High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Isolation and Identification of Environmental Mycobacteria in the Waters of a Hemodialysis Center

The use of poorly treated water during hemodialysis may lead to contamination with nontuberculous mycobacteria (NTM). This study aimed to isolate and identify NTM species in the water of a Brazilian hemodialysis center. We collected 210 samples of water from the hydric system of the unit (post-osmosis system, hemodialysis rooms, reuse system, and hemodialysis equipment) and from the municipal supply network; we isolated the NTM by a classic microbiological technique and identified them by the PCR restriction enzyme pattern of the hsp65 gene (PRA). Fifty-one (24.3 %) of the collected samples tested positive for NTM; both the municipal supply network (2 samples, 3.2 %) and the hydric system of the hemodialysis center (49 samples, 96.1 %) contained NTM. We isolated and identified potentially pathogenic bacteria such as Mycobacterium lentiflavum (59.0 %) and M. kansasii (5.0 %), as well as rarely pathogenic bacteria like M. gordonae (24.0 %), M. gastri (8.0 %), and M. szulgai (4.0 %). The ability of NTM to cause diseases is well documented in the literature. Therefore, the identification of NTM in the water of a Brazilian hemodialysis center calls for more effective water disinfection procedures in this unit.     

Manipulating vibratory devices’ orientation and position enhances proprioceptive disturbance during upright stance

We tested local vibration effects during upright standing considering: (i) the orientation of vibratory devices in relation to muscle fibres; (ii) the muscle region stimulated; and (iii) the number of stimulation spots. Results showed a higher balance disturbance with vibration devices oriented parallel to triceps surae muscle fibres. The single stimulation of the proximal region of the tibialis anterior muscle belly induces the same proprioceptive disturbance as stimulating multiple regions simultaneously.     

Trace element transfer from two contaminated soil series to Medicago sativa and one of its herbivores,Spodoptera exigua

Alfalfa was cultivated in two potted soil series obtained from two sandy soils contaminated by Cu (SM) and metal(loids)/PAH (CD). Shoot production was monitored for 8 weeks. Then, larvae of Spodoptera exigua were reared on alfalfa of both soil series for eight days. A biotest (using Phaseolus vulgaris) was used to assess the soil phytotoxicity. Increasing soil contamination reduced P. vulgaris growth, but alfalfa growth was only reduced on the SM soil series. Exposure to the SM soil was mirrored by shoot Cu and Cr concentrations of alfalfa (respectively, in mg kg ^?1 DW, Cu and Cr ranged from 11.9 and 0.4 in the CTRL soil to 98.5 and 1.2 in the SM one). Exposure to the CD soil series was mirrored by shoot Zn concentrations (i.e., 48–91.6 mg kg^?1 DW). Internal metal(loid) concentrations of S. exigua remained generally steady across both soil series (respectively Cd 0.05–0.16, Cr 0.5–3.3, Cu 5.8–98.5, Ni 0.6–1.6, Pb 0.4–1.3, and Zn 57–337 mg kg^?1 DW), and most of the associated transfer factors were lower than 1. Here, due to the excluder phenotype of alfalfa across our TE contamination gradients, S. exigua could cope with high total metal(loid) concentration in both contaminated soils.     

Trophic structure of coastal freshwater stream fishes from an Atlantic rainforest: evidence of the importance of protected and forest-covered areas to fish diet

The role of riparian forests in the functioning of aquatic ecosystems is well known, and they are recognized as an important food source for riverine fauna. This study investigates the trophic structure of coastal freshwater stream fishes from a large conservation area in an Atlantic rainforest using stomach content and food availability analyses. Four samples were collected from 19 sample sites. Fishes were caught with electrofishing. Prey were sampled with trays, Surber, traps, and electrofishing to evaluate the availability of food resources. The diets of 20 fish species were determined from the stomach contents of 1691 individuals. Terrestrial and aquatic insects and detritus were the most consumed items. Fish diet and prey availability were not seasonally dependent. A cluster analysis showed five trophic functional groups: terrestrial insectivores, aquatic insectivores, detritivores, carnivores, and omnivores. Insectivores predominated in species richness (60%), abundance (47%) and biomass (39%). Allochthonous and autochthonous items were found in similar proportions in the environment; however, allochthonous items were representative for insectivores and detritivores, whereas autochthonous items were important for primarily aquatic insectivores. The preference for certain insects by insectivorous fishes was associated with food selectivity rather than the availability of the resource and demonstrated the strong relationship between feeding behavior and food preference. The absence of seasonal variation in the diets of the fishes was possibly related to the consistent food supply. Our results confirm the role of the forest as a food provider for stream fishes, such as terrestrial insects and plant debris/detritus (also consumed by aquatic insects, which subsequently serve as food for fish), highlighting the importance of conserving the Brazilian Atlantic rainforests.     

Selective suppression of rod signal transmission by cobalt ions of low levels in carp retina

Selective suppression of rod signal transmission by cobalt ions was reported in carp retina. Using 10 μnol/L Co²⁺, rod-driven horizontal cells were hyperpolarized and light responses were completely suppressed in superfused, isolated retina, while cone-driven horizontal cells were almost unaffected. Similarly, scotopic electroretinographic bwave was suppressed by 10 μnol/L Co²⁺, while the photopic b-wave remained unaffected. Furthermore, the glutamate-isolated receptor potential (PIII) was not altered by low Co²⁺ under dark-adapted conditions. Other divalent ions with high affinity to calcium channels, such as cadmium and manganese ions, did not show similar suppressive effect on the rod horizontal cells. When rod horizontal cells were hyperpolarized by 10 μnol/L Co²⁺, the use of 3 mmol/L glutamate caused a significant depolarization of the cells, indicating that Co²⁺ application did not impair the ability of these cells to respond to glutamate. On the other hand, application of 200 μnol/L β-hydroxyaspartate, a glutamate transport blocker, mimicked the effect of low Co²⁺, suggesting a possibility that the low Co²⁺ effect might be related to a blockade of glutamate uptake by rods. Project supported by the State Commission of Science and Technology of China, the National Natural Science Foundation of China, the National Eye Institute, the Human Frontier Science Program.     

Construction, detection and microarray analysis on theShigella flexneri 2asitC mutant

In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442, Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study. With this improved knockout system, we inactivatedsitC gene, which is associated with iron transport inShigella flexneri 2a strain 301, to yield the mutant, MTS. The functional detection of the mutant was performed at the level of culture medium, cell and animal experiment, respectively. The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions. The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150 μmol/L iron chelator DIP (2,2′-dipyridyl). Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium. In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines, or the experiment on keratoconjunctivitis in guinea pigs, MTS showed no obvious changes in virulence compared with the parental strain Sf301. When 65 μmol/L DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS reduced about 51.6% as compared with that of Sf301. The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301. There are 106 more up-regulated genes in MTS than in wild-type strains, which are involved in membrane transportation, amino acid metabolism and uncategorized function genes, while down-regulated genes are mainly involved in energy and carbohydrate metabolism. Under low iron conditions, the expression levels of known iron-transport associated genes generally increased. Additionally, the number of these genes and their increase amplitude in MTS are more than those in Sf301. Together, these results confirmed that Sit iron-transport system is important for the growth ofShigella.     

PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells

Lithium is a well‐established non‐competitive inhibitor of glycogen synthase kinase‐3β (GSK‐3β), a kinase that is involved in several cellular processes related to cancer progression. GSK‐3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK‐3β‐independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment. J. Cell. Biochem. 117: 458–469, 2016. © 2015 Wiley Periodicals, Inc.     

Cloning,Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification.     

Multiple Exposures to Ascaris suum Induce Tissue Injury and Mixed Th2/Th17 Immune Response in Mice

Ascaris spp. infection affects 800 million people worldwide, and half of the world population is currently at risk of infection. Recurrent reinfection in humans is mostly due to the simplicity of the parasite life cycle, but the impact of multiple exposures to the biology of the infection and the consequences to the host’s homeostasis are poorly understood. In this context, single and multiple exposures in mice were performed in order to characterize the parasitological, histopathological, tissue functional and immunological aspects of experimental larval ascariasis. The most important findings revealed that reinfected mice presented a significant reduction of parasite burden in the lung and an increase in the cellularity in the bronchoalveolar lavage (BAL) associated with a robust granulocytic pulmonary inflammation, leading to a severe impairment of respiratory function. Moreover, the multiple exposures to Ascaris elicited an increased number of circulating inflammatory cells as well as production of higher levels of systemic cytokines, mainly IL-4, IL-5, IL-6, IL-10, IL-17A and TNF-α when compared to single-infected animals. Taken together, our results suggest the intense pulmonary inflammation associated with a polarized systemic Th2/Th17 immune response are crucial to control larval migration after multiple exposures to Ascaris.