TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) was found to increase Alzheimer’s disease (AD) risk. This mutation is located at the cleavage site of TREM2 extracellular domain. Ectopic expression of TREM2-H157Y in HEK293 cells resulted in increased TREM2 shedding. However, the physiological outcomes of the TREM2 H157Y mutation remain unknown in the absence and presence of AD related pathologies.The rare p.H157Y variant of Trem2 H157Y knock-in mouse model through CRISPR/Cas9 technology and investigated the effects of Trem2 H157Y on TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathologies by conducting biochemical assays, targeted mass spectrometry analysis of TREM2, hippocampal electrophysiology, immunofluorescent staining, in vivo micro-dialysis, and cortical bulk RNA sequencing.We generated a novel Trem2 H157Y increases TREM2 shedding with elevated soluble TREM2 levels in the brain and serum. Moreover, Trem2 H157Y enhances synaptic plasticity without affecting microglial density and morphology, or TREM2 signaling. In the presence of amyloid pathology, Trem2 H157Y accelerates amyloid-β (Aβ) clearance and reduces amyloid burden, dystrophic neurites, and gliosis in two independent founder lines. Targeted mass spectrometry analysis of TREM2 revealed higher ratios of soluble to full-length TREM2-H157Y compared to wild-type TREM2, indicating that the H157Y mutation promotes TREM2 shedding in the presence of Aβ. TREM2 signaling was further found reduced in Trem2 H157Y homozygous mice. Transcriptomic profiling revealed that Trem2 H157Y downregulates neuroinflammation-related genes and an immune module correlated with the amyloid pathology.Consistent with previous in vitro findings, Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD risk, we speculate TREM2 H157Y in humans might increase AD risk through an amyloid-independent pathway, such as its effects on tauopathy and neurodegeneration which merit further investigation.Taken together, our findings suggest beneficial effects of the The online version contains supplementary material available at 10.1186/s13024-023-00599-3. TREM2) was identified from a relatively small number of carriers and conferred an increased AD risk with an odds ratio (OR) of 11.01 , 0.4%) in a Han Chinese cohort e domain , 17. Thee domain , 18, 19 e domain , 21. In e domain –24. Ectoe domain , 24. Thee domain  and decrTrem2 H157Y knock-in mouse model through CRISPR/Cas9 technology, and investigated effects of this mutation on TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology. We found Trem2 H157Y increased TREM2 shedding, and enhanced synaptic plasticity. In the presence of amyloid pathology, Trem2 H157Y accelerated Aβ clearance, and reduced amyloid burden, toxic Aβ oligomer, dystrophic neurites, and gliosis at the late stage of amyloid pathology. Trem2 H157Y was also found to increase TREM2 shedding and reduce TREM2 signaling in the presence of Aβ. In line with these observations, transcriptomic analysis revealed downregulated immune responses which was correlated with reduced amyloid pathology in the Trem2 H157Y homozygous knock-in mice. Overall, our results imply that Trem2 H157Y might be beneficial to brain function and in reducing amyloid pathology and related toxicity.To address this, we generated a novel Trem2 H157Y knock-in mice were generated via CRISPR/Cas9 by the Hope Center Transgenic Vectors Core of the Washington University [http://crispor.tefor.net/) and synthesized as gBlocks  with the sequence 5’GGAGGTGCTGTgTTCCACTT3’. In vitro target specific gRNA cleavage activity was validated by transfecting N2A cells with PCR amplified gRNA gblock and Cas9 plasmid DNA  using ROCHE Xtremegene HP. Cell pools were harvested 48 h later for genomic DNA prep, followed by sanger sequencing of PCR products spanning the gRNA/Cas9 cleavage site, and TIDE analysis (https://tide.nki.nl/) of sequence trace files. CRISPR sgRNA  and Cas9  proteins were complexed to generate the ribonucleoprotein (RNP) for injection along with a 200 nucleotide ssODN donor DNA , 5’tatatcttgtcctttgctgatctgtttgccctgggacctccatcc gcagtcactgccagggggtctaagaagggaccactactgtacCTGGAGGTGCTGTaTTCCACTTGGGCACCCTCGAAACTCGATGACTCCTCGGGGACCCAGAGATCTCCAGCATCTTGGTCATCTAGAGGGTctgtaatagacaaaccatgagg3’. All animal work were approved by institutional IACUC protocols. B6/CBA F1 mice at 3–4 weeks of age  were superovulated by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin, followed 48 h later by intraperitoneal injection of 5 IU human chorionic gonadotropin . Mouse zygotes were obtained by breeding B6/CBA stud males with superovulated B6/CBA females at a 1:1 ratio. One-cell fertilized embryos were injected into the pronucleus and cytoplasm of each zygote. Microinjections and mouse transgenesis experiments were performed as described previously [http://crispor.tefor.net/) [iversity . CRISPR eviously , 27. Fouor.net/) . Three pTrem2 in all the mice used for experiments was characterized by quantitative PCR (qPCR) with Custom TaqMan SNP Genotyping assays (Thermo Fisher). All the mice were housed in a temperature-controlled environment with a 12-h light–dark cycle and free access to food and water. All animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.The genotype of Trem2 H157Y homozygous mice (Trem2H157Y/H157Y) were crossed with 5xFAD mice  to obtain the 5xFAD; Trem2H157Y/+ offspring. 5xFAD; Trem2H157Y/+ mice were used to setup breeding cages to establish the littermate cohorts with three genotypes including 5xFAD; Trem2+/+, 5xFAD; Trem2H157Y/+, and 5xFAD; Trem2H157Y/ H157Y. The genotype of 5xFAD mice was characterized through probe-based qPCR with the protocol provided by the Jackson Laboratory. All the 5xFAD mice used as breeders or in our experimental cohorts were hemizygous.Trem2 H157Y homozygous mice and WT mice at 6 months of age as previously described [2PO4, 28 mM NaHCO3, 0.6 mM sodium ascorbate, 5 mM glucose, 7 mM MgCl2 and 0.5 mM CaCl2. Field excitatory post-synaptic potentials (fEPSPs) were obtained from area CA1 stratum radiatum with the use of a glass microelectrode (4-6 mΩ) filled with artificial cerebrospinal fluid (aCSF) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM glucose, 1 mM MgCl2 and 2 mM CaCl2. fEPSPs were evoked through stimulation of the Schaffer collaterals using a 0.1 ms biphasic pulse delivered every 20 s. After a consistent response to a voltage stimulus was established, to obtain the profile for input–output curve (I/O curve), the voltage was increased from 0 mV with a step of 0.5-1 mV for 30 sweeps and the inter-sweep interval is 5 s. For each individual slice, all other stimulation paradigms were induced at the same stimulus voltage which produces the 50–60% of the maximum fEPSP amplitude. Paired-pulse facilitation (PPF) was induced with paired-pulses given with an initial delay of 20 ms and the inter-pulse interval incrementally increased 20 ms until a final delay of 400 ms was reached. The fEPSP baseline responses were then recorded for 20 min. The tetanus used to evoke LTP was a theta-burst stimulation (TBS) protocol consisting of five trains of four pulse bursts at 200 Hz separated by 200 ms, repeated six times with an inter-train interval of 10 s. Following TBS, fEPSPs were recorded for 60 min.Electrophysiological recordings were performed with littermates of escribed  with minAll analyses were performed by customized programming in MATLAB (2021b). The fEPSP slope was calculated within the first 1 ms of the descending domain. I/O curve was presented as the fEPSP slope versus fiber volley amplitude responding to increasing stimulus intensities. PPF strength was examined by the ratio of the second fEPSP slope to the first fEPSP slope in each stimulation pair. Long term potentiation profile was assessed in each minute before and after TBS as the mean fEPSP slope normalized to the mean fEPSP slope of baseline recordings.Cortical cells from pups (p1-p3) were isolated, filtered with 100 μm cell strainers , and plated in T75 flasks  with high-glucose DMEM medium  containing 10% Fetal Bovine Serum (FBS). Medium was changed to medium containing 25 ng/mL recombinant mouse GM-CSF  the next day. Tails from each pup were kept for genotyping. Five days after cell plating, medium in each flask was replaced with fresh GM-CSF-containing medium. On day 9 or 10, microglia were collected by shaking the flasks at 200–220 rpm at room temperature (RT) for ~ 20 min, resuspended in non-GM-CSF containing medium, and plated into 6-well plates. After 24 h, medium from each well was collected as conditioned medium. Cells were lysed with RIPA buffer  supplemented with protease inhibitor  and phosphatase inhibitor  followed by mild agitation at 4 °C for 30 min and centrifugation at 20,000 g at 4 °C for 30 min. Supernatant was collected as RIPA lysate.- conjugated beads  followed by magnetic sorting for CD11B+ microglia. Around 300 K microglia were captured per sample and lysed with RIPA buffer supplemented with the protease and phosphatase inhibitors followed by mild agitation at 4 °C for 30 min and centrifugation at 20,000 g at 4 °C for 30 min. Supernatant was collected as RIPA lysate and subject to Western blotting.Microglia were isolated from adult mouse brains as previously described . In brieBlood samples were collected from mice vena cava after isoflurane induced deep anesthesia, stored at 4 °C overnight and subsequently centrifuged at 1000 g for 10 min to collect the supernatant as serum. After blood collection, mice were transcardinally perfused with 0.01 M PBS and the brains were dissected out. Half of the brain was fixed in 4% paraformaldehyde  for 24 h, dehydrated with 30% sucrose (Sigma) for 48 h, embedded in O.C.T. compound (SAKURA) and snap-frozen in liquid nitrogen before cryostat sectioning. The other hemisphere was dissected into cortex, hippocampus, midbrain, and cerebellum which were snap-frozen in liquid nitrogen and stored at -80 °C. The cortices were then pulverized (CP02 cryoPREP Automated Dry Pulverizer) and divided into two aliquots: 20–30 mg for RNA extraction and 55–65 mg for protein extraction.Cortical proteins were extracted sequentially with different lysis buffers. Cortical powder was homogenized in Tris-buffered saline  supplemented with protease inhibitor  and phosphatase inhibitor  and subjected to ultracentrifugation at 100,000 g at 4 °C for 1 h. The supernatant was collected as TBS lysate with a protein concentration ~ 3 µg/µl. The pellets were then resuspended in TBSX  supplemented with protease inhibitor and phosphatase inhibitor, homogenized, and mild agitated at 4 °C for 30 min followed by ultracentrifugation at 100,000 g at 4 °C for 1 h. Supernatant was collected as TBSX lysate with a protein concentration ~ 3.5 µg/µl for non-amyloid bearing mice and ~ 5 µg/µl for amyloid bearing mice. For amyloid bearing mice, the pellet was further resuspended in 5 M guanidine hydrochloride  followed by sonication and centrifuged at 100,000 g for 1 h at 4 °C. The supernatant was collected as GND lysate with a protein concentration ~ 1.6 µg/µl. Total protein concentration in each lysate was measured  before transferring to 96-well storage plates or 1.5 ml tubes and stored at -80 °C until further analysis.The embedded hemispheres were coronally sectioned at a 40 µm thickness. Referencing the mouse brain atlas , sections located from AP -1.7 mm to AP -2.06 mm were selected for the following procedures. First, brain slices were blocked in blocking buffer (5% goat serum plus 0.25% Triton in PBS) for 1 h at RT, then incubated overnight in primary antibody solution at 4 °C. Slices were then incubated in the Alexa Fluor-conjugated secondary antibodies solution  at RT for 2 h. We used the following antibodies against IBA1 , Aβ , CD68 , LAMP1 , APP , and GFAP . To detect TREM2, 5% BSA and 0.25% Triton X-100 in PBS was used for blocking and preparation of TREM2 antibody solution . Fibrillar Aβ plaque staining used free-floating sections from 5xFAD mouse cohorts. Sections were permeabilized with 0.25% Triton X-100 in PBS and stained with 10 µM X34  in a mixture of 40% ethanol and 0.02 M NaOH in PBS . FollowiTo quantify signals of Aβ, X34, IBA1, LAMP1, APP, IBA1, CD68, GFAP and TREM2, images were taken, and stitched using Keyence (BZ-X800) at 20X. For each staining, the hippocampus and the cortex region located above the hippocampus were traced and saved as region of interest (ROI) images in Image J. A unified intensity threshold was applied to all the sample images in each staining. Pixels with the signal intensity above the threshold were used to calculate area percentages of positive immunoreactive signals in ROI through the particle analysis plugin. For Aβ and X34 staining, plaque numbers and sizes were also assessed. Plaque densities were calculated using plaque  numTo assess the interaction of microglia and plaque, we co-stained X34 and IBA1. 30-40 z-stack images per sample were taken under Confocal (Zeiss) at 40X with a zoom factor 0.6. Plaque-centered ROI was traced with a radius of 30 µm and saved through Image J. The number of microglia surrounding each plaque within the radius of 30 µm were manually counted. Colocalization of IBA1 and X34 was decided through ‘Colocalization Threshold’ analysis in Image J.MATLAB (2021b) with the same setting parameters for all the samples. Researchers were blinded to genotypes and groups when performing and quantifying the immunofluorescence staining.All the analyses were conducted in a batch mode through customized macro coding in Image J and Aβ40 and Aβ42 levels in TBS, TBSX, and GND lysates were determined by ELISA as previously described  using anTrem2-KO mice were used as negative controls.TREM2 in cortical TBS, TBSX lysates, conditioned medium and cell lysates of primary microglia, serum were measured as described , 37 withMouse brain tissues were snap-frozen immediately after collection. Brain tissues (2 mg) were lysed in lysis buffer  by sonication. After centrifugation, the supernatant was transferred as brain lysate and subjected to the following procedures. The protein concentration from the brain lysate was determined by BCA assay. Biotinylated anti-mouse TREM2 antibody  was bound to Streptavidin-Dynabeads  by mixing for 30 min at RT. After washing to remove unbound antibody, equal amounts of protein from each sample were incubated with biotinylated anti-mouse TREM2 antibody  bound to Streptavidin-Dynabeads overnight at 4 °C. The automated KingFish Flex System (ThermoFisher Scientific) was used to wash beads with ice-cold PBS and elute of TREM2 with 5% acetic acid with high reproducibility. The eluents containing TREM2 were subjected to in-solution trypsin digestion. The resulting peptides were spiked with 5 femtomoles (fmol) of stable isotope-labeled internal standard (SIL-IS) peptides unique to sTREM2-WT, flTREM2-WT, sTREM2-H157Y, and flTREM2-H157Y (synthesized by the Mayo Clinic Proteomics Core). Parallel reaction monitoring (PRM) analysis was performed on an Orbitrap Exploris 480 mass spectrometer coupled to Ultimate 3000RSLC NANO LC System (ThermoFisher). The acquired mass spectra were analyzed using Skyline –40 to caEqual amounts of protein from the brain TBS and TBSX lysates, or RIPA lysates from isolated microglia were resolved by SDS-PAGE and transferred to PVDF membranes. After blocking, proteins of interest were detected with appropriate primary antibodies. The membrane was then probed with HRP-conjugated or LI-COR secondary antibodies and visualized using the films or Odyssey infrared imaging system (LI-COR). We used the following primary antibodies against: TREM2 , GLUR2 , PSD95 , synaptophysin , SYK , pSYK  and β-actin .RNA from pulverized cortex was extracted and purified according to our previous study . The RNATrem2 H157Y (5xFAD) homozygous female sample was excluded from further analysis due to low gene count percentage (39%) and strand-ness check (0.5009). Genes were filtered out from further analyses if there were not at least four samples with 10 counts of the gene. Trimmed Mean of M-values (TMM) normalization was performed with calcNormFactors from the edgeR R package [RNA sequencing reads were processed through the Mayo Clinic RNA sequencing analytic pipeline, MAP-RSeq Version 2.1.1 . Briefly package .2|fold change (FC)|> 0.25. Hierarchical clustering was performed in MATLAB using the Clustergram function based on standardized Euclidean distance metric. Volcano plots were generated in MATLAB using –log10 (FDR) as y axis and ± log2 (|FC|) as x axis. Pathway analyses of differentially expressed genes were performed through Ingenuity Pathway Analysis  [Differential gene expression analyses were performed in the comparison of Hom vs WT (separately for non-amyloid or amyloid cohorts) using the edgeR quasi-likelihood pipeline . Differenalysis) .Weighted gene co-expression network analysis (WGCNA) was conducted in non-amyloid mice and amyloid mice, respectively, using residual expression values calculated from adjusting for sex, strand, and exonic rate. Based on the relationship between power and scale independence, the power of 12 was chosen to build scale-free topology using signed hybrid network. We set the minimum modules size as 40 and merged modules whose correlation coefficients were greater than 0.6 (mergeCutHeight = 0.4). Each module was summarized by the first principal component of the scaled module expression profiles, termed module eigengene (ME). For each module, the module membership (MM) was defined as the correlation between gene expression values and ME. Intramodular hub genes are genes with the highest connectivity to other genes within a given module, and were selected based on the p values of MM. To assess the correlation of modules to genotype, we defined the WT genotype as 0 and Hom as 1. Modules were annotated using R package anRichment. MEs of selected modules were compared between genotypes through Wilcoxon rank-sum test. Gene–gene connections among top hub genes were visualized using Cytoscape. GO term enrichment analysis was conducted with anRichment R package.The preservation of the modules in amyloid network was tested in the non-amyloid network. Separate module preservation analyses were performed for the two datasets using WGCNA. In all analyses, module definitions from the mouse network were used as reference to calculate the z-summary statistics for each module. Z summary score > 2 suggests moderate preservation and Z summary score > 10 suggests strong preservation.Trem2 mRNA, predesigned primers (IDT) targeting exon 4–5 (Mm.PT.58.45957937.g), and customized primers targeting exon 2 (Forward 5′-GCCCATGCCAGCGTGTGGT-3′ and Reverse 5′-CACTGGTAGAGGCCCGC-3′) were used, respectively. Predesigned primers from IDT were used to quantify the mRNA levels of Tyrobp (Mm.PT.58.6069426), Tmem119 (Mm.PT.58.6766267), Cx3cr1 (Mm.PT.58.17555544), C1qa (Mm.PT.58.5375735). The relative gene expression was normalized to Gapdh  and assessed using the 2−ΔCT method.Purified RNA (1 µg) was used to prepare cDNA iScript™ Reverse Transcription Supermix (Biorad) and qPCR was performed using the QuantStudioTM 7 Flex Real-Time PCR System (ThermoFisher Scientific). To assess the total 2, 1.2 MgSO4, 3 KCl, 0.4 KH2PO4, 25 NaHCO3, and 122 NaCl, pH 7.4) containing 3% bovine serum albumin  filtered through a 0.1 mm membrane was used as microdialysis perfusion buffer. Flow rate was a constant 1.0 ml/min. Samples were collected hourly into a refrigerated fraction collector. The baseline samples were collected for 10 h followed by subcutaneous administration of a 1/2) of Aβ42 is further calculated as T1/2To assess the Aβ clearance, we examined the Aβ level in hippocampal interstitial fluid (ISF) obtained through in vivo microdialysis in awake, free-moving mice as previously described , 49, 50.t test was used since nonparametric tests would have very low power. Specifically, One-Way ANOCOVA with comparison of slopes was used in Fig. t test with Welch’s correction (Welch’s t test) was used Fig. MATLAB. The statistical tests used for each analysis, the sample size and the significance levels were reported in the legend of each figure.All data were reported as mean values ± SEM. Generally, if sample sizes are larger than 7, to ensure that results were valid in the presence of non-normal distributions, or differing variances between groups, Kruskal–Wallis tests with uncorrected Dun’s multiple comparisons or Wilcoxon Rank-sum tests were used. If the sample size ≤ 7 and dataset showed similar variances examined by F-test, unpaired Trem2 H157Y mutation, we introduced a C > T substitution in exon3 through CRISPR/Cas9 technology to create the missense H157Y mutation , heterozygous , and homozygous . Littermates of these three genotypes were used to investigate the impacts of the Trem2 H157Y mutation on TREM2 proteolytic processing, microglial density and morphology, synaptic plasticity, and cognitive function.TREM2-H157 is located where TREM2 undergoes proteolytic shedding to produce sTREM2 –24. To sTrem2 R47H knock-in mouse models generated through CRISPR/Cas9 technologies exhibit aberrant splicing and reduced expression levels of Trem2 mRNA [Trem2 H157Y affects Trem2 transcription. We measured total Trem2 mRNA using primers either targeting the N-terminal (exon 2) or C-terminal part (exon 4–5) of Trem2 in mice of each genotype at 6 months of age. Neither of these primers recognized significant differences of total Trem2 level between genotypes  and did not observe significant differences of these ratios between genotypes  from Hom microglia compared to that from Het and WT microglia  and TBSX (TBS + 1% Triton X-100) and analyzed by N-terminal TREM2-capturing ELISA. Although membrane bound TREM2 in TBSX did not differ between genotypes , phosphorylated SYK (pSYK)  recording in WT and Hom mice of our mouse model at 6 months of age to examine whether the Trem2 H157Y leads to spatial cognitive performance, we conducted a battery of behavioral tests with mice of each genotype at 6 months of age. We did not observe significant performance differences in anxiety .To examine whether this strengthened synaptic capability with ty Fig. SE and assty Fig. SG-H betweTrem2 H157Y on synaptic plasticity and presynaptic function, even though it did not translate into significant enhancement at the behavioral level in our paradigms.Taken together, these results support a beneficial effect of Trem2 H157Y knock-in mice with 5xFAD amyloid model mice. Early-stage amyloid pathologies were assessed in mice of each genotype at 4 months of age [To investigate the effects of H157Y mutation on AD-related amyloid pathogenesis, we crossed + areas in the cortex  and hippocampus  compared to WT mice. No significant differences of IBA1+ areas were observed between Het and WT mice. On the other hand, cortical and hippocampal GFAP+ areas showed no significant differences between genotypes, indicating the astrogliosis is not affected in our animal model.We also assessed the microgliosis and astrogliosis in response to the amyloid pathology, through IBA1 Fig. SA and GFATrem2 H157Y does not affect early-stage amyloid pathologies.Taken together, these results suggest that Next, we assessed the late-stage amyloid pathology in 5xFAD mice of each genotype at 8.5 months of age . InteresTrem2 H157Y on Aβ related neuronal toxicity, we assessed the dystrophic neurites through lysosome-associated membrane protein (LAMP1) immunostaining  and a significant decrease in the hippocampus . Through measuring Aβ40 and Aβ42 by ELISA in different cortical lysates   Fig. A. SignifF4) Fig. B and memF4) Fig. C were obp = 0.05)   Fig. D. Total Trem2 H157Y suppressed TREM2 signaling in the presence of Aβ.Similarly, we isolated microglia from WT and Hom mice with amyloid pathology at 8.5 months of age and examined the TREM2 signaling through the detection of pSYK and SYK Fig. I. We obsTrem2 H157Y facilitates TREM2 shedding, and reduces TREM2 signaling in the brains of 5xFAD mice.These results collectively indicate that To understand how Trem2 H157Y affects brain transcriptional profiles, we conducted bulk RNA sequencing with brain RNA samples from the non-amyloid-bearing mice at 6 months of age and amyloid-bearing mice at 8.5 months of age.Trem2, Tyrobp, Itgax, Cd63, Cst7, Ccl6, Ctsd, Gpnmb, lyz2, etc.) and microglial homeostatic genes  [Trem2, Tyrobp, Tmem119, Cx3cr1, Cd68, C1qa) in Hom mice compared to WT mice by qPCR  identified in the non-amyloid cohort in the comparison of Hom vs. WT mice Fig. SA, 183 DE3, etc.) , 60 wereM2+ Fig. U-W and C. + Fig.  R-T area. + Fig. X-Z areashttp://dropviz.org/) [Trem2, Cd68, Aif1, Ctsz, Ctsd, Cx3cr1, Tmem106a and other 18 genes, suggesting the critical contribution of microglia to the transcriptional changes with the Trem2 H157Y mutation responding to the amyloid pathology. Further, a significant positive correlation was found between the ME of this immune module and the corresponding amyloid pathological readouts from the same mice  to identify gene modules associated with genotype in the non-amyloid and amyloid cohorts, respectively. Although no significant module was identified related to genotype in the non-amyloid cohort Fig. SB, a uniqiz.org/) , all theice Fig. H indicatTrem2 H157Y downregulated microglial genes involved in the neuroinflammation signaling pathway in Hom mice.Taken together, Trem2 H157Y mutation promotes TREM2 shedding in our novel Trem2 H157Y knock-in mouse models. Moreover, we found Trem2 H157Y enhances synaptic plasticity, facilitates Aβ clearance, reduces amyloid burden and gliosis, and downregulates neuroinflammatory pathways.In this study, we provided in vivo evidence that Trem2 H157Y mutation. We did not observe significant changes in the membrane associated flTREM2 levels and TREM2 signaling. Since N-terminal TREM2 ELISA does not distinguish mature and immature flTREM2, we cannot conclude that Trem2 H157Y specifically reduces mature TREM2 in our mouse model as described in the in vitro studies [+ areas, as well as TREM2 signaling in Hom mice compared to WT mice at 8.5 months of age. Despite the reduction of total TREM2 levels, TREM2-H157Y displays higher s/fl ratios compared to TREM2-WT, suggesting that Trem2 H157Y facilitates TREM2 shedding in the 5xFAD mice. In addition, similar to the non-amyloid cohort, we observed elevated serum sTREM2 in Hom mice compared to WT mice. Collectively, our in vivo studies suggest that Trem2 H157Y promotes TREM2 shedding in the absence or presence of Aβ.Consistent with previous in vitro findings , 24, we  studies , 24. On Trem2 H157Y on synaptic functions in our mouse models may be mediated, but not restricted to sTREM2, through a microglia non-cell autonomous mechanism. To address our hypothesis and elucidate the exact roles of TREM2 and sTREM2 in regulating neuronal activity, more comprehensive studies are needed in future studies.Moreover, we observed enhanced presynaptic facilitation and synaptic plasticity in Hom mice and a trending improvement of spatial working memory in Het mice, along with the increased sTREM2 from microglia. It has been shown that sTREM2 was colocalized with neurons  which imTrem2 H157Y knock-in mice. We also did not observe significant cortical transcriptional profile changes in Hom mice compared to WT mice. However, a study from Zhong et al., reported that the application of exogenous sTREM2 increases microglia survival, stimulate microglia to a reactive status, and induce proinflammatory cytokine production [We detected ~ 0.4 and ~ 0.6 ng sTREM2/mg TBS proteins in the TBS lysates from WT and Hom mice, i.e., ~ 0.4 and ~ 0.6 fmol sTREM2/mg brain tissue, respectively. These amounts are at the same order of magnitude as the mass spectrometry measurements in the amyloid mice  with Cutting Frequency Determination (CFD) Score ranging from 0.28 to 0.44 were identified and sequenced with primers accordingly. B. Single peaks were seen at the putative sites , while two signals were seen at the Trem2 H157Y target site . Orange arrows indicate the putative region and direction recognized by gRNA.Additional file 2: Figure S2.Trem2 H157Y does not affect microglia density and morphology. A. Representative images of IBA1 staining are shown for WT, Het, and Hom mice at 6 months of age. Scale, 400 µm. B-C. Cortical microglia (MG) number (B) and cell body size (C) are quantified in Image J for each genotype at 6 months of age. N =11-14 mice per genotype, mixed sex. D-F. Representative confocal images (D) of IBA1 staining were processed (E) and skeletonized (F) in image J for each genotype at 6 months of age. Scale bar for D and E, 50 µm; Scale bar for F 10 µm. G-I. The branch number (G), junction number (H), and total branch length per microglia (MG) (I) were assessed for each genotype at 6 months of age. N = 9-10 mice per genotype, mixed sex. Data are presented as Mean±SEM. Kruskal-Wallis tests with uncorrected Dun’s multiple comparisons were used were used in B-C, G-I. N.S., not significant.Additional file 3: Figure S3.Trem2 H157Y does not affect synaptic integrity, anxiety, working memory and associative memory. A. Synaptophysin, PSD95, and GLUR2 were detected in TBSX lysates. B-D. Synaptophysin (B), PSD95 (C), and GLUR2 (D), were quantified and normalized to WT. N = 9-10 mice per genotype at 6 months of age, mixed sex. E. Open field analysis (OFA) was conducted to examine the anxiety of mice with different genotypes at 6 months of age. N =37-40 mice per genotype, mixed sex. F. Y-maze spontaneous alteration test was conducted to examine the working memory of mice with different genotypes at 6 months of age. N =23-26 mice per genotype, mixed sex. G. Contextual fear conditioning test (CFC) was conducted to examine the associative memory of mice with different genotypes at 6 months of age. N =37-40 mice per genotype, mixed sex. H. Cued fear conditioning test (CFC) was conducted to examine the associative memory of mice with different genotypes at 6 months of age. N =37-40 mice per genotype, mixed sex. B-D, E-H. Data are presented as Mean±SEM. Kruskal-Wallis tests with uncorrected Dun’s multiple comparisons were used. N.S., not significant.Additional file 4: Figure S4.Trem2 H157Y doesn’t affect amyloid pathology in 5xFAD mice at 4 months of age. A-B. Representative images of pan-amyloid  and fibrillar amyloid  staining are shown at 4 months of age. Scale, 400 µm. C-H. Cortical (C-E) and hippocampal (F-H) amyloid plaque area coverages , densities  and sizes  are quantified and normalized to WT for each genotype. I-N. Cortical (I-K) and hippocampal (L-N) fibrillar amyloid plaque area coverages , densities  and sizes  are quantified and normalized to WT. O-T. Aβ40  and Aβ42  were quantified by ELISA and normalized to WT in cortical TBS , TBSX  and GND  for each genotype. C-T. N = 12-18 mice per genotype at 4 months of age, Data are presented as Mean±SEM. Kruskal-Wallis tests with uncorrected Dun’s multiple comparisons were used. N.S., not significant.Additional file 5: Figure S5.Trem2 H157Y does not affect microgliosis and astrogliosis in 5xFAD mice at 4 months of age. A. Representative images of IBA1 staining are shown for each genotype at 4 months of age. Scale, 400 µm. B-C, Cortical (B) and hippocampal (C) IBA1+ area coverages are quantified and normalized to WT. D. Representative images of GFAP staining are shown for each genotype at 4 months of age. Scale, 400 µm. E-F. Cortical (E) and hippocampal (F) GFAP+ area coverages were quantified and normalized to WT for each genotype. B-C, E-F. N = 12-18 mice/genotype at 4 months of age. Data are presented as Mean±SEM. Kruskal-Wallis tests with uncorrected Dun’s multiple comparisons were used. N.S., not significant.Additional file 6: Figure S6. Effects of Trem2 H157Y mutation on Aβ levels, neuronal toxicity, and APP processing at 8.5 months of age. A-D. Aβ40  and Aβ42  were quantified by ELISA, and normalized to WT in cortical TBS , and TBSX  for each genotype. E. Representative images of fibrillar amyloid (X34) staining are shown for each genotype at 8.5 months of age. Scale, 400 µm. F-K. Cortical (F-H) and hippocampal (I-K) fibrillar amyloid plaque area coverages , densities  and sizes  are quantified and normalized to WT. L. Representative images of APP  staining are shown for each genotype at 8.5 months of age. Scale, 400 µm. M-N. Cortical (B) and hippocampal (C) APP+ area coverages were quantified and normalized to WT. O. Representative gel images are shown for synaptophysin and PSD95, in TBSX lysates with β-Actin stain for normalization. P-Q. PSD95 (P) and Synaptophysin (Q), were quantified and normalized to WT. R-S. Soluble APPa , Soluble APPβ  were examined by ELISA, quantified, and normalized to WT in TBS lysates for each genotype. T. CTFβ was examined by ELISA, quantified, and normalized to WT in TBSX lysates for each genotype. A-T. N = 19-24 mice per genotype at 8.5 months of age, mixed sex. Data are presented as Mean±SEM. Kruskal-Wallis tests with uncorrected Dun’s multiple comparisons were used. N.S., not significant. * p<0.05.Additional file 7: Figure S7. Effects of Trem2 H157Y mutation on Aβ levels and neuronal dystrophy in founder 2# linage 5xFAD mice at plateau stage of amyloid development. A-D. Aβ40  and Aβ42  were quantified by ELISA, and normalized to WT in cortical TBS , and TBSX  for each genotype. E. Representative images of fibrillar amyloid (X34) staining are shown for each genotype at 8.5 months of age. Scale, 400 µm. F-K. Cortical (F-H) and hippocampal (I-K) fibrillar amyloid plaque area coverages , densities  and sizes  are quantified and normalized to WT for each genotype. L. Representative images of APP  staining are shown for each genotype at 8.5 months of age. Scale, 400 µm. M-N. Cortical (M) and hippocampal (N) APP+ area coverages are quantified and normalized to WT for each genotype. A-N. N = 8-13 mice per genotype at 8.5 months of age, mixed sex. Data are presented as Mean±SEM. Kruskal-Wallis tests with uncorrected Dun’s multiple comparisons were used. N.S., not significant. * p<0.05.Additional file 8: Figure S8. Identification and measurements of full-length, soluble TREM2-WT and TREM2-H157Y in mouse brain through mass spectrometry. A. Workflow of the TREM2-targeted mass spectrometry is illustrated. B. TREM2 was detected through an N-terminal antibody (5F4) in the input, supernatant and eluent samples from TREM2 immunoprecipitation through a biotinylated N-terminal antibody (BAF1729) in brain lysates of 5xFAD+/- and TREM2-/- mice. C. Signature peptide sequences for flTREM2-WT, sTREM2-WT, flTREM2-H157Y and sTREM2-H157Y are shown. D-G, Annotated MS/MS spectra of four unique peptides from flTREM2-WT (D), sTREM2-WT (E), flTREM2-H157Y (F) and sTREM2-H157Y (G), respectively. H-K. Measurements of flTREM2-WT (H), sTREM2-WT (I), flTREM2-H157Y (J) and sTREM2-H157Y (K) in brain samples from each genotype of 5xFAD mice. N = 3 mice/sex/genotype at 8.5 months of age. Data are presented as Mean±SEM. Ordinary one-way ANOVA with uncorrected Fisher's LSD multiple comparisons were used. N.S., not significant. *p < 0.01. **p < 0.01. ***p < 0.001. ****p < 0.0001.Additional file 9: Figure S9. Effects of Trem2 H157Y on the transcriptome profiles. A. Three DEGs  were identified in the non-amyloid mice. N=5 mice/sex/genotype at 6 months of age. B. No significant modules were identified related to genotype in the non-amyloid cohort. C-G. DEGs, Trem2, Tyrobp, Tmem119, Cx3cr1, and C1q were validated through qPCR in 5xFAD mice. N = 19-24 mice per genotype at 8.5 months of age, mixed sex. Data are presented as Mean±SEM. Kruskal-Wallis tests with uncorrected Dun’s multiple comparisons were used. N.S., not significant. * p<0.05. ** p<0.01. *** p<0.001. **** p<0.0001. H. The magenta module identified in amyloid mice was not preserved in the non-amyloid network revealed by a low preservation Zsummary value (<2).Additional file 10: Figure S10. A working model is illustrated demonstrating the hypothesis that Trem2 H157Y reduces amyloid load through facilitating Aβ clearance mediated by sTREM2 which binds to Aβ oligomers and fibrils, and inhibit plaque formation. The reduced amyloid load leads to downregulated immune responses of microglia with less cytokine released.


The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68-142.86 ng/mL, respectively. This study provided helpful insights into the scientific deployment of tests and recommended the full-scale consideration of the testing purpose, resource availability, cost performance, result rapidity, and accuracy to facilitate a profound pathway toward the long-term surveillance of coronavirus disease 2019 (COVID-19).The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an enormous burden on the global public health system and has had disastrous socioeconomic consequences. Currently, single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests (POCTs), and rapid antigen tests are implemented in different scenarios to detect SARS-CoV-2, but a comprehensive evaluation of them is scarce and remains to be explored. In this study, 3 SARS-CoV-2 inactivated cell culture supernatants were used to evaluate the analytical performance of these strategies. Additionally, 5 recombinant SARS-CoV-2 nucleocapsid (N) proteins were also used for rapid antigen tests. For the wild-type (WT), Delta, and Omicron strains, the lowest inactivated virus concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 10IMPORTANCE In the study, we reported an evaluation of 4 detection strategies implemented in different scenarios for SARS-CoV-2 detection: single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests, and rapid antigen tests. 3 SARS-CoV-2-inactivated SARS-CoV-2 cell culture supernatants and 5 recombinant SARS-CoV-2 nucleocapsid proteins were used for evaluation. In this analysis, we found that for the WT, Delta, and Omicron supernatants, the lowest concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68 to 142.86 ng/mL, respectively. Since December of 2019, the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an enormous burden on the global public health system has had disastrous socioeconomic consequences .Naturally occurring mutations can be selected continuously during virus transmission, which is a dominating obstacle to the prevention and control of COVID-19. Among the variants of concern (VOC) and variants of interest (VOI) designated by the World Health Organization (WHO), based on the transmissibility, pathogenicity, and threat to public health , the DelReal-time reverse transcriptase polymerase chain reaction S(rRT-PCR) is based on the genomic sequences of SARS-CoV-2 and is regarded as the most sensitive and specific method for confirming clinical diagnoses . Driven Regarding the detection methods mentioned above, analytical sensitivity is a fundamental parameter that can facilitate decisions for application and determine their practical use in various scenarios. The performance of the NMPA-approved rRT-PCR assays has been comprehensively analyzed and demonstrated previously; however, there are restricted data on the performance characteristics of these newly approved rapid antigen tests. Additionally, the evaluation of single sampling tests, 20-in-1 pooling tests, nucleic acid POCTs, and rapid antigen tests implemented in different scenarios is scarce and remains to be explored. Therefore, this study aimed to evaluate the analytical sensitivities of these four detection strategies applied in different settings and to provide sufficient research-based evidence for the scientific refinement of the deployment of these tests.2 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL for the wild-type (WT), Delta, and Omicron strains, respectively, corresponding to 7.81 × 103 to 6.25 × 104, 7.81 × 103 to 2.50 × 105, and 7.81 × 103 to 1.25 × 105 copies/mL of the 50 μL original sample. For the 20-in-1 pooling tests and 5 rRT-PCR detection kits, the lowest concentrations ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL for the WT, Delta, and Omicron strains, respectively. Kit N03 reliably detected as few as 102 copies/mL for both sampling strategies, showing the most sensitive performance compared with the other rRT-PCR kits. Unexpectedly, the POCT Kit P01 showed a comparable analytical sensitivity to those of the rRT-PCR kits with the lowest concentration of 1.42 × 103 copies/mL for all three strains  and Omicron  variants below than the claimed LODs, impaired analytical sensitivity was found in the 20-in-1 pooling strategy using Kit N03. In terms of test results among variants, in contrast to the WT strain, the Delta variant adversely affected the analytical sensitivity of Kit N04, both for the single sampling tests  and for the 20-in-1 pooling tests . Also, the Omicron variant had no significant effect on the 5 rRT-PCR kits and the POCT kit.Overall, when detecting the diluted samples higher than the claimed limits of detection (LODs), the 20-in-1 pooling tests could substantially achieve the same detection performance as could the single sampling tests. When it came to the Delta  has ravaged many cities, including Guangzhou, Nanjing, Yangzhou, Putian, Xiamen, and Ejina Banner. The Omicron variant is estimated to have an R0 value of up to 10 and a doubling time of every 2 to 3 days  and rapid antigen tests applied in different scenarios.2 and 5 × 103 copies/mL, with Kit N03 performing the best. Large-scale population screening via the rRT-PCR method plays a crucial role in the identification of COVID-19 positive cases. In the quest for improving overall detection efficiency with an acceptable slight loss of analytical sensitivity, the pooling strategies are proposed, and these can be implemented to expedite the early discovery of community transmission, promote timely infection control measures of SARS-CoV-2, and alleviate the workload of medical staff. By mixing nasopharyngeal and oropharyngeal swabs and testing them as a single pool, the detection kits and turnaround times are largely economized. Catherine et al.  and nucleic acid lateral flow technologies, can detect samples at concentrations as low as 1.42 × 10ocedures , and an ocedures  and can ocedures . However4 to 106 copies/mL for inactivated cell culture supernatants and from 1 to 150 ng/mL for recombinant N proteins, which was essentially consistent with the findings reported in previously published articles (5 to 107 copies/mL) were about 100 times as high as those of the single sampling test (103 to 105 copies/mL). On account of the viral load changing rapidly during the acute phase of infection, rapid antigen tests can only be applied validly in the first week of symptoms  and for the 20-in-1 pooling tests , whereas the Omicron variant had no significant effect on the 5 rRT-PCR kits and the POCT kit, which is in concordance with the findings of a prior study  and rapid antigen testing. The single-center evaluation of analytical sensitivity was based on SARS-CoV-2 cell culture supernatants with determined viral loads. As a complement, recombinant SARS-CoV-2 N proteins were also used for the rapid antigen tests .3 inactivated cell culture supernatants of SARS-CoV-2 WT, Delta, and Omicron strains were provided by the Sinovac Biotech Co., Ltd. (China). For quantification, viral RNAs with proper dilutions were extracted using a QIAamp Viral RNA Minikit  and were reverse transcribed into cDNAs using a PrimeScript RT Reagent Kit . Subsequently, the samples were subjected to droplet digital PCR (ddPCR) on a Bio-Rad QX-200 System (USA), using the N gene assay recommended by China’s Centers for Disease Control (CDC). 5 recombinant SARS-CoV-2 N proteins of the SARS-CoV-2 WT strain (cat. number 40588-V07E), Delta AY.2 sublineage (cat. number 40588-V07E29), Delta AY.1/AY.3 sublineage (cat number 40588-V07E32), Omicron BA.1 sublineage (cat number 40588-V07E34), and Omicron BA.2 sublineage (cat number 40588-V07E35) were obtained from Sino Biological, Inc. (China). The concentrations of the recombinant N proteins were measured using a Qubit 3.0 fluorometer with the Qubit Protein Assay Kit .5, 2.5 × 105, 1.25 × 105, 6.25 × 104, 3.12 × 104, 1.56 × 104, 7.81 × 103, 3.91 × 103, and 1.95 × 103 copies/mL), and 50 μL of them were added to each sample, respectively. Remarkably, this step with positive supernatants diluted in sample preservation solution introduces a predilution effect , leading to a loss of sensitivity as claimed by the reagent manufacturers. This factor was staken into account when calculating the analytical sensitivity afterwards. Nucleic acid was extracted using a Tianlong automatic nucleic acid extraction system , and tested in triplicate using 5 NMPA approved SARS-CoV-2 rRT-PCR kits   . The ret3 copies/mL  complex. Within 15 min, the test results can be interpreted with simple instruments when using the fluorescence method or with the naked eye when using the latex and colloidal gold methods. We included 19 rapid antigen tests approved by the NMPA in our study, including two fluorescence immunochromatography methods , five latex methods , and 12 colloidal gold methods  . 50 μL oThe concentration equivalent of the 50 μL sample was the lowest concentration that achieved a 100% detection rate of the 50 μL original sample before being added to the sample preservation solution. The lowest concentration with a 100% detection rate was that of the diluted sample. The predilution ratios varied according to the volumes of sample preservation solution (3 mL for the single sampling tests and 12 mL for the 20-in-1 pooling tests) or extraction solution (point-of-care test and rapid antigen tests), to which 50 μL samples of known concentrations were added  software package. A


The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family. Here, Waltenspühl et al. report the cryo-EM structure of active human oxytocin receptor in complex with oxytocin and with a heterotrimeric G protein, providing insights into this hormone system critically involved in the regulation of social behaviour and reproduction. This OT/AVP hormone system is highly conserved among many species and exerts a central role in the regulation of social cognition, social behaviour, and sexual reproduction2.The neurohypophyseal hormones oxytocin (OT) and arginine vasopressin (AVP) are cyclic peptides that activate an evolutionary ancient subfamily of class A G protein-coupled receptors (GPCRs)3, anxiety4 and schizophrenia5. While OT itself is an approved peripheral drug in obstetrics6, OT-based treatments of socio-behavioural deficiencies, requiring central administration of the hormone, have yet failed, potentially due to its poor drug-like properties7 and limited penetration through the blood brain barrier8. Despite recent advances in finding a non-peptide agonist that is active in an animal model9, the high demand for such drugs continues. Up to now, the identification and development of OTR-specific molecules with satisfactory pharmacokinetic properties, favourable biodistribution and specificity has been impeded by the lack of structural information on the OTR:OT signalling complex.Currently, several clinical trials are evaluating the efficacy of OTR-mediated signalling through administration of OT to treat malfunctions such as autism-spectrum disordersHere, we now report the single-particle cryo-electron microscopy (cryo-EM) structure of the OT-bound human OTR in complex with a heterotrimeric G protein at a resolution of 3.2 Å.10. This mutation enabled a 50-fold increase in the yield of purified functional receptor, with very similar agonist binding and signalling behaviour 14. For simplicity, the resulting mini-G protein is denoted Go/q henceforth. These combined engineering efforts finally enabled single-particle cryo-EM analysis of the OTR:OT:Go/q:scFv16 complex at a resolution of 3.2 Å . To improve expression and purification yields we included a single stabilising mutation D153Y), which we previously identified by a next-generation sequencing (NGS)-based in-depth analysis of directed evolution experiments53Y, whic9, known to be important for activation15, is located in proximity of residues E421.35 and D1002.65  of transmembrane helices I and II, which are involved in magnesium-dependent modulation of OT binding17. Together with the neighbouring Leu8, the extracellular surface of the orthosteric pocket is lined by Pro7 and Asn5, which pack against extracellular loop 3 (ECL3) and ECL2, respectively. Leu8 is oriented towards the extracellular space, explaining why position 8 is best suited for the attachment of fluorophores in OT18. Gln4 is the only residue pointing out perpendicularly from the ring plane and stabilizes the cyclic ring position through a hydrogen bond to Q2956.55. Ile3 is buried in a hydrophobic pocket formed by side-chain residues of transmembrane helices IV, V, and VI. The critical contribution of this hydrophobic pocket is underlined by the observed reduction in OT potency when mutating the main contact residues I2015.39, I2045.42 and F2916.51 of the receptor to alanine , F2846.44 (PIF motif) and Y3297.53 (NPxxY motif) , and the reorientation of D1363.49 into a position enabling direct contact with the α5 helix of Go/q  Fig. . These ro/q Fig. .Fig. 2Li1, Tyr2 and Gln4 as centrally involved ligand residues in receptor activation. For example, alkylation of the Tyr2 hydroxy group led to decreased agonistic activity20, suggesting an important role of this residue, consistent with our structural data. Remarkably, we observe a local unfolding of helix VII at the extracellular receptor side in the region of L3167.40, creating a pronounced kink which is stabilised by a hydrogen bond formed between Tyr2 of OT with the backbone oxygen of L3167.40   Fig. . While t I Figs. d and 4. 25, we do not observe an interaction of OT with R34N terminus or F103ECL1 of the OTR. This might indicate an allosteric effect, or in the case of R34, which does not show clear density, a more dynamic interaction. We speculate that this could additionally contribute to subtype selectivity in the OT/AVP receptor family.In variance to long-standing models2R are found in the positioning of the helix VII-VIII transition region and the elongation of helix V of V2R compared to OTR .On the intracellular receptor side, the main conformational differences between OTR and Vo/q is bound in a crevice constituted by helices II, III, V, VI and VII at the receptor intracellular side, where the G protein C-terminal residues E350 and Y351 engage in hydrogen bonding with R732.38 and D1363.49 of OTR , codon-optimised for expression in Spodoptera frugiperda (Sf9) , were cloned into a modified pFL vector  for Sf9 expression. The resulting expression constructs contained a melittin signal sequence, followed by a FLAG-tag, a His10-tag, and a human rhinovirus 3C protease cleavage site N-terminal to the gene of interest. To increase OTR purification yield, the mutation D153Y was introduced into the truncated wtOTR sequence, as identified previously10. It shows very similar KD and EC50 for oxytocin (OT) 33 in the C-terminal helix were mutated to corresponding amino acids of Gq. Finally, the engineered Gαo/q chimera sequence was cloned into one pFL vector, together with Gβ1  and with Gγ2, with each gene under the control of its own polyhedrin promoter.The sequences of scFv1617. In brief, 4 L of Sf9 insect cell culture at a density of 3 × 106 cells/ml were infected with baculovirus stocks at a multiplicity of infection ≥5. Cells were harvested 72 h post-infection and stored at −80 °C. Cells were thawed on ice before purification, lysed, and membranes were isolated by repeated Dounce homogenisation in hypotonic buffer containing 10 mM Hepes (pH 7.5), 20 mM KCl, 10 mM MgCl2, and protease inhibitors  and then in hypertonic buffer containing 10 mM Hepes (pH 7.5), 20 mM KCl, 10 mM MgCl2, 1.0 M NaCl, 0.1 mg/ml deoxyribonuclease  and protease inhibitors. Washed membranes were resuspended in hypotonic buffer, and the low-affinity antagonist SSR 149415 (Tocris) was added to a final concentration of 100 μM and the suspension was incubated for 30 min. Then, iodoacetamide  was added to the solution followed by another 30 min of incubation. Subsequently, the receptor was solubilized in buffer containing 30 mM Hepes (pH 7.5), 500 mM NaCl, 10 mM KCl, 5 mM MgCl2, 50 μM SSR 149415, 1% (w/v) n-dodecyl-β-d-maltopyranoside , and 0.2% (w/v) cholesteryl hemisuccinate  for 3 h at 4 °C. Insoluble material was removed by ultracentrifugation at 220,000 g, and the supernatant containing the solubilized receptor was incubated with TALON IMAC resin (Cytiva) at 4 °C.Expression and purification were performed as previously described in detail2, 5 mM imidazole, 10% (v/v) glycerol, 8 mM adenosine triphosphate , supplemented with 0.5% (w/v) DDM, 0.01% (w/v) CHS, and 10 μM SSR 149415. Subsequently, the detergent was exchanged with 16 CVs of wash buffer I supplemented with 1% (w/v) lauryl maltose neopentyl glycol , 0.1% (w/v) CHS, and 10 μM SSR 149415, and the antagonist was replaced with OT (Psyclo Peptide Inc.) with another 16 CVs of wash buffer I supplemented with 0.5% (w/v) LMNG, 0.05% (w/v) CHS, and 100 μM OT. Agonist-bound OTR was finally washed with 25 CVs of wash buffer II containing 50 mM Hepes (pH 7.5), 150 mM KCl, 15 mM imidazole, 10% (v/v) glycerol, 0.05% (w/v) LMNG, 0.005% (w/v) CHS, and 50 μM OT, before eluting the receptor in 4 subsequent fractions of 1 CV of elution buffer containing 50 mM Hepes (pH 7.5), 150 mM KCl, 250 mM imidazole, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, and 50 μM OT.The receptor-bound resin was washed with 20 column volumes (CVs) of wash buffer I containing 50 mM Hepes (pH 7.5), 500 mM NaCl, 10 mM MgClProtein-containing fractions were combined and concentrated to 0.5 ml using a 50-kDa molecular weight cutoff (MWCO) Vivaspin 2 concentrator (Sartorius), and desalted by buffer exchange on a PD MiniTrap G-25 column (Cytiva) equilibrated with G25 buffer containing 50 mM Hepes (pH 7.5), 150 mM KCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, and 50 μM OT. To remove the N-terminal affinity tags and to deglycosylate the receptor, OT-bound receptor was treated with His-tagged 3 C protease and His-tagged peptide N-glycosidase (PNGase) F (both prepared in-house) overnight. To collect cleaved receptor, the reaction was incubated with Ni-nitrilotriacetic acid (Ni-NTA) resin (Cytiva) for 1 h, cleaved receptor was collected as the flow-through, then concentrated to ~3 to 5 mg/ml with a 50-kDa MWCO Vivaspin 2 concentrator, and directly used for complex formation. Protein purity and monodispersity were assessed by LDS–polyacrylamide gel electrophoresis and analytical size exclusion chromatography (SEC) using a Nanofilm SEC-250 column (Sepax).2, and protease inhibitors. Solubilisation and immobilisation on TALON IMAC resin was performed as described above.Purification of the engineered heterotrimeric G protein was carried out similarly to receptor purification, with small adaptations. All buffers used were supplemented with 10 μM guanosine diphosphate  and 100 μM tris(2-carboxyethyl)phosphine) . In contrast to the receptor purification, monovalent cation concentration never exceeded 150 mM, and all buffers were devoid of any receptor ligands and iodoacetamide. Enriched G protein-containing membranes were washed by Dounce homogenisation without high salt concentrations in physiological buffer, containing 10 mM Hepes (pH 7.5), 150 mM NaCl, 20 mM KCl, 10 mM MgCl2, 5 mM imidazole, 10% (v/v) glycerol, 1% (w/v) DDM, 0.2% (w/v) CHS, 10 μM GTP and 100 μM TCEP followed by a wash with 30 CV of detergent exchange buffer containing 50 mM Hepes (pH 7.5), 150 mM KCl, 1 mM MgCl2, 5 mM imidazole, 10% (v/v) glycerol, 1% (w/v) LMNG, 0. 1% (w/v) CHS 8 mM ATP, 10 μM GTP and 100 μM TCEP. G proteins were finally washed with 30 CVs of wash buffer II containing 50 mM Hepes (pH 7.5), 150 mM KCl, 1 mM MgCl2, 15 mM imidazole, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, 10 μM GTP and 100 μM TCEP and eluted stepwise in 4 subsequent fractions of 1 CV of elution buffer containing 750 mM imidazole.The G protein–bound resin was initially washed with 30 CVs of wash buffer I containing 50 mM Hepes (pH 7.5), 150 mM KCl, 10 mM MgClEluted G protein was concentrated using a 100-kDa MWCO Vivaspin 2 concentrator and further purified using a Superdex 200 10/300 (Cytiva) column equilibrated with SEC buffer containing 25 mM Hepes (pH 7.5), 150 mM KCl, 2% (v/v) glycerol, 0.01% (w/v) LMNG, and 0.001% (w/v) CHS. Fractions containing monodisperse G protein were collected, concentrated to 7.1 mg/ml with a 100-kDa MWCO Vivaspin 2 concentrator (Sartorius), frozen in liquid nitrogen, and stored at −80 °C until use.12. Briefly, scFv16 was expressed by secretion from baculovirus-infected Sf9 cells for 72 h. ScFv16-containing expression medium was separated from cells by centrifugation, then pH-adjusted by addition of Hepes (pH 7.5). Metal-chelating agents from the cells and medium were quenched by incubation with 1 mM CoCl2 and 5 mM CaCl2 for 1 h at 22 °C. Resulting precipitates were removed by centrifugation, and the filtrated supernatant was loaded onto a Co2+-loaded HiTrap IMAC HP column (Cytiva). The column was washed with 20 CVs of buffer A containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 4 mM ATP, and 5 mM imidazole, followed by 20 CVs of buffer B containing 20 mM Hepes (pH 7.5), 150 mM NaCl, and 30 mM imidazole. The protein was eluted with buffer C . Monomeric fractions were pooled, concentrated using a 10-kDa MWCO Amicon Ultra concentrator (Merck), and imidazole was removed by applying the concentrate to a PD-10 desalting column (Cytiva) equilibrated with G25-buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, and 0.001% (w/v) CHS. ScFv16 was incubated for 3 h with His-tagged 3 C protease and His-tagged PNGase F for removal of affinity tags and deglycosylation. After incubation with Ni-NTA resin for 1 h, cleaved scFv16 was collected as the flow-through, concentrated with a 10-kDa MWCO Amicon Ultra concentrator, and further purified using a Superdex 200 10/300 column equilibrated with SEC buffer. Monomeric fractions were pooled, concentrated to ~6 to 7 mg/ml, flash-frozen in liquid nitrogen, and stored at −80 °C. Right before complex formation, scFv16 was thawed and the buffer was exchanged on a PD MiniTrapTM G-25 column (Cytiva) equilibrated with G25-buffer.ScFv16 was expressed and purified as described beforeo/qGβ1Gγ2) were mixed in a molar ratio of 1:1.2 in complexation buffer (25 mM Hepes (pH 7.5), 100 mM KCl, 1 mM MgCl2, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, 100 μM OT, and 100 μm TCEP). After 30 min, apyrase  and λ-phosphatase  were added to the mixture. After 2 h, purified scFv16 was added at 4-fold molar excess over receptor, and complex formation was allowed to proceed overnight at 4 °C. Stable complex was isolated by SEC on a Superdex 200 10/300 column (Cytiva) equilibrated with blotting buffer (25 mM Hepes (pH 7.5), 100 mM KCl, 1 mM MgCl2, 100 μM OT, and 100 μm TCEP, 0.001% (w/v) LMNG, and 0.0001% (w/v) CHS). Corresponding peak fractions were concentrated to 1 mg/ml for EM studies, using a 100-kDa MWCO Vivaspin Turbo PES (Sartorius) concentrator.Purified OTR and engineered heterotrimeric G protein , and subsequently vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 100% humidity and 4 °C. Cryo-EM images were acquired by a Titan Krios G3i electron microscope (Thermo Fisher Scientific), operated at 300 kV, at a nominal magnification of 130,000 using a K3 direct electron detector (Gatan) in super-resolution mode, corresponding to a pixel size of 0.325 Å. A BioQuantum energy filter (Gatan) was operated in a zero-loss mode, using 20 eV energy slit-width. A total of 6,450 movies were obtained, with a defocus range of −0.8 to −2.4 µm using automatic data acquisition with EPU software . The total exposure time was 1.79 s with an accumulated dose of ~63.78 electrons/Å2 and a total of 67 frames per micrograph. A second set of 5,217 image stacks were acquired with the same conditions and parameters.For cryo-EM grid preparation, 3 µL of purified OTR:OT:G34 (version 1.4). All images were contrast transfer function corrected using Gctf35 (version 1.06), as implemented in cryoSPARC36(version 3.0.1). Subsequent image processing steps were performed in cryoSPARC. Initial particle selection was done using the automated blob picker on 100 micrographs, using a 100 Å minimum and 150 Å maximum particle diameter, to extract a total of 38,166 particles. Next, the extracted particles were subjected to one round of 2D classification, into 200 classes, from which 7 classes were selected and used as a template for the automatic particle picking process. A total of 3,062,337 particles were extracted from the first 6,450 micrographs, followed by a round of 2D classification that resulted in 200 classes. Finally, a round of 3D reconstructions and classification produced 6 classes.All image stacks were binned to generate a pixel size of 0.65 Å followed by motion-correction and dose-weighting using MotionCor2A total of 3,504,800 particles were extracted from the second data-set of image-stacks, followed by a round of 2D classification, split into 200 classes, and a round of 3D reconstructions and classification into 3 classes. The particles from the best classes from both datasets were then joined together and subjected to one ab-initio round of 3D reconstructions split into 6 classes. A final data-set of 392,370 particles from the best 3D classes were subjected to local and global CTF refinements, followed by a 3D non-uniform refinement. The final density map was resolved to 3.25 Å, after map sharpening, as determined by gold-standard Fourier shell correlation using the 0.143 criterion. Local resolution estimation was performed using cryoSPARC.37 (version 1.15). The following structures from the Protein Data Bank (PDB) were used: OTR (PDB ID: 6TPK), Gαo (PDB ID: 6WWZ), Gβ1γ2, scFv16 (PDB ID: 6OIJ), and AVP (PDB ID: 7DW9). All initial model components were manually rebuilt in Coot38 (version 0.9.7), followed by several rounds of manual real-space refinement in Coot and real-space refinement with the software package Phenix.real_space_refine from Phenix39 (version 1.20.1-4487). The quality of the models was assessed using MolProbity40 before PDB deposition. PyMOL (version 2.5) was used for visual inspection, model comparison and figure preparation.An initial model was created by docking the individual complex components into the cryo-EM map using the “fit in map” routine in UCSF Chimera2 and 95% air. Transient transfections were performed with TransIT-293 (Mirus Bio) according to the manufacturer’s instructions. Full-length OTR, truncated V1AR  and mutants thereof were directly cloned into a mammalian expression vector containing an N-terminal SNAP-tag . Cells were transfected and directly seeded at 7500 cells per well in poly-D-lysine-coated white 384-well plates (Greiner).Ligand-induced IP1 accumulation and ligand-binding experiments were measured using transiently transfected Human Embryonic Kidney (HEK) 293 T/17 cells (American Type Culture Collection). The cells were cultivated in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with penicillin (100 U/ml), streptomycin , and 10% (v/v) foetal calf serum (BioConcept) and maintained at 37 °C in a humidified atmosphere of 5% CO41. The cells were washed 48 h after transfection with phosphate-buffered saline (PBS) and stimulation buffer (Cisbio) and subsequently incubated for 1 h at 37 °C with a concentration range of oxytocin (Psyclo Peptide Inc.) diluted in stimulation buffer. The IP1 accumulation was determined using the HTRF IP-One Kit (Cisbio) according to the manufacturer’s protocol. Fluorescence intensities were measured on a Spark fluorescence plate reader (Tecan). To generate concentration-response curves, data were analysed by a three-parameter logistic equation in GraphPad Prism software (version 9.2.0).To compare IP1 accumulation a homogeneous time-resolved fluorescence (HTRF) signalling assay was performed, adapting a previously described protocol17. Forty-eight hours post-transfection the cells were labelled with 50 nM SNAP-Lumi4-Tb (Cisbio) in labelling buffer (20 mM Hepes (pH 7.5), 100 mM NaCl, 3 mM MgCl2, and 0.05% (w/v) bovine serum albumin (BSA)) for 1.5 h at 37 °C. The cells were washed two times with labelling buffer and two times with assay buffer (20 mM Hepes (pH 7.5), 100 mM KCl, 3 mM MgCl2, and 0.05% (w/v) BSA)) and subsequently incubated for 1 h at room temperature with a concentration range of fluorescently labelled peptide HiLyte Fluor 488-Orn8 oxytocin (Eurogentec) in assay buffer. Fluorescence intensities were measured on a Spark fluorescence plate reader with an excitation wavelength of 340 nm and emission wavelengths of 620 nm and 510 nm for Tb3+ and the fluorophore HiLyte Fluor 488, respectively. The ratio of fluorescence resonance energy transfer (FRET) donor and acceptor fluorescence intensities was calculated (F510 nm/F620 nm). Nonspecific binding was determined in the presence of a 1000-fold excess of unlabelled oxytocin. Data were analysed by global fitting to a one-site saturation binding equation with the GraphPad Prism software.Ligand-binding experiments were performed on whole cells for comparison of affinities for wild-type and receptor mutants using an HTRF binding assay as previously describedFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Pandanus amaryllifolius, commonly recognized as fragrant screw pine due to its characteristic smell, is widely distributed in Southeast Asia and is consumed as a food flavor. In search for potential anti-AD agents from terrestrial sources, P. amaryllifolius was explored for its in vitro anti-amyloidogenic and neuroprotective effects. Thioflavin T (ThT) assay and the high-throughput screening multimer detection system (MDS-HTS) assay were used to evaluate the extracts’ potential to inhibit Aβ aggregations and oligomerizations, respectively. The crude alcoholic extract  and crude base extract  obstructed the Aβ aggregation. Interestingly, results revealed that only CBE inhibited the Aβ nucleation at 100 μg/mL. Both CAE and CBE also restored the cell viability, reduced the level of reactive oxygen species, and reversed the mitochondrial dysfunctions at 10 and 20 μg/mL extract concentrations in Aβ-insulted SY-SY5Y cells. In addition, the unprecedented isolation of nicotinamide from P. amaryllifolius CBE is a remarkable discovery as one of its potential bioactive constituents against AD. Hence, our results provided new insights into the promising potential of P. amaryllifolius extracts against AD and further exploration of other prospective bioactive constituents.Accumulation of amyloid- BP. amaryllifolius extracts to inhibit the Aβ oligomerization was evaluated by the MDS-HTS assay , as previously described [42 (200 μg/mL), the crude extracts (100 μg/mL), and BPL-1  were dissolved in PBS and were utilized, following an enzyme-linked immunosorbent assay method [42 and the extracts in PBST (100 μL) were added to the antibody-coated wells of a 96-well microtiter plate and incubated for 1 h at RT. Detection antibody with conjugated horseradish peroxidase  was employed to spot the Aβ oligomer attached to the capture antibody. After washing with washing buffer, a solution of 3,3′,5,5′-tetramethylbenzidine (100 μL) was added and incubated at RT for 30 min. A stop solution (50 μL) was finally added, and the optical density was measured after 24 h. The significant difference (p < 0.05) against the negative control was determined.The ability of escribed . Aβ42  were obtained from the American Type Culture Collection  and maintained in DMEM supplemented with 10% FBS and 1% kanamycin/penicillin at 37 °C and 5% CO4 cells/well) were plated in 96-well plate and acclimatized for 24 h. Then, the cells were treated with 50, 20, 10, and 1 μg/mL P. amaryllifolius extracts for 24 h. After treatment, cells were washed with PBS, followed by the addition of 100 μL fresh media and incubation for 30 min. The cell viability was assessed following the ATP Luminescence (CellTiter-Glo®)  method as previously described [SH-SY5Y cells  were sub-cultured in 96-well plate and incubated for 24 h. After incubation, cells were then treated with the 20, 10, and 1 μg/mL P. amaryllifolius extracts for 6 h. This was followed by Aβ (10 μM) treatment for 24 h. The % cell viability was determined in triplicate experiments using the ATP Luminescence (CellTiter-Glo®) method as previously described [For the neuroprotective experiment, SH-SY5Y cells  cells were pre-treated with 20, 10, and 1 μg/mL P. amaryllifolius extracts for 6 h and continued with 10 μM Aβ for 24 h. After treatment, cells were then incubated with 25 μM H2DCFDA  for 2 h in 37 °C oven. Fluorescence intensity  was measured, and the ROS level was calculated as a percentage of the control cells in triplicate experiments.After 24 h incubation, SH-SY5Y (1 × 104 cells/well) cells were pre-treated with 20, 10, and 1 μg/mL P. amaryllifolius extracts for 6 h and incubated with 10 μM Aβ for 24 h. The process was followed by the addition of 1 μM TMRE staining solution  and incubation at 37 °C for 1 h. The fluorescence  was measured, and the ∆Ψm was calculated as a percentage of the untreated control (untreated) cells in triplicate experiments.Following 24 h incubation, SH-SY5Y  was subjected to MPLC using 1:1 CHCl3/acetone as eluent to obtain nicotinamide as white crystals (1.8 mg). The schematic diagram of the isolation and identification of nicotinamide from P. amaryllifolius is presented in 1H NMR, 13C NMR, and LCMS spectra of nicotinamide are found in the CBE (3 g) was initially separated by silica gel flash column chromatography using CHCl1H NMR  δ 9.02 , 8.77 , 8.18 , 7.43 ; 13C NMR  δ 167.1 (C=O), 152.8 (CH), 148.2 (CH), 135.5 (CH), 129.6 (C), 123.6 (CH).Nicotinamide: white crystals; 1H-NMR was recorded on an ECZ 400 FT-NMR spectrometer, and 13C-NMR spectra were recorded on an ECZ 600 FT-NMR spectrometer using CDCl3 as solvent and TMS as internal standard. Thin layer chromatography was performed using Merck 60 F254 precoated silica gel plates (0.25 mm thickness). UV254, followed by Dragendorff reagent, was used for visualization. Medium-pressure liquid chromatography (MPLC) was accomplished using a silica gel prepacked column CPS-HS-221-05. PerkinElmer Victor-3® multi-plate reader was used to measure the luminescence, fluorescence, or optical density in the biological assays.p < 0.05.The quantitative data were reported as mean ± SD of at least three experiments. Statistical analysis was determined by one-way ANOVA , with the statistical significance considered at P. amaryllifolius CAE and CBE fractions were elucidated using the ThT and MDS-HTS assays  restored the cell viability. Both extracts at 20 μg/mL showed the highest % cell viabilities at 86.02 ± 4.76% (CAE) and 80.65 ± 4.81% (CBE).In the Aβ-insulted SH-SY5Y cytotoxicity assay , 49.05 ±2DCFDA) reagent. As shown in p < 0.05) in the ROS level was observed when compared to the SH-SY5Y cells treated only with Aβ. CAE exhibited 120.31 ± 3.77% (20 μg/mL) and 123.56 ± 2.04% (10 μg/mL) reductions, while CBE showed 122.63 ± 3.06% (20 μg/mL) and 124.52 ± 1.05% (10 μg/mL) ROS levels. Both extracts did not efficiently lower the ROS level at 1 μg/mL.The amount of intracellular ROS production when SH-SY5Y cells were induced with 10 μM Aβ was determined using 2′,7′-dichlorodihydrofluorescein diacetate (HP. amaryllifolius extracts to restore the MMP was disclosed in p < 0.05) restored the Δψm at 20 μg/mL (86.54 ± 4.61% for CAE and 84.55 ± 2.08% for CBE) and 10 μg/mL (74.43 ± 3.77% for CAE and 78.31 ± 1.06% for CBE). None of the extracts gave a substantial Δψm result at 1 μg/mL.As a result of Aβ induction in the neuroblastoma cells, an increase in ROS level was observed, which may result in mitochondrial dysfunctions. Hence, the capability of P. amaryllifolius CBE led to the isolation of white crystals elucidated as nicotinamide by spectroscopic analyses. This is the first report on the isolation of nicotinamide, a pyridine-containing alkaloid, from a Pandanus species. Nicotinamide was previously identified to influence a flower-inducing activity in Lemna gibba G3 [Mytilus edulis in Mallotus japonicus [Fractionation of gibba G3  and was aponicus . Nicotinaponicus .Although nicotinamide was not subjected to bioassay experiments in this study, foregoing in vitro and in vivo studies, have described its potential effects against AD. Nicotinamide restored cognitive deficits in vivo using the 3x-Tg AD mice model by selectively reducing Thr231 in a Tau protein associated with microtubule depolymerization and increasing the acetylated α-tubulin . In vivoP. amaryllifolius. P. amaryllifolius is an important medicinal plant in Philippine traditional folk medicine. Its ethnomedicinal uses were scientifically validated in pharmacological studies including anti-inflammatory [P. amaryllifolius  from Thailand have been reported [P. amaryllifolius are still unknown. Recently, we described the neuroprotective activity of P. clementis [P. amaryllifolius is also an excellent source of alkaloids, collectively known as Pandanus alkaloids, yet biological studies on these Pandanus alkaloids, including the crude alkaloids extract, are limited [P. amaryllifolius. Natural products, particularly alkaloids, have been the primary source of new scaffolds for the treatment of neurodegenerative diseases, including AD. Interestingly, the current drugs memantine, tacrine, rivastigmine, donepezil, and galantamine approved by the US FDA for AD are all alkaloids.As part of our interest in exploring plant resources with anti-amyloidogenic activity against AD, we have investigated the crude alcoholic and crude alkaloidal extracts of ammatory , antioxiammatory , antiproammatory , hepatopammatory , antidiaammatory , antimicammatory , and antammatory  propertireported . Thus falementis  and the lementis . P. amar limited ,39. HencP. amaryllifolius to effectively inhibit the aggregation and oligomerization of Aβ. As a major contributor leading to AD, these Aβ aggregates lead to Aβ plaques causing various oxidative stressors, neuroinflammation, mitochondrial deterioration, and death. Hence, the neuroprotective effects of P. amaryllifolius extracts on Aβ-insulted neuronal cells were also assessed. As seen in P. amaryllifolius extracts rescued the cell damage caused by Aβ toxicity. This may be explained by reducing the levels of ROS and increasing the mitochondrial membrane potential. To further explore the possible bioactive scaffold, the alkaloid fraction was subjected to chromatographic purification. Unprecedentedly, we were able to isolate nicotinamide, a pyridine-type compound that possesses an array of in vitro and in vivo activities against AD [P. amaryllifolius as a potential medicinal plant against AD. Since nicotinamide was isolated as a minor metabolite comprising only 0.06% of the crude alkaloid fraction, we may speculate that the Pandanus alkaloids may have synergistic inhibitory and neuroprotective effects and other potential bioactive Pandanus alkaloids are present in the extract. Hence, comprehensive isolation of known and possibly new Pandanus alkaloids is being undertaken to assess their in vitro activities against Aβ aggregation and toxicity. Moreover, inhibition of Aβ aggregation and oligomerization was evaluated using protein-based assays. From a future perspective, Aβ will transfect neuronal cells to exploit the anti-aggregation and anti-oligomerization of plant extracts and pure compounds in live cells. Furthermore, validation of the neuroprotective effects of the alkaloids on other neuronal cells and exploring other AD-related mechanistic assays are also warranted.In the present work, we were able to disclose the alkaloidal extract of ainst AD ,32,33. Oainst AD  on P. am


Flavobacterium enshiense R6S-5-6 which was isolated from Ungok (Ramsar) Wetland and conducted whole-genome sequencing to investigate what contribution R6S-5-6 could make to the wetland community. The complete genome sequence of R6S-5-6 has a size of 3,251,289 bp with 37.68% of GC content. Gene annotation revealed that R6S-5-6 has several pathways to break down pollutants, including denitrification, assimilatory sulfate reduction (ASR), and polyphosphate-accumulating process. Furthermore, R6S-5-6 has genes that can have a positive effect on plants living in wetlands, such as storing essential nutrients, promoting plant growth, and protecting plants against pathogens.The wetland is an important ecosystem for purifying pollutants and circulating nutrients. Numerous microorganisms contribute to maintaining this function. We obtained The online version contains supplementary material available at 10.1007/s00284-022-03157-0. Wetlands are among the most productive ecosystems on the planet. They are often referred to as “Earth’s kidneys” because they play a crucial role in managing greenhouse gas emissions, controlling floods and droughts, and purifying pollutants from water . Also, tFlavobacterium, a member of the family Flavobacteriaceae, was first proposed by Bergey et al. [Flavobacterium is widely distributed in the environment, including activated sludge [Flavobacterium not only has a wide range of habitats but also has a wide range of characteristics, such as pathogenicity, which causes fatal damage to both wild and cultured fish, and plant growth-promoting properties [The genus y et al. . Flavobad sludge , stream d sludge , glacierd sludge , and rhid sludge . Flavobaoperties , 11.Flavobacterium enshiense was first reported by Dong et al. in 2013 and consists of only one type strain, designated DK69T [F. enshiense DK69T is a Gram-negative, strictly aerobic, yellow-pigmented, and rod-shaped bacterium isolated from soil collected from the wastewater treatment plant in Enshi, Hubei province, China. In the chemotaxonomic data, F. enshiense DK69T contained menaquinone-6 and phosphatidylethanolamine as the major quinone and polar lipid, respectively. In the whole-genome analysis of F. enshiense DK69T, no pathogenic genes were found [ed DK69T . F. enshF. enshiense, designated R6S-5-6, for a better understanding of the species F. enshiense and bacteria in wetlands. The complete genome sequence of R6S-5-6 is expected to broaden our understanding of the genomic features of F. enshiense and the characteristics of wetland bacteria.Here, we report the complete genome of a novel strain of From the National Institute of Biological Resources Culture Collection (NIBR), we obtained the strain R6S-5-6 which was isolated from Ungok (Ramsar) Wetland located in Gochang, Jeollabuk-do province, the Republic of Korea in 2011 (NIBRBA0000113315). According to the sample details provided, a wetland sample from Gochang was preserved in a 50-ml conical tube in 2011. The sample was serially diluted and plated onto R2A agar. After inoculation, appeared colonies were moved to the same fresh medium at 25 ºC. A purified single colony was confirmed with PCR amplification using universal bacterial 16S rRNA gene primers 27F and 1492R (GenBank accession: JQ928689). The selected colony was termed strain R6S-5-6. The strain was preserved at − 80 ºC until conducting whole-genome sequencing.A genomic DNA was extracted using RBC DNA extraction kit according to the manufacturer instruction. The genomic DNA was purified, sheared, and normalized for PacBio SMRTbell 20 kb Library . Continuhttps://github.com/PacificBiosciences/GenomicConsensus). The dnaA gene in the genome was rotated to the starting point using Circlator (v1.5.5). CheckM (v1.1.3) and BUSCO (v5.2.2) with flavobacteriales_odb10 dataset were used to evaluate the quality of assembly.We used Samtools (v1.13) to convert PacBio BAM file to FASTA and removed short reads under 1000 bp that could affect assembly quality. De novo genome assembly was conducted using Flye (v2.8.3) with –meta option as suggested to be effective for controlling any contamination or artifacts. After excluding contigs of less than 10,000 bp that were suspected of contamination or artifacts, the errors of long circular contig were polished using pbmm2 (v1.4.0) and GCpp (2.0.2) with Arrow algorithm until no more variants were identified  and OrthoFinder (v2.5.4). Multiple sequence alignment for gyrB and orthologs was performed using MAFFT-linsi (v7.487), and they were trimmed using ClipKIT (v.1.3.0) with ‘kpic-smart-gap’ mode. All of alignments were used for a maximum-likelihood tree construction using IQTREE (v2.1.4 beta) under model testing with 1000 ultrafast bootstraps. Protein sequence identity for gyrB gene was calculated through VectorBuilder’s Sequence Alignment tool . Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were calculated using pyani (v0.2.12) and Genome to Genome Distance Calculator , respectively. For further confirmation of the taxonomic assignment, the whole-genome was submitted to the Type Strain Genome Server (TYGS), the Microbial Genome Atlas (MiGA), and the fast bacterial genome identification platform (fIDBAC). Whole-genome-based taxonomic assignment was also conducted using GTDB-Tk (v1.6.0).Phylogenetic analysis of R6S-5-6 was carried out using 16S rRNA gene,  GenBank . For 16Shttps://github.com/tseemann/abricate) with VFDB.Functional annotation was carried out using Prokka, Rapid Annotation using Subsystems Technology (RAST) server, eggNOG-mapper (v2.1.6) with eggNOG 5.0 database, and MicrobeAnnotator (v2.0.5). Other genomic features were investigated using dbCAN2 for Carbohydrate-Active enZYmes (CAZymes), PHASTER for prophage sequences, and IslandViewer4 for genomic islands. Secondary metabolite biosynthesis gene clusters were identified using antiSMASH (v6.0) with “loose” detection strictness. Operons and clustered regularly interspaced short palindromic repeats (CRISPRs) were predicted with Operon-mapper and CRISPRCasFinder, respectively. Enzyme commission number (EC number) was predicted by DeepEC (v0.4.0). Metabolic pathway of enzymes was visualized by iPATH3. Virulence genes were identified using ABRicate (v1.0.1) . The GC content was accounting for 37.68% of the whole-genome. The genome completeness and contamination were determined to be 99.65% and 0.47%, respectively. BUSCO evaluation using flavobacteriales_odb10 showed 100% completeness with no duplicated, fragmented, or missing BUSCOs, revealing high-quality genome can be derived from only using PacBio reads. A total of 3034 genes were predicted, comprising 2940 coding DNA sequences (CDSs), three complete rRNA operons, 33 misc RNAs, 51 tRNAs, and one tmRNA. F. enshiense DK69T (99.65% similarity)  was below the threshold value of 96.5%. MiGA showed an average amino acid identity (AAI) value of 96.58% against F. enshiense and concluded that strain R6S-5–6 possibly even belongs to the species F. enshiense.To investigate the phylogenetic position of R6S-5-6, we first calculated the 16S rRNA gene sequence similarity against type strains. The sequences of all three 16S rRNA genes in R6S-5-6 were identical, and the closest species was ty) Fig. A. The gy69T Fig. B. In the5-6 Fig. C. The 16of 97.2% . TherefoF. enshiense DK69T revealed that ANI based on MUMmer (ANIm) value was 91.01%, dDDH value was 69.3% (C.I. 65.4–72.9%) at formula d0, and 40.4% at d4, and GC content differed by 0.05%  purifying wastewater, (2) reducing eutrophication, or (3) promoting plant growth. We highlight genomic properties of R6S-5-6 that have potential for sustaining wetland ecosystems.Wastewater contains nitrogen, phosphorus, and sulfur, which can cause eutrophication. Wetland ecosystems have the ability to purify water, and bacteria have been found to play a major role in this process . In this2O [3− to NO2− by nitrate reductase (Nar), NO2− to NO by nitrite reductase (Nir), NO to N2O by nitric oxide reductase (Nor), and N2O to N2 by nitrous oxide reductase (Nos) [Denitrifiers in wetlands can control greenhouse effects by removing greenhouse gas N2O . R6S-5-62O . The prose (Nos) . R6S-5-6pstA, pstB, pstC, pstS, and pitA).Excess phosphorus discharged from agriculture and industry adversely affects the ecosystem and causes eutrophication. Accumulating polyphosphate plays an important role in removing soluble phosphorus in wastewater . The abi42− → adenosine-5-phosphosulfate (APS) → 3′-phospho-5′-adenylyl sulfate (PAPS) → SO32− → S2− [cysN/cysD, cysD, cysH, and cysJ/cysI encoding enzymes associated with the ASR pathway .Sulfate present in wastewater is removed through assimilatory sulfate reduction (ASR) pathway . Sulfate2− → S2− . R6S-5-6n = 13), glycosyltransferases (GTs) (n = 44), carbohydrate esterases (CEs) (n = 12), carbohydrate-binding modules (CBMs) (n = 1), and auxiliary activities (AAs) (n = 2). GTs are most abundant, accounting for 61% of the total number of predicted CAZymes. GT families are involved in the biosynthesis of compounds such as oligosaccharides, polysaccharides, and glycoconjugates. The role of this enzyme is important in that it converts potential toxic organic pollutants into low-molecular-weight intermediates or harmless terminal products [Analysis of the genome using dbCAN showed 72 genes encoding for CAZymes, including glycoside hydrolases (GHs) (products . As a reproducts .Flavobacterium spp. have a positive effect on plants. Plant growth-promoting bacteria (PGPB) contribute to plants as follows: (1) storage of essential nutrients for plants, (2) promotion of plant growth, and (3) plant protection. Considering this, several genes of R6S-5-6 emphasize some useful effects on plant ecology in wetlands.Recent studies have shown that some nif genes [nifU), nitrogen fixation regulation protein (FixK), and Global nitrogen regulator (NtcA) associated with nitrogen-fixing.Nitrogen and phosphorus are essential nutrients for plants but they are limited in the rhizosphere . Nitrogeif genes . In thispqqL gene predicted to be related to PQQ production [Alkaline phosphatase (ALP) converts organic phosphorus to available phosphorus for plants . Howeveroduction .Auxin is a plant hormone that promotes plant growth and development . Indole-Bacteria with 1-aminocyclopropane-1-carboxylate (ACC) deaminase absorb some of the ACC synthesized by plants. ACC is decomposed by ACC deaminase and easily metabolized in bacteria. Lowering ACC in plants reduces the amount of ethylene. This prevents the effect of stress ethylene generated in stressful situations and helps plants grow . R6S-5–6Flavobacterium isolated from plants is known to be involved in the biosynthesis of compounds that inhibit plant pathogens [Aquimarina sp. BL5 at 40%.The nonribosomal peptide synthetase and polyketide synthase (NRPS-PKS) gene cluster of athogens . R6S-5-6Flavobacterium. They are pigments that form a yellow colony and are composed of a 2–5 dialkylresorcinol (DAR) with a non-isoprenoid aryl-polyene carboxylic acid ester [Pseudomonas sp. [The antiSMASH results indicated that R6S-5-6 has gene clusters for flexirubin and APE Vf. Flexirubin which are widely distributed in the genus id ester , 39. Altonas sp. .Cell wall dissolving enzymes produced by PGPB can break down the cell walls of plant pathogens. R6S-5-6 has cellulase (EC 3.2.1.4), glucanase (EC 3.2.1.21), and β-1,3-glucanase (EC 3.2.1.39) which are known to contribute to plant defense by degrading the cell walls of plant pathogens , 42.In wetlands where various living things are in harmony, bacteria play a significant role in maintaining the ecosystem, including purifying pollutants and promoting plant growth. However, bacteria and their genomes isolated from wetlands have been scarcely reported. Therefore, in this study, we investigated the genomic characteristics of R6S-5-6 which was isolated from Ungok Wetland by conducting de novo assembly.F. enshiense. In this regard, there have been studies that ANI cannot perfectly reflect the evolutionary distance of all members of bacteria, including that the threshold value for ANI was inflated by using highly redundant genomes [Variovorax paradoxus and Stenotrophomonas maltophilia [Donovan et al. [F. enshiense, and it was isolated from the waste liquid treatment facility of the pharmaceutical company, we think it should be discreet in proposing R6S-5-6 as a novel species. Since pharmaceutical waste has mutagenicity and genotoxicity [F. enshiense located at the species boundary with type strain DK69. We expect that the phylogenetic position will be changed more precisely if the study of F. enshiense continues and additional strains are reported, as in the case where the species Lactobacillus gasseri was recently divided into L. gasseri and Lactobacillus paragasseri due to intraspecies heterogeneity [We performed several analyses to confirm the taxonomic status of R6S-5-6. Interestingly, all metrics except for ANI concluded that R6S-5-6 belongs to  genomes  and thattophilia . Also, an et al. , intrasptoxicity , it is togeneity . In addiF. enshiense R6S-5-6 in the wetland ecosystem, we looked into the pathways related to the purification of wetlands and plant growth. R6S-5-6 has genes involved in denitrifying, polyphosphate-accumulating, and assimilatory sulfate-reducing pathways for wastewater purification. Regarding plant growth in wetland ecosystems, R6S-5-6 has genes that store nitrogen and phosphorus for plant use. R6S-5-6 also has genes encoding tryptophan biosynthesis-related enzymes and ACC deaminase that inhibit plant pathogens. These results suggest that F. enshiense R6S-5-6 has the potential to play a beneficial role in the wetland community.To predict the role of F. enshiense R6S-5-6 in the wetland ecosystem. Unlike previous investigations of Flavobacterium spp., which mainly focused on pathogenic characteristics, we provided information on how R6S-5-6 is involved in the circulation of wetland and specific pathways in plant ecosystems. The genomic characteristics of R6S-5-6 investigated in this study will broaden understanding of species F. enshiense where only one strain has been reported.In this study, we analyzed the role of Supplementary file1 (PDF 157 kb)Below is the link to the electronic supplementary material.