Corrigendum: A review on deep learning applications in highly multiplexed tissue imaging data analysis.

[This corrects the article DOI: 10.3389/fbinf.2023.1159381.].

S3-CIMA: Supervised spatial single-cell image analysis for identifying disease-associated cell-type compositions in tissue.

The spatial organization of various cell types within the tissue microenvironment is a key element for the formation of physiological and pathological processes, including cancer and autoimmune diseases. Here, we present S3-CIMA, a weakly supervised convolutional neural network model that enables the detection of disease-specific microenvironment compositions from high-dimensional proteomic imaging data. We demonstrate the utility of this approach by determining cancer outcome- and cellular-signaling-specific spatial cell-state compositions in highly multiplexed fluorescence microscopy data of the tumor microenvironment in colorectal cancer. Moreover, we use S3-CIMA to identify disease-onset-specific changes of the pancreatic tissue microenvironment in type 1 diabetes using imaging mass-cytometry data. We evaluated S3-CIMA as a powerful tool to discover novel disease-associated spatial cellular interactions from currently available and future spatial biology datasets.

A review on deep learning applications in highly multiplexed tissue imaging data analysis.

Since its introduction into the field of oncology, deep learning (DL) has impacted clinical discoveries and biomarker predictions. DL-driven discoveries and predictions in oncology are based on a variety of biological data such as genomics, proteomics, and imaging data. DL-based computational frameworks can predict genetic variant effects on gene expression, as well as protein structures based on amino acid sequences. Furthermore, DL algorithms can capture valuable mechanistic biological information from several spatial "omics" technologies, such as spatial transcriptomics and spatial proteomics. Here, we review the impact that the combination of artificial intelligence (AI) with spatial omics technologies has had on oncology, focusing on DL and its applications in biomedical image analysis, encompassing cell segmentation, cell phenotype identification, cancer prognostication, and therapy prediction. We highlight the advantages of using highly multiplexed images (spatial proteomics data) compared to single-stained, conventional histopathological ("simple") images, as the former can provide deep mechanistic insights that cannot be obtained by the latter, even with the aid of explainable AI. Furthermore, we provide the reader with the advantages/disadvantages of DL-based pipelines used in preprocessing highly multiplexed images (cell segmentation, cell type annotation). Therefore, this review also guides the reader to choose the DL-based pipeline that best fits their data. In conclusion, DL continues to be established as an essential tool in discovering novel biological mechanisms when combined with technologies such as highly multiplexed tissue imaging data. In balance with conventional medical data, its role in clinical routine will become more important, supporting diagnosis and prognosis in oncology, enhancing clinical decision-making, and improving the quality of care for patients. Since its introduction into the field of oncology, deep learning (DL) has impacted clinical discoveries and biomarker predictions. DL-driven discoveries and predictions in oncology are based on a variety of biological data such as genomics, proteomics, and imaging data. DL-based computational frameworks can predict genetic variant effects on gene expression, as well as protein structures based on amino acid sequences. Furthermore, DL algorithms can capture valuable mechanistic biological information from several spatial "omics" technologies, such as spatial transcriptomics and spatial proteomics. Here, we review the impact that the combination of artificial intelligence (AI) with spatial omics technologies has had on oncology, focusing on DL and its applications in biomedical image analysis, encompassing cell segmentation, cell phenotype identification, cancer prognostication, and therapy prediction. We highlight the advantages of using highly multiplexed images (spatial proteomics data) compared to single-stained, conventional histopathological ("simple") images, as the former can provide deep mechanistic insights that cannot be obtained by the latter, even with the aid of explainable AI. Furthermore, we provide the reader with the advantages/disadvantages of the DL-based pipelines used in preprocessing the highly multiplexed images (cell segmentation, cell type annotation). Therefore, this review also guides the reader to choose the DL-based pipeline that best fits their data. In conclusion, DL continues to be established as an essential tool in discovering novel biological mechanisms when combined with technologies such as highly multiplexed tissue imaging data. In balance with conventional medical data, its role in clinical routine will become more important, supporting diagnosis and prognosis in oncology, enhancing clinical decision-making, and improving the quality of care for patients.

Expression and prognostic significance of chromatin modulators EHMT2/G9a and KDM2b in acute myeloid leukemia.

Epigenetics factors are critical for normal cell function and their regulation is sensitive to malignancy development. EHMT2/G9a and KDM2b are key epigenetics players in different cancer types. However, their expression profiles and related consequences in acute myeloid leukemia (AML) patients have not been known yet. In addition to routine lab work, expression levels of EHMT2/G9a and KDM2b were determined in 110 adult and pediatric patients with De Novo AML. Relations between their expression and patients' clinical data were tested by statistical methods. EHMT2/G9a and KDM2b were highly expressed in AML patients against control cases and associated with the presence of adverse genomic alterations. In response to induction chemotherapy, EHMT2/G9a and KDM2b showed to be significantly high in resistant and relapsed patients in comparison to the complete remission group. KDM2b overexpression was associated with CD11c (integrin alpha X) downregulation. Kaplan-Meier analysis indicated that EHMT2/G9a and KDM2b overexpression was correlated with poor survival status in AML patients. We conclude that EHMT2/G9a and KDM2b expression levels could be used as independent prognostic factors for AML disease.

NRF2-mediated signaling is a master regulator of transcription factors in bovine granulosa cells under oxidative stress condition.

Transcription factors (TFs) are known to be involved in regulating the expression of several classes of genes during folliculogenesis. However, the regulatory role of TFs during oxidative stress (OS) is not fully understood. The current study was aimed to investigate the regulation of the TFs in bovine granulosa cells (bGCs) during exposure to OS induced by H2O2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H2O2 for 40 min. Twenty-four hours later, cells were subjected to various phenotypic and gene expression analyses for genes related to TFs, endoplasmic reticulum stress, apoptosis, cell proliferation, and differentiation markers. The bGCs exhibited higher reactive oxygen species accumulation, DNA fragmentation, and endoplasmic reticulum stress accompanied by reduction of mitochondrial activity after exposure to OS. In addition, higher lipid accumulation and lower cell proliferation were noticed in H2O2-challenged cells. The mRNA level of TFs including NRF2, E2F1, KLF6, KLF9, FOS, SREBF1, SREBF2, and NOTCH1 was increased in H2O2-treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1, were downregulated in the H2O2-challenged group. Moreover, targeted inhibition of NRF2 using small interference RNA resulted in reduced expression of KLF9, FOS, SREBF2, and NOTCH1 genes, while the expression of KLF4 was upregulated. Taken together, bovine granulosa cells exposed to OS exhibited differential expression of various transcription factors, which are mediated by the NRF2 signaling pathway.

Hyaluronic acid and epidermal growth factor improved the bovine embryo quality by regulating the DNA methylation and expression patterns of the focal adhesion pathway.

Focal adhesion pathway is one of the key molecular pathways affected by suboptimal culture conditions during embryonic development. The epidermal growth factor (EGF) and hyaluronic acid (HA) are believed to be involved in the focal adhesion pathway function by regulating the adherence of the molecules to the extracellular matrix. However, regulatory and molecular mechanisms through which the EGF and HA could influence the embryo development is not clear. Therefore, this study aimed to investigate the effect of continued or stage specific supplementation of EGF and/or HA on the developmental competence and quality of bovine preimplantation embryos and the subsequent consequences on the expression and DNA methylation patterns of genes involved in the focal adhesion pathway. The results revealed that, the supplementation of EGF or HA from zygote to the blastocysts stage reduced the level of reactive oxygen species and increased hatching rate after thawing. On the other hand, HA decreased the apoptotic nuclei and increased blastocyst compared to EGF supplemented group. Gene expression and DNA methylation analysis in the resulting blastocysts indicated that, combined supplementation of EGF and HA increased the expression of genes involved in focal adhesion pathway while supplementation of EGF, HA or a combination of EGF and HA during the entire preimplantation period changed the DNA methylation patterns of genes involved in focal adhesion pathway. On the other hand, blastocysts developed in culture media supplemented with EGF + HA until the 16-cell stage exhibited higher expression level of genes involved in focal adhesion pathway compared to those supplemented after the 16-cell stage. Conversely, the DNA methylation level of candidate genes was increased in the blastocysts obtained from embryos cultured in media supplemented with EGF + HA after 16-cell stage. In conclusion, supplementation of bovine embryos with EGF and/or HA during the entire preimplantation period or in a stage specific manner altered the DNA methylation and expression patterns of candidate genes involved in the focal adhesion pathway which was in turn associated with the observed embryonic developmental competence and quality.

Sexual dimorphic expression and release of transcription factors in bovine embryos exposed to oxidative stress.

Sexually dimorphic differences in genome activity, which is orchestrated by transcription factors (TFs), could explain the differential response of male and female embryos to environmental stressors. To proof this hypothesis, the expression of cellular and extracellular TFs was investigated in male and female bovine embryos in vitro cultured either under low (5%) or high (20%) oxygen levels. The intracellular reactive oxygen species (ROS), total cell number, expression of nuclear factor (erythroid-derived 2) factor 2 (NFE2L2), Krüppel-like factor 4 (KLF4), notch receptor 1 (NOTCH1), E2F transcription factor 1 (E2F1), and SREBF2 along with extracellular vesicles (EVs) biogenesis genes were assessed at the blastocyst stage and their released EVs. Low blastocyst rate in both sexes due to oxidative stress (OS) was accompanied by increased ROS accumulation and reduced cell number in female embryos. The messenger RNA and protein levels of NFE2L2, as well as KLF4 expression, were higher in male embryos exposed to OS compared with female embryos. However, the expression of NOTCH1 and E2F1 was higher in female embryos cultured in high oxygen level. Male embryos exposed to OS released more EVs enriched with NFE2L2, superoxide dismutase 1, and NOTCH1 accompanied by elevated expression of EVs biogenesis genes. Accordingly, differential expression of TFs and their release into spent media could partially explain the sexual dimorphic response of bovine embryos to environmental stresses.

Extracellular vesicle-coupled miRNA profiles in follicular fluid of cows with divergent post-calving metabolic status.

Most high-yielding dairy cows enter a state of negative energy balance (NEB) during early lactation. This, in turn, results in changes in the level of various metabolites in the blood and follicular fluid microenvironment which contributes to disturbed fertility. Extracellular vesicles (EVs) are evolutionarily conserved communicasomes that transport cargo of miRNA, proteins and lipids. EV-coupled miRNAs have been reported in follicular fluid. However, the association between postpartum NEB and EV-coupled miRNA signatures in follicular fluid is not yet known. Energy balance analysis in lactating cows shortly after post-calving revealed that the majority of the cows exhibited transiently negative energy balance levels, whereas the remaining cows exhibited either consistently negative or consistently positive energy levels. Metabolic status was associated with EV-coupled miRNA composition in the follicular fluid. Cows experiencing NEB showed reduced expression of a large number of miRNAs while cows with positive energy balances primarily exhibited elevated expression of EV-coupled miRNAs. The miRNAs that were suppressed under NEB were found to be involved in various metabolic pathways. This is the first study to reveal the presence of an association between EV-coupled miRNA in follicular fluid and metabolic stress in dairy cows. The involvement of differentially expressed miRNAs in various pathways associated with follicular growth and oocyte maturation suggest the potential involvement of specific follicular miRNAs in oocyte developmental competence, which may partially explain reduced fertility in cows due to post-calving metabolic stress.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

BACKGROUND: Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. RESULTS: The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. CONCLUSION: In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

Measured and estimated data of non-linear BRAN channels using HOS in 4G wireless communications.

The aim of this research is to develop a non-linear blind estimator able to represents a Broadband Radio Access Networks (BRAN) channels. In the one hand, we have used Higher Order Statistics (HOS) theory to build our algorithm. Indeed, we develop a non-linear method based only on fourth order cumulants for identifying the diagonal parameters of quadratic systems. In the other hand, the developed approach is applied to estimate the experimental channels, BRAN A, C and E data normalized for MC-CDMA, in non-linear case. However, the estimated data will be used in the blind equalization. The simulation results in noisy environment and for different signal to noise ratio (SNR) show the accuracy of develop estimator blindly (i.e., without any information about the input) with non-Gaussian signal input. Furthermore, in part of blind equalization problem the obtained results, using Zero forcing (ZF) and Minimum Mean Square Error (MMSE) equalizers, demonstrate that the proposed algorithm is very adequate to correct channel distortion in term the Bit Error Rate (BER). Finally, these estimated data present a necessary asset for conducting validation experiments, and can be also used as a baseline.

Cellular and exosome mediated molecular defense mechanism in bovine granulosa cells exposed to oxidative stress.

Various environmental insults including diseases, heat and oxidative stress could lead to abnormal growth, functions and apoptosis in granulosa cells during ovarian follicle growth and oocyte maturation. Despite the fact that cells exposed to oxidative stress are responding transcriptionally, the potential release of transcripts associated with oxidative stress response into extracellular space through exosomes is not yet determined. Therefore, here we aimed to investigate the effect of oxidative stress in bovine granulosa cells in vitro on the cellular and exosome mediated defense mechanisms. Bovine granulosa cells were aspirated from ovarian follicles and cultured in DMEM/F-12 Ham culture medium supplemented with 10% exosome-depleted fetal bovine serum. In the first experiment sub-confluent cells were treated with 5 µM H2O2 for 40 min to induce oxidative stress. Thereafter, cells were subjected to ROS and mitochondrial staining, cell proliferation and cell cycle assays. Furthermore, gene and protein expression analysis were performed in H2O2-challenged versus control group 24 hr post-treatment using qRT-PCR and immune blotting or immunocytochemistry assay, respectively. Moreover, exosomes were isolated from spent media using ultracentrifugation procedure, and subsequently used for RNA isolation and qRT-PCR. In the second experiment, exosomes released by granulosa cells under oxidative stress (StressExo) or those released by granulosa cells without oxidative stress (NormalExo) were co-incubated with bovine granulosa cells in vitro to proof the potential horizontal transfer of defense molecules from exosomes to granulosa cells and investigate any phenotype changes. Exposure of bovine granulosa cells to H2O2 induced the accumulation of ROS, reduced mitochondrial activity, increased expression of Nrf2 and its downstream antioxidant genes (both mRNA and protein), altered the cell cycle transitions and induced cellular apoptosis. Granulosa cells exposed to oxidative stress released exosomes enriched with mRNA of Nrf2 and candidate antioxidants. Subsequent co-incubation of StressExo with cultured granulosa cells could alter the relative abundance of cellular oxidative stress response molecules including Nrf2 and antioxidants CAT, PRDX1 and TXN1. The present study provide evidences that granulosa cells exposed to oxidative stress conditions react to stress by activating cascades of cellular antioxidant molecules which can also be released into extracellular environment through exosomes.

Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.