Significant impact of divalent metal ions on the fidelity,sugar selectivity,and drug incorporation efficiency of human PrimPol

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979 nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4 × 10⁻² to 3.8 × 10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57–1800 with Mn²⁺ and 150–4500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).     

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.     

DPHL:A DIA Pan-human Protein Mass Spectrometry Library for Robust Biomarker Discovery

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.     

Synthesis and antiviral properties of novel indole-based thiosemicarbazides and 4-thiazolidinones

A novel series of indolylthiosemicarbazides (6a–6g) and their cyclization products, 4-thiazolidinones (7a–7g), have been designed, synthesized and evaluated, in vitro, for their antiviral activity against a wide range of DNA and RNA viruses. Compounds 6a, 6b, 6c and 6d exhibited notable antiviral activity against Coxsackie B4 virus, at EC₅₀ values ranging from 0.4 to 2.1 μg/mL. The selectivity index (ratio of cytotoxic to antivirally effective concentration) values of these compounds were between 9 and 56. Besides, 6b, 6c and 6d also inhibited the replication of two other RNA viruses, Sindbis virus and respiratory syncytial virus, although these EC₅₀ values were higher compared to those noted for Coxsackie B4 virus. The SAR analysis indicated that keeping the free thiosemicarbazide moiety is crucial to obtain this antiviral activity, since the cyclization products (7a–7g) did not produce any antiviral effect.     

The Amount of BCL6 in B Cells Shortly after Antigen Engagement Determines Their Representation in Subsequent Germinal Centers

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Nuttallia balearicae sp. n., an Avian Piroplasm from Crowned Cranes (Balearica spp.)

An avian piroplasm, Nuttallia balearicae sp. n., is described from captive Balearica p. pavonina and B. p. gibbericeps. Other avian piroplasms and their validity and taxonomic status are discussed. The possible route of transmission of these parasites is also considered.     

Binding of B‐cell maturation antigen to B‐cell activating factor induces survival of multiple myeloma cells by activating Akt and JNK signaling pathways

B‐cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. In this study, we characterized the mechanism underlying the protein kinase B (Akt) and c‐Jun N‐terminal kinase (JNK) pathways and BCMA interactions in regulating multiple myeloma (MM) cell survival. It was found that the expression levels of B cell‐activating factor (BAFF) and BCMA were increased in MM cells as compared with those in normal controls. The proliferation of U266 cells was induced by recombinant human BAFF (rhBAFF) and could also be decreased by BCMA siRNA. The expression of Bcl‐2 protein was up‐regulated, and Bax protein was down‐regulated after rhBAFF treatment, which could be reversed by BCMA siRNA. Similarly, the protein p‐JNK and p‐Akt were activated by rhBAFF and could be changed by BCMA siRNA. In addition, the BCMA mRNA and protein expression levels were decreased after treatment with Akt and JNK pathway inhibitors. These results suggest that Akt and JNK pathways are involved in the regulation of BCMA. A novel BAFF/BCMA signalling pathway in MM may be a new therapeutic target for MM. Copyright © 2016 John Wiley & Sons, Ltd.     

Estimating recent growth in the cuttlefish Sepia officinalis: are nucleic acid-based indicators for growth and condition the method of choice?

A laboratory calibration study was undertaken with juvenile Sepia officinalis (80-85 g initial wet weight) to investigate the effects of different food rations and different starving intervals on RNA/dry weight (DW) ratios and RNA/DNA ratios in cephalopod mantle muscle at two different temperatures. The digestive gland index was also used as an additional indicator of recent growth. High food rations and low temperature went along with high RNA/DW ratios and high RNA/DNA ratios. Starving resulted in a linear decline in growth performance and a concomitant decrease in RNA/DW and RNA/DNA ratio, with RNA/DNA ratios representing the growth data better. RNA/DNA ratios decreased faster at higher temperatures. A fluorimetric assay for nucleic acid analysis was optimized for cephalopod mantle tissues and yielded reproducible RNA/DNA ratios with a relative variance below 10%. Thus, it may be possible to use this estimator of recently encountered feeding regime for the evaluation of mortality rates of early teuthid paralarvae to eventually support stock management. Also, log relative digestive gland weight showed a strong relationship with starving time, but, surprisingly, not with temperature. Data from the two temperatures analyzed could be combined to form a common regression line of relative digestive gland index with starving time. This indicator for recent growth might be especially suitable for large specimens with a well-developed digestive gland.