All DNA2.51H NMR spectroscopy.2O with 0.05% of sodium trimethylsilyl propionate‐d4 (TSP) as the chemical shift reference. All 1H NMR spectra of the brain and colon were carried out on a Bruker AVANCE III 600 MHz NMR spectrometer with a 5‐mm TXI probe (Bruker BioSpin) at 298 K. Acquisition parameters were set: acquisition time, 2.65 s per scan; relaxation delay, 6 s; data points, 64 K; spectral width, 12,000 Hz. Then, all 1H NMR spectra were phase/baseline corrected manually using Topspin software .Water‐soluble metabolites in the striatum, midbrain, cortex, and colon were identified and quantified using After removing the region of 4.60–5.20 ppm to eliminate artifacts related to the residual water resonance, the remaining spectral segments were normalized and typed in SIMCA software  for multivariate recognition analysis, including principal component analysis (PCA) and optimized potentials least squares‐discriminant analysis (OPLS–DA).2.6p < 0.05 accepted as statistically significant.The significance of the difference in alpha diversity measures (the observed species and Shannon index) among the 3 groups was analyzed using non‐parametric Kruskal–Wallis. Beta diversity was calculated by weighted UniFrac distances and visualized by principal coordinates analysis. In addition, Adonis, a permutational analysis of variance, was performed using 999 permutations to test for differences. In addition to microflora sequencing, the significance of group differences for normally distributed data was analyzed using a one‐way analysis of variance (ANOVA) combined with Tukey's multiple comparisons test. Data that do not exhibit a normal distribution were analyzed using the non‐parametric Kruskal–Wallis test, and data normality was tested using the Shapiro–Wilk test. Statistical analysis of group differences was carried out using SPSS for Windows 19 Inc Chicago, with a level of 33.1p < 0.001, Figure p < 0.05, Figure p < 0.001, Figure To assess whether FGF21 can attenuate the MPTP‐induced motor locomotion, and exploration abilities, we treated mice with FGF21 followed by MPTP injection Figure . Spontan3.21H NMR spectra of the striatum, cortex and midbrain are shown in Figure ), whereas the FGF21 treated mice could not be distinguished from CON mice, especially along the first principal component PC1 . Furthermore, the corresponding loading plot of PCA identified that the metabolic difference was ascribed to choline (Cho), Glycerol‐phosphocholine (PC), taurine (Tau), lactate (Lac), 3‐Hydroxybutyrate (3‐HB), and glutamine (Gln). Similarly, the PCA score plots of striatum  were significantly increased by FGF21 , while this reduction was blocked by FGF21 treatment . The abundance of the Muribaculaceae and Bacteroidaceae family was significantly higher in the PD group, while the abundance of Clostridiales, Ruminococcaceae, Lachnospiraceae was significantly higher in the CON group. Most significantly, the Muribaculaceae displayed the strongest correlation with PD disease, with the mean abundance being increased by 3 folds in MPTP groups compared to CON mice (Figure Clostridiales, Ruminococcaceae, and Lachnospiraceae was significantly enriched in the FGF21 group (LDA score >4, Figure Ruminococcaeae family from the Clostridia class was increased by 2.6 folds in the FGF21 group compared with PD mice (Figure LEfSe plots identified that 15 bacterial taxa displayed distinct relative abundances between CON and MPTP groups (LDA score >4.0, Figure e Figure A. Compare Figure B.3.41H NMR spectra from the colon were displayed in Figure To test the hypothesis that FGF21 treatment influences brain functions through the gut microbiota–gut–brain metabolic pathway, we measured metabolites in the colon of mice after FGF21 injection. Typical Firmicutes choline and valine at the phylum level. In contrast, Bacteroidetes displayed a positive correlation with inosine in the colon but a negative correlation with choline and valine (Figure unidentified_Erysupelotrichacrae and Feacecalibaculum with choline, glutamate, and aspartate. On the contrary, there was a negative association between the level of Allpprevotella, Alistipes, and Bacteroides and choline and valine (Figure unidentified_Ruminococcaceae, candidates_saccharimonas, and glutamine (p < 0.05, Figure To further explore the underlying effects of FGF21 on the microbiota–brain metabolic axis, relationships between colonic microbial abundance and colone metabolites were examined with Spearman correlation analysis Figure . The fine Figure . At the e Figure . In addi4While many studies have indicated that gut–brain crosstalk was involved in the pathology of PD, few studies have focused on therapies in the gut microbiota–gut–brain metabolic axis. In the current study, we provided the first evidence that FGF21 treatment alleviated motor and cognitive function in PD mice via modulating gut microbiota and maintaining gut–brain metabolic homeostasis, benefiting from comprehensive investigations on neuronal function, gut microbiota profiling, brain‐region specific metabolic homeostasis, and metabolic profiling in the colon.The neuroprotective effects of FGF21 have been shown to be of clinical value in treating neurodegenerative disease. Recently, we reported that peripheral administration of FGF21 ameliorates MPTP‐induced behavior deficits and neuroinflammation in mice.Previous studies have reported that administration of FGF21 and FGF21 analogs reduce bodyweight in animal models of obesity and in short‐term clinical trials.Epidemiological studies have found males with PD have significantly greater motor disorders and cognitive impairments compared with females.Given the growing body of evidence linking metabolic homeostasis to neuronal function, and the fact that FGF21 is widely recognized as a metabolic regulator,Ruminococcaeae family from the Clostridia class was increased by 2.6 folds in the FGF21 group compared with PD mice. Previous results showed that the Ruminococcaceae and Lachnospiraceae are among the main producers of butyrate, which could modulate brain metabolism and plasiticity.It has been reported that alterations of gut microbiota and neurotransmitter metabolism may be involved in the neuronal apoptosis in the striatum of PD mice.As regards to the gut metabolic homeostasis, our results also showed that FGF21 promotes restoration of colon homeostasis in PD mice. Further multivariate analysis based on colon metabolic profile also identified significant difference in the level of lactate, choline, taurine and 3‐HB in FGF21 mice compared with CON mice. 3‐HB, also known as β‐hydroxybutyrate, is the most prevalent type of ketone in the human body. Recent publications indicate that 3‐hydroxybutyrate plays a neuroprotective role in PD mice model.Faecalibaculum and unidentified _Erysipelotrichaceae was positively correlated with choline, valine, alanine, glutamate and aspartate, while the level of Allpperavotella, Alistipes, and Bacteroides was negatively correlated with choline and valine and glutamate (Figure To illustrate the connection between colonic microbial abundance and metabolic homeostasis, a correlation analysis was conducted between colonic microbial abundance and colon metabolites. At the genus level, we identified that the level of e Figure . Herein,5In summary, our findings support the hypothesis that FGF21 could modulate gut microbiota profile, restore region‐specific metabolic homeostasis and ameliorate behavior deficits in PD mice. Brain regional specific metabolic metabolism, such as choline metabolism, glutamate–glutamine cycle, and energy metabolism were involved in FGF21 protection. These findings also suggested a novel link between gut microbiota and neuronal function via the microbiota–gut–brain metabolic axis under the treatment of FGF21 in PD mice.Changwei Yang: Conceptualization, Writing‐Original draft preparation. Wuqiong Wang: Investigation, Data curation. Pengxi Deng and Xinyi Wang: Investigation. Lin Zhu: correlational analyses and visualization. Liangcai Zhao: Writing‐Reviewing and Editing. Chen Li: Methodology, Project administration. Hongchang Gao: Supervision, Funding acquisition. All the authors read and approved the final manuscript.The authors declare no competing financial interests in this study.Appendix S1Click here for additional data file.


Cntnap2−/− mice (a model of syndromic ASD). We found that both ASD models produced more nose pokes into the rough condition than the smooth condition, suggesting an increased preference for complex tactile stimulation when compared with the neurotypical groups, wherein no differences were observed. Furthermore, we found increased stereotypy and time spent in the center, suggestive of decreased anxiety, only for BTBR mice compared with the other mouse strains. Overall, SNAP is an easy to implement task to assess the degree of preference for complex tactile stimulation in ASD mouse models that can be further modified to exclude possible confounding effects of novelty or anxiety on the sensory preferences.One of the most prevalent deficits in autism spectrum disorder (ASD) are sensitivities to sensory stimuli. Despite the prevalence of sensory deficits in autism, there are few paradigms capable of easily assessing sensory behaviors in ASD-like mouse models. We addressed this need by creating the Somatosensory Nose-poke Adapted Paradigm (SNAP), which consists of an elevated platform with 6 holes in the center, half of which are lined with sandpaper and half are smooth, requiring mice to use their whiskers to sense the texture. The SNAP paradigm assesses tactile sensory preferences as well as stereotypy, anxiety, and locomotion. We used two wild-type  mouse strains, C57BL/6J (C57) inbred and CD-1 outbred mice, and two ASD mouse models, BTBR (a model of idiopathic ASD) and  Cntnap2−/− mice. Both ASD models displayed preferences for rough textures and interstrain differences in stereotypy, anxiety, and locomotion. SNAP is thus an easy to implement test to assess differences in tactile sensory preferences in ASD mouse models.Despite sensory deficits occurring in 90% of individuals with autism spectrum disorder (ASD), there are few behavioral sensory tasks available. To address this need, we developed a tactile sensory task, called the Somatosensory Nose-poke Adapted Paradigm (SNAP) that harnesses innate behavior, is easy to implement, and is not memory dependent. We assessed two neurotypical mouse strains: C57 and CD-1 mice, and two ASD mouse models: BTBR and Autism Spectrum Disorder (ASD) is a heterogenous neurodevelopmental disorder that is found in one out of 44 children . The cliA leading reason why sensory behaviors are understudied in murine models is the lack of an easy to implement and effective sensory behavioral task, as most of the available tactile tasks are complex and have significant drawbacks. For instance, in one task mice are restrained and discriminate between different textures using their whiskers, whereas in another, mice are placed in a maze that they navigate based off of tactile cues . Both taCntnap2−/− mice) and compared them to C57 inbred and CD-1 outbred neurotypical mice. Our novel task detected increased preference for complex tactile stimulation in ASD-like mice when compared with neurotypical mice. This newly developed task together with other behavioral assays may help to better phenotype ASD mouse models.In light of these limitations, we developed a novel tactile based paradigm, the Somatosensory Nose-poke Adapted Paradigm (SNAP), that does not require training, animal restraint, or surgery, is not memory-dependent, isolates whiskers for tactile assessment, and is easy to implement and cost/time effective. Since mice make and explore holes in the ground to seek shelter or food, we created a paradigm that harnesses this innate behavior and allows mice to explore premade holes  that were lined with different textures. This forced the mice to brush their whiskers across either textured or smooth holes each time they made a nose poke. The number of nose pokes made in smooth and textured holes was recorded along with the total number of nose pokes made overall, the total distance traveled, and the time spent in the center region of the chamber. We used the SNAP paradigm to assess potential differences in tactile sensory preferences in a classic idiopathic model of ASD (BTBR mice) as well as in a prominent syndromic model of ASD (Cntnap2tm1Pele/J (Cntnap2−/− mice), and BTBR T+ Itpr3tf/J (BTBR) mice were purchased from The Jackson Laboratory, whereas CD-1 mice were purchased from Charles River. These strains were chosen to assess the validity of our behavioral task in inbred (C57) and outbred  mice as well as in an idiopathic (BTBR) and a syndromic (Cntnap2−/−) ASD model. A total of 81 mice were used: 21 C57BL/6J , 18 CD-1 , 22 Cntnap2−/− , and 20 BTBR mice . Animals were tested during the light cycle, between 1 and 4 P.M. and were five to six weeks old. The mice were group-housed in a climate-controlled colony room on a 12/12 h light/dark cycle with ad libitum access to food and water. All test procedures were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by Yale University’s Institutional Animal Care and Use Committee.C57BL/6J (C57), B6.129(Cg)-Mice were habituated to the testing room for 30 min. They were then individually removed from their home cage and placed on a clear elevated platform (2 inches high) that was made out of acrylic. The platform had six ¾ inches in diameter holes in the center with a depth of 1 cm A–C. Halfpost hoc tests to clarify any significant interactions. ANOVA tests were used to assess stereotypy , distance traveled, and the duration spent in the center of the apparatus (an 8 × 8 inch region). Tukey’s HSD post hoc test was used to clarify any significant interactions for the ANOVAs. A value of p < 0.05 was considered significant for each statistical test, with figures depicting the mean ± SEM.All data were analyzed using IBM SPSS Statistics 21.0 (IBM) or GraphPad Prism 7 software. A repeated measures ANOVA was used to analyze differences in sensory preferences which was followed by Sidak Cntnap2−/− and BTBR ASD-like mice of similar ages were assessed with the SNAP paradigm. They were tested in a chamber containing an elevated platform with six ¾ inches in diameter holes in its center. Half of the holes were lined with 80 coarse-grit sandpaper to create a rough texture and half of the holes remained smooth. Abnormalities in response to sensory stimulation were assessed by quantifying the number of nose pokes made in rough and smooth holes. When assessing sensory preference, we found a main effect for texture  = 78.59, p < 0.001), a main effect of strain  = 34.98, p < 0.001) and a texture by strain interaction F = 39.01, p < .001. No main effect of sex was found  = 0.36, p = . 55) nor was there a texture by sex  = 0.06, p = 0.81), strain by sex  = 0.09, p = 0.97) or texture by strain by sex  = 0.36, p = 0.78) interactions. Male and female data were thus pooled. Sidak post hoc tests found that BTBR and Cntnap2−/− mice produced significantly more nose-pokes into rough holes than smooth holes , whereas both C57 and CD-1 mice displayed no preference  = 36.04, p < 0.001) and a texture by strain interaction  = 4.96, p = 0.003). No main effect of strain  = 1.18, p = 0.32) nor sex  = 1.27, p = 0.26) were found. There were also no texture by sex  = 0.35, p = . 56), strain by sex  = 1.18, p = 0.32), texture by strain by sex  = 1.26, p = 0.30) interactions. Sidak post hoc analyses found that BTBR and Cntnap2−/− mice both produced a higher percentage of rough nose pokes than smooth nose pokes (p < 0.0001) while no differences were found between C57 and CD-1 mice  = 34.98, p < 0.001) but no main effect of sex  = 0.36, p = 0.55), nor any strain by sex interaction  = 0.09, p = 0.97). Tukey’s HSD post hoc analyses revealed that BTBR mice produced more total nose pokes than any other group (p < 0.0001). No differences were found between Cntnap2−/−, CD-1, and C57 mice  = 10.56, p < 0.001) but no main effect of sex  = 0.02, p = 0.90) nor any strain by sex interaction  = 1.20, p = 0.32). A Tukey’s post hoc test found that BTBR mice spent significantly more time in the center of the chamber than the other groups . There were no differences in time spent in the center for C57, CD-1, and Cntnap2−/− mice  = 13.62, p < 0.001) but no main effect of sex  = 0.64, p = 0.43) nor any strain by sex interaction  = 0.011, p = 0.99). Tukey’s HSD post hoc analyses found that C57 mice traveled a significantly shorter total distance than all other groups . No other differences between strains were found  that are widely used ASD behavioral models with strong, but not identical, ASD-like phenotypes. Furthermore, BTBR mice are a model of idiopathic autism whereas Cntnap2−/− mice are a model of syndromic autism . SNAP utilizes natural mouse behaviors to efficiently assess differences in tactile sensory preferences, with an emphasis on complex sensory stimulation . Using SNAP, we investigated the behavior of ASD-like mice  and examine whether the sensory preference remains or goes away; another option is to have a 1-h-long habituation phase (either the same day or an earlier day) with the holes and the different textures before testing. The novelty effect and the design of the apparatus (holes in the center of the platform) could also trigger anxiety and affect the way animals perceive and respond to their environment as well as the amount of time spent in the middle of the arena. We found that BTBR mice spent a large amount of time in the center of the chamber, suggestive of decreased anxiety. This finding is consistent with previously reported decreased anxiety in BTBR mice using a different task , which is principally an assessment of memory. Several murine models of ASD, such as behavior . SNAP utbehavior , whereasbehavior . Conversbehavior . In compbehavior .In conclusion, alterations in sensory behaviors, particularly in tactile sensory behaviors, are arguably one of the most significant yet ambiguous phenotypes of ASD. In a condition that truly exists as a spectrum, and is in large part defined by its symptomatic variability, a surprising 90% of individuals with ASD present with sensory deficits . These d


Altered trace element status is associated with epilepsy in humans and dogs with idiopathic epilepsy (IE).Compare hair element concentrations in epileptic and healthy dogs.Sixty‐three dogs with IE  and 42 controls.Case‐control study using ICP‐MS to determine hair calcium, magnesium, phosphorus, sodium, potassium, iron, copper, manganese, zinc, selenium, chromium, lead, mercury, cadmium, arsenic, aluminum, and nickel concentration. Groups were compared using nonparametric tests. Results were controlled for diet, sex, age, and hair color using generalized linear mixed models.P = .001), higher hair copper , zinc , copper/zinc ratio , selenium , and arsenic . When comparing treated and untreated epileptic dogs with healthy dogs, the differences in phosphorus and selenium remained significant for both groups, whereas the differences in copper, zinc, and arsenic were significant only for treated dogs. Potassium bromide treatment was strongly associated with high hair arsenic (P = .000).Compared to healthy controls, dogs with IE had lower hair phosphorus (mean ± SD; IE: 286.19 ± 69.62 μg/g, healthy: 324.52 ± 58.69 μg/g; Altered trace element status could be involved in the pathophysiology of IE in dogs. Antiseizure drugs might affect trace element and arsenic metabolism. ASDantiseizure drugAlaluminumAsarsenicCacalciumCdcadmiumCrchromiumCucopperEEAHEvidensia Espoo Animal HospitalFeironGLMMgeneralized linear mixed modelHgmercuryHUAHHelsinki University Animal HospitalICP‐MSinductively coupled plasma mass spectrometryIEidiopathic epilepsyKpotassiumKBrpotassium bromideLODlimit of detectionMgmagnesiumMnmanganeseNasodiumNinickelPphosphorusPbleadSeseleniumZnzinc1Epilepsy is considered the most common neurological disorder in dogs with an estimated prevalence of 0.6%‐0.75% in the general dog population.Trace elements are essential for brain health by participating in enzymatic activities, mitochondrial functions, myelination, synaptogenesis and plasticity, and neurotransmission. Their deficiency or excess can result in neurodegeneration, inflammation, and oxidative stress, which contribute to neurological disorders and behavioral changes.To determine trace element status in epileptic people or dogs, most studies have used serum or blood analysis. However, blood‐based samples are prone to rapid fluctuation and homeostatic regulation, and therefore, hair analysis, which provides a reading of elements deposited in the cells and interstitial spaces of the hair over a 2‐3‐month period, has been considered a more stable method for estimating long‐term trace element status.Therefore, the objective of our study was to expand the current knowledge about trace element status in dogs with IE by measuring their concentrations in hair. We hypothesized that epileptic dogs have altered trace element status that might be involved in the pathogenesis of IE and that certain elements might be affected by ASDs.22.1This was a case‐control study that included epileptic and healthy companion dogs living in their home environment in Finland. Dogs with a diagnosis of IE were recruited to give hair samples. Part of the dogs were regular clients of board‐certified neurologists at 2 small animal veterinary hospitals: Evidensia Espoo Animal Hospital (EEAH) and Helsinki University Animal Hospital (HUAH). To increase sample size, dog owners were also asked, via HUAH's Facebook page and other local dog groups, to send hair samples by post from dogs that have been diagnosed with IE. If they also had healthy dogs living in the same household, those dogs were recruited as controls. The minimum criteria for IE diagnosis in our study were a history of recurrent epileptic seizures (minimum 2), the first seizure occurring between 6 months‐6 years of age, and no detectable abnormalities on interictal clinical and neurological examination, complete blood cell count, and serum biochemistry profile. In addition to these minimum criteria, part of the dogs’ diagnostic workups also included additional tests, which are presented in Table 2.2A portion of the hair samples was collected by the main author or another veterinarian in a small animal clinic setting (n = 54) and another part was collected by the dog owners in their home environment (n = 53). In the latter case, dog owners were provided with disposable A12t Dilutus 80% ethanol disinfectant wet wipes , paper envelopes, and detailed written and video instructions on how to take the sample. All samples were taken from the neck area. The hair was cleaned with A12t Dilutus 80% ethanol disinfectant  and cut close to the skin using clean stainless‐steel scissors. The distal part of the sample was discarded, leaving only about the 3 cm of hair that had been closest to the skin. If the dog's hair was shorter than 3 cm, the full length of the hair was used. The total amount of hair in 1 sample was approximately 125 mg. Based on visual classification, the samples were classified as having either dark (70%‐100% of the sample was black or brown) or light  color. This did not however always correspond to the dog's overall coat color. Hair samples were put in individual paper envelopes and, if the sample was taken by a dog owner, it was sent by post to the main author, whereafter all samples were stored at room temperature until analysis. The samples were sent to Accutrace Laboratories, Inc.  for analysis of calcium (Ca), magnesium (Mg), phosphorus (P), sodium (Na), potassium (K), iron (Fe), Cu, Mn, Zn, Se, chromium (Cr), lead (Pb), mercury (Hg), cadmium (Cd), arsenic (As), aluminum (Al), and nickel (Ni). At the laboratory, the samples were placed in plastic cups and cut into small pieces using titanium scissors. Samples were then weighed, and 40 mg of unwashed hair was digested for 10 hours in PTFE reflux tubes containing a combination of nitric and perchloric acid  in an open vessel and using a hot block/plate (Thermo Scientific model 2200) set at 188°C. After digestion, each sample was reconstituted to 2 mL using laboratory grade deionized water, and then analyzed with inductively coupled plasma mass spectrometry . A daily maintenance run was performed on the ICP‐MS instrument before analyzing the samples . To ensu2.3t‐test for age (years) and weight (kg), and a chi‐square test for sex , hair color (light/dark), diet (dry/raw/mixed), living environment , and drinking water (tap/well water). First, a preliminary screen was done to evaluate which elements were significantly associated with diagnosis by using a Mann‐Whitney U test or a Kruskal‐Wallis test followed by pairwise comparisons with the Dunn‐Bonferroni approach. Elements with significant (P < .05) differences between epileptic and healthy dogs were then chosen for further analyses. Data were not normally distributed, and some sample subgroups were small. A generalized linear mixed model (GLMM) was therefore used to determine the associations of selected hair element concentrations as target variables with diagnosis, diet, sex, age, and/or hair color as fixed effects. The selection of tested variables was based on our previous study.P < .05 in all analyses.All statistical analyses were done using SPSS for Windows . Data normality was assessed using a Shapiro‐Wilk test. The characteristics of the study and control groups were compared using an independent sample 33.1A total of 78 epileptic dogs were recruited for this study. Of these, 15 were excluded: 6 because of late seizure onset, 4 because of having had only 1 seizure, 2 because of having an unclear epilepsy diagnosis, 1 because of having hypothyroidism, 1 because of having Chiari malformation and syringomyelia, and 1 because of abnormalities in the complete blood cell count and serum biochemistry profiles. We included 1 dog with seizure onset at 6.6 years of age as this dog's diagnostic workup included a brain MRI without any detected abnormalities. The control group included 31 dogs from our previous study on hair elements in healthy dogsThe characteristics of the study and control groups are presented in Table 3.2P = .001), and this difference was significant also when looking at untreated (P = .02) and treated (P = .01) epileptic dogs separately  and dark‐haired dogs (P < .001). However, after adjusting for these cofactors, the difference in hair P was also significant because of disease status (P = .04).Mean hair element concentrations in the study and control groups are presented in Table y Figure . The GLMP < .001). Both untreated and treated epileptic dogs had higher hair Cu compared to healthy dogs, although the difference reached statistical significance only in the treated (P < .001), but not in the untreated (P = .1) dogs , and the difference was significant in the treated (P < .001), but not in the untreated (P = .74) dogs  after adjusting for hair Zn (P = .01). However, in the Zn model, disease status lost its significance after adjusting for Cu. We calculated the Cu/Zn ratio and found that it was significantly higher in epileptic compared to healthy dogs (P = .003), although only reaching statistical significance in the treated (P = .02), but not in the untreated (P = .15) dogs , and in this case, there was a clear significant difference for both untreated (P = .01) and treated (P < .001) dogs , and when looking at subgroups, this difference was only significant in the treated dogs , whereas disease status lost its significance (P = .142).Epileptic dogs had significantly higher hair As compared to healthy dogs (P = .03), although when looking at subgroups the difference was significant only in the untreated (P = .04) but not in the treated (P = .27) dogs. However, in the GLMM, disease status did not have a significant effect on hair Ni even if it was added to the model alone.Hair Ni was significantly higher in epileptic compared to healthy dogs  which might have affected these results not being statistically significant. Genetic factors did not have any significant effect on the results (data not shown).4The present study supports the role of altered trace element status in dogs with IE. Dogs with IE had lower hair P and higher hair Cu, Zn, Se, and As, as well as a higher Cu/Zn ratio, compared to healthy controls. Alterations in P and Se were significant for both treated and untreated epileptic dogs, suggesting a possible role for these elements in the pathophysiology of IE. Treatment with KBr was associated with a significantly elevated hair As.Epileptic dogs, both treated and untreated, had significantly lower hair P compared to healthy dogs. Phosphorus plays an essential role in energy metabolism as a component of adenosine triphosphate (ATP).Hair Cu was significantly higher in epileptic compared to healthy dogs, although the difference only reached statistical significance in treated, but not in untreated epileptic dogs. This is in line with the study by Vitale et al,Hair Zn was significantly higher in treated epileptic dogs compared to healthy dogs. There is higher serum Zn in uncontrolled epileptic dogs compared to healthy or untreated dogs.The Cu/Zn ratio has been considered both clinically more important than the concentration of either trace element by itself and a marker of oxidative stress and inflammation,Hair Se was significantly higher in epileptic compared to healthy dogs, and this was true for both treated and untreated subgroups. Higher serum Se is detected in treated epileptic dogs.Epileptic dogs treated with KBr had significantly higher hair As compared to those treated with other ASDs and to healthy dogs. They also had elevated hair As compared to data from previous studies, with the mean (1.67 μg/g) being as much as 84 times higher compared to the mean of healthy dogs in our previous study (0.02 μg/g).Our study had several limitations. The main limitation was the small number of dogs included in the untreated epileptic group, which might have interfered with identifying significant relationships from the data. A limitation regarding the dogs that were recruited by post is that we do not know if their diagnosis was made by a certified neurologist or a general practitioner, although they had all received a diagnosis of IE and 7 of them had MRI examination of the brain which is usually done by specialists. Furthermore, since the control dogs were recruited from 3 years of age, we cannot know for sure that they will not develop epilepsy later in life. Another limitation was that we did not specify epileptic syndromes in this study. For example, seizure strength affects body metabolism and might thus potentially also affect trace element metabolism. Finally, we did not assess the dietary intake of the studied elements in our study, which would have made it possible to clarify whether the observed alterations were related to dietary intake or other reasons, such as metabolic or other changes. Since this study does not demonstrate a causal relationship between trace elements and IE in dogs it cannot serve as the basis for altering current treatment approaches.In conclusion, this study strengthens the evidence of altered trace element status in dogs with epilepsy. Hair Se was significantly elevated also in drug‐naïve epileptic dogs, suggesting a role for Se in the pathogenesis of epilepsy in dogs. However, it remains unclear whether the observed alterations represent a contributing factor to developing epilepsy or seizures or if they are a consequence of the disease. The study also suggests that KBr might alter As metabolism in dogs.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Approved by the Animal Experiment Board in Finland .Authors declare human ethics approval was not needed for this study.Supplementary Material 1. ICP daily maintenance and monitoring form.Click here for additional data file.Supplementary Material 2. Canine epilepsy and other health information questionnaire.Click here for additional data file.Supplementary Material 3. Detailed information about study and control dogs.Click here for additional data file.