Determining effects of physiological stress and vaccination type on performance and immune responses in cattle can be a difficult task due to the many factors that contribute to the stress response. Nonetheless, defining alterations caused by the stress response after vaccination upon feedlot arrival is vital in improving vaccination practices in the feedlot industry. Utilizing a stress model with stressors induced by current industry practices to mimic a “high-risk” cattle situation, we found that vaccination with a killed or modified-live virus vaccine induces altered immune responses in cattle that underwent industry stressors when compared to a control group. In addition, performance variables were altered in stressed cattle and those that received modified-live vaccination. We propose the feedlot industry consider these results when implementing a vaccine protocol in “high risk”, or chronically stressed, cattle during arrival processing.n = 12/treatment) including non-stress control (C) with KV (CKV), C with MLV (CMLV), stress model implementation (S) with KV (SKV), and S with MLV (SMLV). The C calves were weaned at the origin ranch on Day −37 and transported 472 km to the study site on Day −21 to allow acclimation. The S calves were weaned on Day −3, transported 460 km to a research facility on Day −2, held overnight, and transported 164 km to the study site on Day −1 to mimic the beef cattle marketing process. Vaccines were administered on Day 0 and KV was revaccinated on Day 14. The animal was the experimental unit and dependent variables were analyzed using PROC MIXED with repeated measures (day). A stress model effect (p = 0.01) existed for DMI from Day 0 to Day 7 with greater DMI for C (6.19 vs. 4.64 kg/day) when compared to S. The MLV groups had reduced (p = 0.05) ADG from Day 0 to Day 56, compared to KV. There was a vaccine type × day (p < 0.01) interaction with increased (p ≤ 0.01) PI3V- and IBRV-specific antibody titers for KV on Day 21; conversely, MLV had increased (p ≤ 0.01) BVDV titers on Days 14, 28, 35, 42, 49, and 56. Increased (p ≤ 0.05) BRSV titers were observed in a stress model × day (p < 0.01) interaction for S on Days 21, 28, 36, and 42; however, C exceeded S in BVDV-specific antibody concentration on Days 21, 28, and 49. A day effect (p < 0.01) was observed for serum haptoglobin with the greatest (p < 0.01) concentration on Day 3. Serum cortisol concentration was greater (p ≤ 0.04) for C vs. S on Days −2, 0, 1, 3, and 5. Total leukocytes were decreased for C vs. S on Days 0, 1, 3, 5, 7, 14, and 21 (p ≤ 0.02). A reduction (p ≤ 0.04) in total leukocytes was observed for MLV on Days 5, 7, and 14 vs. KV. Neutrophils and neutrophil:lymphocyte were markedly increased (p ≤ 0.01) for S on Day −2, whereas neutrophils were decreased (p ≤ 0.01) on Days 1 and 21 for S. Monocytes were decreased on Days 1, 5 and 7 for MLV (p ≤ 0.04) and Days −2 to 14 for S (p ≤ 0.03). Eosinophils were reduced (p = 0.007) for S vs. C on Day −2, yet a distinct rebound response (p = 0.03) was noted for S on Day 0. The results indicate that S and MLV vaccination more profoundly induced immunomodulation in beef calves.The study objective was to determine if a combined weaning and transportation stress model affected performance, antibody, endocrine, or hematological responses to modified-live virus (MLV) or killed virus (KV) respiratory vaccination in beef steers. In total, 48 calves (Day 0 BW = 226 ± 6.2 kg) from a single origin were used in a 2 × 2 factorial to evaluate main effects of stress model, vaccine type, and their interaction, resulting in four treatments ( Cattle may be vaccinated with an array of antigens and two primary antigen types  with the goal of preventing infectious diseases; however, host-dependent vaccine failure may be attributable to parasitic infection, poor health or nutrition status, and/or physiological stress . Acute sThis study was conducted from November 2016 to March 2017 at 2 research sites. The initial Day −2 collection sample reported herein for the stress model was conducted at the USDA-ARS Livestock Issues Research Unit near Lubbock, TX, USA. Subsequent samples were collected at the West Texas A&M University (WTAMU) Research Feedlot in Canyon, TX, USA. Animal procedures and experimental protocols were independently approved by the animal care and use committees at USDA-ARS (protocol # 1603S) and WTAMU (protocol # 2-11-16), specific to procedures conducted at each location.Forty-eight clinically heathy, previously unvaccinated Angus × Hereford beef steers (Day 0 average BW = 226 ± 6.2 kg) that were seronegative on Day −37 to infectious bovine rhinotracheitis (IBRV), bovine viral diarrhea virus (BVDV), parainfluenza-3 virus (PI3V), and bovine respiratory syncytial virus (BRSV) antibodies were acquired for use in the study from a single ranch located in central New Mexico. In addition, on Day −37, non-stress control (C) steers were weaned and placed in an isolated pen at the origin ranch where they were allowed ad libitum access to hay and water until Day −21 when they were transported in a sanitized trailer to the WTAMU Research Feedlot . The C steers were classified as such because they were weaned, relocated, and acclimated prior to vaccination with handling for sample collection being the primary stressor imposed. Upon arrival to WTAMU, C steers were allotted to their assigned pens where they resided for the remainder of the study. Steers allocated to the stress model treatment (S) were not separated from their dams until Day −3 and were transported on Day −2 approximately 450 km in a sanitized trailer to the USDA-ARS Bovine Immunology Research and Development (BIRD) facility . Upon arrival to BIRD, S calves were allowed to rest overnight in covered soil-surfaced pens with ad libitum access to hay and water. The calves were transported approximately 177 km on Day −1 in a sanitized trailer to the WTAMU Research Feedlot. Upon arrival to the WTAMU Research Feedlot, the S calves were held in soil-surfaced pens separate from the C calves. Simultaneous weaning, transportation, handling, and relocation imposed on S calves over a period of three days was intended to mimic the stressors encountered during a typical marketing process that occurs for high-risk beef calves prior to feedlot arrival and associated chronic stress ,13,14.Feed bunks were observed each morning (07:00 h) by trained WTAMU Research Feedlot personnel and daily feed offering was adjusted to promote slick bunk management protocol. At the end of each weekly period, feed refusals were weighed, converted to DM basis using the previous day ration DM determined from drying samples in a forced-air oven at 60 °C for 24 h and weekly DMI was adjusted accordingly on a per pen basis. Calves were fed a common starter diet formulated to meet or exceed nutrient requirements of growing beef cattle  upon arrOn Day 0, steers assigned to C and S were randomly and equally allocated to receive either a pentavalent MLV respiratory vaccine , Duluth, GA, USA) or pentavalent KV respiratory vaccine . Both vaccines were administered s.c. at dose of 2 mL in the neck, per label recommendation, at 06:00 h on Day 0. On Day 14, per label directions, the KV treatment was administered a secondary booster with KV vaccine in the same manner as the primary dose. The MLV treatment was not subsequently revaccinated. The handling and administration of vaccines used in this study closely followed Beef Quality Assurance guidelines. This resulted in four experimental treatments arranged as a 2 × 2 factorial design consisting of: (1) non-stress control (C) with KV (CKV); (2) C with MLV (CMLV); (3) stress model implementation (S) with KV (SKV); and (4) S with MLV (SMLV). Both vaccines consisted of IBRV, BVDV Type I and II, PI3V, and BRSV antigens, but with replicating (MLV) or non-replicating (KV) properties. Treatment randomization was achieved by drawing labeled cards from a hat with treatment designation of KV or MLV resulting in 12 animals assigned to each of the four treatments.After vaccine treatments were administered, steers were allotted to one of eight pens. Each treatment was housed in three consecutive pens, resulting in three pens per treatment with four animals per pen. Between each third pen , a pen was left empty to keep treatment groups separated. This biosecurity consideration was implemented to reduce the risk of transmission of viruses from animals that might have shed vaccine-origin virus between stress model or vaccine type.A 4-mL anti-coagulated blood sample was collected on Day −2 at the WTAMU Research Feedlot for C calves and at the USDA-ARS BIRD facility for S calves via jugular venipuncture into tubes containing 7.2 mg EDTA . The timing of Day −2 blood collection began at 18:00 h and was coordinated between the two research sites. Subsequent anti-coagulated blood samples were collected at the WTAMU Research Feedlot on Days 0, 1, 3, 5, 7, 14, and 21 and analyzed using an automated hematology analyzer  at the WTAMU Animal Health Laboratory to determine complete blood count.g for 20 min at 20 °C. After centrifugation, serum was harvested and stored in triplicate aliquots at −20 °C until subsequent analyses were performed.Jugular blood was also collected into 9-mL evacuated blood tubes with no additive  to harvest serum used to determine haptoglobin and cortisol concentration. All blood samples were placed in an insulated cooler immediately after sample collection without ice to achieve storage temperature of approximately 20 °C during cold ambient temperature and immediately transported to the WTAMU Animal Health Laboratory. The samples were allowed to clot ≥30 min before centrifugation at 1250× One aliquot of the frozen sera was packaged on ice and transported to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) located in Amarillo, TX, US to determine IBRV-, BVDV-, PI3V-, and BRSV-specific antibody titers from Days 0, 7, 14, 21, 28, 35, 42, 49, and 56. Antibody titers were determined using the virus neutralization assay previously described by . Sera frp-value was ≤0.05 and statistical tendency was considered if the resulting p-value was ≤0.10 and ≥0.06. Differences of least squares means were determined using the PDIFF option in SAS with α = 0.05.This 2 × 2 factorial experiment used animal within pen as the experimental unit for average daily gain, body weight, virus-specific antibody response, serum haptoglobin and cortisol, and complete blood count analyses, and pen as the experimental unit for DMI calculations in a completely randomized design . Growth p < 0.01) for DMI, from Day 0 to Day 7 such that C calves had greater (p < 0.01) DMI than S calves. This was probably a consequence of the acclimation period and bunk training C calves were afforded beginning on Day −21, but also differences in stressors imposed upon initiation of the study. A main effect of stress (p < 0.02) on DMI was detected from Day 0 to Day 14 with C exceeding S calves and a tendency (p < 0.08) for C to have greater DMI was also noted from Day 0 to Day 28. A stress model × vaccine type interaction (p < 0.04) existed for ADG from Day 21 to 28 and a tendency (p < 0.10) from Day 0 to Day 28 with CKV having greater (p ≤ 0.05) ADG than CMLV. Additionally, vaccine type affected ADG from Day 28 to Day 35 (p < 0.04), from Day 35 to Day 42 (p < 0.01), from Day 42 to Day 49 (p < 0.02), and from Day 0 to Day 56 (p < 0.05), illustrating consistent increase in ADG for calves administered KV compared to those administered MLV.Only one steer (SMLV treatment) was identified as clinically ill and treated once with an antimicrobial during the study period and this animal died on Day 10; the cause of death was attributed to peritonitis and not determined to be influenced by the experimental treatment. Dry matter intake and ADG are represented in p < 0.01) for PI3V-specific neutralizing antibodies detected in serum such that KV calves had greater (p ≤ 0.01) PI3V antibody titer than MLV from Day 21 to Day 49 (p < 0.01) existed for IBRV-specific neutralizing antibodies such that KV calves exhibited greater (p ≤ 0.02) IBRV antibody titer than MLV from Day 21 to Day 42.All calves used in this study were seronegative to IBRV, BVDV, PI3V, and BRSV on Day −37. We did not detect a stress model × vaccine type interaction or a stress model × vaccine type × day interaction for any of the virus-specific antibody titers evaluated in the current study. There was a vaccine type × day interaction (o Day 49 . Likewisp < 0.01) observed for BVDV-specific antibody ; in contrast with PI3V and IBRV observations, the MLV calves had increased (p ≤ 0.01) BVDV antibody titer on Days 14, 28, 35, 42, 49, and 56. Temporal trends for all vaccine antigens were indicative of a typical vaccine response with antibody becoming detectable between Days 7 and 14, and peak antibody titers observed between Days 28 and 42 after initial vaccine administration. The authors of [There was a vaccine type × day interaction (thors of  reportedthors of  tested sp < 0.01) was noted for serum BRSV-specific antibody titer (p ≤ 0.01) BRSV titer than S on Days 7 and 14, S exceeded (p ≤ 0.05) the C group for BRSV titer on Days 21, 28, 36, and 42 after primary vaccination. This subsequent increase in BRSV titer for the S model may be indicative of greater antibody response in S calves that are experiencing greater immunosuppression. The authors of [p < 0.01) existed for BVDV-specific antibody titer. In contrast to BRSV antibody results, BVDV antibody titer for C exceeded (p ≤ 0.03) S on Days 21, 28, and 49. These results contradict results reported in [A stress model × day interaction  were observed for serum Hp activity, there was a day effect (p < 0.01) resulting in overall serum Hp concentration being increased on Days 3, 5, and 7, and decreased on Days −2, 0, and 14  for serum cortisol such that C had increased (p ≤ 0.04) serum cortisol concentration on Days −2, 0, 1, 3, and 5  observed for total leukocytes (WBC), neutrophils (NEU), lymphocytes (LYM), neutrophil to lymphocyte ratio (N:L), monocytes (MONO), eosinophils (EOS), percent neutrophils (PERNEU), percent lymphocytes (PERLYM), percent monocytes (PERMONO), percent eosinophils (PEREOS), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red cell distribution width (RDW), and platelets (PLT). Increased (P ≤ 0.02) WBC concentrations were observed for C relative to S steers beginning on Day 0 and continued through Day 21 (p ≤ 0.04) for C calves from Day −2 to Day 14 and PERMONO were increased (p ≤ 0.01) for C from Day −2 to Day 7. The greatest (p < 0.01) total WBC was observed in C steers on Day 1 (11.22 K/µL) followed by a decrease until Day 5 (8.45 K/µL). This corresponds with results reported in [p < 0.01) NEU on Day 1 (3.36 K/µL) compared to S (2.00 K/µL) followed by a decrease in NEU for C on Day 3 (1.64 K/µL). However, S calves had previously exhibited a marked increase (p < 0.01) in NEU on Day −2 (3.83 K/µL) followed by a decrease on Day 0 (1.63 K/µL). This response is indicative of a rebound effect on Day 0 for S following the marked increase in NEU on Day −2. Similar results were observed for PERNEU and N:L with the greatest (p < 0.01) N:L for S steers on Day −2 (p < 0.01) on Day 1 for both C and S steers. No further differences were reported for PERNEU or N:L until Day 21 when increased (p ≤ 0.03) PERNEU and N:L were observed for C calves. Decreased (p ≤ 0.04) LYM were observed on Days -2, 1, 5, and 7 for S calves, whereas PERLYM was decreased (p < 0.01) on Day −2 for S calves with increased PERLYM on Days 1 (p < 0.04) and 5 (p < 0.05). A marked increase (p ≤ 0.02) was observed for EOS (p ≤ 0.01) on Day −2. Subsequently, decreased (p ≤ 0.03) EOS in S was observed on Day 1, 3, 5, and 7 and decreased (p ≤ 0.01) PEREOS existed on Day 3 and 7. Additionally, there was >50% decrease (p ≤ 0.01) in EOS and PEREOS from Day 0 to Day 7 for both C and S calves. The eosinopenia after stress model implementation corresponds with results in water restricted and transported steers [p ≤ 0.04) LYM and EOS compared to C calves which may be the result of greater redistribution of LYM to lymphatic tissue, apoptosis and decreased proliferation of LYM [p ≤ 0.01) HGB and HCT concentrations were observed on Day −2 for S. In addition, S calves exhibited decreased (p ≤ 0.05) HGB on Days 7, 14, and 21 and HCT (p < 0.01) on Day 14. Profiles for MCV and MCH were comparable such that S calves consistently exhibited numerically less of each variable than C calves. Decreased (p ≤ 0.03) concentrations were observed for MCHC in S calves on Days -2 and 1 and PLT were decreased (p < 0.02) for S calves on Day 3. Stress model influenced RDW with decreased (p ≤ 0.01) RDW for S calves from Day −2 to Day 14 of the sampling period with similar (p > 0.72) values on Day 21. These RBC component responses were most likely indicative of dehydration and disrupted feed intake experienced by S calves from weaning on Day −3 and transportation on Days -2 and -1. Similar results have been reported such that HCT and HGB [There was a stress model × day interaction  on WBC, LYM, MONO, PERMONO, EOS, PEREOS, HGB, HCT, MCV, MCH, RDW, and PLT. Decreased (p ≤ 0.01) WBC were observed from Day 5 to Day 14 for MLV that was influenced by the decreased (p ≤ 0.04) LYM observed from Day 3 to Day 14 for MLV calves (p ≤ 0.05) MONO and PERMONO on Day 1, and MONO was decreased (p ≤ 0.02) on Days 5 and 7, whereas, MLV had greater (p < 0.01) PERMONO than KV calves on Day 14. Monocytopenia observed for MLV could be from viral replication that existed for MLV, but not KV calves until PERMONO was similar (p > 0.80) on Day 21 for KV and MLV. Responses detected for EOS and PEREOS were similar such that MLV had reduced (p ≤ 0.02) EOS and PEREOS concentrations on Day 5. The decreases in EOS and PEREOS in MLV calves may indicate EOS recruitment to infected sites from viral replication initiated by the replicating antigens in the MLV vaccine. The authors of [There was a vaccine type × day interaction  for RBC (p ≤ 0.05) RBC on Days 0–7 and 21. Additionally, SMLV and SKV calves had increased (p ≤ 0.05) RBC compared to CKV calves on Days −2, 0, and 1. These results could indicate more dehydration among treatments with elevated RBC because, as previously mentioned, RBC are known to increase during water deprivation in cattle [A stress model × vaccine type × day interaction existed ( for RBC . A diffen cattle ,38. In aOur data reveal some alterations in antibody response, APP, endocrine, and hematological variables from implementation of stress model and vaccination with KV or MLV respiratory vaccine. Antigen-specific antibody titer results suggest that KV had a greater antibody response against PI3V and IBRV antigens, whereas MLV had a greater antibody response against BVDV. Stress model implementation yielded inconsistent results to titer concentrations such that BRSV-specific antibody response was delayed in S and BVDV-specific antibody response was greater in C. The stress model may have caused greater stress-induced immunosuppression because increased NEU were observed following stress model implementation and LYM and EOS were reduced, despite overall greater cortisol detected for C. Dry matter intake and ADG was reduced for S and MLV groups and this was probably influenced by differences in inflammation and the associated effect on appetite and growth. Further research investigating the interaction between stress-induced immunosuppression and concurrent use of different vaccine types is needed to better define the safe and efficient use of respiratory vaccines in cattle.


Alzheimer’s Disease (AD) is closely connected to aberrant lipid metabolism. However, how early AD-like pathology synchronously influences brain and plasma lipidome in AD mice remains unclear. The study of dynamic change of lipidome in early-stage AD mice could be of great interest for the discovery of lipid biomarkers for diagnosis and monitoring of early-stage AD. For the purpose, an untargeted lipidomic strategy was developed for the characterization of lipids  perturbation occurring in plasma and brain in early-stage AD mice  by ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. Significant changes were detected in the levels of several lipid species including lysophospholipids, phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and Ceramides (Cers), as well as other related lipid compounds such as fatty acids (FAs), diacylglycerols (DGs) and triacylglycerols (TGs) in AD mice. In this sense, disorders of lipid metabolism appear to involve in multiple factors including overactivation of phospholipases and diacylglycerol lipases, decreased anabolism of lysophospholipids in plasma and PEs in plasma and brain, and imbalances in the levels of PCs, FAs and glycerides at different ages. We revealed the changing panels of potential lipid biomarkers with the development of early AD. The study raises the possibility of developing lipid biomarkers for diagnosis of early-stage AD. Familial AD (an earlier onset < 65 years of age), is induced by mutations in the autosomal dominant genes encoding amyloid precursor protein (APP) and presenilins 1 and 2 (PS1 and PS2), which leads to the subsequent accumulation of β-amyloid (Aβ) in the brain6. The pathological accumulation and deposition of extracellular Aβ and aberrantly phosphorylated tau filaments in brain neurons finally lead to senile plaques and neurofibrillary tangles8. Double-transgenic mouse models co-expressing mutations in the human APP and PS1 genes are widely applied to explore underling pathophysiological mechanism of Aβ in early-stage AD. These mice generated Aβ plaques in the brain at 5–6 months old, although production of Aβ had been found as early as 3 months old9. APP/PS1 mice displayed progressive age-related memory impairments, which appeared as early as 7 months old11. In behavioristics testing, the mice showed significant deficiencies in measuring spatial memory and reference learning11. Thus, 2, 3 and 7-month-old APP/PS1 transgenic mice were selected to perform current study. Although APP/PS1 mice do not model all facets of human AD, they enable longitudinal investigations in laboratory but impossible in a clinical environment.AD is a progressive and chronic fatal neurodegenerative disease and the most common form of dementia, accounting for 60–80% of all dementia patients, and the number of patients with AD will reach 152 million until 205013. Lipidome alteration reflects the situation of AD at certain time. Lipidomics technique has become a considerable potential as a discovery platform for identifying novel diagnostic lipid biomarkers for AD and other neurodegenerative diseases15. Lipidomics studies have revealed a series of biochemical disturbances in APP/PS1 mice18. Previous studies focused on untargeted and targeted metabolomics and lipidomics for AD21. Mass spectrometry has been considered as an important and powerful tool to conduct lipidomic studies for AD, including LC-QTOF/MS, LC-Orbitrap, LC–MS/MS, nano-ESI–MS, GC–MS and HPLC-ELSD29.Lipidomics investigations of AD demonstrated that the pathology unbalanced lipid homeostasis and effected signal transduction17. On the other hand, the majority of previous studies were ill-considered to have the effect of early AD and gender on the lipidome16. In light of the deficiencies described above, the aim of our study was originally proposed to longitudinally study the dynamic change of lipid species in the APP/PS1 transgenic AD model over its lifespan from 2, 3 to 7 months old, and to monitor lipid disturbances close to the initial pathological impairment in brain of female AD mice as well as within the blood circulation, as depicted in Figure Although the abovementioned studies provided some valuable methodology, they suffered from limitations in many metabolomics investigations. For instance, a common limitation was a single time point, providing only the narrowest of windows through which to view and obtain reliable biological informationHPLC-grade isopropanol and acetonitrile were bought from Merck . LC–MS-grade ammonium formate and ammonium acetate were bought from Sigma-Aldrich . HPLC-grade ethyl acetate n-butanol and n-heptane were bought from Macklin . Water (Ω > 18.0) was purified by Milli-pore Milli-Q Ultra-pure water purification system . All lipid standards in current study were bought from Avanti Polar Lipids lnc .Specific-pathogen-free APP/PS1 mice, aged 2, 3 and 7 months and age-matched wild-type (WT) C57/BL6 mice were purchased from Animal Center of Guangdong Province  and kept to acclimate for one week in a light and dark alternate, each cycle for 12 h with regulated temperature (23 ± 2 °C) and humidity (40–70%). APP/PS1 and WT mice were housed under the identical conditions and fed the same rodent maintenance diet. Animal studies were carried out in accordance with the Guiding Principles for Care and Use of Laboratory Animals, and the protocol was approved by the Ethics Committee of Experimental Animals of Jinan University (Certification number SCXK-2013-0002).31. Mice not expressing the transgene (APP/PS1) were used as WT controls. For this study, female AD model groups and age-matched WT controls (n = 8–13) were used.DNA of each mouse was extracted and determined according to the previously reported methodBefore the experiment, all mice were fasted for 12 h to avoid diet disturbance and fed with water. Mouse plasma samples were acquired by centrifuging blood, approximately 0.3 mL  from the oculi chorioideae vein and stored at -80 °C. Mice were immediately sacrificed after withdrawing blood, and then mouse brain samples were instantly taken out from cranial cavity on ice, cleaned with normal saline, dehydrated on tissue surface, weighed and collected into individual new tubes and finally homogenized (50 Hz/s) in twofold volume of ice-cold normal saline for 1.0 min. The tissue homogenates were stored at –80 °C until further use.32. Briefly, each sample (20 μL) (including plasma and brain homogenate samples) was successively treated with 200 μL of solution A  and solution B , and next layered by adding 200 μL of ammonium acetate (50 mM) and further handled by ultrasound  for 10 min. The upper organic layer was obtained by centrifugation  for 10 min 4 °C and transferred to a new tube and dried under vacuum. Finally, all dried samples were reconstituted with acetonitrile/ isopropanol/water , treated by ultrasound  for 5 min and the clear solutions were removed for LC–MS analysis. Equal aliquots of each sample were pooled and mixed to make quality control (QC) samples for further analysis.Lipids were extracted by an optimized butanol-methanol method as described in the previous study to obtain the upper clear organic layer32. The chromatographic separation was achieved on an ACQUITY UPLC CSH C18 column  from Waters Technologies . The column temperature was set at 55 °C. A binary mobile phase system consisted of (A) acetonitrile containing 10 mM ammonium acetate and (B) acetonitrile/isopropanol  containing 10 mM ammonium acetate. Mobile Phase constitution was changed as followed: initiation at 40% B followed by a linear gradient to 43% B over 2 min, turned to 45% B at 2.1 min, turned to 48% B at 12 min, reached 60% B at 12.1 min, rose to 100% B at 18 min, returned to 40% B at 18.1 min and lasted 1.9 min. The flow rate was set at 0.4 mL·min−1. The injection volume was 20 μL. The analysis of mass spectrometry was operated using an electrospray ionization source (ESI) in the negative and positive mode. The optimized parameters were set under the negative mode as follows: capillary voltage, 3.5 kV; charging voltage, 2.0 kV; end plate offset, 500 V; dry temperature, 250 °C; nebulizer pressure, 2.0 bar; dry gas, 6 L·min−1, funnel 1 RF 350 Vpp; funn el 2 RF 600 Vpp; multipole RF 700 Vpp; collision RF 1,200 Vpp; transfer time, 80 μs and deflection delta 70 V; Under the positive ion mode, capillary voltage, 4.5 kV; charging voltage, 2.0 kV; end plate offset, 500 V; dry temperature, 250 °C; nebulizer pressure, 2.0 bar; dry gas, 7 L·min−1, funnel 1 RF 350 Vpp; funnel 2 RF 600 Vpp; multipole RF 1,200 Vpp; collision RF 1,000 Vpp; transfer time, 70 μs and deflection delta 70 V. Nitrogen was used as the cone and desolvation gas, and argon was used as the collision gas. Collision energies were set at 7 eV for low energy under negative and positive modes and linear energy change from 25 to 75 eV with molecule weight changing for high energy, respectively under negative and positive modes for Auto-MS/MS mode  in the respective time slice to acquire their fragment ions. Accurate mass was maintained by introduction of a micro-syringe bump containing sodium formate at a concentration of 10 mM in 50% aqueous isopropanol and a rate of 20 μl·min−1 before the chromatographic separation. Data was acquired and processed by otofControl 5.0 and Data analysis 5.0 software, respectively.A Thermo Scientific Dionex UltiMate 3,000-UHPLC system  coupled with a QTOF/MS spectrometer with an electrospray ion source (ESI)  was utilized in present study. UHPLC-QTOF/MS analysis was performed based on our previous investigation32. Briefly, the cluster peaks  of sodium formate introduced before the chromatographic separation were used to recalibrate the m/z of all peaks in raw data. Next, the corrected peak areas were utilized for further analysis. Progensis QI v2.0  and EZinfo 3.0.3  were employed to visualize, process, and interpret multi-dimension LC–MS data. The corrected LC–MS data were imported to Progensis QI for peak picking and alignment. Data was further normalized using total ion intensity. Analysis was explored by using multivariate statistical methods, including principal components analysis (PCA) and orthogonal partial least square and partial least square-discriminant analysis (OPLS/PLS-DA) and further confirmed using analysis of variance (ANOVA) in EZinfo. The peak height intensity of the differential lipid metabolites was compared by t-test using statistical software to confirm the biomarker alterations between WT and AD groups, and only the intensity with P value threshold set at 0.05, fold change at 1.3, and variable coefficient (CV) at 30 could be considered as the potential biomarkers. Difference between AD and WT groups is considered statistically significant when P value is less than 0.05.Data processing and statistical analysis were performed according to the previous referencehttps://www.metaboanalyst.ca/faces/ModuleView.xhtml) based on database source including KEGG and HMDB, was used to help relevant pathway analysis and visualization of potential biomarkers with its topological characteristics in this work.For visualization of pathway analysis, Metaboanalyst  , PEs and lysophosphatidylethanolamines (lysoPEs)  or phosphoserine moiety and choline (m/z 168.04 for ESI−), ethanolamine (m/z 196.07 for ESI-) and serine derived lipids35. Furthermore, fragmentation of glycerides (di- and triacylglycerol) occurs through the release of fatty acids generating different types of ions, which show characteristic m/z values according to the fatty acid attached to the glycerol backbone36. On the other hand, cholesteryl esters can be easily identified by means of an abundant fragment ion at m/z 369.40 induced upon collision induced dissociation37. Free fatty acids were also confirmed with characteristic fragments described in the literature40.Potential lipid biomarkers were identified through matching the experimental retention time (± 0.1 min), accurate mass and tandem mass spectra (MS/MS), combing with those available in metabolomic databases (HMDB and LIPIDMAPS) with a mass accuracy of ± 5 ppm for parent ions and ± 20 ppm for fragment ions, and, then confirmed with marketable standard compounds when available. Different classes of lipids were confirmed based on characteristic fragmentation patterns previously described, including PCs and lysophosphatidylcholines (lysoPCs)  plot was generated with data from plasma and brain using the different MS techniques (UHPLC-ESI(−/ +)/Qtof/MS) coupled with chromatographic separation  analyzed for plasma Fig.  and braip) < 0.05, VIP ≥ 1.0 at any age. The detailed results were presented in Tables In lipid extraction, recoveries of lipid standards were checked to confirm the efficiency of lipid extraction for latter unbiased lipid analysis and results showed that recoveries of different lipid species were more than 50% (Table 1 (PLA1) and phospholipase A2 (PLA2) and are either hydrolyzed by lysophospholipase or used to regenerate phospholipids in the remodeling pathway41. The most notable finding was an abnormal metabolism of phospholipids regarding changed levels of lysoPCs, lysoPEs and PCs, as well as other related compounds such as DGs, TGs, cholesteryl esters (CEs) and other catabolites in plasma and brain of APP/PS1 mice in current study. Abnormal metabolism of membrane phospholipids caused by over-activated phospolipases activity, mainly PLA2 , has been traditionally considered as a key pathological hallmark in the development of Alzheimer’s disease34. Recent studies performed in serum samples from human AD patients and mice also pointed to the implication of an altered fatty acid composition of phospholipids containing decreasing polyunsaturated fatty acids (PUFA) and increasing saturated species43. During the remodeling of phospholipids, lysophospholipids are transiently generated by the action of PLA2, but they are rapidly acylated with acyl-CoA in the diacylation reacylation cycle for the maintenance of the normal and essential neural membrane composition41. However, it is discovered that lysophospholipids are not only intermediates in metabolism of glycerides, but they serve as mediators in multiple neuronal pathways involved in neurobiology of AD 42. In this sense, we detected overall decrease of different lysophospholipids such as LysoPC(18:2), LysoPC(16:0) and LysoPE(24:6), LysoPE(22:0) , LysoPE(20:5) in plasma from AD mice at different ages  in accordance with previous investigations in serum of transgenic mice of AD and human AD44. Furthermore, substantial decrease of different PEs (including PE(20:3_18:0), PE(22:6_22:1), PE(22:6_22:0), PE(P-16:0_20:4), PE(P-18:1_20:4), PE(P-18:0_18:1)) in plasma and brain of the APP/PS1 model  in this study were observed, which had the similar situation with previous studies in different brain region of AD mouse46. The reason of such decrease in phospholipids may be the stimulation of various isoforms of PLA2 including PE-PLA2, cytosolic PLA2(cPLA2) and secretory PLA2(sPLA2), which leads to the increased activities of PLA2 isoforms in AD brain47. The decrease of PCs (including PC(P-16:0_18:3), PC(14:0_16:0) and PC(18:0_24:1)) in plasma and brain of the APP/PS1 mice with the ages of 2, 3 and 7 months were consistent with the decrease in brain of 6-month-old AD transgenic mice in these published literatures49. At the same time, there were some increased PCs (including PC(16:0_22:6), PC(18:1_22:5) and PC(18:0_22:4)) in AD. The increase of PEs (including PE(20:4_16:0), PE(18:1_18:2), PE(18:1_20:4), PE(22:5_22:6) and PE(15:0_22:4)) and PCs denotes a profound membrane remodeling in the APP/PS1 mice34.These lipid biomarkers in brain and plasma of early-AD mice provided the opportunity to elucidate possible biochemical pathways affected in the APP/PS1 mice, thus allowing a better understanding about the disease pathology. Numerous lipid compounds were significantly disturbed in plasma and brain from APP/PS1 mice. Lysophospholipids are generated by the action of phospholipase A50. There were some mutative ceramides and its analogues including ceramide(d18:1_18:1) (Cer (d18:1_18:1)), 3-O-Sulfogalactosylceramide(d18:1_24:1) ) in brain, glucosyl- or galactosyl-ceramide(d18:1_24:1)) in plasma. It is interesting that the level of Cer (d18:1_18:1) distinctly decreased at 2 and 3 months old and then increased in the brain of AD mice at 7 months old but the previous study reported the level of Cer(d18:1_18:1) significantly increased in blood serum of AD subjects48, suggesting such reverse change at early-stage AD. These results implied that ceramides might be glycosylated and sulfated to form Glc/GalCer(d18:1_24:1) and SulgalCer(d18:1_24:1), respectively and the enzymatic activity involving in these metabolism processes could be changed in APP/PS1 mice. In our study, the significantly decreased Glc/GalCer(d18:1_24:1) indicated that the synthesis of Glc/GalCer (d18:1_24:1) by glucosylceramide/galactosylceramide synthase might be weakened, which was opposite to the case of SulgalCer(d18:1_24:1). These results implied the perturbed sphingolipid metabolism in APP/PS1 mice and revealed these dysregulated lipid metabolic pathways in a more targeted and specific way, which perhaps provide more clues for early-AD pathology.Apart from the glycerolipids and glycerophospholipids, we also found that SM(d18:0_22:1) belonging to sphingolipids, was significantly altered. Sphingolipids are not only structural components of biological membranes, but also involve in many important physiological and pathophysiological processes including cellular growth, cell adhesion, differentiation, migration and apoptosis12. Furthermore, some evidences demonstrated a role of abnormal phospholipase C (PLC) and D (PLD) in processes associated with degradation of phospholipids in AD52. These enzymes hydrolyze phosphoester bonds in the hydrophilic group of phospholipids, thus releasing diacylglycerols. DGs could be further hydrolyzed into monoacylglycerols by monoacylglycerol lipase such as in the process of degradation of PC12. In this context, the elevation of total levels of diacylglycerols in serum from AD patients could be considered as potential marker of over-activated PL C/D54. In our study, the increased DGs (including DG(16:1_22:6), DG(18:2_18:2), DG(14:0_20:2)) were observed in the plasma and brain of AD mice  to support the above-mentioned biological hypothesis.Diacylglycerol lipases have been demonstrated that they have higher (six to eight times) enzymic activity in plasma membrane and synaptosomal plasma membrane from different brain regions of Alzheimer’s patients than normal subjects55. In this study, all of TGs (including TG(16:1_18:2_22:6), TG(16:1_16:1_16:1), TG(16:1_16:1_18:2), TG(16:1_18:2_20: 4), TG(15:0_16:0_20:3), TG(16:0_18:0_18:2) and TG(16:0_18:2_20:1)) were observed to increase in plasma of AD mice, indicating the accumulation of TGs in blood circulation.Moreover, the abovementioned changes and abnormalities in lipid metabolism were also reflected in other compounds not directly related to membrane destabilization processes, including free FA, CE and TG. The accumulation of TG suggests a serious hyperlipidemia, one of the most important vascular risk factors that have been associated with the development of AD. Furthermore, several investigations have proved relationships between Alzheimer’s disease and high levels of lipids, principally triglycerides in both mice and human blood58, indicating inflammation might occur in the brain of AD mice. Decreased free FAs including docosahexaenoic acid, arachidonic acid, hydroxyoctadecanoic acid, stearic acid and tetracosanoic acid were detected in the plasma and brain of AD mice in this investigation. Previous studies also accordingly demonstrated that several free fatty acids decreased in serum of APP/PS1 mice, plasma and brain of AD patients62. The levels of bile acids are clearly disturbed during the development of AD pathology63. In our study, 27,8-HDC significantly increased in plasma of 2- and 3-month-old AD mice.In addition, free fatty acid , cholesteryl ester(CE(20:5), CE (16:1)), cholesterol), prostaglandin D3, leukotriene D4, hydroxy-E4-neuroprostane were also found to have abnormal change in plasma and brain of AD mice in this study. In these metabolites, leukotriene D4, prostaglandin D3 and hydroxy-E4-neuroprostane in AD brain involved in the process of inflammation cascadeAlso, we found that early AD pathology significantly altered lipid constitutes in mouse plasma as well as lipid metabolism in the brain. It is interesting that 11-dehydro-thromboxane B2(11-dehydro-TXB2), prostaglandin D3 and Cer(d18:1_18:1) decreased in AD brain at 2 and 3 months, and increased at 7 months. It is notable that hydroxy-E4-neuroprostane were observed that its level decreased in AD brain at 2 months but increased at 7 months. However, the changed level for leukotriene D4 was just opposite in the brain of early-stage AD mice. The reason of such contrary alterations is unclear and needs to further be confirmed. It is speculated that the pathology of early-stage AD affects the metabolism of multiple pathways containing these abnormal lipid metabolites.67. The levels of PC(14:0_16:0)  and PC(18:0_24:1) (3 month old) decreased in the brain of AD mice in this study, indicating that they undergo flux. However, levels of PC(14:0_16:0) and PC(18:0_24:1) were not found to increase in the plasma. Meanwhile, PE(22:5_22:6) (2 and 7 month old) and PE (15:0_22:4) (7 month old) in the brain undergo influx due to their increased levels. PE(20:4_16:0) (2 month old), PE(18:1_20:4) , PE(18:1_18:2) (7 month old) and PE (22:6_22:1)  in the plasma also increased in AD mice but they had no significant fluctuation in the brain. It is speculated that these significantly altered PCs and PEs in the brain could be diluted in the blood circulation and show no significant difference in the plasma. Such differences of potential biomarkers between brain and plasma of AD mice at different month ages, as discussed previously, could provide an opportunity to view the pathological progression of the early-stage AD.Taken together, much more potential lipid biomarkers in plasma than in brain of AD mice were observed to reflect the pathological state of the early-stage AD. PEs  and PCs (∼45%) are major components of the cell membrane in the brain and play essential roles involved in neural membrane formation, trans-bilayer movement, intraneuronal signal transduction, recognition and engulfment of cells, maintaining normal mitochondrial morphology68. These were just evident from our broader timeframe of longitudinal assessment compared with other studies45.We also found that some disturbances were frequently transient and others were persistent with AD progression at 2, 3 and 7 months, as described above in this study. Therefore, not all potential biomarkers can be selected to serve as decent biomarkers to monitor and diagnose the progression of early AD. Many potential lipid biomarkers in AD mice could not be significantly changed at all early stages  possible because the pathophysiology of early-stage AD slightly and finitely disturbs the lipid metabolism. However, a biomarker panel could be selected to reflect overall situation of the pathophysiology of AD at certain timepointp < 0.05) in lipid metabolism including glycerophospholipid, sphingolipid, arachidonic acid metabolism and biosynthesis of unsaturated fatty acid in AD mice. As presented in Fig. p value; 7.96 E−03, FDR), glycerophospholipid metabolism  and arachidonic acid metabolism  were significantly impacted in early AD mice model. These results were in accordance with the altered metabolic pathway in AD mice and patients48. Arachidonic acid metabolism containing altered arachidonic acid, phosphatidylcholine and leukotriene D4 had significant difference from WT mice, indicating that inflammation might occur in AD mice brain, as known that inflammatory cascade might induce AD as a kind of biological hypothesis69.Metabolic pathway analysis (MPA) and multiple comparison testing by the identified biomarker candidates demonstrated that there were significant changes , Cer(d18:1_18:1), Glc/GalCer(d18:1_24:1), SulgalCer(d18:1_24:1), arachidonic acid, Leukotriene D4 and hydroxy-E4-neuroprostane in APP/PS1 mice at the ages of 2, 3 and 7 months, as depicted in Fig. This study originally provides a systematic approach to investigate pathophysiology of early AD to synergistically disclose dynamic change of potential lipid biomarker panels in mouse plasma and brain, and also provides strong evidence to lipid metabolic alterations in early-stage AD mice. The study enhances the possibility of developing lipid biomarkers for diagnosis and monitoring of early-stage AD.Supplementary Information.


A novel halogen-free flame retardant containing sulfonamide, 1,3,5,7-tetrakis (phenyl-4-sulfonamide) adamantane (FRSN) was synthesized and used for improving the flame retardancy of largely used polycarbonate (PC). The flame-retardant properties of the composites with incorporation of varied amounts of FRSN were analyzed by techniques including limited oxygen index, UL 94 vertical burning, and cone calorimeter tests. The new FR system with sulfur and nitrogen elements showed effective improvements in PC’s flame retardancy: the LOI value of the modified PC increased significantly, smoke emission suppressed, and UL 94 V-0 achieved. Typically, the composite with only 0.08 wt% of FRSN added  can increase the limiting oxygen index (LOI) value to 33.7% and classified as UL 94 V-0 rating. Furthermore, the mechanical properties and SEM morphology indicated that the FRSN has very good compatibility with PC matrix, which, in turn, is beneficial to the property enhancement. Finally, the analysis of sample residues after burning tests showed that a high portion of char was formed, contributing to the PC burning protection. This synthesized flame retardant provides a new way of improving PC’s flame retardancy and its mechanical property. Polycarbonate (PC) is playing an important role in modern industries such as automobile industry, glass assembly, construction, electrical and electronic (E&E) by virtue of its outstanding performance including high mechanical strength, elastic coefficients, transparency and thermal stability ,2,3,4. ANumerous flame retardants (FRs) have been developed for PC, mainly including bromine-based ,9, nitro4) [4 or 0.10 wt% ASN. However, we found the notch impact strength notably decreased, compared to pure PC, and observed FRs agglomeration, indicating their poor dispersion in matrix. It was deduced that the ionic groups in FRs were counterproductive, which is difficult to achieve a better FRs dispersion in polymer matrix.Mechanical properties of PC composites after incorporating FRs usually decrease ,23, whic4)  and 1,3,4)  were demThe objectives of this work are three-folds: firstly, we aimed to design and produce a new halogen-free flame retardant used for effectively improving the flame retardancy performance of PC, and the improvement can be achieved just with a tiny amount of FR addition; secondly, improved mechanical properties of the resulting PC composite with the same FR incorporation are also expected; thirdly, good FR dispersion in PC matrix without agglomeration and thus desirable compatibility between PC and FR would be accomplished. To this end, a sulfur and nitrogen based FR, 1,3,5,7-tetrakis (phenyl-4-sulfonamide) adamantane (FRSN) centered on adamantane  was Polycarbonate pellet 301-10 purchased from Dow Chemical Company  was air-dried at 120 °C for at least 8 h before uses. KSS provided by Guangdong Ever Sun Corp.  was used as a reference FR. Chlorosulfonic acid (99%) and ammonia solution , and 1-bromoadamantane were used as received from Energy Chemical Co. , and Macklin Biochemical Co., Ltd. , respectively. 1,3,5,7-tetraphenyladamantane was synthesized according to our previously reported procedures ,30.The FRSN was successfully synthesized from 1,3,5,7-tetraphenyladamantane through a two-step reaction, as shown in In a 100 mL three-neck flask, 1.63 g 1,3,5,7-tetraphenyladamantane (3.70 mmol) and 25 mL dry dichloromethane were added, and the flask was equipped with a dropping funnel, a tail gas unit and a reflux condenser consisting of calcium chloride drying tube on the top. The flask was placed in an ice bath before 2.5 mL chlorosulfonic acid (37.63 mmol) was added in drops. After stirring for 10 min, the mixture as stirred for 75 min after the temperature was adjusted to reach 35 °C. Once cooling down to room temperature, a light-yellow powder was obtained by centrifuge and rotary evaporation, which was then washed by water. After filtration and vacuum-dried at 45 °C for 12 h, 1,3,5,7-tetrakis (phenyl-4-sulfonyl chloride) adamantane  was obtained.25 mL ammonia solution was placed in the 100-mL three-neck flask placed in an ice bath. 1.1 g (1.32 mmol) 1,3,5,7-tetrakis (phenyl-4-sulfonyl chloride) adamantane dissolved in 25 mL tetrahydrofuran was added dropwise at room temperature. The aqueous layer was obtained with separatory funnel after stirring the mixture for 16 h. By removing water through rotary evaporation and vacuum drying, the final product FRSN, a light-yellow powder, was obtained .3 for UL 94 tests, 100 × 6.5 × 3 mm3 for LOI tests, 100 × 100 × 3 mm3 for cone calorimetry tests, and 80 × 10 × 4 mm3 with A type notches for notch Izod impact tests). Samples incorporating varied FRSN contents were denoted as PC/FRSN (0.06 wt%), PC/FRSN (0.08 wt%), PC/FRSN (0.1 wt%), PC/FRSN (0.12 wt%) and PC/FRSN (0.14 wt%). Pure PC and PC mixed with 0.08 wt% KSS were prepared at same conditions and denoted as PC, PC/KSS (0.08 wt%).Firstly, PC and FRSN were dried overnight in a vacuum oven at 110 °C and 60 °C, respectively. PC and 0.06 wt%–0.12 wt% FRSN were mixed in a HAAKE PolyLab System , including PolyLab QC system, Rheomix 3000 mixer and Rheomex CTW100 QC compounder and feeding system under 50 rpm at 250 °C for 10 min. Hot press molding  was followed, and the size of composites was designed by various test standards  using dimethylsulfoxide-d6 (DMSO-d6) as a standard solvent. The FTIR spectra were obtained from a Thermo Electron Nicolet-6700 spectrometer  after 16 scans at 4 cm−1 spectrum resolution.UL 94 vertical burning tests following the ASTM D3801-10 standard were performed on AG5100A vertical-horizontal burning apparatus .The LOI tests in accordance with the ASTM D2863-010 standard were carried out on a HP6115 type oxygen index apparatus with a magneto-dynamic oxygen analyzer .−2 within aluminum foil. The values of time to ignition , heat release rate , total heat release  specific extinction area , total smoke release , and CO production rate  were recorded, and analyzed in the main text  evaluating flammability of polymeric materials . The retention width at the gap is 8 mm.SEM experiments were performed on a high resolution field-emission scanning electron microscope, Hitachi SU8010 . Sample surfaces were coated with a thin layer of gold prior to collecting SEM images from samples after the UL 94 burning and notch impact measurements.−1 under air atmosphere.Thermogravimetric analysis (TGA) experiments were conducted on a STA409PC thermal analyzer, NETZSCH Group . TGA data were collected when samples were heated from room temperature to 800 °C with a heating of 10 °C·min1H-NMR spectra of FRSN were displayed in −1, respectively. Peaks at 833 cm−1, and 2928 cm−1 and 2854 cm−1 were ascribed to the characteristic absorption peak of para-disubstituted benzene, and −CH2 of adamantine, respectively [−1 and 1163 cm−1 were assigned to the stretching vibrations of S=O from the sulfamide group [−1 and 3258 cm−1 corresponded to the stretching vibration of N-H [−1.FTIR and de group ; and pean of N-H , which a1H NMR spectrum of the flame retardant FRSN. Chemical shift peak at 2.17 ppm  were attributed to H proton of -CH2- in adamantane. The peaks at 7.75—7.85 ppm  were ascribed to the protons on benzene ring, and the chemical shift of -NH2 was observed at 7.31 ppm [m ,37,38. BLOI and UL 94 vertical burning level of the PC/FRSN composites are shown in Such flame retardants are extremely hard to ignite according to JIS K7201 industrial standard. As for the vertical burning test, the UL 94 rating of pure PC lies in the V-2 level. However, as the addition content of FRSN ranged from 0.08 wt% to 0.12 wt%, all composites incorporating FRSN content varied from 0.08 wt% to 0.12 wt% can reach V-0 level of the UL 94 rating standard. Again, the optimum amount of FR addition was 0.08 wt%. As a comparison, adding the same amount of KSS (0.08 wt%) into PC, the PC/KSS composite only passed UL 94 V-2 rating (against V–O rating) and the LOI value only increased to 27.1% (against 33.7%).The cone calorimeter test results were summarized in −2) versus 313 kW·m−2 (pure PC), reduced by 20%. Different from the triangular and single peak of HRR curve at the time range of 100-400 s for pure PC, the HRR curves of PC/FRSN (0.08 wt%) descended more slowly, indicating the lower intensity and stable combustion state. In addition, the THR value for pure PC is 61 MJ·m−2, whilst that for PC/FRSN (0.08 wt%) is 51 MJ·m−2, clearly suggesting that the total heat release is suppressed after addition the FRSN.HRR, in particular the peak of HRR (pk-HRR) value, was applied to evaluate fire safety of samples. 2·kg−1 against 680 m2·kg−1 for pure PC. In addition, the TSP of pure PC was slightly higher in absence of FRSN. The lower values of SEA and TSP revealed that the incorporation of FRSN might retrain the smoke emission of PC composites. Overall, the cone calorimetry analysis suggests that both heat and smoke release of PC combustion could be reduced by adding only 0.08 wt% FRSN.Smoke and toxic gas release performances in fire safety assessment  were als5%), temperature at first and second maximum mass loss rate  and char residues at 800 °C were listed in TGA results under air atmosphere were plotted in 2. The residue would be the char of benzene rings and adamantane. Usually, the decomposition temperature of additive flame retardants must not decompose at processing temperature of their correspondent engineering plastics. The decomposition temperature of the synthesized FRSN in this study is 375 °C, 125 °C higher than PC’s processing temperature (around 250 °C).As shown in 5% and Tmax1 temperatures of PC/FRSN composites were lower than that of pure PC. The more FRSN added to PC, the lower Tmax1 of the resulting composite, suggesting the FRSN’s effect on the PC decomposition. In the oxidation process of sulfonate groups, an acidic compound SO2 is commonly released, which frequently has an impact on the thermal decomposition of PC by promoting the char forming process of PC [It is noted that Tss of PC ,42. It iThe morphology of residual char obtained from pure PC and PC/FRSN (0.08 wt%), after their UL 94 vertical burning tests was further analyzed by SEM to better understand the char forming process during combustion. Comparing SEM images of PC and the composite, shown in −1 and 1402 cm−1 represent the stretching and plane bending vibration of -OH groups, respectively. Peaks at 1632 cm−1 and 1597 cm−1 are attributed to the −C=C− in benzene rings, which indicates that some aromatic alcohol compounds might be produced during combustion. Furthermore, pure PC lost all carbonyl groups after combustion for the reason that the C=O absorption peak at 1771 cm−1 in (b) in The residue char of PC and PC/FRSN specimen was further analyzed by FTIR, as shown in 4 content, using PC/AS4 system [4 composites with increasing the FR content [−2), and the PC/FRSN composite incorporating low FR content exhibited an overall higher notch impact strength. Higher addition of FR, however, may lead to severe agglomeration within matrix, which would weaken the interfacial adhesion or compatibility; thus the impact strength starts to drop when FR content is higher than 0.1 wt%. The maximum impact strength of PC/FRSN (0.10 wt%) is 16.8 kJ·m−2, while that for PC/AS4 (0.08 wt%) is around 16.3 kJ·m−2, indicating that the covalent groups in FRSN have better compatibility with PC than the ionic compounds in AS4 system.Standard notch impact tests were carried out, and the impact strength was evaluated as a function of FRSN or AS4 system  for comp content , is quit4 (0.08 wt%) and PC/FRSN (0.08 wt%). SEM micrographs and EDX mapping picture of the sulfur element at the same location are shown in 4 would agglomerate even at an ultralow addition (0.08 wt%). On the contrary, only small granules can be found in Cross section after the notch impact tests was observed by SEM, along with energy dispersive X-ray (EDX) mapping results. Two composite samples were selected and analyzed, PC/AS4 composites, attention has been paid to details of their structural analysis. Firstly, it is evident that the well distributed FRSN within PC matrix . The noer group a could agroup . Specifically, the LOI value of PC/FRSN composite (0.08 wt%) increased from 25.8% to 33.7%, and UL 94 V–0 rating standard was achieved. Cone calorimetry results showed that the heat and smoke release of PC were reduced with the aid of FRSN, suppressing the process of combustion and toxin emission. The decomposition of PC was promoted by SO2 produced during the oxidation of FRSN, forming protective char layers. With their protective char and favorable dispersion in matrix, this halogen-free flame retardant provides a novel solution to PC for enhancing its flame retardancy and mechanical properties.A novel flame retardant with sulfamide centered on adamantane (FRSN) was designed, successfully synthesized and characterized by


Both materials were tested for the detection of lead (II) ions in aqueous media. Au nanoparticle incorporated CuBTC MOF showed great affinity and selectivity toward Pb2+ ions and achieved a lower detection limit (LOD) of 1 nM/L by differential pulse voltammetry (DPV) technique, which is far below than MCL for Pb2+ ions (0.03 μM/L) suggested by the United States (U.S.) Environmental Protection Agency (EPA) drinking water regulations.In the present investigation, copper benzene tricarboxylate metal organic frameworks (CuBTC MOF) and Au nanoparticle incorporated CuBTC MOF (Au@CuBTC) were synthesized by the conventional solvothermal method in a round bottom flask at 105°C and kept in an oil bath. The synthesized CuBTC MOF and Au@CuBTC MOFs were characterized by structure using X-ray diffraction (XRD) spectroscopic methods including Fourier Transform Infrared spectroscopy, Raman Spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and Energy dispersive spectroscopy (EDS). We also characterized them using morphological techniques such as Field emission scanning electron microscopy (FE-SEM), and electrochemical approaches that included cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We examined thermal stability by thermogravimetric analysis (TG/DTA) and N Heavy metal ions are a major water pollutant caused by sewage from chemical industries and various other sources  due to versatile physical and chemical properties, high porosity, ultrahigh surface area, high electrical transfer rate, high crystallinity, evenly element doping, and abundant metal sites  MOF to synthesize the Au nanoparticles incorporated CuBTC (henceforth referred to as Au@CuBTC). CuBTC MOF contains Cu(II) central metal ion and benzene tricarboxylic acid as an organic linker. CuBTC MOF is the most explored MOF. The CuBTC MOF shows good electrochemical performance and electro catalytic activity which makes them suitable for electrochemical sensors using hydrazine 2,3H2O), and N, N dimethyl formamide (DMF) purchased from Sigma Aldrich. Chloroauric acid, Hydrazine hydrate, hydrochloric acid, sodium acetate, acetic acid, sodium hydroxide, hydrochloric acid, and ethanol purchased from Molychem, India.All the reagents and chemicals were analytical grade and used without purification. Benzene-1,3,5-tricarboxylic acid (BTC), Copper nitrate trihydrate (Cu(NO3)2, 3H2O] was mixed with 50 ml of deionized water and benzene-1,3,5-tricarboxylic acid (BTC) (0.982 gm) in 50 ml of DMF, prepared separately and mixed with constant stirring at room temperature. The precursor solution was transferred into a 250 ml round-bottom flask and kept in an oil bath at 105°C for 4 h. After completion of the reaction, CuBTC MOF crystals were observed at the bottom of the round-bottom flask. After cooling at room temperature, CuBTC MOF crystals were washed with deionized water and ethanol three times each to remove unreacted reactants. The filtered CuBTC MOF was kept for activation at 120°C for 12 h to remove coordinated solvent molecules from the pores  (0.71 mM) was used as the gold (Au) source, mixed with 0.1 M hydrochloric acid (HCl) in D. I. water, with 0.71 mM hydrazine hydrate then added slowly. This solution was stirred for 30 min and sonicated for 5 min. The synthesis procedure for Au@CuBTC MOF is the same as CuBTC MOFs. In the precursor solution of CuBTC, the Au NPs precursor solution was added slowly then stirred for 30 min and sonicated for 5 min. This solution was transferred into a 250 ml round bottom flask and kept in an oil bath for 4 h at 105°C. Au@CuBTC MOF was cooled at room temperature and washed with deionized water and ethanol three times each. The filtered Au@CuBTC MOF activated at 120°C for 12 h and stored for further use.Gold (Au) nanoparticle precursor solution was prepared separately, as reported in other studies  technique in the potential window of −0.35 to 1.5 V at a scan rate of 100 mV/s until stable voltammogram was observed.Glassy carbon electrode (GCE) was used as an electrode for all electrochemical experiments. The GCEs were carefully polished with alumina powder slurries of 1, 0.3, and 0.05 μ, respectively, to get a mirror like surface. Then the GCE was ultrasonicated and washed with acetone and D.I. water sequentially. The preconditioning of bare GCE was performed in 0.5 M of HThe pure CuBTC and Au@CuBTC MOF were properly mixed in diluted nafion in acetone. The prepared suspension of pure CuBTC and Au@CuBTC MOF were drop cast on GCE electrodes and dried at room temperature, then used for electrochemical experiments.2+ ions solution was prepared in acetate buffer at pH 5.0 using Pb(NO3)2 (Pb2+ ions source), Pb(NO3)2 mixed properly, with constant stirring for 30 min. The Pb2+ ions solution was prepared at a higher concentration and used as a stock solution, a lower concentration was prepared by diluting it with the addition of a buffer solution. Electrochemical detection of Pb2+ ions was carried out on CHI 660C electrochemical workstation by the differential pulse voltammetry technique. The GCE modified electrodes of pure CuBTC and Au@CuBTC were used as the working electrode, platinum served as the counter electrode, while Ag/AgCl were used as a reference. Initially, the accumulation of Pb2+ ions on GCE modified electrodes was carried out by dipping the modified electrode for 5 min at constant pH of 5.0, then DPV in another buffer (pH = 5.0) was carried out in the potential from 0 to (−1) volts.The Pb−1. Nitrogen adsorption-desorption isotherm measurements were carried out on Autosorb iQ Station 1 instrument at 77 K and all samples were degassed at 150°C in a helium environment. The X-Ray Photoelectron studies were carried out on the XPS facility available at Raja Ramanna Center for Advanced Technology (RRCAT), Indore, India.Powder X-ray diffraction (XRD) measurements were performed on a Bruker D8 Advance Diffractometer with Cu kα1 radiation (λ = 1.5406). The Fourier Transform Infrared (FTIR) spectrum was recorded on Bruker Alpha FTIR in ATR mode with the ZnSe window in the range of 500 to 4,000 cm−1 at 532 nm laser excitation wavelength. Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) measurements were carried out on a CHI 660C electrochemical workstation with platinum (Pt) foil, Ag/AgCl as counter and reference electrodes, respectively. The CV was recorded in the potential range of −0.8–0.5 V in 10 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl solution at a scan rate of 100 mV/S. EIS measurements were taken in the frequency range of 0.01–0.1 MHz in the 0.1 M KCl solution. Electrochemical sensing experiments were performed in acetate buffer at 5.0 pH by differential pulse voltammetry (DPV) technique on CHI 660C electrochemical workstation having Ag/AgCl and Pt foil as reference and counter electrodes respectively.Thermogravimetric measurements were carried out on Shimadzu to take the DTG-60H model from room temperature to 1,000°C, with a scan rate of 10°C/min in the nitrogen environment. The Raman spectrum of all the samples was recorded on a Renishaw inVia reflex Raman microscope in the spectral range of 90 to 2,000 cm−1 and 1,446 cm−1  species, with peaks at 1,006 cm−1 and 1,606 cm−1 attributed to the ν(C=C) modes of the benzene ring. The vibrational peak at 1,457 cm−1 corresponds to νs(COO−). This confirms the formation of CuBTC MOF , 6.7° (200), 9.5° (220), 11.6° (222), 14.7° (331), 15° (420), 16.5° (422), 17.5° (333), 19,0° (440), 20.2° (442), 21.3° (620) 23.4° (444), 24.1° (551), 26.0° (553), 27.7° (733), 28.7° (660), and 35.2° (951). The crystalline size of both the compounds was calculated by classical Scherrer formula , K is the Scherrer constant (0.89 for cubic shape), θ is the Bragg diffraction angle, and β is the full width at half-maximum height (FWHM). The crystallinity and crystallite size of as synthesized CuBTC were found to be 74.4% and 199.94 nm, respectively, while reduction of the crystallinity and crystallite size in Au nanoparticle incorporated CuBTC MOF observed as 69.9% and 108.41 nm respectively.where The FE-SEM images show distinct changes in morphology, particle size, and the crystalline nature of as-synthesized CuBTC, Au@CuBTC, shown in 2O molecules, while a weight loss of 29.8%, from 133 to 343°C was observed, due to the dislodgement of coordinated DMF molecules from the CuBTC MOF Structure. There was an obvious weight loss above 343°C of ~49% in the CuBTC MOF network, which produced H2O, CO2, and Cu2O, meaning that CuBTC MOF is thermally stable up to 343°C. In Au@CuBTC, a small weight loss (15.16%) started from 40 to 122°C due to the removal of the H2O molecule. A weight loss of (16.9%) from 133 to 180°C was observed, which is due to the dislodgement of coordinated DMF molecules from the Au@CuBTC MOF Structure. An obvious weight loss of ~41.78% above 335°C was observed in Au@CuBTC MOF and produced H2O, CO2, and Cu2O. These results confirm that the thermal stability of both the materials is almost the same up to ~335°C.The thermogravimetric analysis of as-synthesized CuBTC and Au nanoparticles incorporated CuBTC is shown in 2 adsorption-desorption isotherm and BJH Pore size distribution of the as synthesized CuBTC and Au@CuBTC was studied using the Brunauer-Emmett-Teller (BET) technique, shown in NTo analyze the surface chemical state of as synthesized CuBTC and Au@CuBTC, XPS measurements were performed as shown in 6]3−/4− solution containing 0.1 M KCl solution at a scan rate of 100 mV/s, as shown in The electrochemical activity of bare GCE, Au Np, CuBTC, and Au@CuBTC were studied by cyclic voltammetry in a 10 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl. Nyquist plot ist plot  showed a2+ ions solution, and (2) reduction in buffer solution by applying differential pulse voltammetry (DPV), where the accumulated Pb2+ ions can get reduced at particular potential Pb2+ ions (Equation 1). As shown in 2+ ions at a concentration of 0.01 mM/L. However, Au@CuBTC shows a significant sensing response to the Pb2+ ions, which means that after incorporating Au nanoparticles in CuBTC become sensitive toward Pb2+ ions. The Au Np work as a catalyst while the CuBTC MOF provides more surface area and the pore provides more active sites for electrocatalytic activity, which enables highly sensitive detection of Pb2+ ions. Here, in the accumulation step, Pb2+ ions coordinate with Au@CuBTC MOF and form PbO that are trapped on the surface of Au@CuBTC MOF, while in the DPV they are reduced at the reduction potential (Equation 1)  Accumulation in PbMetzger, , which r2+ ions at the concentration of 1 μM/L at the various pH (4–8) and shows the highest sensitivity at pH 5. All the sensing experiments were carried out in acetate buffer at pH 5. The Au@CuBTC modified GCE electrode was tested for Cr3+ ions and Pb2+ ions and shows a more selective response toward Pb2+ ions as shown in 2+ ions in water, as shown in 2+ ions up to a lower detection limit of 1 nM/L, which is below the Maximum Contamination Level (MCL) of 0.03 μM/L as proposed by EPA, USA  as proposed by EPA, USA.In this study successfully synthesized and rigorously characterized CuBTC MOF and Au nanoparticle incorporated CuBTC MOF (Au@CuBTC). We prepared an electrochemical sensor based on Au@CuBTC MOF that showed a significant response and sensitivity toward PbThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GB and BH contributed equally in experimental work, idea generation, and data analysis. MD and HP contributed for electrochemical experiment data analysis. SMS contributed in experimental work. DP and KP contributed for XPS and XRD experimental data collection analysis. MS contributed as corresponding author . All authors contributed to the article and approved the submitted version.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.


PIEZO1 gene have been identified that cause Hereditary Xerocytosis (HX), a rare autosomal dominant haemolytic anemia. PIEZO1 expression is not limited to erythrocytes and expression levels are significantly higher in erythroid precursors, hinting to a role in erythropoiesis. During erythropoiesis, interactions between erythroblasts, central macrophages, and extracellular matrix within erythroblastic islands are important. Integrin α4β1 and α5β1 present on erythroblasts facilitate such interactions in erythroblastic islands. Here we found that chemical activation of PIEZO1 using Yoda1 leads to increased adhesion to VCAM1 and fibronectin in flowing conditions. Integrin α4, α5, and β1 blocking antibodies prevented this PIEZO1-induced adhesion suggesting inside-out activation of integrin on erythroblasts. Blocking the Ca2+ dependent Calpain and PKC pathways by using specific inhibitors also blocked increased erythroid adhesion to VCAM1 and fibronectins. Cleavage of Talin was observed as a result of Calpain and PKC activity. In conclusion, PIEZO1 activation results in inside-out integrin activation, facilitated by calcium-dependent activation of PKC and Calpain. The data introduces novel concepts in Ca2+ signaling during erythropoiesis with ramification on erythroblastic island homeostasis in health and disease like Hereditary Xerocytosis.The non-selective mechanosensitive ion channel PIEZO1 controls erythrocyte volume homeostasis. Different missense gain-of-function mutations in  Informed consent was given in accordance with the Declaration of Helsinki, the Dutch National and Sanquin Internal Ethic Boards.Erythroblasts were expanded as previously described . In shor0.4 (Ibidi) were coated with 10 μg/ml recombinant human VCAM-1  or fibronectin  for 30 min at room temperature. The flow chambers were placed at room temperature on an inverted microscope. Tubes connected a syringe pump, containing CellQuin medium, with one side of the channel and the other side of the channel with a waste reservoir. Tubes and channels were washed with CellQuin medium at a flow speed of 7,5 ml/h. Samples consisted of 4 × 106 erythroblasts and were incubated 10 min at 37°C with or without PKC inhibitor Gö6976 , Calpain 1 and 2 inhibitor (Sigma), integrin α4 antibody , β1 , and α5 , GsMTx4 , or Yoda1 . After incubation, samples were injected into the flow with a syringe and the pump was left running for 15 min unless otherwise stated. Nine pictures were taken across the channel after 15 min and attached erythroblasts were counted with ImageJ. Tubes were washed with water and fresh CellQuin medium in between samples. Data was analyzed with Prism 8 . Unpaired student t test was used to calculate statistical significance between each condition. D’Agostino-Pearson test was used to assess the normality of the samples’ distribution.Channels of the μ-Slide VI2+). Samples were taken at different time points, each containing 200.000 erythroblasts in a volume of 200 μl, and were stained in a 96-well plate. The samples were kept on ice after treatment and during staining. Erythroblasts were incubated for 10 min at 37°C with or without PKC inhibitor Gö6976 (500 nM) or Calpain inhibitor 1 and 2 (1 mM) with gentle mixing. Samples were taken and resuspended into ice-cold PBS. Remaining erythroblasts were incubated at 37°C with or without Yoda1 or Manganese (II) chloride dihydrate solution in water . Samples were taken after 10 and 30 min of incubation and were resuspended into ice-cold PBS. Staining was performed with 30 min incubation in FACS buffer  with the following antibodies antiCD71 , antiCD235a OriGene Technologies, Inc., Rockville, MD, United States), antiCD49d , anti β1 integrin unconjugated . Staining was followed by two washing steps with FACS buffer, followed or not by staining with secondary antibody. After the last staining and wash, the cells were resuspended into 200 μl of FACS buffer. Cells were transferred to FACS tubes before measurement on FACSCanto II . Data was analyzed with FlowJo® software  and Prism 8 .Erythroblasts were treated with or without inhibitor, Yoda1, or Manganese . After centrifugation, samples were washed in PBS and resuspended into 100 μl Extraction Buffer. Samples were incubated  and spun down . Cell lysate was transferred to a new tube and kept on ice. 10x Reaction buffer and Calpain substrate was added to 85 μl cell lysate and samples were transferred to 96-well plate. After incubation in the dark , samples were measured with 400 nm excitation filter and 505 nm emission filter with a plate reader . Positive control  and negative control  were taken along. Data was analyzed with Prism 8. Two-way ANOVA was used to calculate statistical significance between samples.Calpain activity was measured with the Calpain Activity Fluorometric Assay Kit , following manufacturer protocol. Samples were measured in triplicates. In short, the amount of 2 × 10Cells were lysed in CARIN lysis buffer . Following Bradford protein quantification , lysates were boiled in Laemmli sample buffer , subjected to SDS-polyacrylamide gel electrophoresis, blotted using iBlot-PVDF blotting system , and stained as indicated in the figure legends.5 in 0.5 ml CellQuin, were incubated with integrin β1 antibody , DRAQ5TM , and CD235a . Microscopy images were taken with an Axiovert 200 microscope  with bright field, DAPI, Alexa Fluor 488, and Alexa Fluor 647 filters using 40x/1.3 oil objective. Software ZEN2.3  was used to analyze and convert images.Expression of integrin β1 on erythroblasts was determined with live imaging. Erythroblasts, 25 × 10in vitro cultured erythroid cells (ITGA) and five β subunits , α4 (ITGA4), α5 (ITGA5), αE (ITGAE), αV (ITGAV), α6 (ITGA6), and subunits β1 (ITGB1), β2 (ITGB2), β3 (ITGB3), and β4 (ITGB4) were observed albeit with significantly different expression levels  in our id cells . To furtid cells . This ants ITGB; . At the n levels . At the n levels , α4 and 1 VLA-4;  and woul1 VLA-4; . Of note1 VLA-4; . Express1 VLA-4;  and imag1 VLA-4; .2+ dependent signal transduction in erythroblasts  before Yoda1 incubation decreased adhesion of Yoda1 treated erythroblasts to VCAM1  pathways including NFAT and MAPK . Such a condition could lead to stress erythropoiesis (which would explain the reticulocytosis observed in these patients), a situation where support of the erythroblastic island microenvironment has been shown to be important.The role of PIEZO1 during erythropoiesis has been investigated only recently. Increased activation of PIEZO1 was followed by activation of specific (Caand MAPK . We foundy state . We finddy state . NeverthAll datasets presented in this study are included in the article/The studies involving human participants were reviewed and approved by Dutch National and Sanquin Internal Ethic Boards. The patients/participants provided their written informed consent to participate in this study.FA designed the experimental setup, performed and designed the experiments, and wrote the manuscript. NH, MH, and NB designed and performed the specific experiments. EA designed the experimental setup, and supervised and edited the manuscript. All authors contributed to the article and approved the submitted version.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.


Dysfunctions of the NALCN channelosome, NALCN with several auxiliary subunits, are associated with a variety of human diseases. Here, we report the cryo-EM structure of human NALCN in complex with FAM155A at an overall resolution of 3.1 angstroms. FAM155A forms extensive interactions with the extracellular loops of NALCN that may help stabilize NALCN in the membrane. A Na+ ion-binding site, reminiscent of a Ca2+ binding site in Cav channels, is identified in the unique EEKE selectivity filter. Despite its ‘leaky’ nature, the channel is closed and the intracellular gate is sealed by S6I, II-III linker and III-IV linker. Our study establishes the molecular basis of Na+ permeation and voltage sensitivity, and provides important clues to the mechanistic understanding of NALCN regulation and NALCN channelosome-related diseases.NALCN, a sodium leak channel expressed mainly in the central nervous system, is responsible for the resting Na NALCN, a sodium leak channel, plays a key role in regulating the resting membrane potential and controlling neuronal excitability. Here the authors report a cryo-EM structure of human NALCN in complex with FAM155A, that with complementary functional analyses provide insights on its ion selectivity, voltage sensing and specific interactions with auxiliary subunits. In neurons, the resting membrane potential (RMP) is approximately −70 mV, considerably depolarized as compared to the equilibrium potential of K+ of −90 mV. The depolarization is mostly attributable to resting Na+ permeability, which is important for the regulation of neuronal excitability3. The newly identified ion channel NALCN  has a key role in regulating RMP and controlling neuronal excitability. It is primarily expressed in the central nervous system and is mainly responsible for neuronal tetrodotoxin (TTX)-resistant Na+ leak conductance4.The membrane potential across the cell membrane is essential for signal transduction in excitable cells such as neurons and muscle cellsv) channels and voltage-gated calcium (Cav) channels (4 × 6 TM). Each functional repeat of NALCN contains six TMs (S1-S6), with S1-S4 corresponding to the voltage-sensing domain (VSD) in Nav/Cav. S5, S6, and intervening segments, including the pore helices and selectivity filter (SF) from all four repeats, constitute the ion-conducting pore. NALCN shares less than 20% identity with any known Nav or Cav channel, and has been classified as a member of a new subclass of the 4 × 6 TM ion channel family5. Compared to Nav and Cav channels, NALCN contains fewer positively charged residues (arginine or lysine) on S4 segments. In addition, NALCN has a unique ion SF with EEKE residues in the four repeats, which is different from the SFs of Nav channels (DEKA) and Cav channels (EEEE/EEDD)5.NALCN consists of a single polypeptide chain of 24 transmembrane helices (TM) that form four homologous functional repeats connected by intracellular linkers. The topology of NALCN is similar to that of voltage-gated sodium . Together, they form a large protein complex termed the NALCN channelosome. Notably, these auxiliary subunits are unique to NALCN and exhibit no sequence homology to the auxiliary subunits of any other channel. Both UNC80 and UNC79 are large proteins that contribute to the neuronal localization and stabilization of NALCN in C. elegans and D. melanogaster, although their own functional domains and subcellular localization are largely unclear8. UNC80 directly interacts with NALCN and acts as a scaffold protein for UNC7910. FAM155A, also named NLF-1 in C. elegans, was reported to be an endoplasmic reticulum (ER) resident protein that acts as a chaperone to facilitate the folding and promote the axon delivery of NALCN11. In humans, FAM155B, a FAM155A homolog, can functionally substitute for FAM155A, and is likely a component of the NALCN channelosome12. In addition, NALCN channelosome activity is regulated by the Src family of tyrosine kinases (SFKs) and several GPCRs such as the M3 muscarinic receptor (M3R) through direct interactions13. The NALCN channel is also activated by neuropeptides, including substance P and neurotensin, through the SFK-dependent pathway in mouse hippocampal neurons14. How NALCN interacts with and is regulated by its auxiliary subunits remains largely unclear.NALCN is associated with several auxiliary subunits, including UNC80, UNC79, and FAM155A 21, psychomotor retardation and characteristic facies (IHPRF)23, infantile neuroaxonal dystrophy (INAD)24, cervical dystonia, schizophrenia and bipolar disorder27. There are also many diseases related to dysfunctions of the auxiliary subunits of NALCN29.In addition to its role in regulating neuronal excitability, NALCN is also important in many fundamental physiological processes, such as motor function, pain sensitivity and circadian rhythm. For example, NALCN mutant mice die within 24 h after birth due to disrupted respiratory rhythmX. laevis oocytes and the neuronal cell line NG108-1530. NALCN expressed in HEK293 cells was reported to exhibit a linear current–voltage relationship, suggesting that NALCN is a voltage-independent ion channel4. However, other studies suggest that the VSDs of NALCN can exhibit a broader range of gating behaviors and coexpression of NALCN, UNC79, UNC80, and FAM155A resulted in voltage-dependent NALCN currents30. NALCN was originally reported to be non-selective and permeable to Na+, K+, and Ca2+ ions4. Nevertheless, NALCN was recently reported to be selective only for monovalent cations and to be blocked by extracellular divalent cations12.Despite its physiological and pathological importance, the biophysical properties of the voltage sensitivity and ion selectivity of NALCN are still under debate. Electrophysiological studies on NALCN have been performed in heterologous expression systems such as HEK293 cells, 32. These back-to-back works mutually verified the main conclusions and ours also provides several novel points, such as unique lipid binding in repeat I and Na+ binding sites analysis in EEKE SF, as will be described below.To better understand the functional properties and mechanisms of NALCN, we sought to determine the structure of NALCN in complex with its auxiliary subunits. Here, we report the cryo-EM structure of human NALCN in complex with FAM155A at an overall resolution of 3.1 Å. Our structure, along with electrophysiology and molecular dynamics (MD) simulation data, provides important insights into the structure, function and regulation of the NALCN channelosome. It is to our attention that as we were submitting the manuscript, two similar works were reported elsewhere30. The electrophysiological properties of NALCN suggest that it tends to maintain the RMP in a manner reminiscent of Le Chatelier’s principle in chemical equilibria. In comparison, Nav channels always show rapid current activation and inactivation for fast initiating action potentials33, while certain Kv channels such as KCNQ channels show slow activation kinetics to avoid fast repolarization34.We employed patch-clamp recordings in HEK293 cells to characterize the electrophysiological properties of NALCN. The current elicited in response to voltage steps was very small and appeared to be indistinguishable from the mock result when NALCN was expressed alone in HEK293 cells , whereas FAM155A was built de novo. In total, 1278 and 182 residues were built for NALCN and FAM155A, respectively. The complex structure was confirmed by cross-linking mass spectrometry analysis  and rabbit Cav1.1 (PDB: 6JP5) as representative Nav and Cav channels for comparisons with NALCN36. The structures of human Nav1.7 (PDB: 6J8I), rat Nav1.5 (6UZ3) and American cockroach NavPaS (6A95) were also selected for specific discussions39. The structures of NALCN can be superimposed to the α subunit of Nav1.4 and α1 subunit Cav1.1 with root-mean-square deviation (r.m.s.d.) of 2.99 Å over 760 Cα atoms and 3.894 Å over 905 Cα atoms, respectively. In addition to the four homologous repeats, a C-terminal domain (CTD) after repeat IV, which was observed in the structures of Cav1.1 and NavPaS but not in human Nav channels, was also clearly resolved in the structure of NALCN  similar to the structure of the frizzled-like cysteine-rich domain (CRD) of receptor tyrosine kinase MuSK (PDB: 3HKL), which had 84 Cα atoms aligned to FAM155A with an r.m.s.d. of 2.8 Å  was similar    in a pattern of occurring every three amino acids in the S4 segments. As usual, we define the position of the last GC in each S4 as R643. NALCN has two to four GCs in each repeat distributed at positions R2–R6  which consists of a negatively charged residue (An2) and an aromatic occluding residue (F) on S2, and a negatively charged residue on S3, are highly conserved in NALCN except for the occluding residue on VSDIII. The An1 sites, which lie seven residues ahead of the occluding residues on S2 segments of NALCN, are mainly negatively charged or polar residues, similar to those of Nav/Cav channels  in repeat I-III form more extensive hydrogen bond interactions with surrounding negatively charged or polar residues than their counterparts in Nav/Cav channels  that intervene between S5 and S6  in the last residue of P1, while the other residues are not very conserved  in repeat I is the structural determinant of TTX sensitivity in Nav channels39. This residue forms a π–π stacking interaction with TTX-bound in the outer pore of Nav1.7, and replacement of the residue with cysteine in TTX-insensitive Nav1.5 results in much lower TTX affinity  by gravity. The resin was rinsed several times with the Wash buffer, which contains 25 mM Mops (pH 7.4), 500 mM NaCl, 0.01% (w/v) Lauryl Maltose Neopentyl Glycol, and the protease inhibitor cocktail. The target proteins were eluted by the Elution buffer containing 25 mM Mops (pH 7.4), 500 mM NaCl., 0.02% (w/v) glyco diosgenin , and the protease inhibitor cocktail plus 200 μg/ml FLAG peptide (GenScript). The eluent was then concentrated using a 50-kDa cut-off Centricon (Millipore) and further purified by Superose 6 Increase 10/300 GL column  in 25 mM Mops (pH 7.4) 150 mM NaCl, 0.02% (w/v) GDN, and the protease inhibitor cocktail. Fractions were collected and concentrated to about 8.5 mg/ml for cryo-EM analysis.NALCN, UNC-80, UNC-79, and FAM155A were cloned into pCAG vector. HEK 293F cells (Invitrogen) were transfected with the four plasmids and harvested after 60 h. Cells were resuspended in buffer containing 25 mM Mops (pH 7.4), 250 mM NaCl, 0.5% (w/v) Lauryl Maltose Neopentyl Glycol , 0.06% (w/v) cholesteryl hemisuccinate Tris salt  and protease inhibitor cocktail including 2 mM phenylmethylsulfonyl fluoride (PMSF), 1.3 μg/ml aprotinin, 0.7 μg/ml pepstatin, and 5 μg/ml leupeptin, then incubated at 4 °C for 2 h. The insoluble fraction was precipitated by ultracentrifugation at 255,700×d-(+)-glucose (pH 7.4) with NaOH, and intracellular solution contained 136 mM NaCl, 5 mM EGTA, 10 mM Hepes, and 2 mM Na2ATP (adenosine 5′-triphosphate) (pH 7.2) with NaOH. The current amplitude at the steady-state during the last 20 ms of voltage steps was averaged to construct the G–V curve. All recordings were performed under room temperature (~22 °C). Temperature variation was less than 1 °C as monitored by a thermometer. Current signal was sampled at 20 kHz.Patch-clamp recordings were performed with a HEKA EPC10 amplifier with PatchMaster software (HEKA) in whole-cell configuration. Patch pipettes were prepared from borosilicate glass and fire-polished to resistance of ~6 MΩ. For whole-cell recording, serial resistance was compensated by at least 65%. Extracellular solution contained 150 mM NaCl, 10 mM Hepes, and 30 mM HEK-293T cells were grown in Dulbecco’s modified Eagle’s medium (ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a 5% CO2 humidified growth incubator. Cells were transiently transfected by Lipofectamine 3000 (Life Technologies) 48–72 h before patch-clamp recordings. The NALCN, UNC-79, UNC-80, and FAM155A cDNAs were mixed in the mass ratio of 1:1:1:1.To apply solutions containing ligands such as verapamil during patch-clamp recording, a rapid solution changer with a gravity-driven perfusion system was used . Each solution was delivered through a separate tube so that there was no mixing of solutions. Pipette tip with a membrane patch was placed directly in front of the perfusion outlet during recording. Each membrane patch was recorded only once.G/Gmax is the normalized conductance, V1/2 is the half-activation voltage, qapp is the apparent gating charge and F is Faraday’s constant, R is gas constant, and T is the temperature in Kelvin, which is set to 295 K (22 °C).Data from patch-clamp recordings were analyzed in Igor Pro (WaveMatrix). To characterize the steady-state G–V curves, a single-Boltzmann function was used:50, with a 20 eV slit width and a defocus range from −0.5 to −2.5 μm. Each stack consisting of 32 frames was exposed for 2.56 s with 0.08 s per frame, and for ~50 e−/Å2 of the total dose.The purified NALCN complex was concentrated to about 8.5 mg/ml for cryo-sample preparation. Aliquots (3.5 μl) of protein solution were loaded onto glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3), which were blotted for 3.5 s and immersed in liquid ethane cooled by liquid nitrogen using Vitrobot . The grids were exposed through Titan Krios operating at 300 kV equipped with Gatan K3 Summit detector and GIF Quantum energy filter in super-resolution mode . Movie stacks were automatically acquired using AutoEMation51 with 2-fold binning, producing micrographs of 1.087 Å pixel size. Following patch-CTF estimation, around eleven million particles were automatically picked with cryoSPARC v252 from 17,922 micrographs. Three rounds of 2D classification enriched images of good classes, resulting in a total of 905,458 particles being selected. An Ab-initio reconstruction generated a low-resolution initial map for the subsequent homogeneous and non-uniform refinement jobs53, yielding a 3.8-Å reconstruction with identifiable side-chain densities in NALCN pore domain only. To improve map quality, we exported the aligned particles and did a 3D classification without orientation assignment using RELION 3.0 54. One out of five 3D classes represented clear secondary structures of the overall channel complex, and the 65,177 particles were fed back to cryoSPARC v2 for further homogeneous and non-uniform refinements that pushed the resolution to 3.14 Å. Adapted masks for protein regions, extracellular subunit, and intracellular domain were applied in particle subtraction and local refinement tasks, which helped in achieving slightly higher resolution at 3.11, 2.94, and 3.04 Å, respectively. Map resolutions were determined by the gold-standard Fourier shell correlation (FSC) 0.143 criterion using Phenix.mtriage55.Collected movie stacks were motion-corrected by MotionCor256. The template used for the homology modeling was the α1 subunit of Cav1.1 (PDB: 5GJV). The model was firstly docked into the final reconstruction map in Chimera57 and then manually adjusted in COOT58. After building the model of NALCN, an extra density remained unmodelled, which is close to the ECL of NALCN in the extracellular side. The high quality of the map enables us to identify that the extra density belongs to FAM155A. The structure of FAM155A was then de novo built. The sequence assignment was guided by bulky residues such as Phe, Tyr, and Trp. Up to six disulfide bonds and a glycosylation site (Asn217) were identified in FAM155A, further verifying the accuracy of the model. Ten lipid molecules were manually built to fit into the corresponding densities. The density for the C6 helix of CTD is discontinuous and has been tentatively assigned as NALCN (1589-1602) based on the side chain density.An initial model of NALCN was generated using SWISS-MODEL online server55 in real space  with secondary structure and geometry restraints generated by ProSMART59. Overfitting of the overall models was monitored by refining the models in one of the two independent half maps from the gold-standard refinement approach and testing the refined model against the other map60. Statistics of 3D reconstruction and model refinement can be found in Supplementary Table Subsequently, the models were refined against the corresponding maps by PHENIX61 and peptide terminal neutralization by acetyl/methylamine capping groups, water molecules and 150 mM NaCl were added using VMD62, resulting in a system of size 100 × 100 × 105 Å with ~93,000 atoms. The system was parameterized using tleap in AmberTools1863 by ff14SB64 and LIPID17 force fields, and along with TIP3P water model65. All the simulations were performed using OpenMM766. We first applied positional constraints  on protein-heavy atoms for a 5000-step minimization, then released constraints of side chains when heating up the system to 310 K with 2 fs stepsize (H-bonds constraints), 1 ps−1 friction coefficient for Langevin dynamics, Particle Mesh Ewald (PME) method67, and 12 Å non-bonded cut-off. During 50-ns pre-equilibration for membrane relaxation in NPT ensemble, the force constant k of backbone atoms was reduced from 10 to 0.1 kcal/mol/Å2. Three independent 200-ns production runs were conducted constraining Cα atoms more than 15 Å away from the selectivity filter residues (EEKE) with k = 0.1 kcal/mol/Å2. Simulation trajectories were analyzed using MDTraj 1.9.368. After superimposition of the pore region to the initial structure, we recorded the positions of sodium ions within 15 Å of SF residues EEKE every 1 ns for each run.The pore domain of NALCN  was embedded in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer for molecular dynamics simulations. After the determination of side-chain protonation states by PROPKA3.1Equal amount (w/w) of BS3 (bis[sulfosuccinimidyl] suberate) was added to the protein mixture, which was incubated at room temperature for 1 h. The reaction was terminated by adding 0.5 M ammonium bicarbonate to a final concentration of 20 mM for 10 min incubation. The SDS-PAGE was used to separate the crosslinked protein and stained with Coomassie Blue G-250. The gel bands of interest were cut into pieces. Sample was digested by trypsin with prior reduction and alkylation in 50 mM ammonium bicarbonate at 37 °C overnight. The digested products were extracted twice with 1% formic acid in 50% acetonitrile aqueous solution and dried to reduce volume by speedvac.m/z, 60,000 resolution) followed by 20 data-dependent MS/MS scans at 30% normalized collision energy. Each mass spectrum was analyzed using the Thermo Xcalibur Qual Browser and pLink 269 for the database searching and cross-linking analysis.For LC–MS/MS analysis, the peptides were separated by a 65 min gradient elution at a flow rate 0.300 µl/min with the Thermo EASY-nLC1200 integrated nano-HPLC system which is directly interfaced with the Thermo Q Exactive HF-X mass spectrometer. The analytical column was a home-made fused silica capillary column  packed with C-18 resin . Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur 4.1 software and there is a single full-scan mass spectrum in the Orbitrap (300–1800 Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1