Weakly supervised learning and interpretability for endometrial whole slide image diagnosis

Fully supervised learning for whole slide image–based diagnostic tasks in histopathology is problematic due to the requirement for costly and time-consuming manual annotation by experts. Weakly supervised learning that utilizes only slide-level labels during training is becoming more widespread as it relieves this burden, but has not yet been applied to endometrial whole slide images, in iSyntax format. In this work, we apply a weakly supervised learning algorithm to a real-world dataset of this type for the first time, with over 85% validation accuracy and over 87% test accuracy. We then employ interpretability methods including attention heatmapping, feature visualization, and a novel end-to-end saliency-mapping approach to identify distinct morphologies learned by the model and build an understanding of its behavior. These interpretability methods, alongside consultation with expert pathologists, allow us to make comparisons between machine-learned knowledge and consensus in the field. This work contributes to the state of the art by demonstrating a robust practical application of weakly supervised learning on a real-world digital pathology dataset and shows the importance of fine-grained interpretability to support understanding and evaluation of model performance in this high-stakes use case.     

Nature of functional estrogen receptors at the plasma membrane

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.     

Encapsulation of soybean meal with fats enriched in palmitic and stearic acids: effects on rumen-undegraded protein and in vitro intestinal digestibility

Fat coating of soybean meal (SBM) can reduce its protein degradability in the rumen, but the encapsulation of SBM with palmitic (PA) and stearic acids (SA) has not yet been investigated, despite both fatty acids are common energy sources in dairy cow diets. This study aimed to evaluate the effects of applying a novel method, using either 400 or 500 g fat/kg (treatments FL40 and FL50, respectively), which was enriched in PA and SA at different ratios (100:0, 75:25, 50:50, 25:75 and 0:100), on physical and chemical characteristics, ruminal degradability, solubility and in vitro intestinal protein digestibility (IVIPD) of the obtained products. Encapsulation of SBM in fat resulted in greater mean particle size and lower bulk density and protein solubility than unprotected SBM (USBM). Treatment FL50 resulted in increased (p < 0.01) rumen-undegraded protein (RUP) compared to USBM. There were no differences in RUP of SBM when different PA: SA ratios were used. The mean RUP content of treatments FL40 and FL50 (306 and 349 g/kg, respectively) was greater compared to USBM (262 g/kg, p < 0.05), but lower than that for a standard heat-treated SBM (431 g/kg). Values of IVIPD did not differ among SBM, heat-treated SBM and FL40 and FL50 samples, all being greater than 97.8%. In conclusion, encapsulation of SBM with fats enriched in PA and SA proved to be effective in reducing protein solubility and increasing RUP without depressing protein digestibility in the intestine. For validation of the method, in vivo research to investigate the effects of these products on the production of dairy cows is warranted.     

Characterization of the acidic N-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach

Oocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. In the pig, two-dimensional electrophoretic analysis demonstrated an acidic shift of the zona pellucida glycoproteins of about 1.5–2.0 pH units during the maturation process. These findings were corroborated by histological studies that indicated the synthesis of acidic glycoconjugates in the cumulus cells and an increased occurrence of acidic glycans in the zona pellucida after oocyte maturation. In order to provide structural data on prepuberal zona pellucida N-glycosylation, N-glycans were released from prepuberal zona pellucida glycoproteins by N-glycosidase F and studied by mass spectrometry before and after desialylation and treatment with endo-β-galactosidase. Our results verified the presence of high-mannose-type Man₅GlcNAc₂ compounds as well as diantennary N-glycans as major neutral species, whereas sialylated diantennary and triantennary species constituted the dominant non-sulfated acidic sugar chains. The major acidic N-glycans of prepuberal animals, however, represented mono-sulfated diantennary, triantennary and tetraantennary oligosaccharides carrying, in part, N-acetyllactosamine repeating units as well as additional Neu5Ac or Neu5Gc residues. Glycans comprising more than one sulfate residue were not detected. In contrast to the literature data on zona pellucida glycoprotein-N-glycans of cyclic animals, our data thus reveal a lower degree in glycan sulfation of the prepuberal zona pellucida.     

Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress,wounding, and salicylic acid treatment in Carthamus tinctorius

Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.     

Expression pattern of olfactory receptor genes in human cumulus cells as an indicator for competent oocyte selection

Odorant or olfactory receptors are mainly localized in the olfactory epithelium for the perception of different odors. Interestingly, many ectopic olfactory receptors with low expression levels have recently been found in nonolfactory tissues to involve in local functions. Therefore, we investigated the probable role of the olfactory signaling pathway in the surrounding microenvironment of oocyte. This study included 22 women in intracytoplasmic sperm injection cycle. The expression of olfactory target molecules in cumulus cells surrounding the growing and mature oocytes was evaluated by Western blotting and real-time polymerase chain reaction. Additionally, integrated bioinformatics analyses were carried out and 6 ectopic olfactory receptors were selected for further evaluation. The initiation of olfactory transduction cascade in cumulus cells of competent oocytes was confirmed by analyzing the expression of adenylyl cyclase type 3 and olfactory market protein. Moreover, the expression pattern of the selected olfactory receptors was evaluated and OR10H2 was selected due to a high level of expression in mature fertile oocytes. We suggested that OR10H2 could be considered as a reliable biomarker for oocyte selection in assisted reproduction technique programs. However, further studies are required to elucidate the role of olfactory transduction cascade in embryo quality and implantation.     

Analyses of Arabidopsis trr14 T-DNA Insertion Mutants Reveal an Essential Role in Seed Germination

TRR14 is an unknown protein that was first identified as a component of Arabidopsis responses to trehalose treatment. Phylogenic analysis showed that TRR14 belongs to a seven-gene family in Arabidopsis. Close homologues of TRR14 were found in plants and many cyanobacteria. GFP expression analysis showed that TRR14 is located in the chloroplast. GUS::TRR14 expression was found in leaves, flowers, stems and siliques. We investigated the functional roles of TRR14 in Arabidopsis thaliana under salt and drought stress. By a reverse genetic approach, two trr14 T-DNA insertion mutants were isolated from the SALK collection. Functional analysis of the trr14 mutants revealed enhanced sensitivity of the mutants to salt and drought stress, compared with the wild type plants. Further experiments indicated that the trr14 mutants have reduced seed germination, root length, survival rate and chlorophyll content under stress conditions. In addition activity of oxidative enzymes like peroxidase, catalase and polyphenol oxidase was reduced under salt and drought treatments. Thus, the present data indicate that a novel protein, TRR14, is involved in plant salt and drought tolerance.