Search

We had shown Nw-hydroxy-l-arginine (NOHA) as a promising blood-based biomarker for estrogen-receptor-negative (ER−) breast cancer (BC) that differentiates ER− BC based on grade and molecular phenotype. In this in vitro study, we assessed the metabolic relevance for ER− BC-specific NOHA modulation and correlated them with NOHA regulatory responses. This study aids future NOHA clinical utility in ER− BC diagnosis and therapy management and would prove useful for potential drug discovery and development process. more...

Significance In laboratory experiments, Wolbachia ( w Mel strain)-infected Aedes aegypti are refractory to disseminated arboviral infections. Yet previous characterizations of w Mel-mediated blocking have not considered several biologically and ecologically important factors likely to influence the virus–mosquito interaction. After direct feeding on 141 viremic dengue patients, we demonstrate w Mel lowers dengue virus (DENV) transmission potential and lengthens the extrinsic incubation period. Subsequently, using established field populations of wild-type and w Mel-infected Ae. aegypti , we compared field- versus laboratory-rearing conditions on mosquito susceptibility to disseminated DENV infection. The magnitude of w Mel-mediated virus blocking was even greater when mosquitoes developed under field conditions. These clinically and ecologically relevant findings support Wolbachia introgression into Ae. aegypti populations as a biocontrol method to reduce the transmission of DENV and other arboviruses. more...

In our prior study we identified N w-hydroxy-l-arginine (NOHA) as a simple, yet sensitive indicator for estrogen negative (ER−) breast cancer early-prognosis, but not estrogen positive (ER+), and to offer ethnic selectivity for ER− detection. However, the ability of NOHA to assess ER− breast tumor based on disease progression, and tumor severity needs further delineation. Also, the overall NOHA storage stability needs to be validated. To assess the NOHA predictive capability based on disease progression, ER−/ER+ 3D-spheroids (from breast tumor cell lines of human origin) were cultured for 10 weeks. We found only ER− 3D-spheroid cultured for 10 weeks to show a gradual reduction in NOHA (both in culture medium and 3D-spheroid lysates) that correlated with a progressive increase in cellular NOS2 expression and NOS2 activity (measured as total nitrites). We additionally identified the NOHA-NOS2 correlation to be ethnically selective between ER− African American versus ER− Caucasian groups. Interestingly, such NOHA reduction was observed earlier in ER− culture medium (viz., after week 1) than from ER− 3D-spheroids lysates (viz., at the end of 3 weeks). When categorized based on 3D-spheroid grade, we found a ≥ 68% NOHA reduction in ER− spheroids that were ≤ 3 weeks old, that was categorized as “low-grade” (based on tumor size ≤ 250 µm, and with cellular characteristics identical to healthy cells). A substantial reduction in NOHA of ≥ 87% occurred with ER− 3D-spheroids grown for 6 weeks, which were categorized as “intermediate-grade” (with tumor size of ≥ 400 µm, and with less characteristic similarity to control spheroids). These in vitro findings thus suggest a distinct correlation between NOHA reduction and ER− tumor grade. Such distinctive correlation between NOHA and ER− tumor grade was additionally observed in de-identified clinical samples where a onefold higher reduction in NOHA occurred in grade-2 than with grade-1 de-identified patient plasma (when compared with control), and such correlation offered ethnic selectivity between ER− African American and ER− Caucasian groups. Of additional interest, when NOHA overall storage stability was assessed by incubating patient plasma and culture medium spiked with 75 pg/ml NOHA at multiple incubation temperatures and time-points, we found NOHA to maintain its stability for up to 6 weeks in culture medium and for 7 days in plasma at 4 °C and below. These results thus provide the first evidence of NOHA as a stable indicator to monitor ER− disease progression and tumor severity in ethnically distinctive populations. more...

We found a unit error with our LC-MS lower limit of quantitation (LLOQ) measurement in the Amino Acids Journal.

Fibroblast growth factor 21 (FGF21) is an important metabolic regulator expressed predominantly in the liver. In this study, we evaluated the role of LY2405319, an analogue of FGF21, in hepatic stellate cell (HSC) activation and in a methionine and choline-deficient (MCD)-diet induced mouse model of liver fibrosis. During liver injury, HSCs trans-differentiate into activated myofibroblasts which produce alpha-smooth muscle actin (α-SMA) and become a major cell type in hepatic fibrogenesis. Succinate and succinate receptor (GPR91) signaling has emerged as a regulator to promote α-SMA production in MCD diet- induced mice. Treatment with palmitate or MCD medium on LX-2 cells (HSCs) increased succinate concentration in the conditioned medium and cell lysate of LX-2 cells and increased production of GPR91 and α-SMA. However, LY2405319 administration ameliorates palmitate or MCD media-induced succinate production and decreases over-expression of GPR91 and α-SMA in LX2-cells. In an in vivo study, the MCD diet treatment caused increased steatohepatitis and liver fibrosis compared with the control diet in mice. Administration of LY2405319 improved steatohepatitis ameliorated GPR91 and α -SMA production in the liver, decreased succinate concentration in both liver and serum of MCD diet -induced mice. These results suggest that FGF21 reduces production of α-SMA by inhibiting the succinate-GPR91 pathway. We conclude that FGF21 acts as an inhibitor of the succinate-GPR91 pathway to control liver fibrosis. This suggests that FGF21 has therapeutic potential for treating liver fibrogenesis. more...

Background Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix. Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA). Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified. Methods The immortalized human HSCs, LX-2 cells, were used for the in vitro study. For the in vivo study, male C57BL/J6 mice were randomly divided into 3 groups and were fed with a methionine-choline-deficient diet (MCD diet group) as a nonalcoholic steatohepatitis (NASH) mouse model with or without 0.1% metformin for 12 weeks, or were fed a control methionine-choline-sufficient diet (MCS diet group). Results In our study, metformin and 5-aminoimidazole-4-carboxamide 1-β- d -ribofuranoside (AICAR), which is an analog of adenosine monophosphate, were shown to suppress α-SMA expression via enhanced phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of succinate-GPR91 signaling in activated LX-2 cells induced by palmitate- or succinate. Metformin and AICAR also reduced succinate concentration in the cell lysates when LX-2 cells were treated with palmitate. Moreover, metformin and AICAR reduced interleukin-6 and, transforming growth factor-β1 production in succinate-treated LX-2 cells. Both metformin and AICAR inhibited succinate-stimulated HSC proliferation and cell migration. Mice fed a MCD diet demonstrated increased steatohepatitis and liver fibrosis compared to that of mice fed control diet. Metformin ameliorated steatohepatitis, liver fibrosis, inflammatory cytokine production and decreased α -SMA and GPR91expression in the livers of the MCD diet-fed mice. Conclusion This study shows that metformin can attenuate activation of HSCs by activating the AMPK pathway and inhibiting the succinate-GPR91 pathway. Metformin has therapeutic potential for treating steatohepatitis and liver fibrosis. more...

The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL, which belong to the tumor necrosis factor (TNF) receptor-ligand family, mediate osteoclastogenesis. The crystal structure of the RANKL ectodomain (eRANKL) in complex with the RANK ectodomain (eRANK) combined with biochemical assays of RANK mutants indicated that three RANK loops (Loop1, Loop2, and Loop3) bind to the interface of a trimeric eRANKL. Loop3 is particularly notable in that it is structurally distinctive from other TNF-family receptors and forms extensive contacts with RANKL. The disulfide bond (C125-C127) at the tip of Loop3 is important for determining the unique topology of Loop3, and docking E126 close to RANKL, which was supported by the inability of C127A or E126A mutants of RANK to bind to RANKL. Inhibitory activity of RANK mutants, which contain loops of osteoprotegerin (OPG), a soluble decoy receptor to RANKL, confirmed that OPG shares the similar binding mode with RANK and OPG. Loop3 plays a key role in RANKL binding. Peptide inhibitors designed to mimic Loop3 blocked the RANKL-induced differentiation of osteoclast precursors, suggesting that they could be developed as therapeutic agents for the treatment of osteoporosis and bone-related diseases. Furthermore, some of the RANK mutations associated with autosomal recessive osteopetrosis (ARO) resulted in reduced RANKL-binding activity and failure to induce osteoclastogenesis. These results, together with structural interpretation of eRANK-eRANKL interaction, provided molecular understanding for pathogenesis of ARO. more...