Insights into the Mechanical Properties of Ultrathin Perfluoropolyether-Silane Coatings.

Ultrathin perfluoropolyether-silane (PFPE-silane) films offer excellent functionality as antifingerprint coatings for display touchscreens due to their oleophobic, hydrophobic, and good adhesion properties. During smartphone use, PFPE-silane coatings undergo many abrasion cycles which limit the coating lifetime, so a better understanding of how to optimize the film structure for improved mechanical durability is desired. However, the hydrophobic and ultrathin (1-10 nm) nature of PFPE-silane films renders them very difficult to experimentally characterize. In this study, the cohesive fracture energy and elastic modulus, which are directly correlated with hardness and better wear resistance of 3.5 nm-thick PFPE-silane films were, respectively, measured by double cantilever beam testing and atomic force microscopy indentation. Both the cohesive fracture energy and modulus are shown to be highly dependent on the underlying film structure. Both values increase with optimal substrate conditions and a higher number of silane groups in the PFPE-silane precursor. The higher cohesive fracture energy and modulus values are suggested to be the result of the changes in the film chemistry and structure, leading to higher cross-linking density. Therefore, future work on optimizing PFPE-silane film wear resistance should focus on pathways to improve the cross-linking density. Subcritical fracture testing in humid environments reveals that humidity negatively affects the fracture properties of PFPE-silane films.

Protocol for laparoscopic cholecystectomy: Is it rocket science?

Laparoscopic cholecystectomy (LC) does not require advanced techniques, and its performance has therefore rapidly spread worldwide. However, the rate of biliary injuries has not decreased. The concept of the critical view of safety (CVS) was first documented two decades ago. Unexpected injuries are principally due to misidentification of human factors. The surgeon's assumption is a major cause of misidentification, and a high level of experience alone is not sufficient for successful LC. We herein describe tips and pitfalls of LC in detail and discuss various technical considerations. Finally, based on a review of important papers and our own experience, we summarize the following mandatory protocol for safe LC: (1) consideration that a high level of experience alone is not enough; (2) recognition of the plateau involving the common hepatic duct and hepatic hilum; (3) blunt dissection until CVS exposure; (4) Calot's triangle clearance in the overhead view; (5) Calot's triangle clearance in the view from underneath; (6) dissection of the posterior right side of Calot's triangle; (7) removal of the gallbladder body; and (8) positive CVS exposure. We believe that adherence to this protocol will ensure successful and beneficial LC worldwide, even in patients with inflammatory changes and rare anatomies.

[Local excision of T1b anal canal cancer - a case report].

We performed transanal local excision of anal canal cancer in a 51-year-old man. The tumor was detected as an Isp polyp(7 mm)on the dentate line, by colonofiberscopic examination. Pathological findings indicated adenocarcinoma(T1b). The patient desired preservation of anal function, and hence refused abodominoperineal rectal transection with lymph node dissection. We obtained informed consent for recurrence, and observed the patient rigorously. No recurrence or metastasis has been detected 3 years and 3 months after tumor excision. We propose that transanal local excision might be a treatment option for early stage cancer of the anal canal.

[Efficacy and toxicity of transcatheter arterial chemoembolization with Cisplatin suspended in lipiodol for unresectable hepatocellular carcinoma].

In this study we evaluated the efficacy and toxicity of transcatheter arterial chemoembolization (TACE) with Cisplatin (CDDP)-Lipiodol (LIP) suspension in 24 patients with advanced hepatocellular carcinoma (HCC). Eligibility criteria were as follows; unresectable HCC, age <75 years, performance status (PS) 0-2, Child-Pugh A or B and adequate heart and renal function. When TACE was performed, the catheter was placed selectively in feeding arteries of the tumors, and CDDP-LIP suspension (20 mg/mL) was injected followed by gelatin sponge particles. The direct and total effect on tumors were evaluated 3 and 6 months after TACE, respectively. As for a direct effect, complete and partial response rates were 54.2% and 25%, respectively. As for a total effect, complete and partial response rates were 41.7% and 4.1%, respectively. Grade 3/4 drug-related toxicities were as follows: thrombocytopenia (13%), appetite loss (8%) and nausea (4%). These severe side effects disappeared within 10 days after TACE. No renal and hepatic dysfunction was encountered, and no drug-related deaths occurred. TACE with CDDP suspended in LIP may provide some clinical benefits with relatively tolerable toxicities.

An efficient method for protein phosphorylation using the artificially introduced of cognate-binding modules into kinases and substrates.

Protein phosphorylation is a major post-translational modification that regulates cellular signal transduction. The phosphorylation of substrate proteins by kinases requires cognate pairs of substrates and kinases. In addition, phosphorylation is mediated through both indirect and direct interaction between these kinases and substrates, which makes it difficult to effectively prepare large quantities of recombinant phosphorylated proteins. Here, we report a novel protein phosphorylation method involving the artificial introduction of cognate-binding modules into substrates and enzymes. This enhances the local concentration of substrates around enzymes so that the enzymatic reaction proceeds more efficiently. We prepared substrate proteins containing an SH3 domain at their N-terminus, and a kinase containing an SH3-binding motif at its C-terminus. This method was successfully applied to the phosphorylation of CrkII and the Vav DH domain, and we prepared (15)N-labelled phosphorylated CrkII for NMR analysis.

Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK.

CRKI (SH2-SH3) and CRKII (SH2-SH3-SH3) are splicing isoforms of the oncoprotein CRK that regulate transcription and cytoskeletal reorganization for cell growth and motility by linking tyrosine kinases to small G proteins. CRKI shows substantial transforming activity, whereas the activity of CRKII is low, and phosphorylated CRKII has no biological activity whatsoever. The molecular mechanisms underlying the distinct biological activities of the CRK proteins remain elusive. We determined the solution structures of CRKI, CRKII and phosphorylated CRKII by NMR and identified the molecular mechanism that gives rise to their activities. Results from mutational analysis using rodent 3Y1 fibroblasts were consistent with those from the structural studies. Together, these data suggest that the linker region modulates the binding of CRKII to its targets, thus regulating cell growth and motility.

In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes.

It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes.

Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver.

Previously, we reported a system to enrich mouse fetal hepatic progenitor cells (HPCs) by forming cell aggregates. In this study, we sorted two cell populations, CD49f(+)Thy1(-)CD45(-) cells (CD49f-positive cells) and CD49f(+/-)Thy1(+)CD45(-) cells (Thy1-positive cells), from the cell aggregates using a flow cytometer. CD49f-positive cells stained positive for endodermal specific markers such as alpha-fetoprotein (AFP), albumin (ALB), and cytokeratin 19 (CK19), and are thus thought to be HPCs. However, Thy1-positive cells were a morphologically heterogeneous population; reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemical analyses revealed the expression of mesenchymal cell markers such as alpha-smooth muscle actin, desmin, and vimentin, but not of AFP, ALB, or CK19. Therefore, Thy1-positive cells were thought to be of a mesenchymal lineage. When these two cell populations were co-cultured, the CD49f-positive colonies matured morphologically and stored a significant amount of glycogen. Furthermore, real-time RT-PCR demonstrated an increased expression of tyrosine amino transferase and tryptophan oxygenase mRNA, and transmission electron microscopy confirmed that co-cultured cells produced mature hepatocytes. However, when CD49f-positive cells were cultured alone or when the two populations were cultured separately, the CD49f-positive cells did not mature. These results indicate that CD49f-positive cells are primitive hepatic endodermal cells with the capacity to differentiate into hepatocytes, and that Thy1-positive cells promote the maturation of CD49f-positive cells by direct cell-to-cell contact. In conclusion, we were able to isolate CD49f-positive primitive hepatic endodermal cells and Thy1-positive mesenchymal cells and to demonstrate the requirement of cell-to-cell contact between these cell types for the maturation of the hepatic precursors.

Commitment of bone marrow cells to hepatic stellate cells in mouse.

BACKGROUND/AIMS: Recently, several cells found within the liver have been reported to derive from bone marrow (BM). This study sought to examine the commitment of BM cells to hepatic stellate cell (HSC) lineage in mouse liver. METHODS: We transplanted BM cells from green fluorescent protein (GFP) transgenic mice into age-matched C57BL/J mice. Hepatic nonparenchymal cells were isolated from the livers of BM-transplanted mice using density gradient centrifugation with Nycodenz. The expression of lineage markers by the isolated cells was evaluated by RT-PCR and immunostaining. We then examined the histology of liver tissues obtained from BM-transplanted mice with and without carbon tetrachloride-induced injury. RESULTS: GFP-expressing cells with intracytoplasmic lipid droplets comprised 33.4 +/- 2.3% of the cells isolated by density gradient centrifugation. These cells expressed the HSC lineage markers, such as desmin and glial fibrillary acidic protein (GFAP), by both RT-PCR and immunostaining. During a 7-day culture, GFP-positive cells began to express alpha-smooth muscle actin, a marker of activated HSC. In the liver of BM-transplanted mice, GFP-positive nonparenchymal cells expressed GFAP and extended their process around hepatocytes. Upon liver injury, these cells also co-expressed desmin and alpha-smooth muscle actin. CONCLUSIONS: Nonparenchymal cells, derived from transplanted BM, acquired HSC characteristics in both quiescent and activated states.