Effect of influent pH and alkalinity on the removal of chlorophenols in sequential anaerobic–aerobic reactors

This study was carried out to determine the effect of influent pH and alkalinity on the performance of sequential UASB and RBC reactors for the removal of 2-CP and 2,4-DCP from two different simulated wastewaters. The performance of methanogens at low (<6.0) to high (>8.0) pH values and at sufficiently high alkalinity (1500–3500 mg/l as CaCO₃) is described in this paper. Sequential reactors were capable of handling wastewaters with influent pH, 5.5–8.5. However, with influent pH 7.0 ± 0.1 UASB reactor showed best performance for 2-CP (99%) and 2,4-DCP (88%) removals. Increase in alkalinity/COD ratio in the influent (>1.1) caused gradual decrease in the chlorophenol removal in UASB reactors. The UASB reactors could not tolerate wastewater with higher alkalinity/COD ratio (2.6) and showed significant deterioration of its performance in terms of chlorophenols removal achieving only 74.7% 2-CP and 60% 2,4-DCP removals, respectively.     

Accessibility and partner number of protein residues, their relationship and a webserver, ContPlot for their display

Background  

Depending on chemical features residues have preferred locations – interior or exterior – in protein structures, which also determine how many other residues are found around them. The close packing of residues is the hallmark of protein interior and protein-protein interaction sites.     

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.     

Regulated phosphorylation of a major UDP-glucuronosyltransferase isozyme by tyrosine kinases dictates endogenous substrate selection for detoxification

Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE(1)) with barely detectable 17β-estradiol (E(2)) conversion following expression in COS-1 cells. Consistent with the UDP-glucuronosyltransferase requirement for regulated phosphorylation, we discovered that 2B7 requires Src-dependent tyrosine phosphorylation. Y236F-2B7 and Y438F-2B7 mutants were null and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [(33)P]orthophosphate and that 2B7His, previously expressed in SYF-(Src,Yes,Fyn)(-/-) cells, was Src-supported or phosphorylated under in vitro conditions. Unexpectedly, 2B7 expressed in SYF(-/-) and SYF(+/-) cells metabolized 4-OHE(1) at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E(2) metabolism was 16- and 9-fold higher than in COS-1. Because anti-Tyr(P)-438-2B7 detected Tyr(P)-438-2B7 in each cell line, results indicated that unidentified tyrosine kinase(s) (TKs) phosphorylated 2B7 in SYF(-/-). 2B7-transfected COS-1 treated with increasing concentrations of the Src-specific inhibitor PP2 down-regulated 4-OHE(1) glucuronidation reaching 60% maximum while simultaneously increasing E(2) metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E(2) metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF(-/-) is more competent at metabolizing E(2) in cellulo than 2B7 expressed in COS-1. To confirm Src-controlled 2B7 prevents toxicity, we showed that 2B7-transfected COS-1 efficiently protected against 4-OH-E(1)-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE(1), but not E(2), in COS-1, whereas non-Src-phosphorylated 2B7 metabolizes both chemicals. Importantly, we determined that 2B7 substrate selection is not fixed but varies depending upon the TK(s) that carry out its required phosphorylation.     

Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors

Surface modification of adenovirus vectors can improve tissue-selective targeting, attenuate immunogenicity, and enable imaging of particle biodistribution, thus significantly improving therapeutic potential. Currently, surface engineering is constrained by a combination of factors, including impact on viral fitness, limited access to functionality, or incomplete control over the site of modification. Here, we report a two-step labeling process involving an initial metabolic placement of a uniquely reactive unnatural amino acid, azidohomoalanine (Aha), followed by highly specific chemical modification. As genetic modification of adenovirus is unnecessary, vector production is exceedingly straightforward. Aha incorporation demonstrated no discernible impact on either virus production or infectivity of the resultant particles. "Click" chemical modification of surface-exposed azides was highly selective, allowing for the attachment of a wide range of functionality. Decoration of human adenovirus type 5 (hAd5) with folate, a known cancer-targeting moiety, provided an ～20-fold increase in infection of murine breast cancer cells (4T1) in a folate receptor-dependent manner. This study demonstrates that incorporation of unnatural amino acids can provide a flexible, straightforward route for the selective chemical modification of adenoviral vectors.     

Role of caffeine in DNA recognition of a potential food‐carcinogen benzo[a]pyrene and UVA induced DNA damage

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine–BP complexes. While steady‐state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine–BP complex. The picosecond‐resolved emission spectroscopic studies precisely explore the caffeine‐mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time‐gated fluorescence microscopy has been used to monitor caffeine‐mediated exclusion of BP from various cell lines including squamous epithelial cells, WI‐38 (fibroblast), MCF‐7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages. Copyright © 2014 John Wiley & Sons, Ltd.     

Intermittent Right Ventricular Outflow Tract Capture due to Chronic Right Atrial Lead Dislodgement

A 58 year old male, known case of type 2 diabetes and hypertension, had undergone implantation of a dual chamber pacemaker(DDDR) in 2007 for complaints of recurrent syncope and trifascicular block with a normal ejection fraction andnormal coronaries. His post implantation parameters were normal at that time.He now presented to our pacemaker clinic where his ECG done showed two types o fpaced complexes. The first few complexes were consistent with atrial sensed right ventricular apical pacing with left superior axis. Later complexes showed loss of atrial sensing with pacing from right ventricular outflow tract(inferior axis) with subtle oscillation in it''s axis. On application of magnet, two pacemaker spikes were visible withinterspike interval of 120 ms and paced complexes with inferior axis starting from the first spike suggesting that the atrial lead was responsible for RVOT depolarization. On interrogation of the pacemaker, atrial EGM showed sensed activity from atrium followed by large sensed ventricular complex. Fluoroscopy confirmed that the atrial lead was dislodged and was intermittently prolapsing into the RVOT. Since the patient was asymptomatic, he refused any intervention and subsequentlyhis atrial lead was switched off by telemetry. The above case signifies that asymptomatic lead dislodgement is no talways manifested as loss of capture and even subtle variation of the axis o fthe paced complexes can provide us with a clue that can be confirmed by telemetry of the pacemaker and fluoroscopy.     

Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

Purpose

Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.

Methods

The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.

Results

RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.

Conclusions

We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.     

Mitochondrial-targeted antioxidants represent a promising approach for prevention of cisplatin-induced nephropathy

Cisplatin is a widely used antineoplastic agent; however, its major limitation is the development of dose-dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show not only that mitochondrial dysfunction is a feature of cisplatin nephrotoxicity, but also that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently prevented cisplatin-induced renal dysfunction. Mito-CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito-CP did not diminish cisplatin's antineoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Because similar compounds seem to be safe in humans, mitochondrially targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.