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.     

Relationship between Initial Telomere Length,Initial Telomerase Activity,Age, and Replicative Capacity of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs: What Is a Predictor of Replicative Potential?

There is evidence that telomere length (TL), telomerase activity (TA), and age are related to the replicative potential of human nucleus pulposus chondrocytes (NPCs). However, it has not yet been established if any of these factors can serve as predictors of the replicative potential of NPCs. To establish predictors of the replicative potential of NPCs, we evaluated potential relationships between replicative capacity of NPCs, initial TL (telomere length at the first passage), initial TA (telomerase activity at the first passage), and age. Nucleus pulposus specimens were obtained from 14 patients of various ages undergoing discectomy. NPCs were serially cultivated until the end of their replicative lifespans. Relationships among cumulative population doubling level (PDL), initial TL, initial TA, and age were analyzed. Initial TA was negatively correlated with age (r = -0.674, P = 0.008). However, no correlation between initial TL and age was observed. Cumulative PDL was also negatively correlated with age (r = -0.585, P = 0.028). Although the cumulative PDL appeared to increase with initial TL or initial TA, this trend was not statistically significant. In conclusion, age is the sole predictor of the replicative potential of human NPCs, and replicative potential decreases with age. Initial TL and initial TA are not predictors of replicative potential, and can serve only as reference values.     

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif (⁷⁷⁴gpgfgssppGaeaapslRyvPY⁷⁹⁵) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X _2–5PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.     

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.     

Inhibition of Cell Proliferation and Migration by miR-509-3p That Targets CDK2, Rac1, and PIK3C2A

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.     

Pulmonary Toxocariasis Mimicking Invasive Aspergillosis in a Patient with Ulcerative Colitis

A 45-year-old-male who had underlying ulcerative colitis and presented with fever and dry cough. Initially, the patient was considered to have invasive aspergillosis due to a positive galactomannan assay. He was treated with amphotericin B followed by voriconazole. Nevertheless, the patient deteriorated clinically and radiographically. The lung biopsy revealed eosinophilic pneumonia, and ELISA for Toxocara antigen was positive, leading to a diagnosis of pulmonary toxocariasis. After a 10-day treatment course with albendazole and adjunctive steroids, the patient recovered completely without any sequelae. Pulmonary toxocariasis may be considered in patients with subacute or chronic pneumonia unresponsive to antibiotic agents, particularly in cases with eosinophilia.     

Difference in microchip electrophoretic mobility between partially and fully PEGylated poly(amidoamine) dendrimers

The objective of this study was to investigate the difference in electrophoretic mobility between partially and fully poly(ethylene glycol)-conjugated poly(amidoamine) dendrimers (part-PEG-PAMAM and full-PEG-PAMAM, respectively) using a microchip capillary gel electrophoresis (MCGE). While MCGE allowed size-based separation of PEG-PAMAMs prepared with monomethoxy PEG-nitrophenyl carbonate, full-PEG-PAMAMs migrated slower than part-PEG-PAMAMs that were similar in size or larger. When the measured molecular weights obtained from MCGE analysis and the calculated molecular weights were plotted, each part-PEG-PAMAM and full-PEG-PAMAM showed correlation coefficients greater than 0.98. This study indicates that MCGE would be useful for characterizing PEG-PAMAMs with different PEGylation degrees.     

Profilin: At the crossroads of signal transduction and the actin cytoskeleton

Despite its small size, profilin is an amazingly diverse and sophisticated protein whose precise role in cells continues to elude the understanding of researchers 15 years after its discovery. Its ubiquity, abundance and necessity for life in more evolved organisms certainly speaks for its exterme importance in cell function. So far, three ligands for profilin have been well-characterized in vitro: actin monomers, membrane polyphosphoinositides and poly-L-proline. In the years following its discovery, profilin's role in vivo progressed from that of a simple actin-binding protein which inhibits actin polymerization, to one which, as an important regulator of the cytoskeleton, can even promote actin polymerization under the appropriate circumstances. In addition, interactions with components of the phosphatidylinositol cycle and the RAS pathway in yeast implicate profilin as an important link through which the actin cytoskeleton is able to communicate with major signaling pathways.     

Rapid and high-throughput analysis of N-glycans from ovarian cancer serum using a 96-well plate platform

We present a rapid and high-throughput human serum N-glycan preparation technology using 96-well plate-based procedures. The released N-glycans from polyvinylidene fluoride (PVDF) membrane filter plate are subsequently loaded to porous graphitic carbon (PGC) containing a 96-well plate to remove salts and other contaminants without sacrificing accuracy or reproducibility. This robust glycan preparation technology is applied to ovarian cancer diagnosis using 5 μl of patient serum.