Inhibition of Osteocyte Apoptosis Prevents the Increase in Osteocytic Receptor Activator of Nuclear Factor κB Ligand (RANKL) but Does Not Stop Bone Resorption or the Loss of Bone Induced by Unloading

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.     

Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

Minimum sampling effort for reliable non-invasive estimations of excretion abundance of Elaphostrongylus cervi L1 in red deer (Cervus elaphus) populations

Faecal surveys are commonly used as non-invasive means to evaluate population abundance of animals as well as comparable indexes of prevalence and intensity of diseases between populations, especially macroparasites. While faecal surveys are among one of the simplest means to perform these evaluations, they are time consuming and labour intensive. The present study evaluated 80 red deer (Cervus elaphus) faecal samples collected in two study sites for the presence and abundance of first-stage larvae of the nematode Elaphostrongylus cervi and established pools of samples for epidemiological analysis. The analysis of 20-30 individual samples would produce a reliable estimate of the 'true' prevalence, and the error of the smaller sample size only doubled that of the 80 reference samples. The analysis of 5 pools of 5 pellet groups each, or of 4 pools of 10 pellet groups each, provided a reliable estimation of the 'true' excretion abundance estimated by the analysis of all 80 samples. These pools comprise a cost-effective and dependable alternative for measuring and obtaining comparable abundances of parasitic faecal excretion stages in red deer populations, which is very valuable for epidemiological and ecological research as well as for management purposes.     

Are there multiple circadian clocks in plants?

We have reported that Arabidopsis might have genetically distinct circadian oscillators in multiple cell-types.¹ Rhythms of CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are 2.5 h longer in phytochromeB mutants in constant red light and in cryptocrome1 cry2 double mutant (hy4-1 fha-1) in constant blue light than the wild-type.² However, we found that cytosolic free Ca²⁺ ([Ca²⁺]_cyt) oscillations were undetectable in these mutants in the same light conditions.¹ Furthermore, mutants of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) have short period rhythms of leaf movement but have arrhythmic [Ca²⁺]_cyt oscillations. More important, the timing of cab1-1 (toc1-1) mutant has short period rhythms of CAB2 promoter activity (∼21 h) but, surprisingly, has a wild-type period for circadian [Ca²⁺]_cyt oscillations (∼24 h). In contrast, toc1-2, a TOC1 loss-of-function mutant, has a short period of both CAB2 and [Ca²⁺]_cyt rhythms (∼21 h). Here we discuss the difference between the phenotypes of toc1-1 and toc1-2 and how rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations might be regulated differently.Key words: circadian rhythms, TOC1, multiple oscillators, CAB2, Ca²⁺ signalling, arabidopsis, circadian [Ca²⁺]_cyt oscillations, aequorin, luciferase, central oscillatorThe plant circadian clock controls a multitude of physiological processes such as photosynthesis, organ and stomatal movements and transition to reproductive growth. A plant clock that is correctly matched to the rhythms in the environment brings about a photosynthetic advantage that results in more chlorophyll, more carbon assimilation and faster growth.³ One of the first circadian clock mutants to be described in plants was the short period timing of cab1-1 (toc1-1), which was identified using the rhythms of luciferase under a CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter as a marker for circadian period.⁴Circadian rhythms of both CAB2 promoter activity and cytosolic-free Ca²⁺ ([Ca²⁺]_cyt) oscillations depend on the function of a TOC1, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL (TOC1/CCA1/LHY) negative feedback loop.⁵ In tobacco seedlings, CAB2:luciferase (CAB2:luc) rhythms and circadian [Ca²⁺]_cyt oscillations can be uncoupled in undifferentiated calli.⁶ In Arabidopsis, we reported that toc1-1 has different periods of rhythms of CAB2 promoter activity (∼21 h) and circadian [Ca²⁺]_cyt oscillations (∼24 h). The mutant allele toc1-1 has a base pair change that leads to a full protein that has an amino acid change from Ala to Val in the CCT domain (CONSTANS, CONSTANS-LIKE and TOC1).⁷ On the other hand, the mutant toc1-2 has short period of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations (∼21 h).^1,7 This allele has a base pair change that results in changes to preferential mRNA splicing, resulting in a truncated protein with only 59 residues.⁷ Thus, the mutated CCT domain in toc1-1 might lead to the uncoupling of rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations while the absence of TOC1 in toc1-2 causes the shortening of the period of both rhythms. Indeed, zeitlupe-1 (ztl-1) mutants, that have higher levels of TOC1, have long periods of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations.¹ The biochemical function of the CCT domain is unknown but it is predicted to play an important role in protein-protein interactions⁸ and nuclear localization.⁹One model to explain the period difference of CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation is that the toc1-1 mutation has uncoupled two oscillators in the same cell. Uncoupled oscillators are a predicted outcome of certain mutations in the recently described three-loop mathematical model.^10–11 However, both rhythms of TOC1 and CCA1/LHY expression, which would be in uncoupled oscillators accordingly to the model, are described as short-period in toc1-1.⁵ Thus, we have favored the model in which CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation are reporting cell-types with different oscillators that are affected differently by toc1-1.It is possible that TOC1 could interact with a family of cell-type specific proteins. The interaction of TOC1 with each member of the family could be affected differently by the mutation in the CCT domain (Fig. 1). Two-hybrid assays have shown that TOC1 interacts with PIF proteins (PHYTOCHROME INTERACTING FACTOR3 and PIF4) and related PIL proteins (PIF3-LIKE PROTEIN 1, PIL2, PIL5 and PIL6).⁸ In fact, TOC1 interaction with both PIF3 and PIL1 is stronger when the N-terminus receiver domain is taken out and the CCT domain is left intact.⁸ Thus, it is possible that TOC1 and different PIF/PIL proteins interact to regulate the central oscillator. This interaction could be impaired by the Ala to Val change in the toc1-1 mutation, leading to the period shortening. However, lines misexpressing PIF3, PIL1 and PIL6 showed no changes in their circadian rhythms.^12–16Open in a separate windowFigure 1Models of how the toc1-1 mutation might differently affect cell-type specific circadian oscillators. The single mutant toc1-1 have 21 h rhythms of CAB2 promoter activity and 24 h-rhythms of [Ca²⁺]_cyt oscillations. The toc1-1 mutation is a single amino acid change in the CCT domain. The CCT domain is involved in protein-protein interaction and/or nuclear localization. We have proposed that circadian oscillators with different periods are present in different cell-types. The luminescence generated by CAB2 promoter-drived luciferase (from the CAB2:luc) is probably originated in the epidermis and mesophyll cells. In this model, we propose that the mutation on the CCT domain impairs the mutated TOC1 interaction with the hypothetical protein Z in these cells-types. In contrast, in other cell-types, the mutated TOC1 still interacts with other hypothetical proteins (W), despite the mutation in the CCT domain. In those cell-types, the circadian oscillator could still run with a 24 h period for [Ca²⁺]_cyt rhythms (from the 35S:AEQ construct). One possible identity for Z and W are the members of the PHYTOCHROME INTERACTING FACTOR (PIF) related PIF3-LIKE (PIL) family.One possible explanation for the absence of alterations in the period of circadian rhythms in lines misexpressing PIF/PIL is that they only have roles in certain cell-types. As an example, PIL6 and PIF3 are involved with flowering time and hypocotyl growth in red light^12–15 while PIL1 and PIL2 are involved with hypocotyl elongation in shade-avoidance responses.¹⁶ Both hypocotyl growth and flowering time require cell-type specific regulation: vascular bundle cells in the case of the flowering time¹⁷ and the cells in the shoot in the case of the hypocotyl elongation.¹⁶ If TOC1 interaction with certain PIF/PIL is indeed cell-type specific, the mutated CCT domain found in the toc1-1 mutant could affect the clock in different ways, depending on the type of PIF/PIL protein expressed in each cell-type. Therefore, a question that arises is: which cell-types are sensitive to the toc1-1 mutation?There is evidence that CAB2 and CATALASE3 (CAT3) are regulated by two oscillators that respond differently to temperature signals.¹⁸ These genes might be regulated by two distinct circadian oscillators within the same tissues or a single cell.¹⁸ Interestingly, the spatial patterns of expression of CAB2 and CATALASE3 overlap in the mesophyll of the cotyledons.¹⁸ Furthermore, rhythms of CAB2 and CHALCONE SYNTHASE (CHS) promoter activity have different periods and they are equally affected by toc1-1 mutation.¹⁹ Whereas CAB2 is mainly expressed in the mesophyll cells, CHS is mainly expressed in epidermis and root cells.¹⁹ However, rhythms of AEQUORIN luminescence, which reports [Ca²⁺]_cyt oscillation, were insensitive to toc1-1 mutation and appear to come from the whole cotyledon.²⁰ One cell-type which is found in the whole cotyledon but is distinct from either mesophyll or epidermis cells is the vascular tissue and associated cells.Another approach to determine which cell-types are insensitive to toc1-1 mutation is to compare the toc1-1 and toc1-2 phenotypes. The period of circadian [Ca²⁺]_cyt oscillations is not the only phenotype that is different in toc1-1 and toc1-2 mutants. Rhythms in CAB2 promoter activity in constant red light are short period in toc1-1 but arrhythmic in toc1-2.^21,22 COLD, CIRCADIAN RHYTHM AND RNA BINDING 2/GLYCINE-RICH RNA BINDING PROTEIN 7 (CCR2/GRP7) is also arrhythmic in toc1-2 but short period in toc1-1 in constant darkness.^7,22 When the length of the hypocotyl was measured for both toc1-1 and toc1-2 plants exposed to various intensities of red light, only toc1-2 had a clear reduction in sensitivity to red light. Therefore, toc1-2 has long hypocotyl when maintained in constant red light while hypocotyl length in toc1-1 is nearly identical to that in the wild-type.²² These differences may allow us to separate which cell-types are sensitive to the toc1-1 mutation and which not.Hypocotyl growth is regulated by a large number of factors such as light, gravity, auxin, cytokinins, ethylene, gibberellins and brassinosteroids.²³ There is also a correlation between the size of the hypocotyl in red light and defects in the circadian signaling network.^24,25 The fact that toc1-1 has different hypocotyl sizes from toc1-2 suggests that circadian [Ca²⁺]_cyt oscillations could be involved in the light-dependent control of hypocotyl growth. Circadian [Ca²⁺]_cyt oscillations might encode temporal information to control cell expansion and hypocotyl growth.^26–28 toc1-1 have short-period rhythms of hypocotyl elongation, which indicates that the cells in the hypocotyl have a 21 h oscillator.²⁹ However, toc1-1 might also have a wild-type hypocotyl length in continuous red light because cells which generate the signal to regulate hypocotyl growth might have 24 h oscillators.The toc1-1 mutation was the first to be directly associated with the plant circadian clock, revitalizing the field of study.⁴ Now, by either uncoupling two feedback loops or by distinct TOC1 protein-protein interaction in different cell-types, toc1-1 has shown new properties of the circadian clock that may deepen our understanding of this system.     

Patterns of roost site selection and use by Southern Ground-Hornbills in north-eastern South Africa

Different habitats may be used for the needs of various aspects of an animal’s life. Southern Ground-Hornbill Bucorvus leadbeateri groups announce their presence within year-round territories by calling at dawn from their overnight roost sites. Knowledge on ground-hornbill roosting habits is limited. Groups roost in large trees, apparently close to where they end up after daily foraging. We investigated patterns of roost site selection and use for four Southern Ground-Hornbill groups in the Associated Private Nature Reserves, north-eastern South Africa, based on data from GPS-satellite transmitters. The number of roost sites used per month averaged 15.4 ± 4.7 across all groups, indicating little evidence of strong preferences for specific sites. This number was least when groups were breeding, decreasing throughout the early wet season (October–December) and was lowest during the late wet season (January–March) when actively breeding groups frequently roosted close to the nest (54–83% of roosts <1 000 m of the nest). As might be expected, the mean monthly number of nights per roost peaked during the breeding season (December–January). Riparian habitats were preferred for roosting during the breeding season, whereas disturbed areas, as well as Combretumand mopane-dominated habitats were preferred during the dry non-breeding season. Adequate large trees not only for nesting, but also for roosting, particularly in riparian habitats, may therefore be an important and potentially limiting factor for the successful reproduction of Southern Ground-Hornbills.     

Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis

Differential stress/inflammatory responses were characterized at the mRNA and protein levels in mandibular lymph nodes (MLN) and oropharyngeal tonsils of European wild boars (Sus scrofa), naturally infected with Mycobacterium bovis. Suppression-subtractive hybridization combined with immunohistochemistry and/or quantitative real-time RT-PCR were used to identify and characterize abundant stress/inflammatory gene sequences differentially expressed in tuberculous (TB+) wild boars. Genes identified in MLN and tonsils corresponded to serum amyloid A, arginase I, osteopontin, lysozyme, annexin I, and heat shock proteins, respectively. Global protein patterns in MLN and tonsils were compared between TB+ and nontuberculous (TB-) boars by 2-DE and MALDI-TOF MS. Five proteins, including stress/inflammatory proteins annexin V, serum albumin, and apolipoprotein A1 were found at lower levels in MLN of TB+ boars. Manganese superoxide dismutase was found up-regulated in MLN of TB+ boars. Five proteins, including creatine kinase and MHC class II antigens were found up-regulated in tonsils of TB+ boars. These results demonstrated differential stress/inflammatory responses in wild boars naturally infected with M. bovis and suggest possible markers of tuberculosis in this species that may prove useful for future studies of host-pathogen interactions and for diagnostics and vaccine development.     

Gene expression,protein profiling,and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.