Correction to "NMR Assays for Estimating the O-Acetyl Content of Meningococcal Polysaccharide Serogroup A in Quadrivalent Conjugate Vaccine Formulation".

[This corrects the article DOI: 10.1021/acsomega.9b01678.].

NMR Assays for Estimating the O-Acetyl Content of Meningococcal Polysaccharide Serogroup A in Quadrivalent Conjugate Vaccine Formulation.

The use of multivalent glycoconjugate vaccines has dramatically contributed to reduce the incidence of meningococcal infectious disease. The advanced structural characterization of polysaccharide conjugates leads to enhancements in the quality and control of the products. Here, we report a novel nuclear magnetic resonance (NMR) method to confirm the identity and structural conformity (e.g., O-acetyl content) of saccharide antigens that comprise a licensed tetravalent meningococcal serogroups A, C, W, and Y vaccine. For the first time, the NMR methodology is applied on a formulation (licensed vaccine) containing a large excess of excipient (i.e., sucrose) without analytical sample pretreatment. This work confirms the applicability of a rapid and easy NMR assay on a multivalent conjugate vaccine, which might be extended to other combination vaccines that are already licensed or in clinical development.

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy.

In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2ß-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.

Ablation of Protein Kinase CK2ß in Skeletal Muscle Fibers Interferes with Their Oxidative Capacity.

The tetrameric protein kinase CK2 was identified playing a role at neuromuscular junctions by studying CK2ß-deficient muscle fibers in mice, and in cultured immortalized C2C12 muscle cells after individual knockdown of CK2α and CK2ß subunits. In muscle cells, CK2 activity appeared to be at least required for regular aggregation of nicotinic acetylcholine receptors, which serves as a hallmark for the presence of a postsynaptic apparatus. Here, we set out to determine whether any other feature accompanies CK2ß-deficient muscle fibers. Hind limb muscles gastrocnemius, plantaris, and soleus of adult wildtype and CK2ß-deficient mice were dissected, cross-sectioned, and stained histochemically by Gomori trichrome and for nicotinamide adenine dinucleotide (NADH) dehydrogenase and succinate dehydrogenase (SDH) enzymatic activities. A reduction of oxidative enzymatic activity was determined for CK2ß-deficient muscle fibers in comparison with wildtype controls. Importantly, the CK2ß-deficient fibers, muscle fibers that typically exhibit high NADH dehydrogenase and SDH activities, like slow-type fibers, showed a marked reduction in these activities. Altogether, our data indicate additional impairments in the absence of CK2ß in skeletal muscle fibers, pointing to an eventual mitochondrial myopathy.

Synthesis, self-aggregation and bioactivity properties of a cationic aminoacyl surfactant, based on a new class of highly functionalized nucleolipids.

A highly functionalized aminoacyl nucleolipid based on uridine is here proposed as a novel cationic surfactant. To achieve this, a straightforward, high yielding and versatile protocol has been devised, in principle providing synthetic access to a variety of different, related analogs. Self-aggregation properties of this nucleolipid were determined by using a combined approach, including surface tension, conductivity and DLS measurements. Above the critical micellar concentration of 4 × 10(-5) mol kg(-1), large supramolecular assemblies with a counterion condensation degree of 0.25 were observed. The bioactivity profile of this new compound was investigated on cancer and non cancer cell lines.

N(3)-protection of thymidine with Boc for an easy synthetic access to sugar-alkylated nucleoside analogs.

The use of Boc as a nucleobase-protecting group in the synthesis of sugar-modified thymidine analogs is reported. Boc was easily inserted at N(3) by a simple and high-yielding reaction and found to be stable to standard treatments for the removal of Ac and (t) BuMe(2) Si (TBDMS) groups, as well as to ZnBr(2) -mediated 4,4'-dimethoxytrityl (DMTr) deprotection. Boc Protection proved to be completely resistant to the strong basic conditions required to regioselectively achieve O-alkylation, therefore, providing synthetic access to a variety of sugar-alkylated nucleoside analogs. To demonstrate the feasibility of this approach, two 3'-O-alkylated thymidine analogs have been synthesized in high overall yields and fully characterized.

Ruthenium-based complex nanocarriers for cancer therapy.

A new organometallic ruthenium complex, named AziRu, along with three amphiphilic nucleoside-based ruthenium complexes, ToThyRu, HoThyRu and DoHuRu, incorporating AziRu in their skeleton, have been synthesized, stabilized in POPC phospholipid formulations and studied for their antineoplastic activity. Self-aggregation behavior of these complexes was investigated, showing that the three synthesized AziRu derivatives able to form liposomes and, under specific conditions, elongated micelles. The formulations prepared in POPC proved to be stable for months and showed high in vitro antiproliferative activity. The here described results open new scenarios in the design of innovative transition metal-based supramolecular systems for anticancer drugs vectorization.

Cholesterol-based nucleolipid-ruthenium complex stabilized by lipid aggregates for antineoplastic therapy.

A novel ruthenium complex, linked to a cholesterol-containing nucleolipid (named ToThyCholRu), stabilized by lipid aggregates for antineoplastic therapy is presented. In order to retard the degradation kinetics typically observed for several ruthenium-based antineoplastic agents, ToThyCholRu is incorporated into a liposome bilayer formed by POPC. The resulting nanoaggregates contain up to 15% in moles of the ruthenium complex, and are shown to be stable for several weeks. The liposomes host the ruthenium-nucleolipid complex with the metal ion surrounded by POPC lipid headgroups and the steroid moiety inserted in the more external acyl chain region. These ruthenium-containing liposomes are more effective in inhibiting the growth of cancer cells than a model NAMI-A-like ruthenium complex, prepared for a direct evaluation of their anti-proliferative activity. These results introduce new perspectives in the design of innovative transition-metal-based supramolecular systems for anticancer drug vectorization.

Design, synthesis and characterisation of guanosine-based amphiphiles.

A small library of sugar-modified guanosine derivatives has been prepared, starting from a common intermediate, fully protected on the nucleobase. Insertion of myristoyl chains and of diverse hydrophilic groups, such as an oligoethylene glycol, an amino acid or a disaccharide chain, connected through in vivo reversible ester linkages, or of a charged functional group provided different examples of amphiphilic guanosine analogues, named G1-G7 herein. All of the sugar-modified derivatives were positive in the potassium picrate test, showing an ability to form G-tetrads. CD spectra demonstrated that, as dilute solutions in CHCl(3), distinctive G-quadruplex systems may be formed, with spatial organisations dependent upon the structural modifications. Two compounds, G1 and G2, proved to be good low-molecular-weight organogelators in polar organic solvents, such as methanol, ethanol and acetonitrile. Ion transportation experiments through phospholipid bilayers were carried out to evaluate their ability to mediate H(+) transportation, with G5 showing the highest activity within the investigated series. Moreover, G3 and G5 exhibited a significant cytotoxic profile against human MCF-7 cancer cells in in vitro bioassays.

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery.

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.

Identification of Erbin interlinking MuSK and ErbB2 and its impact on acetylcholine receptor aggregation at the neuromuscular junction.

Erbin, a binding partner of ErbB2, was identified as the first member of the LAP family of proteins. Erbin was shown at postsynaptic membranes of the neuromuscular junction (NMJ) or in cultured C2C12 myotubes (1) to be concentrated, (2) to regulate the Ras-Raf-Mek pathway, and (3) to inhibit TGF-beta signaling. In the CNS, Erbin interacts with PSD-95. Furthermore, agrin-MuSK signaling initiates formation of AChR aggregates at the postsynaptic membrane. In search of proteins interacting with MuSK, we identified Erbin as a MuSK binding protein. We verified the interaction of MuSK with Erbin, or both concomitantly with ErbB2 by coimmunoprecipitation, and we mapped the interacting epitopes between Erbin and MuSK. We demonstrated elevated mRNA levels of Erbin at synaptic nuclei and colocalized Erbin and MuSK at postsynaptic membranes. We identified several Erbin isoforms at the NMJ, all of which contained the MuSK binding domain. By knocking down Erbin, we observed agrin-dependent AChR aggregates on murine primary skeletal myotubes and C2C12 cells, and in the absence of agrin, microclusters, both of significantly lower density. Complementary, AChR-epsilon-reporter expression was reduced in myotubes overexpressing Erbin. We show that myotubes also express other LAP protein family members, namely Scribble and Lano, and that both affect physical dimensions of agrin-dependent AChR aggregates and density of microclusters formed in the absence of agrin. Moreover, MuSK-Erbin-ErbB2 signaling influences TGF-beta signaling. Our data define the requirement of Erbin on the cross talk between agrin and neuregulin signaling pathways at the NMJ.