Gene

The orphan receptor DAX-1 plays an essential role in human development, steroid hormone synthesis and the maintenance of embryonic stem cell pluripotency. Recent studies have demonstrated DAX-1 is involved in cancer development, and, depending on the specific cancer type, has a negative or positive effect on cancer growth. In order to better understand the mechanism of DAX-1 gene regulation in various cancer cell lines, the epigenetic regulation of DAX-1 was investigated. Following confirming levels of DAX-1 expression at both the mRNA and protein levels, the overall methylation status of the DAX-1 gene was probed using methylation-sensitive restriction enzyme analysis. To determine the molecular mechanism of DNA methylation of the DAX-1 gene, chromatin immunoprecipitation assays identified key methylating proteins that localize to specific CpG islands in the DAX-1 promoter. In conclusion, this study demonstrates that methylation of key cytosine residues in CpG islands within the DAX-1 promoter play a central role in regulating DAX-1 expression and varying degrees of methylation result in differences in DAX-1 expression in human cancer cell lines.

To investigate alternative splicing capacity, we statistically compared the properties of human protein-coding genes with multiple transcript isoforms (MISOG) and single transcript isoforms (SISOG). Apart from global exon content, differential features are concentrated in the 5 gene regions, with MISOG presenting complex 5 untranslated region architecture and a distinctive flanking environment around first 5 intron. Importantly, we found that 5 exons are more prone to alternative splicing in MISOG. These results unravel previous observations indicating the importance of 5 gene regions in some transcriptional processes and call for their reassessment in light of the MISOG/SISOG profiles.

Methylation at the N6-position of adenosine, m6A, is the most abundant mRNA modification in eukaryotes. It is a highly conserved universal regulatory mechanism controlling gene expression in a myriad of biological processes. The role of m6A methylation in sexual maturation, however, has remained largely unexplored. While the maturation process is known to be affected by many genetic and environmental factors, the molecular mechanisms causing variation in the timing of maturation are still poorly understood. Hence, investigation of whether a widespread mechanism like m6A methylation could be involved in controlling of the maturation timing is warranted. In Atlantic salmon (Salmo salar), two genes associated with the age at maturity in human, vgll3 and six6, have been shown to play an important role in maturation timing. In this study, we investigated the expression of 16 genes involved in the regulation of m6A RNA methylation in the hypothalamus of Atlantic salmon with different homozygous combinations of late (L) and early (E) alleles for vgll3 and six6 genes. We found differential expression of ythdf2.2 which encodes an m6A modification reader and promotes mRNA degradation. Its expression was higher in six6*LL compared to other genotypes as well as immature males compared to matures. In addition, we found that the expression levels of genes coding for an eraser, alkbh5, and for a reader, ythdf1, were higher in the hypothalamus of females than in males across all the different genotypes studied. Our results indicate a potential role of the m6A methylation process in sexual maturation of Atlantic salmon, and therefore, provide the first evidence for such regulatory mechanism in the hypothalamus of any vertebrate. Investigation of additional vertebrate species is warranted in order to determine the generality of these findings.

LncRNA Mrhl has been shown to be involved in regulating meiotic commitment of mouse spermatogonial progenitors and coordinating differentiation events in mouse embryonic stem cells. Here we have characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is predominantly expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid derived radial-glia like neuronal progenitor cells. Mrhl levels are significantly down regulated in postnatal brains and in maturing neurons. In neuronal progenitors, a master transcription factor, PAX6, acts to regulate the expression of Mrhl through direct physical binding at a major site in the distal promoter, located at 2.9kb usptream of the TSS of Mrhl. Furthermore, NFAT4 occupies the Mrhl proximal promoter at two sites, at 437bp and 143bp upstream of the TSS. ChIP studies reveal that PAX6 and NFAT4 interact with each other, suggesting co-regulation of lncRNA Mrhl expression in neuronal progenitors. Our studies herewith are crucial towards understanding how lncRNAs are regulated by major lineage-specific TFs towards defining specific development and differentiation events. Summary statementTranscriptional regulation of lncRNA Mrhl by multiple lineage-specific transcription factors in neuronal progenitors highlights context-dependent regulation important for lineage specification.

Breast cancer cells enter into the cell cycle following progestin exposure by the activation of signalling cascades involving a plethora of enzymes, transcription factors and co-factors that transmit the external signal from the cell membrane to chromatin, ultimately leading to a change of the gene expression program. Although many of the events within the signalling network have been described in isolation, how they globally team up to generate the final cell response is unclear. In this study we use antibody microarrays and phosphoproteomics to provide a dynamic global signalling map that reveals new key regulated proteins and links between previously known and novel pathways. Detailed analysis of the data revealed intriguing changes in protein complexes involved in nuclear structure, EMT, cell adhesion, as well as transcription factors previously not associated with breast cancer proliferation. As different post-translational modifications can mediate complex crosstalk mechanisms and massive PARylation is also rapidly induced by progestins, we provide details of important chromatin regulatory complexes containing both phosphorylated and PARylated proteins. This study contributes an important resource for the scientific community, as it identifies novel players and connections meaningful for breast cancer cell biology and potentially relevant for cancer management. (197 w)

PURPOSEMTHFR is a pivotal enzyme in the folic acid cycle. In specific populations, two functional SNPs in the encoding gene, i.e. rs1801133 (677C/T) and rs1801131 (1298A/C), have shown associations with different diseases including cancers. In the present study, the role of these SNPs was analyzed in breast cancer cases from Pakistani population. PARTICIPANTS AND METHODSThe pilot study includes 187 participants with 124 breast cancer patients and 63 matched individuals as controls. PCR-RFLP methods validated by Sanger sequencing were used for the polymorphic investigations. Hardy-Weinberg Equilibrium was tested by chi-squared goodness-of-fit test. Independent and combined associations were evaluated by Cochran-Armitage trend-test. Semi-parameteric haplotype analysis was carried out and odds ratios with 95% confidence interval were tabulated. Linkage disequilibrium between two loci was calculated. p-values <0.05 were significant. RESULTSBoth MTHFR 677C/T and MTHFR 1298A/C SNPs were in Hardy-Weinberg Disequilibium. Cochran-Armitage trend test showed lack of independent associations of these SNPs with the risk for breast cancers. The diplotype analysis revealed that 677CC+1298AC increased the risk of breast cancers significantly [OR: 2.553 (95% CI: 1.177-5.541)], while 677CT+1298AA had a protective effect [OR: 0.537 (95% CI: 0.404-0.713)]. Haplotype analysis did not show any significant association. Interestingly, despite the proximity, these loci were not linked (r2 = 0.042 and 0.046 in cases and controls, respectively). CONCLUSIONHere, we report association analysis of two putative candidate SNP markers in the MTHFR gene with breast cancers in a South-Asian population. To the best of our knowledge, two diplotype combinations show unique associations with breast cancer susceptibility in this population, which have not been reported earlier. The study implies translational potentials of these polymorphisms for breast cancer management.

Transcription elongation factor A (TCEA) is a eukaryotic transcriptional molecule, required for a formation of initiation and elongation of gene transcription-mediated RNA polymerase II (RNAPII) complex, to promote transcription-coupled nucleotide excision repair (TC-NER) after RNAPII backtracking recovery. TCEA shares three isoforms in which TCEA1 is ubiquitously expressed among all eukaryotic cells. We found a spermatogenesis TCEA1 and TCEA2 expression profile has a unique transcriptional programme, compared with embryogenesis. Moreover, the testis-specific TCEA2 profile correlates with gene transcription, whereas TCEA1 specifically correlates with genes transcribed for Nuclear excision repair (NER) during human spermatogenesis. We also found that the expression activation of RNF20, a TCEA1 inhibitor, leads to expressional TCEA1 reduction, but having no direct impact on TCEA2 expression, implying the potential RNF20-dependent transcriptional switching of TCEA2 in transcriptional regulation during spermatogenesis. Our analysis defined a transcriptional bursting event where transcription-coupled repair (both Base excision repair and Nuclear excision repair) is a major pathway highly expressed in early spermatogenesis, supporting the transcriptional scanning hypothesis of which mutation of transcribed genes is effectively repaired as proposed by Xia B., et al. (2020).

The Adenylate-Uridylate Rich elements (AREs) are adenine and uracil abundant sequences, established in ephemeral mRNAs, principally present in the 3 untranslated region (3 UTR). AREs are widely accepted as a cause of high turnover of the mRNAs containing them (Bakheet, 2001; Barreau, 2005; Shaw and Kamen, 1986). The mammalian ARE-mRNAs primarily translate into nuclear transcription factors, oncoproteins, cytokines, and G-protein-coupled receptors which indicates their indispensable role in the regulation of gene transcription during cell growth and differentiation, and the immune response (Chen and Shyu, 1995; Wilson et al., 1999). The present study is an attempt to comprehensively analyze the eloquent presence of AU rich elements in genome, transcriptome and 3UTR of three important plant species (Arabidopsis thaliana, Oryza sativa and Zea mays) and compare the statistics of putative ARE motifs in plants with H.sapiens. Statistical analysis of genome-wide putative AU-rich elements revealed the explicit presence of ARE motifs in plants. This is the first study that analyses the presence of Adenylate-Uridylate Rich elements (AREs) in three different plants which can be further validated through experiments.

BackgroundDUXC is a multi-copy transcription factor gene found within a long tandem repeat locus in several Laurasiatherians. It is suggested to be functionally similar to human DUX4 because of its shared C-terminal domain and its close phylogenetic relationship to DUX4. DUX family genes are transiently expressed in preimplantation embryos of placental mammals. However, early embryo-derived cDNA proof for DUXC, which is needed for its further functional characterization, has not been reported so far. ResultsOur study provides a full-length sequence of DUXC mRNA, derived from the 8-cell stage in vitro fertilization (IVF) bovine embryos, containing double homeobox and 9aa transactivation domain (9aaTAD)-encoding sequences. Identified DUXC sequence uncovered a first exon that was not previously annotated. We showed that DUXC mRNA levels are independent of the embryonic transcription at the 2-, 4-, and 8-cell stage, whereas its decline, observed from the 8-cell stage onwards, is minor embryonic genome activation (EGA)-dependent. We also investigated the genomic organisation of the DUXC array in eight different cattle breed assemblies, revealing polymorphic internal repeats flanked by an incomplete distal unit at the telomeric end and a much shorter unit at the proximal end of the DUXC array. Despite the presence of a putative polyadenylation signal downstream the distal unit, we presented evidence for the expression of internal but not distal DUXC in early bovine IVF embryos. ConclusionsDUXC is a potential bovine EGA inducer, supported by its expression at peak levels at pre-EGA stages followed by a decrease with a dependency on minor EGA.

Biological functions of micro RNAs (miRNAs) in the early stages of vertebrate development remain largely unknown. In zebrafish, miRNA miR-92a-3p is abundant in the germ cells throughout gonadal development, as well as in ovulated oocytes. Previously, we demonstrated that inhibition of miR-92a-3p in mature ovaries resulted in developmental arrest at the 1-cell stage upon fertilization of the affected oocytes. This suggested functions of miR-92a-3p in early development. In the present study, we identified wee2, an oocyte-specific protein tyrosine kinase, as a target of maternal miR-92a-3p during the early stages of zebrafish embryogenesis. Spatiotemporal co-presence of both miR-92a-3p and wee2 during early embryo development was confirmed by absolute quantification and in situ hybridization. Targeted knockdown of miR-92a-3p in embryos resulted in retarded embryonic development over the first 24 hours. Target validation assays demonstrated that miR-92a-3p interacted with the predicted wee2 3UTR binding site, which was strongly suppressed by endogenous miR-92a-3p. Our results suggest that miR-92a-3p regulates the abundance of wee2, a cyclin-dependent kinase 1 inhibitor, thus having important role in regulation of the cell cycle during cleavage stages in zebrafish.\n\nSummary statementIn zebrafish, maternal miR-92a-3p was demonstrated to suppress translation of wee2, a cyclin-dependent kinase 1 inhibitor which regulates cell cycle progression during the early stages of embryogenesis.

Heterochromatin protein 1 (HP1) is widespread in several organisms playing a role in control of gene expression by heterochromatin formation and maintenance of silent chromatin. Schistosoma mansoni is a human parasite that is responsible for Schistosomiasis, a tropical neglected disease in the tropical and subtropical areas in the world, where the intermediate host Biomphalaria glabrata is present. In this study we attempted to investigate if the SmHP1 is enriched in S. mansoni chromatin in cercariae larvae stage, compared with another larvae stage sporocysts and its importance for S. mansoni life cycle progression and parasite fitness. We used ChIPmentation with commercial antibody ab109028 that passed in-house quality control. Our data show that S. mansoni HP1 enrichment is non-canonical with a peak at the transcription end sites of protein coding genes. We did not find strong differences in SmHP1 chromatin landscapes between sporocysts and cercariae. Knock-down of SmHP1 in schistosomula and in vivo experiments in mice unexpectedly increased parasite fitness. Our results suggest that SmHP1 may influence chromatin structure in a non-canonical way in S. mansoni stages and may play a role in regulation of parasite fitness.

Approximately half of the human genome is comprised of transposable elements (TEs), which are genetic elements capable of amplifying themselves within the genome. Throughout the course of human life, TEs are expressed in germ cells, the preimplantation embryo, and the placenta but silenced elsewhere. However, the functions of TEs during embryonic development are poorly understood. Trophoblast stem (TS), embryonic stem (ES), and extraembryonic endoderm stem (XEN) cells are cell lineages derived from the preimplantation embryo and known to have different TE silencing mechanisms. Thus, it is likely distinct TEs are expressed in each lineage and that proteins coded by these TEs have lineage-specific functions. The purpose of this research was to determine which TEs are expressed in each of these stem cell lineages and to compare expression levels between lineages. Each lineages transcriptome was analyzed by quantifying TE expression in RNA-sequencing data from mouse stem cells. Expression data were then used for differential expression analyses performed between the cell types. It was found that certain families of TEs are distinctly expressed in certain lineages, suggesting expression of these families may be involved in the differentiation and development of each lineage, the understanding of which can lead to improved stem cell therapies and capacity to study human embryonic development.

Epigenetic regulation plays a central role in the developmental control and antigenic variation of Plasmodium falciparum, yet the functions of many chromatin-modifying enzymes remain poorly understood. Here, we investigated the role of the putative Jumonji C (JmjC) domain-containing enzyme PfJmjC2 (PF3D7_0602800) in maintaining chromatin organization and histone modification balance during the asexual blood stages of the parasite. Using a conditional glmS ribozyme-mediated knockdown system, we achieved a [~]70% reduction in PfJmjC2 transcript and protein levels, which resulted in a marked delay in DNA replication and intraerythrocytic development. Immunoprecipitation assays revealed that PfJmjC2 physically interacts with histone H2A, suggesting an association with the nucleosome core. Upon PfJmjC2 depletion, the balance of epigenetic marks was disrupted, indicating a shift toward a more compact and transcriptionally repressed chromatin state. MNase-ChIP-PCR analysis further showed that PfJmjC2 associates with nucleosome-dense regions, including var gene promoters, although its reduction did not alter var gene transcription or switching patterns. Together, these findings indicate that PfJmjC2 contributes to chromatin organization and nucleosome stability rather than direct transcriptional control, highlighting its potential role in maintaining chromatin compaction and epigenetic homeostasis in P. falciparum.

The Ko-Osnac02 mutant matured earlier than the wild type (WT) in 15 days obviously. The result showed that the Ko-Osnac02 chalky with white-core and white-belly mature endosperm. The seed filling rate plays a major role in determining the yield and quality of rice (Oryza sativa L. versus japonica). Interestingly, in the Ko-Osnac02 seeds, higher amylose content (AC) was observed in vegetative endosperm (5DAP), while lower amylose content (AC) and higher soluble sugar content were observed in the mature endosperm. RNA-Seq analysis of N2/N3 seed (3DAPs) and the WT revealed that among the top differentially expressed genes (DEGs), the OsBAM2 (LOC_Os10g32810) expressed significantly high which involved in starch degradation. In addition, seven genes were expressed at a lower-level in pro-pro interactions inducing the chalky endosperm formation in N2 mutant seeds (3 DAP), which could be verified by heatmap diagrams based on DEGs of N2 versus WT. The cell cycler controlling the Tubulin genes downregulated their expression together with the MCM family genes MCM4 ( {downarrow} ), MCM7 ( {uparrow} ), which may cause white-core in the early endosperm development. In conclusion, the developing period shorten significantly in the Ko-Osnac02 mutants may cause the chalkiness in seeds during the early endosperm development. ONE SENTENCE SUMMARYThe gene OsNAC02 which controls a great genetic co-network for cell cycle regulation in early development, and Ko-Osnac02 mutant shows prematurity and white-core in endosperm.

In carcinomas, dissemination of cancer cells via blood or lymph circulation constitutes an early event. E-cadherin is a transmembrane calcium dependent adhesion protein. Cellular de-differentiation and plasticity, underlying metastasis, is attributed to the loss of function of E-cadherin (cdh1) gene. The loss of gene expression may arise from promoter hypermethylation, which has been reported in multiple cancers. In the present pilot project, sixty (60) blood samples were collected from the breast cancer patients at a tertiary care hospital in Karachi, Pakistan. DNA was isolated from the cells circulating in the peripheral blood of the participants. Promoter hypermethylation was investigated through sodium-bisulfite treatment of DNA followed by methylation-specific PCR. In 53.3% of the patients, E-cadherin gene promoter hypermethylation was observed. Promoter hypermethylation of E-cadherin has been reported in DNA isolated from the tissue specimen. However, to the best of our knowledge this is the first report of E-cadherin promoter hypermethylation in cells isolated from the peripheral blood of breast cancer patients from a geographically specific population. The results have important implications in tumour staging and selection of treatment regimens.

The last decade has witnessed great progresses regarding the molecular basis of breast cancer with discovery of different nuclear susceptibility genes; in addition investigations and researches regarding mitochondrial DNA (mtDNA) mutations in breast cancer have been started. Mitochondrial haplogroup determinants (single nucleotide polymorphism SNP) and somatic mitochondrial mutations have recently been studied as possible risk factors for carcinogenic processes in different tissues, hence in order to identify breast cancer related SNPs and haplogroups among the population of Sulaimaniyah city/Iraq, the entire mitochondrial genome of 20-breast cancer samples and comparable controls were sequenced. Haplogrep 2.0 was used for haplogroup identification; Chi-square and Fishers exact test were applied to assess relational significance. HV haplogroup in the cancer samples appeared to be a risk factor for breast cancer compared to the most common H haplogroup in control samples with a p-values of 0.002 and 0.006 respectively and an Odd Ratio (OR) = 28.00. Besides, SNP (A8860G) was also identified as a risk factor for breast cancer as compared to other randomly selected SNPs (A750G, A1438G and C7028T) with p values {square}0.05 and OR >1.

As an oocyte-specific growth factor, bone morphogenetic protein 15 (BMP15) plays a critical role in controlling folliculogenesis. However, the mechanism of BMP15 action remains elusive. Using zebrafish as the model, we created a bmp15 mutant using CRISPR/Cas9 and demonstrated that bmp15 deficiency caused a significant delay in follicle activation and puberty onset followed by complete arrest of follicle development at previtellogenic stage without yolk accumulation. The mutant females eventually underwent female-to-male sex reversal to become functional males, which was accompanied by a series of changes in secondary sexual characteristics. Interestingly, the blockade of folliculogenesis and sex reversal in bmp15 mutant could be rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic stage with yolk accumulation and the fish maintained their femaleness without sex reversal. Transcriptome analysis revealed up-regulation of pathways related to TGF-{beta} signaling and endocytosis in the double mutant follicles. Intriguingly, the expression of inhibin/activin {beta}Aa subunit (inhbaa) increased significantly in the double mutant ovary. Further knockout of inhbaa in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-) resulted in the loss of yolk granules again in the oocytes although the follicles could continue to grow beyond the size range of previtellogenic stage. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly in bmp15 single mutant females, returned to normal in the double mutant (bmp15-/-;inha-/-), but reduced again significantly in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-). E2 treatment could rescue the vitellogenic follicles in bmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation in bmp15-/-;inha-/- fish. In summary, the present study provided comprehensive genetic evidence for the interaction of bmp15 pathways and the activin-inhibin system in regulating folliculogenesis, in particular E2 production from the follicle, Vtg biosynthesis in the liver and its update by the developing oocytes.

Trans-Himalayan Ladakh has witnessed complex cultural movements and demographic changes since the Neolithic period, which is still continue despite the harsh, inhospitable and cold climate. Although geographically isolated from mainland South Asia, Ladakh has historic trade routes and is well connected and accessible to travelers from Tibet and Central Asia. Despite its rich cultural heritage, a detailed description of the genetic landscape of the Ladakh region is completely lacking, particularly with regard to genome-wide analysis and larger sample sizes. Therefore, in the current study, we genotyped 80 individuals from Kargil and Leh districts of the Union Territory of Ladakh, India. Here, we performed a comprehensive genetic analysis based on allele frequency and haplotype sharing. Our analysis revealed the presence of two distinct genetic lineages in the region with quite distinct genetic composition. The population of Leh Region is more similar to East Asian and Southeast Asian populations. In contrast, the population of the Kargil Region (LDKLA) is more similar to Indo-European populations. Demographic modeling suggests that the Leh group shares a genetic history with Tibetans, while the Kargil group showed great affinities with Kashmiri Muslims, Gujjars and Nepalese Brahmins, and both showed recent admixture. Both groups have experienced a founder event around during 11th to 22nd generations ago, the duration of which coincided with the Mughal invasion. The genome-wide scan for a signal of positive selection revealed genetic signatures of high-altitude adaptation (EPAS1 and ELMO2) in the Leh population, while in the Kargil population the key gene signatures were associated with immunity and female fertility.

The molecular mechanism of temperature-dependent sex determination (TSD) in reptiles has been drawn great interest from biologists for several decades. However, which genetic factors are essential for TSD remain elusive, especially for the female sex determination process. Cyp19a1, encodes an enzyme of aromatase catalyzing the conversion of testosterone to estrogen, has been confirmed to modulate steroid hormones involved in the sexual differentiation of many species, but whether it has a critical role in determining the gonadal sexual fate in TSD is still to be elucidated. Here, we identified that Cyp19a1 expression exhibited a temperature-dependent, sexually dimorphic expression pattern, preceding gonadal sex differentiation in a TSD turtle Mauremys reevesii. Cyp19a1 expression in gonads increased dramatically when embryos developed at high female-producing temperatures (FPT), but were extremely low throughout embryogenesis at low male-producing temperatures (MPT). Cyp19a1 expression increased rapidly in response to the temperature shift from MPT to FPT in developing gonads. The sexual phenotype of turtles was successfully reversed by aromatase inhibitor treatment at FPT, and by estrogen treatment at MPT, accompanied with the rapid upregulation of Cyp19a1. These results demonstrate that Cyp19a1 is essential for the female sex determination process in M. reevesii, indicating its vital role in the female pathway of TSD.

Ovulation is a fundamental prerequisite for achieving successful reproduction. In vertebrates, ovulation is controlled by the cyclical action of hormones, particularly the gonadotropins such as follicle stimulating hormone (FSH) and luteinizing hormone (LH). A critical component of the intracellular activity of these two hormones is relayed by the second messenger cAMP. Although it is well established that a family of transcription factors facilitate cAMP mediated gene expression, it remains unknown how these factors directly affect ovulation. In particular, the Inducible cAMP Early Repressor (ICER) has been implicated in the transcriptional repression of FSH inducible genes during folliculogenesis. Here we show, using an ovarian-specific transgenic mouse model that ICER potentiates ovulation. We observed a twofold rate increase in ovulation for transgenic mice when compared to the wild type in response to exogenous gonadotropin treatment. Furthermore, mature cycling transgenic mice display a significantly enhanced ovulation rate compared to the wild-type. The observed changes in ovulation in the transgenic females are accompanied by altered gonadotropins production and gene expression. Most significantly, the expression of inhibin alpha subunit (INHA) was found to be about 5-fold higher in the transgenic mice. These observations may aid in unraveling some of the molecular mechanisms underlying ovulation and be relevant to the development of novel reproductive technologies. Summary SentenceGeneration of an FSH inducible ovarian specific FLAG-ICER-II(C) Tg Mice results in hyper-ovulation upon gonadotropin stimulation.