RANKL induces heterogeneous DC‐STAMPlo and DC‐STAMPhi osteoclast precursors of which the DC‐STAMPlo precursors are the master fusogens

Osteoclasts (OC) are multinucleated bone resorbing cells that form via RANKL‐induced fusion of heterogeneous mononuclear OC precursors (OCP). Currently, there are no unique surface markers to distinguish these OCP populations, which are diagnostic for erosive and metabolic bone diseases using culture assays. Thus, we investigated expression of DC‐STAMP, a surface receptor required for OCP fusion, during osteoclastogenesis in vitro using a novel monoclonal antibody (1A2). Immunoprecipitation‐Western blot analysis of OCP membrane proteins detected 106 kDa dimeric and 53 kDa monomeric DC‐STAMP in non‐denaturing and denaturing conditions, respectively, with greater sensitivity versus rabbit anti‐sera (KR104). 1A2 also detected 99.9% of undifferentiated monocytes as a single population by flow cytometry with a MFI 100‐fold over background, while KR104 was not useful in this assay. Functionally, 1A2 inhibited OCP fusion in vitro. RANKL stimulation of OCP induced DC‐STAMP^lo and DC‐STAMP^hi cells, which mature into OC and mononuclear cells respectively as determined by fluorescent microscopy and TRAP assays. Addition of DC‐STAMP^hi cells to purified DC‐STAMP^lo cultures produced larger, more nucleated OC vs. pure DC‐STAMP^lo cultures. RT‐qPCR analysis of these two populations showed that OC markers (Trap and Oc‐stamp) and fusogenic gene expression (Cd9 and Cd47), were significantly increased in DC‐STAMP^lo vs. DC‐STAMP^hi cells. Collectively, these results demonstrate that DC‐STAMP is expressed on OCP as a dimer, which is efficiently detected by 1A2 via flow cytometry. RANKL induces osteoclastogenesis by stimulating DC‐STAMP internalization in some OCP, and these DC‐STAMP^lo cells display the “master fusogen” phenotype. In contrast, DC‐STAMP^hi OCP can only act as mononuclear donors. J. Cell. Physiol. 223: 76–83, 2010. © 2009 Wiley‐Liss, Inc.     

Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollenin monomer biosynthesis

The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors.     

Evolution of Sindbis Virus with a Low-Methionine-Resistant Phenotype Is Dependent Both on a Pre-Existing Mutation and on the Methionine Concentration in the Medium

SVlm21 is a mutant of Sindbis virus which was isolated by serial passage of virus in mosquito cells maintained in low-methionine medium; it therefore has a low-methionine-resistant (LMR) phenotype. This phenotype requires mutations at nts 319 and 321; these mutations result in Arg to Leu and Ser to Cys changes at positions 87 and 88 respectively in the viral methyl transferase, nsP1. To better understand the genesis of SVlm21, we carried out serial passages of viruses having only one of these amino acid changes, but in mosquito cells maintained in normal methionine-medium. Whether the passage was begun with SV319 or with SV321, the dominant virus population which emerged always acquired the second SVlm21 amino acid change. However, when the passage was begun with virus having neither the nt 319 or the nt321 mutation, even after many passages neither of these mutations was seen in the passaged virus population. Virus with the LMR phenotype emerged earlier when the virus encoded a wild-type RDRP (passage 4) rather than the mutant RDRP encoded by SVpzf (passage 7). When the methionine concentration in the medium of mosquito cells was increased to 250 µM, more than 20 passages were required until the LMR phenotype predominated. Competition experiments were carried out to compare the relative fitness of SVlm21, SVwt, SV319 and SV321 to each other. Our results indicated that SVlm21 was dominant to SVwt, as well as to both SV319 and SV321. However, SV319 and SV321 were able to co-exist with SVwt implying that in these mixed infection the presence of SVwt inhibited the emergence of SVlm21. Finally, our experiments highlight how a virus population by mutation and selection can adapt to the intracellular concentration of a simple metabolite, S-adenosylmethionine.     

Onychomycosis and <Emphasis Type="Italic">Tinea Pedis</Emphasis> in Athletes from the State of Rio Grande Do Sul (Brazil): A Cross-Sectional Study

Onychomycosis and tinea pedis are common superficial infections caused primarily by dermatophytes. The aim of this investigation was to study the epidemiology, etiological agents, and potential risk factors for infection based on comparison of athletes and non-athletes from a northern region of Rio Grande do Sul (Brazil). Each group consisted of 100 male individuals with ages ranging from 18 to 40 years. After a clinical examination, samples were taken from individuals presenting signs of onychomycosis and/or tinea pedis for direct microscopic examination and culture. Among the athletes, the frequency of onychomycosis and/or tinea pedis was 32%, and for the control group, it was 20%. The athletes presented 16% of onychomycosis, 12% of tinea pedis, and 4% of onychomycosis and tinea pedis together. The distribution in the control group was 10% of onychomycosis, 7% of tinea pedis, and 3% of this association. The pathogens identified were dermatophytes (84.8%) and yeasts (15.2%), and the most commonly identified organism was Trichophyton rubrum, followed by Trichophyton mentagrophytes var. interdigitale. No significant differences were found when the frequency of species distribution in the athletes and non-athlete groups was compared. Risk factors for onychomycosis in athletes included familial cases of fungal infection, contact with domestic animals, and nail trauma, while the risk factors in non-athletes included the habit of not using sandals in public bathrooms and nail trauma. For tinea pedis, the habit of not using sandals in public bathrooms was a predisposing factor in both groups, while hyperhydrosis was a risk factor only in non-athletes. This study concludes that despite the higher number of fungal infections in athletes, there is no significant difference between these groups.     

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.     

Anti-Prion Activity of a Panel of Aromatic Chemical Compounds: In Vitro and In Silico Approaches

The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrP^C) into the scrapie form (PrP^Sc) is the hallmark of TSEs. Once formed, PrP^Sc aggregates and catalyzes PrP^C misfolding into new PrP^Sc molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed in vivo due to poor pharmacokinetic profiles. In this work we propose a combined in vitro/in silico approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP^Sc (ScN2a) for their ability to inhibit PK-resistant PrP (PrP^Res) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP^Res in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted in silico, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP^Res from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP^109–149). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined in vitro/in silico approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP^Res in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.     

Pivotal role of Toll-like receptors 2 and 4, its adaptor molecule MyD88, and inflammasome complex in experimental tubule-interstitial nephritis

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process.