Inhibition of CD73 improves B cell-mediated anti-tumor immunity in a mouse model of melanoma

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8(+) T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20(+) B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti-IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8(+) T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.     

Some reproductive traits of the Tristan klipfish, Bovichtus diacanthus (Carmichael 1819) (Notothenioidei: Bovichtidae) from Tristan da Cunha (South Atlantic)

The reproductive biology of the Tristan klipfish, Bovichtus diacanthus, was investigated by macroscopic and histological analyses of the gonads. Fish samples were collected in tide pools at Tristan da Cunha in July 2004. Most specimens of both sexes were developing, or sexually mature, with a gonadosomatic index (GSI) of 7.0–9.2% in females and 0.2–0.6% in males. Histologically, testes showed a random distribution of spermatogonia along the lobules, a condition defined as the unrestricted spermatogonial type. Ripe males exhibited lobules with all spermatogenic stages of development from spermatogonia to spermatozoa. In mature females, the ovarian follicles consisted of three main cohorts of oocytes of different sizes; the smaller one represented by previtellogenic oocytes of 15–150 μm and the other two by yolked oocytes measuring, respectively, 300–1000 and 800–1500 μm. The overlap between the stock of advanced yolked oocytes and the early yolked oocytes was low, decreasing progressively with final maturation. As a result, B. diacanthus was considered a batch spawner, with a spawning season extending from July to August onward. Batch fecundity, based on the most advanced yolked oocytes, was 2,047–8,317 mature oocytes/female, whereas the relative fecundity was 77–141 mature oocytes/g. In the light of the phyletically basal position of bovichtids in the suborder, the reproductive traits of B. diacanthus were compared with those previously described in other Antarctic and non-Antarctic notothenioids.     

Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

In Vitro and In Vivo Antitumor Activity of [Pt(O,O′-acac)(γ-acac)(DMS)] in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy highly resistant to chemotherapy. There is an urgent need for effective therapy inasmuch as resistance, intrinsic and acquired, to conventional therapies is common. Among Pt(II) antitumor drugs, [Pt(O,O′-acac)(γ-acac)(DMS)] (Ptac2S) has recently attracted considerable attention due to its strong in vitro and in vivo antiproliferative activity and reduced toxicity. The purpose of this study was to examine the efficacy of Ptac2S treatment in MPM. We employed the ZL55 human mesothelioma cell line in vitro and in a murine xenograft model in vivo, to test the antitumor activity of Ptac2S. Cytotoxicity assays and Western blottings of different apoptosis and survival proteins were thus performed. Ptac2S increases MPM cell death in vitro and in vivo compared with cisplatin. Ptac2S was more efficacious than cisplatin also in inducing apoptosis characterized by: (a) mitochondria depolarization, (b) increase of bax expression and its cytosol-to-mitochondria translocation and decrease of Bcl-2 expression, (c) activation of caspase-7 and -9. Ptac2S activated full-length PKC-δ and generated a PKC-δ fragment. Full-length PKC-δ translocated to the nucleus and membrane, whilst PKC-δ fragment concentrated to mitochondria. Ptac2S was also responsible for the PKC-ε activation that provoked phosphorylation of p38. Both PKC-δ and PKC-ε inhibition (by PKC–siRNA) reduced the apoptotic death of ZL55 cells. Altogether, our results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.     

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE*

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N_cyt/C_exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.     

The Brain of the Domestic Bos taurus: Weight,Encephalization and Cerebellar Quotients,and Comparison with Other Domestic and Wild Cetartiodactyla

The domestic bovine Bos taurus is raised worldwide for meat and milk production, or even for field work. However the functional anatomy of its central nervous system has received limited attention and most of the reported data in textbooks and reviews are derived from single specimens or relatively old literature. Here we report information on the brain of Bos taurus obtained by sampling 158 individuals, 150 of which at local abattoirs and 8 in the dissecting room, these latter subsequently formalin-fixed. Using body weight and fresh brain weight we calculated the Encephalization Quotient (EQ), and Cerebellar Quotient (CQ). Formalin-fixed brains sampled in the necropsy room were used to calculate the absolute and relative weight of the major components of the brain. The data that we obtained indicate that the domestic bovine Bos taurus possesses a large, convoluted brain, with a slightly lower weight than expected for an animal of its mass. Comparisons with other terrestrial and marine members of the order Cetartiodactyla suggested close similarity with other species with the same feeding adaptations, and with representative baleen whales. On the other hand differences with fish-hunting toothed whales suggest separate evolutionary pathways in brain evolution. Comparison with the other large domestic herbivore Equus caballus (belonging to the order Perissodactyla) indicates that Bos taurus underwent heavier selection of bodily traits, which is also possibly reflected in a comparatively lower EQ than in the horse. The data analyzed suggest that the brain of domestic bovine is potentially interesting for comparative neuroscience studies and may represents an alternative model to investigate neurodegeneration processes.     

Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis

? The aim of this study was to investigate Ca(2+) responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. ? Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca(2+) responses in host root tissues. ? Ca(2+) spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca(2+) spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. ? These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca(2+) response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals.     

Maximal lactate steady-state independent of recovery period during intermittent protocol

Barbosa, LF, de Souza, MR, Corrêa Caritá, RA, Caputo, F, Denadai, BS, and Greco, CC. Maximal lactate steady-state independent of recovery period during intermittent protocol. J Strength Cond Res 25(12): 3385-3390, 2011-The purpose of this study was to analyze the effect of the measurement time for blood lactate concentration ([La]) determination on [La] (maximal lactate steady state [MLSS]) and workload (MLSS during intermittent protocols [MLSSwi]) at maximal lactate steady state determined using intermittent protocols. Nineteen trained male cyclists were divided into 2 groups, for the determination of MLSSwi using passive (VO(2)max = 58.1 ± 3.5 ml·kg·min; N = 9) or active recovery (VO(2)max = 60.3 ± 9.0 ml·kg·min; N = 10). They performed the following tests, in different days, on a cycle ergometer: (a) Incremental test until exhaustion to determine (VO(2)max and (b) 30-minute intermittent constant-workload tests (7 × 4 and 1 × 2 minutes, with 2-minute recovery) to determine MLSSwi and MLSS. Each group performed the intermittent tests with passive or active recovery. The MLSSwi was defined as the highest workload at which [La] increased by no more than 1 mmol·L between minutes 10 and 30 (T1) or minutes 14 and 44 (T2) of the protocol. The MLSS (Passive-T1: 5.89 ± 1.41 vs. T2: 5.61 ± 1.78 mmol·L) and MLSSwi (Passive-T1: 294.5 ± 31.8 vs. T2: 294.7 ± 32.2 W; Active-T1: 304.6 ± 23.0 vs. T2: 300.5 ± 23.9 W) were similar for both criteria. However, MLSS was lower in T2 (4.91 ± 1.91 mmol·L) when compared with in T1 (5.62 ± 1.83 mmol·L) using active recovery. We can conclude that the MLSSwi (passive and active conditions) was unchanged whether recovery periods were considered (T1) or not (T2) for the interpretation of [La] kinetics. In contrast, MLSS was lowered when considering the active recovery periods (T2). Thus, shorter intermittent protocols (i.e., T1) to determine MLSSwi may optimize time of the aerobic capacity evaluation of well-trained cyclists.