Synthesis, structures and antitumor activity of the first crown ester-linked bipyridyl platinum complexes.

Three new crown ester-linked bipyridine homologs with three, four or five ethylene glycol units, which are bulky and soluble in both hydrophilic and lipophilic media, were synthesized. The reaction of the appropriate macrocycles with K2PtCl4 in water gave yellow cisplatin analogs in good yield. These complexes were converted to carboplatin analogs by exchange of the leaving group. All the compounds were characterized by elemental analysis and various spectroscopic methods. Carboplatin analogs showed good solubility in both hydrophilic and lipophilic media. The crystal structure of 2c, the carboplatin analog with macrocycles containing five ethylene glycol units, was determined by X-ray diffraction: space group P1, a = 9.798(1), b = 12.580(3), c = 13.945(2) A, alpha = 108.61(2), beta = 94.59(1), gamma = 97.42(2) degrees, Z = 2, R = 0.0618. Some of platinum complexes showed a moderate cytotoxic effect on both murine leukemia L1210 and P388 even though they do not have any NH proton.     

Theoretical basis for stabilizing messenger RNA through secondary structure design

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term ‘superfolder’ mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.     

Delta-aminolevulinate dehydratase activity in red blood cells of rats infected with Trypanosoma evansi

The aim of this study was to evaluate the activity of delta-aminolevulinate dehydratase (δ-ALA-D) in red blood cells of rats infected with Trypanosoma evansi and establish its association with haematocrit, serum levels of iron and zinc and lipid peroxidation. Thirty-six male rats (Wistar) were divided into 2 groups with 18 animals each. Group A was non-infected while Group B was intraperitoneally infected, receiving 7·5×106 trypomastigotes per animal. Each group was divided into 3 subgroups of 6 rats and blood was collected during different periods post-infection (p.i.) as follows: day 5 (A1 and B1), day 15 (A2 and B2) and day 30 PI (A3 and B3). Blood samples were collected by cardiac puncture to estimate red blood cell parameters (RBC), δ-ALA-D activity and serum levels of iron, zinc and thiobarbituric acid reactive substances (TBARS). Rats in group B showed a significant (P<0·05) reduction of RBC count, haemoglobin concentration and haematocrit at days 5 and 15 p.i. The activity of δ-ALA-D in blood was significantly (P<0·001) increased at days 15 and 30 p.i. δ-ALA-D activity in blood had a significant (P<0·05) negative correlation with haematocrit (r=-0·61) and haemoglobin (r=-0·70) at day 15 p.i. There was a significant (P<0·05) decrease in serum iron and zinc levels and an increase in TBARS levels (P<0·05) during infection. The δ-ALA-D activity in blood was negatively correlated with the levels of iron (r=-0·68) and zinc (r=-0·57) on day 30 p.i. It was concluded that the increased activity of δ-ALA-D in blood might have occurred in response to the anaemia in remission as heme synthesis was enhanced.     

Investigation of Oncogenic Cooperation in Simple Liver-Specific Transgenic Mouse Models Using Noninvasive In Vivo Imaging

Liver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. Here, we report a simple methodology by which oncogenic cooperation between cancer-related genes can be efficiently investigated in the liver. We developed various non-germline transgenic mouse models using hydrodynamics-based transfection which express HrasG12V, SmoM2, and a short-hairpin RNA down-regulating p53 (shp53) individually or in combination in the liver. In this transgenic system, firefly luciferase was co-expressed with the oncogenes as a reporter, allowing tumor growth in the liver to be monitored over time without an invasive procedure. Very strong bioluminescence imaging (BLI) signals were observed at 4 weeks post-hydrodynamic injection (PHI) in mice co-expressing HrasG12V and shp53, while only background signals were detected in other double or single transgenic groups until 30 weeks PHI. Consistent with the BLI data, tumors were observed in the HrasG12V plus shp53 group at 4 weeks PHI, while other transgenic groups failed to exhibit a hyperplastic nodule at 30 weeks PHI. In the HrasG12V plus shp53 transgenic group, BLI signals were well-correlated with actual tumor growth in the liver, confirming the versatility of BLI-based monitoring of tumor growth in this organ. The methodology described here is expected to accelerate and facilitate in vivo studies of the hepatocarcinogenic potential of cancer-related genes by means of oncogenic cooperation.     

The role of calcium channel blockers and resveratrol in the prevention of paraquat-induced parkinsonism in Drosophila melanogaster: a locomotor analysis

Studies have suggested that neuronal loss in Parkinson's disease (PD) could be related to the pacemaker activity of the substantia nigra pars compacta generated by L-type Ca(v) 1.3 calcium channels, which progressively substitute voltage-dependent sodium channels in this region during aging. Besides this mechanism, which leads to increases in intracellular calcium, other factors are also known to play a role in dopaminergic cell death due to overproduction of reactive oxygen species. Thus, dihydropyridines, a class of calcium channel blockers, and resveratrol, a polyphenol that presents antioxidant properties, may represent therapeutic alternatives for the prevention of PD. In the present study, we tested the effects of the dihydropyridines, isradipine, nifedipine, and nimodipine and of resveratrol upon locomotor behavior in Drosophila melanogaster. As previously described, paraquat induced parkinsonian-like motor deficits. Moreover, none of the drugs tested were able to prevent the motor deficits produced by paraquat. Additionally, isradipine, nifedipine, resveratrol, and ethanol (vehicle), when used in isolation, induced motor deficits in flies. This study is the first demonstration that dyhidropyridines and resveratrol are unable to reverse the locomotor impairments induced by paraquat in Drosophila melanogaster.     

Assembly of the capsid protein of red-spotted grouper nervous necrosis virus during purification,and role of calcium ions in chromatography

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.     

Modulation of ethanol effect on hepatocyte proliferation by polyamines

An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.     

Advantages of Single-Molecule Real-Time Sequencing in High-GC Content Genomes

Next-generation sequencing has become the most widely used sequencing technology in genomics research, but it has inherent drawbacks when dealing with high-GC content genomes. Recently, single-molecule real-time sequencing technology (SMRT) was introduced as a third-generation sequencing strategy to compensate for this drawback. Here, we report that the unbiased and longer read length of SMRT sequencing markedly improved genome assembly with high GC content via gap filling and repeat resolution.