Enhanced viral transmission and escape from vaccine–elicited neutralizing antibodies drive worldwide spread of SARS-CoV-2 variants and promote disease progression. However, the impact of specific spike mutations that are carried by different viral variants on viral infectivity and neutralization sensitivity has not been completely defined. Here, we use pseudoviruses to assess the contribution of spike mutations within the Receptor Binding Domain (RBD) and the Furin Cleavage Site (FCS), and appear in circulating viral variants, on viral infectivity and neutralization potential against sera that was drawn from fully vaccinated individuals. Our functional analysis demonstrates that single, P681H, P681R or A701V–FCS mutations do not play a role in viral infectivity and neutralization potential. However, when in conjunction with the RBD–N501Y mutation, viral infectivity is enhanced. Similarly, combining the E484K–RBD mutation to the spike that carries FCS mutations reduces neutralization sensitivity with no effects on viral infectivity. Employing a similar approach onto the spike from Delta or Lota SARS-CoV-2 variants further reveals that specific RBD mutations affect neutralization sensitivity or viral infectivity differently. Our results validate the efficacy of the Pfizer third dose vaccine against Delta and Lota SARS-CoV-2 variants, and outline the significance of distinct RBD mutations in promoting viral infectivity and neutralization sensitivity to post–vaccination sera. In late 2019, an emergent betacoronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was identified as the cause for severe respiratory COVID-19 disease in humans. One year into this outbreak, it is clear that the pandemic is here to stay, leading to approximately 4% mortality and causing global effects on both economic and quality of life ,12,13,14681PRRAR/SV687 cleavage site in SARS-CoV-2 spike at the S1/S2 boundary serves as a Furin Cleavage Site (FCS) has been suggested to increase viral transmissibility by facilitating maturation of the spike precursor through furin-like proteases in producer cells prior to virion release [The viral spike (S) glycoprotein is a trimeric class I fusion transmembrane glycoprotein with S1 and S2 subunits that are non–covalently bound. Following attachment of S1 subunit to ACE2 receptor, two steps of proteolytic cleavage are executed; one at the S1/S2 boundary and the other at the S2′ site, which promotes membrane fusion between envelopes of the virus and its cell target. A polybasic  release . Followi release ,25.681PRRAR/SV687 FCS mutations—P681H found in Alpha or Omicron, V701A in Beta or P681R in Delta—in affecting neutralization and infectivity levels have not been explored. This polybasic motif in spike has recently been shown to enhance membrane fusion and syncytia formation [While the involvement of mutations within the Receptor Binding Domain (RBD) of spike are extensively being tested for effects on antibody-neutralization and viral infectivity, the role of ormation ,27,28,29ormation . Removalormation ,36,37,38ormation ,30.Following a long period of dominance of the Delta variant, in November 2021, another variant of concern has emerged in South Africa and Botswana—BA.1/B.1.1.529 also designated Omicron. Omicron has since then quickly over-powered Delta SARS-CoV-2. Its genome harbors 59 mutations, 36 of them are located within the spike, and 15 specifically within the RBD. Like Alpha, Omicron carries a P681H mutation within its FCS region. Recent reports show that Omicron partially escapes immunity elicited by infection or vaccines, with boosted individuals presenting waned vaccine efficacy over time. These reports also show that, compared to Delta, Omicron results in lower incidences of severe disease ,42,43,44Here we used pseudoviruses to monitor the effects of FCS-spike mutations of either Alpha/Omicron (P681H), Beta (A701V), or Delta (P681R) on viral infectivity and neutralization sensitivity against sera that was drawn from fully vaccinated individuals. Single mutations within the FCS region were inserted to the wild-type Wuhan spike, as we demonstrated that single, P681H, V701A, and P681R FCS mutations have no role in viral infectivity or neutralization sensitivity. However, adding a single N501Y-RBD mutation into the wild type spike carrying FCS mutations substantially enhanced viral infectivity relative to wild-type SARS-CoV-2, with no effect on neutralization sensitivity. Similarly, adding the E484K-RBD mutation to pseudoviruses that carry an FCS-mutation decreased neutralization sensitivity against our tested sera, with no effects on viral infectivity.We also tested the role of single mutations within Delta RBD-L452R, T478K, and the double L452R/T478K—on pseudovirus infectivity and neutralization sensitivity. Our analysis shows that, similarly to Delta pseudoviruses that carry L452R, T478K, and P681R, pseudoviruses with single L452R or K478T RBB mutations display a moderate reduction in neutralization sensitivity relative to wild-type SARS-CoV-2. In addition, viral infectivity of Delta SARS-CoV-2 pseudovirus is increased ×2-fold, while its single RBD mutations, L452R, and T478K or double L452R/T478K has no effect on infectivity levels. We employed this functional analysis approach by introducing RBD mutations that appear within the spike of lota SARS-CoV-2. Lota carries S477N/E484K mutations within its RBD, as well as a A701V-FCS mutation. Our analysis shows that the single S477N-RBD mutation has no impact on viral infectivity or neutralizing sensitivity to post vaccination sera. However, combining the RBD-N501Y mutation to S477N spike, enhances pseudoviral infectivity. Furthermore, inserting the E484K mutation into the S477N spike reduces the neutralizing sensitivity of these pseudoviruses to post vaccinated sera, with no effects on viral infectivity. We therefore conclude that, while FCS spike mutations have no impact on viral infectivity and neutralization potential, distinct RBD mutations respectively drive viral infectivity and neutralization sensitivity to post vaccination sera of SARS-CoV-2 variants. Furthermore, we validate the efficacy of the Pfizer third dose vaccine against Delta and Lota SARS-CoV-2 variants.Resources and reagents generated in this study are available from the Lead Contact with completed Materials Transfer Agreement. Further information and requests for resources and reagents should be directed to the Lead Contact, and will be fulfilled by the lead contact, Ran Taube (rantaube@bgu.ac.il).The study was conducted in compliance with ethical principles of the Declaration of Helsinki and approved by the Soroka Medical Center Institutional Review Board (protocol 0281-20-SOR). Sera was collected from a cohort of 10 individuals that were vaccinated in the first or second Pfizer vaccine. Samples were from either a 21-day post first dose, or 9–11 days post second dose. 2 incubator. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) high glucose , supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX (Gibco), and 100 U/mL penicillin-streptomycin. HEK-ACE2 expressing cells were generated by stable transduction with lentivirus expressing human ACE2. Our pseudoviruses were standardized for equal loads by monitoring p24 levels by ELISA. E. coli DH5 bacteria were used for transformation of plasmids coding for lentivirus packaging DNA and SARS-CoV-2 spike. A single colony was picked and cultured in LB broth with 50 g penicillin at 37 °C at 200 rpm in a shaker for overnight.HEK-ACE2 stable cells were cultured at 37 °C in a 5% COhACE2  was re-cloned into a lentiviral expression vector. Lentiviral particles were produced as described previously ,46. BrieA QuikChange Lightening Site-Directed Mutagenesis kit was used to generate amino acid substitutions in the pCDNA wild-type spike plasmid carrying a D614G , following the manufacturer’s instructions . For each mutant, the relative oligos that harbored the required mutation were employed. 50-50% inhibitory titers concentration.Pseudotyped viruses were generated in HEK293T cells. Briefly, LTR-PGK luciferase lentivector was transfected into cells together with other lentiviral packaging plasmids coding for Gag, Pol Tat Rev, and the corresponding wild-type or mutate spike envelopes. Transfections were done in a 10 cm format, as previously described and supernatant containing virus were harvested 72 h post transfection, filtered and stored at −80 °C . NeutralTo determine the titers of pseudoviruses, 1e5 ACE2 stable HEK cells were plated in a 12-well plate. In addition, 24 h later, decreased serial dilutions of pseudovirus were used to transduce cells. Furthermore, 48 h post transduction, cells were harvested and analyzed for their luciferase readouts. p24 ELISA measurements were conducted to ensure equal loads. t test—p ≤ 0.001. Error bars throughout all figures represent one standard deviation. Specific details on statistical tests and experimental replicates can be found in the figure legends.Statistical analyses were performed using GraphPad Prism. Measured statistical significance was calculated between experiments by a two-tailed Student’s n = 20) of the BNT162b2-Pfizer vaccine and assessed their potential to neutralize infection of SARS-CoV-2 pseudoviruses of HEK-target cells that express the human ACE2 receptor. Neutralization potential of these samples was monitored against pseudoviruses that harbored mutations within the Furin-Cleavage Site (FCS) or RBD of spike. We acquired blood samples from a cohort of post-vaccination individuals who received three doses  spike. These included either Alpha (P681H), Beta (A701V), or Delta (P681R) mutations. Omicron spike also exhibits a P681H-FCS mutation and was tested as well. Our findings show that pseudoviruses carrying each of these single FCS mutations at position P681 were similarly neutralized by post-vaccination sera as wild-type SARS-CoV-2 pseudoviruses A.We next monitored infectivity levels of our pseudoviruses by testing their ability to infect HEK-ACE2 target cells B. As ourAdditionally, as the FCS region has been suggested to play an important role in TMPRSS2-mediated viral infection and fusion between the virus and its target cells, we tested the effects of TMPRSS2 host protease in pseudovirus infection. TMPRSS2 has been suggested to play a key role in enhancing SARS-CoV-2 infectivity by promoting S2 cleavage ,34. HEK-We further generated pseudoviruses that carried in addition to a unique FCS-P681H or A701V mutations, a single N501Y-RBD mutation within the wild-type spike. Previous reports show that the N501Y mutation increases affinity of the viral spike to its ACE2 human receptor ,54. We tWe next monitored the infectivity levels of the single FCS-N501Y pseudoviruses and the double P681H/N501Y or A701V/N501Y pseudoviruses B. Our anWe further generated pseudoviruses that carried single E484K RBD mutation, as well as double mutated pseudoviruses that carried P681H/E484K, or A701V/E484K mutations, and tested their neutralization sensitivity against our post-vaccination cohort. Previous reports show that E484K is critical for increased neutralization resistance of SARS-CoV-2 variants ,48,56,57When analyzing the infectivity levels of the pseudoviruses, with either P681H/E484K or A701V/E484K spike mutations, these exhibited a moderate increase in infectivity levels which was ×1.5-fold higher than a wild-type or single E484K SARS-CoV-2 pseudovirus B. We theTo further evaluate the functional significance of spike RBD mutations in neutralization potential and viral infectivity, we generated pseudoviruses that exhibited within their spike specific mutations that appear within Delta SARS-CoV-2. The Delta variant of concern was initially identified in India and has become dominant before Omicron emerged and took over. The Delta spike carries unique RBD mutations including L452R, T478K, and P681R. We initially tested neutralization sensitivity of Delta pseudoviruses against post-vaccinated sera and demonstrated a reduced ×2-fold decrease in their ability to neutralize post-vaccinated sera A. These We then tested the infectivity levels of our engineered pseudoviruses. While Delta pseudoviruses exhibited a ×2-fold increase in their viral infectivity relative to the Wuhan wild-type SARS-CoV-2 pseudovirus, the single L452R mutated pseudovirus had no effects on viral infectivity. Furthermore, the double L452R/T478K pseudoviruses exhibited only moderate enhancement in infectivity levels (×1.2-fold) relative to wild-type SARS-CoV-2 B. We thus conclude that L452R and T478K RBD mutations play a role in the moderate decrease in the ability of Delta to neutralize post vaccination sera or in its infectivity. To further evaluate the functional significance of RBD mutations, we employed a similar functional approach on pseudoviruses that carry spike mutations that correspond to the lota-B.1.526 variant . Lota SAWe initially tested neutralization sensitivity of pseudoviruses that carried a single S477N spike RBD mutation against our post-vaccinated sera. We showed that S477N-pseudoviruses exhibited an insignificant reduction of neutralization potential relative to wild-type SARS-CoV-2-×1.2 fold A. Upon mWe next tested the impact of adding N501Y-RBD mutation to pseudoviruses that already carried S477N-RBD mutation A. Our anWe next monitored the neutralization sensitivity and infectivity of pseudoviruses that carried double S477N/E484K-RBD spike mutations. We documented a ×2.7-fold decrease in neutralization sensitivity relative to wild-type pseudoviruses, again showing the significant role of E484K in antibody escape of SARS-CoV-2 A. MoreovCurrently administrated vaccines that have been developed in a relatively short time frame have successfully limited the COVID-19 pandemic, reducing viral spread and limiting hospitalization. These vaccines target the spike of SARS-CoV-2 and efficiently induce the development of neutralizing antibodies that inhibit viral infection. Despite these achievements, the overwhelming spread of the virus constantly drives the emergence of new viral variants that resist vaccine-induced neutralization. Alpha, Beta, Delta, and Omicron variants all carry numerous mutations in their spike, specifically within the RBD, which overall enhance viral transmission and mediate escape from vaccine-elicited or monoclonal neutralizing antibodies. As such, these variants were defined variants of interest or concern by the WHO, as they potentially compromise vaccine efficiency. Along RBD mutations that are extensively studied for their effects on viral evasion against vaccine-elicited antibodies, circulating variants also carry mutations within their polybasic Furin Cleavage site (FCS). FCS mutations include P681H in Alpha and Omicron, A701V in Beta, and P681R in Delta—indicating that the P681 residue plays a critical role in viral fitness. However, effects of FCS mutations on viral infectivity and neutralization escape have not been thoroughly investigated and their combined contribution to RBD mutations has also not been employed.In this study, we used pseudoviruses that express spike proteins from different SARS-CoV-2 variants to monitor the effects of mutations within FCS of either Alpha and Omicron (P681H), Beta and P1 (A701V), and Delta (P681R) on viral infectivity and neutralization sensitivity to post-vaccination sera drawn from fully vaccinated individuals. Our study has few limitations as it relies on our conclusions on pseudoviruses and not live viruses. However, our questions focus only on the first step of the virus life cycle-attachment of the viral particle to the ACE2 cell receptor and entry into the target cells-where pseudoviruses are a suitable model. Moreover, pseudoviuses are lentiviral-based single round replication viruses, and, as such, with no ability to extend their use into late stages of virus production and release. Nevertheless, the pseudovirus system has been broadly tested in the literature for measuring vaccine neutralization efficiency against SARS-CoV-2. Numerous studies have demonstrated high correlation between vaccine-neutralization titers measured against pseudovirus and live SARS-CoV-2 ,51,52,53We show that FCS mutations within a spike have no impact on either neutralization sensitivity or viral infectivity . HoweverIt is important to note that our study relates only to the effects of FCS mutations on viral infectivity and to post-vaccination neutralization response in a pseudovirus system. Recent work has confirmed that the basic PRRAR motif in FCS is important for viral transmission, as it provides an advantage for the virus to enter its target cells at the cell surface, promoting membrane fusion between the virus and the cell, primarily in lung and in human epithelial cells ,61. In tWe also determined the effects of RBD mutations that appear within the spike of the Delta SARS-CoV-2 variant of concern. Single L452R and T478K RBD mutations moderately impact the neutralization potential, reaching about ×2 fold reduction . The samThe significance of RBD mutations was also demonstrated with the Lota SARS-CoV-2 that carries S477N/E484K within its spike. Our data showed that, while S477N-RBD mutation alone had no impact on both viral infectivity or neutralizing sensitivity to post vaccination sera, the addition of N501Y or E484K spike mutations enhanced viral infectivity and decreased neutralizing sensitivity to post vaccinated sera .In this work, only sera that drawn from vaccinated individuals who received the third dose of the Pfizer-BTN162b2 mRNA vaccine has been tested. However, several other vaccines, specifically Moderna mRNA-1273 and the Ad26 CoV2.S from Janssen/J&J, have been developed and successfully applied for immunization against SARS-CoV-2. These vaccines exhibit high efficiency in preventing viral infection and disease progression ,13,21,64Taken together, our results highlight the importance of specific residues within the RBD of spike in driving neutralization sensitivity and viral infectivity of different SARS-CoV-2 variants. N501Y L452R, E484K S477S, and T478K distinct RBD mutations potentially compromise the efficacy of administrated vaccines.


Background: LncRNA is closely associated with the progression of human tumors. The role of lncRNA TNFRSF10A-AS1 (T-AS1) in gastric cancer (GC) is still unclear. We aim to investigate the functional significance and the underlying mechanisms of T-AS1 in the pathogenesis and progression of GC.Experimental Design: The clinical impact of T-AS1 was assessed in 103 patients with GC. The biological function of T-AS1 was studied in vitro and in vivo. T-AS1 downstream effector were identified by RNA sequencing and RNA pulldown assay.Results: T-AS1 was upregulated in GC cell lines and GC tissues as compared to adjacent non-cancer tissues . Multivariate analysis showed that GC patients with T-AS1 high expression had a significantly shortened survival . T-AS1 significantly promoted GC cell proliferation, cell-cycle progression, and cell migration/invasion abilities, but suppressed cell apoptosis. Silencing of T-AS1 in GC cells exerted opposite effects in vitro. Knockout of T-AS1 significantly inhibited xenograft tumor growth in nude mice. Mechanistically, T-AS1 directly bound to Myelin Protein Zero Like 1 (MPZL1). MPZL1 showed an oncogenic function in GC by promoting cell proliferation, migration and invasion but inhibiting cell apoptosis. High expression of MPZL1 was associated with poor survivor of GC patients. Knockdown of MPZL1 could abrogate the effect of T-AS1 in the tumor-promoting function.Conclusions: T-AS1 plays a pivotal oncogenic role in GC and is an independent prognostic factor for GC patients. The oncogenic function of T-AS1 is dependent on its direct downstream effector MPZL1. Gastric cancer (GC) is the fourth-leading cause of cancer-related mortality globally Aberrant expression of long non-coding RNAs (lncRNA) have been reported to be involved in cell proliferation, tumor progression, invasion, and metastasis We recently discovered that a novel lncRNA TNFRSF10A-AS1 (T-AS1) was overexpressed in GC Figure ). Upregulation of T-AS1 in GC tumors was confirmed in two different cohorts. T-AS1 expression was significantly upregulated in primary gastric tumors as compared with adjacent normal tissues (n = 47) as determined by qRT-PCR (P < 0.001) in our cohort. Consistently, T-AS1 was upregulated in GC paired samples  and unpaired samples  from TCGA cohort . These results demonstrated that T-AS1 was commonly overexpressed in GC.T-AS1 expression in GC cells and primary GC tissues was determined by RT-PCR and qRT-PCR, T-AS1 was highly expressed in all 6 GC cell lines , but low expressed in the normal gastric cell GES1 (Table S1), multivariate Cox regression analysis showed that T-AS1 expression was an independent poor prognostic factor for GC patients  . Kaplan-Meier survival curves showed that GC patients with high T-AS1 expression had significantly shorter survival than those with low T-AS1 expression (P <0.05) .We further evaluated the clinicopathologic feature and the mortality risk of T-AS1 expression in GC patients. In primary GC tissues from 103 Chinese patients . Conversely, T-AS1 knockdown by RNA interference in MGC803 and AGS cells markedly inhibited cell viability and clonogenicity . To investigate the effect of T-AS1 on subcutaneous xenograft tumors, CRISPR/Cas9 was used to knock out the T-AS1 exon in the gene and T-AS1 stable knockout was constructed in MGC803 cells. Four sgRNAs was used to knock out Exon 1 and part of Exon 2 of T-AS1 gene . The knockout effect was confirmed by qRT-PCR and gene sequencing . We subcutaneously injected MGC803 cells stably knock out T-AS1-KO or NC into the left and right dorsal flanks of nude mice, respectively. As shown in Figure , knockout of T-AS1 markedly decreased the size (P < 0.0001) and weight (P < 0.05) of MGC803 xenograft tumors . Ki-67 staining further confirmed that knockout of T-AS1 inhibited cell proliferation evidenced by reduced Ki-67 positive cells .Overexpression were performed on BGC823 and GES1 cells which expressed T-AS1 at low level, and knockdown were performed on MGC803 and AGS cells which with high level of T-AS1 and more commonly used in gastric cancer research. P < 0.05) , while knockdown of T-AS1 significantly increased apoptosis cells of MGC803 and AGS cells (both P < 0.05) . The T-AS1 induced decrease in apoptosis was confirmed by the elevated protein expression of key apoptosis markers , while knockdown of T-AS1 showed the converse effects .Apoptosis analysis by flow cytometry showed that T-AS1 significant reduced total apoptotic cells in BGC823 and GES1 cells transfected with T-AS1 compared to the control cells .To understand the molecular basis of the oncogenic property of T-AS1, RNA-sequencing was performed in T-AS1 knockdown and control GC cells (MGC803 and AGS). KEGG pathway enrichment analysis showed that knockdown of T-AS1 significantly activated tumor suppressive p53 signaling (P < 0.01) and GES1 cells . Conversely, knockdown of T-AS1 by RNA interference in MGC 803 and AGS cells decelerated the G1-S cell cycle transition . The enhanced G1/S cell cycle by T-AS1 was confirmed by the upregulation of the key G0/G1 phage regulators CDK4 and cyclin D1, and knockdown of T-AS1 showed opposite effect . Our results indicated that T-AS1 could promote cell cycle and inhibit cell apoptosis by activating p53 signaling .We next investigated the effect of T-AS1 on cell cycle progression by flow cytometry. Ectopic expression of T-AS1 accelerated G1/S phase progression in BGC823 (P < 0.001) and GES1 cells . On the other hand, knockdown of T-AS1 markedly suppressed the migration and invasion abilities of MGC803 and AGS cells . Protein expression of epithelial-to-mesenchymal transition markers was investigated by western blot. T-AS1 overexpression upregulated the expression of Snail and Slug , while knockdown of T-AS1 had the opposite effects . These findings demonstrated that the oncogenic effect of T-AS1 was also associated with its role in promoting cell migration and invasion of GC cells.Wound-healing and Matrigel invasion assays were performed to determine the effects of T-AS1 on cell migration and invasion. Ectopic expression of T-AS1 significantly increased the migration and invasiveness abilities of BGC823 cells . T-AS1 expression was positively correlated with MPZL1 mRNA expression on cells  and in TCGA cohort . Moreover, T-AS1 expression was also correlated with MPZL1 at protein level by Western blot . lncLocator analysis showed that T-AS1 mainly located in cell cytoplasm . RNA FISH analysis using eight probes of T-AS1 gene (Table S2) confirmed that T-AS1 was mainly located in the cytoplasm in AGS cells and MGC803 cells . RNA pull-down assays confirmed that T-AS1 could bind to MPZL1 .To elucidate the molecular mechanisms underlying the pro-tumorigenic action of T-AS1, gene expression profiles were analyzed by RNA sequencing in T-AS1 knockdown MGC803 and AGS cells. MPZL1 was the most significantly downregulated gene in T-AS1 knockdown cells . MPZL1 mRNA expression was significantly higher in GC tumor tissues compared to adjacent normal tissues of paired samples ; and in GC tumor tissues compared to normal gastric tissues of unpaired samples  . Kaplan-Meier analysis showed that high MPZL1 expression was correlated with poor overall survival of GC patients . To investigate the function of MPZL1 in GC, knockdown of MPZL1 by RNA interference in MGC803 and AGS cells, low expression of MPZL1 was confirmed by qRT-PCR and western blot . Moreover, MPZL1 expression promoted cell proliferation and clonogenicity in MGC803 and AGS cells . MPZL1 also promoted migration and invasion in MGC803 and AGS cells .MPZL1 mRNA expression was upregulated in 5 GC cell lines  but not in BGC823 and GES1 cell lines . MPZL1 knockdown in BGC823 and GES1 cells significantly abolished the promoting effect of T-AS1 on cell viability  and clonogenicity . Knockdown of MPZL1 significantly blunted the promoting effects of T-AS1 overexpression on cell migration and invasion in BGC823 and GES1 cells . These results indicated that the tumor-promoting effect of T-AS1 is at least in part dependent on MPZL1 in GC.We then assessed whether the oncogenic function of T-AS1 in GC was dependent on MPZL1. BGC823 and GES1 cells stably transfected with T-AS1 or control vector were co-transfected with siRNA against MPZL1  and one normal gastric epithelial cell line GES1 were used in this study. AGS was obtained from the ATCC. BGC823, MGC803, HGC27 and GES1 were obtained from Cell Research Institute, Shanghai, China. MKN74 was obtained from the Japanese Collection of Research Bioresources Cell Bank. These cell lines were obtained between 2014 and 2015, and cell authentication of cells we performed the function and mechanisms study  was verified by short tandem repeat profiling. Cells were cultured in DMEM . Routine mycoplasma testing was performed by PCR. Cells were grown for no more than 20 passages in total for any experiment.6 cells) stably transfected with sgNC or sgT-AS1 were injected subcutaneously into the left and right flanks of 4-week-old male Balb/c nude mice (n = 10 per group). Tumor volume (mm3) was calculated using the longest and shortest diameters of the tumor as previously described To assess the effect of T-AS1 on tumor growth, MGC803 cells  for 16h at 37 °C. Chromosomes were stained with DAPI in PBS for 5 min. Immunofluorescence images were taken with a fluorescence microscope .T-AS1 expression in GC cells was detected using Stellaris RNA FISH with 8 independent probes  on cell slides according to the protocol provided by the manufacturer. Probe sequences are listed in A 3-μg sample of RNA was used as the input material for RNA sample preparation. All samples had RNA integrity values > 6.8. Sequencing libraries were generated using the IlluminaTruSeq RNA Sample Preparation Kit  following the manufacturer's recommendations. The libraries were sequenced using the Illumina HiSeq X-ten platform as per the manufacturer's instructions .in vitro using the Pierce RNA 3′ End Desthiobiotinylation Kit . RNA pull-down assays were performed using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). Desthiobiotin-labelled RNA (50 pM) was mixed with 50 μL of magnetic beads and incubated with AGS cell protein lysates for 60 min at 4 °C with rotation. The beads were washed briefly 4 times, boiled in SDS buffer and the RNA-binding proteins were detected by western blot.Full-length T-AS1 was transcribed using T7 MEGAscript kits  and labelled Table S3. After transfection, MGC803 cells were selected by puromycin and distributed into 192-well plates. sgT-AS1 in single stable cells was verified by PCR using the detection primer shown in Table S4. Control cells were treated with an empty vector containing nonsense sgRNA.The lentiviral sgT-AS1 construct, as reported previously 2 test was used to compare patient characteristics and determine distributions of expression and covariates by vital status. The Mann-Whitney U test or Student's t test was used to compare variables between two groups. The cutoff value was determined by survival significance analysis using the tool Cutoff Finder (http://molpath.charite.de/cutoff/) P < 0.05 was considered statistically significant, and all tests were two-tailed.Data are expressed as the mean ± SD. Statistical analysis was performed using the Statistical Package for the Social Sciences . The χSupplementary methods, figures and tables.Click here for additional data file.


O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranoside (spiroconazol A), a steroidal saponin, from the flowers of Dioscorea bulbifera L. var. sativa. In the present study, we aimed to investigate the effects of spiroconazol A on autophagy and its underlying mechanisms in A549 and NCI-H358 human non-small cell lung cancer (NSCLC) cells. Spiroconazol A inhibited the proliferation of NSCLC cells in a concentration- and time-dependent manner. To determine the type of programmed cell death induced by spiroconazol A, we performed a characterization of apoptosis in spiroconazol A-treated A549 cells. Our results showed that spiroconazol A significantly suppressed A549 cell viability but did not influence cell apoptosis because phosphatidylserine and caspase activation were not detected. Furthermore, spiroconazol A treatment upregulated the expression of LC3-II and autophagy-related Beclin-1 protein, suggesting that spiroconazol A induces autophagy in A549 cells. Moreover, spiroconazol A activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but did not affect the phosphorylation of Janus kinase or ERK1/2. Notably, SB203580, a p38 MAPK inhibitor, had a significant inhibitory effect on spiroconazol A-induced autophagic cell death in A549 cells. Our results indicated that spiroconazol A-induced autophagy is dependent on p38 MAPK signaling and has potential as a therapeutic target in NSCLC.In our previous study, we reported the isolation of pennogenin-3- Lung cancer is a major cause of mortality worldwide, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases . AlthougAutophagic cell death, another cell death pathway, is considered an alternative therapeutic approach for cancer cells . AutophaDioscorea bulbifera L. var. sativa and the stem bark of Dracaena mannii Baker, and the cytotoxic activity of various isolated steroidal saponins against bladder carcinoma cells and antiedema activity was determined in a carrageenan-induced rat model [O-α-L-rhamnopyranosyl-(1->3)-β-D-glucopyranoside) and spiroconazol A (pennogenin 3-O-α-L-rhamnopyranosyl-(1->2)-[α-L-rhamnopyranosyl-(1->3)]-β-D-glucopyranoside). Based on the findings of our previous study on the structure–cytotoxicity relationship of hederagenin glycosides isolated from Kalopanax pictus Nakai, we acknowledge that the linkage of sugar moieties is very important for biological activities [Steroidal saponins isolated from medicinal herbs have been studied for their pharmacological effects, including antifungal, cytotoxic, and antitumor effects, owing to their structural diversity and important biological activities, such as cell cycle arrest, apoptosis, and autophagy ,8,9. In at model ,11. Spirtivities ,13. Of tD-glucose in mannioside A) had a potent cytotoxic effect , while pennogenin did not exhibit any cytotoxic effect on various cancer cells at a dose of up to 100 μM (D-glucose in pennogenin A) showed moderate cytotoxicity . Because spiroconazol A exerted the maximum potent cytotoxic effect on human lung cancer cells (A549 IC50: 2.07 ± 0.18 μM and NCI-H358 IC50: 2.17 ± 0.08 μM) while spiroconazol A had lower cytotoxicity in the normal cell , as described in our previous study ,11, we fo 100 μM . Mannios1 phase, which represented cell death, also increased in a time-dependent manner, but the cell cycle arrest was not affected in A549 cells  by flow cytometry. Cells in the sub-G49 cells A. To inv49 cells B. Furthe49 cells C.To better understand the caspase-independent cell death induced by spiroconazol A, we investigated other cellular responses related to cell death following spiroconazol A treatment. As shown in During autophagy, the degraded cytoplasmic contents are engulfed by double-membrane vesicles called autophagosomes, which can be observed on microscopy by the punctate formation . To confMAPKs play an essential role in eukaryotic cells by controlling cellular processes including cell proliferation, differentiation, migration, and programmed cell death such as autophagy . In partD. bulbifera L. var. sativa was determined by the MTT assay; spiroconazol A showed a potent cytotoxic effect (IC50 = 6.6 μM) on the urinary bladder carcinoma cell line ECV-304 [The cytotoxicity of the 16 isolated steroidal saponins from the flowers of  ECV-304 . HoweverD-glucose in pennogen-in A) had a significantly improved cytotoxic effect when compared with the pennigonin A. Furthermore, spiroconazol A contains an additional rhamnose moiety attached at site C-2 of D-glucose in mannioside A, and this moiety led to a markedly higher activity against various cancer cells  than those of mannioside A . An appropriate concentration of spiroconazol A could reduce the viability of NSCLC cells but did not influence apoptosis induction. Annexin V-positive cells and caspase activity, which are pivotal biomarkers of cancer cell apoptosis [Medicinal plants containing valuable substances with therapeutic effects play an essential role in the treatment of multiple diseases including cancer . Most ofThe retention of intracellular homeostasis is regulated by the balance between cell survival and death. In particular, autophagy maintains cell homeostasis, including cell survival (cytoprotective autophagy) ,23 and cMAPK pathways, which include ERK1⁄2, JNK, and p38 MAPK, are involved in various biological processes such as cell differentiation, proliferation, and death . The ERK2DCFDA staining (N-acetylcysteine (NAC) suppressed spiroconazol A-induced cell death, indicating that NAC probably contributed to preventing spiroconazol A-induced autophagy by suppressing ROS production . The mobile phase was adjusted to pH 5.7 with phosphoric acid and delivered at a rate of 0.5 mL/min. The column was eluted into a Waters 2996 photodiode array detector (PDA)  with the wavelength set at 210 nm. Data processing was performed with Empower Pro® software . Fetal bovine serum (FBS), penicillin/streptomycin (PS), and RPMI 1640 medium in cell culture were obtained from Life Technologies Inc. . The z-VAD-fmk was purchased from R&D Systems  and Bradford solution was obtained from Bio-Rad Laboratories . Antibodies for Bcl-2, Bcl-xL, β-actin, MEK3/6, p-ERK1/2, ERK1/2, p-JNK, JNK1, and p38 mitogen-activated protein kinase (MAPK) were purchased from Santa Cruz Biotechnology . LC3 I/II, p-MEK3/6, and p-p38 MAPK were purchased from Cell Signaling Technology . A FITC (fluorescein isothiocyanate)-Annexin V Apoptosis Detection Kit I was purchased from BD Bioscience pharmingen . MTT, 4′,6-diamidino-2-phenylindole (DAPI), PI, sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), 3-MA, and other chemicals were purchased from Sigma Aldrich .Pennogenin, mannioside A, and spiroconazol A  were obtHuman cancer cells  were obtained from the Korean Cell Line Bank  and were cultured according to the institution’s guideline.50 of cytotoxicity was calculated with the results of the MTT assay, as previously described [The ICescribed .To evaluate the spiroconazol-induced cell death, PI staining analysis was used, as previously described .To identify apoptotic cells, spiroconazol A-treated cells were incubated with 100 μL of annexin V binding buffer containing PI and FITC-conjugated annexin V for 15 min in the dark. The cells were then analyzed using a FACS cytometer .After the extraction of cells , the extHuman LC3-II cDNA was amplified from A549 cells by PCR with PrimeStar Taq DNA polymerase . The primers including the Kozak consensus sequence (GCC ACC) were constructed based on the previous literature . The PCRTo measure spiroconazol A-induced intracellular ROS level, we used DCFH-DA, which is the most widely used fluorescent probe for the detection of intracellular oxidative stress. The spiroconazol A-treated cells were incubated with 20 μM of DCFH-DA for 30 min at 37 °C. The intracellular ROS level was measured by flow cytometry.t-test, and p values of < 0.05 were considered statistically significant.Data are presented as the mean ± SD of triplicate experiments. Statistical analysis was performed using the Student’s In summary, spiroconazol A-induced cell death was mainly due to autophagy induction, which was associated with Beclin-1 and MAPK signaling. Therefore, we speculated that spiroconazol A may be a molecular-targeted therapeutic drug for the treatment of NSCLC. However, the mechanism underlying the relationship between p38 MAPK and ROS-related signaling responsible for autophagy induced by spiroconazol A remains uncertain. Hence, further studies are required to clarify the rational mechanism of spiroconazol A treatment in an animal model of NSCLC.


G93Adl and TDP-43 (TAR6/6) mice [G298S mouse model to study the interaction of motor neuron pathophysiology, muscle denervation and NF-L levels. TDP-43G298S mutant mice show decreased performance in grip strength and motor activity compared to controls [Elevated levels of neurofilament light chain (NF-L) in CSF and blood are linked to the presymptomatic and symptomatic phase of patients suffering from amyotrophic lateral sclerosis (ALS). However, whether the NF-L level in extracellular liquids like serum or CSF is a marker of destruction or NF-L is secreted actively outside the cell is not known so far. NF-L levels in CSF and blood clearly separate ALS patients and controls , servingDP-43 TAR/6 mice [controls .G298S mice  . WT mice needed 12 months longer to reach the same plateau as TDP-43G298S mice, thus potentially pointing to a premature aging phenotype or to early MN degeneration in TDP-43G298S mice. These findings are in line with previous studies demonstrating increased NF-L levels at 9 and 27 weeks of age in transgenic TDP-43 (TAR6/6) and SOD1G93A mice, respectively [G298S mouse model compared to controls and are in line with clinical data from ALS patients [Serum NF-L level measurements were carried out with the ultrasensitive single molecule array (Simoa) technique and showed a significant age- and genotype-dependent increase of NF-L in both WT and TDP-43DP-43 TAR/6 and SOG298S mice differed significantly from WT mice showing reduced MN counts comparable to the MN count of WT mice at older ages like 10 or 18–20 months  , genotype (*p < 0.05) and interaction of both (***p < 0.001)) .To study whether destructive processes in MN pathophysiology might be the underlying cause of NF-L level increase, the number and area of MNs in the ventral horn of the lumbar spinal cord were determined. Again, as also seen for NF-L levels, already at the age of 2 months TDP-43G298S mice, we performed immunohistochemistry and oberserved that the overlap between TDP-43 and nuclear staining is less pronounced in TDP-43G298S tg animals than in WT mice  . In addition, we conducted RNA sequencing of the cortex of these mice for all age groups and identified genes that were significantly altered in each respective group, even at the early age of 2 months .To further characterize ALS related alterations in TDP-43G298S mice recapitulate elevated serum NF-L levels as seen in ALS patients. Simultaneously to NF-L elevation MN loss and muscle denervation were detected suggesting that NF-L elevation might be due to MN degeneration. We conclude that this mouse model is suitable for biomarker research in ALS with regard to the specific mechanisms involved in the disease associated release of NF-L.Together, the present study demonstrates that ALS TDP-43Supplementary file1 (PDF 331 KB)Below is the link to the electronic supplementary material.


The key insight disclosed here will underpin the ongoing development of increasingly sophisticated branch selective Murai hydroarylations.Quaternary benzylic centers are accessed with high atom and step economy by Ir-catalyzed alkene hydroarylation. These studies provide unique examples of the use of non-polarized 1,1-disubstituted alkenes in branch selective Murai-type hydro(hetero)arylations. Detailed mechanistic studies have been undertaken, and these indicate that the first irreversible step is the demanding alkene carbometallation process. Structure-reactivity studies show that the efficiency of this is critically dependent on key structural features of the ligand. Computational studies have been undertaken to rationalize this experimental data, showing how more sterically demanding ligands reduce the reaction barrier  Quaternary benzylic centers are accessed with high atom and step economy by intermolecular Ir-catalyzed hydroarylation of non-polarized 1,1-disubstituted alkenes. This would then provide a viable method where feedstock reactants are used directly, and C–C bond formation and stereocenter creation are united.9,10 To achieve this, we envisaged initiating the reaction by metal-catalyzed C–H activation of the arene rather than by Friedel–Crafts-type activation of the alkene  C–H bond activation regioselectivity, (2) branched : linear regioselectivity11 and (3) enantioselectivity.11 In this report, we outline efforts to address these objectives, which have resulted in an efficient C–H activation protocol for the hydro(hetero)arylation of non-polarized 1,1-disubstituted alkenes. Our results make a significant contribution to the area of Murai-type hydroarylations,11 provide an enabling cross-coupling methodology and set the stage for the development of an enantioselective method.As outlined above, state of the art cross-coupling methods require substantial levels of prefunctionalization, and the desired stereochemistry is established in an earlier step, thereby detracting from atom and step economy. Consequently, we considered whether the Friedel–Crafts approach might be re-engineered to offer broader scope  selectivity (entry 1). As part of extensive optimization efforts, we varied the electronics of the flanking biaryl units of the ligand. Replacement of the t-Bu groups of L-1 with chloride, methoxy or trifluoromethyl groups (L2–4) resulted in lower yields (entries 2–4). Pleasingly, L-5 (Y = H) was effective, and this allowed 2a to be isolated in 73% yield (entry 5). Higher catalyst loadings led to diminished yields and 1,4-dioxane was found to be the optimal solvent.15 Ir-precatalysts possessing more strongly coordinating counterions (e.g. triflate) were less effective (entry 6). Replacement of the N,N-diethyl groups of 1a with other substituents gave targets 2b–e in lower yields (entries 7–10). The attempted use of weaker 6-membered anilide-based chelates or stronger 5-membered 2-pyridyl-based chelates was not successful. As discussed later, reaction efficiency is specifically dependent on the structural features of the central biaryl unit of L-5, such that we have been unable to identify commercially available ligands that offer comparable efficiencies. Additionally, dFppb, a ligand developed previously for branch selective hydroarylations of monosubstituted alkenes,16 is ineffective.We have previously developed an Ir-system modified with bisphosphite ligand L-1 for the enantioselective hydroarylative cross-coupling of anilide lstyrene . Remarkaortho-alkylation of the arene occurred. The latter is consistent with initial monoalkylation causing the directing group to twist from the plane of the arene, such that alkylation of the remaining ortho-position cannot occur. Significantly, the process extends to aliphatic alkenes; for example, 2j was accessed in 57% yield. Substitution at the meta- and para-positions of the benzamide is tolerated (2k–m). For 2l, C–C bond formation was highly selective for the less hindered ortho-position, and this presumably reflects the steric demands of L-5. Very electron poor arenes (e.g. R1 = p-NO2) are not suitable, perhaps because these systems are less effective at stabilizing the cyclometallated Ir(iii)-intermediate (vide infra).As outlined in N,N-diisopropyl directing group is more efficient than an N,N-diethyl variant , both styrenic and “all-aliphatic” 1,1-disubstituted alkenes participated to provide 8a–c in 54–86% yields. As with 6g, the trisubstituted alkene associated with 8c did not undergo hydroheteroarylation.To explore scope further, we examined hydroheteroarylations with thiophene 7, which possesses a C2 directing group . Althougdeuterio-9 with furan 3a resulted in deuterium transfer to C4–H of deuterio-4i, and scrambling in recovered deuterio-9′  that reversible alkene hydrometallation is operative (III to IV and V) and (c) that the alkene isomerizes under the reaction conditions. Based on these data, C–C bond formation could occur either via C–C reductive elimination from V or via carbometallation from III. To distinguish these options, natural abundance 13C KIE experiments were undertaken on alkene 9 using the Singleton method, which establishes which carbon centers are involved in the first irreversible step.17 This revealed significant KIEs at both C2 (1.013) and C1 (1.035), as well as a large inverse KIE at C3 (0.981)  even carbon-13 distribution is established. To confirm this, alkene 9 was exposed to the reaction conditions in the absence of arene 3a; integration of the 13C NMR spectra of recovered 9 revealed a relative enhancement at C1 (1.020) and a depletion at C3 (0.984), with the average approximating unity as expected , and the large inverse KIE at C3 in 13 and likely reflects the increased steric demands of 1,1-disubstituted variants. Branched selectivity is presumably favored during carbometallation because the bulky Ir-center ends up at the less hindered position. The processes described here require 5-membered amide-based chelates, which are presumably sufficiently tractable to be intercepted by the hindered alkene. Weaker 6-membered anilide-based chelates are not suitable.13A series of experiments support the working mechanistic hypothesis in terio-9′ . These r (0.981) . The KIEexpected . In Sche1 = Me, R2 = t-Bu) affords 4i in 86% yield (entry 1). Reducing the size of the R2 substituent  decreased the yield slightly (76%), whereas removal of this unit (L-7) significantly lowered the yield (19%) (entry 2–3). Removal of the R1 substituent  had a less significant effect (entry 4). NMR profiling indicated that the yields are reflected in the reaction rates  was modelled with DFT using Gaussian 16 -center. The lowest energy conformations of each species and the relative barriers to transition state TS-III from intermediate III are provided in iii) reaction center is nearly identical for each ligand . Consequently, the method can be considered “enabling”, and so we anticipate that it will be of broad interest. Ongoing efforts towards (a) an enantioselective variant and (b) catalysts that can accommodate internal alkenes will be guided by the intriguing ligand design insights outlined here.In summary, we report unique examples of branch selective Murai-type hydro(hetero)arylations of non-polarized 1,1-disubstituted alkenes. The chemistry is enabled by key structural features of L-5, a ligand that is accessed in just one step. Experimental trends in yield over a variety of ligands were examined by DFT calculations, which revealed how more sterically demanding ligands reduce the reaction barrier Compound characterisation data and Gaussian 16 output files for all computed structures are openly available in the ESI.†P. C., A. G. D, T. P. A., S. G. and E. L. performed the experimental work, which was directed by L. J. F., P. D. K. and J. F. B. The computational studies were conducted by E. H. E. F. and M. N. G. The manuscript was written by J. F. B. and M. N. G. with contributions from all authors.There are no conflicts to declare.SC-013-D2SC02790A-s001SC-013-D2SC02790A-s002


Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ER) agonists or antagonists in a tissue-specific manner. ERs exert effects via nuclear actions but can also utilize membrane-initiated signaling pathways. To determine if membrane-initiated ERα (mERα) signaling affects SERM action in a tissue-specific manner, C451A mice, lacking mERα signaling due to a mutation at palmitoylation site C451, were treated with Lasofoxifene (Las), Bazedoxifene (Bza), or estradiol (E2), and various tissues were evaluated. Las and Bza treatment increased uterine weight to a similar extent in C451A and control mice, demonstrating mERα-independent uterine SERM effects, while the E2 effect on the uterus was predominantly mERα-dependent. Las and Bza treatment increased both trabecular and cortical bone mass in controls to a similar degree as E2, while both SERM and E2 treatment effects were absent in C451A mice. This demonstrates that SERM effects, similar to E2 effects, in the skeleton are mERα-dependent. Both Las and E2 treatment decreased thymus weight in controls, while neither treatment affected the thymus in C451A mice, demonstrating mERα-dependent SERM and E2 effects in this tissue. Interestingly, both SERM and E2 treatments decreased the total body fat percent in C451A mice, demonstrating the ability of these treatments to affect fat tissue in the absence of functional mERα signaling. In conclusion, mERα signaling can modulate SERM responses in a tissue-specific manner. This novel knowledge increases the understanding of the mechanisms behind SERM effects and may thereby facilitate the development of new improved SERMs. Estrogen treatment protects against osteoporosis-related fractures, has favorable effects on several metabolic parameters, and alleviates postmenopausal symptoms. However, due to adverse effects, estrogen treatment is avoided. Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ER) agonists in some tissues, and as antagonists in others, have been developed to avoid these adverse effects. SERMs are mainly used to prevent and treat osteoporosis and breast cancer and to alleviate postmenopausal symptoms, but also to maintain a beneficial lipid profile in postmenopausal women (Igf1) (Lasofoxifene (Las) and Bazedoxifene (Bza), which are third-generation SERMs, have agonistic effects in bone and can prevent both vertebral and non-vertebral fractures in humans (Igf1) . There aSERMs exert effects by binding to ERs. ERs can, in addition to nuclear actions, also exert membrane-initiated effects. ERα is the major mediator of many estrogenic effects in the body and palmitoylation of cysteine 451 (C451) in the murine ERα is required for association of the receptor to the membrane .All animal experiments were approved by the Gothenburg Ethical Committee for Animal Research. Transgenic C451A mice with a point mutation at the palmitoylation site C451 in ERα has been described before (Ovariectomy was performed on 12-week-old C451A and WT (control) littermate mice. After one-week recovery the mice received daily subcutaneous injections for 3 weeks with either vehicle , 17β-estradiol-3-benzoate (estradiol (E2); 0.3 μg/mouse/day; Sigma-Aldrich), Lasofoxifene , or Bazedoxifene . The doses were chosen based on the substances’ ability to protect against ovariectomy-induced bone loss  were performed using a Lunar PIXImus mouse densitometer .High-resolution microcomputed tomography (μCT) analysis was performed on the vertebra (L5) and femur using an 1172 model μCT  as previously described (Igf1: Mm00439559_m1), progesterone receptor (Pgr: Mm00435628_m1), lactotransferrin (Ltf: Mm00434787_m1), cytokeratin 8 (Krt8: Mm04209403_g1), and 18S: (4310893E). The relative gene expression values were calculated using the ΔΔCt method.RNA was isolated from uterus using the RNeasy Mini Kit (Qiagen). The RNA was reversed transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit . Amplifications were performed using the Applied Biosystem StepOnePlus Real-Time PCR System (ThermoFisher Scientific) and Assay-on-Demand primer and probe sets (ThermoFisher Scientific), labeled with the reporter fluorescent dye FAM. Predesigned primers and probes labeled with the reporter fluorescent dye VIC, specific for 18S ribosomal RNA, were included in the reaction as an internal standard. The assay identification numbers were insulin-like growth factor-1 . In the tables, values are given as mean ± s.e.m. The statistical differences between veh and E2, Las, and Bza were calculated using one-way ANOVA followed by Dunnett’s post hoc test separately for each genotype (GraphPad Prism version 9.2.0). To determine if there was a statistically significant difference in the treatment responses between C451A and control mice, the interaction P value from two-way ANOVA for each treatment was used. Logarithmic transformations were used when appropriate to ensure normal distribution of data.In the figures, all individual values are presented with mean  and P  < 0.001, interaction Pvalue from two-way ANOVA). In contrast, treatment with the SERMs Las and Bza increased the uterus weight to a similar extent in controls and C451A mice , were unchanged in both control and C451A mice after treatment with E2 or SERMs . The perP = 0.08), while no significant treatment effects were found for any of the treatments in C451A mice  signaling is required for normal E2 effects on the uterus. This statement is supported by studies using mice with a deletion of activation function two in ERα, which is required for nuclear actions of ERα .This work was supported by the Swedish Research Council (2017-01286), the Swedish state under the agreement between the Swedish government and the county councils (ALF-agreement) (ALFGBG721581), the Gustaf V 80-years fund (FAI-2018-0466), the Swedish Rheumatism Association (R664771), the Emil and Wera Cornell Foundation (100.Islander), the IngaBritt and Arne Lundberg Foundation (LU2017-0076), the Ragnar Söderberg’s Foundation (M133/12), the Knut and Alice Wallenberg Foundation (2015-0317), and the K L G, C O and M K L conducted the study design. K L G, S M S, H H F, C E, P H, K H N, J M S, E S, U I and M K L were responsible for acquisition of data and K L G, M K L, E R L, and C O performed the analysis and interpretation of data. M K L, K L G and C O wrote the main manuscript text and K L G and M K L prepared the figures. All authors reviewed the manuscript.


Reaching the K2S phase in potassiation is the essential reason for obtaining the ultrahigh capacity. Nonetheless, systematic kinetics analyses demonstrate that the K2S involved depotassiation deteriorates the charge kinetics. The density functional theory (DFT) calculation revealed that the nitrogen-rich micropore surface facilitated the sulfur reduction for K2S but created a higher energy barrier for the K2S decomposition, which explained the discrepancy in kinetics modification effect produced by the porosity engineering.Potassium–sulfur batteries (KSBs) are regarded as a promising large-scale energy storage technology, owing to the high theoretical specific capacity and intrinsically low cost. However, the commercialization of KSBs is hampered by the low sulfur utilization and notorious shuttle effect. Herein, we employ a porosity engineering strategy to design nitrogen-rich carbon foam as an efficient sulfur host. The tremendous micropores magnify the chemical interaction between sulfur species and the polar nitrogen functionalities decorated carbon surface, which significantly improve the sulfur utilization and conversion. Meanwhile, the abundant mesopores provide ample spaces, accommodating the large volume changes of sulfur upon reversible potassation. Resultantly, the constructed sulfur cathode delivers an ultrahigh initial reversible capacity of 1470 mAh g A cathode ,7,8,9,10 cathode ,14,15,16elopment . Consequelopment ,19,20,21elopment . For insrication .−30 S cm−1) and the severe volume expansion (up to 300%) of LSBs and NSBs. Second, analogous to LSBs and NSBs, long-chain potassium polysulfides (KPSs) can be dissolved in electrolytes, especially ether-based electrolytes. Soluble KPSs will shuttle to the anode side, leading to the loss of active material, rapid capacity decay, and poor cycle stability, which is the notorious shuttle effect. Moreover, the K2S3 phase with low solubility in ether-based electrolytes is easily deposited and the further potassiation of K2S3 is a solid-phase reaction [2S2 and K2S on the cathode, causing sluggish kinetics and terminating the discharge process with high overpotential [Like the LSBs and NSBs, KSBs also face several critical problems ,25,26,27reaction . This maotential . All of To address the aforementioned issues, the design of cathode materials for KSBs is critical. Similar to LSBs and NSBs, various carbonaceous materials with excellent electrical conductivity, precisely controlled porosity, low cost, and simple preparation have been used to host sulfur for KSBs ,30. The 2 activation. The enormous micropores enhance the chemical interaction between sulfur species and carbon host decorated with polar nitrogen functionalities, which greatly facilitate the utilization and conversion of sulfur. Furthermore, the rich mesopores provide sufficient spaces to accommodate the huge volume expansion of sulfur during reversible potassation. The S@P-NCF cathode delivers excellent electrochemical performances with an ultrahigh reversible specific capacity of 1470 mAh g−1  and a superior rate capacity of 560 mAh g−1 at 2 C. More K2S phase in the discharge product is the reason for the ultrahigh capacity achieved by S@P-NCF. The kinetics analyses show that S@P-NCF exhibits superior discharge kinetics but inferior charge kinetics. DFT calculation suggested that the nitrogen-rich micropore surface boosted the formation of K2S but simultaneously produced a higher energy barrier for the decomposition of K2S, which explains the different modification effect towards K-S redox kinetics by porosity engineering.Herein, we employ a porosity engineering strategy towards nitrogen-rich carbon foam as a distinguished sulfur host (named P-NCF). The P-NCF with abundant porosity was created by pre-pyrolysis lyophilization and post-pyrolysis CO3N4 as both a nitrogen source and sacrificial template in the starch gelatinization enables the preparation of nitrogen-doped carbon materials with abundant pores. Following this methodology, nitrogen-rich carbon foam (NCF) based on porosity engineering strategy (P-NCF) was synthesized and the schematic process is summarized in 3N4 was obtained via pyrolysis of urea in static air [3N4 degrades automatically and creates porous structures and nitrogen heteroatoms in the carbon matrix [3N4 was dispersed in deionized water and heated to 100 °C under vigorous stirring. Subsequently, the starch solution was added into the aforementioned mixture under continuous stirring, and the mixture gradually thickened. After the starch gelatinization process, the viscous mixture is cooled to room temperature and freeze dried to obtain starch xerogel. Subsequently, the starch xerogel was carbonized at 900 °C under Ar atmosphere, and then activated at 800 °C under CO2 atmosphere to form the final product P-NCF. In contrast, the sample without CO2 activation is named as NCF. The final cathode materials S@P-NCF and S@NCF were obtained by an initial sulfur vapor-infiltrated method at 500 °C and a following heat treatment at 200 °C to remove the surficial sulfur. The morphologies of the carbon hosts and sulfur-impregnated composites were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As shown in The introduction of g-Catic air . During n matrix . The g-CP/P0 and an obvious hysteresis loop at medium P/P0 indicate the existence of micropores and mesopores, respectively. The Brunauer−Emmett−Teller (BET) surface areas of P-NCF and NCF are 1167.41 and 619.95 m2 g−1, respectively (3 g−1) than NCF (0.14 cm3 g−1). 2 activation process. Evidently, after sulfur loading, the BET surface area and pore volume of both samples sharply reduced , disorder (D), amorphous (B), and graphitic (G) [ID/IG) can reflect the graphitization degree of carbon [ID/IG values of P-NCF, S@P-NCF, NCF, and S@NCF are 2.26, 2.89, 2.57, and 3.08, respectively  patterns of carbon hosts and composites are presented in itic (G) . The aref carbon . The ID/ectively . The lown degree . Increasn degree . In addiretching . As show1/2 and S 2p3/2. The small peak at 162.4 eV indicates a partial charge transfer between the carbon matrix and sulfur [The composition and surface chemical states were further investigated by X-ray photoelectron spectroscopy (XPS). The peaks of O, N, C, and S elements are observed in the survey XPS spectra of S@P-NCF and S@NCF . High-red sulfur , which cd sulfur . The pead sulfur , demonstd sulfur . The cond sulfur . Therefo6 in EC/DEC = 1/1 by volume) electrolyte. The cyclic voltammograms (CV) curves of S@P-NCF were recorded at a scan rate of 0.1 mV s−1 in the voltage window of 0.5–3.0 V (vs. K+/K) (−1) within the voltage window of 0.5–3.0 V. After the first cycle, two long discharge plateaus at ~1.5 V and ~0.8 V and a long charge plateau at ~1.6 V can be observed, which are consistent with the CV results. Most importantly, S@P-NCF exhibits an ultrahigh initial reversible capacity (1470 mAh g−1), much higher than S@NCF (850 mAh g−1). The above-mentioned results indicate that richer micropores and larger polar surface area enhance the interaction between sulfur and carbon matrix, improve the utilization and conversion of sulfur, and reduce the polarization of S@P-NCF.In order to examine the electrochemical properties of the S@P-NCF cathode, K-S coin cells were assembled utilizing K metal anode, glass fiber separator, and carbonate solution (0.8 M KPFs. K+/K) a. During−1 at 0.2, 0.3, 0.5, 1, and 2 C, respectively. On the contrary, S@NCF only delivers capacities of 680 and 400 mAh g−1 at current densities of 0.2 and 2 C, respectively, much lower than that of S@P-NCF. However, at small current densities S@P-NCF exhibits a more rapid capacity decay than S@NCF. −1. After 100 cycles, the remaining capacity is 832 mAh g−1 and the Coulombic efficiency is ~100%. S@NCF only delivers an initial reversible capacity of 690 mAh g−1 and remaining capacity of 556 mAh g−1 after 100 cycles. The higher specific capacity of S@P-NCF demonstrates that more abundant micropores and larger polar surface area can enhance the interaction between sulfur and carbon matrix, and thus promote the conversion of sulfur species and realize higher utilization of sulfur. In addition, during the cycling process, the rich mesopores provide ample space for the volume expansion of sulfur species [−1. The electrochemical performance of S@P-NCF-based K-S batteries under high areal loading conditions has also been attempted to be measured. −2 at 0.1 C. Of interest is that S@P-NCF delivers a high initial reversible capacity (1100 mAh g−1) even at a high sulfur areal loading of 2.0 mg cm−2. To further demonstrate the stable long-term cycling performance of the electrodes, the cell impedances of S@P-NCF and S@NCF in the charged states after the 1st and 100th cycles were analyzed by electrochemical impedance spectroscopy (EIS) (Rct) through the electrode/electrolyte interface. The equivalent circuit model and fitted electrode resistance data are shown in Rct from 1411 Ω (1st) to 4901 Ω (100th), compared to S@P-NCF from 383.7 to 1429 Ω. The lower Rct of S@P-NCF after the first cycle indicates that porosity engineering and polar nitrogen functionalities provide the fast ion transport and excellent electrical conductivity, which contribute to sulfur utilization and conversion. In addition, the smaller Rct increment of S@P-NCF after 100 cycles indicates the superior cycling stability and structural stability of S@P-NCF. The above electrochemical tests show that S@P-NCF has higher specific capacity, sulfur utilization, and better long-term cycling performance at a high current density than S@NCF. Obviously, the ultrahigh initial reversible capacity of S@P-NCF is related to the tight binding between sulfur in the micropores and carbon matrix, which effectively increases the utilization of sulfur. In addition, the larger polar surface area, higher content of pyridinic-/pyrrolic-N, and more abundant mesopores of P-NCF also make an essential contribution to the high capacity and excellent cycling performance.The rate capabilities of sulfur cathodes were measured at current densities from 0.2 to 2 C c. The S@ species . As showpy (EIS) . The sem−1 of S@P-NCF and S@NCF, respectively. These CV curves exhibit a similar profile, except for a noticeable shift as the scan rate increases. As the scan rate increases, two major peaks  for both S@P-NCF and S@NCF can be observed. The reaction mechanisms of S@P-NCF and S@NCF can be evaluated based on an established formula of log i = b log v + log a, where i is the peak current, v is the scan rate of the CV and b is a constant ranging from 0.5 to 1. When the b value is approaching 0.5 or 1, the system is under the diffusion dominated or surface capacitive-controlled process, respectively [b values of S@P-NCF for Peak-R and Peak-O, which are fitted to be 0.905 and 0.747, respectively. This indicates that the reduction conversion reaction of S@P-NCF is a rapid dynamic pseudocapacitance behavior. The b values of S@NCF for Peak-R and Peak-O are 0.857 and 0.782, respectively  were employed to demonstrate the kinetics of the sulfur cathodes. The CVs at different scan rates were further examined for the in-depth kinetic analyses. ectively . Figure ectively d. Intereetention .2S phase. For S@NCF, a lot of K2S2 nanocrystals can be observed in the HRTEM images  and DFT calculation demonstrate that the microporosity with nitrogen functionalities contribute to the sulfur reduction for the end member of K2S, which nonetheless deteriorates the oxidation (charge) kinetics by increasing the energy barrier of K2S decomposition.In this report, we employ a porosity engineering strategy to a nitrogen-rich carbon foam to construct a sulfur host for high-performance room temperature K-S batteries. The abundant microporosity greatly enlarges the polar surface area of the carbon host, thereby reinforcing the affinity of sulfur species on the electrode, both at the pristine state and during electrochemical cycling. Meanwhile, the mesoporosity create ample room to buffer the large volume change of sulfur upon repeated potassiation. Ultra-high capacity of 1470 mAh g


In this study, C57BL/6J mice were fed a vitamin D-deficient diet for 13 weeks to establish a vitamin D-deficient mice model. The vitamin D-deficient mice were then orally gavaged with vitamin D (VD), folic acid (FA), and vitamin B12 (VB12) alone or together for eight weeks. Following the gavage, the learning and memory ability of the mice were evaluated by Morris Water Maze and Novel object recognition test. The CYP27A1-related gene and protein expressions in the liver and brain were determined by qRT-PCR. The serum level of 27-OHC was detected by HPLC-MS. Serum levels of 25(OH)D, homocysteine (Hcy), and S-Adenosylmethionine (SAM) were measured by ELISA. After feeding with the vitamin D-deficient diet, the mice performed longer latency to a platform (p < 0.001), lower average speed (p = 0.026) in the Morris Water Maze, a lower time discrimination index (p = 0.009) in Novel object recognition, and performances were reversed after vitamin D, folic acid and vitamin B12 supplementation alone or together (p < 0.05). The gene expressions of CYP27A1 in the liver and brain were upregulated in the vitamin D-deficiency (VDD) group compared with the control (CON) group (p = 0.015), while it was downregulated in VDD + VD and VDD + VD-FA/VB12 groups compared with the VDD group (p < 0.05), with a similar trend in the protein expression of CYP27A1. The serum levels of 27-OHC were higher in the VDD group, compared with CON, VDD + VD, and VDD + VD-FA/VB12 group (p < 0.05), and a similar trend was found in the brain. The serum 25(OH)D levels were significantly decreased in the vitamin D-deficiency group (p = 0.008), and increased in the vitamin D-supplemented group (p < 0.001). The serum levels of SAM were higher in the B vitamins-supplemented group, compared with CON and VDD groups (p < 0.05). This study suggests that CYP27A1 expression may be involved in the mechanism of learning and memory impairment induced by vitamin D deficiency. Co-supplementation with vitamin D, folic acid, and vitamin B12 significantly reverses this effect by affecting the expression of CYP27A1, which in turn regulates the metabolism of 27-OHC, 25(OH)D, and SAM.The cholesterol-oxidized metabolite 27-hydroxycholesterol (27-OHC) is synthesized by CYP27A1, which is a key factor in vitamin D and oxysterol metabolism. Both vitamin D and 27-OHC are considered to play important roles in Alzheimer’s disease (AD). The study aims to research the effects of co-supplementation of vitamin D, folic acid, and vitamin B An increasing number of studies have proved that vitamin D deficiency may play an important role in the pathological progression of Alzheimer’s disease (AD) ,2. VitamAs one of the important components of the brain, cholesterol plays an important role in maintaining normal brain functions. Increasingly, studies have confirmed that impaired cholesterol homeostasis is involved in the development of AD, epidemiological evidence revealed that individuals with a high level of blood cholesterol are more likely to develop AD, and animal experiments showed that high cholesterol diets induced AD-like pathology . HoweverCYP27A1 is an enzyme involved in the activation of vitamin D and the production of 27-OHC. Vitamin D can be activated by the enzyme CYP27A1 to produce 25-hydroxyvitamin D (25(OH)D), which is then activated by the enzyme CYP27B1 to produce 1alpha,25-dihydroxyvitamin D 2D) ,12. Mean12 also play important roles in AD. Recently, a case-control study investigated serum vitamins and the risk of AD in the Chinese population and found that lower levels of vitamin D and B vitamins were associated with higher risks of AD, especially folic acid and vitamin B12 [12 are inadequate [12 participate in the formation of S-Adenosylmethionine (SAM), which is the methyl donor for methylation reactions and may improve cognitive function in AD [12 co-supplementation may be better than either alone [12 co-supplementation in improving cognitive function need to be further explored, as the three vitamins alone show a limited improvement in cognitive function and remain controversial.In addition to vitamin D, folic acid and vitamin Bamin B12 . A meta-amin B12 . Homocysadequate . Folic aon in AD . A retroer alone . The syn12 co-supplementation may play a joint role in the process by regulating the levels of Hcy and SAM. The results of this study may provide a new direction for the discovery of the mechanism involved in vitamin D deficiency and AD. Hence, we hypothesized that vitamin D deficiency may lead to a decline in learning and memory ability by affecting CYP27A1. Vitamin D intervention can downregulate the expression of CYP27A1, then reduce the levels of 27-OHC, which then reverses the decline in learning and memory ability induced by vitamin D deficiency. Besides, folic acid and vitamin BMale C57BL/6J mice used in this study were around 8 months old and were purchased from Animal Research Center of Capital Medical University . The mice were housed in a SPF facility with light/dark cycles (12 h/12 h), temperature-controlled (22 ± 2 °C), with free access to water. All animal experiments were performed after receiving approval from the ethics committee of Capital Medical University . 12 supplemented group (VDD + FA/VB12), Vitamin D deficient and vitamin D supplemented group (VDD + VD), Vitamin D deficient and vitamin D, folic acid/vitamin B12 supplemented group (VDD + VD-FA/VB12). Control mice were fed an AIN93 standard diet, and VDD mice were fed a vitamin D-deficient diet for 13 weeks to establish a vitamin D-deficient mice model [12 (80 ug/kg), respectively, by oral gavage, the control group was maintained with the same volumes of saline and corn oil, four times a week and this lasted for 8 weeks. The flow chart is shown in After adaptive feeding for 2 weeks, forty mice were randomly and equally divided into five groups based on initial body weights evenly. The five groups are as follows: Control group (CON), Vitamin D deficient group (VDD), Vitamin D deficient and folic acid/vitamin Bce model . FollowiMorris water maze test was used to determine the spatial learning and memory ability of the mice . A whiteNovel object recognition was used to detect the short-term memory of mice . In the g for 10 min. Finally, a total of 75 µL sample was injected into the HPLC-MS system and the quantification of 27-OHC was performed.The concentration of 27-OHC in serum and brain was measured using HPLC–MS . In brieg for 15 min at 4 °C and stored at −80 °C until measurement. Then the ELISA kits were used to measure serum 25(OH)D , serum Hcy , and serum SAM  in accordance with the manufacturers’ instructions.The serum levels of 25(OH)D, Hcy, and SAM were detected by ELISA according to previously published research ,27. BlooThe total mRNA was extracted from livers and brains by using the SV Total RNA Isolation System  followed by reverse transcription using the Reverse Transcription System . qRT-PCR was used to analyze the mRNA expression levels of CYP27A1, CYP27B1and VDR in liver and brain. Primers were designed specifically based on the NCBI database, the forward and reverse primer sequences used in this study are shown in g for 10 min at 4 °C. The supernatant was reserved and the protein concentrations were determined with BCA assay. An equal amount of protein was separated through SDS-PAGE running and then transferred to PVDF membranes . The antibodies used were as follows: CYP27A1 1:5000 , CYP27B1 1:1000 , VDR 1:1000 , β-actin 1:5000 , and Goat anti-rabbit IgG 1:3000 . Fluorchem FC 2 software was used to analyze the image and the relative expression of target proteins and normalized to β-actin protein.Liver or brain tissues were lysed with RIPA buffer containing protease inhibitor (PMSF) on ice for 30 min and centrifuged at 12,000× p < 0.05 was considered to be statistically significant.SPSS 23.0 and GraphPad Prism 9.1.1 were used for statistical analysis. All data were expressed as mean ± standard error (mean ± SEM). One-way ANOVA  and least significance difference post-hoc test were used to evaluate the significant differences between groups. Two-way ANOVA analysis was used for repeated measurement data in Morris water maze test. p < 0.001, p < 0.001). Compared with the CON group, the escape latency of the VDD (p < 0.001), VDD + FA/VB12 (p < 0.001), VDD + VD (p = 0.001), and VDD + VD-FA/VB12 (p = 0.001) groups were significantly increased on day four and dayfive. On day four, the escape latency of the mice in the VDD group (p = 0.035) and VDD + FA/VB12 (p = 0.002) group, but not the VDD + VD group and VDD + VD-FA/VB12 group, were significantly longer than the CON group. On day five, only the VDD group (p = 0.036) had a higher escape latency compared to the CON group. There were no interaction effects between factors of training time and group in the training period . The average speed of mice was different among groups (p = 0.019), significantly slower speeds were observed in the VDD (p = 0.049), VDD + FA/VB12 (p = 0.001), and VDD + VD-FA/VB12 groups (p = 0.014) in comparison to the CON group (Ps p > 0.05)  c–e. The p < 0.001). Compared with the CON group, the TDI of the VDD group (p = 0.001) was significantly decreased, while VDD + FA/VB12 (p = 0.001), VDD + VD (p = 0.001), and VDD + VD-FA/VB12 (P = 0.048) groups revealed significant increased TDI. Mice in VDD + FA/VB12, VDD + VD, and VDD + VD-FA/VB12 groups had higher TDI than that in the VDD group (p < 0.001). Additionally, although no significant difference was found in FDI among groups, VDD + FA/VB12, VDD + VD and VDD + VD-FA/VB12 groups showed higher FDI than the VDD group. These results revealed that the supplement of vitamin D and/or B vitamins reversed the decline in short-term memory due to vitamin D deficiency.The novel object recognition test was performed to measure the short-term memory of the mice. Details of the results are shown in p = 0.002). The serum 25(OH)D levels of the VDD + VD group and VDD + VD-FA/VB12 group were significantly higher than the VDD group and VDD + FA/VB12 group (p < 0.001). The serum Hcy level of the VDD group was higher than the other groups and VDD + FA/VB12 group was lower, however, the differences were not statistically significant. The serum level of SAM showed a significant increase in the VDD + FA/VB12 group and VDD + VD-FA/VB12 group compared with the CON group  and VDD group . These results suggested that vitamin D supplementation increased the serum 25(OH)D concentrations in all treated mice. B vitamins supplementation alone or in combination with vitamin D also increased serum SAM levels.To investigate the mechanisms by which vitamin D and B vitamins restore memory capacity in mice, we measured the levels of their related factors in the serum. As shown in p = 0.020) and decreased in VDD + FA/VB12 group (p = 0.030). The 27-OHC concentration in VDD + FA/VB12 group (p = 0.005), VDD + VD group (p = 0.008), and VDD + VD-FA/VB12 group (p = 0.031) was significantly decreased when compared with that in the VDD group. The 27-OHC level in the brain performed a similar trend to that in serum, although it was not statistically significant. The above results demonstrated that the supplementation of vitamin D and/or B vitamins may inhibit the increase of 27-OHC induced by vitamin D deficiency.In order to investigate the mechanism of memory improvement in vitamin D-deficient mice by vitamin D and/or B vitamins co-supplementation, the concentration of 27-OHC in serum and brain was measured for its important role in the progress of AD. As shown in p = 0.015) was upregulated, while the VDR (p = 0.032) was downregulated in the liver of mice in the VDD group. Compared with the VDD group, the mRNA expression of CYP27A1 and CYP27B1 were downregulated in the VDD + FA/VB12 group , VDD + VD group  and VDD + VD-FA/VB12 group  in the liver, while the mRNA expression of VDR was upregulated in the VDD + FA/VB12 group (p = 0.003) and VDD + VD-FA/VB12 group (p = 0.005). The mRNA expression of CYP27A1 and CYP27B1 were upregulated in the VDD group (p < 0.001), VDR had a decreasing trend compared with the CON group in the brain; while the mRNA expression of CYP27A1  and CYP27B1  were downregulated, VDR  was upregulated in the VDD + VD group and VDD + VD-FA/VB12 group compared with the VDD group in the brain. These results revealed that the mRNA expression of CYP27A1 and CYP27B1 were upregulated, while VDR was downregulated in the liver and brain of mice under vitamin D deficient conditions. Intervention using vitamin D alone or in combination with B vitamins may reverse this situation.Vitamin D and 27-OHC metabolism-related gene expressions in the liver and brain were significantly different among groups. Results are shown in p = 0.021) were upregulated, and CYP27B1 protein expression had a similar trend in the liver. Compared with the VDD group, the protein expression of CYP27A1 was downregulated in the VDD + VD group (p = 0.006) and VDD + VD-FA/VB12 group (p = 0.001) in the liver. The protein expression CYP27B1 was upregulated in the VDD group (p < 0.001) and VDR was downregulated compared with the CON group in the brain, CYP27A1 also had an increased expression trend in the VDD group. The protein expression of VDR was upregulated in the VDD + VD group (p = 0.013) and VDD + VD-FA/VB12 group (p = 0.010) compared with the VDD group in the brain. Overall, the protein expression of CYP27A1, CYP27B1, and VDR followed a similar trend to mRNA expression.In order to clarify the effect of vitamin D deficiency and intervention on the above indicators, their expression on protein levels in livers and brains was also detected using western blot. Details of the results are shown in 3) mice exhibited impaired spatial learning and memory ability in 6-month-old APP/PS1 mice, suggesting that vitamin deficiency could accelerate cognitive impairment [3) mice could induce impairment in learning and memory ability, and longer latency to find the platform [Vitamin D deficiency is a widespread nutritional deficiency. Among the elderly Chinese population, 34.1% of men and 44.0% of women have vitamin D deficiency according to the analysis of the China national nutrition and health survey (CNNHS) . Vitaminpairment . The resplatform . In maleplatform . These s12 co-supplementation may have the same or even better effect than vitamin D supplementation alone. An animal study observed that vitamin D deficiency damaged neurogenesis both in wild-type and 5XFAD transgenic mice, and high vitamin D supplementation was efficient in improving learning and memory ability and neurogenesis in the brain [12 group slowed down the decline of overall cognitive function to a certain extent, in addition to preventing osteoporosis [12, and it was also confirmed in this study. Thus, the co-supplementation of vitamin D, folic acid, and vitamin B12, may have a better positive effect than vitamin D supplementation alone in cognitive function, and it should be further explored and verified in population research.Therefore, intervention in vitamin D deficiency may play an important role in preventing AD at the early stages. In the present study, the results showed that vitamin D supplementation in vitamin D-deficient mice could reverse the impaired ability of learning and memory. Moreover, we found that vitamin D, folic acid, and vitamin Bhe brain . Previouoporosis . Althoug12 were supplemented in the diets. In contrast, expression of VDR was downregulated in mice consuming a vitamin D-deficient diet and upregulated after vitamin D, folic acid, and vitamin B12 supplementation. A previous study using high-fat diet animal models found that vitamin D deficiency could also upregulate the expression of CYP27A1, and vitamin D supplements reversed it, which suggests that there may be an interaction between high cholesterol and vitamin D [3) mice performed downregulated expression of VDR, then further reduced the biological effects of vitamin D, consistent with our results [Vitamin D can convert cholecalciferol to 25(OH)D by the enzyme CYP27A1, while, 25(OH)D is further hydroxylated by CYP27B1 to produce the active vitamin D metabolite . Vitaminitamin D . CYP27B1itamin D . An anim results . TherefoBesides, as a cholesterol-oxidized metabolic enzyme, CYP27A1 is also critical for the synthesis of 27-OHC, which is one of the most important risk factors in the pathology of AD. A meta-analysis indicated that the increased levels of 27-OHC may be a potent biomarker for the evaluation or recognition of MCI and AD . Meanwhi12 deficiencies, which induced potential negative consequences on cognitive function [12 in vitamin D-deficient mice and showed that co-supplementation produced better results in improving cognition and increasing serum levels of SAM. SAM was found to decrease in the population with AD in a cross-sectional study [12 might alleviate bioenergetic defects and oxidative stress, and delay beta-induced paralysis by regulating the methionine/SAM cycle [25(OH)D is often measured in serum to determine vitamin D status. In the presence of vitamin D deficiency, the level of 25(OH)D could decrease significantly, which was further confirmed in the present study. A large-scale aging cohort study found that the aging population has a high burden of vitamin D, folic acid, and vitamin Bfunction . Therefoal study . An animal study . A randoal study . AnotherAM cycle . Thus, a12 having been widely considered as potential treatment strategies for cognitive impairment, no study has been conducted to investigate their potential interactions, especially in an animal model [12 co-supplementation on learning and memory ability in vitamin D-deficient mice. The results demonstrated that supplementation with vitamin D, folic acid, and vitamin B12 alone or together was efficient in improving learning and memory ability in vitamin D-deficient mice, and co-supplementation may be more effective. Besides, the current study provided a new perspective that CYP27A1 may be a mediator for vitamin D, 27-OHC, SAM, and learning and memory ability. However, whether CYP27A1 plays a decisive role in vitamin D-related pathology of AD needs to be verified in further studies. A graphic abstract is shown in To the best of our knowledge, despite single vitamin D, folic acid, and vitamin Bal model ,44,45. H12 may collectively exert effects on learning and memory ability by lowering the expression of CYP27A1 and regulating the levels of 27-OHC and SAM. These findings will provide further insights into the involvement of vitamin D, folic acid, and vitamin B12 co-supplementation in cognitive function, and the potentially significant role of the key factor CYP27A1 in the pathogenesis of MCI and AD. In addition, this study could also be referenced in the development of strategies to address vitamin D deficiency and prevent or mitigate adverse consequences. In summary, the present study indicated that vitamin D deficiency was closely associated with learning and memory impairment in mice, probably in part by inducing 27-OHC metabolism disorders through upregulating the expression of CYP27A1. A combined supplementation of vitamin D, folic acid, and vitamin B


We assessed HIV responses in persons with HIV pre antiretroviral therapy (ART) (n = 17) and post-ART (n = 18) and compared these responses to 15 controls without HIV . Using the Activation Induced Marker (AIM) assay, we quantified HIV-specific total CD4+, memory CD4+, circulating T follicular helper, total CD8+ and memory CD8+ T cells. We also compared the Class I and Class II HIV MPs to commercially available HIV gag peptide pools. Overall, HIV Class II MP detected HIV-specific CD4+ T cells in 21/35 (60%) HIV positive samples and 0/15 HIV negative samples. HIV Class I MP detected an HIV-specific CD8+ T cells in 17/35 (48.6%) HIV positive samples and 0/15 HIV negative samples. Our innovative HIV MPs are reflective of the entire HIV genome, and its performance is comparable to other commercially available peptide pools. Here, we detected HIV-specific CD4+ and CD8+ T cell responses in people on and off ART, but not in people without HIV.Reagents to monitor T cell responses to the entire HIV genome, based on well characterized epitopes, are missing. Evaluation of HIV-specific T cell responses is of importance to study natural infection, and therapeutic and vaccine interventions. Experimentally derived CD4 The HIV RNA load was 120,000 copies/ml  with a median estimated date of infection of 98 days  at the time of sample collection. The post-ART median CD4+ T cell count was 825 cells/mm3 . The HIV RNA load was below the limit of detection with a median of 667 days  from beginning ART and a median of 236 days [162–644] from starting ART till an undetectable HIV viral load (VL).Men who have sex with men with HIV were enrolled, from whom blood was taken pre-ART (n = 17) and post-ART (n = 18) . One chronically infected person had a detectable VL of 9,695 copies/mL at time of blood draw. The duration of ART for the 10 early infected patients was 551 days (range of 368 to 762 days from time of ART start date to date of blood draw). None of the early infected persons had detectable VL.+ T cells in HIV positive and HIV negative samples. In HIV-specific CD4+ T cells, there is lower 41BB expression, attributed to T-cell exhaustion from chronic infection which precludes use of 41BB by AIM assay [+ T cells by a combination of OX40 and PD-L1 by AIM assay  [+ T cells responses were detected for pre-ART (p = 0.0012) and post-ART samples (p = 0.013) compared to HIV negative samples . There was no difference in magnitude of responses between paired pre- and post-ART samples (p = 0.24). With a limit of sensitivity of 0.022%, higher proportions of responders were detected for pre-ART  and post-ART  samples compared to HIV negative samples (2/15); there was no difference in the proportion of responders detected between paired pre- and post-ART samples (p = 0.51). Compared to HIV negative samples, this limit yielded a sensitivity of 57.14% and a specificity of 86.67%.Using a Class II HIV MP consisting of immunodominant epitopes from the entire HIV proteome, we first tested its ability to detect HIV-specific CD4IM assay . AdditioIM assay . We thus . Higher + T cells. We identified HIV-specific total memory CD4+ T cells and circulating T follicular helper (Tfh) cells. We defined total memory CD4+ T cells as a combination of central memory + effector memory + TEMRA . Higher magnitudes of HIV-specific memory CD4+ T cell responses were detected in both pre-ART (p = 0.005) and post-ART HIV-infected individuals (p = 0.025) compared to HIV negative individuals . There was no difference in the magnitudes of responses between paired pre and post-ART samples (p = 0.23). There was a higher proportion of responders pre-ART  and post-ART  samples compared to HIV negative samples (2/15). There was no difference in the proportion of responders detected between paired pre- and post-ART samples (p = 0.51).As the memory T cell compartment is important in chronic infection, we quantified memory CD4+ T cells which provide help to B cells to promote the development high affinity antibodies [+CD45RA- CD4+ T cells . Similarly, higher magnitudes of HIV-specific cTfh tells were detected in pre-ART (p = 0.0001) and post-ART HIV-infected individuals (p = 0.0003) compared to HIV negative individuals . There was no difference in the magnitudes of responses between paired pre and post-ART samples (p = 0.19). There was no difference in the proportion of responders pre-ART  and post-ART  samples compared to HIV negative samples (1/15). There was no difference in the proportion of responders detected between paired pre- and post-ART samples (p = 1.00).Circulating Tfh (cTfh) cells are CD4tibodies . We defi+ T cells in HIV positive and HIV negative samples. By co-expression of CD69 and PD-L1 by AIM assay , we detected HIV-specific CD8+ T cells. The Class I HIV MP detected higher magnitudes of HIV-specific CD8+ T cell responses were detected in pre-ART (p = 0.009) samples but comparable post-ART HIV-infected individuals (p = 0.25) compared to HIV negative individuals . With a limit of sensitivity of 0.025%, there was no difference in the magnitudes of responses between paired pre and post-ART samples (p = 0.73). There was a higher proportion of responders pre-ART  but comparable proportion of responders post-ART  samples compared to HIV negative samples (2/15). There was no difference in the proportion of responders detected between paired pre- and post-ART samples (p = 0.22).Using a Class I HIV MP consisting of immunodominant epitopes from the entire HIV proteome, we tested its ability to detect HIV-specific CD8+ T cells, HIV Class I MP detected comparable magnitudes of HIV-specific memory CD8+ T cell responses in pre-ART (p = 0.22) and post-ART HIV samples (p = 0.061) compared to HIV negative individuals . There was no difference in the magnitudes of responses between paired pre and post-ART samples (p = 0.84). There was a comparable proportion of responders pre-ART  and post-ART  samples compared to HIV negative samples (1/14). There was no difference in the proportion of responders detected between paired pre- and post-ART samples (p = 1.00). The Class I HIV MP had a 47.1% sensitivity and 86.7% specificity.For memory CD8+ T cells, memory CD4+ T cells, and cTfh were comparable when using the Class II MP, JPT gag, or NIH gag peptide pools , as clade B is predominantly found in North America and Europe. The frequencies of HIV-specific total CD8+ T cells and memory CD8+ T cells were also comparable .The detection of gag-specific T cells is the most common way to quantify HIV-specific T cells. We therefore compared the Class I and II HIV MP to 2 commercially available clade B gag peptide pools from JPT and NIH. The frequencies of HIV-specific CD4+ T-cells. The magnitudes of CMV-specific CD4+ T cells were comparable pre- and post-ART for total CD4+ T cells, memory CD4+ T cells, and cTfh . Using the Class I CMV MP, we additionally tested the magnitudes of CMV-specific CD8+ T cells pre- and post-ART. There was no difference between the magnitudes of total and memory CMV-specific CD8+ T cell among pre-ART and post-ART specimens and HIV negative individuals .To demonstrate our ability to detect antigen-specific T cells, as a control, we tested for CMV-specific T cells in these same specimens , 26. Usi+, memory CD4+ T, cTfh, CD8+ T, or memory CD8+ cells in 20/21 (95%) of PWH. Prior studies have demonstrated that 81% of individuals with acute HIV infection had detectable gag-specific CD4+ T cells [+ T cells [+ T cells. Using the HIV MPs, we were unable to detect HIV-specific T cells or their subsets in 1 pre-ART specimen and 3 post-ART specimens. This is consistent with prior studies, in which ART treated individuals have lower frequencies of HIV-specific CD4+ and CD8+ T cells [+, memory CD4+ T, cTfh, CD8+ T, or memory CD8+ cells. Overall, our HIV Class I and Class II MP were comparable in their ability to detect HIV-specific responses to the gag peptide pools alone, regardless of acute or chronic infection.The HIV MP was designed to encompass all HIV proteins instead of only gag, as other HIV proteins such as nef , 27, 28, T cells  whereas  T cells , 31. For+, memory CD4+ T, cTfh, CD8+ T, or memory CD8+ cells in 21/21 (100%) of PWH.PWH are almost universally co-infected with CMV . Our HIV+ T cells vary with duration of viremia and therefore antigen exposure, particularly if the patient is suppressed on ART which removes detectable antigen from the bloodstream versus an elite controller [Limitations of this study include a small sample size comprising entirely of men who have sex with men. These individuals comprised both acute infections which were treated early and chronic infections. The frequency of HIV-specific CD4ntroller . Our coh+ and CD8+ T cell dynamics. The AIM assay allows for easy quantification of antigen-specific T cells. This methodology permits sorting of live antigen-specific T cells for further functional analyses, allowing for sequencing of CD4+ and CD8+ subsets, including Tfh cells [+ and CD8+ T cells could be sorted and sequenced in hyperacute infections [In summary, we describe an innovative Class I and Class II HIV MP, reflective of the entire HIV proteome, which can be used in future studies to better understand the full repertoire of HIV-specific CD4fections  and/or Hfections  and compfections .S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Fig+ T cells in pre-ART  and post-ART  specimens, HIV-specific memory CD4+ T cells in pre-ART  and post-ART  specimens, and HIV-specific cTfh cells in pre-ART  and post-ART  specimens. Data was background subtracted. Data presented represents mean ± standard error of the mean. P-values by Wilcoxon matched-pairs signed rank test.Comparison of responses between HIV Class II MP, JPT gag, and NIH gag for HIV-specific total CD4(EPS)Click here for additional data file.S2 Fig+ T cells in pre-ART  and post-ART  specimens and HIV-specific memory CD8+ T cells in pre-ART  and post-ART  specimens. Data was background subtracted. Data presented represents mean ± standard error of the mean. P-values by Wilcoxon matched-pairs signed rank test.Comparison of responses between HIV Class I MP, JPT gag, and NIH gag for HIV-specific total CD8(EPS)Click here for additional data file.S3 Fig+ T cells, (B) memory CD4+ T cells, and (C) cTfh cells. CMV Class I MP detects CMV-specific (D) total CD8+ T cells and (E) memory CD8+ T cells. Data was background subtracted. Data presented represents mean ± standard error of the mean. P-values by Mann-U Whitney test.Class II MP detects CMV-specific (A) total CD4(EPS)Click here for additional data file.S1 DatasetLOD = 0.005.(XLSX)Click here for additional data file.