Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.     

The complete nucleotide sequence of the mitochondrial genome of Tetraodon nigroviridis.

The fresh water pufferfish Tetraodon nigroviridis is a model organism for studying evolution of genome and gene functions, but its mitochondrial genome (mtDNA) sequence is still not available. We determined the complete nucleotide sequence of its mtDNA using shotgun sequencing. The T. nigroviridis mtDNA was 16,462 bp, and contained 13 protein coding genes, 22 tRNAs, 2 rRNAs and a major non-coding region. The gene order was identical to the common type of vertebrate mtDNA, whereas the G + C content in the sense strand was 46.9%, much higher than most other fish species. One hundred and three SNPs were detected in the control region of the mtDNA of 35 individuals, a majority of SNPs were detected in the 5' end of the control region. A phylogenetic study including 21 fish species was performed on concatenated amino acid sequences of 12 protein coding genes, and revealed that the T. nigroviridis was clustered with Fugu rubripes into a group. The complete mtDNA sequence and SNPs in its control region will be useful in studying fish evolution, in differentiating different Tetraodon species and in analyzing genetic diversity within T. nigroviridis.     

Plasma Catechols in Familial Dysautonomia: A Long-term Follow-up Study

This study tested whether familial dysautonomia (FD) involves progressive loss of noradrenergic nerves. Plasma levels of catechols, including dihydroxyphenylglycol (DHPG), norepinephrine (NE), dopamine (DA), and DOPA, were measured in 7 adult patients with FD and 50 healthy control subjects. FD patients were re-tested after a mean follow-up period of 13 years. Compared to controls, FD patients had low plasma levels of DHPG (P < 0.001), high DOPA and DA levels (P = 0.01, P = 0.0002), and high NE:DHPG (P < 0.0001), DA:NE (P = 0.0003), and DOPA:DHPG (P < 0.0001) ratios. At follow-up there were no changes in plasma levels of individual catechols; however, there were further increases in DOPA:DHPG ratios (mean 24 ± 7%, P = 0.01). In FD, plasma catechol profiles are sufficiently stable, at least over a decade, to be used as a biomarker of disease involvement. An increasing DOPA:DHPG ratio suggests slight but consistent, progressive loss of noradrenergic neurons.     

Methodological Framework for World Health Organization Estimates of the Global Burden of Foodborne Disease

Background

The Foodborne Disease Burden Epidemiology Reference Group (FERG) was established in 2007 by the World Health Organization to estimate the global burden of foodborne diseases (FBDs). This paper describes the methodological framework developed by FERG''s Computational Task Force to transform epidemiological information into FBD burden estimates.

Methods and Findings

The global and regional burden of 31 FBDs was quantified, along with limited estimates for 5 other FBDs, using Disability-Adjusted Life Years in a hazard- and incidence-based approach. To accomplish this task, the following workflow was defined: outline of disease models and collection of epidemiological data; design and completion of a database template; development of an imputation model; identification of disability weights; probabilistic burden assessment; and estimating the proportion of the disease burden by each hazard that is attributable to exposure by food (i.e., source attribution). All computations were performed in R and the different functions were compiled in the R package ''FERG''. Traceability and transparency were ensured by sharing results and methods in an interactive way with all FERG members throughout the process.

Conclusions

We developed a comprehensive framework for estimating the global burden of FBDs, in which methodological simplicity and transparency were key elements. All the tools developed have been made available and can be translated into a user-friendly national toolkit for studying and monitoring food safety at the local level.     

Stereochemical Control in the Still-Wittig Rearrangement Synthesis of Cyclohexyl (Z)-Alkene Inhibitors of Pin1

Three stereoisomeric inhibitors of Pin1: (2R,5S)-, (2S,5R)- and (2S,5S)-Ac–pSer–Ψ[(Z)CH = C]–pipecolyl(Pip)–2-(2-naphthyl)ethylamine 1, that mimic L-pSer–D-Pro, D-pSer–L-Pro, and D-pSer–D-Pro amides respectively, were synthesized by a 13-step route. The newly formed stereogenic centers in the pipecolyl ring were introduced by Luche reduction, followed by stereospecific [2,3]-Still-Wittig rearrangement. The (Z)- to (E)-alkene ratio in the rearrangements were consistently 5.5 to 1. The stereochemistry at the original Ser α-carbon controlled the stereochemistry of the Luche reduction, but it did not affect the stereochemical outcome of the rearrangement, which consistently gave the (Z)-alkene. The epimerized by-product, (2S,5S)-10, resulting from the work-up after Na/NH₃ debenzylation of (2S,5R)-9, was carried on to the (2S,5S)-1 isomer. Compound (2S,5S)-10 was resynthesized from the Luche reduction by-product, (2R,3R)-3, and the stereochemistry was confirmed by comparison of the optical rotations. The IC₅₀ values for (2R,5S)-1, (2S,5R)-1 and (2S,5S)-1 Pin1 inhibition were: 52, 85, and 140 μM, respectively.     

Inhibition of Janus Kinase 1 synergizes docetaxel sensitivity in prostate cancer cells

Prostate cancer (PCa) is the second most common malignancy and is the fifth leading cause of cancer mortality among men globally. Docetaxel-based therapy remains the first-line treatment for metastatic castration-resistant prostate cancer. However, dose-limiting toxicity including neutropenia, myelosuppression and neurotoxicity is the major reason for docetaxel dose reductions and fewer cycles administered, despite a recent study showing a clear survival benefit with increased total number of docetaxel cycles in PCa patients. Although previous studies have attempted to improve the efficacy and reduce docetaxel toxicity through drug combination, no drug has yet demonstrated improved overall survival in clinical trial, highlighting the challenges of improving the activity of docetaxel monotherapy in PCa. Herein, we identified 15 lethality hits for which inhibition could enhance docetaxel sensitivity in PCa cells via a high-throughput kinome-wide loss-of-function screen. Further drug-gene interactions analyses identified Janus kinase 1 (JAK1) as a viable druggable target with existing experimental inhibitors and FDA-approved drugs. We demonstrated that depletion of endogenous JAK1 enhanced docetaxel-induced apoptosis in PCa cells. Furthermore, inhibition of JAK1/2 by baricitinib and ruxolitinib synergizes docetaxel sensitivity in both androgen receptor (AR)–negative DU145 and PC3 cells, but not in the AR-positive LNCaP cells. In contrast, no synergistic effects were observed in cells treated with JAK2-specific inhibitor, fedratinib, suggesting that the synergistic effects are mainly mediated through JAK1 inhibition. In conclusion, the combination therapy with JAK1 inhibitors and docetaxel could be a useful therapeutic strategy in the treatment of prostate cancers.     

Annexin A2 Reduces PCSK9 Protein Levels via a Translational Mechanism and Interacts with the M1 and M2 Domains of PCSK9

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9''s C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.     

A microsatellite linkage map of Barramundi, Lates calcarifer

Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its compact genome (approximately 700 Mb) is among the smallest genomes of food fish species. We established a first-generation genetic linkage map of Barramundi with a mapping panel containing three parents (two males and one female) and 93 progeny. A total of 240 microsatellite markers were mapped into 24 linkage groups. Among these markers, 10 were located in ESTs and known genes. The total lengths of the female and male maps were 873.8 and 414.5 cM with an average marker spacing of 6.20 and 4.70 cM, respectively. Comparing the flanking sequences of the 240 Barramundi microsatellites with the assembled whole-genome sequences of Tetraodon nigrovidiris revealed 55 homologous sequences located in 19 of the 21 chromosomes of T. nigrovidiris. The map will not only enable the mapping of quantitative trait loci, but also provide new resources for understanding the evolution of fish genomes.     

Organic/inorganic complex pigments: ancient colors Maya Blue

Maya Blue is an ancient blue pigment composed of palygorskite clay and indigo. It was used by the ancient Maya and provides a dramatic background for some of the most impressive murals throughout Mesoamerica. Despite exposure to acids, alkalis, and chemical solvents, the color of the Maya Blue pigment remains unaltered. The chemical interaction between palygorskite and indigo form an organic/inorganic complex with the carbonyl oxygen of the indigo bound to a surface Al(3+) in the Si-O lattice. In addition indigo will undergo an oxidation to dehydroindigo during preparation. The dehydro-indigo molecule forms a similar but stronger complex with the Al(3+). Thus, Maya Blue varies in color due to the mixed indigo/dehydroindigo complex. The above conclusions are the result of application of multiple techniques (X-ray diffraction, differential thermal analysis/thermal gravimetric analysis, high resolution transmission electron microscopy, scanning electron microscopy, infrared and Raman spectroscopy) to the characterization of the organic/inorganic complex. A picture of the bonding of the organic molecule to the palygorskite surface forming a surface complex is developed and supported by the results of density functional theory calculations. We also report that other organic molecules such as thioindigo form similar organic/inorganic complexes thus, opening an entirely new class of complex materials for future applications.     

Cross-talk between T cells and innate immune cells is crucial for IFN-gamma-dependent tumor rejection

Though the importance of IFN-gamma in tumor immunity has been well-demonstrated, little is known about its source and how it is induced. By using various bone marrow chimeric mice, we show here that IFN-gamma essential for tumor immunity is solely produced by hemopoietic cells. Surprisingly, IFN-gamma derived from T cells was not necessary for tumor immunity in this model. In the immunized mice, in which only innate immune cells have the IFN-gamma-producing potential, tumors were efficiently rejected. The innate immune cells, such as NK1.1(+) cells and CD11b(+) cells, can provide sufficient amounts of IFN-gamma which requires, however, the help of T cells. The close cooperation between T cells and innate immune cells during tumor regression is likely mediated by IL-2. Together, our results clearly illustrate how T cells cooperate with innate immune cells for IFN-gamma-mediated tumor rejection and this may have important indications for clinical trials of tumor immunotherapy.