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The greatest challenges for therapeutic efficacy of many macromolecular drugs that act on intracellular are delivery to key organs and tissues and delivery into cells and subcellular compartments. Transport of drugs into critical cells associated with disease, including those in organs protected by restrictive biological barriers such as central nervous system (CNS), bone, and eye remains a significant hurdle to drug efficacy and impacts commercial risk and incentives for drug development for many diseases. These limitations expose a significant need for the development of novel strategies for macromolecule delivery. RTB lectin is the non-toxic carbohydrate-binding subunit B of ricin toxin with high affinity for galactose/galactosamine-containing glycolipids and glycoproteins common on human cell surfaces. RTB mediates endocytic uptake into mammalian cells by multiple routes exploiting both adsorptive-mediated and receptor-mediated mechanisms. In vivo biodistribution studies in lysosomal storage disease models provide evidence for the theory that the RTB-lectin transports corrective doses of enzymes across the blood–brain barrier to treat CNS pathologies. These results encompass significant implications for protein-based therapeutic approaches to address lysosomal and other diseases having strong CNS involvement.

GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid β-galactosidase (β-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1−/− cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes. Keywords: High-throughput imaging, GM1-gangliosidosis, Acid β-galactosidase, Cholera toxin B subunit

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called “hard-to-treat” tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treats organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1–4 h, both β-gal:RTB and RTB:β-gal were processed to the ~64 kDa “activated” β-gal form; the RTB lectin was cleaved and rapidly degraded. The activated β-gal was still detected at 48 h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis – delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.

In Antarctica, crabeater seals tend to strand as immature animals with disorientation, due to their inexperience, given as the probable cause. In 2012 and 2013, we examined a group of 80 mummified crabeater seals on Seymour Island (Marambio). The age and gender of 28 seals was determined, and virology and stomach content analyses were performed in order to determine the cause of stranding. Around 82% of the seals examined were adults and 79% were females, some of which were pregnant. All of the seals sampled tested negative for Morbillivirus, suggesting that the stranding was not related to the mass mortality event reported in the 1950s in the region. Most seals had empty stomachs and thin blubber suggesting that they died from starvation. The state of the carcasses suggests multiple stranding events. Most of the seals were located along an ice-covered stream, suggesting that this may act as a ‘natural trap’, isolating the seals from the open ocean. This is exceptional as it is the first report of mostly adult female seals to strand in Antarctica and refutes the theory that only young animals are prone to stranding. Fil: Negrete, Javier. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soibelzon, Leopoldo Héctor. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. División Paleontología Vertebrados; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soibelzon, Esteban. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. División Paleontología Vertebrados; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Márquez, Maria E. I.. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina Fil: Loza, Cleopatra Mara. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. División Paleontología Vertebrados; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Acosta, Walter. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina Fil: Lusky, Jorge. Ministerio de Relaciones Exteriores, Comercio Internacional y Culto. Direccion Nacional del Antártico; Argentina Fil: Pecoraro, Marcelo Ricardo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

A fractured femur of a giant fossil penguin from the Anthropornis nordenskjoeldi Biozone (Late Eocene), Isla Marambio (Seymour Island), Antarctica (La Meseta Formation) is described. Palaeoecological, palaeopathological and taphonomical implications derived from the analysis of the kinds of fractures identified are also discussed. The main fracture has irregular margins, indicating there was collagen in the bone at the time of the impact. In this fracture, a mineral deposit was also identified as a fracture hematoma. According to the antemortem classification of fractures, it was produced by an indirect mechanism, provocating a “butterfly wing” or “third fragment” fracture. The remaining fractures are assigned to times of biostratinomic and fossil diagenesis.

Enzyme replacement therapies have revolutionized patient treatment for multiple rare lysosomal storage diseases but show limited effectiveness for addressing pathologies in “hard-to-treat” organs and tissues including brain and bone. Here we investigate the plant lectin RTB as a novel carrier for human lysosomal enzymes. RTB enters mammalian cells by multiple mechanisms including both adsorptive-mediated and receptor-mediated endocytosis and thus provides access to a broader array of organs and cells. Fusion proteins comprised of RTB and human α-L-iduronidase, the corrective enzyme for Mucopolysaccharidosis type I, were produced using a tobacco-based expression system. Fusion products retained both lectin selectivity and enzyme activity, were efficiently endocytosed into human fibroblasts and corrected the disease phenotype of mucopolysaccharidosis patient fibroblasts in vitro. RTB-mediated delivery was independent of high-mannose and mannose-6-phosphate receptors, which are exploited for delivery of currently approved lysosomal enzyme therapeutics. Thus, the RTB carrier may support distinct in vivo pharmacodynamics with potential to address hard-to-treat tissues.

Arctotherium angustidens Gervais and Ameghino, 1880 (the South American giant short-faced bear) is known for being the earliest (Ensenadan Age, early to middle Pleistocene) and largest (body mass over 1 ton) of five described Arctotherium species endemic to South America. Here we assess the diet of this bear from multiple proxies: morphology, biomechanics, dental pathology, stable isotopes and a previous study using geometric morphometric methodology. Results favor the idea of animal matter consumption, probably from large vertebrates in addition to vegetable matter consumption. Most probably, active hunting was not the unique strategy of this bear for feeding, since its large size and great power may have allowed him to fight for the prey hunted by other Pleistocene carnivores. However, scavenging over mega mammal carcasses was probably another frequent way of feeding. South American short-faced bears adjusted their size and modified their diet through Pleistocene times, probably as a response to the diversification of the carnivore guild (from the few precursory taxa that crossed the Panamanian Isthmus during the Great American Biotic Interchange). Fil: Soibelzon, Leopoldo Héctor. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Departamento Científico de Paleontología de Vertebrados; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Grispan, Gustavo A.. Universidad de la República; Uruguay Fil: Bocherens, Hervé. University Tübingen; Alemania Fil: Acosta, Walter Gustavo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina Fil: Jones, Washington. Universidad de la República; Uruguay Fil: Blanco, Ernesto R.. Universidad de la República. Facultad de Ciencias; Uruguay Fil: Prevosti, Francisco Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina

Sixteen human cases of Fasciola hepatica infection are described. The liver was involved in 13 cases, the gall bladder in 9 cases and the stomach in 2 cases. Lesions containing parasitic remnants or fluke eggs were rarely seen. Surface scarring of the liver, scar tracks and granulomas within organs were the most characteristic changes seen and were the most useful for the histopathological diagnosis of the disease. The associated liver, bile and gastric lesions are briefly discussed.

In a South American lineage of 668 subjects, mainly Uruguayan, ninety-seven members were afflicted with one or more components of a triad consisting of nephritis, deafness and various eye disorders. These components are described. Men were more severely and more often fatally affected than women. Among the various genetic hypotheses, autosomal association and preferential segregation of X chromosome seems perhaps the most likely, although the data indicate that an additional unknown factor is involved.