Chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy

We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleotides at the 3′-overhang region were easily prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified miR-143 duplexes carrying the urea/thiourea compounds in the 3′-end of each strand were able to inhibit the growth of human bladder cancer T24 cells.     

Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.     

Change of the Gluconeogenic Capacity of Rat Kidney Cortex Slices with Dietary Components

Substituting protein for carbohydrate in diets significantly enhanced the rates of glucose formation from pyruvate, glutamate, or glycerol in rat kidney cortex slices. The tissue, however, increased slightly its gluconeogenic capacity in response to low carbohydrate, high fat diet. The rates of glucose taken up per unit weight of kidney cortex of rats fed a high carbohydrate diet was higher than those of rats fed diets high in protein or fat. Kidney weight in g per 100 g body weight (relative kidney size) of rats fed diets high in protein was significantly higher than that of rats fed diets high in carbohydrate or fat.     

A passive integrated transponder tag implanted by a new alternative surgical method: effects on the oriental weather loach (Misgurnus anguillicaudatus) and application in a small irrigation system

We validated the effects of a passive integrated transponder (PIT) tagging process on the oriental weather loach Misgurnus anguillicaudatus. Laboratory experiments were conducted to assess the effects of PIT tagging on fish survival, growth, wound healing, and tag omission. Two tagging protocols, standard syringe injection versus insertion through a small hole pierced by a fine needle-shaped awl, were compared using a 12.5 × 2.07 mm² tag. A control group was also included. In comparison with the awl technique, syringe injection heightened the mortality of the loach and delayed healing of the wound caused by tag insertion. No effects of either PIT tagging method were detected on the growth of surviving loach. We also field-tested similarly tagged populations within a river-based irrigation system of Sado Island, Japan. Two different sized tags (long, 12.5 × 2.07 mm²; short, 8.5 × 2.12 mm²) were compared by using antenna loggers which detected fish movement through gates and automatically logged tagged fish’s tag IDs and timestamps. By comparing logged data and actual fish collection surveys both below and above the gates, 77% and 30% of actual loach movements were confirmed to have been successfully logged for the long and short tags, respectively. The awl insertion technique with the longer tag is therefore recommended for use in similar studies of smaller fish species.     

Identification of Highly Selective and Potent Histone Deacetylase 3 Inhibitors Using Click Chemistry-Based Combinatorial Fragment Assembly

To find histone deacetylase 3 (HDAC3)-selective inhibitors, a series of 504 candidates was assembled using “click chemistry”, by reacting nine alkynes bearing a zinc-binding group with 56 azide building blocks in the presence of Cu(I) catalyst. Screening of the 504-member triazole library against HDAC3 and other HDAC isozymes led to the identification of potent and selective HDAC3 inhibitors T247 and T326. These compounds showed potent HDAC3 inhibition with submicromolar IC₅₀s, whereas they did not strongly inhibit other isozymes. Compounds T247 and T326 also induced a dose-dependent selective increase of NF-κB acetylation in human colon cancer HCT116 cells, indicating selective inhibition of HDAC3 in the cells. In addition, these HDAC3-selective inhibitors induced growth inhibition of cancer cells, and activated HIV gene expression in latent HIV-infected cells. These findings indicate that HDAC3-selective inhibitors are promising candidates for anticancer drugs and antiviral agents. This work also suggests the usefulness of the click chemistry approach to find isozyme-selective HDAC inhibitors.     

Expression of Cell Competition Markers at the Interface between p53 Signature and Normal Epithelium in the Human Fallopian Tube

There is a growing body of evidence regarding cell competition between normal and mutant mammalian cells, which suggest that it may play a defensive role in the early phase of carcinogenesis. In vitro study in the past has shown that overexpression of vimentin in normal epithelial cells at the contact surface with transformed cells is essential for the cell competition involved in epithelial defense against cancer. In this study, we attempted to examine cell competition in human tissue in vivo by investigating surgically resected human fallopian tubes that contain p53 signatures and serous tubal intraepithelial lesions (STILs), a linear expansion of p53-immunopositive/TP53 mutant tubal epithelial cells that are considered as precursors of pelvic high grade serous carcinoma. Immunofluorescence double staining for p53 and the cell competition marker vimentin was performed in 21 sections of human fallopian tube tissue containing 17 p53 signatures and 4 STILs. The intensities of vimentin expression at the interface between p53-positive cells at the end of the p53 signature/STIL and adjacent p53-negative normal tubal epithelial cells were compared with the background tubal epithelium. As a result, the average vimentin intensity at the interfaces relative to the background intensity was 1.076 (95% CI, 0.9412 – 1.211 for p53 signature and 0.9790 (95% CI, 0.7206 – 1.237) for STIL. Thus, it can be concluded that overexpression of the cell competition marker vimentin are not observed in human tissue with TP53 alterations.     

Identifying indicator species for bryophyte conservation in fragmented forests

It is important to conserve bryophyte diversity in fragmented forests, due to the vulnerability of this group to environmental change. In this study, the utility of bryophyte functional groups (taxonomic classes, substrate classes, and life-forms) was examined as indicators for planning urban area conservation of bryophyte diversity. The study sites comprised 27 fragmented forests in Kyoto City, Japan. Using linear regression models, it was found that the species richness of almost all functional groups was positively correlated with patch size. Furthermore, two types of bryophyte life-form (fans and thalloid mats) were significantly correlated with environmental factors considered important for conservation planning. The species richness of fan bryophytes was positively correlated with the presence of natural forest patches and was negatively correlated with distance from mountainous areas. Similarly, the species richness of thalloid mat bryophytes was negatively correlated with maintenance practices. These results may be explained by the vulnerability of these two bryophyte groups to environmentally caused drought stress, accompanied by decreasing patch size, maintenance practices, disturbance, and/or loss of natural vegetation. Considering that drought stress represents a major threat to bryophyte diversity in fragmented forests, the species richness of hygrophilous life-forms (e.g., fans and thalloid mats) may be used as an indicator of fragmented forests that are less affected by drought stress, and these species should be preferentially conserved to maintain high levels of bryophyte diversity.     

Age- and Sex-Dependency of Laser Speckle Flowgraphy Measurements of Optic Nerve Vessel Microcirculation

Purpose

To investigate the relationship between various characteristics of a normal population and laser speckle flowgraphy (LSFG) measurements of mean blur rate (MBR) in the optic nerve head (ONH).

Methods

A total of 189 eyes of 189 normal subjects (93 male, 96 female, mean age 45 ± 14 years old, age range: 20–72) without any history of hypertension, hyperlipidemia or diabetes were enrolled. ONH microcirculation was measured with LSFG and overall MBR (MA), vessel-area MBR (MV), and tissue-area MBR (MT) were derived from these measurements. The statistical association of these measurements with characteristics such as sex, age, intraocular pressure (IOP) and systolic blood pressure (SBP) was then determined.

Results

There was a trend towards decreased IOP and MV and increased SBP with age (P = 0.002, P = 0.035, and P = 0.006, respectively). Furthermore, IOP, MV and SBP were correlated with age (r = -0.23, P = 0.011; r = -0.24, P < 0.001; and r = 0.30, P < 0.001, respectively). Separate multiple regression analyses of independent contributing factors revealed that sex and IOP contributed to MA (P < 0.001 and P = 0.002, respectively), sex, IOP, and age contributed to MV (P < 0.001, P = 0.003, and P = 0.024, respectively), while only IOP contributed to MT (P = 0.003).

Conclusion

In a normal population, MBR was affected by IOP in both the large vessel and capillary areas of the ONH, but not by SBP. MV was also affected by age and sex, while MT was stable independent of age or sex.     

ESA Hyporesponsiveness Is Associated with Adverse Events in Maintenance Hemodialysis (MHD) Patients,But Not with Iron Storage

Objective

It has been reported that hyporesponsiveness to erythropoiesis-stimulating agent (ESA) is associated with adverse events in patients on maintenance hemodialysis (MHD). However, it has not been determined whether higher iron storage is associated with an improved response, including better survival, to ESA.

Design and Method

We measured serum ferritin, hemoglobin (Hb), and transferrin saturation (TSAT) levels every three months for two years in 1,095 MHD patients. The weekly dose of ESA to Hb ratio was also calculated as an index of ESA responsiveness (ERI).

Results

A significant correlation (p<0.001, R = 0.89) between ferritin and Hb was only observed in the patients with ferritin levels <50 ng/mL. High-dose (≥50 mg/week) intravenous iron administration, female sex, low serum albumin, and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker use were significant predictors of a high ERI value (>280); however, serum ferritin and TSAT levels did not predict a higher ERI. In the time-dependent Cox hazard model, the risk for a composite event in the patients with a high ERI (≥280) and a high ferritin level (≥100 ng/mL) was significantly greater (hazard ratio [HR], 2.09, P = 0.033) than that for patients with a high ERI and a low ferritin (<100 ng/mL) level.

Conclusion

Hb was dependent upon ferritin levels in patients with ferritin levels <50 ng/mL but not in patients with ferritin levels ≥50 ng/mL. Patients with hyporesponsiveness to ESA had a greater risk of composite events, but ERI was unrelated to iron storage.     

Role of IGFBP7 in Diabetic Nephropathy: TGF-β1 Induces IGFBP7 via Smad2/4 in Human Renal Proximal Tubular Epithelial Cells

Tubular injury is one of the important determinants of progressive renal failure in diabetic nephropathy (DN), and TGF-β1 has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. The aim of this study was to identify novel therapeutic target molecules that play a role in the tubule damage of DN. We used an LC-MS/MS-based proteomic technique and human renal proximal epithelial cells (HRPTECs). Urine samples from Japanese patients with type 2 diabetes (n = 46) were used to quantify the candidate protein. Several proteins in HRPTECs in cultured media were observed to be driven by TGF-β1, one of which was 33-kDa IGFBP7, which is a member of IGFBP family. TGF-β1 up-regulated the expressions of IGFBP7 mRNA and protein in a dose- and time-dependent fashion via Smad2 and 4, but not MAPK pathways in HRPTECs. In addition, the knockdown of IGFBP7 restored the TGF-β1-induced epithelial to mesenchymal transition (EMT). In the immunohistochemical analysis, IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells in diabetic kidney. Urinary IGFBP7 levels were significantly higher in the patients with macroalbuminuria and were correlated with age (r = 0.308, p = 0.037), eGFR (r = −0.376, p = 0.01), urinary β₂-microglobulin (r = 0.385, p = 0.008), and urinary N-acetyl-beta-D-glucosaminidase (NAG) (r = 0.502, p = 0.000). A multivariate regression analysis identified urinary NAG and age as determinants associated with urinary IGFBP7 levels. In conclusion, our data suggest that TGF-β1 enhances IGFBP7 via Smad2/4 pathways, and that IGFBP7 might be involved in the TGF-β1-induced tubular injury in DN.