Complete Mitochondrial Genome and Phylogeny of Pleistocene MammothMammuthus primigenius

Phylogenetic relationships between the extinct woolly mammoth(Mammuthus primigenius), and the Asian(Elephas maximus) and African savanna(Loxodonta africana) elephants remain unresolved. Here, we report the sequence of the complete mitochondrial genome (16,842 base pairs) of a woolly mammoth extracted from permafrost-preserved remains from the Pleistocene epoch—the oldest mitochondrial genome sequence determined to date. We demonstrate that well-preserved mitochondrial genome fragments, as long as ~1,600–1700 base pairs, can be retrieved from pre-Holocene remains of an extinct species. Phylogenetic reconstruction of the Elephantinae clade suggests thatM. primigenius andE. maximus are sister species that diverged soon after their common ancestor split from theL. africana lineage. Low nucleotide diversity found between independently determined mitochondrial genomic sequences of woolly mammoths separated geographically and in time suggests that north-eastern Siberia was occupied by a relatively homogeneous population ofM. primigenius throughout the late Pleistocene.     

Molecular-dynamics simulations of pyronine 6G and rhodamine 6G dimers in aqueous solution

We have carried out molecular-dynamics (MD) simulations on dimers of the positively charged laser dyes pyronine 6G (P6G) and rhodamine 6G (R6G) in aqueous solution, generating trajectories of 2.5 ns for various computational protocols. We discuss how the choice of atomic partial charges and the length of the trajectories affect the predicted structures of the dimers and compare our results to those of earlier MD-simulations, which were restricted to only 0.7 ns. Our results confirm that monomers of P6G easily undergo relative rotations within the dimer, but we found new conformations of the R6G dimer at longer simulation times. In addition, we analyzed in detail the energy change during the formation of dimers. With suitable corrections, the electrostatic energy from an Ewald treatment agrees with the results from an approach relying on a residue-based cutoff. For P6G, we show that the strong solvent-mediated electrostatic attraction between the monomers is counteracted by an almost equally large solvent-induced entropy contribution to yield a small driving force to dimer formation, in very good agreement with the free-energy change from a thermodynamic-integration procedure. Thus, earlier rationalizations of the dimer formation, based only on energy arguments, yield a qualitatively wrong picture.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. M?ssbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θ(D0)~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c(2), and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.     

Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics

Novel acetone and aldimine covalent adducts were identified on the N‐termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled ¹³C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified–free glycine and found to correspond to acetone linked to the N‐terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from ¹³C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO₂ through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.     

A Modified Lentivirus-Based Reporter for Magnetic Separation of Cancer Stem Cells

Molecular Biology - Cancer stem cells (CSCs) are the most malignant subpopulation of tumor cells that possess a tumorigenic potential and resistantance to chemotherapy. These properties make CSCs a...     

Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template

The RNA-dependent RNA polymerase of the severe acute respiratory syndrome coronavirus 2 is an important target in current drug development efforts for the treatment of coronavirus disease 2019. Molnupiravir is a broad-spectrum antiviral that is an orally bioavailable prodrug of the nucleoside analogue β-D-N⁴-hydroxycytidine (NHC). Molnupiravir or NHC can increase G to A and C to U transition mutations in replicating coronaviruses. These increases in mutation frequencies can be linked to increases in antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis have not been reported. Here we studied the effects of the active compound NHC 5’-triphosphate (NHC-TP) against the purified severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase complex. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates followed the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), indicating that NHC-TP competes predominantly with CTP for incorporation. No significant inhibition of RNA synthesis was noted as a result of the incorporated monophosphate in the RNA primer strand. When embedded in the template strand, NHC-monophosphate supported the formation of both NHC:G and NHC:A base pairs with similar efficiencies. The extension of the NHC:G product was modestly inhibited, but higher nucleotide concentrations could overcome this blockage. In contrast, the NHC:A base pair led to the observed G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. Together, these biochemical data support a mechanism of action of molnupiravir that is primarily based on RNA mutagenesis mediated via the template strand.     

Supramolecular complexes of the <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> virulence protein VirE2

Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2 proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution in the form of complexes of 2–4 protein molecules of 12–18 nm size. We used computer methods to design models of complexes consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering. We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial length of the ssDNA.     

Actin cytoskeleton remodeling by the alternatively spliced isoform of PDLIM4/RIL protein

RIL (product of PDLIM4 gene) is an actin-associated protein that has previously been shown to stimulate actin bundling by interacting with actin-cross-linking protein α-actinin-1 and increasing its affinity to filamentous actin. Here, we report that the alternatively spliced isoform of RIL, denoted here as RILaltCterm, functions as a dominant-negative modulator of RIL-mediated actin reorganization. RILaltCterm is regulated at the level of protein stability, and this protein isoform accumulates particularly in response to oxidative stress. We show that the alternative C-terminal segment of RILaltCterm has a disordered structure that directs the protein to rapid degradation in the core 20 S proteasomes. Such degradation is ubiquitin-independent and can be blocked by binding to NAD(P)H quinone oxidoreductase NQO1, a detoxifying enzyme induced by prolonged exposure to oxidative stress. We show that either overexpression of RILaltCterm or its stabilization by stresses counteracts the effects produced by full-length RIL on organization of actin cytoskeleton and cell motility. Taken together, the data suggest a mechanism for fine-tuning actin cytoskeleton rearrangement in response to stresses.     

Oncolytic enteroviruses

The growing body of knowledge concerning the molecular biology of viruses and virus-cell interactions provides possibilities to use viruses as a tool in an effort to treat malignant tumors. As a rule, tumor cells are highly sensitive to viruses, which can be used in cancer therapy. At the same time, the application of viral oncolysis in cancer treatment requires that the highest possible safety be ensured for both the patient and environment. Human enteroviruses are a convenient source for obtaining oncolytic virus strains, since many of them are nonpathogenic for humans or cause mild disease. The current progress in genetic engineering enables the development of attenuated enterovirus variants characterized with high safety and selectivity. This review focuses on the main members of the Enterovirus genus, such as ECHO, coxsackievirus, and vaccine strains of poliovirus as a promising source for the development of oncolytic agents applicable for cancer therapy. We have summarized the data concerning recently developed and tested oncolytic variants of enteroviruses and discusses the perspectives of their application in cancer therapy, as well as problems associated with their improvement and practical use.     

Elevated proline content in maize plants expressing a fragment of the proline dehydrogenase gene in antisense orientation

A fragment of the first exon of the proline dehydrogenase gene of arabidopsis (Arabidopsis thaliana L.) in antisense orientation was transferred to maize (Zea mays L.) genome by agrobacterial transformation in planta to elevate the content of free proline in maize cells. The presence of genetic structure carrying an antisense sequence of the fragment of the proline dehydrogenase gene (ASPG) from the genome of diploid maize seedlings was corroborated by PCR method. T-DNA insertions comprising ASPG were found in the genomes of 30 plants (1.2% of 2409 examined seedlings) belonging to the generation T0. A reliable 4.6-fold increase in proline content was registered in the leaves of transformed maize plants.