Effects of Supplementation with Oregano Essential Oil during Late Gestation and Lactation on Serum Metabolites, Antioxidant Capacity and Fecal Microbiota of Sows.

A total of 20 healthy white × landrace sows were evenly and randomly divided into two groups, and fed basal diets unsupplemented or supplemented with 500 g/t Meriden-Stim® from day 100 of gestation until day 21 of lactation. Serum and fecal samples were collected from the sows on the final day for subsequent analysis. Compared to the control group, there were no significant differences in the sows' performances; however, an increase was observed in the piglets' weight at weaning (p = 0.08). Moreover, oregano essential oil (OEO) significantly reduced the levels of urea (UREA) (p < 0.01), total cholesterol (TC) (p < 0.05), low-density lipoprotein (LDL-C) (p < 0.05) and alanine aminotransferase (ALT) (p < 0.05) in serum. In terms of antioxidant indexes in serum, the catalase (CAT) and glutathione (GSH) levels showed significant increases (p < 0.05) while the malondialdehyde (MDA) level exhibited a decrease tendency (p = 0.09). 16S rRNA analysis identified the specific bacteria taxa in feces. OEO significantly decreased the relative abundance of Proteobacteria and Actinobacteria at the phylum level (p < 0.05). At the genus level, OEO significantly increased the relative abundance of Lactobacillus and Prevotellaceae UCG 003 and UCG 005, while decreasing that of Escherichia-Shigella (p < 0.05). Taken together, OEO supplementation in maternal diets during late gestation and lactation improved serum metabolites, antioxidant capacity and regulated the intestinal-flora balance of sows, thereby tending to increase the piglets' weight at weaning.

Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels.

Many voltage-dependent ion channels are regulated by accessory proteins. We recently reported powerful regulation of Kv1.2 potassium channels by the amino acid transporter Slc7a5. In this study, we report that Kv1.1 channels are also regulated by Slc7a5, albeit with different functional outcomes. In heterologous expression systems, Kv1.1 exhibits prominent current enhancement ('disinhibition') with holding potentials more negative than -120 mV. Knockdown of endogenous Slc7a5 leads to larger Kv1.1 currents and strongly attenuates the disinhibition effect, suggesting that Slc7a5 regulation of Kv1.1 involves channel inhibition that can be reversed by supraphysiological hyperpolarizing voltages. We investigated chimeric combinations of Kv1.1 and Kv1.2, demonstrating that exchange of the voltage-sensing domain controls the sensitivity and response to Slc7a5, and localize a specific position in S1 with prominent effects on Slc7a5 sensitivity. Overall, our study highlights multiple Slc7a5-sensitive Kv1 subunits, and identifies the voltage-sensing domain as a determinant of Slc7a5 modulation of Kv1 channels.

L-type amino acid transporter 1, LAT1, in growth hormone-producing pituitary tumor cells.

Pituitary tumors (PTs) can cause significant mortality and morbidity due to limited therapeutic options. L-type amino acid transporters (LATs), in particular, the LAT1 isoform, is expressed in a variety of tumor cells. Pharmacological inhibition or genetic ablation of LAT1 can suppress leucine transport into cancer cells, resulting in suppression of cancer cell growth. However, roles of LAT1 in PTs have not been elucidated. Therefore, we assessed LAT1 expression in PTs and evaluated a LAT1-specific inhibitor, JPH203, on rat somatomammotroph tumor cells, GH4 cells. GH4 cells dominantly express LAT1 mRNA rather than other LAT isoforms, whereas LAT2 transcripts were most abundant in normal rat pituitary tissues. JPH203 inhibited leucine uptake and cell growth in GH4 cells in a concentration-dependent manner, and appeared to be independent of the mechanistic target, the rapamycin pathway. Although JPH203 did not induce apoptosis, it suppressed growth hormone production in GH4 cells. Also, genetic downregulation of LAT1 showed similar effects on cell growth and hormone production. These results indicated that restriction of LAT1 substrates by JPH203 modulated both cell growth and hormone production. In conclusion, LAT1 may be a new therapeutic target for PTs because its inhibition leads to suppression of cell growth as well as hormone production. JPH203 may represent a promising drug for clinical use in patients with PTs, with the potential of hormonal control and tumor suppression.

Synergic "Click" Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells.

Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 µM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low µM concentrations, in neutral buffer, with a rate constant of 9 M-1 s-1 as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 µM, thus confirming the potential of this system in biological applications.

MiR-124 acts as a tumor suppressor by inhibiting the expression of sphingosine kinase 1 and its downstream signaling in head and neck squamous cell carcinoma.

By analyzing the expression profile of microRNAs in head and neck squamous cell carcinomas (HNSCC), we found that the expression level of miR-124 was 4.59-fold lower in tumors than in normal tissues. To understand its functions, we generated a miR-124-expressing subline (JHU-22miR124) and a mock vector-transfected subline (JHU-22vec) by transfecting the mimic of miR-124 into JHU-22 cancer cells. Restored expression of miR-124 in JHU-22miR124 cells led to reduced cell proliferation, delayed colony formation, and decreased tumor growth, indicating a tumor-suppressive effect of miR-124. Subsequent target search revealed that the 3'-UTR of SphK1 mRNA carries a complementary site for the seed region of miR-124. SphK1 was also detected to be overexpressed in HNSCC cell lines, but down-expressed in JHU-22miR124 cells and tumor xenografts. These results suggest that SphK1 is a target of miR-124. To confirm this finding, we constructed a 3'-UTR-Luc-SphK1 vector and a binding site-mutated luciferase reporter vector. Co-transfection of 3'-UTR-Luc-SphK1 with miR-124 expression vector exhibited a 9-fold decrease in luciferase activity compared with mutated vector, suggesting that miR-124 inhibits SphK1 activity directly. Further studies on downstream signaling demonstrated accumulation of ceramide, increased expression of the pro-apoptotic Bax, BAD and PARP, decreased expression of the anti-apoptotic Bcl-2 and Bcl-xL, and enhanced expression of cytochrome c and caspase proteins in JHU-22miR124 compared with JHU-22vec cells and tumor xenografts. We conclude that miR-124 acts as a tumor suppressor in HNSCC by directly inhibiting SphK1 activity and its downstream signals.

Peptide microarray patterning for controlling and monitoring cell growth.

The fate of cells is influenced by their microenvironment and many cell types undergo differentiation when stimulated by extracellular cues, such as soluble growth factors and the insoluble extracellular matrix (ECM). Stimulating differentiation by insoluble or "immobilized" cues is a particularly attractive method because it allows for the induction of differentiation in a spatially-defined cohort of cells within a larger subpopulation. To improve the design of de novo screening of such insoluble factors, we describe a methodology for producing high-density peptide microarrays suitable for extended cell culture and fluorescence microscopy. As a model, we used a murine mammary gland cell line (NMuMG) that undergoes epithelial to mesenchymal transition (EMT) in response to soluble transforming growth factor beta (TGF-ß) and surface-immobilized peptides that target TGF-ß receptors (TGFßRI/II). We repurposed a well-established DNA microarray printing technique to produce arrays of micropatterned surfaces that displayed TGFßRI/II-binding peptides and integrin binding peptides. Upon long-term culture on these arrays, only NMuMG cells residing on EMT-stimulating areas exhibited growth arrest and decreased E-cadherin expression. We believe that the methodology created in this report will aid the development of peptide-decorated surfaces that can locally stimulate defined cell surface receptors and control EMT and other well-characterized differentiation events. STATEMENT OF SIGNIFICANCE: Scope of work: This manuscript aims to accelerate the development of instructive biomaterials decorated with specific ligands that target cell-surface receptors and induce specific differentiation of cells upon contact. These materials can be used for practical applications, such as fabricating synthetic materials for large scale, stem cell culture, or investigating differentiation and asymmetric division in stem cells. Specifically, in this manuscript, we repurposed a DNA microarray printer to produce microarrays of peptide-terminated self-assembled monolayers (SAMs). To demonstrate the utility of these arrays in phenotypic assays with mammalian cells, we monitored the induction of epithelial to mesenchymal transition (EMT) in murine mammary epithelial cells using specific peptide ligands printed on these arrays. Novelty: We, and others, have published several strategies for producing peptide-based arrays suitable for long-term phenotypic assays. Many reports relied on patterning steps that made adaptation difficult. The use of a DNA microarray printer as the sole production tool simplified the production of peptide microarrays and increased the throughput of this technology. We confirmed that simplification in production did not compromise the performance of the array; it is still possible to study short-term adhesion, long-term growth, and complex phenotypic responses, such as EMT, in the cells. EMT was studied using immunofluorescent staining after four days of culture. IMPACT: This methodology will serve as a foundation for future screening of instructive biomaterials in our research group. As DNA printers are broadly available in academic institutions, we foresee rapid adaptation of this approach by academic researchers.

Enforced expression of miR-101 inhibits prostate cancer cell growth by modulating the COX-2 pathway in vivo.

It is commonly agreed that there is an association of chronic inflammation with tumorigenesis. COX-2, a key regulator of inflammation-producing prostaglandins, promotes cell proliferation and growth; thus, overexpression of COX-2 is often found in tumor tissues. Therefore, a better understanding of the regulatory mechanism(s) of COX-2 could lead to novel targeted cancer therapies. In this study, we investigated the mechanism of microRNA-101 (miR-101)-regulated COX-2 expression and the therapeutic potential of exogenous miR-101 for COX-2-associated cancer. A stably expressing exogenous miR-101 prostate cancer cell line (BPH1(CmiR101)) was generated by using lentiviral transduction as a tool for in vitro and in vivo studies. We found that miR-101 inhibited COX-2 posttranscriptional expression by directly binding to the 3'-untranslated region (3'-UTR) of COX-2 mRNA. The regulatory function of miR-101 was also confirmed by using antisense DNA. As a result, exogenous miR-101 is able to effectively suppress the growth of cultured prostate cancer cells and prostate tumor xenografts. The average tumor weight was significantly lower in the BPH1(CmiR101) group (0.22 g) than the BPH1(Cvec) group (0.46 g). Expression levels of the cell growth regulators, such as cyclin proteins, PCNA (proliferating cell nuclear antigen), EGFR (epidermal growth factor receptor), were also studied. In conclusion, COX-2 is a direct target in miR-101 regulation of posttranscription. Exogenous miR-101 suppresses the proliferation and growth of prostate cancer cells in vitro and in vivo. These data suggest that exogenous miR-101 may provide a new cancer therapy by directly inhibiting COX-2 expression.

Improvement of prostate cancer detection by integrating the PSA test with miRNA expression profiling.

Prostate-specific antigen (PSA) test is limited in prostate cancer diagnosis due to its inaccuracy. A new approach which integrates the PSA test with miRNA profiling was investigated to improve prostate cancer diagnosis. Six prostate cancer-related miRNAs (miR-16, -21, -34c, -101, -125b, -141) were tested in five cultured prostate cell lines and 20 human prostate specimens. We found that the miRNA expression profiles were significantly different between nontumorigenic and tumorigenic cell lines and specimens. Positive predictive value analysis of prostate cancer was increased from 40% to 87.5% by integrating patient PSA blood levels with miR-21 and miR-141 profiles.

Combination effects of salvianolic acid B with low-dose celecoxib on inhibition of head and neck squamous cell carcinoma growth in vitro and in vivo.

Head and neck squamous cell carcinoma (HNSCC) development is closely associated with inflammation. Cyclooxygenase-2 (COX-2) is an important mediator of inflammation. Therefore, celecoxib, a selective inhibitor of COX-2, was hailed as a promising chemopreventive agent for HNSCC. Dose-dependent cardiac toxicity limits long-term use of celecoxib, but it seems likely that this may be diminished by lowering its dose. We found that salvianolic acid B (Sal-B), isolated from Salvia miltiorrhiza Bge, can effectively suppress COX-2 expression and induce apoptosis in a variety of cancer cell lines. In this study, we report that combination of Sal-B with low-dose celecoxib results in a more pronounced anticancer effect in HNSCC than either agent alone. The combination effects were assessed in four HNSCC cell lines (JHU-06, JHU-011, JHU-013, and JHU-022) by evaluating cell viability, proliferation, and tumor xenograft growth. Cell viability and proliferation were significantly inhibited by both the combined and single-agent treatments. However, the combination treatment significantly enhanced anticancer efficacy in JHU-013 and JHU-022 cell lines compared with the single treatment regimens. A half-dose of daily Sal-B (40 mg/kg/d) and celecoxib (2.5 mg/kg/d) significantly inhibited JHU-013 xenograft growth relative to mice treated with a full dose of Sal-B or celecoxib alone. The combination was associated with profound inhibition of COX-2 and enhanced induction of apoptosis. Taken together, these results strongly suggest that combination of Sal-B, a multifunctional anticancer agent, with low-dose celecoxib holds potential as a new preventive strategy in targeting inflammatory-associated tumor development.

Salvianolic acid B inhibits growth of head and neck squamous cell carcinoma in vitro and in vivo via cyclooxygenase-2 and apoptotic pathways.

Overexpression of cyclooxygenase-2 (COX-2) in oral mucosa has been associated with increased risk of head and neck squamous cell carcinoma (HNSCC). Celecoxib is a nonsteroidal anti-inflammatory drug, which inhibits COX-2 but not COX-1. This selective COX-2 inhibitor holds promise as a cancer preventive agent. Concerns about cardiotoxicity of celecoxib, limits its use in long-term chemoprevention and therapy. Salvianolic acid B (Sal-B) is a leading bioactive component of Salvia miltiorrhiza Bge, which is used for treating neoplastic and chronic inflammatory diseases in China. The purpose of this study was to investigate the mechanisms by which Sal-B inhibits HNSCC growth. Sal-B was isolated from S. miltiorrhiza Bge by solvent extraction followed by 2 chromatographic steps. Pharmacological activity of Sal-B was assessed in HNSCC and other cell lines by estimating COX-2 expression, cell viability and caspase-dependent apoptosis. Sal-B inhibited growth of HNSCC JHU-022 and JHU-013 cells with IC(50) of 18 and 50 microM, respectively. Nude mice with HNSCC solid tumor xenografts were treated with Sal-B (80 mg/kg/day) or celecoxib (5 mg/kg/day) for 25 days to investigate in vivo effects of the COX-2 inhibitors. Tumor volumes in Sal-B treated group were significantly lower than those in celecoxib treated or untreated control groups (p < 0.05). Sal-B inhibited COX-2 expression in cultured HNSCC cells and in HNSCC cells isolated from tumor xenografts. Sal-B also caused dose-dependent inhibition of prostaglandin E(2) synthesis, either with or without lipopolysaccharide stimulation. Taken together, Sal-B shows promise as a COX-2 targeted anticancer agent for HNSCC prevention and treatment.

Visualizing head and neck tumors in vivo using near-infrared fluorescent transferrin conjugate.

Transferrin receptor (TfR) is overexpressed in human head and neck squamous cell carcinomas (HNSCCs). This study was carried out to investigate the feasibility of imaging HNSCC by targeting TfR using near-infrared fluorescent transferrin conjugate (TfNIR). Western blot analysis of four HNSCC cell lines revealed overexpression of TfR in all four lines compared with that in normal keratinocytes (OKFL). Immunocytochemistry further confirmed the expression of TfR and endocytosis of TfNIR in JHU-013 culture cells. Following intravenous administration of TfNIR (200 microL, 0.625 microg/microL), fluorescent signal was preferentially accumulated in JHU-013 tumor xenografts grown in the lower back (n=14) and oral base tissues (n=4) of nude mice. The signal in tumors was clearly detectable as early as 10 minutes and reached the maximum at 90 to 120 minutes postinjection. The background showed an increase, followed by a decrease at a much faster pace than tumor signal. A high fluorescent ratio of the tumor to muscle was obtained (from 1.42 to 4.15 among tumors), usually achieved within 6 hours, and correlated with the tumor size (r=.74, p=.002). Our results indicate that TfR is a promising target and that Tf(NIR)-based optical imaging is potentially useful for noninvasive detection of early HNSCC in the clinic.

Hyperhomocysteinemia and the MTHFR C677T mutation in Budd-Chiari syndrome.

Hyperhomocysteinemia (HH) is a factor that predisposes individuals to thrombosis, and the C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) is known to give increased plasma homocysteine. However, little is known about their roles in Budd-Chiari syndrome (BCS). This study evaluated the roles of HH and the MTHFR C677T mutation in patients with BCS. We compared 41 BCS patients with 80 sex- and age-matched healthy controls. The mean plasma homocysteine level was significantly higher in patients with BCS (20.15 +/- 5.78 micromol/L) compared with normal controls (15.80 +/- 6.58 micromol/L), P < 0.01. HH (>19.5 micromol/L in men and >15.0 micromol/L in women) was detected in 15 (36.59%) patients and in 14 (17.5%) controls (odds ratio [OR], 2.72; 95% confidence internal [CI], 1.17-6.32). The prevalence of the mutated MTHFR 677TT genotype and the 677T allele in normal controls was 10.0% and 31.3%, respectively. The mutant 677T homozygotes and alleles were more frequent in patients with BCS than in controls (22.0% vs. 10.0%, 0.025 < P < 0.05; 45.1% vs. 31.3%, 0.025 < P < 0.05). The relative risk of BCS among the carriers of 677TT was significantly increased (OR, 3.3; 95% CI, 1.1-10.0). The mutant MTHFR heterozygous 677C/T carriers were not significantly increased in patients with BCS compared with controls (46.3% vs. < 2.5%, P > 0.05). The relative risk OR of BCS among carriers of 677C/T was 1.6 (95% CI, 0.7-3.6). This study suggests that both HH and the homozygous C677T mutation in the MTHFR gene are important risk factors of BCS.