2-(Trimethylammonium) Ethyl (R)-3-Methoxy-3-oxo-2-Stearamidopropyl Phosphate Suppresses Osteoclast Maturation and Bone Resorption by Targeting Macrophage-Colony Stimulating Factor Signaling

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.     

Comparative growth,cross stress resistance,transcriptomics of Streptococcus pyogenes cultured under low shear modeled microgravity and normal gravity

Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV–Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5.     

Heterogeneity of Regional Brain Atrophy Patterns Associated with Distinct Progression Rates in Alzheimer’s Disease

We aimed to identify and characterize subtypes of Alzheimer’s disease (AD) exhibiting different patterns of regional brain atrophy on MRI using age- and gender-specific norms of regional brain volumes. AD subjects included in the Alzheimer''s Disease Neuroimaging Initiative study were classified into subtypes based on standardized values (Z-scores) of hippocampal and regional cortical volumes on MRI with reference to age- and gender-specific norms obtained from 222 cognitively normal (CN) subjects. Baseline and longitudinal changes of clinical characteristics over 2 years were compared across subtypes. Whole-brain-level gray matter (GM) atrophy pattern using voxel-based morphometry (VBM) and cerebrospinal fluid (CSF) biomarkers of the subtypes were also investigated. Of 163 AD subjects, 58.9% were classified as the “both impaired” subtype with the typical hippocampal and cortical atrophy pattern, whereas 41.1% were classified as the subtypes with atypical atrophy patterns: “hippocampal atrophy only” (19.0%), “cortical atrophy only” (11.7%), and “both spared” (10.4%). Voxel-based morphometric analysis demonstrated whole-brain-level differences in overall GM atrophy across the subtypes. These subtypes showed different progression rates over 2 years; and all subtypes had significantly lower CSF amyloid-β_1–42 levels compared to CN. In conclusion, we identified four AD subtypes exhibiting heterogeneous atrophy patterns on MRI with different progression rates after controlling the effects of aging and gender on atrophy with normative information. CSF biomarker analysis suggests the presence of Aβ neuropathology irrespective of subtypes. Such heterogeneity of MRI-based neuronal injury biomarker and related heterogeneous progression patterns should be considered in clinical trials and practice with AD patients.     

Protein–DNA photo-crosslinking with a genetically encoded benzophenone-containing amino acid

The photo-crosslinking amino acid, p-benzoyl-l-phenylalanine (pBpa), was genetically incorporated into Escherichia coli catabolite activator protein (CAP) in bacteria in response to an amber nonsense codon using an orthogonal tRNA/aminoacyl-tRNA synthetase pair. The mutant CAP (CAP-K26Bpa) containing pBpa formed a covalent complex with a DNA fragment containing the consensus operator sequence upon UV irradiation. Because this amino acid can be genetically incorporated into any DNA-binding protein in E. coli, yeast or mammalian cells with minimal perturbation of protein structure, this method should be generally useful for investigating DNA–protein interactions.     

Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.     

Block copolymers modify the internalization of micelle-incorporated probes into neural cells.

An important therapeutic concern is rate and extent of internalization of drugs into cells. Hydrophilic agents often internalize poorly and slowly, and highly lipophilic ones too rapidly. The incorporation of drugs into micelles allows regulation of their internalization parameters, and newly-described block copolymers can be selectively tailored to suit specific drugs. This report compares internalization of Cell Tracker CM-DiI (DiI), a highly lipophilic non-cytotoxic fluorescent probe in common use in biology, from the freely-presented (non-micelle-incorporated) and micelle-incorporated states. DiI was effectively incorporated (>60%) into 25-50 nm diameter spherical micelles made from polycaprolactone-b-polyethylene oxide block copolymer. Confocal microscopy was used to evaluate the internalization of DiI into mixed neuron-glia cultures (2-14 days in vitro, 2DIV-14DIV). Incorporation of DiI into micelles strikingly reduced the rate and extent of its internalization in both 2DIV and 14DIV cultures. Both the age of the cultures and the block copolymer employed to construct the micelles significantly influence the internalization of micelle-incorporated probe.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Efficient and accurate analysis of microRNA using a specific extension sequence

Molecular Biology Reports - We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay...