Ehmt2 cKO, Ehmt1 cKO, Ehmt1/2 cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 in Ehmt1 cKO and Ehmt1/2 cDKO oocytes recapitulated meiotic defects observed in the Ehmt2 cKO; however, there was a significant impairment in oocyte maturation and developmental competence in Ehmt1 cKO and Ehmt1/2 cDKO oocytes beyond that observed in the Ehmt2 cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)–based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome in Ehmt1/2 cDKO than Ehmt2 cKO oocytes, with transcriptional derepression leading to increased protein abundance and local changes in genic DNA methylation in Ehmt1/2 cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence.EHMT1  is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 . Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models ( The mammalian germline is the context for widespread epigenetic changes, in which the somatic epigenetic signature is erased and replaced by a germline signature that is distinct in sperm and oocytes. Before the onset of gametogenesis, primordial germ cells undergo extensive erasure of many epigenetic marks, such as DNA methylation  and histEhmt2 knockout (KO) and Ehmt1 KO embryonic stem cells (ESCs)  8.5 and E12.5  and EHMT2  are best known as histone methyltransferases, although they modify nonhistone targets as well . They prs (ESCs) . Moreovend E12.5 , 2005. And E12.5 .Zp3-Cre transgene driver) impairs maturation and meiosis, with consequences for preimplantation development, leading to partial embryonic lethality  (Ehmt1 (Ehmt1 cKO) (Ehmt2 and Ehmt1 (Ehmt1/2 cDKO) were crossed with a Zp3-Cre driver, which is expressed exclusively in growing oocytes after postnatal day 5 . In Ehmt2 cKO oocytes, EHMT2 protein was lost in NSN oocytes, whereas EHMT1 remained readily detectable  , Ehmt1 (mt1 cKO) , or bothal day 5 . During al day 5 . In conttectable A. In con oocytes A, indica oocytes . This fiZp3-Cre driver has been shown to affect oocyte maturation and meiosis . Spindle abnormalities included collapsed, monopolar, and multipolar spindles. No significant differences were observed between the different cKO models. Taken together, the results show that loss of EHMT2 had mild effects on oocyte maturation compared with the significant impairment caused by loss of EHMT1, whereas effects on meiosis were similar.To determine whether EHMT1 is required for meiosis, we analyzed spindle conformation and chromosome alignment of ovulated metaphase II (MII) oocytes after hormonal stimulation. All three cKO models showed an increase in abnormal chromatin configuration and spindle alignment compared with the controls C. VariouEhmt1/2 cDKO blastocysts significantly reduced, and the proportion of dead and unfertilized oocytes in maternal Ehmt1/2 cDKO embryos significantly increased.To assess whether loss of EHMT1 from growing oocytes also compromises their competence, we examined developmental progression after fertilization. Embryos were collected at E3.5 from cKO females naturally mated with wild-type (WT) C57Bl/6Babr males and scored according to developmental stage. The majority of embryos from control females had reached the blastocyst stage D. In comEhmt1/2 cDKO again showed the strongest phenotype, having significantly fewer two-cell embryos than the controls . Fertilized embryos (one and two cell) were selected for in vitro culture: None of the embryos from Ehmt1 cKO or Ehmt1/2 cDKO oocytes developed to blastocysts, arresting at earlier stages (one- to four-cell stage) by E3.5 . In contrast, 42.9% maternal Ehmt2 cKO embryos developed at least to morulae by E3.5 . However, this was significantly less than the controls, in which 91.1% of embryos reached morula or blastocyst stages by E3.5. These findings reveal that embryos derived from Ehmt2 cKO oocytes show reduced survival through preimplantation development, whereas the preimplantation developmental competence of Ehmt1 cKO and Ehmt1/2 cDKO oocytes was severely impaired.To determine the stage of embryo arrest, embryos were collected from superovulated, naturally mated cKO females at E1.5 and cultured in vitro for 3 d until E4.5. Already at E1.5, a difference was observed, with all three cKO models having a higher proportion of unfertilized oocytes and one-cell embryos than the controls. The Ehmt1 cKO oocytes did progress in vivo to blastocysts; therefore, we examined the implantation and development of embryos after natural mating. In line with findings above, there were significantly fewer implanted embryos at E6.5 from Ehmt1 cKO and Ehmt1/2 cDKO oocytes than from Ehmt2 cKO and the controls . In contrast, although some abnormalities  were observed among E8.5 embryos from the Ehmt2 cKO oocytes, most appeared normal . By E12.5, 55% (11/25) of the embryos from Ehmt2 cKO oocytes were grossly abnormal or showed developmental delay . In Ehmt1/2 cDKO females, with one exception, all the embryos had died and only resorption sites were observed .A small percentage of embryos from controls E. At E8.Ehmt1 cKO or Ehmt1/2 cDKO females, but Ehmt2 cKO females did give birth to a mean of 3.46 healthy pups per litter, a significantly reduced litter size compared with that of the controls . H3K9me2-enriched domains in d25 GV oocytes were defined by merging consecutive 10-kb windows with a Log2RPKM > 2.5  . To link H3K9me2 enrichment to transcription levels, we used published, deeply sequenced RNA-seq data spanning oocyte growth . There was no difference in transcription level between genes localized to H3K9me2-enriched domains or random domains; thus, H3K9me2 enrichment does not appear linked to transcriptional repression in oocytes.With H3K9me1 and H3K9me2 affected by loss of EHMT2 and/or EHMT1, we sought to evaluate the genomic localization of these marks by ultra-low-input native ChIP-seq (ULI-nChIP-seq). Given the low-level, broad enrichment for H3K9me2 in GV oocytes, we performed the ChIP-seq experiments at two time points in WT GV oocytes from 15- and 25-d-old females to ensure we were obtaining robust, reproducible data sets. These time points represented an immature and mature GV oocyte state, respectively, with regards to accumulation of epigenetic marks, oocyte diameter, and NSN/SN state . We weree growth  to definEhmt1/2 cDKO model, which had a slightly more severe phenotype than the Ehmt1 cKO. As this small difference between Ehmt1 cKO and Ehmt1/2 cDKO oocytes might be caused by residual traces of EHMT2 in the Ehmt1 cKO oocytes, using Ehmt1/2 cDKO oocytes allowed us to make a clearer distinction between oocytes depleted of both EHMT2 and EHMT1 or of only EHMT2 (Ehmt2 cKO). To assess the loss of H3K9me2 upon depletion of EHMT1 in the oocyte, we generated ChIP-seq libraries of GV oocytes from d25 Ehmt2 cKO, Ehmt1/2 cDKO, and littermate control females. Notably, H3K9me2 libraries from control oocytes were 4.94% of total chromatin, whereas H3K9me2 libraries from Ehmt2 cKO and Ehmt1/2 cDKO oocytes were only 0.58% and 0.33%, respectively . When comparing enrichment across H3K9me2-enriched domains (±5 kb), Ehmt2 cKO and Ehmt1/2 cDKO showed significant loss of H3K9me2 compared with that of controls, with enrichment comparable to that in the IgG control and input . Gene Ontology (GO) analysis showed enrichment for processes involved in cellular localization and organization, as well as protein folding, metabolic processes, and fertilization . Furthermore, among the top 50 most abundant proteins, we detected oocyte-specific proteins such as members of the subcortical maternal complex , the zona pellucida , as well as proteins known to be highly abundant in the oocyte .Beside their role as histone methyltransferases, EHMT1 and EHMT2 are known to methylate nonhistone proteins and, by doing so, can potentially modulate their function, localization, or stability. To assess whether loss of EHMT1 affects protein abundance in the oocyte, we performed low-input (200 oocytes) quantitative whole-proteome mass spectrometry isobaric labeling analysis of control, Ehmt1/2 cDKO with control oocytes, we identified 187 proteins with a significant change in abundance (P < 0.05 and Log2FC > 0.3) . In contrast, there were only 38 differentially abundant proteins in Ehmt2 cKO oocytes, of which 21 overlapped with those in Ehmt1/2 cDKO oocytes . The majority of changing proteins increased in abundance in both Ehmt2 cKO  and Ehmt1/2 cDKO  oocytes . Furthermore, proteins involved in oocyte maturation (ERMP1) and fertilization (ASTL) showed significantly increased abundance, as well as the candidate oocyte transcriptional regulator HMGB3. Increased abundance of these proteins may contribute to the meiotic spindle abnormalities, impaired oocyte maturation, and poor fertilization rates observed in Ehmt1/2 cKO oocytes. In line with observations in oocyte and embryo development, the more severe effect in Ehmt1/2 cDKO oocytes suggests unique roles for EHMT1. The similar but lesser, nonsignificant changes in protein abundance observed in Ehmt2 cKO oocytes suggests that loss of EHMT2 can be partially compensated by EHMT1.We detected five known EHMT1/2 targets : None of these were significantly altered in cKO oocytes, but this is not entirely unexpected as protein methylation does not necessarily influence abundance but rather may affect activity. GO analysis did not detect any significant enrichment terms among the differentially abundant proteins. Even so, we identified several proteins for which change in abundance may be related to the observed oocyte phenotypes. Among these, two were meiotic factors according to the MGI Gene Ontology Browser (CDC25B and SIRT1), whereas literature research linked a further eight  to meiosis in the oocyte , which resulted in relatively small libraries with an average read number of 1.9 million; however, quality control analysis showed a high correlation between libraries . Differential gene expression was determined by DESeq2 analysis followed by filtering for genes with Log2FC ≥ 1.5. In line with what was observed in the proteome, the vast majority of differentially expressed genes (DEGs) were up-regulated in the Ehmt1/2 cDKO. Again, Ehmt1/2 cDKO oocytes were more severely affected than Ehmt2 cKO oocytes, with 330 DEGs  , in contrast to 79 DEGs in Ehmt2 cKO oocytes   down-regulated in both Ehmt2 cKO and Ehmt1/2 cDKO, (2) up-regulated in both genotypes, and (3) uniquely up-regulated in Ehmt1/2 cDKO , zygotic genome activation (Zscan4d), and embryo development  . Furthermore, several genes important for oocyte maturation, meiosis, and fertilization were deregulated , which may underlie some of the phenotypic effects we see. Consistent with the larger number and unique set of DEGs in the cDKO, the expression of long-terminal repeats (LTRs) of endogenous retroviral elements (ERVs) was up-regulated specifically in Ehmt1/2 cDKO oocytes .To assess whether the proteome changes may be a consequence of transcriptional changes, we evaluated gene expression by RNA-seq of 15 down) D. Of the1/2 cDKO E. GO anaEhmt2 cKO and Ehmt1/2 cDKOs . An explanation for this apparent discrepancy is that the genomic regions assessed by the two methods differ: IF signal reports both euchromatic and heterochromatic fractions of the genome, whereas BS-seq data are enriched in euchromatic and nonrepetitive regions that can be uniquely mapped and analyzed. These differences highlight the value of using both methods to understand genome-wide patterns of DNA methylation and may explain previous contradictory findings (We then explored changes in DNA methylation in greater resolution by whole-genome bisulfite sequencing (BS-seq) of control, findings .Ehmt1/2 cDKO oocytes , of which 4184 (45.5%) were hypermethylated (mean methylation difference: 35.1%) and 5003 (54.5%) hypomethylated (mean methylation difference 25.6%) . Methylation was more affected in Ehmt1/2 cDKO than in Ehmt2 cKO oocytes, with greater than seven times more DMRs identified: There were 1252 DMRs in the Ehmt2 cKO, of which 432 (34.5%) were hypermethylated (mean methylation difference: 39.4%) and 820 (65.5%) hypomethylated (mean methylation difference: 33.8%) . The majority of Ehmt2 cKO DMRs overlapped Ehmt1/2 cDKO DMRs . Furthermore, not only were there more DMRs, the methylation difference of these DMRs was greater in Ehmt1/2 cDKO oocytes  for analysis. Differential methylation analysis identified 9187 DMRs in  oocytes C. This iSupplemental Fig. S7A–C; Supplemental Table S4). In total, there were 707 hypomethylated and 869 hypermethylated domains in Ehmt1/2 cDKO oocytes, with fewer in the Ehmt2 cKO (140 hypomethylated and 66 hypermethylated domains). To see whether the additional changes in the Ehmt1/2 cDKO are unique or whether similar (nonsignificant) trends are present in the Ehmt2 cKO, we performed unsupervised cluster analysis   D,E. ThisSupplemental Fig. S6E). This is expected, as hypomethylated DMRs are found in regions that are methylated in control oocytes, and it is well established that DNA methylation localizes predominantly to transcribed genes in oocytes . These trends were similar in Ehmt2 cKO oocytes . This shows that the transcriptional changes observed in Ehmt1/2 cDKO oocytes impact the associated genic DNA methylation. However, this association does not explain most of the DNA methylation changes in Ehmt1/2 cDKO oocytes; thus, other mechanisms likely underlie the majority of changes observed.As transcription is linked to the deposition of gene-body DNA methylation in the mouse oocyte, we analyzed the correlation between expression and DNA methylation changes. Consistently, expression changes of DEGs positively correlated with gene-body DNA methylation changes in  oocytes G. In conUhrf1 cKO oocytes . As EHMT2 is required for noncanonical imprinting in embryos . Gab1 also showed up-regulated expression in both Ehmt2 cKO and cDKO oocytes  consistent with loss of repressive chromatin leading to increased transcription and gain of DNA methylation. Smoc1 and Sfmbt2 were also up-regulated, but to a lesser magnitude, and not accompanied by altered DNA methylation . Finally, examination of the maternal germline DMRs of canonical imprinted genes indicated the expected high levels of methylation , which is in line with previous reports . To explore this possibility, we compared the RNA-seq data from the three studies. To mitigate differences in RNA-seq library type, sequencing depth, and oocyte stage, we called DEGs separately in each data set using the same criteria. In brief, this analysis revealed a reasonable overlap between DEGs in our and the Au Yeung Ehmt2 cKO, but a lower overlap with DEGs from the Meng Ehmt2 cKO . The overlap between the Meng DEGs and DEGs in our Ehmt1/2 cDKO was also limited. Moreover, there was a strong bias toward up-regulated DEGs in our Ehmt2 cKO and Ehmt1/2 cDKO and the Au Yeung Ehmt2 cKO , but much less so in the Meng Ehmt2 cKO (59%). Finally, Ccnb3 as a major cause of the meiotic defects in their Ehmt2 cKO. We find that Ccnb3 mRNA is down-regulated in both our Ehmt2 cKO and Ehmt1/2 cDKO oocytes , although it was not identified as DEG with the cut-offs used in our analysis. However, the reduction in Ccnb3 transcript level in our Ehmt2 cKO is more profound than in the Ehmt1/2 cDKO oocytes, suggesting that defective Ccnb3 expression does not explain the more strongly impaired developmental competence of the cDKO oocytes. Altogether, these findings suggest that the severity of the Meng Ehmt2 cKO indicates a critical function of EHMT2 very early in oocyte growth, whereas EHMT1 has a separate critical function later in oocyte growth.While our paper was under review, a study was published in which ransgene . EarlierEhmt2 cKO and Ehmt1/2 cDKO oocytes, some genes, proteins, and genomic regions are progressively affected; others, only in Ehmt1/2 cDKO oocytes. The possibility of a unique, EHMT2-independent role for EHMT1 is supported by a recent study that reported that EHMT1 interacts with PRC2 in zygotes to establish H3K27me2 in the paternal pronucleus  VP diet  ad libitum and received seeds  at the time of cage cleaning as part of their environmental enrichment. All experimental procedures were performed under licenses issued by the Home Office (United Kingdom) in accordance with the Animals (Scientific Procedures) Act 1986 and were approved by the Animal Welfare and Ethical Review Body at the Babraham Institute.Zp3-Cre driver in addition with floxed alleles for Ehmt2  were selected for culture in M16 medium (Sigma-Aldrich MR-016-D) under mineral oil (Sigma-Aldrich M8410) at 37°C and 5% COEhmt2 cKO = 3, Ehmt1 cKO = 2, Ehmt1/2 cDKO = 3).GV oocytes from 12-wk-old females were stained with DAPI and scored according to their maturation stage. Absence of a ring around the nucleolus was counted as “NSN,” a partial ring as “intermediate.” and a full ring “SN.” Between 116 and 247 oocytes collected from several different females were analyzed per genotype , pan-histone (chromatin), and DAPI (heterochromatic DNA). Between 46 and 57 MII oocytes from four to seven mice were analyzed per genotype. For H3K9me1, H3K9me2, H3K9me3, 5mC, and 5hmC, two or three replicate experiments were performed, each with 10–15 oocytes from different litters, to control for batch effects. Samples were analyzed on a Zeiss LSM780 confocal microscope (63× oil-immersion objective). Spindle conformation and chromosome alignment of MII oocytes were scored using the categories illustrated in Supplemental Figure S1C. For analysis of H3K9me and DNA methylation, Z-stacks of single optical sections were captured, and semiquantification of fluorescence intensity was performed using Volocity 6.3 (Improvision).IF was performed after antibody staining as previously described . PrimaryQ-value < 0.01. For downstream data analysis, the R package qPLEXanalyzer was used (Oocytes were lysed in 20 µL dissolution buffer containing 100 mM triethylammonium bicarbonate (Sigma-Aldrich T4708) and 0.1% sodium dodecyl sulfate (SDS), followed by water bath sonication and boiling for 5 min at 90°C. Proteins were reduced with tris-2-carboxyethyl phosphine  for 1 h at 60°C at a final concentration of 5 mM, followed by cysteine blocking for 10 min at room temperature using methyl methanethiosulfonate  at final concentration of 10 mM. Samples were digested overnight at 37°C with trypsin (Pierce 90058), and the next day peptides were labeled with TMT11plex reagents (0.4 mg per sample) according to the manufacturer's instructions (Thermo Fisher Scientific). To quench the reaction, 3 µL of 5% hydroxylamine (Thermo Fisher Scientific) was added for 15 min and samples combined and dried with centrifugal vacuum concentrator. The TMT mix was fractionated with reversed-phase spin columns at a high pH (Pierce 84868). Nine fractions were collected using different elution solutions in the range of 5%–50% ACN and were analyzed on a Dionex UltiMate 3000 UHPLC system coupled with the nano-ESI Fusion-Lumos (Thermo Fisher Scientific) mass spectrometer. Samples were loaded on the Acclaim PepMap 100, 100 μm × 2 cm C18, 5 μm, 100 Å trapping column with the ulPickUp injection method at loading flow rate 5 μL/min for 10 min. For peptide separation, the EASY-Spray analytical column 75 μm × 25 cm, C18, 2 μm, 100 Å column was used for multistep gradient elution. Full scans were performed in the Orbitrap in the range of 380–1500 m/z at 120 K resolution and peptides isolated in the quadrupole with isolation window 1.2 Th, HCD collision energy 38%, and resolution 50 K. Raw data were processed with the SequestHT search engine in Proteome Discoverer 2.1 software and searched against a UniProt database containing mouse reviewed entries. The parameters for the SequestHT node were precursor mass tolerance 20 ppm and fragment mass tolerance 0.02 Da; dynamic modifications were oxidation of M (+15.995 Da), deamidation of N, Q (+0.984 Da); and static modifications were TMT6plex at any N terminus, K (+229.163 Da), and methylthio at C (+45.988 Da). The consensus workflow included TMT signal-to-noise (S/N) calculation, and the level of confidence for peptide identifications was estimated using the percolator node with decoy database search. Strict FDR was set at was used .ChIP-seq libraries were prepared using ULI-nChIP as previously described . AntibodSupplemental Table S6). BS-seq libraries were sequenced to an average of 16 million paired-end reads for Ehmt2 cKO and 26 million reads for Ehmt1/2 cDKO oocytes of 100–125 bp read-length. RNA-seq libraries were sequenced to an average single read number of 1.9 million for Ehmt2 cKO and Ehmt1/2 cDKO oocytes of 50-bp read-length. Raw FASTQ files were processed with Trim Galore!  and then mapped to the mouse GRCm38 genome. Mapping of ChIP-seq data was performed using Bowtie 2 (https://bowtie-bio.sourceforge.net/bowtie2/index.shtml), RNA-seq data by HISAT2 v2.1.0 (http://daehwankimlab.github.io/hisat2/main/) guided by known splice sites, and BS-seq data with Bismark v0.19.0 (https://www.bioinformatics.babraham.ac.uk/projects/bismark/). Total sequencing read numbers and uniquely aligned read numbers are listed in Supplemental Table S6.Libraries were sequenced on Illumina MiSeq, HiSeq 2500, and NextSeq 500 systems. ChIP-seq libraries were sequenced to an average of 57 million paired-end reads of 75-bp read-length (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). For ChIP-seq analysis, 10-kb running windows  were quantified as reads per kilobase per million (RPKM). Windows were filtered to exclude mapping artifacts, defined as RPKM > 6 in at least one replicate set of 10% input libraries (N = 408). H3K9me2 enrichment was defined as Log2RPKM > 2.5 in d25 GV oocytes . A set of random windows was sampled from all 10-kb windows . H3K9me2-enriched and random windows were then merged with adjacent windows within 10 kb, resulting in 12,154 H3K9me2-enriched domains and 26,077 random domains. Genic and intergenic regions were defined as overlapping or not overlapping oocyte transcripts, respectively, and promoters were defined as ±500 bp around transcription start sites of oocyte transcripts . Differentially methylated regions (DMRs) were defined by statistical comparison of DNA methylation levels for each 100-CpG window between control and Ehmt2 cKO or control and Ehmt1/2 cDKO oocytes using the edgeR function in SeqMonk. To assess overlap of DMRs with genomic features, CGI and oocyte gene annotations were used from https://CRAN.R-project.org/package=pheatmap).BS-seq data were analyzed using a tile-based approach of 100 CpGs for each consecutive genome window, ensuring equal CpG content in all windows. Methylation values were quantified using the bisulfite-sequencing pipeline quantification, which calculates per-base methylation percentages and averages these within each window. Filters were applied to ensure a minimum coverage of 10 or more observed cytosines per window. Only windows with this minimal coverage in all samples were taken into account, allowing assessment of 86.2% of 100-CpG windows ; ERVs within 2 kb of a gene were excluded to avoid spurious ERV calls from normal exonic gene expression, and then ERV counts were quantified as a percentage of total library counts. Publicly available ChIP-seq, RNA-seq, and BS-seq data accessed from the NCBI Gene Expresion Omnibus  under accession numbers GSE93941, GSE112320, GSE153611, GSE97778, and DRA005849 were used to develop our studies.For analysis of RNA-seq data, the expression of oocyte genes  was quan package . The ranX-square test was used to analyze meiosis abnormalities and the frequency of overlap of genic/intergenic regions with DMRs, histone modifications with DMRs, and UHRF1 hypomethylated regions with DMRs. P-value was adjusted using Bonferroni correction to control for multiple comparisons.Statistical analysis was conducted in GraphPad Prism, R, and VassarStats. SN proportion was compared using two-way ANOVA (Stage × Genotype) with Šídák's multiple comparisons test. The proportion of embryo stages at E3.5, number of implantation sites at E6.5, and mean fluorescence intensity of confocal IF images were analyzed using one-way ANOVA with Tukey's post hoc test. Litter sizes were compared using a nested one-way ANOVA with mouse as nested factor. Overlap of DEGs with histone modifications was analyzed by comparing DEGs to random probes using a two-tailed Fisher's exact test. A http://www.proteomexchange.org/) via the PRIDE partner repository (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE191026.The mass spectrometry proteomics data generated in this study have been submitted to the ProteomeXchange Consortium (pository  under ac


A successful interprofessional faculty development program was transformed into a more clinically focused professional development opportunity for both faculty and clinicians. Discipline-specific geriatric competencies and the Interprofessional Education Collaborative (IPEC) competencies were aligned to the 4Ms framework. The goal of the resulting program, Creating Interprofessional Readiness for Complex and Aging Adults (CIRCAA), was to advance an age-friendly practice using evidence-based strategies to support wellness and improve health outcomes while also addressing the social determinants of health (SDOH). An interprofessional team employed a multidimensional approach to create age-friendly, person-centered practitioners. In this mixed methods study, questionnaires were disseminated and focus groups were conducted with two cohorts of CIRCAA scholars to determine their ability to incorporate learned evidence-based strategies into their own practice environments. Themes and patterns were identified among transcribed interview recordings. Multiple coders were used to identify themes and patterns and inter-coder reliability was assessed. The findings indicate that participants successfully incorporated age-friendly principles and best practices into their own work environments and escaped the silos of their disciplines through the implementation of their capstone projects. Quantitative data supported qualitative themes and revealed gains in knowledge of critical components of age-friendly healthcare and perceptions of interprofessional collaborative care. These results are discussed within a new conceptual framework for studying the multidimensional complexity of what it means to be age-friendly. Our findings suggest that programs such as CIRCAA have the potential to improve older adults’ health by addressing SDOH, advancing age-friendly and patient-centered care, and promoting an interprofessional model of evidence-based practice. The Age-Friendly Health Systems Initiative, born out of a partnership between the John A. Hartford Foundation, the Institute for Healthcare Improvement, the American Hospital Association, and the Catholic Health Association of the United States, was created as a way to advance the practice of providing age-friendly care . This inCIRCAA, which stands for Creating Interprofessional Readiness for Complex and Aging Adults, is a faculty and clinician development program for healthcare professionals. The program was developed by an interprofessional team of faculty and clinicians who had appointments at or affiliations with an urban research university in the mid-Atlantic region of the United States. This interprofessional group meets twice a month to oversee a variety of interprofessional geriatrics training initiatives, including CIRCAA. The CIRCAA curriculum was based on the Faculty Development Program (FDP), an interprofessional curriculum grounded in evidence-based practices for healthcare professionals with a faculty appointment. The program was based on a model offered by the University of California, San Francisco, and the curriculum was guided by the Partnership for Health in Aging Workgroup on Multidisciplinary Competencies in Geriatrics ,3,4,5. ATo create the CIRCAA curriculum, the interprofessional faculty group first gathered geriatrics competencies from each of their respective fields and professional organizations. These competencies as well as the Interprofessional Education Collaboration’s (IPEC) ,9 competThe first year, CIRCAA was offered in person until March, 2020, when the COVID-19 pandemic forced the program to pivot to virtual education. The second year, the program was offered entirely virtually, with each session shortened from five hours to three, with additional materials made available for self-studying. The capstone project was sustained during the pandemic, but in-person practicum experiences were curtailed. The curriculum outline is in The conceptual themes surrounding CIRCAA training included incorporating health disparities and inequities in care delivery and the role of SDOH and their impact on healthy community living. The foundational FDP maintained an academic didactic format of training focused on preparing interprofessional health profession faculties to incorporate geriatrics into their clinical or classroom teaching. The transition to the CIRCAA format created opportunities to provide evidence-based strategies for translation to practice and then to evaluate the learners’ ability to incorporate this training into their own practice settings. Using the 4M’s framework as a guide, the program is able to focus on the concepts that are most important to improving care delivery and outcomes for older adults across practice settings. The onset of the COVID-19 pandemic required a rapid conversion to a hybrid format in order to sustain the program. Converting to a hybrid format presented initial challenges but also provided an opportunity to recruit from a broader geographic region. In prior years, participants were required to attend in person and to live within driving distance for the on-site monthly sessions. With the conversion to a virtual format, participants were able to join the program from as far away as California. The virtual format also required a transition in the types of learning strategies to be more interactive during group learning sessions with less intensive time commitments while still maintaining the integrity of the program and accomplishing the established goals and objectives. Teaching strategies were adapted and revised over the two years of the COVID-19 pandemic using the Plan Do Study Act (PDSA) method of evaluation and continual refinement, including increased small group learning activities and case studies ,11,12.Educational strategies, practice innovation, and evaluation methods were incorporated into the training to help scholars develop their capstone projects, a curriculum enhancement or practice intervention that addresses one of the 4Ms and fills a need in their workplace. Capstone projects could be either a practicum/practice intervention or curriculum-based. CIRCAA scholars implemented practice-based projects to address identified needs in their work settings. Each project was designed to advance at least one of the 4Ms in an interprofessional manner. Many of the projects concerned the 4Ms broadly, while several specifically focused on medication or mentation. Project topics included improving communication for diabetes self-management, de-prescribing in home health, and ageism in health profession training.n = 3), nurse practitioner (n = 1), occupational therapy (n = 1), oral health (n = 1), pharmacy (n = 2), physical therapy (n = 2), and public health (n = 1). Eleven scholars completed the CIRCAA program as part of the 2021 cohort and represented the following disciplines: gerontology (n = 1), health care administration (n = 1), medicine (n = 1), nursing (n = 3), occupational therapy (n = 2), physical therapy (n = 1), physician assistant (n = 1), and social work (n = 1).As CIRCAA was developed, program assessment activities to formatively and summatively evaluate the program were also planned. These activities included an end-of-program focus group with scholars in addition to a pre-program and post-program evaluation questionnaire. The 2020 cohort consisted of 11 scholars, 8 of whom fully completed the program; the remaining 3 scholars partially completed the program and had to reduce engagement due to work constraints caused by the COVID-19 pandemic. The 2020 cohort of scholars represented the following disciplines: nursing  and interprofessional values (t(19) = 3.22, p < 0.01, d = 0.72) subscale scores. Additionally, as can be seen in The average rating on the Interprofessional Interaction subscale before CIRCAA was 4.03 (SD = 0.73), and ratings increased to 4.47 (SD = 0.52) after CIRCAA concluded. Similarly, the average rating on the Interprofessional Values subscale was 4.40 (SD = 0.54), but ratings increased to 4.67 (SD = 0.49) after CIRCAA concluded. Notably, the evaluation data from the pre and post program questionnaires revealed a significant increase in both the interprofessional interactions (t(19) = 3.40, Two main categories of themes emerged from the analysis of the focus group data. The first category, creating age-friendly readiness, included four themes: modeling, innovating, leveraging, and challenging. The second category, understanding and implementing an interprofessional age-friendly framework, established two themes: impacting and cultivating. Each of the categories and their accompanying themes are described in detail below.One core question that was asked during the focus group sessions that concentrated on participants discussed the strengths and challenges of being trained as age-friendly professionals working within an interprofessional environment. The themes emerging from this discussion were modeling, innovating, leveraging, and challenging.The scholars described the clinical practice and teachings of the interprofessional team they had during their year-long time as scholars as exhibiting the professionalism that they want to model in their own practices and teachings. The scholars were exposed to videos, case studies, and experiential learning opportunities where they could observe best practices. The faculty and clinicians who worked with the scholars streamlined information that can be complex and overwhelming into a simplified framework—the 4Ms. In the 2020 cohort, four of the six scholars had the 4Ms focus on their capstone projects, and, in 2021, five of the eight scholars focused on the 4Ms.CIRCAA enhanced the interprofessional experiences for the scholars. Prior to CIRCAA, they described their experience as “lacking”—lacking the chance to experience the reality of professionals from multiple disciplines having opportunities to work with each other. Visiting practice sites and seeing interprofessional teams interact “on-the-job” also enhanced their learning. The scholars felt like they were partnering with the faculty and clinicians teaching them instead of only learning and collaborating with them. Scholars felt exhilarated from the interprofessional training they received in CIRCAA because it provided the “big picture” and was a blending of the interprofessional perspective from both clinicians and faculty researchers. One scholar stated:“The health care climate is changing so rapidly that it is important to remain open minded and learn from each other”.The scholars expressed the importance of the clinicians observing what is going on in a faculty person’s classroom. At the same time, what is being learned in the classroom needs to be practiced in the community:“Person-centered care needs to be practiced. We learned to feel comfortable using the term ‘what matters’ instead of values and preferences. It is important to just plain ask ‘what matters to you?’”CIRCAA shifted the way the scholars normally teach students about working with older adults and provided new ways to deliver challenging content. Using the 4Ms framework was innovating because it provided room to learn about what matters to the client. It allowed the scholars to escape from their known and comfortable silos:“I thought starting off the program using the 4Ms and emphasizing that in the beginning was a great framework to do interprofessional education because there are these four aspects, they involve all of us and it reframes and refocuses our goal”.CIRCAA was innovating for the scholars because the interprofessional learning and the 4Ms framework strengthened their practice.It is important to build on the positive experiences the CIRCAA scholars had and use their testimonials to leverage the sustainability of the program. The scholars expressed a sincere interest in seeing CIRCAA sustained over time and discussed how the program was a good choice over participating in a traditional certificate program. Though often overwhelmed with the amount of content provided, scholars appreciated the sharing of resources. As stated by one of the scholars: “I learned a lot of stuff!”The CIRCAA scholars learned to push past their comfort zones. The challenges noted by the scholars during their CIRCAA training included receiving a plethora of resources—they suggested less volume and content. It was also suggested to use a flipped classroom approach where the scholars are provided readings ahead of time and have more discussions and experiential learning opportunities to apply what they were reading. The scholars expressed that it is important to teach students how to motivate older adults from an interdisciplinary perspective so that changes and improvements can occur. Participants wanted to learn more, but they were unsure of what to do or how to do it until they participated as a CIRCAA scholar. Older adults and professionals need to work together to make a difference in the lives of older adults. Life’s complexities often created some challenges with completing the CIRCAA program. As one scholar stated: “This is my last hurrah. I wanted to be challenged and you have certainly done that!”Generally, professionals learn to practice within their disciplines. The concept of siloing emerged from the data and is being defined as a limiting perspective based on one’s discipline. One scholar stated that exposing yourself to other disciplines’ perspectives is important:“You don’t realize how limited and limiting [your perspective] is until you perhaps, for various reasons, and this was an opportunity here, are exposed to other disciplines and see how the state of healthcare is what it is because of the disconnect in the relationships that need to be occurring for better outcomes to take place”.In addition to discussing the strengths and challenges of CIRCAA, another core focus group question pertained to the 4Ms and the value of the 4Ms framework. Two themes emerged from this discussion: impacting and cultivating.Scholars completed CIRCAA with a perspective of receiving something of “tremendous value”. They expressed an understanding of the importance of interprofessional perspectives. One participant stated that an interprofessional perspective “gives me street cred”. Another scholar expressed how impactful CIRCAA was by stating:“We came in with an expertise in a quadrant. Right from the first case I felt like, OK, I’m going to hone in on what matters to this person. But we had a PT that was raising issues about the motor and movement and mobility. We had a pharmacist that was bringing in all the issues around medication. And then we had people that were working either in Alzheimer’s or nursing with cognition. It was just so neat for us all to come and share those perspectives together, because I think what happened was we all got an appreciation for the contributions that each of those areas have because of our own comfort level and expertise in those areas”.CIRCAA gave the scholars an opportunity to be exposed to interprofessional practice by seeing the impact the interprofessional team of faculty and practitioners had on each other during each training: “I am gaining a lot from watching the faculty and clinicians [instructors] work interprofessionally”. The scholars worked to apply what they were learning during their time in CIRCAA instead of waiting until their scholar experience was complete:“I also want to emphasize something that was really big that I’m implementing more is the social determinants of health, implementing some of the questionnaires that we had talked about, like food security into my practice that has a lot to do with diabetes, things that I hadn’t thought to include but now feel are vital to include”.The 4Ms framework is a model that was easy to grasp and emphasized the importance of person-centered care and integrating what matters to their clients in their daily practice. One scholar emphasized the importance of escaping the silos of your own discipline by stating:“When I think back to about October, I was in K’s group and she was coming all at it from the pharmacist perspective and we were saying, ‘now wait, there’s other things to consider.’ We were like, ‘we need a social worker. We need that information for this case.’ So recruit social workers!”The scholars cultivated relationships with each other as well as with the instructors “making friends while learning”. One scholar was at the end of her career, and her experience in CIRCAA gave her a chance to cultivate future opportunities, even after retiring.“I know OT really well, but not OT with a geriatric population. I really hope I can contribute even in some guest speaking lectures and things like that, because I’ve learned so much”.As a clinician, she had years of experience but not with an older population. She wants to give back to others after her experience as a CIRCAA scholar in a different way than she had anticipated.Our quantitative findings revealed significant gains in scholars’ perceptions of interprofessional care as well as knowledge about critical components of age-friendly care. Quantitative results from this study showed a concrete improvement in the team skills necessary for quality in the person-to-person interactions occurring in an interprofessional context. Two major themes emerged from the qualitative analysis, both individual preparations to become an interprofessional team member and a comprehensive understanding of the 4Ms framework for advancing age-friendly health systems. Scholars valued the opportunity to share perspectives and enjoyed participating in a program that allowed them to become an interprofessional cohort. The scholars discussed innovative ways to cultivate an atmosphere where multi-disciplinary perspectives are allowed to be normalized. They also expressed the importance of having a stronger skill set after the program to better make interdisciplinary work happen where it is not happening. Our qualitative results indicate that the competency-based curriculum developed for CIRCAA ignited a spark that subsumed the 4Ms individually and conceptually. There was an increased awareness of the SDOH operating within an aging individual’s lifespace and an acknowledgement of the various contexts at work over the course of life.The significant increase in interprofessional interactions and values and the increase in knowledge of the 4Ms framework, age-friendly health systems, person-centered care, and interprofessional roles in aging care underpin the themes that emerged in creating readiness and understanding and implementing age-friendly interprofessionalism. These themes enrich the ongoing revision process of the CIRCAA program and encourage the integrated delivery of content, with a dedicated focus on the use of the 4Ms as a universally understood interprofessional framework. A partnership between program faculty and scholars through modeling an interprofessional practice in the delivery of teachings, the application of knowledge in practice, and observation in community settings within the 4Ms framework fosters accomplishment of this mutual goal. This frame of reference allows a common ground to develop interprofessional person-centered goals for aging adults and fosters escape from siloed practice. This does not come without challenges, as evidenced by scholars in health settings without readily accessible interprofessional connections. Debriefing after each CIRCAA learning experience creates the space for scholars and program faculty to discuss gaps, create solutions, and reveal what is lacking from an interprofessional perspective in meeting the challenges of addressing what matters most for aging clients and caregivers. These opportunities emphasize and reinforce the competencies scholars need to cultivate and incorporate age-friendly interprofessionalism into their practices.The quantitative and qualitative results reinforce the importance and usefulness of PDSA in creating awareness and implementation of age-friendly practice in ongoing revisions of the CIRCAA program. The greatest knowledge gains pertained to the 4Ms (90%) and age-friendly, person-centered care (80%), while collaborating with people outside the scholars’ disciplines was the most diminished self-efficacy improvement (65%). Program faculty continuously evaluate the CIRCAA program after each session and each cohort. This process directly informs both what needs to be performed before the next session and what changes are necessary for subsequent cohort years. Learning from each other in this way helps foster the strong relationships that result from CIRCAA participation. Many of the faculty remain engaged with the scholars, both because of ongoing capstone projects and because of a mutual passion for geriatrics education. Currently, 8 of the 25 plenary members are former scholars.While we find that both quantitative and qualitative analyses are helpful in planning future CIRCAA cohorts through PDSA, there are some limitations, which include those imposed by COVID and the small sample size. The first cohort participated in person until the pandemic began in March, 2020, and the second cohort participated virtually. The first cohort attended two in-person retreats and had opportunities to network within the cohort and with the faculty that were not possible after the pandemic arrived. Session length was also shortened from five hours of teaching to three hours, with extended preparation for the virtual sessions. However, monthly session evaluations were similarly positive when comparing the two cohorts. We also noticed benefits of pivoting to a virtual format, which provided opportunities for scholars to participate from other states and while traveling.The focus of this paper is an evaluation of individuals participating in CIRCAA, and how they applied this training to their practice. We also show how interdisciplinary curriculum in geriatrics can be adapted to successfully incorporate age-friendly principles in clinical practice and share them as a model for creating interprofessional learning experiences for a variety of disciplines. The findings suggest that programs such as CIRCAA have the potential to improve older adults’ health by addressing the SDOH, advancing age-friendly and patient-centered care, and promoting an interprofessional model of evidence-based practice. Whereas the previous FDP focused on geriatrics, CIRCAA states “the goal of this program is to create age-friendly practitioners who are knowledgeable in person-centered care”. These are significant changes that point CIRCAA toward age-friendly and patient-centered holistic care and beyond the FDP’s emphasis on geriatrics education and curriculum design.Many barriers exist when implementing the 4Ms in practice, including who is missing and what is missing; who should be part of the team and how do we get them on the team. CIRCAA provides a streamlined framework for modeling how an interprofessional team works and what this team looks like in order to provide true person-centered care. By refocusing and integrating 4Ms content into the CIRCAA curriculum, which had been informed by geriatric and gerontology competencies, scholars were allowed to escape their silos in order to experience a true person-centered care model. The next steps are to address these barriers and provide incentives to develop healthcare settings such as the Program for All Inclusive Care for the Elderly (PACE) so that professionals can more easily escape these silos in practice through the application of the 4Ms framework.The National Academy of Medicine’s 2021 Vital Directions for Health and Health Care: Priorities for 2021 initiative  include Moving forward, to further evaluate the impact of CIRCAA or other geriatrics faculty and clinician programs, it would be worthwhile to follow-up with scholars after their participation. For example, it would be good to assess the inclusion of the 4Ms in participating scholars’ practice and/or teaching in the months and years following CIRCAA completion. Tracking the number and types of practice and teaching sites that these projects touch would also inform the program’s impact. Our curriculum highlighted the utility of making connections between patients and the wealth of resources available in the community. As a consequence, community resources were an aspect woven throughout the mapping of competencies for CIRCAA. Investigations into how these connections are made and sustained as a way to promote age-friendly health care are warranted. CIRCAA emphasized the scholars’ roles as neutral brokers in this process and aimed to make them more adept at locating and referring patients to community services. Essentially, further studies into how geriatrics education programs for faculty and clinicians can contribute to the age-friendly ecosystem are needed. Establishing linkages among healthcare systems and community-based organizations is an equally important aspect of the age-friendly initiative . Replica


Aeromonas hydrophila via intraperitoneal injection. Our findings indicated that both innate and adaptive immune responses were effectively elicited after A. hydrophila infection. Upon bacterial infection, IgM+ B cells were strongly induced in the gut and head kidney, and bacteria-specific IgM responses were detected in high levels both in the gut mucus and serum. Moreover, our results suggested that IgM responses may vary in different infection strategies. Overall, our findings revealed that the infected common carp exhibited high resistance to this representative enteropathogenic bacterium upon reinfection, suggesting that IgM plays a key role in the defense mechanisms of the gut against bacterial invasion. Significantly, the second injection of A. hydrophila induces strong local mucosal immunity in the gut, which is essential for protection against intestinal pathogens, providing reasonable insights for vaccine preparation.Immunoglobulin (Ig) M is an important immune effector that protects organisms from a wide variety of pathogens. However, little is known about the immune response of gut mucosal IgM during bacterial invasion. Here, we generated polyclonal antibodies against common carp IgM and developed a model of carp infection with  Teleost fish are an important link in vertebrate evolution and an indispensable component of comparative immunology, which has both innate and adaptive immune systems . The ada+ B lymphocytes of teleost fish are important subsets of B cells involved in innate and adaptive immune responses (Cyprinus carpio) has been cloned . The heavy chain constant domains  were amplified with primers F/R (F:5’-CGCGGATCCGATGTTCGCGCAACCGTT-3’ R:5’-CCGCTGGAGTTACGGTTTACAAAACGCCGG-3’) and 2×Taq Master Mix . The PCR products were purified using the FastPure® Gel DNA Extraction Mini Kit , digested with restriction enzymes BamH I and Xho I , and ligated to the pET-32a vector. Then the constructed plasmid was transformed into Escherichia coli BL21 (DE3) , inoculated on Luria-Bertani (LB) solid medium, and cultured at 37°C overnight. The expression and purification of recombinant IgM protein (rIgM) expression were performed by referring to the previous method of Wang et al. with minor modifications (Completed IgM heavy chains of common carp cDNA sequences were obtained from NCBI (GenBank accession no. MH352354.1) and were synthesized artificially. We predicted the functional domains through the IMGT tool  twice per day and acclimated at 28°C for at least 2 weeks. The feeding was terminated 48 h before injection and sampling. Animal procedures were approved by the Animal Experiment Committee of Institute of Hydrobiology, Chinese Academy of Sciences and carried out according to the relative guidelines.A. hydrophila strain AH1 was obtained from the Laboratory of Aquatic Animal Medicine of the College of Fisheries at Huazhong Agricultural University . The AH1 strain was streaked consecutively on LB solid medium and cultured at 28°C for 24 hours. Then, single colonies were picked and inoculated into LB liquid medium and cultured at 28°C for 12 h with 200rpm shaking. A. hydrophila suspension was centrifuged at 5000 g for 10 min, bacteria were resuspended with PBS and the concentration was adjusted to 1.6 × 107 CFU/ml for challenge use. We designed two types of challenge experiments. The first was an infection of common carp by intraperitoneal injection with 100 μl of A. hydrophila (1.6 × 107 CFU/ml), tissues  from at least six fish were collected at 0.5, 1, 2, 4, 7, 14, 28 days post-infection (DPI), respectively, and fluids (serum and gut mucus) were taken on 28 DPI  and 35  days post-primary infection. As the control group, the fish were injected with 100 μl of PBS. Tissue samples were used to evaluate morphological change and measure the expression of the immune-related genes, and serum and gut mucus were used to detect the changes in the IgM by western blot.g for 10 min at 4°C and the mucus suspension was then stored at -80°C before use.For common carp sampling, fish were anesthetized with MS-222. The blood was taken from the tail vein, centrifuged at 5000 g for 10 min to remove the plasma, collected the serum, and immediately stored at -80°C before use. To collect skin mucus, the mucus was gently scraped from the surface of common carp skin and then placed in a sterile Petri dish as previously described . To collMucus and serum samples were resolved on 4-15% SDS-PAGE Ready Gel . PVDF membranes  were first activated with methanol and the gels were transferred onto PVDF membranes for western blot analysis. PVDF membranes were blocked with 8% skim milk after the transfer and then incubated with anti-carp IgM (0.2 μg/ml) antibody. As isotype, the rabbit IgG (purified from rabbit blood before immunization) was used at the same concentrations. After washing four times, added HRP Goat Anti-Rabbit IgG (H+L)  and incubated for 45 min at RT. After washing four times, the ECL Substrate  was incubated with PVDF membranes. For quantitative analysis of IgM in serum and gut mucus, the PVDF membranes were scanned under the Amersham Imager 800 Imaging System , and the signal intensity of each band was analyzed with ImageQuant TL software . Thereafter, the concentration of IgM was determined by plotting the obtained signal strength values on a standard curve generated for each blot using known amounts of purified carp IgM. For purification of native IgM in common carp serum, the same procedure was used as for the purification of pAb as described above. Briefly, the affinity column was prepared by coupling anti-carp IgM pAb to CNBr-activated Sepharose 4B . Then a serum sample pooled from several individual carp was diluted 1:2 in PBS (pH 7.2) was applied to the column equilibrated in PBS. The bound IgM was eluted in the same manner, and the concentration was determined using the Micro BCA™ Protein Assay Kit according to the manufacturer’s instructions .+ B cells were detected according to the method previously described by Yu et al. with minor modifications  staining was performed according to the previous method of Kong et al. . Images ications . Briefly® Blood & Tissue Kit  according to the manufacturer’s protocol. Briefly, the tissues were cut up and mixed with 180 μl ATL and 20 μl proteinase K. The tissues were incubated at 56°C until they were completely lysed. Add 200 μl AL and incubate at 56°C for 10 min and then add 200 μl anhydrous ethanol. Transfer supernatant to DNeasy Mini spin column and centrifuge at 7000 g for 1 min. After washing the hybrids with AW1 and AW2, elution was performed with Buffer AE, collected the eluted DNA. The concentration of DNA was determined using a Nanodrop 2000 spectrophotometer . To test bacterial loads, we selected Act virulence genes from A. hydrophila for qPCR. Total RNA was extracted by homogenization in 1 ml TRIzol  using steel beads and shaking  following the manufacturer’s instructions. Extracted RNA was measured by spectrophotometer for concentration and checked for integrity with agarose gel . To eliminate differences in gene expression levels for each sample, RNA was reverse transcribed to cDNA and then analyzed by qPCR according to the method of Yu et al.  to elute bound proteins and mix well, boil at 95°C for 8 min. The prepared samples were separated by SDS-PAGE and then detected for the presence of IgM using western blot and the polyclonal antibody against common carp IgM as described above.Using the pull-down assay as described previously , 43, we t-test, one-way analysis of variance (ANOVA) with Bonferroni correction, and log-rank (Mantel-Cox) test . A value of p < 0.05 or less was considered statistically significant.The data were shown as the mean ± SEM and normality and homogeneity of variance were checked before the statistical analysis. The statistical differences between groups were analyzed using an unpaired Student’s ;To prepare polyclonal antibodies against common carp IgM, we first obtained the complete cDNA sequence of IgM heavy chain from NCBI (GenBank accession: no. MH352354.1) and cloned the CH2-CH4 domains into the prokaryotic expression plasmid  than in the mucosal tissues   genes , inflammatory genes , matrix metalloproteinase 9 (mmp-9), and immunoglobulin (Ig) heavy chain genes . In this study, we found that most of the genes increased significantly in foregut, midgut, hindgut, spleen, and head kidney tissues after A. hydrophila infection  and then increased significantly. The higher expression levels of proinflammatory cytokines, such as il1-β, il6, and il8, were also expressed at 0.5 and 1 DPI. However, the inflammatory cytokine il2 was significantly upregulated at the initial phase of inflammation in common carp gut, as well as at the later phase of tissue remodeling  and hindgut mucus (∼3.5-fold) dilution from 28DPI-S fish and up to 1:100 in dilutions of midgut mucus and hindgut mucus in 35DPI-S fish, an increase of ∼2.8-fold and ∼2.1-fold, respectively, compared to control fish . We found that bacteria-specific IgM binding was detected at 1:10 (∼2.2-fold) foregut mucus dilution in the 28DPI-S fish, in contrast to midgut and hindgut results where the binding was detected only at 1:40 (∼3.1-fold) foregut mucus dilution in the 35DPI-S fish . In addition, there was a significant increase in binding of bacteria-specific IgM in the diluted serum of the 28DPI-S fish and 35DPI-S fish compared with control fish, reaching 1:1000 (∼1.8-fold) and 1:4000 (∼2.8-fold), respectively . In a word, these results indicated that IgM plays an important role in the gut tissue against bacterial invasion.To further study the gut bacteria-specific IgM responses in common carp after A. hydrophila compared with those of the first infected fish   and two light chains (~25 kDa)  , 51–53. ~25 kDa) . Moreove results , 40, 55.A. hydrophila infection model in common carp via intraperitoneal injection. As expected, the fish exhibited the typical symptoms of A. hydrophila infection, including body surface hemorrhage, abdominal dropsy, and enteritis , plays a crucial role in host defense against bacterial invasion , a member of the zinc-dependent endopeptidase family, is associated with vital inflammatory processes in mammals , and these cells increased their IgM secretion  and a grant from the State Key Laboratory of Freshwater Ecology and Biotechnology (2022FB13).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.


M = 37.23, SD = 10.66) completed a battery of instruments before and after the intervention. Statistical analyses showed that users who participated in the reduced version of the program presented a significantly higher rate of improvement in their sexual behaviors compared to those who participated in the extended version , as well as a significantly higher decrease in the risk of suffering sexual abuse . However, no statistically significant differences in sexuality knowledge were obtained with respect to the improvement between the two versions . Meanwhile, the professionals who applied the program found that those who participated in the reduced version, although they presented a significantly greater increase in their knowledge about privacy , did not improve their concerns about their inappropriate sexual behaviors as much as the users of the extended version . Thus, although both versions were effective, the reduced version seems to do so to a greater extent and in a shorter time, which makes it the more recommendable option.Recently, with the increase in demand, multiple intervention proposals aimed at improving the sexual health of people with intellectual disabilities have emerged. Among them is the SALUDIVERSEX program, which takes a positive approach to sexuality. It has an extended version, consisting of 16 sessions and whose efficacy has already been proven, and a reduced version of 10 sessions. Thus, the present study aimed to test the differential efficacy of the two versions. A total of 208 participants (103 women and 105 men) aged between 19 and 67 years ( The affective-sexual education of people with intellectual disabilities (ID) has gone from being a conflictive aspect, due to the controversial attitudes of family members and professionals, to become an increasingly demanded aspect not only by the users themselves but also by the centers they attend  Self-care, which deals with sexual hygiene, autoeroticism and sexual diversity; (III) The self in relationship, in which training is provided on the initiation, maintenance and rupture of affective-sexual relationships, sexual assertiveness, sexual practices and safe sex, prevention of infections and unwanted pregnancies, love and maltreatment, and prevention of sexual abuse; and (IV) My sexuality, which includes only one session of integration and consolidation of learning. In the reduced version, although the basic concepts, skills and attitudes of the original program have been maintained, the depth with which the modules on body image, prevention of sexually transmitted infections and contraceptive methods, and inappropriate behaviors in social networks have been addressed has been reduced. This adaptation is based on the results obtained in the evaluation of the effectiveness of the extended version of the program  and on the feedback provided by the professionals who applied it and by the participants themselves. Thus, while the validation of the original version of the SALUDIVERSEX program reported the high starting levels of the subjects in some of these modules and the small margin for improvement they showed, the professionals who applied them and the users themselves, assessing the real characteristics and needs of the target group, considered that it was unnecessary to go so deeply into these contents.The reduced version is made up of 10 sessions that are grouped into the same 4 blocks of the original program: (I) In relation to the teaching methodology, the shorter version has limited theoretical explanations through the use of expository techniques, although visual material has continued to be used to support them (slides and short and attractive videos), activities that invite reflection and guided debates. On the other hand, playful dynamics and role-playing games have become the star technique to encourage user participation. Also, following the feedback from the participants and instructors of the extended version of the program, although the number of activities within each session is the same, the number of exercises within each session was reduced in order to better address the doubts that arose during their implementation.In view of the above and given the importance of knowing whether these modifications have an impact on user learning, the objective of this work is to analyze the differential efficacy of both versions. If the results of the reduced version were better or at least similar to those of the extended version, we would propose the use of the reduced version given the benefits that, as has been justified, a smaller number of sessions entails.n = 152), and centers of the other received the reduced version of the SALUDIVERSEX program (n = 56).The total sample in the present study was 208 participants who attend two support service networks for people with ID. Participants were taken from multiple support centers within each network. To determine eligibility for the research, we established the following inclusion criteria: (1) age 18 years old or over; (2) have an Intellectual Disability as outlined by the DSM-5 criteria; and (3) have enough communication and reading skills to carry out the activities of the study. Centers of one of the support service networks for people with ID received the extended version of the SALUDIVERSEX program (n = 79) were women and 48% (n = 73) were men, with ages ranging from 19 to 67 years old . The large majority of participants lived in their parents or guardians’ homes , followed by 5.9% (n = 9) living in nursing home/hospital settings for people with ID, the same percentage of participants lived in community facilities with different degrees of supervision, and only 2% (n = 3) of participants lived alone.Among participants in the extended version of the program, 52% (n = 24) were women and 53.14% (n = 32) were men, and their ages ranged between 21 and 64 years old . The majority lived with their parents or guardians , followed by 7.55% (n = 4) living in community facilities with different degrees of supervision, the same percentage of participants who lived alone, and only 1.88% (n = 1) of participants lived in nursing home/hospital settings for people with ID.Among participants in the reduced version of the program, 42.86% .Gender and age were self-reported by the participants, whereas the professionals responsible for their care at the support services were asked about the participants’ residence type.sexual response, or reaction to excitation ; worry, or concerns of people with ID about issues related to sex or interpersonal relationships ; sex practices, or sexual activities that people with ID might engage in ; and condom use, or safe sex practices . The reliability of the factors ranged from 0.64 to 0.72, being similar for men and women.The SEBECOMID-S is a self-administered instrument to assess sexual behavior and concerns of people with mild ID that includes 14 questions. The questions have different response formats depending on the content: a frequency scale ranging from never to always  and dichotomous questions with yes/no answers . This instrument includes four main aspects: α = 0.51) and for the four factors (α ranging between 0.5 and 0.71). It could be understood that these reliability coefficients are not as high as expected due to the limitations involved in the validation of measurement instruments in this population. The poor metacognition and communication skills, the problems of reasoning, planning and abstract thinking, and the high social desirability of this group, lead us to consider lower reliability coefficients acceptable  for people with ID. The scale consists of 19 items, which are grouped into four factors: (1) the denial of the risk of SA by people in the victim's immediate environment , (2) the perception of invulnerability to SA associated with places , (3) the presence of risk indicators associated with drug use or lack of parental supervision and mastery of coping skills , and (4) the person's knowledge about what constitutes a potential threat to personal space . All items are dichotomous  and include an illustration exemplifying the content of the question, in order to help people with ID understand the content of the item and provide reliable responses. Reliability analysis of the DSARss found acceptable internal consistency for the total scale (α = 0.76) and acceptable for the six factors (α ranging between 0.54 and 0.67). Furthermore, this instrument proved to be reliable in assessing sexual knowledge in men and women.The ISK-ID is a scale designed to be applied as a self-report measure and provides a measure of sexual knowledge across six different sexuality domains: (a) knowledge about what kind of sexual activities may be considered sexual or not depending on the context ; (b) knowledge about how to have a positive body image and communicate sexually ; (c) knowledge about the nature of different sexual practices, such as masturbation, oral sex, or vaginal and anal intercourse ; (d) knowledge about sexual diversity ; (e) knowledge about how to interact with a romantic/sexual partner in the context of an intimate relationship ; and (f) knowledge about how to prevent STIs and unwanted pregnancy . It is composed of 34-item that are written in an easy-to-read format and rated on a dichotomous scale (Yes/No), thus helping individuals with ID to understand the item content and provide reliable responses. It takes about 20 min to complete. With respect to its psychometric properties, reliability analysis of the ISK-ID found good internal reliability for the total scale , perception of user’s knowledge about privacy and social norms , perception of user's knowledge about sexuality , and concerns about the user's sexuality . The items have a dichotomous “Yes” or “No” response format. The reliability of the factors ranged from 0.59 to 0.74, being equivalent in both genders.The head of the research project (M. D. Gil-Llario) was contacted by two support service networks for people with ID given the detected needs and their interest in their users receiving an appropriate affective-sexual education adapted to their characteristics. Once the demand was confirmed, our research team got involved in the study of the needs of this group in this area of development. This project allowed us to lay the foundations for the development of an intervention program. The principal investigator held several meetings with the heads of the main support service networks to present the objectives of the project and explain how the intervention would be tested.Once both institutions agreed to participate, 22 occupational centers (clusters) within the two institutions selected to participate in this research. To ensure sample representativeness, we followed a stratified random sampling procedure based on population density to select these centers Lohr, . We prioThe users of the 17 selected occupational centers belonging to the first support service network for people with ID that contacted us became the experimental group, while the users of the 5 occupational centers belonging to the support service network that contacted us second became the wait-list group. Thus, initially, the first 17 occupational centers received the extended version of the SALUDIVERSEX program. However, at the end of this implementation, some changes were made to this version of the program with the aim of improving it and adapting it to the real context of people with ID, including changes suggested by professionals and participants. These changes were very positively received when we resumed the research after the Covid-19 pandemic since, the fact of reducing the number of sessions, facilitated the implementation of the program in post-pandemic. The contents on which, according to feedback from professionals and users and the results of the analysis of the effectiveness of the extended version of the program, were not necessary to go into such depth were eliminated, and theoretical explanations and the use of reading and writing-laden activities were reduced to a minimum. This reduced version of the SALUDIVERSEX program was implemented in the 6 centers that until then were part of the wait-list group.However, regardless of whether they received the extended or reduced version of the program, all participants were assessed twice: before the implementation of the intervention (pre-test) and two weeks after finishing the program (post-test). As for the assessment procedure, two members of the research team with extensive experience in the assessment and treatment of people with ID carried out the data collection. Each participant was assessed individually in a private and quiet room of the support service. Only the participant and one of the evaluators were present while completing the assessment and evaluators kept their distance from participants to respect their privacy and create a comfortable situation. Participants were given a brief explanation about how to complete the assessment tools and, if needed, support was provided while they were filling them in . The two professionals from each center also received the necessary copies of the questionnaire to be filled in about each user, and were given a couple of weeks to complete them (adjusting to their workload), during which time they could contact the principal investigator if they had any doubts.We consulted participants’ educational supervisors in order to assess their reading skills. Participants were excluded from the research only if they did not have high enough comprehension and communication skills to carry out the assessment procedure with the researcher’s assistance. Participants were also excluded from the study if their guardians did not sign the informed consent.The implementation of the program was carried out under the same conditions regardless of the version of the program. It was applied by the caretakers of the support services in groups of no more than 10 people. Before the implementation of the program, these caretakers received extensive instruction on how to apply the program and were provided all required materials. The sessions were held in large spaces, set up with tables and chairs in order to provide a work area for participants, as well as another open area where role-playing could take place. Privacy was also ensured so that participants could express themselves freely without fear of being overheard by people outside the group. Prior to the implementation of the program, all participants agreed to maintain the privacy of what was said by their peers during the sessions. Sessions occurred once a week. Each session began with a set of “pre-questions” which focused on participants’ previous knowledge. These “pre-questions” were followed by the development section, which included activities and explanations. Special attention was paid to the adaptation of the contents according to the autonomy and abilities of each participant. Finally, at the end of each session a summary sheet was presented with the main takeaways from the session. This summary sheet was shown again at the beginning of the next session and periodically throughout the program in order to help participants consolidate what they had learned.All participants filled out an informed consent form and were informed about the confidentiality of their answers and the purpose for which the data were to be used. The study complied with the ethical principles of the Declaration of Helsinki and was approved by the Experimental Research Ethics Committee of the University of Valencia (Spain).t tests for numerical variables after examining normality assumptions.We obtained descriptive statistics of the sample, i.e., means and SDs for numerical variables and percentages for categorical variables by groups at pre-test and at post-test. Inferential statistics of the outcomes were calculated comparing both groups using chi-squares for categorical variables and independent sample d  of the raw mean differences were obtained, using the standardized mean difference,  Hedges, , experim Hedges, . FollowiMultilevel models were conducted to evaluate the efficacy of the intervention controlling for the dependence of the participants who belong to the same occupational center, i.e., individuals nested within the same cluster. One level of random effects, occupational center-random effects was included in the model to account for the dependence within the occupational centers. The intervention effect at post-test was defined as a difference in changes from baseline between the intervention and control group. Goodness of fit of the models was compared using Akaike's Information Criterion (AIC), Schwarz's Bayesian Criterion (BIC) and -2 Restricted Log Likelihood. Mixed-effects regression models were ran using MIXED macro from SPSS v.24 . However, with respect to their concerns (“worry”), although this increase is also greater in the group that participated in the reduced version, these differences were not significant .Regardless of the version of the program in which they participated, users generally obtained higher mean scores after participating in the program (post-test) on all sexual behaviors and concerns assessed, and effect sizes comparing both versions were in favor of the reduced program Table . We obselack of awareness of intimacy rules. The comparison between both versions of the program in the multilevel model controlling for pre-test and cluster dependence . There was a significant decrease in the total score of risk of experiencing SA among the participants of the reduced compare to the long version . In the case of the lack of awareness of intimacy rules, although the difference in improvement between the two versions is not significant, it is very close to being so, in favor of the reduced version .As we can see in Table ce Table  shows a body image and sexual communication, whose effect was medium and significant in favor of the extended version   decreased for the group receiving the extended version and went up for those receiving the reduced version. Likewise, professionals stated that participants in both versions increased their knowledge about privacy and social norms, although in the extended version it was minimal, as well as their knowledge about sexuality after participating in the program. The ESs comparing extended vs. reduced version of the program showed a large and significant effect for the domains related to the professionals' perception of user’s knowledge about privacy and social norms  and about the interpretation of their sexuality and their risk of possessing misconceptions or experiencing loneliness and/or sexual abuse . The ESs showed moderate and significant effect for the professionals' perception of user’s knowledge about sexuality  , with the exception of the user's knowledge about sexuality  , the reduced version obtains a greater range of improvement, although in the case of concerns about aspects related to sexuality or interpersonal relationships this difference between the two versions is not significant. In other words, with 10 sessions, users are able to obtain the self-knowledge necessary to know their sexual response, improve their sexual practices and acquire basic skills of self-protection by performing safe sex. This could be due to the fact that in the reduced version of the program these aspects were substantially changed. The information provided regarding some partner sexual practices  was reduced and more information was provided on autoerotic practices, which are more frequent in this group. In addition, the sexual response was explained on the basis of masturbation, which may have helped to understand it better. Theoretical content related to some less common STIs  and some contraceptive methods with low frequency of use in this group  was also reduced, devoting more attention to the use of condoms. Also, the main concepts were always accompanied by visual material, and the activities were reformulated to emphasize self-pleasure and the use of condoms. This remodeling, eliminating content that does not allow to focus on what is important, seems to facilitates access to knowledge for this group, which, as several studies show, needs to address these areas. This can be seen in the study of Jahoda and Pownall  who founRegarding the risk of sexual abuse, the two versions of the program, both the reduced and extended versions, improve the protection of users, with lower post-test scores on all factors. That is, after the intervention, the acceptance of abuse due to affection decreases , the denial of risk associated with places decreases  and the awareness of privacy norms improves . Improving these aspects is of vital importance, given the high prevalence of sexual abuse in this group, mostly perpetrated by acquaintances  (Mitra et al., There are no significant differences between the two versions in the knowledge of sexuality. This is also an endorsement that reducing the number of sessions has not resulted in a decrease in the amount of relevant knowledge conveyed through the program. Work has continued to the point that course participants have assimilated the most relevant basic aspects, such as the concept of sexuality, communication as a fundamental tool for sexual relations, how and where to perform basic sexual practices safely, the concept of intimacy and assertiveness, having also acquired healthy attitudes of tolerance toward non-majority sexual orientation options, that is, prevention of homophobia mainly. In fact, in the reduced version there have been more notable improvements in all the scales, although, as indicated, these improvements do not reach the level of statistical significance. Such improvements are not shown, as is evident, only in those contents that were suppressed for the reasons given in the introduction, such as body image.In summary, and observing all the data as a whole, we can see that both the extended and the reduced version obtain higher average scores in the post-test of all the factors, which confirms the effectiveness of both versions.Finally, as regards the professional's perspective as a measure of validity of the program's effectiveness, previous research has shown that there is some congruence between the perception of professionals and that of users in various aspects related to sexuality and the quality of life of the latter (Caballero-Gascón et al., Our findings show that both interventions have been shown to be effective. However, the reduced version of the program is more effective and efficient, given the better results obtained in most of the indicators. Improvements in sexual behavior, users' level of knowledge, as well as in the prevention of sexual abuse are noteworthy. To the extent that it is difficult for the managers of occupational centers to integrate extra activities with the permanent ones, this reduced version, offering the same or even better results in a shorter intervention time, seems the most advisable option.