Influence of osmotic condition on secondary cell wall formation of xylem vessel cells induced by the master transcription factor VND7.

Xylem vessels, which conduct water from roots to aboveground tissues in vascular plants, are stiffened by secondary cell walls (SCWs). Protoxylem vessel cells deposit cellulose, hemicellulose, and lignin as SCW components in helical and/or annular patterns. The mechanisms underlying SCW patterning in the protoxylem vessel cells are not fully understood, although VASCULAR-RERATED NAC-DOMAIN 7 (VND7) has been identified as a master transcription factor in protoxylem vessel cell differentiation in Arabidopsis thaliana. Here, we investigated deposition patterns of SCWs throughout the tissues of Arabidopsis seedlings using an inducible transdifferentiation system that utilizes a chimeric protein in which VND7 is fused with the activation domain of VP16 and the glucocorticoid receptor (GR) (VND7-VP16-GR). In slender- and cylinder-shaped cells, such as petiole and hypocotyl cells, SCWs that were ectopically induced by the VND7-VP16-GR system were deposited linearly, resulting in helical and annular patterns similar to the endogenous patterns in protoxylem vessel cells. By contrast, concentrated linear SCW deposition was associated with unevenness on the surface of pavement cells in cotyledon leaf blades, suggesting the involvement of cell morphology in SCW patterning. When we exposed the seedlings to hypertonic conditions that induced plasmolysis, we observed aberrant deposition patterns in SCW formation. Because the turgor pressure becomes zero at the point when cells reach limiting plasmolysis, this result implies that proper turgor pressure is required for normal SCW patterning. Taken together, our results suggest that the deposition pattern of SCWs is affected by mechanical stimuli that are related to cell morphogenesis and turgor pressure.

Kidney Disease: Improving Global Outcomes in neonates with acute kidney injury after cardiac surgery.

BACKGROUND: Acute kidney injury (AKI) after cardiac surgery (CS-AKI) in children with congenital heart disease is a serious complication closely associated with high morbidity and mortality. Kidney Disease: Improving Global Outcomes (KDIGO) AKI staging demonstrates high sensitivity for detecting AKI and predicting associated in-hospital mortality. However, neonatal-modified KDIGO criteria (n-KDIGO), recently introduced as a standard diagnostic tool, for CS-AKI have not been fully validated. Here, we evaluated the incidence of risk factors and postoperative outcomes of neonatal CS-AKI. METHODS: We retrospectively studied 114 consecutive neonates who underwent cardiac surgery at the Kagoshima University Hospital. CS-AKI was classified using the n-KDIGO criteria. Risk adjustment in congenital heart surgery (RACHS-1) score was used to predict the complexity-adjusted mortality and % fluid overload (%FO) was used to monitor fluid balance in pediatric cardiac surgery. RESULTS: Among 81 patients, neonatal CS-AKI occurred in 57 (70.4%) patients according to n-KDIGO criteria. Of these, 28 (34.6%) patients reached n-KDIGO 1, 17 (21.0%) reached n-KDIGO 2, and 12 (14.8%) reached n-KDIGO 3. Patients with CS-AKI had significantly higher vasoactive-inotropic score levels, longer operative times, and higher %FO than patients without CS-AKI. Notably, increased duration of cardiopulmonary bypass times and %FO were risk factors for the development of neonatal CS-AKI. The n-KDIGO-based severe AKI grade had higher risk of in-hospital mortality; however, the n-KDIGO-based mild AKI grade was not associated with any postoperative outcomes. CONCLUSIONS: CS-AKI based on n-KDIGO criteria is common in neonates and is closely associated with higher mortality, especially in patients with severe CS-AKI.

Cytological Features Associated with Ureaplasma Urealyticum in Pap Cervical Smear

Purpose: Ureaplasma urealyticum is associated with several obstetric complications and increases the importance of risk management in pregnant women. Furthermore, U. urealyticum has been identified as a cofactor that interacts with human papillomavirus infection in cervical cancer onset. The aim of this study was to assess specific cytological features of U. urealyticum infection in Pap smears to determine whether additional microbiological testing should be performed for pregnant women with a high possibility of U. urealyticum infection. Methods: Liquid-based cytology specimens (LBC) from cervical swabs of a total of 55 women, including 33 pregnant women who were negative for intraepithelial lesion or malignancy (NILM) on Pap testing and with U. urealyticum diagnosed without any other infectious microbes and 22 U. urealyticum-negative controls, were used in this study. We evaluated the localization of U. urealyticum by immunofluorescence, cytological features of secondary changes in squamous cells caused by inflammation, and the specimen background in Pap smears. Results: Based on analysis of Pap smears, a significant relationship was observed between U. urealyticum infection and cannonballs (p < 0.05) as well as predominance of coccoid bacteria (p < 0.05). A large number of U. urealyticum were detected in cannonballs by immunofluorescence. Conclusion: In the present study, cytological features in Pap smears of U. urealyticum infected samples, which have hardly been understood thus far, were assessed. The cytological features included cannonballs and predominance of coccoid bacteria. Our results might help in determining whether additional microbiological testing should be performed for pregnant women with a high possibility of U. urealyticum infection.

Filter paper-assisted cell transfer (FaCT) technique: A novel cell-sampling technique for intraoperative diagnosis of central nervous system tumors.

BACKGROUND: Intraoperative diagnosis of central nervous system (CNS) tumors provides critical guidance to surgeons in the determination of surgical resection margins and treatment. The techniques and preparations used for the intraoperative diagnosis of CNS tumors include frozen sectioning and cytologic methods (squash smear and touch imprint). Cytologic specimens, which do not have freezing artifacts, are important as an adjuvant tool to frozen sections. However, if the amount of submitted tissue samples is limited, then it is difficult to prepare both frozen sections and squash smears or touch imprint specimens from a single sample at the same time. Therefore, the objective of this study was to derive cells directly from filter paper on which tumor samples are placed. METHODS: The authors established the filter paper-assisted cell transfer (FaCT) smear technique, in which tumor cells are transferred onto a glass slide directly from the filter paper sample spot after the biopsy is removed. RESULTS: Cell yields and diagnostic accuracy of the FaCT smears were assessed in 40 CNS tumors. FaCT smears had ample cell numbers and well preserved cell morphology sufficient for cytologic diagnosis, even if the submitted tissues were minimal. The overall diagnostic concordance rates between frozen sections and FaCT smears were 90% and 87.5%, respectively (no significant differences). When combining FaCT smears with frozen sections, the diagnostic concordance rate rose to 92.5%. CONCLUSIONS: The current results suggest that the FaCT smear technique is a simple and effective processing method that has significant value for intraoperative diagnosis of CNS tumors. Cancer Cytopathol 2017;125:277-282. © 2016 American Cancer Society.

Immunohistochemical expression profiles of solute carrier transporters in alpha-fetoprotein-producing gastric cancer.

AIMS: Alpha-fetoprotein (AFP)-producing gastric cancer (GC) is an aggressive tumour with high rates of liver metastasis and poor prognosis, and for which a validated chemotherapy regimen has not been established. Drug uptake by solute carrier (SLC) transporters is proposed as one of the mechanisms involved in sensitivity to chemotherapy. In this study, we aimed to develop important insights into effective chemotherapeutic regimens for AFP-producing GC. METHODS AND RESULTS: We evaluated immunohistochemically the expression levels of a panel of SLC transporters in 20 AFP-producing GCs and 130 conventional GCs. SLC transporters examined were human equilibrative nucleoside transporter 1 (hENT1), organic anion transporter 2 (OAT2), organic cation transporter (OCT) 2, OCT6 and organic anion-transporting polypeptide 1B3 (OATP1B3). The rates of high expression levels of hENT1 (hENT1high ) and OAT2 (OAT2high ) were statistically higher in AFP-producing GC, compared with conventional GC. When analysing hENT1 and OAT2 in combination, hENT1high /OAT2high was the most particular expression profile for AFP-producing GC, with a greater significance than hENT1 or OAT2 alone. However, no significant differences in OCT2, OCT6 or OATP1B3 levels were detected between AFP-producing and conventional GCs. However, immunoreactivity for hENT1, OAT2 and OCT6 tended to be increased in GC tissues compared with non-neoplastic epithelia. CONCLUSIONS: Because hENT1 and OAT2 are crucial for the uptake of gemcitabine and 5-fluorouracil, respectively, our results suggest that patients with AFP-producing GC could potentially benefit from gemcitabine/fluoropyrimidine combination chemotherapy. Increased expression of hENT1, OAT2 and OCT6 may also be associated with the progression of GC.

Rapid immunocytochemistry with simple heat-induced antigen retrieval technique for improvement in the quality of cytological diagnosis.

Rapid immunocytochemistry (ICC) can improve the accuracy of intraoperative cytological diagnoses; however, it is usually applied without heat-induced antigen retrieval (HIAR). We established a HIAR method for rapid ICC and evaluated its efficacy and reliability. Rapidly fixed smear samples were immunostained using 35 antibodies. We compared the results of HIAR by boiling in a pot or heating in an electric kettle. The smears were incubated for 3 min with each primary antibody and immuno-enzyme polymer reagent, and for 1 min with diaminobenzidine solution. HIAR for 1 min using the kettle method yielded the best cellular integrity. For 32 out of the 35 antibodies, results achieved using rapid ICC within 11 min were comparable to that achieved using standard ICC. HIAR was essential for 13 antibodies. For two of the antibodies, HIAR was not required when standard ICC was applied, but consistent staining with rapid ICC was obtained only with HIAR. In conclusion, we established a rapid ICC procedure using a simple HIAR method, which allowed efficient immunostaining of a panel of antigens, including nuclear antigens, within only 11 min. The combined use of this rapid ICC technique with other staining techniques could be useful for improving intraoperative cytological diagnoses.

Optimal antigen retrieval for ethanol-fixed cytologic smears.

BACKGROUND: Antigen retrieval, a crucial technique for immunostaining, is often carried out on formalin-fixed, paraffin-embedded (FFPE) tissue sections. The role of antigen retrieval in immunostaining of ethanol-fixed smears remains unclear. The authors evaluated the effects of 2 common antigen retrieval procedures, heat-induced antigen retrieval and protease-induced antigen retrieval, for immunostaining using a broad panel of antibodies. METHODS: Papanicolaou-stained ethanol-fixed smears from 36 surgical specimens were immunostained with 43 antibodies. Three widely used heat-induced antigen retrieval solutions, namely, citrate buffer (pH 6.0 and pH 7.0) and ethylenediaminetetraacetic acid solution (pH 8.0) for heat-induced antigen retrieval, and pronase were used. The staining results were compared between the ethanol-fixed smears and the corresponding FFPE tissue sections. RESULTS: Heat-induced antigen retrieval was essential for all the 9 antibodies examined against nuclear antigens, and for 7 of 26 antibodies against cytoplasmic and cell membrane antigens. Superior results were obtained using lower-pH heat-induced antigen retrieval solutions for ethanol-fixed smears than was the case for FFPE tissue sections; use of citrate buffer (pH 6.0) was optimal for most antibodies. For 17 antibodies against cytoplasmic/cell membrane antigens, satisfactory results were obtained even without antigen retrieval on the ethanol-fixed smears, whereas antigen retrieval was necessary for detection on the FFPE tissue sections. Protease-induced antigen retrieval frequently exerted deleterious effects on ethanol-fixed smears. Despite antigen retrieval, detection of 2 lymphocytic markers failed on ethanol-fixed smears. This limitation was overcome by heat-induced antigen retrieval on formalin vapor-fixed smears. CONCLUSIONS: In ethanol-fixed smears, most of the antibodies can be immunostained successfully without antigen retrieval treatment or mild heat-induced antigen retrieval using citrate buffer (pH 6.0). The optimal antigen retrieval condition for each antibody must be individually determined.

Significance of cell proliferation markers (Minichromosome maintenance protein 7, topoisomerase IIalpha and Ki-67) in cavital fluid cytology: can we differentiate reactive mesothelial cells from malignant cells?

The aim of this study was to evaluate whether immunocytochemical expressions of proliferation markers, such as minichromosome maintenance protein 7 (MCM 7), topoisomerase IIalpha (topo IIalpha), and Ki-67, in reactive mesothelial cells and malignant cells obtained from cavital fluids could be useful for their differential diagnosis. Samples diagnosed as reactive mesothelial cells (14 cases) or malignant tumors (28 cases) in cavital fluids were examined. Immunocytochemical staining of MCM 7, topo IIalpha, and Ki-67 was performed with the universal immunoperoxidase polymer method. In reactive mesothelial cells, MCM 7 was stained in a fine granular pattern and its distribution was uniform in the nuclei. Topo IIalpha and Ki-67 were stained in a coarse granular pattern and the distributions were the same as MCM 7. In contrast, in malignant cells, MCM 7 was stained in an irregular and fine granular pattern, and topo IIalpha and Ki-67 were stained in a uniform and coarse granular pattern. Labeling indices of MCM 7 (cut-off value; 30%, sensitivity; 100%, and specificity; 100%), topo IIalpha (cut-off value; 15%, sensitivity; 89.3%, and specificity; 92.9%) and Ki-67 (cut-off value; 30%, sensitivity; 64.3%, and specificity; 92.9%) of malignant cells were significantly higher than those of reactive mesothelial cells. MCM 7, topo IIalpha, and Ki-67 are different types of cell proliferation markers. MCM 7 and topo IIalpha, in particular, could be reliable tools for differential diagnosis between reactive mesothelial cells and malignant cells.