Lactobacillus probiotic and herbal blend was safe and well-tolerated in healthy volunteers and asthmatic patients.Dysbiosis of the gut microbiome may augment lung disease via the gut-lung axis. Proteobacteria may contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic inflammation. To study the effects of probiotics across the gut-lung axis, we sought to determine if a We conducted a 1-month randomized, open-label clinical trial in Cork, Ireland with healthy and asthmatic patients who took the blend twice a day. The primary endpoint was safety with exploratory endpoints including quality of life, lung function, gut microbiome ecology, and inflammatory biomarkers.Escherichia coli. Administration of the probiotic maintained overall microbial community architecture with the only significant difference being an increase in absolute abundance of the probiotic strains measured by strain-specific PCR.All subjects tolerated the blend without adverse events. Asthmatic subjects who took the blend showed significant improvements in lung function as measured by forced expiratory volume and serum short chain fatty acid levels from baseline to Week 4. The gut microbiome of asthmatic subjects differed significantly from controls, with the most prominent difference in the relative abundance of the proteobacteria Lactobacillus probiotic plus herbal blend to act on the gut-lung axis. However, due to the lack of a control group, a longer blinded, placebo-controlled study will be warranted to confirm the efficacy improvements observed in this trial.This study supports the safety and efficacy potential of a https://clinicaltrials.gov/, identifier NCT05173168. Patients with respiratory diseases often have gastrointestinal symptoms and show distinct imbalanced gut microbiomes compared to healthy individuals . InhaledLactobacillus strains have been individually clinically studied for supporting gut and lung health in improving and preventing infections  in both healthy and diseased populations  pathway activity, neutrophil recruitment, and pro-inflammatory biomarkers in preclinical in vitro and in vivo models of lung inflammation  7/9/2021 and ECM 5 (7) 10/26/2021 and ECM 3 (lll) 11/16/2021].n = 11 healthy participants and n = 11 participants diagnosed with asthma. Within these populations, active smoking status was noted . Informed consent was obtained from all subjects involved in the study. First patient first visit was November 23, 2021, and last patient last visit was January 20, 2022.This study was an open-label, exploratory pilot study to assess the safety of 4 weeks of dosing a probiotic and herbal blend  in healthy and asthmatic participants. Forty participants were screened to identify 22 eligible subjects. The study population consisted of The study protocol consisted of four onsite visits over a 6-week period. Participants were pre-screened with an online questionnaire and invited for an onsite screening visit (Visit 1) to confirm their eligibility. A urine drug test was conducted and a urine test for pregnancy was performed for individuals of childbearing potential. No changes were made to the methods after the trial started.2.1.1.Male and female participants ages 18–65 were recruited who were either in general good health at the discretion of the investigator or had asthma and were on stable medication for at least 3 months. Exclusion criteria included pregnancy, acute or chronic illness which by the investigator’s judgment precluded them from participating in the study, ≤2 hospital admissions in the past 6 months, and use of antibiotics, probiotics, immunosuppressive medications, or oral steroids (>10 mg/day) for >3 days in the previous 12 weeks. Participants were also excluded if they had made any major dietary changes or changed medications or supplements in the 30 days prior to enrollment.Participants were enrolled and conducted site visits at a single site: Atlantia Food Trials, Heron House Office, Blackpool Retail Park, in Cork, Ireland.2.1.2.9 CFU ®Lactobacillus plantarum RSB11, 7.9×109 CFU ®Lactobacillus acidophilus RSB12, 6.4 × 109 CFU ®Lactobacillus rhamnosus RSB13, 48.0 mg vasaka (Adhatoda vasica root) extract, 42.0 mg holy basil (Ocimum sanctum leaf) extract, and 30.0 mg turmeric (Curcuma longa root) extract. Total CFU count at the time of the last patient out was 2.6 × 1010 CFU/capsule. At Visit 1, participants were provided with a 30-day supply plus 2 days of overage. Study product was stored refrigerated on-site, and participants were instructed to store it refrigerated at home as well. Supplementation started after completion of Visit 2, where participants were instructed to consume one capsule twice daily with food and water at home. At Visits 3 and 4, participants returned all unused investigational product so that compliance could be calculated. Participants had to consume at least 75% (45 capsules) of their supply to be deemed compliant.All 22 participants took the supplement twice daily for 4 weeks (28 days). One capsule contains 8.25 × 102.1.3.The primary objective of this trial was to measure safety of the blend in healthy and asthmatic study participants. Safety was measured by number of participants experiencing at least one adverse event (AE); number of AEs including causality, severity, and seriousness assessments; number of participants with discontinuations due to AEs; change in systolic blood pressure, diastolic blood pressure, heart rate, and body temperature from baseline to Week 2 and Week 4; change in blood safety parameters via serum chemistry profile and hematology profile from baseline to Week 2 and Week 4.Exploratory objectives for this trial included: change in gut microbiota (16 s) sequencing from baseline to Week 4; change in lung function measured by spirometry [Forced Expired Volume in 1 s (FEV1) and Forced Vital Capacity (FVC)] from baseline to Week 4; change in oxygen levels (% pulse oxygen levels) from baseline to Week 4; change in SGRQ score from baseline to Week 4.At Baseline and Week 4, participants had blood drawn for biomarker analysis, spirometry measured, and completed the Saint George’s Respiratory Questionnaire (SGRQ). Participants supplied a stool sample at Visit 2 and Visit 4. Vitals, safety blood parameters, and adverse events/severe adverse events (AE/SAE) were monitored at each visit. Participants completed an end-of-study product questionnaire at Visit 4. There were no changes to the study protocol after the study initiated.2.1.4.This was an open label study where study personnel and the participants were aware of the product they were taking; blinding did not apply.2.1.5.The primary method of analysis was descriptive statistics. Summary statistics for continuous measures were provided for the actual measurements at each visit and change from baseline to each visit. These tables included sample size, minimum and maximum statistics, mean, median, quartiles, and standard deviations. Key variables were categorized into clinical ranges. In the summary tables, counts and percentages were used in the frequency tables.t-tests were conducted to assess within group changes from baseline to Week 4 in the healthy and asthmatic populations separately. Participants were further segmented into smokers vs. non-smokers in assessing the changes from baseline to Week 4 in serum SCFA using paired t-tests. All analyses requiring significance testing were two-sided at a 5% significance level. Results were viewed as statistically significant if the value of p was less than or equal to 0.05.Inferential statistics were run to provide information on the trends within the data. D’Agostino and Pearson test of normality was run to determine if the data was normally distributed. Post-hoc paired 2.2.Microbial DNA was extracted from human stool samples and 16S amplicon sequencing was performed using the Illumina MiSeq platform at the University of Alabama at Birmingham Microbiome Resource Core Facility under the direction of Dr. Casey Morrow. Sequencing data quality control, alignment and demultiplexing were perform by the using a custom script built using MOTHUR and QIIME 2 with amplicon sequence variants (ASVs) identified using SILVA. Processed ASV tables were imported into MicrobiomeAnalyst for further analysis and data visualization , 26. ASV2.3.DNA was re-extracted from same fecal material used in MiSeq with Zymobiomics DNA Mini-Prep kit (Zymo Research). PCR (Qiagen Fast Cycling PCR kit) was performed on samples with at least 3 ng/μL total DNA by pico green quantification . A common 16S gene-based forward primer was utilized with strain-specific reverse primers at a similar site (0.4 μM), in a 25 μL reaction with 30 ng DNA template, using SYBR Green dye (LTi) to monitor in real time. Analysis of PCR reactions was performed on 2% agarose gels. Densitometry  was calculated from the imaged PCR gels using a GS-900 densitometer and Image Lab software . Bands were gel extracted (Qiagen Qiaquick Gel Extaction Kit) and sequenced to confirm species identity. ZymoBiomics Gut Microbiome Standard (negative control) was used to demonstrate lack of nonspecific amplification.2.4.Serum SCFA levels were measured by GC–MS at Creative Proteomics . The following SCFAs were analyzed: acetic acid (C2:0), propionic acid (C3:0), butyric acid (C4:0), isobutyric acid (C4:0i), valeric acid (C5:0); isovaleric acid (C5:0i), hexanoic acid (C6:0). Free short chain fatty acids were derivatized using methyl chloroformate in 1-propanol yielding propyl esters before subsequent liquid–liquid extraction into hexane and analysis on a SLB-5 ms (30 × 0.25 × 1.0 μm) (Supelco) column and detection using GC-EI-MS in SIM-mode. Instrumental analysis was performed on an Agilent 7890 GC coupled to an Agilent 5977 MSD (Agilent Technologies). Quantification was performed against a 5-point calibration curve.3.3.1.n = 11) or were healthy (n = 11) who met study eligibility criteria  because of its well-documented impact on lung health and susceptibility to disease. Three asthmatic participants also reported having a history of allergic rhinitis. Participants who met the eligibility criteria and successfully completed the Screening Visit were enrolled in the trial. Subjects took the probiotic blend twice a day for 4 weeks, and assessments were conducted at Baseline and the end of Week 2 and Week 4.Forty total participants were screened to randomize 22 participants with asthma , FVC . In asthmatic participants, average FEV1% increased significantly (p = 0.018) and FVC trended up (p = 0.082) from Baseline to Week 4 (p = 0.099) or FVC (p = 0.387), and neither group had a significant improvement in FEV/FVC ratio . Three patients showed a decrease in average FEV1 or FVC, but the change was <10% . Of the participants that smoke, have asthma, or have asthma and smoke, 36% noted an improvement in their overall health, 43% noted less frequent coughing, 43% noted fewer instances of feeling short of breath, and 29% noted fewer cough or breathing-related sleep disturbances.Participants responded to the Saint George’s Respiratory Questionnaire (SGRQ) at Baseline, Week 2, and Week 4 to evaluate the supplement’s effect on quality of life as measured through symptoms, impact, and total score . Across 3.4.In an end of trial survey, 100% of participants who smoke would recommend the probiotic and herbal blend to friends and family. Ninety percent of asthmatic participants and 82% of healthy participants would recommend the same.3.5.Serum was analyzed for changes in short chain fatty acid (SCFA) levels between baseline and Week 4. Participants with asthma showed significant changes in serum SCFA levels. Propionic acid increased across all asthmatics , propion3.6.p = 0.644, t-test; Chao1 Richness Index, p = 0.665, t-test, p = 1, R2 = 0.007, PERMANOVA; p = 0.279, PERMDISP, To examine the effects of the probiotic on the intestinal microbiome for potentially adverse signals, we collected stool samples at baseline and Week 4 and analyzed for participants’ gut microbiome signatures. The overall alpha (intra-sample) and beta (inter-sample) diversity was not significantly altered between baseline and after 4 weeks of probiotic administration , Bacteroidetes dorei (Log2FC 24.2), and B. ovatus (Log2FC 21.7)  .3.7.L. plantarum at baseline (19%), but this signature was augmented in most samples by administration of the probiotic and herbal blend . L. acidophilus colonization was more common at baseline (37%) and was detected in most of the post-exposure samples . Similarly, L. rhamnosus was detected in most individuals before and after probiotic administration .We performed qPCR using primers validated for strain-specificity. Most samples were not colonized with L. plantarum increased significantly and L. acidophilus and L. rhamnosus trended upward from baseline to Week 4 after taking the probiotic and herbal blend  did not show a significant change. The dosing period of the trial was only 4 weeks which did not allow for enough time to capture changes in perceived symptoms as a result of taking the blend.There were slight decreases in average FEV1 and/or FVC in 3 asthmatic patients: 2 smokers and 1 non-smoker. However, the differences from baseline to Week 4 were small, with increases <10% of the lower Week 4 value. Additionally, it was noted that 10 patients had a > 10% difference between their two FEV or FVC readings which could have skewed the average value. Several asthma patients did report increases in SGRQ scores from baseline to Week 4; however, two of the participants with large increases (AN2 and AN4) both contracted lower respiratory tract infections unrelated to taking study product during the course of the trial which may have affected the results of the questionnaire. A third asthma patient with an increase in SGRQ scores has allergic rhinitis, which may have played a role, although they reported no AE or SAEs. Across all participants, there were no related respiratory AEs reported and no SAEs of any kind reported.SCFA levels were not significantly changed in the healthy population from baseline to Visit 4. However, SCFA levels were significantly upregulated in the serum of the asthmatic participants, suggesting a pre-existing deficiency that was addressed through supplementation. SCFAs are known to affect immune cell function and a variety of inflammatory pathways including TNF-α, IL-2, IL-6, and IL-10 . We obseLactobacillus administration and immune regulation of Th1/Th2 response, reducing allergic inflammation characteristic of asthma , although it is unlikely that an effect would have been observed after 2 weeks. The magnitude of change in SCFAs required to affect biomarkers is unknown, as we did not assess asthma-specific markers of inflammation in serum such as eosinophils, leukotrienes, or IL-5. Future trials will include a placebo arm and a longer administration period in order to be able to draw stronger conclusions about clinical efficacy endpoints.Information on the type or severity of asthma was not collected. If a participant had intermittent or non-seasonal asthma, their resolution of symptoms or improvement in lung function could have been reflective of the regular course of the disease. In regard to allergic asthma, three of the asthmatic participants reported having allergic rhinitis; however, none reported allergic symptoms during the trial. If a participant did have an allergic asthma attack, it could have skewed the SGRQ or lung function results, providing either a false improvement or false worsening result. However, due to the season in which the trial was conducted, a seasonal attack was less likely. The study was conducted in its entirety between November 23, 2021, and January 20, 2022, a time of the year during which seasonal allergies are rare and do not begin until mid-late January in Ireland.Lactobacillus probiotic and herbal blend was found to be safe in healthy and asthmatic subjects, and improvements in lung function were accompanied by potentially beneficial increases in SCFA in asthmatic subjects. Based on the results of this preliminary clinical study, we propose that this blend may improve lung function and inflammation by supplementing microbes in the gut to increase SCFA production in systemic circulation. Clinical relevance of these findings affects both patients with and without existing respiratory conditions who are seeking a means to support their lung health.In summary, the https://www.ncbi.nlm.nih.gov/bioproject/PRJNA922968, PRJNA922968.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found: The studies involving human participants were reviewed and approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals of University College Cork. The patients/participants provided their written informed consent to participate in this study.CL: study conception and strategy. NW, LQ, TN, ZN, XX, AG, and CL: study design. NW, TN, KW, NA, AG, CL, and BH: manuscript preparation and interpretation. NW, TD, AG, and CL: clinical trial design and execution. TN, ZN, LQ, GD, CK, AG, CL, BH, KT, ME, and IM: clinical data analysis. All authors reviewed, advised, and approved the final version submitted for publication and agree to be accountable for the work.Research reported in this article was supported by the National Heart, Lung and Blood Institute of the National Institutes of Health under award number K08 HL141652 (CL) and K08 HL151907 (KW). resB Lung Support for clinical trial use was gifted by ResBiotic Nutrition, Inc.ResBiotic Nutrition Inc. is a university startup out of the University of Alabama at Birmingham of which CL is the Founder, AG is the Chief Medical Officer, and NA and KW are Advisors.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Persea americana Mill.) varieties . The results showed that drying methods had significant effect on the avocado oil yield and its composition. Among the three drying methods the highest yield was obtained by freeze drying, and Hass showed the highest yield in the three avocado varieties with its oil owning the lowest peroxide and anisidine value. Reed oil owned the highest levels of functional micronutrients . Vacuum drying resulted in higher concentrations of tocopherols, phytosterols, phenolics, squalene, and thus rendered greater DPPH and ABTS scavenging activity. These results are important to improve the quality of Chinese avocado oil.Avocado oil has gained a lot of favor in foods and cosmetics because of its high-quality fatty acid composition and bioactive components. This study aimed to compare the effect of various predry-treatments on the yield and quality of avocado oil from three Chinese avocado ( Persea Americana Mill.) is nutritious fruit widely distributed in tropical and subtropical regions. Its cultivation and processing are gaining popularity due to the multi-faceted salutary benefits associated with its consumption. The last several years have witnessed the growing demand for its oil because of its great potential applications in human nutrition, cosmetic and pharmaceutical industries  growing in China whose pulp was subjected to three drying pretreatments . The impact of drying treatments on the extraction efficiency and quality of AO were investigated. Moreover, the physicochemical properties and the functional components of AO were systematically analyzed. The combination of drying pretreatment and avocado variety in China may produce AO with various characteristics to better fit individual preferences.Therefore, the objective of this study was to evaluate the quality of AO extracted from three avocado cultivars , phytosterols, squalene, fatty acid methyl esters (37 FAME mix), 2,2′-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Sigma-Aldrich . All other solvents were of chromatography grade and ultrapure water  was used throughout the experiments.6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), tocopherols  were harvested from Hainan, Chain in February 2020 and stored at room temperature until full maturity before manually separated into seeds, pulp and peel. The weight of the whole fruit, pits, peel and pulp of more than 50 avocado fruits were evaluated. The pulp was immersed into citric acid solution  for 15 min to avoid possible enzymatic darkening (Avocado fruits (arkening .Avocado pulp was pulped into puree in a beater with a creamy consistency and then either oven-dried or vacuum-dried or freeze-dried as detailed below. The puree was spread onto stainless steel trays (80 × 100 cm) and then either dried in an oven  with forced air circulation at 60°C or in a vacuum drier  at 40°C and 600 mbar for 24 h. The temperatures were chosen according to the results of dos Santos et al.  was measured using a Bruker Minispec (Model Mq 20) pulse nuclear magnetic resonance (NMR) spectrometer . Samples in the NMR tubes were heated at 80°C for 60 min, and then held at the corresponding measuring temperatures. After 30 min, SFC measurements were taken at 5°C intervals over the range of 0–40°C.The contents of tocopherols were determined by HPLC as previously described . Folin–CDPPH − AAOA)/ADPPH × 100%. DPPHA and AOA are the absorbance of the DPPH solution and the test solution, respectively.Antioxidant activity was assessed via DPPH assay as the method described by Sun et al.  with somp values less than 0.05 were considered statistically significant.All experiments were carried out in triplicate. Results are presented as mean ± standard deviations. Analysis of variance and multiple comparison was performed with Duncan’s tests. Reed showed the largest fruit and seed. Hass presented the lowest pulp percentage (60.59%), which agreed with the data reported by Costagli and Betti  and Reed (14.25 ± 0.99%). This data was similar to a previous study that reported the lipids content in avocado pulp to range from 10 to 15%, showing its dependence on genotype as well as other factors   . Unlike ditions) . Furtherble oils . TherefoHass variety, which was 12.10 and 8.23% higher than those of Pinkerton and Reed varieties, respectively. Therefore, freeze drying treatment could increase oil yield and Hass showed the highest extraction efficiency among the three varieties investigated in the present study. This agreed well with some earlier works . The yields of the five oils are demonstrated in er works , 24.Hass oil (FHO), freeze-dried Pinkerton oil (FPO) and freeze-dried Reed oil (FRO) at 0°C were 13.36, 7.30 and 7.25%, respectively, and decreased almost to 0% at 40°C, showing consistency to previous findings , vacuum-dried Hass oil (VHO) and FHO intertwined with each other in a small range, suggesting no obvious difference was induced from drying treatments. Therefore, Hass can be a promising base material in making cream and lotion for skin nourishment. Reed and Pinkerton, on the other hand, could be used for making sauce, marinade and salad dressing. In general, these results illustrate that the variety but not predry-treatment affects the SFC in AO. With respect to the viscosity profile , followed by palmitic (C16:0), linoleic  and palmitoleic  acids. Correspondingly, stearic (C18:0) and α-linolenic  acids were barely found, which agreed with the observation of Yanty et al.  were found to have a rather low content (< 0.3%), for which their results are not shown here. Interestingly, the content of the monounsaturated oleic acid  was lower than that in the oils from avocado harvested from other parts of the world (45.9–54.5%)   , 31. Thialmitic C:0, linol9–54.5%) . The fat9–54.5%) . However (3–10%) .Hass oil either by oven or vacuum drying pretreatment and Pinkerton as well as Reed oil exceeded the threshold of 4 mg KOH/g oil, which abided by the international standards of crude, cold-pressed oil and indicated that there was no considerable hydrolysis or oxidation of lipids during pulp processing and oil extraction. This agreed with the results of Krumreich et al.  showed the highest contents of tocopherols. The main sterols in the AOs were campesterol, Δ5-avenasterol, β-sitosterol and stigmasterol, where β-sitosterol was the most predominant one. Variety, planting location and different test methods could induce the high variation in total sterols content. The results also revealed significant variation in the total phenolic content of the AOs with respect to both predry-treatment and variety. With respect to the unsaponifiable fraction, squalene was the major component (205.58–611.27 mg/kg), whose content was higher that of total tocopherols and phenolics. FRO had higher squalene content (611.27 mg/kg) than FPO (306.15 mg/kg) and FHO (218.93 mg/kg). These AOs present much greater squalene content in comparison to that in the oils from Brazilian, Mexican and Australian avocados  was investigated in this study. It was found that both avocado varieties and drying methods had significant influence on the oil composition and oil quality. The best extraction yield of AO was obtained by the combination of freeze drying and The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.JW: conceptualization, methodology, writing – original draft, writing – review and editing, and funding acquisition. HY: formal analysis, investigation, visualization, and writing – original draft. PW, JZ, WM, YL, and JL: conceptualization, supervision, and writing – review and editing. All authors contributed to the article and approved the submitted version.This work was financially supported by Key R&D Projects of Hainan Province (ZDYF2021XDNY162) and Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 16300920210010).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Current guidelines recommend that cytotoxic chemotherapy be considered first in non-small cell lung cancer (NSCLC) patients with multiple metastases, and whole-brain radiotherapy (WBRT) is not initially recommended even if brain metastases are present. However, cytotoxic chemotherapeutic agents are less effective in brain metastases due to poor blood–brain barrier permeability. We investigated the effect of WBRT in combination with cytotoxic chemotherapy on survival in NSCLC patients who were EGFR, ALK, and PD-L1 negative, had an ECOG PS of 2, and had multiple metastases including brain metastases. From January 2005 to December 2018, histologically confirmed NSCLC patients who were EGFR, ALK, and PD-L1 negative, had an ECOG PS of 2, and had multiple metastases including brain metastases were included in this study. Patients were classified into two groups based on receiving WBRT prior to or concurrently with administration of first-line chemotherapeutic agents or receiving chemotherapy only. We compared intracranial progression-free survival (iPFS) and overall survival (OS). Of the 240 NSCLC patients with brain metastases at diagnosis and an ECOG PS of 2, 67 patients were EGFR, ALK, and PD-L1 negative with multiple metastases including brain metastases. Among those patients, 43 (64.2%) received WBRT prior to or concurrently with platinum-based chemotherapy. Patients who received WBRT prior to or concurrently with chemotherapy had better iPFS  and OS  than those who did not receive WBRT. In multivariate analyses, WBRT was significantly associated with iPFS  and OS . In NSCLC patients who are EGFR, ALK, and PD-L1 negative, have an ECOG PS of 2, and have multiple metastases including brain metastases, WBRT prior to or concurrently with chemotherapy could improve iPFS and OS. Therefore, the combination of WBRT with cytotoxic chemotherapy should be considered in these patients. Patients with NSCLC with brain metastases often have a poor prognosis, with the median survival ranging between 4 and 6 months2. Whole-brain radiotherapy (WBRT) is often considered for patients in whom surgery or stereotactic radiosurgery is not recommended. However, it is unclear whether WBRT provides clinically significant benefits for NSCLC patients with brain metastases3. Therefore, current guidelines recommend that systemic chemotherapy be considered first in NSCLC patients with multiple metastases, and WBRT is not initially recommended, even if brain metastases are present4.Non-small cell lung cancer (NSCLC) with brain metastases is the most common central nervous system malignancy, accounting for as much as 20% of every brain metastasis case5. Approximately 30–50% of NSCLC patients are PD-L1 low, and half of those patients are epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) negative7. Therefore, given the considerable proportion of patients who are recommended to be treated with cytotoxic chemotherapeutic agents alone under the current guidelines, it is necessary to assess whether WBRT can provide a clinically significant benefit for patients with brain metastases.If tumors are negative for druggable molecular markers and are PD-L1 low (< 1%), systemic chemotherapeutic agents, including cytotoxic agents and immune checkpoint inhibitors (ICI), are considered rather than targeted agents. Specifically, only cytotoxic chemotherapeutic agents are recommended in patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 2. However, unlike ICI, cytotoxic chemotherapeutic agents are less effective in brain metastases due to poor blood–brain barrier (BBB) permeabilityIn this study, we investigated the effect of WBRT in combination with cytotoxic chemotherapy on survival in NSCLC patients who were EGFR, ALK, and PD-L1 negative, had an ECOG PS of 2, and had multiple metastases including brain metastases.4. Patients who did not receive cytotoxic chemotherapy (n = 65) were excluded. Among the remaining patients, those who were positive for EGFR, ALK, or PD-L1 (≥ 1%) (n = 49) were excluded. In total, 67 NSCLC patients who were EGFR, ALK, and PD-L1 negative, had an ECOG PS of 2, and had multiple metastases including brain metastases were included in this study.A total of 240 patients with histologically confirmed NSCLC who were diagnosed between January 2005 and December 2018 at Inha University Hospital  were initially considered for this study  obtained from pretreatment imaging tests. Considering the fact that brain metastases usually have a sphere-like shape, the formula for the volume of a spheroid: 4/3 × π × (CC/2) × (LR/2) × (AP/2) was used to simplify volume assessment10. All patients received platinum-based chemotherapy as the first line chemotherapeutic agent up to six cycles unless patients underwent disease progression or death. All information was collected prospectively from electronic medical records of the Lung Cancer Cohort of Inha University Hospital11. This study was approved by the Institutional Review Board of Inha University Hospital. The need for informed consent was waived by Institutional Review Board of Inha University Hospital (IRB number: 2021-07-032).Information such as age, gender, smoking history, histology, organs of metastasis, and chemotherapeutic agents were analyzed. The stage of all patients was estimated according to the eighth edition of the TNM classification systemPatients were classified into two groups based on whether they received WBRT. WBRT was delivered prior to or concurrently with administration of first-line chemotherapeutic agents. For WBRT, three-dimensional conventional radiotherapy was done by a linear accelerator . Patients were received WBRT dose of 30 Gy in 10 fractions using 4–6 MV X-ray. Each patient was treated in the supine position while wearing a head immobilization mask to ensure that daily positioning was reproducible. The ocular lens was shielded from the direct beam at all times using collimators. Hippocampal sparing was not applied. The planning target volume of WBRT was defined as the whole-brain parenchyma plus 5 mm margins as visualized on the simulator or portal films to account for beam penumbra and day-to-day set-up variation. A dosimetrist optimized the multileaf collimator shape used in the treatment based on the CT image in the treatment planning system of Eclipse.12. The objective response rate (ORR) of intracranial lesions included the combination of complete response (CR) and partial response (PR), and the disease control rate (DCR) of intracranial lesions included CR, PR, and stable disease (SD).Chest and abdomen computed tomography (CT), 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) or bone scan, and brain magnetic resonance imaging (MRI) were conducted using standard protocols at the time of diagnosis and approximately every 12 weeks thereafter. The response of intracranial lesions to chemotherapy with or without WBRT was evaluated by brain MRI that was included in the tests for the re-evaluation. In patients received WBRT, brain MRI for the first re-evaluation of intracranial lesion was performed about 1–2 months after completion of WBRT. Responses were evaluated using RECIST  1.1 criteriaTo assess the association between WBRT and clinical variables, Pearson’s chi-square tests were conducted. Mann Whitney U-test was used to compared the number and volume of brain metastases between WBRT group and non-WBRT group. Overall survival (OS) was defined as the time from the start of chemotherapy to death. Intracranial progression-free survival (iPFS) was defined as the time from the start of chemotherapy to radiologically confirmed intracranial progression or death. The Kaplan–Meier method was adopted to estimate OS and iPFS, and survival curves were compared using log-rank tests. To assess the effect of WBRT, we performed univariate and multivariate analyses using the Cox proportional hazards model. Variables that had a value of p ≤ 0.1 in univariate analyses were included in a multivariate Cox proportional hazards model. Statistical significance was considered as two-sided p values ≤ 0.05. All statistical analyses were performed by using a statistical software package .This study was approved by the Institutional Review Board of Inha University Hospital. The need for informed consent was waived by Institutional Review Board of Inha University Hospital (IRB number: 2021-07-032). All methods were performed in accordance with relevant guidelines and regulations.13 between the two groups . Except for the number and volume of brain metastases, no statistically significant differences were found in the baseline characteristics between the two groups.In total, 67 NSCLC patients who were EGFR, ALK, and PD-L1 negative, had an ECOG PS of 2, and had multiple metastases including brain metastases received a combination of platinum-based chemotherapy as first-line treatment. Among these patients, 43 patients received WBRT prior to or concurrently with administration of first-line chemotherapeutic agents. Of the 43 patients who underwent WBRT, 7 patients (16.3%) received WBRT concurrently with chemotherapy and 36 patients (83.7%) prior to chemotherapy. Table Intracranial responses could be evaluated by brain MRI in 55 patients (82.1%). In the other patients (n = 12), follow-up of intracranial lesions by brain MRI was not performed due to their short OS. Among the 37 patients in the WBRT group, assessment of intracranial lesions indicated that 1 patient (2.7%) had CR, 22 patients (59.5%) had PR, 11 patients (29.7%) had SD, and 3 patients (8.1%) had progressive disease (PD). Whereas 1 patient (5.6%) had PR, 10 patients (55.6%) had SD, and 7 patients (38.9%) had PD among the 18 patients in the non-WBRT group. In the non-WBRT group, 6 of 7 patients with intracranial PD received WBRT after confirmation of intracranial PD. Intracranial ORR was 62.2% for the WBRT group and only 5.6% for the non-WBRT group (p < 0.001). Intracranial DCR was 91.9% for the WBRT group and 61.1% for the non-WBRT group (p = 0.005).The median iPFS and OS times were 6.8 months (95% CI 5.1–8.5 months) and 8.2 months (95% CI 6.0–10.4 months). Patients who received WBRT prior to or concurrently with chemotherapy had better iPFS  and OS  than those who did not receive WBRT  < 70 or ECOG PS ≥ 2, showed no benefit from WBRT23. Considering that not only WBRT but also chemotherapy can affect cognitive function25, it is necessary to investigate changes in cognitive function before and after treatment in the WBRT with chemotherapy group and chemotherapy alone group in the future. Third, because of the small number of patients, it was difficult to conduct a subgroup analysis such as dividing into patients received WBRT concurrently with chemotherapy and those received WBRT prior to with chemotherapy. We believe that a study with a larger number of patients is needed in the future.There are limitations of this study. First, this study was conducted at a single center and external confirmatory studies should be conducted in the future. However, all clinical information was obtained prospectively from the Inha Lung Cancer Cohort. Second, although it is important to consider the quality of life and the toxicity of treatment, these considerations could not be investigated in this studyWBRT prior to or concurrently with chemotherapy improved iPFS and OS in NSCLC patients who were EGFR, ALK, and PD-L1 negative, had an ECOG PS of 2, and had multiple metastases including brain metastases. Therefore, we suggest that the combination of WBRT with cytotoxic chemotherapy should be considered in these patients. The combination of WBRT with cytotoxic chemotherapy should be carefully individualized for each patient with consideration of each patient’s situation, such as characteristics of metastatic lesions and general health conditions.


As in the FIT clinical trials, we confirm better responses to fostamatinib when used in the second or third line. However, its use should not be excluded in patients with longer and more complicated drug histories. Given the different mechanism of action of fostamatinib compared to TPO-RAs, it would be interesting to identify predictive factors of responsiveness applicable to all patients.Immune thrombocytopenia (ITP) is immune-mediated platelet loss due to increased destruction and insufficient production. Treatment guidelines provide for first-line steroid-based therapies followed by thrombopoietin receptor agonists (TPO-RAs) and fostamatinib for chronic ITP. Fostamatinib demonstrated efficacy in phase 3 FIT trials (FIT1 and FIT2) mainly in second-line therapy resulting in the maintenance of stable platelet values. Here, we describe two patients with extremely heterogeneous characteristics that responded to fostamatinib after two and nine previous treatments. Responses were complete with stable platelet counts ≥50,000/ Differential diagnosis is important because ITP is confirmed when all other causes have been ruled out. It can arise at any age, but incidence increases with age and has a slight female preponderance.Immune thrombocytopenia (ITP) is a common autoimmune disease with an estimated incidence rate of 1.6 to 3.9 per 100,000 adult patient years and an annual prevalence of 9.5 cases per 100,000 [μL), but not on therapeutic algorithms. First-line therapies include steroids+/-intravenous immunoglobulin (IVIG), followed by thrombopoietin receptor agonists (TPO-RAs), splenectomy, rituximab, or immunosuppressants for chronic forms. Recent updates recommend the spleen tyrosine kinase (SYK) inhibitor, fostamatinib, and TPO-RAs  in patients with chronic refractory ITP [International guidelines agree on the goal of treatment (reduced bleeding risk) and the minimum platelet values to start therapy  and phase 3 studies (FIT1 (NCT02076399) and FIT2 (NCT02077192)) in which just under 50% of patients achieved a stable response as defined for these trials  during weeks 14–24 of the study) [The efficacy of fostamatinib was demonstrated in phase 2 NCT0070642 and phμL) [We report the cases of two patients with chronic ITP and treated with fostamatinib in the Hematology Division of the Garibaldi Hospital in Catania. μL) . This stμL) and was followed at another hematologic center until December 2021. Other diseases responsible for thrombocytopenia were excluded. Medical history includes hypertension well controlled with an angiotensin II inhibitor and ascending aorta ectasia (maximum diameter 40 mm).A 54-year-old Caucasian/white male came to our clinic in January 2022. He was originally found to have moderate thrombocytopenia in 2011  were positive (1 : 80), as were antinuclear ribonucleoprotein antibodies (ENA-RNP) and anti-DNA antibodies . At that time, he started steroid therapy with prednisone (PDN) 50 mg/day, which he continued for ∼4-5 months with a good hematological response , but treatment was complicated by hypertension and headache. Due to a loss of response after steroid dosage reduction, at the end of 2016 he started second-line treatment with eltrombopag at an initial dose of 25 mg/day. This low initial dose was due to platelet count of 45,000/μL at the start of therapy. The dose was progressively increased to 50–75 mg/day, and the patient obtained a durable response.In 2016, his platelet count was <30,000/In September 2021, the patient was referred to a rheumatologist regarding the appearance of Raynaud's phenomenon. They suspected scleroderma, but this was excluded. In January 2022, the rheumatologist started the patient on PDN 50 mg/day for 10 days, followed by 37.5 mg/day for 10 days and then PDN 6.75 mg/day from January 20, 2022.μL without additional dyscrasias. Looking at the patient's laboratory values from the previous months, his platelets were becoming progressively lower from 70,000–50,000/μL to 30,000/μL between October and December 2021, despite continuing eltrombopag at the maximum dosage of 75 mg/day. This was compatible with a loss of response to eltrombopag after 6 years of continuous treatment. Other laboratory tests showed no changes in hemocoagulation parameters or renal function. The patient denied noteworthy symptoms, referring exclusively to rare posttraumatic bruising. On physical examination, there were no superficial clinically relevant adenopathies noted and no hepatosplenomegaly. The severity of the hemorrhagic syndrome was quantified based on the SMOG parameters developed by the International Working Group on ITP [For concomitant worsening of ITP, the patient came to our attention on January 25, 2022. Complete blood cell counts (CBC) showed platelets at 23,000/μL), and improved at one month . At the end of the third month, there were no adverse events, blood pressure was controlled, and a CR was confirmed . To date, after a year of continuous treatment with fostamatinib at 100 mg twice a day, the patient maintains a CR with platelets always >100,000/μL.After discussing the available therapeutic alternatives (romiplostim and fostamatinib) with the patient, he preferred the oral formulation. Informed of potential side effects and after acquisition of written informed consent, therapy with fostamatinib was started on February 2, 2022, at 100 mg twice a day for 4 weeks. Platelet count was evaluated weekly, showed a good response after two weeks , and spontaneous ecchymosis spread throughout the soma (SMOG 2.0.0.). For medical history, she reported a hemithyroidectomy in 2000 due to multinodular goiter followed by hormonal replacement therapy (levothyroxine 75 mcg daily), hypertension since 2014 controlled with beta blockers, hypercholesterolemia since 2018 treated with statin therapy, renal lithiasis, and gallstones since 2016, and Parkinson's disease treated with dopaminergic agents for about three years. On admission, laboratory tests showed hepatorenal function, inflammatory indexes, and coagulative parameters within normal limits. Viral serological tests showed previous EBV and CMV infections, negative hepatitis B and C tests, and negative HIV tests. Autoimmunity testing revealed positive ANA (1 : 80) and negative antiplatelet antibodies.An 80-year-old Caucasian/white female was diagnosed with acute ITP in March 2016, with isolated severe thrombocytopenia . Dexamethasone was chosen due to the faster response and fewer side effects compared to prednisone , 8. The μL, revealing no response to dexamethasone. Treatment was initiated with intravenous immunoglobulins (IVIG) 1 g/kg/day for two consecutive days. A partial response  was seen on day two, so two additional IVIG cycles were given every 21 days. However, the IVIG response quickly faded after each cycle.The patient came to our clinic one month after ITP diagnosis. She was continuing steroid treatment . On admission, her platelet count was 23,000/μg/kg/week. After an initial response , her platelet counts gradually decreased despite maximal dosing . We decided to change the patient to second TPO-RA, eltrombopag, which was initiated on December 2016 . After one month of eltrombopag treatment, the patient's refractoriness was confirmed with PLT <10,000/μL at the maximum dosage of 75 mg daily.Therefore, at 6 months after diagnosis (October 2016), we started her on a TPO-RA, romiplostim 4 μL), and eltrombopag was restarted with no improvement. In sequence, IVIG cycles and weekly rituximab were given for four doses, followed by high-dose steroids, danazole, and then continuing with bimonthly cycles of IVIG + prednisone or dexamethasone until March 2021. In the meantime, in April 2018, we performed a bone marrow revaluation confirming the diagnosis.In January 2017, the patient underwent a splenectomy. The patient proved refractory to this procedure . Weekly intravenous infusions were given for 8 weeks. The patient obtained a minimal response . During the subsequent off-treatment observational period of 16 weeks, the patient remained severely thrombocytopenic .At our center a double-blind trial with the monoclonal antibody anti-CD38, TAK-079, was ongoing, and the patient was considered eligible. The patient was screened and enrolled. On May 18, 2021, she started TAK-079 treatment . Platelet count was evaluated weekly, showing a mild response at one month . Fostamatinib was increased to 150 mg twice a day giving a stable response after 6 weeks at the full dose . Stable response is defined here as in the fostamatinib FIT1 and FIT2 trials  [μL [μL) and no bleeding has been reported. After six months of treatment, the patient's complete response was confirmed  and the dosage of fostamatinib was reduced to 100 mg twice a day. To date, after a year of continuous treatment with fostamatinib, the dosage was reduced at 100 mg per day due to an excessive increase in platelets .After the approval of fostamatinib for patients with refractory chronic ITP, we started treatment at 100 mg twice a day for 4 weeks  [μL . At threThese two cases illustrate how seemingly unfavorable characteristics  do not necessarily impact therapeutic outcomes. Moreover, fostamatinib can stabilize platelet counts without vigorous platelet responses, reduce the risk of bleeding, and reduce the need for rescue therapy , 5. EvenCurrent American Society of Hematology guidelines briefly mention fostamatinib as it had only recently been approved when the guidelines were issued and had been studied primarily in a third-line setting . RecommeDespite a modest platelet response, early use of fostamatinib as a second- or third-line agent can be justified in patients with underlying prothrombotic conditions or with previous thrombosis for whom TPO-RAs may not be safe and anti-CD antibodies (rituximab) or splenectomy are inadvisable due to the risk of immunosuppression or surgical complications. However, the lack of randomized, comparative clinical trials does not allow effective evaluation of the best treatment sequence in ITP patients. Extrapolation from case reports and comparisons between studies do not provide the highest quality data to drive therapeutic decisions.μL are recommended [In addition, the best responses obtained from the second line onwards should stimulate clinicians to stratify patients according to risk factors adapting therapy to their clinical conditions. If thrombotic events are a concern, a procoagulant profile may be useful. Increases in prothrombin fragments 1 and 2, plasminogen activator inhibitor-1 (PAI-1), and D-dimer could increase thrombotic risk . This stommended . In thesommended .


We identified 3 subpopulations of adipocytes, including PDE4D+/PDE7B+ (in HLW and LLW), DGAT2+/SCD+ (mostly in HLW) and FABP5+/SIAH1+ cells (mostly in HLW). Moreover, we showed that fibro/adipogenic progenitors could differentiate into IMF cells and contribute to 43.35% of adipocytes in mice. In addition, RNA-seq revealed different genes involved in lipid metabolism and fatty acid elongation. Our study provides new insights into the cellular and molecular signatures of marbling formation; such knowledge may facilitate the development of new strategies to increase IMF deposition and the lipo-nutritional quality of high marbled pork.Pork is the most consumed meat in the world, and its quality is associated with human health. Intramuscular fat (IMF) deposition  is a key factor positively correlated with various quality traits and lipo-nutritional values of meat. However, the cell dynamics and transcriptional programs underlying lipid deposition in highly marbled meat are still unclear. Here, we used Laiwu pigs with high (HLW) or low (LLW) IMF contents to explore the cellular and transcriptional mechanisms underlying lipid deposition in highly-marbled pork by single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing. The HLW group had higher IMF contents but less drip loss than the LLW group. Lipidomics results revelled the changes of overall lipid classes composition  between HLW and LLW groups. SnRNA-seq revealed nine distinct cell clusters, and the HLW group had a higher percentage of adipocytes (1.40%  Many quality aspects can be used to define ‘better meat’, such as eating quality, including flavour, tenderness, and juiciness3. Marbling  deposition or accumulation) is important for estimating the potential eating quality of pork loins, and it gives meat its particular flavour, succulence and tenderness5. In humans, fat infiltration in skeletal muscle is often accompanied by ageing and metabolic or nonmetabolic diseases, such as obesity, diabetes, and myosteatosis8. However, the cell sources and regulatory mechanism of IMF deposition are still unknown. Hence, it is necessary to explore the regulatory mechanism of IMF deposition and its impact on lipid metabolism in skeletal muscle.Meat is an important source of protein for humans, and it directly impacts human health and is closely associated with a better life for humans. People are pursuing ‘less but better’ meat to tackle the sustainability challenges of meat production and consumption9 and spatial transcriptomics (ST)10. They have been used to identified several cell types that contribute to the formation of IMF, including satellite cells (SCs), fibro/adipogenic progenitors (FAPs), mesenchymal stem cells (MSCs), fibroblasts, endothelial cells (ECs), pericytes, side population cells (SPs), PW1+/Pax7− interstitial cells (PICs), and myeloid-derived cells. Due to the size of myotubes and myofibers, it is difficult to detect larger cell types in skeletal muscle. Interestingly, different to scRNA-seq and ST, single-nucleus RNA-seq (snRNA-seq)11 is another high-resolution transcriptome method that can assay both mononuclear and multinucleated cells together. However, recent studies using by snRNA-seq have mostly focused on mice13, and there is still no study on livestock and poultry using snRNA-seq.Pork is the most produced and consumed meat worldwide. In particular, China is the world’s largest pork market and survey results show that nearly 60% of Chinese consumers eat pork at least three to five times a week. However, obese and lean-type pig breeds show obvious differences in lipid deposition, and the IMF content of lean-type pig breeds is always low. Hence, how to appropriately increase IMF content while ensuring lean meat percentage and without increasing subcutaneous fat content to produce high-quality pork is a key challenge in pig production. With the continuous development of high-throughput sequencing, an increasing number of technologies have been reported to identify the cell sources and transcriptional heterogeneity in skeletal muscle, including single-cell RNA-seq (scRNA-seq)Local Chinese pig species always have a higher IMF content than lean breeds of pigs, and they can serve as good models for studying the IMF deposition mechanism. When the IMF content is larger than 8%, the pork is always considered snowflake pork (high marbled pork). Laiwu pig is an excellent local Chinese pig species; it has a high IMF content, and the average IMF content is 11.6%. However, the IMF content of Laiwu pigs varies greatly among individuals, and the differential mechanism is still unclear. Hence, it can serve as an appropriate animal model for studying the cell heterogeneity and regulatory mechanism of IMF deposition. In this study, we used Laiwu pigs with high and low IMF content to serve as a study model, and we explored the lipid dynamics, myonuclear heterogeneity, transcriptomic profiles, and differential mechanism of IMF in skeletal muscle through multiomics . Our findings will provide new insights into the cytological and molecular mechanisms of marbling formation, which may provide a foundation for the production of high-quality snowflake pork.Beef, pork and chicken constitute nearly 90% of global meat production and in the past five years, pork is still the number one meat consumed in the world (average 40.67%) despite the impact of African swine fever Fig. . In Chin14. We found the HLW group had a higher pH (1 h and 24 h after slaughter), L (1 h and 24 h after slaughter), and b (1 h and 24 h after slaughter) value  than the LLW group ; DGs (P < 0.01); monoglycerides , a type of glycerolipid; ceramides , monohexose ceramide , a type of sphingolipid; and stigmasterol esters , a type of serol lipid, were increased in the HLW group. In addition, there was no significant difference in other lipid classes , 448 diglycerides (DGs), 406 phosphatidylcholines (PCs), 324 phosphatidylethanolamines (PEs), and other lipid classes Fig. . The ortses Fig. . At the ses Fig. .Fig. 2ChWe next explored the acyl chain content in TGs, which indicated the fatty acid composition in lipids. As shown in Supplementary Fig. DMD and CAPN3), FAPs/fibroblasts (PDGFRA), muscle satellite cells (MuSCs) (PAX7), ECs (PECAM1), pericytes (RGS5), myeloid derived cells (MRC1), immune cells (CD3E), adipocytes (ADIPOQ), and SPs (JAM2)   Fig. . The tSNvs. 79.02%) and a higher percentage of MuSCs (4.59% vs. 3.56%). Importantly, the HLW group had a higher percentage of adipocytes (1.40% vs. 0.17%) than the LLW group. In addition, for other nonmyofiber nuclei, such as FAPs/fibroblasts, ECs, and SPs we also found increases in these cell types in the HLW group   Fig. .PPARG, PPARA, CEBPA, and CEBPB), mature adipocyte marker genes , and lipid metabolism-related genes  was increased, but the expression of LPL was decreased in the HLW group. Additionally, we also observed that the expression of fatty acid metabolism-related genes such as ACAA1, FABP3, FABP4, FABP5, CPT1A, ACSL4, and ELOVL1 was upregulated but HACD1 was downregulated in the HLW group  between the two groups and different cell clusters , type IIa myonuclei (MYH2), type IIx myonuclei (MYH1), type IIb myonuclei (MYH4), neuromuscular junction 15 and myotendinous junctions 16  while the percentage of type IIb myonuclei had a decreased tendency (38.56% vs. 43.75%) in the HLW group  had relatively higher expression levels, but glycolysis-related genes  had relatively lower expression levels in the HLW group  were also relatively different between the two groups , adipogenic master genes , mature adipocyte marker genes , and lipid metabolism-related genes  were all increased in the HLW group  and sphingolipid metabolism-related genes  was upregulated in the HLW group , DGAT2+/SCD+ (mostly in HLW) and FABP5+/SIAH1+ subclusters (mostly in HLW), in which different subclusters play different roles in IMF deposition and we speculated the difference in IMF deposition mainly relies on DGAT2+/SCD+ subclusters and FABP5+/SIAH1+ subclusters. meansFunctional enrichment analyses using KEGG pathways revealed a significant enrichment of the PPAR signalling pathway, insulin signalling pathway, and AMPK signalling pathway in adipocytes Fig. ; the cal18. Hence, we first applied subcluster analysis on FAPs/fibroblasts. In total, we identified 3 subclusters in FAPs/fibroblasts according to the expression of specific markers, including FAPs (PDGFRA), fibroblasts (COL1A1), and PDE4D+/PDE7B+ subclusters (PDE4D and PDE7B) , adipogenic master genes (PPARG and PPARA), mature adipocyte marker genes , lipid metabolism-related genes , and FAP master genes  between the two groups  Fig. . We foun7B) Fig. . At the +/PDE7B+ subclusters  and adipogenic master genes (PPARG)  Fig. . MoreoveP-value < 0.05 and |log2(fold change) | > 1, we found a total of 1034 DEGs, of which 534 were increased and 500 genes were reduced  and lipid metabolism-related genes  was significantly increased but the expression level of LPL was significantly decreased in the HLW group  while others  are mononuclear, it has been technically challenging to explore potential functional distinctions and transcriptional heterogeneity in skeletal muscle. SnRNA-seq is a new high-resolution transcriptome method and is able to assay both mononuclear and multinucleated cells together11. In this study, we revealed the different cell type populations, gene expression profiles, and potential mechanisms of snowflake pork by using snRNA-seq combined with lipidome and RNA-seq.Marbling is one of the important meat quality values, and it affects the sensory properties of pork, including juiciness, tenderness, and flavour19 and Luchuan pigs had higher TGs and DGs compared to Duroc pigs20. Also, abundant TGs, phosphatidylinositols and phosphatidylserines were found to be upregulated in LDM of Xidu black pigs with higher IMF contents21. Besides, our previous study has found dietary conjugated linoleic acid supplement could induce lipid dynamics in muscle of Heigai pigs22. In our Laiwu pig model, we found that the overall composition of lipid classes had significant heterogeneity. A high IMF content is always accompanied by an increase in glycerolipids and sphingolipids. Previous studies have clarified that adipose tissue has much more glycerolipids than skeletal muscle23. In humans with insulin resistance independent of obesity, the sphingolipid metabolism gene pathway was upregulated in muscle24. Sphingolipids regulate a plethora of cell biological processes including growth regulation, cell migration, adhesion, apoptosis, senescence and inflammatory responses. They are related to diseases such as cancers, metabolic disorders, immune function, cardiovascular disorders and skin integrity at the tissue and organismal levels25. In muscle, intramuscular lipid droplets primarily comprise TGs26. In our study, we found higher contents of TGs and Cers in the HLW group. In addition, snRNA-seq and RNA-seq results showed that the expression of fatty acid and sphingolipid metabolism-related genes was significantly changed. Another local Chinese pig species, Luchuan pigs, had upregulated contents of TGs and Cers compared with Duroc pigs20. Similarly, in GLY-injected mice, the contents of total TGs and Cers were also significantly increased in muscle9. However, previous studies found that obesity induces upregulated levels of Cers and that increased Cers correlate with the pathogenesis of metabolic diseases27. Except for TGs and Cers, we also found that the content of StEs was also higher in the HLW group. It has been reported that stigmasterol could protect against high-fat Western diet-induced nonalcoholic fatty liver disease28. Besides, we found there is a reduced tendency in glycerophospholipids, including PEs, PSs, and PCs. Previous study has found adipose tissue had a lower concentration of glycerophospholipids, including PGs, PSs, PEs, and PCs23. However, there is still a blank in the association between glycerophospholipids and meat quality. These results indicated that lipids may serve as new biomarkers for marbling formation and that IMF deposition is always accompanied by the increased contents of TGs and Cers but decreased contents of glycerophospholipids in muscle. However, the physiological functions of these lipids need to be further explored.Fat deposition and lipid composition in skeletal muscle are affected by a variety of factors. Previous studies have found that breed is an important factor to affect lipid composition in skeletal muscle of pigs, which is manifested in Laiwu pigs contained more TGs than Yorkshire pigs30 and ST31. However, it is challenging to assay larger cell types by scRNA-seq and ST due to the size of myotubes and myofibers. Another new high-resolution transcriptome method, snRNA-seq, can profile the transcriptomes of mature muscle cells such as myotubes or myofibers at the whole cell or nucleus level16. Here, we used snRNA-seq to analyse the dynamics of cell populations in muscle of pigs. Although we have reduced the problem of cell debris affecting the number of captured nuclei through CellBender, we found there is still a difference in collected nuclei between two groups. We identified 9 cell populations in the skeletal muscle of Laiwu pigs, including myofibers, FAPs/fibroblasts, MuSCs, ECs, pericytes, myeloid-derived cells, immune cells, adipocytes, and SPs. HLW groups had the lower myofiber nuclei and the higher adipocytes nuclei, MuSCs nuclei and FAPs nuclei. FAPs are the main source of IMF32. and previous study found some MuSCs can differentiate into adipogenic cells33. Besides, adipocytes are easier to digest than muscle, hence, we think the difference in collected nuclei may result from the more resistant to enzyme digestion of LLW groups. Likewise, Dos Santos et al. and Petrany et al. identified many cell types, including SCs, FAPs, tenocytes, endothelial cells, smooth muscle cells, pericytes, B/T cells immune cells, and adipocytes in adult mouse skeletal muscle by snRNA-seq34. In a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, Chemello et al. identified three clusters of nuclei related to skeletal muscle regeneration including cluster MuSC, cluster Myog, and cluster RegMyon11.Skeletal muscle is the largest tissue in the human body and comprises many cell types. The synergistic action of these single cells together determines the biological function of skeletal muscle. Recently, an increasing number of studies have focused on exploring the cell sources and heterogeneity of skeletal muscle in human and animal models by using high throughput single-cell and subcellular resolution transcriptomics such as scRNA-seq36. Four major myonuclei types with distinct myosin compositions have been identified in the skeletal muscle of mice, including slow myofibers with type I myonuclei (Myh7), fast myofibers with type IIa myonuclei (Myh2), type IIx myonuclei (Myh1), and type IIb myonuclei (Myh4)34. In our study, we identified four muscle fiber type in pigs, including type I myonuclei (MYH7) and type IIa myonuclei (MYH2), type IIx myonuclei (MYH1), and type IIb myonuclei (MYH4). We found that the HLW group had a relatively higher content of type IIa myonuclei but a relatively lower content of type IIb myonuclei. Previous study has found MyHC IIa expression was negatively related to muscle fiber diameter but MyHC IIb expression was postively related to muscle fiber diameter37. Besides, the diameter of slow myofiber was always smaller than the diameter of fast myofiber which could explain why the HLW group had a smaller muscle fiber diameter. A previous study found that slow-twitch oxidative type I myonuclei have higher activities in myoglobin and mitochondrial oxidative metabolic enzymes while fast-twitch glycolytic type II myonuclei have higher activities in glycogen and glycolytic enzymes36. This is consistent with our findings that the expression of oxidation-related genes was relatively upregulated while glycolysis-related genes were relatively downregulated in the HLW group. Overall, our results indicate that the skeletal muscle of pigs has great heterogeneity and that muscle fiber types are associated with muscle fiber diameter and IMF deposition.Skeletal muscle comprises slow and fast myofibers which are classified by MYH (myosin heavy chain) expression+/SCD+ subclusters and FABP5+/SIAH1+ subclusters in adipocytes clusters. DGAT1 and DGAT2 are important for fatty acid esterification into TGs38 and are always accompanied by increasing TG levels39. A previous study found that during adipogenesis in white preadipocytes, the expression of DGAT2, SCD, and FABP5 was significantly higher in Cluster 1, as detected by snRNA-seq and scRNA-seq40. These results indicated that DGAT2+/SCD+ subclusters and FABP5+/SIAH1+ subclusters may be the key differentiated cell populations between the HLW and LLW groups.Marbling is one of the important indexes to evaluate the pork quality and is associated with flavour, tenderness, and juiciness. Interestingly, we found that compared with the LLW group, the HLW group had a higher proportion of DGAT2+ or CD140α+ progenitor cells is the main source of intramuscular adipocytes during muscle regeneration in mice, and they are defined as FAPs32. However, there is very little research on FAPs in pigs. In our study, we identified three subclusters in FAPs/fibroblasts, including FAPs, fibroblasts, and PDE4D+/PDE7B+ subclusters. Interestingly, we found that PDE4D+/PDE7B+ subclusters are similar to one of the subclusters in adipocytes and trajectory analysis indicated that in the HLW group, more FAPs differentiated into PDE4D+/PDE7B+ subclusters compared to LLW group. In addition, we confirmed that FAPs have the capacity to differentiate into adipocytes in vitro. Hence, we speculated that PDE4D+/PDE7B+ subclusters might be the main cell populations in the process of preadipocyte differentiation in differential IMF deposition. However, further studies need to verify our hypothesis. Unexpectedly, although FAPs contribute to IMF formation, we found that less than 50% of adipocytes are derived from FAPs, suggesting that other types of cells may also be involved in marbling formation. However, the proportion of FAPs during the regeneration phase and the effects of injection time and sustainability of tamoxifen on cell proportion of differentiated IMF cells should be considered in further studies.Uezumi et al. verified that a subpopulation of PDGFRα43. Overall, our results indicate that the heterogeneity of adipocytes and adipogenic cell populations such as FAPs, ECs, SPs, and pericytes is the potential regulatory mechanism of IMF deposition. However, future work should reveal the functional significance of different cell populations during IMF deposition.Notably, we observed that the expression of preadipocyte-related genes was higher in EC, SP, and pericyte populations of the HLW group and adipocytes mainly interacted with SPs, FAPs/fibroblasts, ECs, and pericytes in turn. Existing evidence supports that some ECs, SPs, and pericytes have the potential for adipogenic differentiation in vitro44. In our study, we found that the expression of FABP4, FABP5, LIPE, ELOVL1, and CPT1A was positively associated with IMF deposition, but LPL expression was negatively associated with IMF deposition. Lipoprotein lipase (LPL) is an enzyme that catalyzes the hydrolysis of protein-linked TGs and plays a key role in TG metabolism. In Italian Large White pigs, LIPE and LPL gene expression has been reported to be related to IMF deposition45. However, further studies are required to explore the special regulatory mechanism of LPL in IMF deposition. In addition, we discovered that FABP5 was highly expressed in one of the adipocytes subclusters. FABP5, together with FABP4, is the main FA-binding protein in adipose tissue46. Previous studies demonstrated that septin 11 could bind to FABP5 to regulate adipocyte metabolism in 3T3-L1 cells47. ELOVL1 has been suggested to be involved in the elongation of SFAs and MUFAs and to regulate lipid metabolism48. For signalling pathways, we found that DEGs were enriched in glycerophospholipid metabolism, the cGMP-PKG signalling pathway, the calcium signalling pathway, and the Notch signalling pathway. Consistent with our findings, Xu et al. found that the MAPK signalling pathway and calcium signalling pathway were significantly associated with IMF deposition44. Notch signalling plays an important role in regulating metabolic disorders and participates in beige adipocyte formation, adipocyte dedifferentiation and function50. We also found that the significant changes in genes in the glycerolipid pathway resulted in the alteration of TG contents. Likewise, in our previous GLY-induced IMF deposition mice, the glycerolipid pathway and glycerophospholipid pathway were also changed9. However, the functions of these signalling pathways in IMF deposition need to be further explored.Marbling formation is a complex process, and the regulatory mechanism of IMF deposition involves genes related to lipid metabolism signaling pathways+/PDE7B+, DGAT2+/SCD+ and FABP5+/SIAH1+ cells) of adipocytes and found that FAPs could differentiate into IMF cells and contribute to less than 50% of adipocytes. Our data also described different genes involved in lipid metabolism and fatty acid elongation. Our findings could provide a foundation for developing new strategies to increase IMF deposition and the lipo-nutritional quality of snowflake pork.In conclusion, we provide detailed insights into the cellular and molecular signatures of marbling formation by using a Laiwu pig model with a high IMF content. We identified 3 subpopulations (PDE4D22. and the IMF content are shown as the weights of fat or protein per 100 g of freeze-dried LDM (g per 100 g). Based on the determination of IMF content in Laiwu pigs, we chose six Laiwu pigs with higher IMF content and six Laiwu pigs with lower IMF content then divided them into two groups: the HLW group (high IMF content) and the LLW group (low IMF content). Approximately 200 g of LDM was rapidly collected, frozen in liquid nitrogen immediately, and subsequently stored at −80 °C for lipidomics determination. The two most representative samples from each group were selected for later snRNA-seq and four samples from each group were selected for RNA-seq analyses. A block of LDM was removed from the body and placed into a fixative solution for haematoxylin-eosin (H&E) staining.All the procedures involving mice were approved by Zhejiang University Animal Care and Use Committee. Laiwu pigs from the original Laiwu pig breeding farm and Laiwu Pig Breeding Co., LTD were used for the study. Laiwu pigs were fed full-price compound feed with the same nutritional content according to their age and provided with a 12 h light/dark cycle and free access to water and food. Laiwu pigs were slaughtered at the same slaughterhouse when they weighed approximately 100 kg and were humanely sacrificed after fasting for 12–16 h. Samples of longissimus dorsi muscle (LDM) were obtained from the 3rd to 11th rib on the right side of the carcass for meat quality measurement. Carcass traits and meat quality were measured as previous describrdPdgfrα-CreER (stock no. 018280) and ROSAmT/mG (stock no. 007676) mice were purchased from Jackson Laboratory , and Pdgfrαcre-ER/ROSAmTmG mice were generated. PCR genotyping was performed using protocols described by the supplier. All mice were anaesthetized and injected with 50 μL 50% GLY into the tibialis anterior (TA) muscle after being fed standard rodent chow food for 14 days as previously described9. At 14 days postinjection (DPI), the TA samples were carefully sampled, placed into a mould, buried in OCT, frozen immediately in liquid nitrogen, and stored at −80 °C for subsequent sectioning. The freezing slicer setting program was temperature −20 °C and section thickness 8 μm.For the lineage tracing experiment, C57BL6/J mice were housed under standard laboratory conditions with a 12 h light/dark cycle and free access to water and standard rodent chow. LDM from Laiwu pigs were fixed in 4% paraformaldehyde for 24 h at room temperature. Then, the tissues were removed from the fixative solution for paraffin embedding and microtome sectioning. Paraffin sections were dewaxed in water: the sections were placed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 80% alcohol for 5 min, and 70% alcohol for 5 min and washed in tap water. The sections were stained with haematoxylin for 15 min and eosin solution for 3–5 min and after anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, dimethyl I for 5 min, and xylene II for 5 min; sealed with neutral gum; and mounted for microscopic examination.The dewaxed sections were immersed in sodium citrate and placed in a microwave oven for 15 min for antigen retrieval. After the sections cooled to room temperature, they were fixed in 4% paraformaldehyde for 10 min, permeated with 0.5% Triton-X100 for 10 min and blocked with blocking buffer  for 1 h. Then, the sections were incubated with Perilipin  and MF20  primary antibodies diluted in blocking buffer overnight. After washing with PBS three times for 10 min, the samples were incubated with secondary antibodies and DAPI for 6 min at room temperature. After sealing with glycerol, fluorescent images were captured as single-channel greyscale images using a Leica DM 6000B fluorescence microscope with a ×20 objective (NA 0.70).g for 15 min at 10 °C) to extract lipids. The upper organic layer was dried in a vacuum centrifuge. Prior to analysis, lipid extracts were resuspended in 200 μL of isopropanol acetonitrile 9:1 (v/v). In brief, lipids were separated on a Waters ACQUITY PREMIER CSH C18 Column . MS detection was performed using a Thermo Scientific™ Q Exactive mass spectrometer mass spectrometer, equipped with an ESI ion source. Data were acquired in both positive and negative ion modes, respectively. Data-dependent acquisition methods were used for MS/MS analyses of lipidome. Lipidsearch 4.0 software was used for peak detection and annotation of lipids or internal standards.200 μL cold water and 20 μL lipid internal standard mixture were added to 60 mg LDM tissue, then the samples were homogenized on a MP homogenizer at 4 °C . Following this, 800 μL cold methyl tert-butyl ether and 240 μL methanol were added to the samples and vortexed for 30 s, sonicated at 4 °C for 20 min and stand for 30 min, then centrifuged , filtered through a 20 μm cell strainer , and counted.Longissimus dorsi muscle nuclei were isolated with Nuclei EZ Lysis buffer  supplemented with protease inhibitor  and RNase inhibitor . Samples were cut into 2 mm pieces and homogenized using a Dounce homogenizer  in 2 ml of ice-cold Nuclei EZ Lysis buffer, and they were incubated on ice for 5 min with an additional 2 ml of lysis buffer. The homogenate was filtered through a 40 mm cell strainer  and then centrifuged at 500 × Single-cell suspensions were loaded onto 10X Genomics Chromium to capture 5000 single cells according to the manufacturer’s instructions for the 10X Genomics Chromium Single-Cell 3’ kit (V3). The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system  by LC-Bio Technology Co., Ltd.  at a minimum depth of 20,000 reads per cell.https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome, version 6.1.2), and snRNA-seq data were aligned to Ensmebl Sus scrofa genome v96 as the reference genome. A total of 56108 single cells captured from 2 HLW pigs and 2 LLW pigs were processed using 10X Genomics Chromium Single Cell 3’ Solution. The Cell Ranger output was loaded into Seurat (version 4.1.0) for dimensional reduction, clustering, and analysis of snRNA-seq data. Overall, 47273 cells passed the quality control threshold: the number of genes expressed per cell >500 and the percent of mitochondrial DNA-derived gene expression <25%. The DoubletFinder package (version 2.0.3) was used to remove doublets. We also performed batch correction using Harmony for data integration between samples.The sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software (version 2.20). Sample demultiplexing, barcode processing and single-cell 3’ gene counting were performed using the Cell Ranger pipeline ≥0.26.To visualize the data, we further reduced the dimensionality of all 47273 cells using Seurat and used t-SNE to project the cells into 2D space. The steps include the following: 1. The LogNormalize method of the “Normalization” function of Seurat software was used to calculate the expression value of genes. 2. Principal component analysis (PCA) was performed using the normalized expression value. Among the PCs, the top 10 PCs were used to perform clustering and t-SNE analysis. 3. To find clusters, the weighted shared nearest neighbour (SNN) graph-based clustering method was selected. Marker genes for each cluster were identified with the Wilcoxon rank-sum test with default parameters via the FindAllMarkers function in Seurat. This selects marker genes that are expressed in more than 10% of the cells in a cluster, http://cole-trapnell-lab.github.io/monocle-release/docs, version 2.22.0). For cell communication analysis, we used CellPhoneDB  to further speculate on the underlying cellular interaction mechanisms.To model differentiation trajectories, we performed trajectory analysis using Monocle 2  for 60 min at 37 °C. The cells were then filtered from debris through 70 μm filters, centrifuged at 600 × g for 6 min and cultured in growth media  on collagen-coated dishes. The medium was refreshed every two days. For magnetic cell sorting, primary FAPs were isolated from LDM in 3-day-old piglets. Muscles were minced and digested in 0.2% type I collagenase at 37 °C for 60 min. The cells were then filtered from debris through sterilized gauze and centrifuged at 500 × g for 6 min. After the supernatant was removed, red blood cell lysate  was added to split at 4 °C for 5 min. Then, the cells were incubated at room temperature for 15 min with a Dead Cell Removal Kit  and centrifuged at 400 × g for 5 min. CD140a antibody  was added, followed by incubation at 4 °C for 5 min and centrifugation at 400 × g for 4 min. Then, 20 μL antibiotin microbeads  were added, followed by incubation at 4 °C for 15 min and centrifugation at 400 × g for 4 min. After the magnetic column, the cells on the adsorption column were PDGFRα+ cells. For adipogenic differentiation and 3D cluture, the cells were induced to differentiate when they reached 90% confluence by induction medium  and 500 μM 3-isobutylmethylxanthine (IBMX)) for 3 days upon confluence and then differentiated in differentiation medium  for 2 days until the adipocytes matured.Primary IMF cells were isolated from hind limb skeletal muscle in −ΔΔCT method was used to analyse the relative changes in gene expression normalized against 18 S ribosomal RNA as an internal control.Total RNA was extracted from FAPs using TRIzol Reagent , and the purity and concentration of total RNA were measured by a spectrophotometer  at 260 and 280 nm. Random primers and a ReverAid First Strand cDNA Synthesis Kit (Thermo Fisher) were used to reverse RNA samples. qPCR was performed with an Applied Biosystems StepOnePlus Real-Time PCR System using Hieff qPCR SYBR® Green Master Mix (Yeasen) and gene-specific primers  content greater than 1% and reads with Qphred ≤20 accounting for more than 50% of the entire read length. Clean reads were mapped to the pig reference genome (Sscrofa11.1) and gene model annotation file (Ensembl Genes 103) used by HISAT2 (version 2.0.5). The number of reads mapped to each gene was calculated using featureCounts (1.5.0-p3), and the FPKM of each gene was calculated according to its length. Gene expression values of the transcripts were computed by StringTie (version 1.3.3b). Differentially expressed genes (DEGs) were identified between the two groups by using the package DESeq2 (version 1.12.4). Genes with a P < 0.05 were considered significantly different.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify which DEGs were significantly enriched in GO terms or metabolic pathways. GO terms and KEGG pathway analyses using the hypergeometric test were performed to identify significantly enriched metabolic pathways or signal transduction pathways enriched in DEGs. GO terms and KEGG pathways with false discovery rates t tests. Differences were considered significant at P < 0.05.Experimental data are presented as the means±standard errors of the means (SEM) from at least three independent experiments. GraphPad (Prism 8.3.0) and R software (version 4.1.3) were used for data analyses and data visualization. Comparisons were made by unpaired two-tailed Student’s Supplementary Information


Women with PD had higher NMF scores especially in depression/anxiety, sleep/fatigue and dysautonomia domains. Our study reported the presence of a gender-related pattern in the frequency of NMS and NMF in PD patients, with female gender associated with a higher risk of developing NMF, highlighting the need for personalized treatment strategies when addressing NMF.Non-motor symptoms (NMS) and Non-motor fluctuations (NMF) in Parkinson’s Disease (PD) are common, involving several domains and affecting quality of life. Aim of the study is to estimate the burden of NMF in PD patients and to evaluate the possible gender effect. PD patients fulfilling the MDS-PD diagnostic criteria attending the “Parkinson’s Disease and Movement Disorders Centre” of the University of Catania were evaluated using the Non-Motor Fluctuations Assessment (NoMoFA) Questionnaire. NoMoFA items were also grouped into the following domains: cognitive, mood, sleep/fatigue, dysautonomia, hallucination/perception and miscellaneous domains were identified. One-hundred and twenty-one patients with PD  were evaluated. All PD patients reported at least one NMS, whereas 87 (71.9%) also reported NMF. “Feel sluggish or had low energy levels” (47.2%) along with “Feel excessively sleepy during the day” (40.0%) were the most common NMF reported in the whole sample. The majority of PD patients reported the presence of NMF during the OFF state . At multivariate analysis, NMF were positively associated with the female gender (adjusted OR 3.13; 95%CI 1.21–8.11 The online version contains supplementary material available at 10.1007/s00702-023-02679-6. Despite the classic clinical motor features peculiar to the diagnosis  and gender differences in PD patients assessed by the NoMoFA questionnaire.Patients with a diagnosis of PD according to the Movement Disorder Society (MDS)-PD diagnostic criteria . Then it is asked if the NMS is worse during ON or during OFF time or if there is no difference. The “total NMF subscore” is the sum of the subscores “NMF ON” and “NMF OFF”, while the total (static) NMS score is the sum of all “NMS no difference” scores. The total NoMoFA score is calculated using the formula: Total NoMoFA = Total NMF (ON + OFF) + Total (static) NMS.Additionally, considering all the 27 items, the following domains have been identified: attention (items 1–4), memory (item 15), language (item 5), depression/anxiety (items 6–10), impulsiveness (items 12–14 and 16), apathy (item 17), hallucination/perception (items 11 and 23), sleep/fatigue (items 18–19), dysautonomia  and miscellaneous .Data were analyzed using STATA 16 software packages . Quantitative variables were described using mean and standard deviation. Qualitative variables were expressed as number and percentage. Differences between means and proportions were evaluated and t-test and the chi-square test, respectively. Normal distribution was assessed using the Shapiro–Wilk test and, in case of a not normal distribution, appropriate non-parametric tests were performed. When needed, variables have been dichotomized according to the median value of the pooled distribution.p-value = 0.10. The model was manually constructed using the likelihood ratio test (LRT) to compare the log-likelihood of the model with and without a specific variable. The significance level was set at 0.05 and the 95% confidence intervals (CI) were calculated. Correction for multiple comparisons according to false discovery rate (FDR) was also applied. Finally, in order to compare our results to the original validation study of the NoMoFA scale, in agreement with the original study  and demographic and clinical characteristics. Multivariate logistic regression analysis was performed adjusting for age and gender considered as a priori confounders and for each variable associated with the outcome at univariate analysis with a threshold of p-value 0.03), motor fluctuations  and use of benzodiazepines  , while a significant negative association was found between age and presence of motor complications  (Supplementary table 1).One-hundred and twenty-one patients with PD were consecutively enrolled . Out of the 121 patients according to items 32–39 of the UPDRS part IV, 67 (55.4%) had motor complications, of whom 57 (47.1%) motor fluctuations, and 42 (34.7%) dyskinesias. There were no differences between men and women with PD in demographic and clinical characteristics, except for the presence of motor complications , while the mean total (static) NMS score was significantly higher among men  as shown in Table All 121 enrolled PD patients (100%) reported at least one NMS, while 87 reported NMF leading to a prevalence of 71.9% (95% CI 63.1–79.8). The mean total NoMoFA score was 20.5 ± 10.3 without significant differences between men and women  and “Difficulty finding the right words when speaking” 71.9%) Table , without.9% Tableversus 41 men [38.8%]; p-value 0.005), Hoehn–Yahr stage and the presence of motor complications (motor fluctuations and/or dyskinesia), as shown in Table A total of 87 PD patients (71.9%) reported at least one non-motor symptom in OFF or ON state and then were considered as non-motor fluctuating (NMF +). There were no demographic or clinical differences between NMF + and NMF-, except for gender distribution  along with “Feel excessively sleepy during the day” (40.0%) were the most common NMF reported. Frequency of each single item according to the presence of fluctuations is reported in Table % along wp-value 0.005) at univariate analysis. A close association was found in multivariate analysis adjusting by age, Hoehn–Yahr stage and the presence of motor complications  as shown in Table p-value 0.01) as shown in Table p-value 0.05), while no association was found between NMF and UPDRS-ME, disease duration and LEDD , as previously demonstrated . Gender-related difference in NMF was also found in several domains and in particular, depression/anxiety, apathy, sleep/fatigue, dysautonomia and miscellaneous were the non-motor fluctuating domains more frequently reported among women with PD. Furthermore, women also presented a significantly higher NMF total score. In our sample presence of NMF was strongly associated with the female gender with an Adjusted OR of 3.13. The higher frequency of NMF among women is in agreement with a previous observation reported by Picillo and coll   rather than in ON state (35.5%). It should be noted that the questionnaire was administered in OFF condition, thus we cannot exclude that ON or OFF states may also influence the perception of the NMS. In other words, we cannot exclude that NMS in ON state may be underestimated if recorded during the OFF phase or that may be underreported, as for dyskinesia, because perceived as a general greater well-being with respect to the OFF phase.In our sample motor complication (presence of motor fluctuations and/or dyskinesia) were significantly positively associated with the NMF. As previously demonstrated  is not always well recognized by PD patients and misinterpretation is possible, also considering that NMS such as cognition and cardiovascular dysfunction might influence each other Supplementary file2 (DOCX 19 kb)Supplementary file3 (DOCX 20 kb)Below is the link to the electronic supplementary material.