Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Serum markers of hepatocyte death and apoptosis are non invasive biomarkers of severe fibrosis in patients with alcoholic liver disease

Background

Quantification of hepatotocyte death is useful to evaluate the progression of alcoholic liver diseases. Our aims were to quantify and correlate the circulating levels of Cytokeratin 18 (CK18) and its caspases-generated fragment to disease severity in heavy alcoholics.

Methodology/Principal Findings

CK18 and CK18-fragment were evaluated in the serum of 143 heavy alcoholics. Serum levels of markers of hepatocyte death (CK18), apoptosis (CK18 fragment) and necrosis (CK18 -CK18 fragment) increased in patients with severe fibrosis compared to patients with mild fibrosis. These markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, fibrosis and with hepatic TNFα and TGFβ assessed in the liver of 24 patients. Elevated levels of serum hepatocyte death and apoptotic markers were independent risk factors in predicting severe fibrosis in a model combining alkaline phosphatase, bilirubin, prothrombin index, hyaluronate, hepatocyte death and apoptotic markers. The level of markers of hepatocyte death and apoptosis had an area under the receiving operator curve that predicted severe fibrosis of 0.84 and 0.76, respectively.

Conclusion/Significance

Death of hepatocytes can be easily evaluated with serum markers and correlated with severe fibrosis in heavy alcohol drinkers. These biomarkers could be useful to rapidly evaluate liver injuries and the efficacy of therapies.     

The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1)

Isoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)- and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling.     

Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains.     

The Evolutionary Divergence of Shiga Toxin-Producing Escherichia coli Is Reflected in Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Spacer Composition

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the “big six” serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.     

Incidence,survival and mortality rates of stage-specific bladder cancer in United States: A trend analysis

PurposeTo examine the overall and stage-specific age-adjusted incidence, 5-year survival and mortality rates of bladder cancer (BCa) in the United States, between 1973 and 2009.Materials and methodsA total of 148,315 BCa patients were identified in the Surveillance, Epidemiology and End Results database, between years 1973 and 2009. Incidence, mortality, and 5-year cancer-specific survival rates were calculated. Temporal trends were quantified using the estimated annual percentage change (EAPC) and linear regression models. All analyses were stratified according to disease stage, and further examined according to sex, race, and age groups.ResultsIncidence rate of BCa increased from 21.0 to 25.5/100,000 person-years between 1973 and 2009. Stage-specific analyses revealed an increase incidence for localized stage: 15.4–20.2 (EAPC: +0.5%, p < 0.001) and distant stage: 0.5–0.8 (EAPC: +0.7%, p = 0.001). Stage-specific 5-year survival rates increased for all stages, except for distant disease. No significant changes in mortality were recorded among localized (EAPC: ?0.2%, p = 0.1) and regional stage (EAPC: ?0.1%, p = 0.5). An increase in mortality rates was observed among distant stage (EAPC: +1.0%, p = 0.005). Significant variations in incidence and mortality were recorded when estimates were stratified according to sex, race, and age groups.DiscussionAlbeit statistically significant, virtually all changes in incidence and mortality were minor, and hardly of any clinical importance. Little or no change in BCa cancer control outcomes has been achieved during the study period.     

Anticancer and Antimicrobial Activities of Some Antioxidant-Rich Cameroonian Medicinal Plants

Traditional remedies have a long-standing history in Cameroon and continue to provide useful and applicable tools for treating ailments. Here, the anticancer, antimicrobial and antioxidant activities of ten antioxidant-rich Cameroonian medicinal plants and of some of their isolated compounds are evaluated.The plant extracts were prepared by maceration in organic solvents. Fractionation of plant extract was performed by column chromatography and the structures of isolated compounds (emodin, 3-geranyloxyemodin, 2-geranylemodin) were confirmed spectroscopically. The antioxidant activity (AOA) was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) bleaching method, the trolox equivalent antioxidant capacity (TEAC), and the hemoglobin ascorbate peroxidase activity inhibition (HAPX) assays. The anticancer activity was evaluated against A431 squamous epidermal carcinoma, WM35 melanoma, A2780 ovary carcinoma and cisplatin-resistant A2780cis cells, using a direct colorimetric assay. The total phenolic content in the extracts was determined spectrophotometrically by the Folin–Ciocalteu method. Rumex abyssinicus showed the best AOA among the three assays employed. The AOA of emodin was significantly higher than that of 3-geranyloxyemodin and 2-geranylemodin for both TEAC and HAPX methods. The lowest IC₅₀ values (i.e., highest cytotoxicity) were found for the extracts of Vismia laurentii, Psorospermum febrifugum, Pentadesma butyracea and Ficus asperifolia. The Ficus asperifolia and Psorospermum febrifugum extracts are selective against A2780cis ovary cells, a cell line which is resistant to the standard anticancer drug cisplatin. Emodin is more toxic compared to the whole extract, 3-geranyloxyemodin and 2-geranylemodin. Its selectivity against the platinum-resistant A2780cis cell line is highest. All of the extracts display antimicrobial activity, in some cases comparable to that of gentamycin.     

Human APOBEC3A Isoforms Translocate to the Nucleus and Induce DNA Double Strand Breaks Leading to Cell Stress and Death

Human APOBEC3 enzymes deaminate single stranded DNA. At least five can deaminate mitochondrial DNA in the cytoplasm, while three can deaminate viral DNA in the nucleus. However, only one, APOBEC3A, can hypermutate genomic DNA. We analysed the distribution and function of the two APOBEC3A isoforms p1 and p2 in transfected cell lines. Both can translocate to the nucleus and hypermutate CMYC DNA and induce DNA double strand breaks as visualized by the detection of ©H2AX or Chk2. APOBEC3A induced G1 phase cell cycle arrest and triggered several members of the intrinsic apoptosis pathway. Activation of purified human CD4+ T lymphocytes with PHA, IL2 and interferon α resulted in C->T hypermutation of genomic DNA and double stranded breaks suggesting a role for APOBEC3A in pro-inflammatory conditions. As chronic inflammation underlies many diseases including numerous cancers, it is possible that APOBEC3A induction may generate many of the lesions typical of a cancer genome.