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Plastination is a late 20th century preservation methodology which replaces tissue fluid within a specimen with a curable polymer, such as silicone. Plastination yields superb, beautiful, well-preserved specimens each with their own unique qualities. Silicone polymer is used around the world to preserve macroscopic cadavers or portions/organs thereof. Plastination was conceived by Dr. Gunther von Hagens, Universität Heidelberg, Heidelberg, Germany prior to 1977. Silicone polymer was the primary polymer which emerged initially for plastination. The Biodur ® line of silicone polymer and additives was chosen and manufactured because it has consistently produced the best plastinates since the inception of plastination. Since the discovery of silicone, generic and similar silicone polymers are known and used around the World by many industries and used in numerous products. The plastination process has four steps: Specimen preparation, Specimen dehydration and degreasing, Vacuum-forced impregnation of specimens and Specimen hardening., (© 2019 Blackwell Verlag GmbH.)

Plastination was a game-changing invention for macroscopic anatomical preparation. The method yielded dry, odourless, tangible and durable specimens which allowed new exhibition and teaching set-ups and paved the way for sophisticated preparations and spectacular positioning of specimens. Despite the impact of the new method, there have been similar techniques in place before. Exsiccation techniques, polymer embeddings and specimen impregnation with hardening substances were earlier methods which already included the main concepts that were later combined and refined in plastination. S10 silicone plastination, the technique most commonly known and applied, was followed by plastination methods suitable for research and sectional anatomy teaching. Numerous variations of sheet plastination techniques allow research applications and new ways of presenting topographic relations and mesoscopic insights. Besides the development of plastination techniques in sensu stricto, related techniques had a renaissance with new applications and developments, including corrosion casting and diaphonization methods. This brief review shall provide a historical context of plastination including some anecdotal spotlights on the ideas and innovations that lead to nowadays plastination techniques., (© 2019 Blackwell Verlag GmbH.)

For 20 years, the cold temperature/S10/von Hagens' plastination technique was used to preserve biological specimens without challenge. It became the "gold standard" for preservation of beautiful, dry biological specimens. Near the end of the 21st century, a group from the University of Michigan and environs and Dow Corning™, USA, combined silicone ingredients, similar to the von Hagens' plastination products, however in a different sequence. The new polymer (Cor-tech) was combined with the cross-linker to design the "impregnation mix" which would invade the cellular structure of the specimen and yet was stable at room temperature. Later, curing would be by application of the catalyst onto the impregnated specimen. This unique sequencing of products would become the "Room temperature/Dow Corning™/Corcoran-Silicone plastination technique." The results of this room temperature technique provided similar plastinates, beautiful and practical for demonstration, containing no toxic chemical residues and forever preserved. As the name implies, impregnation of this silicone mix could be done at room temperature, without having to be kept cold. Both processes (cold and room temperature) required the same four basic steps for plastination. As well, both processes used similar basic polymers and additives to produce plastinates. However, they were combined in a different sequence. Cold temperature combines polymer and catalyst/chain extender, which is not stable and therefore must be kept colder than -15°C, while room temperature combines polymer with cross-linker which is stable, and likely forever., (© 2019 Blackwell Verlag GmbH.)

In the early days of plastination, plastinate Color was the usual grey/brown familiar to formalin-fixed biological specimens. Initially, trials with Kaiserling's, Klotz, Jore's and McCormick's fixative solutions were disappointing. Vascular injections with Colored epoxy were a great breakthrough in the 1980s. Biodur AC10 ® stain was the first stain of note to be applied to gross specimens to be plastinated and was applied in the last acetone bath. As plastination became more popular, specimen Color became an important and necessary aspect. Reactivation of the normal Color of red blood cells within a formalin-fixed specimen was introduced as a mechanism to restore Color to plastinated specimens. Painting of plastinated vessels was tried with some success, and finally, a superior new proprietary type of silicone coloration was developed. More recently, a versatile red pigment stain was developed. All of these have added aesthetically to the plastination processes and will certainly be a reality in the years to come. The various methodologies to Color plastinates are presented. Time will tell how effective these may or may not be., (© 2019 Blackwell Verlag GmbH.)

In 1977, plastination was unveiled, which replaced tissue fluid with a curable polymer. Today preservation via plastination of various animal and plant tissues, organs, and whole bodies is an extremely useful technique to display such and help educate vast arrays of both allied science students and the lay public across the planet. The diversity of applications of plastination techniques seems to be without limits. In fact, the only real limitation to plastination is one's imagination! The size of plastinates during the early years of plastination was comparatively small and dictated primarily by the size of the available plastination kettle/chamber, 35 L Heidelberg plastination kettle (49 cm H × 34.5 cm diam.). In the 1990s larger chambers were designed and slowly became available:150-210 cm (long) × 65-80 cm (wide) and 83-92 cm (high). Today a few large vacuum chambers are in service which will accommodate whole bodies of man and domestic or exotic animals. Today, at least two gigantic chambers are available to impregnate massive specimens. These are 3.5 m × 2 m × 1.5 m (Dalian) and 4 m × 3 m × 2.2 m (Guben). Also, the need for larger quantities of acetone and impregnation mix, not to mention the great increase in specimen preparation time, makes this a major investment. The "cold temperature process" is used to impregnate these massive creations. The room temperature technique could be used. The same four plastination steps are necessary for larger and massive specimens. Besides their tremendous size, the slippery silicone polymer is a reckoning force., (© 2019 Blackwell Verlag GmbH.)

The ramus communicans, neural connection between medial and lateral plantar nerves of the horse, was transected to determine the degree to which medial and lateral plantar nerves contribute to the plantar ramus. After 2 months, sections of plantar nerves immediately proximal and distal to the communicating branch were collected and processed for electron microscopy. All examined nerves had undergone Wallerian degeneration and contained regenerating and mature fibers. Layers of the myelin sheath were separated by spaces and vacuoles, indicating demyelination of medial and lateral plantar nerves. Shrunken axons varied in diameter and were surrounded by an irregular axolemma. Shrunken axoplasm of both myelinated and non-myelinated fibers contained ruptured mitochondria and cristae, disintegrating cytoskeleton, and vacuoles of various sizes. The cytoplasm of neurolemmocytes contained various-sized vesicles, ruptured mitochondria within a fragile basal lamina and myelin whorls of multilayered structures indicative of Wallerian degeneration. These ultrastructural changes, found proximal and distal to the ramus in medial and lateral plantar nerves, suggest that axonal flow is bi-directional through the ramus communicans of the pelvic limbs of horses, a previously unreported finding. As well, maturity of nerves proximal and distal to the ramus indicates that all nerve fibers do not pass through the ramus.

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle., Competing Interests: The authors declare no conflict of interest.

In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical-basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis.

The retinoblastoma tumor suppressor (RB) and its related family members p107 and p130 regulate cell proliferation through the transcriptional repression of genes involved in cellular G1 to S phase transition. However, RB proteins are functionally versatile, and numerous genetic and biochemical studies point to expansive roles in cellular growth control, pluripotency, and apoptotic response. For the vast majority of genes, RB family members target the E2F family of transcriptional activators as an integral component of its gene regulatory mechanism. These interactions are regulated via reversible phosphorylation by Cyclin/Cyclin-dependent kinase (Cdk) complexes, a major molecular mechanism that regulates transcriptional output of RB/E2F target genes. Recent studies indicate an additional level of regulation involving the ubiquitin-proteasome system that renders pervasive control over each component of the RB pathway. Disruption of the genetic circuitry for proteasome-mediated targeting of the RB pathway has serious consequences on development and cellular transformation, and is associated with several forms of human cancer. In this review, we discuss the role of the ubiquitin-proteasome system in proteolytic control of RB-E2F pathway components, and recent data that points to surprising non-proteolytic roles for the ubiquitin-proteasome system in novel transcriptional regulatory mechanisms., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Use of complete mitochondrial genomes (mitogenomes) can greatly increase the resolution achievable in phylogeographic and historical demographic studies. Using next-generation sequencing methods, it is now feasible to efficiently sequence mitogenomes of large numbers of individuals once a reference mitogenome is available. However, assembling the initial mitogenomes of nonmodel organisms can present challenges, for example, in birds, where mtDNA is often subject to gene rearrangements and duplications. We developed a workflow based on Illumina paired-end, whole-genome shotgun sequencing, which we used to generate complete 19-kilobase mitogenomes for each of three species of North Pacific albatross, a group of birds known to carry a tandem duplication. Although this duplication had been described previously, our procedure did not depend on this prior knowledge, nor did it require a closely related reference mitogenome (e.g. a mammalian mitogenome was sufficient). We employed an iterative process including de novo assembly, reference-guided assembly and gap closing, which enabled us to detect duplications, determine gene order and identify sequence for primer positioning to resolve any mitogenome ambiguity (via minimal targeted Sanger sequencing). We present full mtDNA annotations, including 22 tRNAs, 2 rRNAs, 13 protein-coding genes, a control region and a duplicated feature for all three species. Pairwise comparisons supported previous hypotheses regarding the phylogenetic relationships within this group and occurrence of a shared tandem duplication. The resulting mitogenome sequences will enable rapid, high-throughput NGS mitogenome sequencing of North Pacific albatrosses via direct reference-guided assembly. Moreover, our approach to assembling mitogenomes should be applicable to any taxon., (© 2014 John Wiley & Sons Ltd.)

The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. To investigate whether the unique features of RBf2 contribute to diverse roles in gene regulation, we performed chromatin immunoprecipitation sequencing for both RBf2 and RBf1 in embryos. A previous model for RB-E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; however, we found that RBf2 targets approximately twice as many genes as RBf1. Highly enriched among the RBf2 targets were ribosomal protein genes. We tested the functional significance of this finding by assessing RBf activity on ribosomal protein promoters and the endogenous genes. RBf1 and RBf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. We carried out bioinformatic analysis to investigate the basis for differential targeting by these two proteins and found that RBf2-specific promoters have distinct sequence motifs, suggesting unique targeting mechanisms. Association of RBf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both RBf1 and RBf2 require dE2F2/dDP. The presence of unique RBf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family., (Copyright © 2015 Wei et al.)

The ultrastructure of the developing bronchiolar cell was studied in six age groups: prenatal (60 d post-conception); postnatal (1-, 7-, 14- and 21-day-old); and adult. Following intratracheal fixation, the lung tissue was processed for scanning and transmission electron microscopy. The lining of terminal bronchioles consists of cuboidal to columnar nonciliated bronchiolar cells (NBCs) and ciliated with or without microvilli. NBCs were recognized by indented centrally located nucleus. The apical surface extended beyond the surface of neighboring cells and was covered by minute microvilli, except in prenatal kittens. The NBCs of the adult were characterized by abundant mitochondria and glycogen inclusions. In prenatal kittens, the cytoplasm was filled with patches of alpha and beta form of glycogen. Postnatally, glycogen was reduced in quantity, became scattered throughout the cytoplasm and was predominantly of the beta form. Islands of cytoplasm, separated from the apical cytoplasm were observed in the lumen of adult bronchioles. This suggests an apocrine mode of secretion. The NBCs attain maturity by three weeks of age.

The RB tumor suppressor, a regulator of the cell cycle, apoptosis, senescence, and differentiation, is frequently mutated in human cancers. We recently described an evolutionarily conserved C-terminal "instability element" (IE) of the Drosophila Rbf1 retinoblastoma protein that regulates its turnover. Misexpression of wild-type or non-phosphorylatable forms of the Rbf1 protein leads to repression of cell cycle genes. In contrast, overexpression of a defective form of Rbf1 lacking the IE (ΔIE), a stabilized but transcriptionally less active form of the protein, induced ectopic S phase in cell culture. To determine how mutations in the Rbf1 IE may induce dominant effects in a developmental context, we assessed the impact of in vivo expression of mutant Rbf1 proteins on wing development. ΔIE expression resulted in overgrowth of larval wing imaginal discs and larger adult wings containing larger cells. In contrast, a point mutation in a conserved lysine of the IE (K774A) generated severely disrupted, reduced wings. These contrasting effects appear to correlate with control of apoptosis; expression of the pro-apoptotic reaper gene and DNA fragmentation measured by acridine orange stain increased in flies expressing the K774A isoform and was suppressed by expression of Rbf1ΔIE. Intriguingly, cancer associated mutations affecting RB homologs p130 and p107 may similarly induce dominant phenotypes.

The retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications; however, we lack a general understanding of how distinct modifications coordinately impact both of these properties. Previously, we showed that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, we assessed the impact of Cyclin-Cdk kinase regulation on Rbf1. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response and showed a dominant impact on turnover and activity; however, specific residues in the C-terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved Lys-774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. Our data support a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The gross anatomical structure of the ringed seal (Pusa hispida) gastrointestinal tract is poorly described and often veterinary anatomical terminology is not used. Although the basic abdominal visceral pattern corresponded to domestic carnivores, significant differences were noted. The stomach was an elongated sharply bent tube (u-shaped) with the pylorus and fundus juxtaposed. The elongated jejunum measured up to 15.6 times body length and had 37 jejunal arteries from the cranial mesenteric artery. The pancreas was asymmetrical with a small right lobe and a large left lobe. The unusually short greater omentum negated formation of deep and superficial leaves. The most remarkable difference was the separation of the liver parenchyma into three physically separate masses, held together by hepatic ducts, veins and arteries. The topography and position of the liver was dependent on the amount of blood in the hepatic sinus (distended hepatic veins and hepatic portion of vena cava). Thus, as the hepatic sinus filled, the lateral liver masses separate from the central mass by moving caudolaterally. This was facilitated by modified coronary and triangular ligaments which did not attach directly to the liver, but instead to the hepatic sinus. These anatomical adaptations are apparently advantageous to ringed seal's survival in a deep marine environment., (© 2013 Blackwell Verlag GmbH.)

Objective: To assess outcome after neurectomy of the deep branch of the lateral palmar nerve (DBLPaN) as a treatment for horses with persistent lameness associated with chronic proximal suspensory desmitis (PSD) of the thoracic limb., Study Design: Case series., Animals: Adult, mixed-breed horses (n = 4), weighing 510-585 kg, used for amateur show-jumping., Methods: Records of 4 horses chronically lame because of PSD of one or both thoracic limbs that were treated by neurectomy of the DBLPaN were reviewed. The site of pain causing lameness was localized using regional anesthesia. The proximal aspect of the suspensory ligament of the affected limb(s) of all horses were enlarged on ultrasonographic examination, but fiber disruption was not observed. All horses remained lame after conservative therapy. Neurectomy was performed with the horses anesthetized and positioned in dorsal recumbency., Results: All 4 horses were sound at 6 weeks and remained sound for at least 12 months after neurectomy., Conclusion: Lameness in horses caused by chronic PSD can be resolved by neurectomy of the DBLPaN in horses that are refractory to conservative management., (© Copyright 2012 by The American College of Veterinary Surgeons.)

Multiple myeloma (MM) is a malignant disorder of differentiated B-cells for which standard care involves the inhibition of the proteasome. All clinically used proteasome inhibitors, including the chemotherapeutic drug bortezomib, target the catalytic active sites of the proteasome and inhibit protein proteolysis by competing with substrate binding. However, nearly all (~97%) patients become intolerant or resistant to treatments within a few years, after which the average survival time is less than 1 year. We describe herein the inhibition of the human proteasome via a noncompetitive mechanism by the imidazoline scaffold, TCH-13. Consistent with a mechanism distinct from that of competitive inhibitors, TCH-013 acts additively with and overcomes resistance to bortezomib. Importantly, TCH-013 induces apoptosis in a panel of myeloma and leukemia cell lines, but in contrast, normal lymphocytes, primary bone marrow stromal cells (hBMSC), and macrophages are resistant to its cytotoxic effects. TCH-013 was equally effective in blocking MM cell growth in co-cultures of MM cells with hBMSC isolated from CD138 negative bone marrow (BM) samples of MM patients. The cellular activity translated well in vivo where TCH-013 delayed tumor growth in an MM xenograft model to a similar extent as bortezomib.

The retinoblastoma (RB) tumor suppressor protein regulates multiple pathways that influence cell growth, and as a key regulatory node, its function is inactivated in most cancer cells. In addition to its canonical roles in cell cycle control, RB functions as a global repressor of RNA polymerase (Pol) III transcription. Indeed, Pol III transcripts accumulate in cancer cells and their heightened levels are implicated in accelerated growth associated with RB dysfunction. Herein we review the mechanisms of RB repression for the different types of Pol III genes. For type 1 and type 2 genes, RB represses transcription through direct contacts with the core transcription machinery, notably Brf1-TFIIIB, and inhibits preinitiation complex formation and Pol III recruitment. A contrasting model for type 3 gene repression indicates that RB regulation involves stable and simultaneous promoter association by RB, the general transcription machinery including SNAPc, and Pol III, suggesting that RB may impede Pol III promoter escape or elongation. Interestingly, analysis of published genomic association data for RB and Pol III revealed added regulatory complexity for Pol III genes both during active growth and during arrested growth associated with quiescence and senescence. This article is part of a Special Issue entitled: Transcription by Odd Pols., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Stressors associated with human activities interact in complex ways to affect marine ecosystems, yet we lack spatially explicit assessments of cumulative impacts on ecologically and economically key components such as marine predators. Here we develop a metric of cumulative utilization and impact (CUI) on marine predators by combining electronic tracking data of eight protected predator species (n=685 individuals) in the California Current Ecosystem with data on 24 anthropogenic stressors. We show significant variation in CUI with some of the highest impacts within US National Marine Sanctuaries. High variation in underlying species and cumulative impact distributions means that neither alone is sufficient for effective spatial management. Instead, comprehensive management approaches accounting for both cumulative human impacts and trade-offs among multiple stressors must be applied in planning the use of marine resources.

The retinoblastoma (RB) tumor suppressor family functions as a regulatory node governing cell cycle progression, differentiation, and apoptosis. Post-translational modifications play a critical role in modulating RB activity, but additional levels of control, including protein turnover, are also essential for proper function. The Drosophila RB homolog Rbf1 is subjected to developmentally cued proteolysis mediated by an instability element (IE) present in the C terminus of this protein. Paradoxically, instability mediated by the IE is also linked to Rbf1 repression potency, suggesting that proteolytic machinery may also be directly involved in transcriptional repression. We show that the Rbf1 IE is an autonomous degron that stimulates both Rbf1 ubiquitination and repression potency. Importantly, Rbf1 IE function is promoter-specific, contributing to repression of cell cycle responsive genes but not to repression of cell signaling genes. The multifunctional IE domain thus provides Rbf1 flexibility for discrimination between target genes embedded in divergent cellular processes.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The retinoblastoma (RB) tumor suppressor protein is a transcriptional cofactor with essential roles in cell cycle and development. Physical and functional targets of RB and its paralogs p107/p130 have been studied largely in cultured cells, but the full biological context of this family of proteins' activities will likely be revealed only in whole organismal studies. To identify direct targets of the major Drosophila RB counterpart in a developmental context, we carried out ChIP-Seq analysis of Rbf1 in the embryo. The association of the protein with promoters is developmentally controlled; early promoter access is globally inhibited, whereas later in development Rbf1 is found to associate with promoter-proximal regions of approximately 2000 genes. In addition to conserved cell-cycle-related genes, a wholly unexpected finding was that Rbf1 targets many components of the insulin, Hippo, JAK/STAT, Notch, and other conserved signaling pathways. Rbf1 may thus directly affect output of these essential growth-control and differentiation pathways by regulation of expression of receptors, kinases and downstream effectors. Rbf1 was also found to target multiple levels of its own regulatory hierarchy. Bioinformatic analysis indicates that different classes of genes exhibit distinct constellations of motifs associated with the Rbf1-bound regions, suggesting that the context of Rbf1 recruitment may vary within the Rbf1 regulon. Many of these targeted genes are bound by Rbf1 homologs in human cells, indicating that a conserved role of RB proteins may be to adjust the set point of interlinked signaling networks essential for growth and development.

The E2F family of transcription factors contributes to oncogenesis through activation of multiple genes involved in cellular proliferation, a process that is opposed by the Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by inhibiting its proteasome-mediated degradation, but the consequences of this post-translational regulation of E2F1 remain unknown. To better understand the mechanism of E2F stabilization and its physiological relevance, we examined the streamlined Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that modulates E3 ubiquitin ligase activity. Here, we report that the COP9 signalosome also protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels, enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1. We further show that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1 cell cycle-target genes are distinct properties. Removal of an evolutionarily conserved Rbf1 C terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This mutant form of Rbf1 also enhanced G(1)-to-S phase progression when expressed in Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate gain-of-function properties relevant to cellular transformation. Consistent with this idea, several studies have identified mutations in the homologous C terminal domains of RB and p130 in human cancer.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The urethral fold of 30 mares was split transversely into dorsal and ventral shelves, and the ventral shelf was used to help create a urethral extension. The dorsal shelf was stretched caudally and sutured to the roof of the extension so that it covered at least the cranial half of the extension. For 20 mares, a relaxing, vaginal incision was created cranial to the external urethral orifice to enable the dorsal shelf to be retracted further caudally. Ten of the 30 mares (33.3 per cent) developed a defect, but none developed a defect in that portion covered by the dorsal shelf of the urethral fold. Two of the 30 mares (6.7 per cent) developed a defect so small that the defect could be detected only by inserting a dye, under pressure, into the tunnel. The total number of mares that developed only a grossly visible and palpable defect was eight of 30 (26.6 per cent). Four of the 10 mares that did not receive the relief incision and six of 20 mares that did receive the relief incision developed a defect in the extension. Modifying the McKinnon technique by transversely splitting the urethral fold and retracting the dorsal half helps prevent a defect from forming in the cranial portion of the extension. The dorsal shelf can be retracted further caudally by creating a relief incision on the floor of the vagina.

Purpose: To assess the intrarenal arteries injuries after cranial pole nephrectomy in a pig model to compare these findings with those in humans., Materials and Methods: Polyester resin was injected through the ureter and the renal artery to make three-dimensional casts of 61 pig kidneys. The cranial pole of the kidneys was sectioned at four different sites before the solidification of the resin, and the casts were examined for arterial damage., Results: Section performed through the hilus (15 kidneys): The cranial division of the renal artery was sectioned in two (13.33%) cases, the ventral branch of the cranial division of the renal artery was sectioned in 13 (86.7%) cases, and the dorsal branch of the cranial division of the renal artery was sectioned in 11 (73.34%) cases. Section at 0.5 cm cranial to the hilus (16 kidneys): The cranial division of the renal artery was sectioned in 1 (6.25%) case, the ventral branch of the cranial division of the renal artery was sectioned in 14 (87.5%) cases, and the dorsal branch of the cranial division of the renal artery was sectioned in 13 (81.25%) cases. Section at 1.0 cm cranial to the hilus (15 kidneys): The ventral branch of the cranial division of the renal artery was sectioned in five (33.33%) cases, and the dorsal branch of the cranial division of the renal artery was injured in five (33.33%) cases. Section at 1.5 cm cranial to the hilus (15 kidneys): No lesions were found in the main arteries, only in the interlobular branches., Conclusions: As previously demonstrated in humans, sections at 1.0 cm or more cranially to the hilus in pigs also showed a significant decrease in damage to the major intrarenal arteries. Therefore, as regards arterial damage, the pig kidney is a useful model for partial nephrectomy in the cranial (upper) pole.

The respiratory tracts of seven grey short-tailed opossums were histologically examined. Six opossums were prepared by perfusion with buffered formalin. Opossum seven was perfused with gluteraldehyde. Samples taken from the respiratory passages and lungs of specimens 1-6 were stained with haematoxylin and eosin. A mixture of methylene and azure blue was used for specimen 7. The trachea and right and left principal bronchi are lined with a pseudostratified ciliated columnar epithelium with occasional goblet cells. The secondary and tertiary bronchi and the primary and secondary bronchioles are lined by a simple ciliated columnar epithelium. The terminal bronchioles and a portion of the respiratory bronchioles are lined by a simple ciliated cuboidal epithelium. The terminal portion of the respiratory bronchioles and the alveolar ducts are lined with simple squamous epithelium. Alveoli are lined by type I and II pneumocytes. Tracheal glands are present in the tela submucosa. The fibromusculocartilaginous tunic of the trachea consists of c-shaped cartilage rings and the trachealis muscle. A lamina muscularis mucosa begins in the intrapulmonary portion of the principal bronchus and continues into the respiratory bronchioles. Bronchial glands are present in the propria submucosa and tela submucosa of the principal bronchi. The musculocartilaginous tunic is localized to the extrapulmonary portion of the principal bronchus. The bronchial cartilages are irregular shaped plates and limited to the extrapulmonary portion of the principal bronchus. The visceral pleura is a simple squamous mesothelium covering the outer surface of the lung., (© 2011 Blackwell Verlag GmbH.)

The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks.

Our aim was to compare plastinated sections of the canine heart with corresponding two-dimensional (2D) echocardiographic images. Thirteen dog hearts were fixed by dilation and then processed by the S10 silicon plastination method (Biodur). Two dogs without evidence of cardiac disease were imaged using 2D echocardiography so as to obtain a complete series of the standard right and left parasternal images, which were compared with corresponding plastinated slices obtained by knife sectioning of the hearts. The plastinated slices revealed the internal anatomy of the heart with great detail and were particularly useful to display the spatial relationship between complex anatomic structures. The plastinated slices corresponded accurately with the echocardiographic images. Because of the dilation of the right heart during the fixation process, it was not possible to obtain plastinated specimens in ventricular systole. This paper may be a reference atlas for assisting 2D echocardiography interpretation.

DNA damage induced by ionizing radiation activates the ataxia telangiectasia mutated pathway, resulting in apoptosis or DNA repair. The serine/threonine checkpoint kinase (Chk2) is an important transducer of this DNA damage signaling pathway and mediates the ultimate fate of the cell. Chk2 is an advantageous target for the development of adjuvant drugs for cancer therapy, because inhibition of Chk2 allows normal cells to enter cell cycle arrest and DNA repair, whereas many tumors bypass cell cycle checkpoints. Chk2 inhibitors may thus have a radioprotective effect on normal cells. We report herein a class of natural product derived Chk2 inhibitors, exemplified by indoloazepine 1, that elicit a strong ATM-dependent Chk2-mediated radioprotection effect in normal cells and p53 wt cells, but not p53 mutant cells (>50% of all cancers). This study represents the first example of a radioprotective effect in human cells other than T-cells and implicates a functional ATM pathway as a requirement for IR-induced radioprotection by this class of Chk2 inhibitors. Several of the hymenialdisine-derived analogues inhibit Chk2 at nanomolar concentrations, inhibit autophosphorylation of Chk2 at Ser516 in cells, and increase the survival of normal cells following ionizing radiation.

Here we report the first measurements of polybrominated diphenyl ethers (PBDE 47, 99, and 153) alongside 11 organochlorine pesticides (OCPs) and 28 polychlorinated biphenyls (PCBs) in the plasma of albatross from breeding colonies distributed across a large spatial east-west gradient in the North Pacific Ocean. North Pacific albatross are wide-ranging, top-level consumers that forage in pelagic regions of the North Pacific Ocean, making them an ideal sentinel species for detection and distribution of marine contaminants. Our work on contaminant burdens in albatross tissue provides information on transport of persistent organic pollutants (POPs) to the remote North Pacific and serves as a proxy for regional environmental quality. We sampled black-footed (Phoebastria nigripes; n = 20) and Laysan albatross (P. immutabilis; n = 19) nesting on Tern Island, Hawaii, USA, and Laysan albatross (n = 16) nesting on Guadalupe Island, Mexico. Our results indicate that North Pacific albatross are highly exposed to both PCBs and OCPs, with levels ranging from 8.8 to 86.9 ng/ml wet weight and 7.4 to 162.3 ng/ml wet weight, respectively. A strong significant gradient exists between Laysan albatross breeding in the Eastern Pacific, having approximately 1.5-fold and 2.5-fold higher levels for PCBs and OCPs, respectively, compared to those from the Central Pacific. Interspecies levels of contaminants within the same breeding site also showed high variation, with Tern black-footed albatross having approximately threefold higher levels of both PCBs and OCPs than Tern Laysan albatross. Surprisingly, while PBDEs are known to travel long distances and bioaccumulate in wildlife of high trophic status, we detected these three PBDE congeners only at trace levels ranging from not detectable (ND) to 0.74 ng/ml wet weight in these albatross., (Copyright © 2011 SETAC.)

Thorough dehydration is a key for good plastination and invariably it leads to shrinkage. Shrinkage during plastination has been studied to lesser extent. Shrinkage was studied in 10 pig kidneys including regional shrinkage (cortex, medulla, sinus) and at which stages of the process (dehydration, impregnation, curing) shrinkage occurred. Kidneys were fixation by perfusion of 10% neutral buffered formalin solution via the renal artery. The vessels and ureter were filled with colored silicone (Dow Corning, Silastic E RTV Silicone Rubber) and the kidneys were cut into one centimeter transverse slices. Two slices of each kidney were plastinated via the classic von Hagens' method. Slices were photographed at the same focal length after preparation and at the end of each stage of plastination. Slice surface area was determined by a point-counting planimetry method. Post dehydration shrinkage of the kidney was 10.21% while post impregnation 10.11%. After completion of plastination, total area of kidney slice shrinkage was 19.72%. Cortical area shrunk 12.81% after dehydration and 13.16% after impregnation. After plastination, cortical area had shrunk 24.28%. No significant shrinkage occurred in the medulla and sinus. Results demonstrate that kidney shrinkage during impregnation is as intense as during dehydration. Significant shrinkage occurred in the renal cortex but not in the medulla and sinus. This demonstrates that different tissue types, even in the same specimen, have different rates of shrinkage during dehydration and impregnation. Therefore, plastinated specimens should be used carefully in research where obtaining measures is important., (Copyright © 2011 Wiley-Liss, Inc.)

Pelagic marine predators face unprecedented challenges and uncertain futures. Overexploitation and climate variability impact the abundance and distribution of top predators in ocean ecosystems. Improved understanding of ecological patterns, evolutionary constraints and ecosystem function is critical for preventing extinctions, loss of biodiversity and disruption of ecosystem services. Recent advances in electronic tagging techniques have provided the capacity to observe the movements and long-distance migrations of animals in relation to ocean processes across a range of ecological scales. Tagging of Pacific Predators, a field programme of the Census of Marine Life, deployed 4,306 tags on 23 species in the North Pacific Ocean, resulting in a tracking data set of unprecedented scale and species diversity that covers 265,386 tracking days from 2000 to 2009. Here we report migration pathways, link ocean features to multispecies hotspots and illustrate niche partitioning within and among congener guilds. Our results indicate that the California Current large marine ecosystem and the North Pacific transition zone attract and retain a diverse assemblage of marine vertebrates. Within the California Current large marine ecosystem, several predator guilds seasonally undertake north-south migrations that may be driven by oceanic processes, species-specific thermal tolerances and shifts in prey distributions. We identify critical habitats across multinational boundaries and show that top predators exploit their environment in predictable ways, providing the foundation for spatial management of large marine ecosystems., (©2011 Macmillan Publishers Limited. All rights reserved)

The retinoblastoma (RB) transcriptional corepressor and related family of pocket proteins play central roles in cell cycle control and development, and the regulatory networks governed by these factors are frequently inactivated during tumorigenesis. During normal growth, these proteins are subject to tight control through at least two mechanisms. First, during cell cycle progression, repressor potential is down-regulated by Cdk-dependent phosphorylation, resulting in repressor dissociation from E2F family transcription factors. Second, RB proteins are subject to proteasome-mediated destruction during development. To better understand the mechanism for RB family protein instability, we characterized Rbf1 turnover in Drosophila and the protein motifs required for its destabilization. We show that specific point mutations in a conserved C-terminal instability element strongly stabilize Rbf1, but strikingly, these mutations also cripple repression activity. Rbf1 is destabilized specifically in actively proliferating tissues of the larva, indicating that controlled degradation of Rbf1 is linked to developmental signals. The positive linkage between Rbf1 activity and its destruction indicates that repressor function is governed in a manner similar to that described by the degron theory of transcriptional activation. Analogous mutations in the mammalian RB family member p107 similarly induce abnormal accumulation, indicating substantial conservation of this regulatory pathway.

Objective: To evaluate the intrarenal anatomy of kidneys obtained from cattle and to propose a new classification for the renal collecting system of cattle., Sample Population: 37 kidneys from 20 adult male mixed-breed cattle., Procedures: Intrarenal anatomy was evaluated by the use of 3-D endocasts made of the kidneys. The number of renal lobes and minor renal calyces in each kidney and each renal region (cranial pole, caudal pole, and hilus) was quantified., Results: The renal pelvis was evident in all casts and was classified into 2 types (nondilated [28/37 {75.7%}] or dilated [9/37 {24.3%}]). All casts had a major renal calyx associated with the cranial pole and the caudal pole. The number of minor renal calices per kidney ranged from 13 to 64 (mean, 22.7). There was a significant correlation between the number of renal lobes and the number of minor renal calices for the entire kidney, the cranial pole region, and the hilus region; however, there was not a similar significant correlation for the caudal pole region. Major and minor renal calices were extremely narrow, compared with major and minor renal calices in pigs and humans., Conclusions and Clinical Relevance: The renal collecting system of cattle, with a renal pelvis and 2 major renal calices connected to several minor renal calices by an infundibulum, differed substantially from the renal collecting system of pigs and humans. From a morphological standpoint, the kidneys of cattle were not suitable for use as a model in endourologic research and training.

During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.

Abstract A systematic study of the morphometry and the collecting system of the canine kidney is presented and compared with previous findings in humans. Renal measurements (kidney length, width, and thickness) were recorded. In addition, 110 three-dimensional endocasts of the kidney collecting system were produced and studied. Anatomic details, important to research and surgical training in endourology, were observed and recorded in canine kidneys. Dogs whose height was more than 70 cm at the withers presented similar kidney measurements to those found in the adult human. The collecting system consisted only of a renal pelvis with a variable number of recesses around its perimeter. The dog kidney is not a good model for experimental studies that consider the morphology of the collecting system. Kidneys from dogs taller than 70 cm, however, might be useful as a model in experimental studies in which renal volume is an important aspect, such as shockwave lithotripsy and endourology.

The ringed seal [Pusa (Phoca) hispida], as well as other seals, exhibits unique anatomical properties when compared to its terrestrial counterparts. In the ringed seal, the most conspicuous marine adaptation is the aortic bulb. This large dilatation of the ascending aorta is comparable to that found in other seal species and marine mammals. The branches of the ascending aorta (brachiocephalic trunk, left common carotid artery and left subclavian artery) are similar to those of higher primates and man. The peculiarities of the venous system are: three pulmonary veins, a pericardial venous plexus, a caval sphincter, a hepatic sinus with paired caudal vena cavae and a large extradural venous plexus. Generally, three common pulmonary veins (right, left and caudal) empty into the left atrium. The pericardial venous plexus lies deep to the mediastinal pericardial pleura (pleura pericardica) on the auricular (ventral) surface of the heart. The caval sphincter surrounds the caudal vena cava as it passes through the diaphragm. Caudal to the diaphragm, the vena cava is dilated (the hepatic sinus), and near the cranial extremity of the kidneys, it becomes biphid. The azygos vein is formed from the union of the right and left azygos veins at the level of the 5th thoracic vertebra. Cardiovascular physiological studies show some of these anatomical variations, especially of the venous system and the ascending aorta, to be modifications for diving. This investigation documents the large blood vessels associated with the heart and related structures in the ringed seal.