Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures: IMPLICATIONS FOR GENOMIC INSTABILITY*

The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose “C_(d)C_(S)C_(S)” (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability.     

Characterization of G-quadruplex antibody reveals differential specificity for G4 DNA forms

Accumulating evidence suggests that human genome can fold into non-B DNA structures, when appropriate sequence and favourable conditions are present. Among these, G-quadruplexes (G4-DNA) are associated with gene regulation, chromosome fragility and telomere maintenance. Although several techniques are used in detecting such structures in vitro, understanding their intracellular existence has been challenging. Recently, an antibody, BG4, was described to study G4 structures within cells. Here, we characterize BG4 for its affinity towards G4-DNA, using several biochemical and biophysical tools. BG4 bound to G-rich DNA derived from multiple genes that form G-quadruplexes, unlike complementary C-rich or random sequences. BLI studies revealed robust binding affinity (K_d = 17.4 nM). Gel shift assays show BG4 binds to inter- and intramolecular G4-DNA, when it is in parallel orientation. Mere presence of G4-motif in duplex DNA is insufficient for antibody recognition. Importantly, BG4 can bind to G4-DNA within telomere sequence in a supercoiled plasmid. Finally, we show that BG4 binds to form efficient foci in four cell lines, irrespective of their lineage, demonstrating presence of G4-DNA in genome. Importantly, number of BG4 foci within the cells can be modulated, upon knockdown of G4-resolvase, WRN. Thus, we establish specificity of BG4 towards G4-DNA and discuss its potential applications.     

Best Practices for the Development,Scale-up,and Post-approval Change Control of IR and MR Dosage Forms in the Current Quality-by-Design Paradigm

In this whitepaper, the Manufacturing Technical Committee of the Product Quality Research Institute provides information on the common, best practices in use today in the development of high-quality chemistry, manufacturing and controls documentation. Important topics reviewed include International Conference on Harmonization, in vitro–in vivo correlation considerations, quality-by-design approaches, process analytical technologies and current scale-up, and process control and validation practices. It is the hope and intent that this whitepaper will engender expanded dialog on this important subject by the pharmaceutical industry and its regulatory bodies.     

Synthesis of N4-(substituted phenyl)-N4-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines and identification of new microtubule disrupting compounds that are effective against multidrug resistant cells

A series of fourteen N⁴-(substituted phenyl)-N⁴-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cyclization of 2-amino-6-hydrazinylpyrimidin-4(3H)-one with cyclohexanone, followed by oxidation, chlorination and displacement with appropriate anilines. Compounds 6, 14 and 15 had low nanomolar potency against MDA-MB-435 tumor cells and depolymerized microtubules. Compound 6 additionally had nanomolar GI₅₀ values against 57 of the NCI 60-tumor panel cell lines. Mechanistic studies showed that 6 inhibited tubulin polymerization and [³H]colchicine binding to tubulin. The most potent compounds were all effective in cells expressing P-glycoprotein or the βIII isotype of tubulin, which have been associated with clinical drug resistance. Modeling studies provided the potential interactions of 6, 14 and 15 within the colchicine site.     

Enhancement of the chiroptical response of α-amino acids via N-substitution for molecular structure determination using vibrational circular dichroism

The chiroptical response in the form of vibrational circular dichroism (VCD) in the midinfrared region is found to be enhanced when a hydrogen of amino group of l -tryptophan is substituted with acetyl, acryloyl, or maleyl group. The order of preference for VCD enhancement is found to be acryloyl > acetyl > maleyl group. The resulting experimental VCD spectra are also found to be satisfactorily reproduced by the quantum mechanical (QM) predicted spectra. The QM predicted spectra were simulated using the conformer populations, (a) predicted by Gibbs energies and (b) optimized to maximize the similarity between experimental and predicted VCD spectra. It is found that the conformer populations predicted by Gibbs energies do not yield the maximum possible similarity between experimental and the QM predicted spectra. This work identifies the N-substitution of α-amino acids and determining the conformer populations that best reproduce the experimental spectra as two new approaches for molecular structure determination.     

Illegal and unmanaged aquaculture,unregulated fisheries and extreme climatic events combine to trigger invasions in a global biodiversity hotspot

Biological Invasions - Focusing on extreme climatic events in India’s Western Ghats Biodiversity Hotspot, we demonstrate that unmanaged aquaculture and unregulated fisheries can often combine...