The relationship between autoimmune diseases and the gut microbiome has been intensively studied, and several autoimmunity-associated bacterial taxa have been identified. However, much less is known about the roles of the gut virome in autoimmune diseases.Here, we performed a whole gut virome analysis based on the shotgun sequencing of 476 Japanese which included patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis and healthy control subjects.Podoviridae significantly decreased in the gut of the patients with SLE. To understand how these viruses affected the bacteriome, we performed a quantitative virus–bacterium association analysis and clustered regularly interspaced short palindromic repeat-based virus–bacterium interaction analysis. We identified a symbiosis between Podoviridae and Faecalibacterium. In addition, multiple bacterial targets of crAss-like phages were identified .Our case–control comparison of the viral abundance revealed that crAss-like phages, which are one of the main components of a healthy gut virome, significantly decreased in the gut of the patients with autoimmune disease, specifically the patients with RA and SLE. In addition, Our data suggest that the gut virome can affect our body either directly or via bacteria. Our analyses have elucidated a previously missing part of the autoimmunity-associated gut microbiome and presented new candidates that contribute to the development of autoimmune diseases. Alteration of the gut microbiome has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and multiple sclerosis. However, much less is known about the roles of the gut virome in the development of these diseases.Our case–control comparison of the viral abundance revealed that crAss-like phages, which are one of the main components of a healthy gut virome, significantly decreased in the gut of the patients with autoimmune disease, mainly in the patients with RA and SLE.Ruminococcus spp) from the quantitative virus–bacterium association analysis and clustered regularly interspaced short palindromic repeat based virus–bacterium interaction analysis.Multiple bacterial targets of crAss-like phages were identified , gut virome studies have used virus-like particle (VLP) sequencing or whole-metagenome shotgun sequencing that require complex data processing and computational resources. In addition, the quantification methods used in bacterial analysis are not transferable to virome analysis because the current reference viral genome databases cover only a limited fraction of the actual gut virome populations.In addition to the quantification of the gut viral abundance, the identification of the viral host is necessary to reveal the effect of bacteriophages on the gut microbial community. Although the coabundance analysis was useful in conferring virus–host interaction, it was not sufficient to show the evidence of viral infections.A case–control discrepancy of the gut virome was reported for only a limited number of intestinal,N = 111), SLE (N = 47) and MS (N = 29) and healthy control (HC) subjects (N = 289). This is the largest case–control comparison of the viral abundance to date. We evaluated changes in the gut virome and whether it was specific to each autoimmune disease or shared across multiple autoimmune diseases. In addition, we performed a virus–bacterium association analysis based on the abundance data and a CRISPR-based virus–bacterium interaction analysis to reveal the virus–bacterium interaction mediated by disease-associated viruses identified in this study.Here, we performed a whole gut virome analysis of 476 Japanese which included the patients with RA  were not included in the dataset. All subjects provided written informed consent before participation. Those who took antibiotics within a month was reported as the patients treated with antibiotics. The characteristics of the subjects are shown in 10.1136/annrheumdis-2021-221267.supp1Supplementary dataFaecal samples were collected in tubes containing RNAlater (Ambion). After the weights of the samples were measured, RNAlater was added to produce 10-fold dilutions of homogenates. Faecal samples were stored at −80°C within 24 hours after collection. Bacterial DNA was extracted according to a previously described method. Briefly, 0.3 g glass beads (diameter: 0.1 mm) (BioSpec) and 500 µL EDTA-Tris-saturated phenol were added to the suspension, and the mixture was vortexed vigorously using a FastPrep-24  at 5.0 power level for 30 s. After centrifugation at 20 000 g for 5 min at 4°C, 400 µL of supernatant was collected. Subsequently, phenol–chloroform extraction was performed, and 250 µL of supernatant was subjected to isopropanol precipitation. Finally, DNAs were suspended in 100 µL EDTA-Tris buffer and stored at −20°C.A shotgun sequencing library was constructed using the KAPA Hyper Prep Kit (KAPA Biosystems), and 150 bp paired-end reads were generated on HiSeq 3000 and NovaSeq 6000 for sequencing batches 1–4 and batch 5, respectively. The sequencing data from the patients with RA in sequencing batch 3 were newly obtained for this study, while other sequencing data were derived from previous studies,We followed a series of steps to maximise the quality of the datasets. The main steps in the quality control (QC) process were as follows: (1) trimming of low-quality bases, (2) identification and masking of human reads and (3) removal of duplicated reads. We marked duplicate reads using PRINSEQ-lite . We trimmed the raw reads to clip Illumina adapters and cut-off low-quality bases at both ends using the Trimmomatic . We discarded reads less than 60 bp in length after trimming. Next, we performed duplicate removal by retaining only the longest read among the duplicates with the same sequences. As a final QC step, we aligned the quality-filtered reads to the human reference genome (hg38) using bowtie2 (V.2.3.5) with default parameters and BMTagger (V.3.101). We kept only the reads of which both paired ends failed to align in either tool. The average QC-passed total sequencing base pairs were 25.5, 7.8, 6.7, 7.0 and 8.3 Gb for sequencing batches 1–5.et al.https://portal.nersc.gov/dna/microbial/prokpubs/EarthVirome_DP/),The de novo assembly of the filtered paired-end reads into contigs was conducted using MEGAHIT . Following assembly, contigs whose 5’ and 3’ terminals have ≥50 bp overlap were marked as circular contigs. Linear contigs of ≥5 kbp and circular contigs of ≥1.5 kbp were subjected to VirSorter (V.1.0.6),https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/) as reference. Although the taxonomic information of viral genomes was based on NCBI taxonomy data, family-level classification of crAssphage (NC_024711.1) was modified to crAss-like phage according to recent publications.−10, nucleotide identity of >95%, and coverage of contigs of ≥85%. Viral contigs were assigned to higher than or equal to species level taxonomy based on the megablast hit with the highest bit score. Contigs not assigned taxonomy in the previous step based on the nucleotide-level comparison proceeded to protein-level comparison for annotation of family-level taxonomy. First, we predicted open reading frames (ORFs) in viral contigs using MetaProdigal (V.2.6.3) with -p meta option. To detect crAss-like phages, we compared ORFs in viral contigs and crAss-like phage signature genes.crAssphage (NC_024711.1) were queried against the ORFs in viral contigs using blastp with E-value of <10−5 and alignment length of ≥350. Viral contigs with blastp hits were assigned family-level taxonomic annotation as crAss-like phage. Then, the remaining unclassified contigs were proceed to taxonomic annotation by a voting system.−5. Based on the DIAMOND blastp hits with the highest bit score, the ORFs were assigned taxonomic annotation. We then summarised all the taxonomic assignments of the ORFs for each contig, and assigned taxonomic information higher than or equal to family level based on the majority taxonomic assignment among the annotated ORFs. Viral contigs without a majority taxonomic assignment were regarded as unclassified viruses. Viral contigs with less than two annotated ORFs were also regarded as unclassified viruses.We first classified viral contigs using complete viral RefSeq genomes  was used to remove the reads mapped at <95% nucleotide identity to the contigs. Then, CoverM trimmed_mean (parameters: --trim-min 0.05 --trim-max 0.95) was used to calculate the average read depth across the viral populations. The read depth of each viral population was divided by the total sequenced length of each sample for normalisation. The normalised read depth of each viral population was summed up by each sample, and the normalised abundance of each clade was calculated at different taxonomic ranks. Then, we detected outlier samples using principal component analysis (PCA).After viral quantification, we extracted nonviral reads to obtain bacterial abundance. We used the previously constructed bacterial reference dataset composed of curated 7881 genomes.We normalised the viral abundances using log transformation. For family-level analysis, we retained only those clades that were detected in all the sequencing batches and ≥10% of the samples. The detection ratios of the QC-passed viral clades are indicated in Ncase=111 and Ncontrol=111). Sequencing batches 2, 3 and 5 were used in the analysis for SLE (Ncase=201 and Ncontrol=47). Sequencing batch 4 was used in the analysis for MS (Ncase=29 and Ncontrol=74). We adopted sex, age, age2, sequencing batch, and total sequenced length as covariates. To evaluate the effect of potential confounding factors, we performed sub-analysis with datasets from which (1) males were removed, (2) non-new onset patients were removed, (3) those treated with antibiotics were removed, (4) those treated with proton pump inhibitors were removed, (5) those treated with steroids were removed or (6) those treated for autoimmune diseases were removed. Case–control comparisons per sequencing batch were performed to evaluate the consistency of the results between the sequencing batches. In addition, comparisons between (1) the treated patients and the untreated patients, (2) the new onset patients and the non-new onset patients, and (3) those with high vs low disease activity were performed. For the meta-analysis, we used the metafor (V.3.0–2) package for R. As the case–control analyses for RA and SLE had significant overlap in control samples, we used only subjects from sequencing batch five for SLE case–control comparison. As for the random-effect model, ‘REML’ option was used for fitting parameters with restricted maximum likelihood estimation.Case–control association tests were performed separately for each standardised clade abundance using the glm function in the R (V.4.0.1), and the effect size of the viral abundance was evaluated. Sequencing batches 1, 2 and 3 were used in the analysis for RA .2, sequencing batch, total sequenced length and the top 10 principal components of the normalised bacterial abundance as covariates. The false discovery ratio was calculated using the Benjamini-Hochberg procedure. To evaluate the effect of potential confounding factors, we performed subanalysis with datasets from which (1) male subjects were removed, (2) non-new-onset patients were removed, (3) only HC subjects were retained, (4) those treated with antibiotics were removed, (5) those treated with proton pump inhibitors were removed, (6) those treated with steroids were removed and (7) those treated for autoimmune diseases were removed. In addition, disease-specific virus–bacterium associations were evaluated by adding and evaluating virus × RA, virus × SLE, or virus × MS terms in the regression formula. The sample sets were defined as in the case–control comparison for each disease.Virus–bacterium association tests were performed separately for each virus–bacterium pair using the lm function in the R, and the effect size of the viral abundance was evaluated. We evaluated the association between eight viral families that passed clade QC described above and 802 bacterial clades. We adopted sex, age, age−10, nucleotide identity of >95%, and coverage of contigs of ≥90%. Based on the megablast hit with the highest bit score, bacterial taxonomy was assigned to the contigs. Spacers within the predicted CRISPR sequences were queried against the viral contigs recovered from the same sample using blastn with E-value of <10−5, nucleotide identity of >95%, and coverage of spacers of ≥90%. Based on the blastn hit with the highest bit score, viral taxonomy was assigned to the spacers. Then, we summarised the virus–bacterium pair based on taxonomy annotated to the spacers and the contigs, collapsed within each sample, and summarised across all the samples.We extracted contigs of ≥5 kbp that were not classified as viral ones. Then, we predicted the CRISPR sequences in these contigs with MinCED (V.0.4.2),This research was done without patient and public involvement. Patients and public were not invited to comment on the study design and were not consulted to develop patient relevant outcomes or interpret the results.Caudovirales including Siphoviridae, Myoviridae, Podoviridae, and unclassified Caudovirales were highly abundant among the gut virome .An overview of our study is shown in 2, sex, sequencing batch and total sequencing length. We found that crAss-like phages significantly decreased in the metagenome of the patients with RA , suggesting that the significant decrease in crAss-like phages was driven by RA and SLE. A comparison between the treated patients and the untreated patients, or the new-onset patients and the non-new onset patients showed no significant difference  with non-significant heterogeneity among the diseases (Q = 3.03 and P for Q = 0.22). The Association between crAss-like phages and combined autoimmune diseases was detected even when the viral abundance was transformed to the absence/presence state , we found that the abundance of crAss-like phages was significantly decreased in the combined autoimmune diseases , table 2P = 0.0036 and P = 0.019, respectively; In our study, some crAss-like phages, which had nucleotide-level similarity to NC_024711.1, were identified. We performed a case–control comparison of NC_024711.1 and other crAss-like phages in combined autoimmune diseases, which revealed that both NC_024711.1 and other crAss-like phages were significantly associated with combined autoimmune diseases . Even after removing male subjects, non-new onset patients, patients with combined autoimmune disease, or those who took medications such as antibiotics, proton pump inhibitors, steroids and any therapeutics for autoimmune diseases, the effect sizes of the association between Faecalibacterium and Podoviridae were consistent . Faecalibacterium is a bacterial genus that produces short-chain fatty acids, which have anti-inflammatory activity.Faecalibacterium were frequently reported in immune-related diseases or conditions.Podoviridae and Faecalibacterium is important to keep the homeostasis of the immune system.In our case–control comparison of the viral abundance, with SLE , table 1with SLE . In the To further elucidate the virus–bacterium interaction, we analysed the CRISPR loci of bacterial contigs from all samples to identify infected viruses. We detected 28,347 CRISPR spacer sequences in total (601.6 CRISPR spacer sequences per sample) by MinCED,Caudovirales such as Siphoviridae, Myoviridae and Podoviridae were frequently observed. In addition, the preferred hosts for each viral family  were also consistent with the previous report.Consistent to a previous report of healthy Japanese subjects,Podoviridae . This result supported the symbiotic relationship between Podoviridae and Faecalibacterium, which was suggested from the quantitative virus–bacterium association analysis.To reveal the autoimmunity-associated virus–bacterium interaction, we searched the hosts of autoimmunity-associated viruses, crAss-like phages and oviridae . As for crAssphage (NC_024711.1), infected to Bacteroidetes from multiple lines of evidence.Bacteroides vulgatus. Fujimoto et al reported that crAss-like phages, which had nucleotide-level similarity to NC_024711.1 dominantly infected to Bacteroidetes, while other crAss-like phages could also infect to Firmicutes.Firmicutes in our analysis. Among the virus–host interaction involving crAss-like phages, Ruminococcus spp infection was detected in three independent samples. Therefore, we detected several hosts of the bacteriophages which decreased in autoimmune diseases.It was reported that crAss-like phages, especially those closely related to the first identified Podoviridae also decreased in the gut of the patients with SLE. Our quantitative virus–bacterium association analyses revealed a positive correlation between Podoviridae and Faecalibacterium abundance, and a direct evidence of phage infection was provided by CRISPR-based virus–bacterium interaction analyses. Bacteroidetes and Firmicutes, including Ruminococcus spp, were detected as hosts of crAss-like phages, which were replicated in multiple samples.In this study, we revealed the disease-associated changes in the gut virome of Japanese patients with autoimmune disease. We discovered that crAss-like phages in the gut of the patients with RA and SLE decreased. In addition, Caudovirales that included Siphoviridae, Myoviridae and Podoviridae, was mostly consistent with previous reports,Microviridae was lower than that in previous studies performed with VLP-sequencing,Microviridae was strongly dependent on the library preparation method, specifically the multiple displacement amplification technique that is frequently used in VLP-sequencing and is known to show an upward bias of the ssDNA viral abundance.et al compared the VLP-sequencing data with the shotgun sequencing data and revealed that there was a large difference in the reconstructed viral populations, and the composition of ssDNA viruses recovered from shotgun sequencing data was consistently lower than that of the VLP-sequencing data.We obtained the gut virome abundance data from the whole-metagenome shotgun sequencing reads. The resulting viral composition, characterised by highly abundant crAssphage was first identified in 2014 through the cross-assembly of shotgun sequencing data from multiple faecal samples.crAssphage frequently existed in the healthy gut virome, a large fraction of its proteins showed no similarity to the already existing viral proteins. Thus, crAssphage was considered as comprising a new taxonomic group (crAss-like phage). Since crAss-like phages were discovered, its biological features including taxonomy and infective hosts,crAssphage and disease states have been warranted, there had been no reports on the relationships between crAssphage and diseases.Bacteroides vulgatus and various Firmicutes were detected as hosts of crAss-like phages. Bacteroidetes was originally predicted as a host of crAss-like phages by read co-occurrence analysis and CRISPR-based analysis.Bacteroides vulgatus as a host of crAssphage (NC_024711.1) was reasonable. Although not detected in our CRISPR-based analysis, Bacteroides intestinalis was also a host of crAss-like phages, which was validated in vitro.Bacteroides intestinalis was nominally correlated (effect size = 0.083 and Pvirus-bacterium = 0.0078), suggesting that the virus–host relationship between the two existed in our dataset. In previous bacterial case–control comparisons,Bacteroides intestinalis and SLE , although the association was not significant for RA .In our CRISPR-based virus–bacterium interaction analysis, Ruminococcus spp was detected in three independent samples. The 16S rRNA sequencing study of the patients with SLE,Ruminococcus was related to the SLE and RA pathology.Among the virus–host interactions involving crAss-like phages, infection of et al performed a CRISPR-based virus–host association analysis for crAss-like phages,Prevotella copri, which associated with the pathogenesis of RA,Recently, Yutin Podoviridae significantly decreased in the gut metagenome of the patients with SLE. Both the quantitative and CRISPR-based virus–bacterium interaction analyses suggested the symbiosis of Podoviridae and Faecalibacterium. Faecalibacterium is known beneficial bacteria as they produce short-chain fatty acids.Faecalibacterium, Faecalibacterium prausnitzii was the most intensively studied and is associated with many conditions, including autoimmune diseases.Podoviridae affected Faecalibacterium and vice versa, the effect of Podoviridae on the biological property of Faecalibacterium should be experimentally tested in the future.In summary, our whole gut virome case–control study revealed a previously unknown part of the link between the gut microbiome and autoimmune diseases. A comprehensive understating of the gut microbiome, including its virome, deepens the insights of the pathogenesis of autoimmune diseases.https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software/downloads.htmlbcl2fastq, http://www.usadellab.org/cms/?page=trimmomatic bowtie2, http://bowtie-bio.sourceforge.net/bowtie2/index.shtmlTrimmomatic, ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/bmtagger/BMTagger, http://prinseq.sourceforge.net/PRINSEQ, https://github.com/voutcn/megahitMEGAHIT, https://github.com/simroux/VirSorterVirSorter, https://github.com/jessieren/VirFinderVirFinder, https://busco.ezlab.org hmmer, http://hmmer.org/download.html ncbi-blast-plus, https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=DownloadBUSCO, https://github.com/bbuchfink/diamond coverM, https://github.com/wwood/CoverMDIAMOND, https://github.com/hyattpd/ProdigalProdigal, https://github.com/ctSkennerton/mincedMinCED, http://www.htslib.org/download/ bedtools, https://github.com/arq5x/bedtools2Samtools, https://www.r-project.orgR, https://www.python.org/downloads/release/python-376/Python, https://bioinf.shenwei.me/seqkit/download/Seqkit, https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/RefSeq (Virus), https://portal.nersc.gov/dna/microbial/prokpubs/EarthVirome_DP/VPF,


The essential amount of powders was milled to yield different compositions such as AA8079, AA8079-5 wt.%B4C, AA8079-10 wt.%B4C, and AA8079-15 wt.%B4C. The influence of powder metallurgy parameters on properties’ density, hardness, and compressive strength was examined. The green compacts were produced at three various pressures: 300 MPa, 400 MPa, and 500 MPa. The fabricated green compacts were sintered at 375 °C, 475 °C, and 575 °C for the time period of 1, 2 and 3 h, respectively. Furthermore, the sintered samples were subjected to X-ray diffraction (XRD) analysis, Energy Dispersive Analysis (EDAX), and Scanning Electron Microscope (SEM) examinations. The SEM examination confirmed the uniform dispersal of B4C reinforcement with AA8079 matrix. Corrosion behavior of the composites samples was explored. From the studies, it is witnessed that the rise in PM process parameters enhances the density, hardness, compressive strength, and corrosion resistance.In this paper, Al-Fe-Si-Zn-Cu (AA8079) matrix composites with several weight percentages of B B4C possesses better wear and impact opposition, a higher maximum melting point, and better resistance to chemical agents [4C has been extensively utilized as cements and armor plates, despite its less specific gravity, better hardness value, and maximum elastic modulus value [4C/Al nanocomposites produced through the PM method, and stated that maximum hardness and compression strength was achieved for Al-5% B4C composites [2O3 nanocomposites produced through mechanical alloying and PM route and described the mechanical properties. Anil Kumar Bodukuri et al. synthesized B4C/SiC/Al powder metallurgic composites and studied the mechanical behavior [2O3 reinforced nanocomposite manufactured via mechanical alloying [2–Gr composites, and observed the presence and dispersal of reinforcement particles with matrix [Metal based composites have extensive uses in numerous engineering areas owing to its extreme properties, namely superior precise stiffness, strength/weight ratio, and wear opposition . Traditil agents ,8. B4C hus value . Hamid Amposites . Jeyasimmposites  examinedbehavior . Sivasanalloying . Ravichah matrix . Mohammeh matrix . H.M. Zah matrix .2O3 hybrid composites and stated that inclusions of Al2O3 and Gr particles improve the mechanical behavior of the AMCs considerably. Nazli Akcamli [4C particles results in significant enhancement in mechanical behavior of the produced composites. Vipin Kumar Sharma [2O3-SiC-CeO2 composites by PM route and concluded that a rise in reinforcement wt.% enhances the composites mechanical behavior drastically. Mohd Bilal Naim Shaikh [2O3 nanocomposites through the PM process and observed that superior hardness and strength properties were attained at integration of 10 wt.% Al2O3. Erdemir et al. [4C hybrid composites prepared by the PM route, and concluded that increase in reinforcement particles results in enhancement in mechanical properties. Fathy et al. [2O3/Gr hybrid composites produced via the PM process and witnessed that a rise in reinforcement weight percentage increases the micro hardness gradually. Stalin et al. [3 composites synthesized by the PM route; from the experimentation it has been concluded that corrosion opposition of the composites enhanced with the addition of a MoO3 particle.Norul AmierahBinti Nor Zamani et al.  studied  Akcamli  developer Sharma  synthesim Shaikh  exploredm Shaikh  producedr et al.  producedr et al.  exploredy et al.  exploredy et al.  investign et al.  studied 4C particle. From the literature review it has been found that the development of aluminum alloy is a challenging task. Hence, ball milling was used to develop aluminum alloy in the present work. However, the mixing of alloying element and reinforcement particle with the major constituent could be possible by selecting suitable ball milling and powder metallurgy process parameters. Furthermore, this work has made an effort to synthesize AA8079-B4C composites at different powder metallurgy process parameters to analyze the microstructure, mechanical, and corrosion behavior. The effect of parameters on the hardness, density, CS, corrosion properties, and microstructure have been analyzed in detail.From the detailed literature review, it could be understood that very little research work has been completed in the development of aluminum alloys of 8xxx series using PM technique. Additionally, present work aims to develop composite with the AA8079 and B4C was purchased from kemphasol, Mumbai, India. The other powders, such as copper, iron, silicon, and zinc were purchased from Lobachemi, Mumbai, India. SEM image of the as procured Al and B4C powders are displayed in AA8079 was manufactured via mixing the 99.5% elemental powders aluminum (100 μm), copper (10 μm), iron (10 μm), silicon (8 μm), and zinc (10 μm). Boron carbide of size 10 μm was utilized as reinforcement. The aluminum and BThe composite powders were synthesized using high energy ball mill for 10 h . The drum speed was 100 rpm. A steel ball with 10 mm diameter was used. The ball to powder ratio was 5:1. To avoid the temperature rising, a cooling process was carried out every 10 min as per . The gre4C sintered preforms to study the phase identification using X-ray diffractometer (Broker Eco D 8). The chemical compositional examination was examined via energy dispersive analysis (via EDAX-AMETEK-TSL). Then, the green compacts were sintered at various temperatures of 375 °C, 475 °C, and 575 °C for a period of 1, 2, and 3 h, respectively, using electric muffle furnace (HITECH India) below controlled atmosphere to evade oxidation; the sintered samples were retained in the furnace until it reaches the room temperature . By foll4C composite samples with 1.0 cm2 surface area are wide-open to corrosion medium of 3.5% NaCl solutions. The potentio-dynamic current–potential curves were obtained by polarizing the specimen from −0.1 V to +0.1 V on open circuit potential at a scan rate of 0.05 mV/s. EIS measurements was conducted utilizing a small amplitude AC signal of 10 Mv over a frequency of 100 kHz–0.01 Hz [The microstructure analysis of sintered composite samples was performed by SEM (ZESIS model). The micro Vickers hardness test was carried out using micro Vickers hardness tester (Model: MV-1 PC), test was carried out at a load of 0.3 kg and a stay time of 10 s, as per ASTM standard E384-08. The compressive test was carried using computer controlled universal testing machine (Tinius Olsen) having a capacity of 50 kN in accordance with ASTM standard E9-89a. Electrochemical measurement was achieved by utilizing Versa STAT MC. Later, for polarization examinations, electrodes were utilized for the electrochemical impedance spectroscopy (EIS) examination deprived of any surface treatment. The AA8079/B–0.01 Hz ,31. The This section explains the microstructure and characterization studies of sintered composite preforms, and the effect of PM parameters on the density, hardness, CS, corrosion behavior, and the microstructure of the composites after compression test.4C, AA8079-10 wt.%B4C, and AA8079-15 wt.%B4C composites preforms has been studied using SEM. 4C content, and it can be seen that pores are witnessed for unreinforced AA8079. Furthermore, no pore has been found in the composite preforms, due to occupation of B4C particle in the matrix. 4C and the presence of B4C particles are evident.The microstructure analysis of the as-sintered AA8079, AA8079-5 wt.%B4C particle can be seen, and no pores or crack has been found. Enhanced interfacial connection was attained amid the matrix and B4C particles. 4C, and from the image no agglomeration of particles was observed.From 4C in Al matrix. However, in this work, we overcome that problem by selecting the suitable ball milling parameters [4C with matrix is also of note. The SEM revealed the occurrence of the distributed phase, which is the B4C is dispersed evenly in the matrix.Some researchers reported agglomeration issues for the inclusion of 10 wt.% of Brameters ,33,34. D4C was predicted with fine dispersal with matrix.The EDAX analysis of the sintered composites preforms are displayed in 4C peaks reveals the occurrence of B4C in (110), (104), (021), (211), and (205) planes. The intensity of B4C peaks enhances with the raise in weight percentage of B4C in the composites. XRD results confirm the occurrence of aluminum in the major peak, and the occurrence of B4C, exposed by small peak, and it ensures the respective weight percentage of the composites. Furthermore, it has been confirmed that no intermetallic compounds were formed during the sintering process as reported by the previous researchers [earchers ,36. Peak4C composites with respect to CP, S.Temp, and ST. The increase in CP, S.Temp, and ST increases the density of the AA8079-5 wt.%B4C composites. For AA8079-5 wt.%B4C composites, maximum density of 2.96 g/cm3 was attained at CP of 500 MPa, S.Temp of 575 °C, and ST of 3 h. Densification is proportional to CP, S.Temp, and ST. The rate of dispersal enhances, while increase in S.Temp offers good densification at high temperature.The influence of compaction pressure (CP), sintering temperature (S.Temp) and sintering time (ST) on the density are provided in 4C composites. The increase in CP, S.Temp, and ST increases the density of the AA8079-10 wt.%B4C composites. For AA8079-10 wt.%B4C composites, maximum density of 3.25 g/cm3 was obtained at CP of 500 MPa, S.Temp of 575 °C, ST of . Enhancement in ST offers much time for pore closure in the matrix; henceforth, densification is perceived to rise with the rise in ST. At 575 °C, the density enhances due to a decrease in pores. Amid the sintering process, reduction in the samples occurs, despite the volume of diffusion of atoms from the grain boundary sources to the voids, which results in density enhancement.4C composites. The increase in CP, S.Temp, and ST increases the density of the AA8079-15 wt.%B4C composites. For AA8079-15 wt.%B4C composites, maximum density of 3.45 g/cm3 was obtained at CP of 500 MPa, S.Temp of 575 °C, and ST of 3 h. It could be understood that an increase in CP, S.Temp, and ST enhances the density of the AA8079-B4C composites. This is due to the fact that diffusion of particles and decrease in porosity occurred; it results in improved density as reported by Patel et al. [4C composites at a maximum density, the specimen would be compacted to 500 MPa and sintered at 575 °C temperature for 3 h.l et al. . General4C composites with respect to CP, S.Temp, and ST. 4C composites. Increasing the CP, S.Temp, and ST results in hardness enhancement due to maximum densification. When the PM process parameters increased, pores and voids present in the samples were completely occupied by the B4C particles. This could be one of the reasons for hardness increment.4C is the third hardest material, due to the fact that the hardness of the composites increased gradually. These outcomes are all around concurred with the earlier findings of different researchers [4C with AA8079 occurred, it resulted in maximum hardness enhancement. At maximum sintering temperature, particle-to-particle binding takes place, forming a better bond by the diffusion of atoms in a solid-state bonding method. Improved ductility, dispersion strengthening mechanism, and refinement of grain size result in enhanced hardness [The applied load results in particle deformation; however, alterations in particle size and shape improve the hardness. The hardness upsurges while enhancing the CP, S.Temp, and ST. It is obvious that when the CP, S.Temp, and ST increase, the hardness of the composites enhances due to particle-to-particle appropriate bonding. Furthermore, Bearchers ,8,37,38.hardness ,40,41.4C composites with respect to CP, S.Temp, and ST. 4C wt.% rises could be ascribed to the dispersal strengthening effect [Additionally, the compressive strength of the composites totally relies upon the PM parameters, which create the enhancement in properties conceivable. As indicated by this examination, the most elevated compressive strength was noticed for the specimen compacted at 500 MPa, and sintered at 575 °C for 3 h. The enhancement in the compressive strength may be accredited to the shifting of load from matrix to the hard reinforcement ,48. The g effect . The maxg effect .4C particle into the matrix alloy. It is clear from the SEM images in 4C particles are reoriented in the way of metal flow during compression process.The microstructure of the preforms after the compression test are shown in 4C weight percentage. Hence, galvanic influence amid them is detached. The witnessed increase in corrosion resistance for composites is dispensed to probable electrochemical decoupling between B4C particles and AA8079 matrix [4C [4C particles perform as physical protectors to stop the actuation and rate of development for pitting corrosion. The anodic polarization curves for AA8079 and AA8079-B4C display the endurance in corrosion current density, representing the exposure of pitting corrosion. The B4C particles which are utilized as reinforcing elements impede the creation of oxide layer and thus reduce the composites corrosion rate expressively.The corrosion behavior of composite samples has been studied by using electroanalytical techniques such as polarization and impedance measurements (EIS). The polarization curve of samples are displayed in 9 matrix . Soorya trix [4C . In inor4C, AA8079-10 wt.%B4C, and AA8079-15 wt.%B4C composites are shown in 4C particle; perhaps the opposition of the surface oxide film on the samples upsurges with the enhancement in B4C reinforcement. The uneven semicircle displays a non-ideal electrochemical performance on the electrode surface, which is despite the frequency distribution, roughness of the metal surface, and inhomogeneity. The Nyquist plot displays capacitive loop which is linked to the behavior of double layer capacitance, along with the charge transfer process amid electrolyte and metal surface. The diameter of the semicircle decreases with an increase in acid concentration, indicating an increase in the corrosion rate. A rise in the diameters of the Nyquist plots indicates the improved protective nature of the inhibitor against damage of material in the corrosive solution [4C weight percentage.The Nyquist plots observed in 3.5% Nacl solution for the AA8079, AA8079-5 wt.%Bsolution . It coul4C as reinforcements were successfully fabricated at different PM process parameters, and the subsequent conclusions were obtained:4C particles with the AA8079 matrix has been observed;From the SEM examination, fine dispersal and occurrence of B4C particles with minor peaks;XRD analysis shows the presence of B4C particles with AA8079 matrix and the respective elemental powders of the AA8079 matrix;The EDAX analysis of the sintered samples witnessed the existence of BThe density, hardness, and compressive strength of the composite was increased while increasing the reinforcement weight percentage from 5 to 15 wt.% with respect to an increase in PM process parameters, compaction pressure, sintering temperature, and time;4C reinforcement with AA8079 matrix without pores and grain boundaries;The SEM micrographs, after compression testing, exposed the homogenous dispersal of B4C composites corrosion resistance rose with a rise in weight percentage of B4C reinforcement with AA8079 matrix.The AA8079-BAA8079 matrix composites containing different weight percentage of B


The probability of infection changes nonlinearly with the amount of respiratory matter to which a person is exposed. If most people in the wider community wear even simple surgical masks, then the probability of an encounter with a virus particle is even further limited. In indoor settings, it is impossible to avoid breathing in air that someone else has exhaled, and in hospital situations where the virus concentration is the highest, even the best-performing masks used without other protective gear such as hazmat suits will not provide adequate protection.The effectiveness of masks in preventing the transmission of severe acute respiratory syndrome coronavirus 2 has been debated since the beginning of the COVID-19 pandemic. One important question is whether masks are effective despite the forceful expulsion of respiratory matter during coughing and sneezing. Cheng Science, abg6296, this issue p. 1439 Face masks are highly effective at preventing SARS-CoV-2 transmission because, in most environments, airborne virus abundance is limited. Airborne transmission by droplets and aerosols is important for the spread of viruses. Face masks are a well-established preventive measure, but their effectiveness for mitigating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission is still under debate. We show that variations in mask efficacy can be explained by different regimes of virus abundance and are related to population-average infection probability and reproduction number. For SARS-CoV-2, the viral load of infectious individuals can vary by orders of magnitude. We find that most environments and contacts are under conditions of low virus abundance (virus-limited), where surgical masks are effective at preventing virus spread. More-advanced masks and other protective equipment are required in potentially virus-rich indoor environments, including medical centers and hospitals. Masks are particularly effective in combination with other preventive measures like ventilation and distancing. Airborne transmission is one of the main pathways for the transmission of respiratory viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , surgical and similar masks exhibit higher and more variable penetration rates (~30 to 70%)  has the same chance to infect a person, Pinf can be calculated by a single-hit model of infectionPsingle represents the infection probability for a single virus and Nv represents the total number of viruses to which the person is exposed . This very large number implies that indoor environments are usually in a respiratory particle–rich regime. Surgical masks with particle collection efficiencies of ~50% cannot prevent the release of millions of particles per person and their inhalation by others  are typically low, with mean values of ~53 for coronaviruses , ~38 for influenza viruses (A and B), and ~96 for rhinoviruses  for the number of potentially inhaled viruses , assuming a characteristic infectious dose of IDv,50 = 100 or 1000 viruses, respectively , influenza viruses (A and B), rhinoviruses, and SARS-CoV-2 . We find that usually just a minor fraction of exhaled respiratory particles contains viruses. In contrast to the high number of emitted respiratory particles, the number of viruses in 30-min samples of exhaled air . For this purpose, we modeled Nv for SARS-CoV-2 as lognormally distributed with standard deviations (σ) in the range of ~1 to 2 on the basis of recently reported distributions of the viral load of SARS-CoV-2 in respiratory fluids   and average daily numbers of human contacts (c ≈ 10 to 25 contacts per day)  is plotted against the infection probability without masks . It shows that surgical masking achieves large reductions in infection probability when the maskless infection probability is low but increasingly smaller reductions when the maskless infection probability is high. Pinf,pop/Pinf,pop = / Pinf,pop]. It decreases slowly with increasing Pinf,pop in the virus-limited regime, exhibits a steep decrease upon transition into the virus-rich regime as Pinf,pop approaches unity, and goes to zero at Pinf,pop = 1. The nonlinear dependence of mask efficacy on infection risk differs from the assumption that the percentage change of infection probability as a result of mask use would be proportional to the percentage change of inhaled particle number . Accordi8 per milliliter of respiratory fluid, which is realistic and higher than the estimated infectious dose for SARS-CoV-2   cannot really protect them from inhaling these particles. However, as only few respiratory particles contain viruses and most environments are in a virus-limited regime, wearing masks can keep the number of inhaled viruses in a low-


This study set out to investigate how incentives for mothers, health workers and boda–boda riders can improve the community-based referral process and deliveries in the rural community of Busoga region in Uganda. Both the monetary and non-monetary incentives have been instrumental in the improvement of deliveries at health centres.The study was a 2 arm cluster non-randomized control trial study design; with intervention and control groups of mothers, health workers and boba–boda  riders from selected health centres and communities in Busoga region. Among the study interventions was the provision of incentives to mothers, health workers (midwives and VHTs) and boda–boda riders for a duration of 6 months. Monetary and non-monetary incentives were applied in this study, namely; provision of training, training allowances, refreshments during the training, payment of transport fares by mothers to boda–boda riders, free telephone calls through establishment of a pre-paid Closed Caller User Group (CUG) and provision of bonus airtime to all registered CUG participants and rewards to best performers. The study used a mixed methods design. Descriptive statistical analysis was computed using STATA version 14 for the quantitative data and thematic analysis for qualitative data.Findings revealed that incentives improved community-based referrals and health facility deliveries in the rural community of Busoga. The proportion of mothers who delivered from health centres and used boda–boda transport were 70.5% in the intervention arm and only 51.2% in the control arm. Of the mothers who delivered from the health centres, majority (69.4%) were transported by trained boda–boda riders while only 30.6% were transported by un-trained boda–boda riders. And of the mothers transported by the boda boda riders, 21.3% in the intervention arm reported that the riders responded to their calls within 20 min, an improvement from 4.3% before the intervention. Mothers who were responded to between 21–30 min increased from 31.4% to 69.6% in the intervention arm while in the control arm, it only increased from 37.1% to a dismal 40.3%. Interestingly, as the time interval increased, the number of boda–boda riders who delayed to respond to mothers’ calls reduced. In the intervention arm, only 6.2% of the mothers stated that boda–boda riders took as many as 31–60 min’ time interval to respond to their calls in post intervention compared to a whopping 54.9% in the pre intervention time. There was little change in the control arm from 53.2% in the pre intervention to 41.2% in the post intervention.Incentives along the maternal health chain are key and the initiative of incentivising the categories of stakeholders  has demonstrated that partnerships are very critical in achieving better maternal outcomes  as a result of proper referral processes. Maternal and child health has become a priority area of focus among health policy makers and planners. Several interventions are being tried in an effort to increase deliveries in health facilities in order to realise better health outcomes. This study focused on how incentives for mothers, health workers and boda–boda riders can improve the community-based referral process and deliveries in the rural community of Busoga region in Uganda.Two groups of boda–boda riders, mothers and health workers were followed up. One group was given incentives while the other was not. The incentives included training and training allowances, refreshments during training, payment of transport fares by mothers to boda–boda riders, free telephone calls and provision of bonus airtime to all registered CUG participants and rewards to best performers.The results of this study showed that incentives help to boost deliveries from health facilities hence improving maternal outcomes. In intervention group, 70.5% of mothers who deliveries from health facilities were transported by boda–boda riders whereas this was only 51.2% in the control group. The response time of boda–boda risers to calls for transport by expecting mothers improved tremendously in the intervention group when compared to the control group. All the targeted parties—boda–boda riders, mothers, midwives and Village Health Teams were more active and motivated to work towards improving maternal health of the mothers. Maternal and child health continues to be a largely overlooked aspect of health care system leading to major risks associated with pregnancy and child birth . This co. [. [. [Both monetary and non-monetary incentives have been instrumental in the improvement of deliveries at health centres. According to Ekirapa-Kiracho et al. , the vou. [. , non-mon. [. [. , in Camb. [Most of the studies which involved training of different stakeholders to improve maternal indicators did not consider much of the monetary incentives. The main focus of the studies was to provide knowledge as a non-monetary incentive though monetary incentives in form of allowances were later discovered to be vital. The Hunger Project  in Ghana.  on trainTo establish the role of incentives in improving deliveries in health units in Busoga region;To examine the influence of incentives on the boda–boda riders’ responsiveness to the call by mothers for referral transport;To find out the effect of time interval on deliveries at health facilities.The goal of this study was to establish the role of incentives to the mothers, health workers and boda–boda riders in improving community-based referrals and health facility-based deliveries in Busoga Region of Eastern Uganda. More specifically, the study aimed to achieve the following objectives:The overall justification of this study was based on the need to establish the role that monetary and non-monetary incentives play in improving maternal referrals and discovery of how raising awareness and interest through creating a collaborative partnership between mothers, boda–boda riders and the health workers would increase maternal referrals and deliveries in health facilities hence improving maternal health.The study was a 2 arm cluster non-randomized control trial study design; with an intervention group and a control group from selected health centres and communities. The study interventions involved providing incentives to mothers, health workers (midwives and VHTs) and boda–boda riders and this was done in a period of 6 months.The study was carried out in the rural districts of Busoga in Uganda. Local level participants came from the districts of Bugiri and Iganga where study projects were conducted in selected Sub-Counties that made up the intervention and control arms. These sub counties included; Nambale, Nabitende, Nawandala in Iganga district and Budaya Sub County in Bugiri district for the intervention arm while Nawaningi, Ibulanku and Makuutu in Iganga district and Nabukalu in Bugiri district formed the control arm. The intervention health facilities included; Bugono health centre IV, Kasambika health centre III, Nambale health centre III, Nawandala health centre III, and Namusiisi health centre III in Iganga district and Mayuge health centre III in Bugiri. While the control arm included; Makuutu health centre III, Bunyiiro health centre III, Busesa health centre IV in Iganga district and Nabukalu health centre III in Bugiri district.The reason for choice of study area was that motorcycle ambulances had once been operating under a funded project and had not been sustained. Therefore the study aimed at testing a local initiative that is more sustainable since it used locally available motorcycle (boda–boda) groups mobilised with the aim of referring mothers for further management at the different levels of health centres.The participants in Table The mothers were recruited from the antenatal register based on their willingness and meeting the inclusion criteria and in the selected study villages and sub counties. Mothers in their third trimester were recruited purposely to determine the impact of the incentives. Mothers were recruited in both the intervention and control arms.The boda–boda riders were recruited basing on their operating stage (work station), activeness and willingness to take part in the study and be trained. Boda–boda riders who were from boda–boda stations within the study sub counties who had no registered crime with police were selected.The midwives from the selected health facilities were recruited basing on their expressed interest and experience in research. They must have worked with the health facility for over 6 months. The VHTs were recruited purposively based on their presence in the selected sub counties together with their willingness to take part in the study. Also, very active VHTs were considered for this study.Consent was sought from all the participants  before they were initiated into the study.For this study, purposive and random sampling techniques were employed. Study participants were identified from different data sources such as delivery register, ANC register and PNC registers in the selected health centres, VHTs registers at the district health office and boda–boda riders’ register at the village, sub county or district levels. Random methods were used to select from a given register to give each qualifying member a chance.One of the cross-cutting interventions was training. Four training sessions were conducted in the selected four sub counties for the intervention group for the boda–boda riders and health workers. The training lasted five days. Mothers on the other hand were trained from the health centres during the ANC visits. Participants in the training were facilitated and compensated for the days in training since some of the riders did not own motorcycles and were supposed to deposit their commissions to the motorcycle owners. Sessions were conducted for a relatively short period of time (9 am–2 pm) daily to allow the riders continue with their daily work after the training since there was demand for their services in the community. The VHTs and midwives were also facilitated with allowances in the training.Training sessions of mothers at health centres took a shorter time of around 1–2 h. These were mothers in their third trimester. As an incentive for the training, mothers were given only refreshments on every ANC visit.Another intervention was use of the closed caller user groups (CUG). Participants who benefited from this were the mothers, boda–boda riders, VHTs and midwives. Participants in the control arm did not benefit from the CUG. On consent, participants in the intervention arm were registered by the MTN telecommunication company to benefit from the free communication calls between all the members in the group.As another strategy, a bonus airtime of 10,000 Uganda shillings was given to each member of the group for communicating to other members who were not in the group during times of emergency. This was in a situation where the boda–boda rider was far and would use the bonus airtime to call another boda–boda rider at the stage to stand in for him regardless of whether he was in the CUG or not.In order for mothers to fully benefit from the boda–boda services, they were continuously encouraged to save money in the savings boxes for a sustainable motivation of boda–boda riders, as an intervention. This money was to pay for mothers’ transport fares for boda–boda services. It motivated the boda–boda riders because they were assured of their payments for the services. There was increased access and quality of health care services in local communities.The terms of payment for boda–boda transport services were mutually agreed upon between the mothers and the boda–boda riders in accordance with the distance travelled to reach the health facility. The study commenced with a baseline survey in both groups so as to get statistics for comparison after the intervention. This was important for determining the extent of the changes brought by the intervention.Questionnaires: We had baseline and exit interview questionnaires that were filled at the health facility for the mothers who used boda–boda transport in to the health centres.Interview guide: Data was also collected using the key informant and in-depth interview guides for the boda–boda riders, mothers, VHTs and midwives in selected health facilities of the intervention and control arms.Focus group discussions (FGDs): This was done at baseline as an entry point and later on when executing the study. It consisted of different stakeholders in the study.Document review checklist: this was designed to capture data from the secondary source. These included; delivery registers, ANC registers and PNC registers.p-value set at 0.05 and confidence interval at 95%.Data analysis of descriptive statistics was computed using STATA version 14 for the quantitative data. Paired t-tests of independence were used to determine the statistical significance of the different variables with In order for the study to be informative, difference-in-difference (DID) framework was used. The simplest form of the DID design is a special case in which there are only two groups observed in two time periods. DID was used to determine the change effect based on the average value and its statistical significance.Atlas Ti version 7 was used for qualitative analysis. It involved re-reading the interview transcripts to identify themes and sub themes that emerged from the respondents’ answers during the FGDs, Key Informant Interviews (KIIs) and In-depth Interviews (IDIs). The arrangement for analysis was based on the topics and questions formulated for the interviews in order to synthesize the answers to the proposed questions.The results are presented in terms of the intervention strategies that were applied to different participants in the intervention arm; and how they affected the maternal health outcomes. These included: the training component, closed caller group, effect of the bonus airtime and other general incentives in form of allowances to training participants, payment for services as well as non-monetary incentives such as recognition and rewarding of best performers during meetings. The analysis shows how these different intervention strategies impacted the maternal health outcomes.Training incentives included; training allowances to midwives, boda–boda riders and VHTs. Also, mothers were given refreshments at the health centre during ANC visits. The proportion of mothers who delivered from health centres and were transported by boda–boda transport was 70.5% in the intervention arm and only 51.2% in the control arm. And of these 70.5% of the mothers who delivered from the health centres, 69.4% were transported by trained boda–boda riders and only 30.6% of the mothers were transported by un-trained boda–boda riders. This gives a basis to suggest that incentivising and training of the boda–boda riders while linking them to the mothers had an impact on boosting deliveries in health facilities.“...I have learnt a lot from the training, for example the importance to take mothers to the health facility in time. Since you stated that communication was simplified for mothers to contact us, then we shall also respond very fast”This is also supported by the qualitative information which was provided by the different stakeholders during and after the training. It appears that a mix of excitement, friendship creation and participatory learning were witnessed during and after the training. One of the participants (a boda–boda rider) noted;The health workers noted the need for team work and commitment by all those involved in the study would enable them to meet the needs of mothers who are in dire conditions of child birth.The excited VHTs who participated in the training indicated that they should not only enjoy the allowances which were given to them but also emphasised that this training was very timely especially for the boda–boda riders.“...Knowledge building and sharing on understanding of MCH is now seen on your faces, especially you the boda–boda riders who now know the need to support the mothers by timely transporting them to the health centres for delivery. Let us be practical.”One of the VHTs in the training room emphasised to the boda–boda riders that they should put into practice what was learned:Mothers were encouraged to save money in small boxes which were broken at the time they were experiencing labour pains. Boda–boda riders were able to respond to mothers’ calls very fast because they were very sure of their payments when they offered the transport services. As seen before, 70.5% of the mothers who delivered from health facilities were transported by the trained boda–boda riders.“...what we have learnt today is important and I am committed to … save mothers, even if it means taking them to the health facility on credit. However, since they are saving it will not be a challenge for them to pay us.”One of the stage coordinators of the boda–boda riders indicated during an in-depth interview that in the past, he did not consider it urgent to provide transport to mothers in labour or mothers with birth related complications to the health centres. This is because he never had sufficient knowledge of the urgency and the labouring mothers did not have money to pay them on arrival at the health centres.The incentive for the CUG was the free calls and the bonus airtime. Participants who majorly benefited from the CUG included; mothers, health workers and boda–boda riders. The free calls and bonus airtime enjoyed in CUG contributed to the reduction in time boda–boda riders took to respond to mothers’ calls. The results are presented in Table The bonus airtime and free calls were important in the coordination of mothers’ transport to health centres. The mothers in the CUG could easily contact boda–boda riders for transport services because of free calls. Similarly, boda–boda riders were able to call back the mothers and health workers.Of the mothers who were transported by the trained boda–boda riders to health centres for delivery, those who stated that boda–boda riders’ response to their calls took 5–20 min improved from 4.3% to 21.3% in the intervention arm. Those who were responded to between 21 and 30 min also improved from 31.4% to 69.6% in the intervention arm; this compared to a small increase from 37.1% to only 40.3% in the control arm. This shows how training of boda–boda risers impacted their response to mothers’ call for transport.Actually as the time interval increased, the number of boda–boda riders who delayed to respond to mothers’ calls reduced. For instance, Table According to the DID model of analysis, the intervention was impactful. The time interval boda–boda riders took to respond to mothers’ calls for transport kept on reducing. The average value for the time interval boda–boda riders took to arrive when contacted was 0.391. This was found to be statistically significant to the change effect as shown in Table Regression analysis of the time interval boda–boda riders took to respond to mothers’ calls had a statistically significant influence on the deliveries at health centres. The results in Table The incentives to mothers, boda–boda riders and other stakeholders increased their motivation and improved deliveries in health centres. The motivation of these stakeholders reduced the time boda–boda riders took to respond on the mothers’ calls when contacted for transport to health centres. Results show that there was a statistically significant influence of time interval on health facility-based deliveries. Pregnant mothers who contacted boda–boda riders at the different intervals for transport to health centres in the intervention arm were 1.173 times more likely to deliver from health centres compared to those in the control arm .Considering the statistical significance of the time intervals, the 5–20 min interval which boda–boda riders took to arrive when contacted by mothers had no association with deliveries at health centres. The other time intervals .“…Do not take much time to go to mothers whenever they call you. Just know, that is a hard moment especially when in labour. Mothers cannot walk at that time. These are our relatives, wives, daughters and sisters. Learn to rush to them.” One of the members of the group urged the colleagues.During an FGD in the intervention arm specifically in the pre-intervention phase, boda–boda riders were encouraged to be quick and respond whenever a mother contacts them. They were assured of immediate payments on delivery of mothers at the health centres. Mothers had saved money in the saving boxes.“...In these meetings we are able to present our challenges we face in the transportation of mothers such that solutions are discussed here.” One of the boda–boda riders stated.Quarterly meetings in the different sub counties in the intervention arm were conducted. In these meetings, discussions were focussed on the improvement of community-based referrals and increased deliveries in health centres. Boda–boda riders who transported the biggest number of mothers were rewarded with helmets and recognition during meetings. The majority of the boda–boda riders appreciated the importance of the meeting.“…I’m not the best boda–boda rider pronounced in this meeting and this has given me the zeal to work hard such that next time I become one of the best riders in my sub county”.Another one said;“...I did not know that by the humble interaction with the local boda–boda riders I was doing something appreciated by most of them. This has opened my mind and now I know the reason for the increasing number of deliveries at my health centre”. A midwife who was excited stated in one of the meetings.Similarly, in addition to the routine allowances which were given to the different midwives for the recruitment and registration of mothers in the CUG, boda–boda riders gave a feedback of the best midwives who were cooperative. These were rewarded and recognised before the boda–boda riders in the meetings.. [. [Incentives are a key component of performance in this study. All the different categories of participants were motivated in one way or the other with incentives. The different participants were given incentives to take part in the study; these participants are the health workers, the boda–boda riders, and the pregnant women in their third trimester. This was not different from other studies. According to Ekirapa-Kiracho et al.  in the v. [.  study, s. [. [The introduction of the non-monetary incentives in the rural Busoga sub region study like it was in the Malawi study, resulted in the improvement in the deliveries at the health centres. In the rural Busoga sub region study, incentives included recognitions during meetings unlike in Malawi where soap, baby blanket and traditional baby wraps were distributed according to Akker et al. , but had. [. . The stuMothers were able to call members of the group even without airtime or credit on their phones. This made it easy to link the mothers to the VHTs, boda–boda riders and the health workers (midwives) in the health centres for participating sites and cheap to call the boda–boda riders to take the mothers to the health facilities without any delays. The free calls and bonus airtime led to improved communication time and response for taking the mothers to the health facilities. In situations where the boda–boda rider was far, a mother would use the bonus airtime to call another boda–boda rider at the stage regardless of whether he was in the caller group or not. This increased the number of health facility deliveries from 52% to 70.5% in the sub region.While for the VHTs, referral was made easier since they were able to call the midwife in the study facility and give their quick assessment of the situation of the mother before she reaches the health centre. The midwife prepared depending on the information shared by the VHT who was referring the mother from the community. Timely and functional referral system for mothers in the community is important as it backs up antenatal care, labour and delivery at low and high level health facilities . This wa. [Like the voucher system in the Ekirapa-Kiracho et al.  study, tAlthough we tried to reach all the members in the study population and several categories of stakeholders who might influence the intervention, we were unable to interview some key stakeholders such as a few opinion leaders. Another limitation of this study is that the views of those interviewed may not be generalised to the entire institutions they serve or work for. Further still the views of stakeholders may change with time. Since some of the incentives were monetary, its sustainability is still questionable. However, despite the inherent limitations, stakeholder analysis is a vital tool in informing the design of any form of health systems research or intervention.The incentives have proved to be an effective way to improve maternal and neonatal outcomes especially in rural communities in the less developed countries. Incentives along the maternal health chain are key and the initiative of incentivising the three categories of stakeholders  with the closed caller group for free calls, bonus airtime and an extra allowance for the health workers has demonstrated that partnership are very critical in achieving better results or maternal outcomes especially the health facility-based deliveries.The incentives have the potential to increase the interest and commitment of boda–boda riders in transporting mothers to the health facilities as a priority to other customers since they feel obliged. This is a key component in addressing the challenges of complications for women who are at the verge of child birth.It also addresses the other challenges of response time as it is clear that in developing countries most maternal and neonatal deaths can be prevented if there is a robust system to refer, transport and manage the delivery process especially for mothers with complications.We recommend that Government should support riders through credit extension to buy and own motorcycles as those who were using hired ones had more challenges. All mothers in the region should be encouraged to start savings boxes during the first ANC visit in all health centres to gather enough money for transport fare and other necessities during time of delivery. Boda–boda riders should be branded for easy identification since they are all willingly transporting mothers to health centres to deliver.


This method has not been previously used for these viruses.Virus production is an essential part of virology. The quality of virus preparation, in terms of purification and yield, can affect the outcome of experiments. Purified virus is critical for assays, as contamination from cell culture supernatants may affect results. Purified virus is also needed for vaccine preparations. The traditional process of gradient purification of virus is time consuming, with multiple steps spanning over days, and produces significant amounts of biohazard waste. Developing a more efficient alternative that provides a purified virus product is highly desirable. In this study, we performed magnetic bead purification of Zika virus (ZIKV) and Mayaro virus (MAYV) with the Mag4C LV kit from OZ Biosciences. Currently, this kit is only indicated for use with lentiviruses and retroviruses. We found that we were able to successfully concentrate and purify both ZIKV and MAYV using the kit. Therefore, we concluded that the Mag4C LV kit provides a quick and simple alternative to traditional virus purification methods.  Specifications tableVirus production is an essential part of virology experiments and the quality of virus preparation (in terms of purification and yield) can affect the results of these experiments. Purified virus is critical for assays where contamination from the cell culture, e.g. cell culture medium additives, products of cell lysis , cytokines, and chemokines, may affect the results of the assays Magnetic beads can be used for many biological applications, such as immunoprecipitation •Minimal Eagle's Medium (MEM) •Newborn calf serum (NCS) •Penicillin/streptomycin •5-layer cell culture flasks •50 mL conical tubes •1.5 mL screw-cap tubes •15 mL conical tubes •1 mL pipette, 200 µL pipette and tips •Refrigerated centrifuge set to 4°C•0.22 micron vacuum filter/storage system •○Mag4C LV magnetic beads○Elution bufferMag4C LV kit •Magnetic separation rack •Biosafety cabinet•Incubator with 5% CO2 set to 37 °C•1X PBS(1)Seed Vero cells in 5-layer cell culture flasks and grow to confluence in complete medium (MEM + 10% NCS + 1% P/S).(2)Inoculate cells with MAYV or ZIKV and allow virus to multiply for 2 or 4 days, respectively.(3)Decant supernatant from multi-layer flasks into 50 mL conical tubes.(4)Centrifuge at 4 °C at 3500 rpm for 30 min.(5)Vacuum filter (0.22 µm) the supernatants and store at 4 °C.(6)Add 2 mL of clarified supernatant to a 15 mL conical tube.(7)Add 20 µL of Mag4C LV beads to the 2 mL of clarified supernatant.(8)Incubate for 30 min at room temperature.(9)Place tube on magnetic separation rack for 30 min.(10)At this point, the beads should be held on the wall of the tube. Remove the supernatant and save as a sample (leftover clarified supernatant) at −80 °C.(11)Remove the tube from the magnetic stand.(12)aAt this point, the sample can be transferred to a smaller tube if desired.Resuspend the beads in 200 µL of elution buffer.(13)Incubate for 10 min at room temperature.(14)Place tube on magnetic separation rack for 30 min.(15)Collect the supernatant (this contains the eluted virus) and save as a sample (eluted virus) at -80 °C.(16)Remove the tube from the magnetic stand.(17)Resuspend the beads in 100 µL 1X PBS and store as a sample (leftover beads) at −80 °C.Each sample collected during the magnetic bead purification process was subjected to protein estimation by bicinchoninic acid (BCA) protein assay, protein gel, western blot, and determination of the median tissue culture infectious dose (TCID50/mL) The qualitative detection of total protein quantities  was performed using protein gel analysis. 10 µg of protein per sample was separated on a 4–12% Bis-Tris gel  under reducing conditions. The protein gel results correspond to the BCA results; specifically, as more purification steps are performed, less excess protein is observed on the gel, while the virus-specific proteins remain. It is also demonstrated that there are negligible amounts of protein remaining on the beads at the end of the magnetic bead purification process, indicating that the majority of virus has successfully been eluted.The binding of virus to virus-specific monoclonal antibodies  was evaluated by Western blot. Western blot analysis of the ZIKV samples using the ZIKV specific antibody ZV54 showed that the antibody was able to bind to the purified sample, indicating the presence of proteins characteristic of ZIKV. The intensity of the band decreases as more purification steps are performed, indicating possible loss of virus, which is consistent with the titer data . Similar–1, indicating loss of virus particles during the purification process. The process was performed on small volumes for ZIKV  with minimum instrumentation need yet produces purified virus. However, the loss of virus with each purification step may limit the use of this approach where very high concentration and yield may be needed in downstream applications.Magnetic bead purification successfully separates ZIKV and MYAV from various impurities and contaminants , and theOpinions expressed herewith are those of the author(s) and are not necessarily representative of those of the USUHS, DoD, or the United States Army, Navy, or Air Force.


Taxus species under salicylic acid (SA) and methyl jasmonate treatment. However, the molecular mechanism of its accumulation remains unclear. We investigated TcWRKY33, a nuclear-localized group I WRKY transcription factor, as an SA-responsive regulator of taxol biosynthesis. Overexpression and RNA interference of TcWRKY33 confirmed that TcWRKY33 regulates the expression of most taxol biosynthesis genes, especially 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) and taxadiene synthase (TASY), which were considered as key enzymes in taxol biosynthesis. Transient overexpression of TcWRKY33 in Taxus chinensis leaves resulted in increased taxol and 10-deacetylbaccatin accumulation by 1.20 and 2.16 times compared with the control, respectively. Furthermore, TcWRKY33, DBAT, and TASY were confirmed to respond positively to SA signals. These results suggested that TcWRKY33 was the missing component of taxol biosynthesis that responds to SA. The sequence analysis identified two W-box motifs in the promoter of DBAT but not in the TASY. Yeast one-hybrid and dual-luciferase activity assays confirmed that TcWRKY33 can bind to the two W-boxes in the promoter of DBAT, upregulating its expression level. Hence, DBAT is a direct target of TcWRKY33. Furthermore, TcERF15, encoding a TASY activator, also contains two W-boxes in its promoter. Yeast one-hybrid and dual-luciferase activity assays further confirmed that TcWRKY33 can upregulate TASY expression through the activation of TcERF15. In summary, TcWRKY33 transmits SA signals and positively regulates taxol biosynthesis genes in two ways: directly and through the activation of other activators. Therefore, TcWRKY33 is an excellent candidate for genetically engineering regulation of taxol biosynthesis in Taxus plants.Taxol is a rare secondary metabolite that accumulates considerably in  Taxus species that functions as an anticancer drug  is an important endogenous hormone in plants. It is involved in the regulation of the biosynthesis of secondary metabolites including terpenoids, alkaloids, and flavonoids  to upregulate its expression in T. chinensis. Lenka et al. (phenylpropanoyltransferase (BAPT), by binding to G- and E-boxes. In addition, TcJAMYC2 and TcJAMYC4 induce reporter gene expression under the control of taxadiene 5-alpha hydroxylase (T5H) and taxadiene synthase (TASY) promoters, respectively . We subsequently investigated the molecular regulation mechanism of TcWRKY33 through yeast one-hybrid assays, dual-luciferase reporter systems, and evaluation of expression patterns. Our results broaden our understanding of the positive roles of TcWRKY33 transcription factors in regulating the biosynthesis of taxol in plants.In this study, we evaluated the potential functions of the group I WRKY transcription factor Taxus chinensis cell line #48 was maintained in modified Gamborg's B5 medium (62# medium) , and the first chain of cDNA was transcribed by using the RevertAid First Strand cDNA Synthesis Kit .Fresh leaves of TcWRKY33 gene was obtained from the multi-omics analysis data. It was found to harbor two WRKY domains in its deduced amino acid sequences, and its full-length was amplified by using reverse transcription-PCR. The primers named TcWRKY33-1F/R are shown in T. chinensis cDNA, PCR products were T/A cloned into the pMD18-T vector  and sequenced.The sequence of http://www.ncbi.nlm.nih.gov/BLAST/) was used for the homology search from the Swiss-Prot database. ClustalW online  at the default setting was used to align and evaluate the percentage identity of TcWRKY33 with known WRKYs. The alignment results were sent to ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) for coloring  without termination codon was amplified using the primers TcWRKY33-2F/R in SacI and BamHI restriction sites, and then recombinant vector was transformed into GV3101. The onion epidermal cells with a dimension of 1 × 1 cm were infected by positive clones and controls (pCAMBIA1300-35S-sGFP) in 1/2 MS liquid medium at 25°C in dark for 2 days. The infected onion epidermal cells were then plated on the MS medium for 2 days at 25°C in darkness. The GFP fluorescence was observed by using the laser scanning confocal microscope FV1000 .The full-length TcERF15 of T. chinensis was aligned with Taxus baccata genome sequences, suggesting that it was highly conserved in both Taxus spp. In addition, the 5′-flanking sequence of this gene of T. baccata was obtained. To obtain and validate the similarity of flanking sequences of this gene in T. chinensis, the forward primer was designed according to the homolog sequence of T. baccata, while the reverse primer was designed according to the ORF sequence in T. chinensis  of hinensis . After aTcERF15 was inserted into pBI121 with HindIII and BamHI restriction sites, so that the β-glucuronidase (GUS) reporter was under the control of the promoter of TcERF15. The construct was transformed into onion epidermis cells mediated by GV3101 affection , PLACE  and Nsite (http://www.softberry.com/berry.phtml?topic=nsite&group=programs&subgroup=promoter).Then, the 5′-flanking sequence was analyzed to locate basic TcWRKY33 was ligated into pGADT7-Rec2 with EcoRI and BamHI restriction sites, resulting in a TcWRKY33::pGADT7-Rec2 recombinant vector  until OD600 = 0.6–0.8, and then they were collected and resuspended with 20 ml of half-strength MS liquid medium containing 200 μM of acetosyringone. The fresh leaves were gathered from blade tips on the 10-year-old T. chinensis potted plants. After surface disinfection, small incisions were made with a sterile scalpel in the inner surface of the leaves to facilitate Agrobacterium infection. Then, the injured leaves were cultured in a shaker at 125 rpm for 12 h in the dark at 28°C. After that, the infected T. chinensis leaves were plated on the 1/2 MS solid medium for 24 h at 28°C in darkness. Finally, the part of leaves was placed into the liquid nitrogen and then stored in the fridge at −80°C for the following experiments, and the others were dipped into sterile water shaking at 120 rpm for 6 days to test the contents of the taxol and 10-DAB.For transient transformations of t-test. The treated leaves were dried at 60°C for 24 h and crushed to 40 meshes, 0.1 g of powder was first weighed accurately and put into a 10-ml centrifuge tube, and then 3 ml of methanol were added. The extraction procedure was conducted by using an ultrasonic machine  for 30 min, centrifuged for 10 min at 8,000 r/min, and repeated three times, and the upper phase was collected and dried using N-Evap . The taxane samples were diluted by high-performance liquid chromatography (HPLC)-grade methanol and then passed through membrane filters (0.22 μm) before HPLC detection. Each experiment was conducted in triplicate. The extraction compounds were analyzed using the Agilent 1290 HPLC system combined with a phenyl-C18 column . Chromatographic separation was performed by the gradient elution process of mobile phase water (A) and acetonitrile (B) as follows: 0–10 min, 5–40% B; 10–15 min, 40–50% B; 15–30 min, 50–80% B; 30–35 min, 80–95% B. The flow rate was 1.0 ml/min with 20 μl of injection volume. In addition, the column temperature and the detection wavelength were kept at 30°C and 227 nm, respectively.Quantitative real-time PCR was conducted as previously described .For dual-luciferase activity assay, the original 383 bp r vector . Then, TDBAT and TASY respond to SA and MJ signals , taxane 2-alpha-O-benzoyltransferase, 3′-N-debenzoyltaxol N-benzoyltransferase (DBTNBT), and taxadienol acetyltransferase (TAT) were increased by 2.26-, 1.47-, 2.29-, 2.49-, 3.10-, 3.60-, and 3.99-folds, respectively, at 6 h after SA treatment compared with control , T5H, T10H, TAT, and DBTNBT were 3.85-, 2.27-, 2.81-, 2.01-, 1.74-, 2.84-, and 4.19-fold higher in leaves overexpressing TcWRKY33 than in controls , TAT, and DBTNBT, were 0.50, 0.67, 0.19, 0.35, 0.37, 0.42, and 0.41 times those in the control; however, T10H displayed expression levels 2.00-fold those of the control  using HPLC in T. chinensis leaves transiently overexpressing TcWRKY33 or with TcWRKY33 expression knocked down through RNA interference. Taxol accumulated to levels in TcWRKY33-overexpression leaves of up to 1.20 times higher than that in controls  within −368 bp of the DBAT promoter; these W-boxes are the functional sites of TcWRKY1, a group IIa WRKY factor  and 5′-TTGACG-3′ at −452 bp (named W2-box). We quadrupled each 6-bp fragment from the TcERF15 promoter and mutant fragments and ligated these separately into pHIS2.1; these constructs were, respectively, co-transformed with a TcWRKY33-AD fusion vector to detect interaction in a yeast one-hybrid system. Positive clones containing the TcWRKY33-AD fusion vector and W-box bait vectors grew on Trp-, Leu-, and His-deficient media with 40 mM 3-AT, whereas control clones did not. This indicated that TcWRKY33 interacts with W-boxes in the promoter of the TcERF15 gene  in the promoter of DBAT are the main SA-responsive elements , ZCT2, and ZCT3 (Suttipanta et al., MdHY5, MdMYB10, and MdMYB11, regulating their activity. It also binds to the promoter of UFGT, encoding a key structural gene in the anthocyanin biosynthesis pathway, to upregulate the expression of UFGT. Hence, MdWRKY11 is involved in MdHY5-mediated anthocyanin biosynthesis and regulates the expression of MdMYB10, MdMYB11 transcription factors, and UFGT in apple fruit (Liu et al., TcWRKY33 in taxol biosynthesis.TcWRKY33 clearly responds to SA signals and transmits these SA signals to downstream genes. In addition, TcWRKY33 appears to regulate downstream genes, such as DBAT and TASY, in two ways. DBAT responds to SA signals, which are transmitted directly to DBAT via TcWRKY33 interacting with W-boxes. The TASY gene is more sensitive to MeJA stimuli, and this mechanism has been clarified extensively (Lin et al., TASY expression can be induced by SA, although the related mechanism remains unclear. In this study, we showed that TcWRKY33 exerts considerable positive effects on TASY expression, suggesting that TcWRKY33 may play important roles in the response of TASY to SA. As an activator of TASY, TcERF15 is a direct target of TcWRKY33. TcWRKY33 activates TASY by first regulating TcERF15 after the induction of SA. Thus, TcWRKY33 transmits SA signals to DBAT and TASY to regulate taxol biosynthesis (ynthesis .We identified an SA-induced activator in the biosynthesis of taxol and provide references for the role of WRKY transcription factors in the regulation of taxol biosynthesis.MW928850.The original contributions presented in the study are publicly available. This data can be found at: NCBI repository, accession number: TcWRKY33, taxanes content detection, subcellular localization, yeast one-hybrid, dual-luciferase reporter experiment, and so on. YC and HZ analyzed the data and wrote the manuscript. All authors read and approved the manuscript.CF, CZ, and LY conceived the research. YC and CZ designed the study. YC, HZ, MZ, WZ, ZO, and ZP conducted the experiments, including overexpression and RNA interference of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Blood samples are minimally invasive and can be collected repeatedly, but they are far from the site of disease and the target molecules are diluted by the large amount of blood. Therefore, we performed lipidomics using immunoprecipitation as a method to enrich specific fractions of serum. In this study, a CD9 antibody was immobilized on magnetic beads to enrich CD9-containing components in the serum for lipidomics. The percentages of phospholipids recovered from serum by methanol and isopropanol extractions were not significantly different, but triglycerides were barely recovered from serum by methanol extraction, requiring the use of isopropanol. However, once the serum was enriched with CD9 magnetic beads, triglycerides, and phospholipids were recovered at similar levels in both methanol and isopropanol extractions. Therefore, it is possible that the triglyceride fraction of the whole serum and the triglyceride fraction were enriched in CD9 magnetic beads differ in localization and properties. In addition, the variation per disease was small in general serum lipidomics; however, the difference per disease appeared larger when CD9 magnetic bead enrichment was employed. Lipidomics is a field of research that focuses on whole lipids in cells, often using high-performance liquid chromatography (HPLC) or gas chromatography as a separation method and mass spectrometry (MS) as a detection method. There are thousands of lipids in the human body, and the expression and localization of lipid molecules can change in response to changes in the cellular environment due to factors such as exercise, diet, and health conditions. Measuring these changes and understanding lipid-related pathways are important for understanding cellular metabolism, and lipids are also important biomarkers when considering disease mechanisms ,2,3. In Blood samples, such as serum and plasma, are taken by minimally invasive means, are repeatable, and reflect the circulation of the body; therefore, they are thought to reflect the states of various diseases to a certain extent . TherefoThe tetraspanin superfamily is a highly conserved protein family, with at least 33 members identified in humans, including CD9, CD63, and CD81 . In partWe immobilized CD9 antibodies on magnetic beads and examined the lipid composition of serum enriched by CD9-magnetic beads to determine which lipid classes are more likely to be enriched by these CD9-magnetic beads, and how the results differ from general serum lipidomics.We tested whether CD9 antibody-immobilized magnetic beads could be used to enrich CD9 from human serum. Non-specific immunoglobulin G (IgG)-immobilized magnetic beads were used for comparison. One hundred microliters of human serum diluted threefold in phosphate buffered saline (PBS) was mixed with a CD9 antibody or IgG immobilized magnetic beads slowly for 2 h at 4 °C. The beads were collected using a magnet, the supernatant was discarded, and the beads were washed with 500 µL PBS. Binding components were eluted with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer, separated by SDS-PAGE, and subjected to western blotting using a CD9 antibody. The results are shown in The molecular weights are shown on the left. From left to right, the lanes are for the CD9 antibody-enriched, IgG-enriched, and human serum fractions (without enrichment). The samples were separated by gel electrophoresis, the bands were stained with the CD9 antibody, and detected by chemiluminescence.The difference in the amount of lipids enriched by CD9 magnetic beads and IgG magnetic beads was larger when washed twice with PBS . Figure After mixing the magnetic beads with human serum, the number of times the beads were washed with PBS  and the total peak area detected by LC/MS by lipid class is shown on the vertical axis. The CD9 antibody-immobilized magnetic beads and IgG-immobilized magnetic beads were operated twice, and the average values are shown in the graph.  In The Bligh and Dyer or Folch procedure for the extraction of lipids is the gold standard, but it is not easy to operate because it is a two-layer separation and lipids are in the lower layer. Protein removal by addition of organic solvents is simple, but the recovery rate of lipids varies greatly depending on the solvent ,21. To cLipids are grouped by each lipid class and arranged on the horizontal axis, and the vertical axis shows the peak area of each lipid molecule. The left column (blue) shows the lipid profile of direct MeOH extraction of serum, the middle column (orange) shows the lipid profile of direct IPA extraction of serum, and the right column (green) shows the lipid profile of CD9 antibody-immobilized magnetic bead extraction of serum. Each extraction method was repeated three times, and their lipid profiles are shown in vertical order . .This result suggests that the CD9 magnetic beads may enrich low-density lipoprotein (LDL), which is present in the human serum, together with TGs and phospholipids. Therefore, we checked whether LDL and HDL were enriched by the CD9 magnetic beads. The cholesterol contents of HDL and LDL in the serum were 1.8 mM and 2.7 mM, respectively. However, in CD9- and IgG-immobilized magnetic bead enriched fractions, both LDL and HDL were below the detection limit (<50 µM), which was less than one tenth of that in serum.We investigated the possibility that the lipid profile enriched by CD9-immobilized magnetic beads might differ depending on the disease. We collected samples from 18 patients each diagnosed with Alzheimer’s disease, Parkinson’s disease, cerebral stroke, major depression, or schizophrenia who visited the University of Tokyo Hospital for examination, and prepared three mixed samples for each disease by mixing six serum samples for each disease . The results of IPA extraction of serum and lipid profile for each serum sample  and the lipid profile enriched on CD9 magnetic beads are shown in Because enrichment by CD9 magnetic beads is thought to have a significant effect on TG , we focuThe bar graphs from TG(52:1) to TG(52:4) in Serum and plasma are relatively easy samples to use for routine diagnosis, and since they circulate throughout the body, they are thought to reflect various disease states ; at the The CD9 antibody (clone number 12A12) was obtained from Cosmo Bio Co. . HDL and LDL/VLDL cholesterol assay kits were purchased from Cell Biolabs, Inc. . HEPES, MES, ethanolamine, EDTA·2Na, polyoxyethylene lauryl ether, and normal mouse IgG were obtained from Sigma-Aldrich . Lipid standards were obtained from the Cayman Chemical Company  and Avanti . Special grade reagents of ammonium bicarbonate and PBS and HPLC grade solvents of MeOH, IPA, and acetonitrile were purchased from Fujifilm , and ultrapure water was obtained from a Milli-Q System . https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/, our last access was 24 November 2021), revised in 2013. Diagnoses in the electronic medical record system were recorded within two years before the date of blood sampling was used. All samples were stored at −80 °C prior to analysis. This study was conducted with the approval of the ethics committee of the University of Tokyo Hospital and in compliance with the relevant guidelines and regulations. A pooled serum sample was generated by mixing 300 randomly selected patient residual samples after completion of the requested clinical laboratory tests at the University of Tokyo Hospital . Japanese neurodegenerative and psychiatric disease specimens were obtained from residual samples from patients who had been examined at the University of Tokyo Hospital . Informed consent for participation in the study was obtained using an opt-out process on the web page of the University of Tokyo Hospital. The investigations were carried out in accordance with the rules of the Declaration of Helsinki of 1975  was added to disperse the beads, which were centrifuged at 10,000× g for 5 min before the supernatant was discarded. We then added 100 µL each of 1 mg/mL CD9 antibody and IgG to Eppendorf tubes containing the magnetic beads. We added 100 µL of 25 mM MES buffer (pH 6) and suspended the mixture. The mixture was agitated slowly at 4 °C for 4 h. The mixture was then centrifuged at 10,000× g for 5 min, and the supernatant was discarded. Subsequently, 300 µL of a solution with final concentrations of 0.1 M HEPES buffer, 1 M ethanolamine, and 1% polyoxyethylene was adjusted to pH 8 with hydrochloric acid and added to the tube containing the magnetic beads, which was mixed slowly at 4 °C overnight. The sample was centrifuged at 10,000× g for 5 min, the supernatant was discarded and 300 µL of 25 mM HEPES solution (pH 8) containing 2 mM EDTA was added. The magnetic beads were washed, centrifuged at 10,000× g for 5 min, and the supernatant was discarded. Finally, we added 400 µL of 25 mM HEPES solution (pH 8) containing 2 mM EDTA and stored the tube at 4 °C. The protein immobilized bead concentration was approximately 0.1 mg/20 µL.First, 100 µL (2 mg) of HM NHS beads were added to a 1.5 mL Eppendorf tube, which was centrifuged at 10,000× Then, 200 µL of PBS was added to 100 µL of serum, and 7 µL of protein-immobilized magnetic beads were added while suspending them well. The mixture was slowly mixed for 2 h at 4 °C. The water adhering to the back cover of the tube was also dropped into the tube, the magnetic beads were collected using a magnet, and the supernatant was discarded. After adding 500 µL of PBS, the magnetic beads were mixed well, collected with a magnet, and discarded. Another 500 µL of PBS was added and the magnetic beads were mixed well. After transfer to a new tube, the magnetic beads were collected using a magnet, and the supernatant was discarded. MeOH or IPA (50 µL) was added and mixed, the magnetic beads were collected with a magnet, and the supernatant was analyzed by LC/MS.4HCO3/water (mobile phase A), acetonitrile (mobile phase B), and isopropanol (mobile phase C). The pump gradient was programmed as follows [time (%A/%B/%C)]: 0 min (95/5/0), to 8 min (70/30/0), 16 min (30/35/35), 28 min (6/47/47), 35 min (6/47/47), 35.1 min (95/5/0); it was then held for 38 min for equilibration. The flow rate was 0.35 mL/min, and the column temperature was 47 °C. The injection volume was 5 µL. SRM analysis was performed using positive/negative ion-switching mode ESI, with a collision energy of 46 eV. All data were analyzed using Microsoft Excel 2016. The LC and MS measurement conditions were as follows: Serum samples were subjected to a Nexera UHPLC system and LCMS-8060 triple quadrupole mass spectrometer . An Acquity UPLC BEH C8 column  was used with the following mobile phase compositions: 5 mM NHFor serum-enriched fractions using the CD9 antibody and IgG-immobilized magnetic beads, 30 µL of SDS-PAGE sample buffer was added after washing with PBS. Serum was diluted 50-fold with SDS-PAGE sample buffer because the of the excessive amount of protein. They were heated at 95 °C for 3 min. Fifteen microliters per lane was added to the gel and separated by SDS-PAGE (10–20% gradient gel), and then transferred to a polyvinylidene difluoridemembrane (pore size 0.2 µm). After blocking and washing with 1% skim milk, biotin-labeled CD9 antibody (1 µg/mL) was used as the primary antibody and Neutralite Avidin-HRP (Horseradish peroxidase)  as the secondary antibody, and Lumitera  as chemiluminescence reagents were used.g for 20 min. The supernatant was used for the HDL assay, and the precipitate was used for the LDL assay. The supernatant was diluted with 1× assay diluent (50-fold dilution for serum and 25-fold dilution for magnetic beads) for measurement, and the precipitates were dissolved by adding 400 µL of PBS and then diluted with 1× assay diluent (50-fold dilution for serum and 25-fold dilution for magnetic beads) for measurement. For each 50 µL, 50 µL of the cholesterol reaction reagent (HDL and LDL/VLDL cholesterol assay kit) was added and incubated at 37 °C for 45 min before measurement at 530 nm for excitation and 590 nm for fluorescence.The serum-enriched fractions using CD9 antibody and IgG-immobilized magnetic beads were washed with PBS, the supernatant was discarded, and 200 µL of 1× assay diluent (from the HDL and LDL/VLDL cholesterol assay kit) was added to 40 µL each, and then 200 µL of LDL Precipitation Reagent (HDL and LDL/VLDL cholesterol assay lit) was added to each of them. They were mixed well, allowed to stand at room temperature for 10 min, and centrifuged at 2000× In this study, antibodies against CD9 were covalently immobilized on magnetic beads, and the lipid components of human serum that interacted with CD9 antibodies were analyzed by LC/MS. Interestingly, MeOH extraction of serum recovered most of the phospholipids at the same level as IPA extraction, but almost no triglycerides were recovered. However, after enrichment with CD9 magnetic beads, triglycerides were measurable after MeOH extraction, as well as other phospholipids. The CD9 magnetic beads did not enrich LDL or HDL. At present, the implications of these findings remain, but it is possible that there are differences in the distribution and localization of triglycerides in serum and those enriched by CD9 magnetic beads.