A new Southern Ocean species in the remarkable and rare amphipod family Podosiridae (Crustacea: Amphipoda) questions existing systematic hypotheses

The amphipod family Podosiridae is unusual in that it combines morphological elements of the disparate families Podoceridae and Eusiridae. Here, we describe a new species in the family from specimens collected from the Southern Ocean in the vicinity of the South Orkney Islands and South Shetland Islands. We present mitochondrial (COI and 16S) and nuclear (18S) nucleic acid sequences for this and a congeneric species and use these to investigate the phylogenetic placement of Podosiridae within the Amphipoda. Our results do not provide evidence for a close relationship between Podosiridae and Podoceridae or Eusiridae, suggesting that the superficial similarity between these families is the result of morphological convergence. Instead, it is likely that Podosiridae are more closely related to families within Amphilochidira, such as Stenothoidae. Definitive placement of Podosiridae in the Amphipoda awaits further specimen collection, additional nucleotide data (including sequences from the Hyperiopsidae and the Vitjazianidae) and a more directed analysis of relationships within this portion of the amphipod phylogeny.

Tandem paired nicking promotes precise genome editing with scarce interference by p53

SummaryTargeted knock-in mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knock-in. Here, we demonstrate that tandem paired nicking, a method for targeted knock-in involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knock-in largely equivalent to that of the Cas9 nuclease-based approach. Tandem paired nicking seems to accomplish targeted knock-in via DNA recombination analogous to Holliday’s model, and creates intended genetic changes in the genome without introducing additional nucleotide changes such as silent mutations. Targeted knock-in through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes

Genetic code expansion has largely relied on two types of the tRNA—aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus alvus (CMaPylRS) is a single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS—tRNAPyl pair from C. M. alvus can incorporate lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs in Escherichia coli cell-based and cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.

How human serum albumin recognizes DNA and RNA

We show here for the first time that HSA possesses two nucleic acid-(NA) binding sites and we estimated the relative contributions of the nucleotide links of (pN)nto their total affinity for these binding sites with higher and lower affinity for NAs. The minimal ligands of these binding sites areorthophosphate (Kd=3.0 and 20.0 mm), various dNMPs (5.6–400 μmand 0.063–18 mm) and different rNMPs (4.9–30 μmand 14–250 μm). Maximal contribution to the total affinity of all NAs to the first and second sites was observed for one nucleotide and was remarkably lower for three additional nucleotide units of (pN)n(n=1–4) with a significant decrease in the contribution at n=5–6, and at n≥7–8 all dependencies reached plateaus. For d(pA)nand r(pA)na relatively gradual decrease in the contribution to the affinity at n=1–6 was observed, while several d(pN)n, demonstrated a sharp increase in the contribution at n=2–4. Finally, all (pN)n>10demonstrated high affinity for the first (1.4–150 nm) and the second (80–2400 nm) sites of HSA. Double-stranded NAs showed significantly lower affinity comparing with single-stranded ligands. The thermodynamic parameters characterizing the specific contribution of every nucleotide link of all (pN)1−9(ΔG°) to their total affinity for HSA were estimated.

UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall Partly by Inhibition of UDP-Galactopyranose Mutase

ABSTRACT Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a K i of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs. IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs.

Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase

ABSTRACTCyclopropavir (CPV) is a promising antiviral drug against human cytomegalovirus (HCMV). As with ganciclovir (GCV), the current standard for HCMV treatment, activation of CPV requires multiple steps of phosphorylation and is enantioselective. We hypothesized that the resulting CPV triphosphate (CPV-TP) would stereoselectively target HCMV DNA polymerase and terminate DNA synthesis. To test this hypothesis, we synthesized both enantiomers of CPV-TP [(+) and (−)] and investigated their action on HCMV polymerase. Both enantiomers inhibited HCMV polymerase competitively with dGTP, with (+)-CPV-TP exhibiting a more than 20-fold lower apparentKithan (−)-CPV-TP. Moreover, (+)-CPV-TP was a more potent inhibitor than GCV-TP. (+)-CPV-TP also exhibited substantially lower apparentKmand somewhat higher apparentkcatvalues than (−)-CPV-TP and GCV-TP for incorporation into DNA by the viral polymerase. As is the case for GCV-TP, both CPV-TP enantiomers behaved as nonobligate chain terminators, with the polymerase terminating DNA synthesis after incorporation of one additional nucleotide. These results elucidate how CPV-TP acts on HCMV DNA polymerase and help explain why CPV is more potent against HCMV replication than GCV.

Sparfloxacin Resistance in Clinical Isolates ofStreptococcus pneumoniae: Involvement of Multiple Mutations in gyrA and parC Genes

ABSTRACT Antimicrobial susceptibility testing revealed among 150 clinical isolates of Streptococcus pneumoniae 4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, ≥32 μg/ml; MIC of sparfloxacin, ≥16 μg/ml). Gene amplification and sequencing analysis of gyrA andparC revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 μg/ml, respectively, nucleotide changes were detected at codon 81 in gyrA and codon 79 in parC; these changes led to an Ser→Phe substitution in GyrA and an Ser→Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 μg/ml, revealed multiple mutations in both gyrA and parC. These strains exhibited nucleotide changes at codon 85 leading to a Glu→Lys substitution in GyrA, in addition to Ser-79→Tyr and Lys-137→Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp→Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in S. pneumoniae.

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.