Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Safety Evaluation of Lipid Nanoparticle-Formulated Modified mRNA in the Sprague-Dawley Rat and Cynomolgus Monkey.

The pharmacology, pharmacokinetics, and safety of modified mRNA formulated in lipid nanoparticles (LNPs) were evaluated after repeat intravenous infusion to rats and monkeys. In both species, modified mRNA encoding the protein for human erythropoietin (hEPO) had predictable and consistent pharmacologic and toxicologic effects. Pharmacokinetic analysis conducted following the first dose showed that measured hEPO levels were maximal at 6 hours after the end of intravenous infusion and in excess of 100-fold the anticipated efficacious exposure (17.6 ng/ml) at the highest dose tested.24 hEPO was pharmacologically active in both the rat and the monkey, as indicated by a significant increase in red blood cell mass parameters. The primary safety-related findings were caused by the exaggerated pharmacology of hEPO and included increased hematopoiesis in the liver, spleen, and bone marrow (rats) and minimal hemorrhage in the heart (monkeys). Additional primary safety-related findings in the rat included mildly increased white blood cell counts, changes in the coagulation parameters at all doses, as well as liver injury and release of interferon γ-inducible protein 10 in high-dose groups only. In the monkey, as seen with the parenteral administration of cationic LNPs, splenic necrosis and lymphocyte depletion were observed, accompanied with mild and reversible complement activation. These findings defined a well-tolerated dose level above the anticipated efficacious dose. Overall, these combined studies indicate that LNP-formulated modified mRNA can be administered by intravenous infusion in 2 toxicologically relevant test species and generate supratherapeutic levels of protein (hEPO) in vivo.

Targeting the protein ubiquitination machinery in melanoma by the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924).

Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC50 < 0.3 µM) and induced apoptosis in a subset of melanoma cell lines. Sensitive and resistant cell lines exhibited distinct gene expression profiles; sensitive models were enriched for genes involved in DNA repair, replication and cell cycle regulation, while immune response and cell adhesion pathways were upregulated in resistant models. Pevonedistat also reduced tumor growth in melanoma cell line xenografts and PDTX with variable responses. An accumulation of pevonedistat-NEDD8 adduct and CDT1 was observed in sensitive tumors consistent with its mechanism of action. Conclusions This study provided preclinical evidence that NAE inhibition by pevonedistat has anti-tumor activity in melanoma and supports the clinical benefits observed in recent Phase 1 trials of this drug in melanoma patients. Further investigations are warranted to develop rational combinations and determine predictive biomarkers of pevonedistat.

Louis Stromeyer (1804-76): German orthopaedic and military surgeon and his links with Britain.

Louis Stromeyer was a pioneer of orthopaedic surgery and military medicine in Germany. The 200th anniversary of his birth was celebrated on 6 March 2004. He was much admired in Britain, which he visited on three occasions and where he met many of the country's leading surgeons. He was a friend and colleague of the London surgeon W J Little (1810-94), whose clubfoot he corrected by the operation he developed, and which Little introduced to Britain: subcutaneous tenotomy. A bibliography of Stromeyer's publications is appended.

Phylogeography of Lake Malawi cichlids of the genus Pseudotropheus: significance of allopatric colour variation.

One of the most compelling features of the cichlid fishes of the African Great Lakes is the seemingly endless diversity of male coloration. Colour diversification has been implicated as an important factor driving cichlid speciation. Colour has also been central to cichlid taxonomy and, thus, to our concept of species diversity. We undertook a phylogeographical examination of several allopatric populations of the Lake Malawi cichlid Pseudotropheus zebra in order to reconstruct the evolutionary history of the populations, which exhibit one of two dorsal fin colours. We present evidence that populations with red dorsal fins (RT) are not monophyletic. The RT population defining the northern limit of the distribution has evidently originated independently of the southern RT populations, which share a common ancestry. This evidence of species-level colour convergence is an important discovery in our understanding of cichlid evolution. It implies that divergence in coloration may accompany speciation, and that allopatric populations with similar coloration cannot be assumed to be conspecific. In addition to this finding, we have observed evidence for introgression, contributing to current evidence that this phenomenon may be extremely widespread. Thus, in species-level phylogenetic reconstructions, including our own, consideration must be given to the potential effects of introgression.

Effects of immunizing school children with 2009 influenza A (H1N1) monovalent vaccine on absenteeism among students and teachers in Maine.

The overall and indirect effects of immunizing school children with influenza A (H1N1) 2009 pandemic virus vaccine prior to and during the peak of virus circulation were evaluated on student and teacher school absenteeism. We used records collected from late 2009 through early 2010 from schools in four Maine counties. Mixed logistic regression models were used to estimate the daily association between school-level immunization coverage and absenteeism by level of influenza activity, after adjusting for the proportion of students receiving reduced-cost lunches, student minority status, absences adjacent to weekends and Thanksgiving, rural school location, and the circulation of other respiratory viruses. Increasing student immunization coverage was associated with reduced absenteeism during periods of high influenza activity. For example, as immunization coverage during the peak week of pandemic virus circulation increased from 38% to 69% (the 10th and 90th percentiles of observed coverage, respectively), relative reductions in daily absenteeism among all students, unimmunized students, and teachers were 8.2% (95% confidence interval [CI]: 6.5, 9.9), 5.7% (95% CI: 4.2, 7.3), and 8.7% (95% CI: 1.3, 16), respectively. Increased vaccination coverage among school-aged Maine children had modest overall and indirect effects on student and teacher absenteeism, despite vaccination occurring just prior and during peak pandemic virus circulation.

Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression.

Wnt signaling plays critical biological roles during normal embryonic development and homeostasis in adults. In the canonical pathway, binding of Wnt ligands to the Frizzled (Fzd) receptor and the low density lipoprotein-related receptor (LRP) 5 or LRP6 coreceptor initiates downstream signaling events leading to gene activation by beta-catenin and the T-cell factor (TCF)-lymphoid enhancer factor (LEF) family transcription factor complex. In this study, we provide several lines of evidence that the mouse Cristin/R-spondin family proteins function as Fzd8 and LRP6 receptor ligands and induce the canonical Wnt/beta-catenin signaling pathway, leading to TCF-dependent gene activation. First, conditioned medium containing Cristin/R-spondin proteins effectively induced reporter activity in a TCF-binding site-dependent manner. Second, stimulation of cells with Cristin/R-spondin was accompanied by stabilization of endogenous beta-catenin proteins and induction of canonical Wnt target genes. Third, Cristin/R-spondin proteins physically interacted with the extracellular domains of the LRP6 and Fzd8 receptors in vivo and in vitro. Interestingly, unlike canonical Wnt ligands, Cristin/R-spondin failed to form a ternary complex with both LRP6 and Fzd8 receptors, suggesting that R-spondin may activate the canonical Wnt signaling pathway by different mechanisms. Furthermore, Cristin/R-spondin proteins possess an intriguing positive modulatory activity on Wnt ligands, possibly through a direct interaction. Our findings expand the repertoire of ligands that induce beta-catenin/TCF-dependent gene activation and implicate the presence of active beta-catenin-dependent gene activation in a Wnt-free biological context.

Direct detection of weakly interacting massive particles using non-cryogenic techniques.

A flux of weakly interacting neutral particles incident on the Earth would produce occasional nuclear-recoil events in any material. These could be detected as scintillation or ionization pulses in suitable target materials. Expected rates are in the range 1-10(-4) events kg(-1) d(-1). These must be distinguished from much higher rates due to backgrounds from gamma and beta background, even in deep underground locations. Methods of uniquely identifying nuclear recoils are described using crystal scintillators, scintillation and ionization processes in liquid xenon, and ionization tracks in gases. World progress on these techniques and future prospects are summarized. A possible future weakly interacting massive particle detector based on mechanical recoil of suspended microgranules is proposed which, with advances in nanotechnology, could eventually be extended to the detection of low-energy relic neutrinos.

Hybrid origin of a cichlid population in Lake Malawi: implications for genetic variation and species diversity.

The importance of species recognition to taxonomic diversity among Lake Malawi cichlids has been frequently discussed. Hybridization - the apparent breakdown of species recognition - has been observed sporadically among cichlids and has been viewed as both a constructive and a destructive force with respect to species diversity. Here we provide genetic evidence of a natural hybrid cichlid population with a unique colour phenotype and elevated levels of genetic variation. We discuss the potential evolutionary consequences of interspecific hybridization in Lake Malawi cichlids and propose that the role of hybridization in generating both genetic variability and species diversity of Lake Malawi cichlids warrants further consideration.