A sustainable and effective bioprocessing approach for improvement of acid phosphatase production and rock phosphate solubilization by Bacillus haynesii strain ACP1.

There is indeed a tremendous increase in biotechnological production on a global scale, more and more innovative bioprocesses, therefore, require to perform ideally not only in a small lab- but also on large production scales. Efficient microbial process optimization is a significant challenge when accomplishing a variety of sustainable development and bioengineering application objectives. In Egypt's mines, several distinct types of rock phosphate (RP) are utilized as a source of phosphate fertilizers in agriculture. It is more ecologically beneficial to utilize RP bio-solubilization than acidulation. Therefore, this work aimed to strategically scale up the acid phosphatase (ACP) production and RP bio-solubilization by the newly-discovered Bacillus haynesii. The use of consecutive statistical experimental approaches of Plackett-Burman Design (PBD), and Rotatable Central Composite Design (RCCD), followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor revealed an innovative medium formulation. These approaches substantially improved ACP production, reaching 207.6 U L-1 with an ACP yield coefficient Yp/x of 25.2 and a specific growth rate (µ) of 0.07 h-1. The metals Na, Li, and Mn were the most efficiently released from RP during the solubilization process by B. haynesii. The uncontrolled pH culture condition is the most suitable setting for simultaneously improving the ACP and organic acids production. The most abundant organic acid produced through the cultivation process was lactic acid, followed by glutamic acid and hydroxybenzoic acid isomer. The findings of TGA, DSC, SEM, EDS, FTIR, and XRD analysis emphasize the significant influence of organic acids and ACP activity on the solubilization of RP particles.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains. RESULTS: Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter. DISCUSSION: The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application.

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65 °C, respectively, and is stable at 55 °C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0 was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.

Transmembrane prostatic acid phosphatase (TMPAP) delays cells in G1 phase of the cell cycle.

BACKGROUND: Prostate adenocarcinoma is the most common form of prostate cancer. We have previously shown in a murine model that prostatic acid phosphatase (PAP) deficiency leads to increased cell proliferation and development of prostate adenocarcinoma. The association between PAP and prostate cancer has been reported. Indeed, high PAP enzymatic activity is detected in the serum of patients with metastatic disease while its expression is reduced in prostate cancer tissue. However, the molecular mechanisms behind the onset of the disease remains poorly understood. We previously identified a novel transmembrane prostatic acid phosphatase (TMPAP) isoform, which interacts with snapin. TMPAP is expressed on the plasma membrane, as well as endosomal/lysosomal and exosomal membrane vesicles by means of a tyrosine-based lysosomal targeting motif (Yxxϕ). METHODS: We used stable overexpression of the secreted isoform (SPAP) and TMPAP in LNCaP cells, live cell imaging, microarray and qRT-PCR analyses, and fluid phase uptake of HRP and transferrin. RESULTS: Our results indicate that the stable overexpression of TMPAP, but not SPAP in LNCaP cells reduces cell growth while increasing endo/exocytosis and cell size. Specifically, cells overexpressing TMPAP accumulate in the G1 phase of the cell cycle, and show altered gene expression profile. CONCLUSIONS: Our data suggests that TMPAP may function as a non-canonical tumor suppressor by delaying cell growth in G1 phase of the cell cycle.

Testicular acid phosphatase induces odontoblast differentiation and mineralization.

Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in ß-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active ß-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active ß-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through ß-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration.

Association of Tartrate-Resistant Acid Phosphatase-Expressed Macrophages and Metastatic Breast Cancer Progression.

Infiltrating neutrophils, lymphocytes, macrophages, and cytokines constitute a state of chronic inflammation within the tumor microenvironment. Tartrate-resistant acid phosphatase 5a (TRACP5a) protein, a novel product of activated macrophage, is postulated to be a biomarker for systemic inflammatory burden in states of chronic inflammation. We aimed to investigate the clinical significance of TRACP5a expression in tumor-infiltrating macrophages and serum TRACP5a in patients with metastatic breast cancer (BC). We retrospectively analyzed the clinical data from 34 BC patients with confirmed skeletal/visceral metastasis upon or during first-line palliative treatment. Patients were stratified into 3 groups based on the therapeutic responses and follow-up disease course. The association of TRACP5a protein with other inflammatory and cancer biomarkers was assessed among the clinically distinct group of patients. Higher TRACP5a protein was significantly correlated with earlier disease progression and survival (P = 0.0045) in comparison to other inflammatory markers, CRP or IL-6. Patients with higher serum TRACP5a level and shorter survival and treatment refractoriness also had more TRACP+ tumor-infiltrating macrophages. Our data support a hypothesis that serum TRACP5a protein can potentially be a predictive and prognostic marker to evaluate disease progression and therapeutic response in BC patients with bone/visceral metastasis. The associations between overall survival and TRACP expression by macrophages require further prospective investigation.

Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging.

Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)-cSrc-phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression.

Isolation and cytochemical characterization of melanomacrophages and melanomacrophage clusters from goldfish (Carassius auratus, L.).

Pigmented or "melano-" macrophages are prominent in lymphoid and non-lymphoid tissues of poikilotherms. Though they have been extensively studied in situ only recently has a means to isolate them from other cell types been established. We provide the first in vitro characterization of isolated melanomacrophage cytochemistry and survival in culture. Unlike non-pigmented tissue macrophages melanomacrophages do not adhere to polystyrene surfaces making them easy to separate from tissue macrophages. In vitro goldfish melanomacrophages are distinguishable from tissue macrophages and neutrophils by being Sudan Black B positive (unlike tissue macrophages) and non-specific esterase positive (unlike neutrophils). Like tissue macrophages they also express acid phosphatase and CSF-1R. As sorted cells melanomacrophages only survive a few days in culture. However in coarsely disaggregated spleen and kidney tissues melanomacrophages survive for at least 3 weeks. Furthermore after 5 days culture disaggregating tissue clumps revealed encapsulated melanomacrophage clusters that remained intact for at least another week. The encapsulated clusters were resilient enough to allow for their isolation for further imaging and isolation of RNA. In some cases the clusters had either melanomacrophages or non-fluorescent cells protruding and in the latter case these could initiate outgrowths onto the plates with subsequent collapse of the cluster. These approaches for the isolation of melanomacrophages and melanomacrophage clusters should allow further study into specific cell and cluster functions.

M-CSF priming of osteoclast precursors can cause osteoclastogenesis-insensitivity, which can be prevented and overcome on bone.

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.

Manual and Rotary Instrumentation Ability to Reduce Enterococcus faecalis Associated with Photodynamic Therapy in Deciduous Molars

This aim of this study was to assess the ability of manual or rotary instrumentation associated with photodynamic therapy (PDT) to reduce Enterococcus faecalis using three combinations of light/photosensitizers: toluidine blue O/laser, fuchsin/halogen light and fuchsin/LED. Twenty deciduous molars were selected and contaminated with Enterococcus faecalis (McFarland 0.5 scale). Working length determination was performed by visual method. The teeth were randomly divided into two groups: G1 (n=10): manual instrumentation (Kerr-type files) and G2 (n=10): rotary instrumentation (ProTaper system). The bacteria were collected three times using sterile paper cones compatible with the anatomic diameter of the root canal for 30 s before and after instrumentation and after PDT. The samples were diluted in peptone water, seeded on blood agar plates and incubated in an oven at 37 °C for colony-forming units counting. The decrease of E. faecalis counts after instrumentation and after PDT was compared using the Wilcoxon test, t-test and Kruskal Wallis test. A significant reduction of E. faecalis occurred after manual and rotary instrumentation and after PDT using the three combinations of light/photosensitizer (p<0.05). It may be concluded that both rotary and manual instrumentation reduced E. faecalis. Fuchsin with halogen light or LED irradiation and toluidine blue O with laser irradiation can be used to reduce E. faecalis in root canals of primary molars. PDT can be used as an adjuvant to conventional endodontic treatment.

O objetivo do presente estudo foi avaliar a redução de Enterococcus faecalis após instrumentação manual ou rotatória associada à terapia fotodinâmica (PDT) utilizando 3 combinações luz/fotossensibilizante: azul de toluidina O/laser, fucsina/luz halógena e fucsina/LED. Foram selecionados 20 molares decíduos que foram contaminados com Enterococcus faecalis (escala 0,5 de McFarland). A odontometria foi feita através do método visual. Os dentes foram divididos aleatoriamente em dois grupos: G1 (n=10): instrumentação manual (limas tipo Kerr) e G2 (n=10): instrumentação rotatória (sistema ProTaper). Foram realizadas coletas com cone de papel estéril compatível com o diâmetro anatômico do canal durante 30 s antes e após a instrumentação e a PDT. As amostras foram diluídas em água peptonada, semeadas em placas de agar-sangue e incubadas em estufa a 37 °C para contagem das unidades formadoras de colônias. As comparações antes da redução de E. faecalis após a instrumentação e após a realização da PDT foram realizadas pelo teste de Wilcoxon, teste t e Kruskal Wallis. Houve redução significante de E. faecalis após a instrumentação manual ou rotatória e após realização da PDT com as três combinações de luz/fotossensibilizante (p<0,05). Pode-se concluir que a instrumentação rotatória e manual acarretou a redução de E. faecalis. A fucsina irradiada com luz halógena ou led e o azul de toluidina irradiado com laser podem ser utilizados para redução de E. faecalis do sistema de canais radiculares de molares decíduos. A terapia fotodinâmica pode ser utilizada como coadjuvante ao tratamento endodôntico convencional.

The mineral dissolution function of osteoclasts is dispensable for hypertrophic cartilage degradation during long bone development and growth.

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5(-/-) mice have increased trabecular bone mass. We used Dock5(-/-) mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5(-/-) mice. We demonstrate that Dock5(-/-) mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5(-/-) growing mice as compared to Dock5(+/+) animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5(-/-) osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation.

Clinical significance of tartrate-resistant acid phosphatase type-5 expression in human gastric cancer.

AIM: The present study investigated the clinical significance of tartrate-resistant acid phosphatase type-5 (ACP5) expression in gastric cancer. MATERIALS AND METHODS: In 150 specimens of gastric cancer and adjacent normal mucosa, expression of ACP5 protein and mRNA and was determined by immunohistochemical staining and quantitative real-time polymerase chain reaction, respectively. RESULTS: Expression of ACP5 mRNA was significantly higher in cancer tissues than in adjacent normal mucosa. Elevated ACP5 mRNA was associated with lymph node metastasis and peritoneal dissemination. Logistic regression analysis revealed that elevated ACP5 expression was an independent risk factor for peritoneal dissemination and was associated with shorter survival. Immunohistochemical staining of primary carcinomas showed ACP5 to be expressed mainly in the cytoplasm. CONCLUSION: ACP5 is predictive of peritoneal dissemination in patients with gastric cancer, and might play a crucial role in the establishment of peritoneal dissemination.

Cloning of PaAtg8 and roles of autophagy in adaptation to starvation with respect to the fat body and midgut of the Americana cockroach, Periplaneta americana.

Starvation, in particular amino acid deprivation, induces autophagy in trophocytes (adipocytes), the major component of the fat body cell types, in the larvae of Drosophila melanogaster. However, the fat body of cockroach has two additional cell types: urocytes depositing uric acid in urate vacuoles as a nitrogen resource and mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, which can synthesize amino acids from the metabolites of the stored uric acid. These cells might complement the roles of autophagy in recycling amino acids in the fat body or other organs of cockroaches under starvation. We investigate the presence of autophagy in tissues such as the fat body and midgut of the American cockroach, Periplaneta americana, under starvation by immunoblotting with antibody against Atg8, a ubiquitin-like protein required for the formation of autophagosomes and by electron microscopy. Corresponding changes in acid phosphatase activity were also investigated as representing lysosome activity. Starvation increased the level of an autophagic marker, Atg8-II, in both the tissues, extensively stimulating the formation of autophagic compartments in trophocytes of the fat body and columnar cells of the midgut for over 2 weeks. Acid phosphatase showed no significant increase in the fat body of the starved cockroaches but was higher in the midgut of the continuously fed animals. Thus, a distinct autophagic mechanism operates in these tissues under starvation of 2 weeks and longer. The late induction of autophagy implies exhaustion of the stored uric acid in the fat body. High activity of acid phosphatase in the midgut of the fed cockroaches might represent enhanced assimilation and not an autophagy-related function.

The RSC chromatin remodeling complex has a crucial role in the complete remodeler set for yeast PHO5 promoter opening.

Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo. Now we show that remodels the structure of chromatin (RSC) is crucially involved in PHO5 promoter opening. Further, the isw1 chd1 double deletion also delayed chromatin remodeling. Strikingly, combined absence of RSC and Isw1/Chd1 or Snf2 abolished for the first time promoter opening on otherwise sufficient induction in vivo. Together with previous findings, we recognize now a surprisingly complex network of five remodelers (RSC, SWI/SNF, INO80, Isw1 and Chd1) from four subfamilies (SWI/SNF, INO80, ISWI and CHD) as involved in PHO5 promoter chromatin remodeling. This is likely the first described complete remodeler set for a physiological chromatin transition. RSC was hardly involved at the coregulated PHO8 or PHO84 promoters despite cofactor recruitment by the same transactivator and RSC's presence at all three promoters. Therefore, promoter-specific chromatin rather than transactivators determine remodeler requirements.

Macrophage expression of tartrate-resistant acid phosphatase as a prognostic indicator in colon cancer.

Recent research has indicated that separate populations of macrophages are associated with differing outcomes in cancer survival. In our study, we examine macrophage expression of tartrate-resistant acid phosphatase (TRAP) and its effect on survival in colon cancer. Immunohistochemical analysis on colorectal adenocarcinomas confirmed macrophage expression of TRAP. Co-localization of TRAP with CD68, a pan-macrophage marker, revealed that TRAP is present in some but not all sub-populations of macrophages. Further co-localization of TRAP with CD163, an M2 marker, revealed that TRAP is expressed by both M2 and non-M2 macrophages. TRAP expression was then measured using the AQUA method of quantitative immunofluorescence in a tissue microarray consisting of 233 colorectal cancer patients seen at Yale-New Haven Hospital. Survival analysis revealed that patients with high TRAP expression have a 22 % increase in 5-year survival (uncorrected log-rank p = 0.025) and a 47 % risk reduction in disease-specific death (p = 0.02). This finding was validated in a second cohort of older cases consisting of 505 colorectal cancer patients. Patients with high TRAP expression in the validation set had a 19 % increase in 5-year survival (log-rank p = 0.0041) and a 52 % risk reduction in death (p = 0.0019). These results provide evidence that macrophage expression of TRAP is associated with improved outcome and implicates TRAP as a potential biomarker in colon cancer.

Deceased expression of prostatic acid phosphatase in primary sensory neurons after peripheral nerve injury.

Prostatic acid phosphatase (PAP) is expressed in nociceptive dorsal root ganglion (DRG) neurons and functions as an ectonucleotidase that dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine to suppress pain via activating A1-adenosine receptor (A1R) in dorsal spinal cord. However, the effect of peripheral nerve injury on the expression of PAP has not been reported until now. In the present study we found that PAP expression in DRG neurons is significantly decreased from the 2nd day after peripheral nerve injury and reaches the bottom at the 14th. In addition, intrathecal PAP injection can reduce mechanical allodynia induced by spared nerve injury. Our findings suggest that the decrease of PAP is involved in pathophysiological mechanisms of neuropathic pain.

The multifunctional protein fused in sarcoma (FUS) is a coactivator of microphthalmia-associated transcription factor (MITF).

The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a signaling effector engaged by macrophage colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). MITF exerts its regulatory functions through its association with cofactors. Discovering the identity of its various partners will provide insights into the mechanisms governing gene expression during osteoclastogenesis. Here, we demonstrate that the proto-oncogene fused in sarcoma (FUS), the chromatin remodeling ATPase BRG1, and MITF form a trimeric complex that is regulated by phosphorylation of MITF at Ser-307 by p38 MAPK during osteoclast differentiation. FUS was recruited to MITF target gene promoters Acp5 and Ctsk during osteoclast differentiation, and FUS knockdown abolished efficient transcription of Acp5 and Ctsk. Furthermore, sumoylation of MITF at Lys-316, known to negatively regulate MITF transcriptional activity, inhibited MITF interactions with FUS and BRG1 in a p38 MAPK phosphorylation-dependent manner. These results demonstrate that FUS is a coregulator of MITF activity and provide new insights into how the RANKL/p38 MAPK signaling nexus controls gene expression in osteoclasts.

Overexpression of phyA and appA genes improves soil organic phosphorus utilisation and seed phytase activity in Brassica napus.

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg(-1) seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.

Valvular osteoclasts in calcification and aortic valve stenosis severity.

BACKGROUND: Bone remodeling in calcified aortic valves is thought to originate from microfractures at multiple sites of the valve, at which osteoclasts and osteoblasts are recruited. The aim of the present study was to assess circulating mediators of bone homeostasis, correlate them to the severity of stenosis and explore the spatio-temporal distribution of bone turnover in different parts of calcified aortic valve tissue. METHODS AND RESULTS: Plasma and explanted aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery. Plasma levels of tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear-κB (RANK) ligand and Runt-related transcription factor 2 (Runx2/Cbfa1) exhibited a significant correlation to the severity of aortic stenosis. mRNA levels in normal, thickened and calcified parts of aortic valves assessed by quantitative real-time PCR were significantly elevated in calcified parts of valves for TRAP (5.08 ± 1.6-fold, P<0.001) RANK ligand (8.6 ± 4.2-fold, P<0.001) and RANK (1.98 ± 0.78-fold, P=0.015). In an age, gender and aortic valve anatomy-adjusted multivariable regression analysis the local transcript levels of TRAP correlated significantly with echocardiographic parameters quantifying stenosis severity in early stages, whereas the expression level of Runx2/Cbfa1 was a predictor of the stenosis severity in advanced stages. CONCLUSIONS: These findings suggest a critical role of bone turnover as a determinant of aortic stenosis severity.

Foreign body giant cells and osteoclasts are TRAP positive, have podosome-belts and both require OC-STAMP for cell fusion.

Macrophages have the ability to fuse and form multinucleated giant cells such as Osteoclast (OCs) and FBGCs. Osteoclast stimulatory transmembrane protein (OC-STAMP) is an important cell surface protein involved in the formation of OCs. This study sought to determine if OC-STAMP also regulates formation of FBGCs using expression analysis and subsequent inhibition studies. qPCR and Western blot analysis showed that OC-STAMP expression is significantly higher in FBGCs compared to control monocytes (P < 0.05). Four days following cell culture, OCs were positive for TRAP and F-actin ring formation, but FBGCs were not. In contrast, FBGCs were positive for TRAP and showed podosome belts comprised of F-actin on Day 8. FBGCs were subsequently plated onto dentine, but despite presenting some morphologic features of OCs (OC-STAMP expression, TRAP reactivity, and podosome belts) they failed to resorb bone. To evaluate a role for OC-STAMP in FBGCs, we inhibited this cell surface protein with anti-OC-STAMP antibody and observed that cell fusion and podosome belt formation was inhibited in both OCs and FBGCs. Our data support the hypothesis that OC-STAMP is a regulatory molecule for FBGCs; and that they are functionally distinct from OCs, despite similarities in gene expression profile, podosome belt formation, and TRAP expression.