Novel protein extraction approach using micro-sized chamber for evaluation of proteins eluted from formalin-fixed paraffin-embedded tissue sections

We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR

Mutual interaction of kisspeptin, estrogen and bone morphogenetic protein-4 activity in GnRH regulation by GT1-7 cells

Reproduction is integrated by interaction of neural and hormonal signals converging on hypothalamic neurons for controlling gonadotropin-releasing hormone (GnRH). Kisspeptin, the peptide product of the kiss1 gene and the endogenous agonist for the GRP54 receptor, plays a key role in the regulation of GnRH secretion. In the present study, we investigated the interaction between kisspeptin, estrogen and BMPs in the regulation of GnRH production by using mouse hypothalamic GT1-7 cells. Treatment with kisspeptin increased GnRH mRNA expression and GnRH protein production in a concentration-dependent manner. The expression levels of kiss1 and GPR54 were not changed by kisspeptin stimulation. Kisspeptin induction of GnRH was suppressed by co-treatment with BMPs, with BMP-4 action being the most potent for suppressing the kisspeptin effect. The expression of kisspeptin receptor, GPR54, was suppressed by BMPs, and this effect was reversed in the presence of kisspeptin. It was also revealed that BMP-induced Smad1/5/8 phosphorylation and Id-1 expression were suppressed and inhibitory Smad6/7 was induced by kisspeptin. In addition, estrogen induced GPR54 expression, while kisspeptin increased the expression levels of ER alpha. and ER beta, suggesting that the actions of estrogen and kisspeptin are mutually enhanced in GT1-7 cells. Moreover, kisspeptin stimulated MAPKs and AKT signaling, and ERK signaling was functionally involved in the kisspeptin-induced GnRH expression. BMP-4 was found to suppress kisspeptin-induced GnRH expression by reducing ERK signaling activity. Collectively, the results indicate that the axis of kisspeptin-induced GnRH production is bi-directionally controlled, being augmented by an interaction between ER alpha/beta and GPR54 signaling and suppressed by BMP-4 action in GT1-7 neuron cells

Masked CKD in hyperthyroidism and reversible CKD status in hypothyroidism

IntroductionWhile it is well known that thyroid function may affect kidney function, the transition of the chronic kidney disease (CKD) status before and after treatment for thyroid disorders, as well as the factors affecting this change, remains to be explored. In the present study, we focused on the change in kidney function and their affecting factors during the treatment for both hyperthyroidism and hypothyroidism. Methods Eighty-eight patients with hyperthyroidism and fifty-two patients with hypothyroidism were enrolled in a retrospective and longitudinal case series to analyze the changes in kidney function and their affecting factors after treatment for thyroid disorders. Results Along with the improvement of thyroid function after treatment, there was a significant decrease in estimated glomerular filtration rate (eGFR) in hyperthyroidism (an average Delta eGFR of -41.1 mL/min/1.73 m(2)) and an increase in eGFR in hypothyroidism (an average Delta eGFR of 7.1 mL/min/1.73 m(2)). The multiple linear regression analysis revealed that sex, eGFR, free thyroxine (FT4) and free triiodothyronine (FT3) could be considered independent explanatory variables for Delta eGFR in hyperthyroidism, while age, eGFR, and FT3 were detected as independent explanatory variables in hypothyroidism. In addition, the stratification by kidney function at two points, pre- and post-treatment for thyroid disorders, revealed that 4.5% of the participants with hyperthyroidism were pre-defined as non-CKD and post-defined as CKD, indicating the presence of "masked" CKD in hyperthyroidism. On the other hand, 13.5% of the participants with hypothyroidism presented pre-defined CKD and post-defined non-CKD, indicating the presence of "reversible" CKD status in hypothyroidism. Conclusions We uncovered the population of masked CKD in hyperthyroidism and reversible CKD status in hypothyroidism, thereby re-emphasizing the importance of a follow-up to examine kidney function after treatment for hyperthyroidism and the routine evaluation of thyroid function in CKD patients as well as the appropriate hormone therapy if the patient has hypothyroidism

Mutual interaction of kisspeptin, estrogen and bone morphogenetic protein-4 activity in GnRH regulation by GT1-7 cells

a b s t r a c t Reproduction is integrated by interaction of neural and hormonal signals converging on hypothalamic neurons for controlling gonadotropin-releasing hormone (GnRH). Kisspeptin, the peptide product of the kiss1 gene and the endogenous agonist for the GRP54 receptor, plays a key role in the regulation of GnRH secretion. In the present study, we investigated the interaction between kisspeptin, estrogen and BMPs in the regulation of GnRH production by using mouse hypothalamic GT1-7 cells. Treatment with kisspeptin increased GnRH mRNA expression and GnRH protein production in a concentrationdependent manner. The expression levels of kiss1 and GPR54 were not changed by kisspeptin stimulation. Kisspeptin induction of GnRH was suppressed by co-treatment with BMPs, with BMP-4 action being the most potent for suppressing the kisspeptin effect. The expression of kisspeptin receptor, GPR54, was suppressed by BMPs, and this effect was reversed in the presence of kisspeptin. It was also revealed that BMP-induced Smad1/5/8 phosphorylation and Id-1 expression were suppressed and inhibitory Smad6/7 was induced by kisspeptin. In addition, estrogen induced GPR54 expression, while kisspeptin increased the expression levels of ERa and ERb, suggesting that the actions of estrogen and kisspeptin are mutually enhanced in GT1-7 cells. Moreover, kisspeptin stimulated MAPKs and AKT signaling, and ERK signaling was functionally involved in the kisspeptin-induced GnRH expression. BMP-4 was found to suppress kisspeptin-induced GnRH expression by reducing ERK signaling activity. Collectively, the results indicate that the axis of kisspeptin-induced GnRH production is bi-directionally controlled, being augmented by an interaction between ERa/b and GPR54 signaling and suppressed by BMP-4 action in GT1-7 neuron cells

Oncocytic intraductal carcinoma of salivary glands: a distinct variant with TRIM33–RET fusions and BRAF V600E mutations

Aims: Salivary gland intraductal carcinoma (IDC) is a complex ductal neoplasm surrounded by a layer of myoepithelial cells. Recent insights have shown that there are three different types: intercalated duct-like, with frequent NCOA4-RET fusions; apocrine, with salivary duct carcinoma-like mutations; and mixed intercalated duct-like/apocrine, with RET fusions, including TRIM27-RET. In addition, an oncocytic IDC has been described, but it remains unclear whether it represents a fourth variant or simply oncocytic metaplasia of another IDC type. Our aim was to more completely characterize oncocytic IDC. Methods and results: Six IDCs with oncocytic changes were retrieved from the authors' archives, from three men and three women ranging in age from 45 to 75 years (mean, 63 years). Five arose in the parotid gland, with one in an accessory parotid gland. Four patients with follow-up were free of disease after 1-23 months. Several immunostains (S100, mammaglobin, androgen receptor, and p63/p40) and molecular tools (RNA sequencing, RET fluorescence in-situ hybridisation, BRAF V600E VE1 immunohistochemistry, and Sanger sequencing) were applied. Histologically, the tumours were variably cystic with solid intracystic nodules often difficult to recognise as intraductal. In all, tumour ducts were positive for S100 and mammaglobin, negative for androgen receptor, and completely surrounded by myoepithelial cells positive for p63/p40. Molecular analysis revealed TRIM33-RET in two of six cases, NCOA4-RET in one of six cases, and BRAF V600E in two of six cases. One case had no identifiable alterations. Conclusions: Oncocytic IDC shares similarities with intercalated duct-like IDC. Although additional verification is needed, the oncocytic variant appears to be sufficiently unique to be now regarded as the fourth distinct subtype of IDC. Because of its indolent nature, oncocytic IDC should be distinguished from histological mimics

Case-based similar image retrieval for weakly annotated large histopathological images of malignant lymphoma using deep metric learning

In the present study, we propose a novel case-based similar image retrieval (SIR) method for hematoxylin and eosin (H&E)-stained histopathological images of malignant lymphoma. When a whole slide image (WSI) is used as an input query, it is desirable to be able to retrieve similar cases by focusing on image patches in pathologically important regions such as tumor cells. To address this problem, we employ attention-based multiple instance learning, which enables us to focus on tumor-specific regions when the similarity between cases is computed. Moreover, we employ contrastive distance metric learning to incorporate immunohistochemical (IHC) staining patterns as useful supervised information for defining appropriate similarity between heterogeneous malignant lymphoma cases. In the experiment with 249 malignant lymphoma patients, we confirmed that the proposed method exhibited higher evaluation measures than the baseline case-based SIR methods. Furthermore, the subjective evaluation by pathologists revealed that our similarity measure using IHC staining patterns is appropriate for representing the similarity of H&E-stained tissue images for malignant lymphoma

On the Correlation between Hydration of the Stratum Corneum and the Nutritional Status of Dialysis Patients

　Stratum corneum (SC) prevents evaporation of internal moisture and maintains dermal flexibility and fluidity. Malnutrition is known to decrease SC hydration, but involvement of nutritional status in SC hydration of dialysis patients is unclear. In this study, we examined the association between SC hydration and nutritional status on dialysis patients. We studied 16 control subjects and 81 dialysis patients to examine SC hydration of the neck, upper arm and lower leg. In the 81 patients, we measured serum albumin (Alb), transferrin, geriatric nutritional risk index (GNRI) and normalized protein catabolic rate (nPCR). We then classified the dialysis patients into a 21-patients group, with both Alb ≥ 3.6 g/dl and nPCR ≥ 0.9 g/kg/ day, and a 60-patients group with either Alb ＜3.6 g/dl or nPCR ＜0.9 g/kg/day. SC hydration in the lower leg and the Alb value in the 81 patients were significantly lower than those in the control subjects. SC hydration in the upper arm and lower leg and the values of transferrin and GNRI in the 60-patients group were significantly lower than those in the 21-patients group. This study demonstrates for the first time that nutritional status is correlated with maintenance of SC hydration on dialysis patients

Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice.

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells

Let-7 MicroRNA Family Is Selectively Secreted into the Extracellular Environment via Exosomes in a Metastatic Gastric Cancer Cell Line

Background: Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene downregulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. Methodology/Principal Findings: Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. Conclusions/Significance: The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosome