Quadrupedal locomotor performance in two species of arboreal squirrels: predicting energy savings of gliding

Gliding allows mammals to exploit canopy habitats of old-growth forests possibly as a means to save energy. To assess costs of quadrupedal locomotion for a gliding arboreal mammal, we used open-flow respirometry and a variable-speed treadmill to measure oxygen consumption and to calculate cost of transport, excess exercise oxygen consumption, and excess post-exercise oxygen consumption for nine northern flying squirrels (Glaucomys sabrinus) and four fox squirrels (Sciurus niger). Our results indicate that oxygen consumption during exercise by flying squirrels was 1.26–1.65 times higher than predicted based on body mass, and exponentially increased with velocity (from 0.84 ± 0.03 ml O₂ kg⁻¹ s⁻¹ at 0.40 m s⁻¹ to 1.55 ± 0.03 ml O₂ kg⁻¹ s⁻¹ at 0.67 m s⁻¹). Also, cost of transport in flying squirrels increased with velocity, although excess exercise oxygen consumption and excess post-exercise oxygen consumption did not. In contrast, oxygen consumption during exercise for fox squirrels was similar to predicted, varying from 0.51 (±0.02) ml O₂ kg⁻¹ s⁻¹ at 0.63 m s⁻¹ to 0.54 (±0.03) ml O₂ kg⁻¹ s⁻¹ at 1.25 m s⁻¹. In addition, the cost of transport for fox squirrels decreased with velocity, while excess exercise oxygen consumption and excess post-exercise oxygen consumption did not. Collectively, these observations suggest that unlike fox squirrels, flying squirrels are poorly adapted to prolonged bouts of quadrupedal locomotion. The evolution of skeletal adaptations to climbing, leaping, and landing and the development of a gliding membrane likely has increased the cost of quadrupedal locomotion by >50% while resulting in energy savings during gliding and reduction in travel time between foraging patches.     

MicroRNA‐125a promotes resistance to BRAF inhibitors through suppression of the intrinsic apoptotic pathway

Melanoma patients with BRAF^V600E‐mutant tumors display striking responses to BRAF inhibitors (BRAFi); however, almost all invariably relapse with drug‐resistant disease. Here, we report that microRNA‐125a (miR‐125a) expression is upregulated in human melanoma cells and patient tissues upon acquisition of BRAFi resistance. We show that miR‐125a induction confers resistance to BRAF^V600E melanoma cells to BRAFi by directly suppressing pro‐apoptotic components of the intrinsic apoptosis pathway, including BAK1 and MLK3. Apoptotic suppression and prolonged survival favor reactivation of the MAPK and AKT pathways by drug‐resistant melanoma cells. We demonstrate that miR‐125a inhibition suppresses the emergence of resistance to BRAFi and, in a subset of resistant melanoma cell lines, leads to partial drug resensitization. Finally, we show that miR‐125a upregulation is mediated by TGFβ signaling. In conclusion, the identification of this novel role for miR‐125a in BRAFi resistance exposes clinically relevant mechanisms of melanoma cell survival that can be exploited therapeutically.     

Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker

Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV) stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs) and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1) localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.     

Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus.

To identify the molecular determinants of neurovirulence, we constructed an infectious simian immunodeficiency virus (SIV) molecular clone, SIV/17E-Fr, that contained the 3' end of a neurovirulent strain of SIV, SIV/17E-Br, derived by in vivo virus passage. SIV/17E-Fr is macrophage tropic in vitro and neurovirulent in macaques. In contrast, a molecular clone, SIV/17E-Cl, that contains the SU and a portion of the TM sequences of SIV/17E-Br is macrophage tropic but not neurovirulent. To identify the amino acids that accounted for the replication differences between SIV/17E-Fr and SIV/17E-Cl in primary macaque cells in vitro, additional infectious molecular clones were constructed. Analysis of these recombinant viruses revealed that changes in the TM portion of the envelope protein were required for the highest level of replication in primary macaque macrophages and brain cells derived from the microvessel endothelium. In addition, a full-length Nef protein is necessary for optimum virus replication in both of these cell types. Finally, viruses expressing a full-length Nef protein in conjunction with the changes in the TM had the highest specific infectivity in a sMAGI assay. Thus, changes in the TM and nef genes between SIV/17E-Cl and SIV/17E-Fr account for replication differences in vitro and correlate with replication in the central nervous system in vivo.     

Role of Ca2+ and Mg2+ in neutrophil hexose transport

The influence of extracellular Ca²⁺ and Mg²⁺ on the transport of 2-deoxy-[³H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[³H]glucose uptake stimulated by two chemotactic factors (C5a and $\text{N-}\text{formylmethionylleucylphenylalanine}$) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca²⁺ and Mg²⁺ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca²⁺, but not Mg²⁺, supported optimal enhanced hexose transport induced by stimuli.Activation of 2-deoxy-D-[³H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca²⁺ into the cell (as exemplified by the action of the two ionophores), such Ca²⁺ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca²⁺, may be involved in mediating the activation of hexose transport induced by the latter stimuli.     

Frasnian vertebrate taphonomy and sedimentology of macrofossil concentrations from the Langsēde Cliff,Latvia

Luk?evi?s, E., Ahlberg, P.E., Stinkulis, ?., Vasi?kova, J. & Zupi??, I. 2011: Frasnian vertebrate taphonomy and sedimentology of macrofossil concentrations from the Langsēde Cliff, Latvia. Lethaia, Vol. 45, pp. 356–370. The siliciclastic sequence of the Upper Devonian of Kurzeme, Western Latvia, is renowned for abundant vertebrate fossils, including the stem tetrapods Obruchevichthys gracilis and Ventastega curonica. During the first detailed taphonomic study of the vertebrate assemblage from the Ogre Formation cropping out at the Langsēde Cliff, Imula River, abundant vertebrate remains have been examined and identified as belonging to one psammosteid, two acanthodian and three sarcopterygian genera; the placoderm Bothriolepis maxima dominates the assemblage. Besides fully disarticulated placoderm and psammosteid plates, separate sarcopterygian scales and teeth, and acanthodian spines, partly articulated specimens including complete distal segments of Bothriolepis pectoral fins, Bothriolepis head shields and sarcopterygian lower jaws have been found. The size distribution of the placoderm bones demonstrates that the individuals within the assemblage are of approximately uniform age. Distinct zones have been traced within the horizontal distribution of the bones. These linear zones are almost perpendicular to the dominant dip azimuth of the cross‐beds and ripple‐laminae and most probably correspond to the depressions between subaqueous dunes. Concavity ratio varies significantly within the excavation area. The degree of fragmentation of the bones and disarticulation of the skeletons suggest that the carcasses were reworked and slightly transported before burial. Sedimentological data suggest deposition in a shallow marine environment under the influence of rapid currents. The fossiliferous bed consists of a basal bone conglomerate covered by a cross‐stratified sandstone with mud drapes, which is in turn overlain by ripple laminated sandstone, indicating the bones were buried by the gradual infilling of a tidal channel. All the Middle–Upper Devonian vertebrate bone‐beds from Latvia are associated with sandy to clayey deposits and have been formed in a sea‐coastal zone during rapid sedimentation episodes, but differ in fossil abundance and degree of preservation. □Agnathans, Devonian, facies analysis, fish, fossil assemblage, palaeoenvironment.     

Cyclin-Dependent Kinase 5 (CDK5) Controls Melanoma Cell Motility,Invasiveness, and Metastatic Spread—Identification of a Promising Novel therapeutic target

Despite considerable progress in recent years, the overall prognosis of metastatic malignant melanoma remains poor, and curative therapeutic options are lacking. Therefore, better understanding of molecular mechanisms underlying melanoma progression and metastasis, as well as identification of novel therapeutic targets that allow inhibition of metastatic spread, are urgently required. The current study provides evidence for aberrant cyclin-dependent kinase 5 (CDK5) activation in primary and metastatic melanoma lesions by overexpression of its activator protein CDK5R1/p35. Moreover, using melanoma in vitro model systems, shRNA-mediated inducible knockdown of CDK5 was found to cause marked inhibition of cell motility, invasiveness, and anchorage-independent growth, while at the same time net cell growth was not affected. In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases. Inhibition of lung metastasis was further validated in a syngenic murine melanoma model. CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype. Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells. Thus, experimental data presented here provide strong evidence for a crucial role of aberrantly activated CDK5 in melanoma progression and metastasis and establish CDK5 as promising target for therapeutic intervention.     

CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice

Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vβ4⁺ CD8⁺ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8⁺ T cells and MHV68 epitope specific CD8⁺ T cells to the lungs—despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vβ4⁺ CD8⁺ T cell expansion, eliminated MHV68-induced fibrosis—further implicating the activated Vβ4⁺ CD8⁺ T cell population in the induction of fibrosis. We further addressed the role that CD8⁺ T cells play in the induction of fibrosis by depleting CD8⁺ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vβ4⁺ CD8⁺ T cells as mediators of fibrotic disease in IFNγR-/- mice.     

A large-subunit mitochondrial ribosomal DNA sequence translocated to the nuclear genome of two stone crabs (Menippe)

Two DNA sequences that appear to be homologous to large-subunit mitochondrial ribosomal RNA genes have been identified in the stone crabs Menippe mercenaria and M. adina. Amplification from whole genomic DNA by polymerase chain reaction (PCR) with oligonucleotide primers based on conserved portions of large-subunit mitochondrial rRNA genes consistently amplified two products of similar length (565 and 567 bp). These products differed at 3% of their nucleotide bases, and could be distinguished by a HindIII site. Only one of these sequences (designated the A sequence) was detected by PCR in purified mitochondrial DNA. The other (designated the B sequence) hybridized to total genomic DNA at a level consistent with a nuclear genome location. It is unlikely that the type B product would have been recognized as a nuclear copy by examination of its sequence alone. This is the first report of a mitochondrial gene sequence translocated into the nuclear genome of a crustacean.