Essential requirements for robust signaling in Hfq dependent small RNA networks

Bacteria possess networks of small RNAs (sRNAs) that are important for modulating gene expression. At the center of many of these sRNA networks is the Hfq protein. Hfq's role is to quickly match cognate sRNAs and target mRNAs from among a large number of possible combinations and anneal them to form duplexes. Here we show using a kinetic model that Hfq can efficiently and robustly achieve this difficult task by minimizing the sequestration of sRNAs and target mRNAs in Hfq complexes. This sequestration can be reduced by two non-mutually exclusive kinetic mechanisms. The first mechanism involves heterotropic cooperativity (where sRNA and target mRNA binding to Hfq is influenced by other RNAs bound to Hfq); this cooperativity can selectively decrease singly-bound Hfq complexes and ternary complexes with non-cognate sRNA-target mRNA pairs while increasing cognate ternary complexes. The second mechanism relies on frequent RNA dissociation enabling the rapid cycling of sRNAs and target mRNAs among different Hfq complexes; this increases the probability the cognate ternary complex forms before the sRNAs and target mRNAs degrade. We further demonstrate that the performance of sRNAs in isolation is not predictive of their performance within a network. These findings highlight the importance of experimentally characterizing duplex formation in physiologically relevant contexts with multiple RNAs competing for Hfq. The model will provide a valuable framework for guiding and interpreting these experiments.     

CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.     

Autophagy and KRT8/keratin 8 protect degeneration of retinal pigment epithelium under oxidative stress

Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.     

Suprafenacine,an Indazole-Hydrazide Agent,Targets Cancer Cells Through Microtubule Destabilization

Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents (TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we report the identification of a novel cell permeable, tubulin-destabilizing molecule - 4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This led to G₂/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss of mitochondrial membrane potential, JNK - mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3. Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our results suggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.     

Incidence,Trends and Ethnic Differences of Oropharyngeal,Anal and Cervical Cancers: Singapore, 1968-2012

In recent decades, several Western countries have reported an increase in oropharyngeal and anal cancers caused by human papillomavirus (HPV). Trends in HPV-associated cancers in Asia have not been as well described. We describe the epidemiology of potentially HPV-related cancers reported to the Singapore Cancer Registry from 1968–2012. Analysis included 998 oropharyngeal squamous cell carcinoma (OPSCC), 183 anal squamous cell carcinoma (ASCC) and 8,019 invasive cervical cancer (ICC) cases. Additionally, 368 anal non-squamous cell carcinoma (ANSCC) and 2,018 non-oropharyngeal head and neck carcinoma (non-OP HNC) cases were included as comparators. Age-standardized incidence rates (ASR) were determined by gender and ethnicity (Chinese, Malay and Indian). Joinpoint regression was used to evaluate annual percentage change (APC) in incidence. OPSCC incidence increased in both genders (men 1993–2012, APC = 1.9%, p<0.001; women 1968–2012, APC = 2.0%, p = 0.01) and was 5 times higher in men than women. In contrast, non-OP HNC incidence declined between 1968–2012 among men (APC = -1.6%, p<0.001) and women (APC = -0.4%, p = 0.06). ASCC and ANSCC were rare (ASR = 0.2 and 0.7 per 100,000 person-years, respectively) and did not change significantly over time except for increasing ANSCCs in men (APC = 2.8%, p<0.001). ICC was the most common HPV-associated cancer (ASR = 19.9 per 100,000 person-years) but declined significantly between 1968–2012 (APC = -2.4%). Incidence of each cancer varied across ethnicities. Similar to trends in Western countries, OPSCC incidence increased in recent years, while non-OP HNC decreased. ICC remains the most common HPV-related cancer in Singapore, but Pap screening programs have led to consistently decreasing incidence.     

Evaluation of pilot-scale modified A2O processes for the removal of nitrogen compounds from sewage

In this study, pilot-scale experiments for the removal of nitrogen from sewage obtained from a county Y sewer system were performed using modified A₂O processes. Using this approach, the total amount of nitrogen discharged during denitrification of the influent was average 38.6 mg/L and a level of average 10.8 mg/L was maintained throughout the denitrification process, which resulted in an average removal efficiency that was greater than 72%. The nitrogen components in the effluent water consisted of 22% ammonia nitrogen, 6% nitrite nitrogen and 72% nitrate nitrogen, reaching a nitrification efficiency of 94%. In conclusion, since these advanced treatment methods, which involve modified A₂O processes, were successfully employed to remove nitrogen from sewage discharge, they hold promise for wide spread use by treatment plants.     

Fluorescence analysis of receptor-G protein interactions in cell membranes

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.     

Effect of dietary pectin on the production of immunoglobulins and cytokines by mesenteric lymph node lymphocytes in mouse colitis induced with dextran sulfate sodium

The present study explores the dietary effect of pectin on the MLN lymphocyte functions of mice with dextran sulfate sodium (DS)-induced colitis. We found that the immunoglobulin (Ig)A level in mesenteric lymph node (MLN) lymphocytes was high, while the IgE level was lower, in mice fed with pectin than in those fed with cellulose. Interestingly, the fecal IgA concentration of the pectin-fed mice was significantly higher than that of the cellulose-fed mice. The concentrations of interferon-gamma and interleukin (IL)-2 treated with concanavalin A (ConA) were significantly higher in the pectin-fed group than in the cellulose-fed group. Although dietary pectin did not affect the IL-4 and IL-10 levels, the activation-induced IL-4 and IL-10 secretion was lower in MLN cells of the pectin-fed mice than of the cellulose-fed mice following DS-induced colitis. Based on these findings, we propose that the effect of dietary pectin on mice with DS-induced colitis is mediated by the manipulation of Th1 cells. Furthermore, the inhibitory effect of IL-4 and IL-10 by dietary pectin may play an important role in promoting a change in Th1/Th2 balance toward Th1-dominant immunity.     

Atrial Arrhythmia in Ageing Spontaneously Hypertensive Rats: Unraveling the Substrate in Hypertension and Ageing

Background

Both ageing and hypertension are known risk factors for atrial fibrillation (AF) although the pathophysiological contribution or interaction of the individual factors remains poorly understood. Here we aim to delineate the arrhythmogenic atrial substrate in mature spontaneously hypertensive rats (SHR).

Methods

SHR were studied at 12 and 15 months of age (n = 8 per group) together with equal numbers of age-matched normotensive Wistar-Kyoto control rats (WKY). Electrophysiologic study was performed on superfused isolated right and left atrial preparations using a custom built high-density multiple-electrode array to determine effective refractory periods (ERP), atrial conduction and atrial arrhythmia inducibility. Tissue specimens were harvested for structural analysis.

Results

Compared to WKY controls, the SHR demonstrated: Higher systolic blood pressure (p<0.0001), bi-atrial enlargement (p<0.05), bi-ventricular hypertrophy (p<0.05), lower atrial ERP (p = 0.008), increased atrial conduction heterogeneity (p = 0.001) and increased atrial interstitial fibrosis (p = 0.006) & CD68-positive macrophages infiltration (p<0.0001). These changes resulted in higher atrial arrhythmia inducibility (p = 0.01) and longer induced AF episodes (p = 0.02) in 15-month old SHR. Ageing contributed to incremental bi-atrial hypertrophy (p<0.01) and atrial conduction heterogeneity (p<0.01) without affecting atrial ERP, fibrosis and arrhythmia inducibility. The limited effect of ageing on the atrial substrate may be secondary to the reduction in CD68-positive macrophages.

Conclusions

Significant atrial electrical and structural remodeling is evident in the ageing spontaneously hypertensive rat atria. Concomitant hypertension appears to play a greater pathophysiological role than ageing despite their compounding effect on the atrial substrate. Inflammation is pathophysiologically linked to the pro-fibrotic changes in the hypertensive atria.