Insulin Promotes Glucose Consumption via Regulation of miR-99a/mTOR/PKM2 Pathway

Insulin is known to regulate multiple cellular functions and is used for the treatment of diabetes. MicroRNAs have been demonstrated to be involved in many human diseases, including Type 2 diabetes. In this study, we showed that insulin decreased miR-99a expression levels, but induced glucose consumption and lactate production, and increased the expression of mTOR, HIF-1α and PKM2 in HepG2 and HL7702 cells. Forced expression of miR-99a or rapamycin treatment blocked insulin-induced PKM2 and HIF-1α expression, and glucose consumption and lactate production. Meanwhile, knockdown of HIF-1α inhibited PKM2 expression and insulin-induced glucose consumption. Taken together, these findings will reveal the role and mechanism ofinsulin in regulating glycolytic activities via miR-99a/mTOR.     

Endothelin Receptor A Blockade Is an Ineffective Treatment for Adriamycin Nephropathy

Endothelin is a vasoconstricting peptide that plays a key role in vascular homeostasis, exerting its biologic effects via two receptors, the endothelin receptor A (ETA) and endothelin receptor B (ETB). Activation of ETA and ETB has opposing actions, in which hyperactive ETA is generally vasoconstrictive and pathologic. Selective ETA blockade has been shown to be beneficial in renal injuries such as diabetic nephropathy and can improve proteinuria. Atrasentan is a selective pharmacologic ETA blocker that preferentially inhibits ETA activation. In this study, we evaluated the efficacy of ETA blockade by atrasentan in ameliorating proteinuria and kidney injury in murine adriamycin nephropathy, a model of human focal segmental glomerulosclerosis. We found that ETA expression was unaltered during the course of adriamycin nephropathy. Whether initiated prior to injury in a prevention protocol (5 mg/kg/day, i.p.) or after injury onset in a therapeutic protocol (7 mg/kg or 20 mg/kg three times a week, i.p.), atrasentan did not significantly affect the initiation and progression of adriamycin-induced albuminuria (as measured by urinary albumin-to-creatinine ratios). Indices of glomerular damage were also not improved in atrasentan-treated groups, in either the prevention or therapeutic protocols. Atrasentan also failed to improve kidney function as determined by serum creatinine, histologic damage, and mRNA expression of numerous fibrosis-related genes such as collagen-I and TGF-β1. Therefore, we conclude that selective blockade of ETA by atrasentan has no effect on preventing or ameliorating proteinuria and kidney injury in adriamycin nephropathy.     

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment

High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions – provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results.     

Hydrophylita (Lutzimicron) emporos Shih & Polaszek (Hymenoptera: Trichogrammatidae) from Taiwan,Parasitising Eggs,and Phoretic on Adults,of the Damselfly Psolodesmus mandarinus mandarinus (Zygoptera: Calopterygidae)

Hydrophylita emporos n. sp. reared from eggs of Psolodesmus mandarinus mandarinus McLachlan (Zygoptera: Calopterygidae) in Taiwan is described. This is the first species of Hydrophylita to be described from the Old World, and the first record of phoresy in the genus. Adult females were observed aggregating at the base of the female damselfly’s abdomen. When the damselfly begins ovipositing, females move to the tip of the abdomen, enter the water and quickly locate eggs for parasitising. The article contains links to video footage of this process.     

p16 Stimulates CDC42-Dependent Migration of Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Tumor dissemination to the extra-hepatic region of the portal vein, lymph nodes, lungs or bones contributes to the high mortality seen in HCC; yet, the molecular mechanisms responsible for HCC metastasis remain unclear. Prior studies have suggested a potential link between accumulated cytoplasm-localized p16 and tumor progression. Here we report that p16 enhances metastasis-associated phenotypes in HCC cells – ectopic p16 expression increased cell migration in vitro, and lung colonization after intravenous injection, whereas knockdown of endogenous p16 reduced cell migration. Interestingly, analysis of p16 mutants indicated that the Cdk4 interaction domain is required for stimulation of HCC cell migration; however, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. Intriguingly, we found that in p16-positive HCC samples, p16 protein is predominantly localized in the cytoplasm. In addition, we identified a potential role for nuclear-cytoplasmic shuttling in p16-stimulated migration, consistent with the predominantly cytoplasmic localization of p16 in IHC-positive HCC samples. Finally, we determined that p16-stimulated cell migration requires the Cdc42 GTPase. Our results demonstrate for the first time a pro-migratory role for p16, and suggest a potential mechanism for the observed association between cytoplasmic p16 and tumor progression in diverse tumor types.     

Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model

Objective

Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.

Methods

Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO₂ cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.

Results

Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.

Conclusions

These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.     

Reduction of Decoy Receptor 3 Enhances TRAIL-Mediated Apoptosis in Pancreatic Cancer

Most human pancreatic cancer cells are resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. However, the mechanisms by which pancreatic cancer cells utilize their extracellular molecules to counteract the proapoptotic signaling mediated by the TNF family are largely unknown. In this study, we demonstrate for the first time that DcR3, a secreted decoy receptor that malignant pancreatic cancer cells express at a high level, acts as an extracellular antiapoptotic molecule by binding to TRAIL and counteracting its death-promoting function. The reduction of DcR3 with siRNA unmasked TRAIL and greatly enhanced TRAIL-induced apoptosis. Gemcitabine, a first-line drug for pancreatic cancer, also reduced the level of DcR3. The addition of DcR3 siRNA further enhanced gemcitabine-induced apoptosis. Notably, our in vivo study demonstrated that the therapeutic effect of gemcitabine could be enhanced via further reduction of DcR3, suggesting that downregulation of DcR3 in tumor cells could tip the balance of pancreatic cells towards apoptosis and potentially serve as a new strategy for pancreatic cancer therapy.     

The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.     

The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment

Atomic force microscopy provides a novel technique for differentiating the mechanical properties of various cell types. Cell elasticity is abundantly used to represent the structural strength of cells in different conditions. In this study, we are interested in whether physical or physiological cues affect cell elasticity in Atomic force microscopy (AFM)-based assessments. The physical cues include the geometry of the AFM tips, the indenting force and the operating temperature of the AFM. All of these cues show a significant influence on the cell elasticity assessment. Sharp AFM tips create a two-fold increase in the value of the effective Young’s modulus (E_eff) relative to that of the blunt tips. Higher indenting force at the same loading rate generates higher estimated cell elasticity. Increasing the operation temperature of the AFM leads to decreases in the cell stiffness because the structure of actin filaments becomes disorganized. The physiological cues include the presence of fetal bovine serum or extracellular matrix-coated surfaces, the culture passage number, and the culture density. Both fetal bovine serum and the extracellular matrix are critical for cells to maintain the integrity of actin filaments and consequently exhibit higher elasticity. Unlike primary cells, mouse kidney progenitor cells can be passaged and maintain their morphology and elasticity for a very long period without a senescence phenotype. Finally, cell elasticity increases with increasing culture density only in MDCK epithelial cells. In summary, for researchers who use AFM to assess cell elasticity, our results provide basic and significant information about the suitable selection of physical and physiological cues.     

The Post-Resuscitative Urinalysis Associate the Survival of Patients with Non-Traumatic Out-of-Hospital Cardiac Arrest

Objective

To analyze whether urine output and urinalysis results are predictive of survival and neurologic outcomes in patients with non-traumatic out-of-hospital cardiac arrest (OHCA).

Methods

Information was obtained from 1,340 patients with non-traumatic OHCA who had achieved a sustained return of spontaneous circulation (ROSC). Factors that were associated with survival in the post-resuscitative period were evaluated. The association between urine output and fluid challenge in the early resuscitative period was analyzed and compared between the survivors and the non-survivors. The results of the initial urinalysis, including the presence of proteinuria and other findings, were used to evaluate the severity of vascular protein leakage and survival. The association between proteinuria and the neurologic outcomes of the survivors was also analyzed. The clinical features of capillary leakage were examined during the post-resuscitative period.

Results

Of the 1,340 patients, 312 survived. A greater urine output was associated with a higher chance of survival. The initial urine output increased in proportion to the amount of fluid that was administered during early resuscitation in the emergency department for the survivors but not for the non-survivors (p<0.05). In the initial urinalysis, proteinuria was strongly associated with survival, and severe proteinuria indicated significantly poorer neurologic outcomes (p<0.05 for both comparisons). Proteinuria was associated with a risk of developing signs of capillary leakage, including body mass index gain and pitting edema (both p<0.001).

Conclusion

The severity of proteinuria during the early post-resuscitative period was predictive of survival.