Delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC) Shows No Change in Cartilage Structural Composition after Viscosupplementation in Patients with Early-Stage Knee Osteoarthritis

Introduction

Viscosupplementation with hyaluronic acid (HA) of osteoarthritic (OA) knee joints has a well-established positive effect on clinical symptoms. This effect, however, is only temporary and the working mechanism of HA injections is not clear. It was suggested that HA might have disease modifying properties because of its beneficial effect on cartilage sulphated glycosaminoglycan (sGAG) content. Delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) is a highly reproducible, non-invasive surrogate measure for sGAG content and hence composition of cartilage. The aim of this study was to assess whether improvement in cartilage structural composition is detected using dGEMRIC 14 weeks after 3 weekly injections with HA in patients with early-stage knee OA.

Methods

In 20 early-stage knee OA patients (KLG I-II), 3D dGEMRIC at 3T was acquired before and 14 weeks after 3 weekly injections with HA. To evaluate patient symptoms, the knee injury and osteoarthritis outcome score (KOOS) and a numeric rating scale (NRS) for pain were recorded. To evaluate cartilage composition, six cartilage regions in the knee were analyzed on dGEMRIC. Outcomes of dGEMRIC, KOOS and NRS before and after HA were compared using paired t-testing. Since we performed multiple t-tests, we applied a Bonferroni-Holm correction to determine statistical significance for these analyses.

Results

All KOOS subscales (‘pain’, ‘symptoms’, ‘daily activities’, ‘sports’ and ’quality of life’) and the NRS pain improved significantly 14 weeks after Viscosupplementation with HA. Outcomes of dGEMRIC did not change significantly after HA compared to baseline in any of the cartilage regions analyzed in the knee.

Conclusions

Our results confirm previous findings reported in the literature, showing persisting improvement in symptomatic outcome measures in early-stage knee OA patients 14 weeks after Viscosupplementation. Outcomes of dGEMRIC, however, did not change after Viscosupplementation, indicating no change in cartilage structural composition as an explanation for the improvement of clinical symptoms.     

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter

Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins.     

The herpes simplex virus 1 IgG fc receptor blocks antibody-mediated complement activation and antibody-dependent cellular cytotoxicity in vivo

Herpes simplex virus 1 (HSV-1) glycoprotein E (gE) mediates cell-to-cell spread and functions as an IgG Fc receptor (FcγR) that blocks the Fc domain of antibody targeting the virus or infected cell. Efforts to assess the functions of the HSV-1 FcγR in vivo have been hampered by difficulties in preparing an FcγR-negative strain that is relatively intact for spread. Here we report the FcγR and spread phenotypes of NS-gE264, which is a mutant strain that has four amino acids inserted after gE residue 264. The virus is defective in IgG Fc binding yet causes zosteriform disease in the mouse flank model that is only minimally reduced compared with wild-type and the rescue strains. The presence of zosteriform disease suggests that NS-gE264 spread functions are well maintained. The HSV-1 FcγR binds the Fc domain of human, but not murine IgG; therefore, to assess FcγR functions in vivo, mice were passively immunized with human IgG antibody to HSV. When antibody was inoculated intraperitoneally 20 h prior to infection or shortly after virus reached the dorsal root ganglia, disease severity was significantly reduced in mice infected with NS-gE264, but not in mice infected with wild-type or rescue virus. Studies of C3 knockout mice and natural killer cell-depleted mice demonstrated that the HSV-1 FcγR blocked both IgG Fc-mediated complement activation and antibody-dependent cellular cytotoxicity. Therefore, the HSV-1 FcγR promotes immune evasion from IgG Fc-mediated activities and likely contributes to virulence at times when antibody is present, such as during recurrent infections.     

An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.     

GSK-3β Protein Phosphorylates and Stabilizes HLXB9 Protein in Insulinoma Cells to Form a Targetable Mechanism of Controlling Insulinoma Cell Proliferation

Insulinomas (pancreatic islet β cell tumors) are the most common type of functioning pancreatic neuroendocrine tumors that occur sporadically or as a part of the MEN1 syndrome that is caused by germ line mutations in MEN1. Tissue-specific tumor predisposition from germ line mutations in ubiquitously expressed genes such as MEN1 could occur because of functional consequences on tissue-specific factors. We previously reported the proapoptotic β cell differentiation factor HLXB9 as a downstream target of menin (encoded by MEN1). Here we show that GSK-3β inactivates the proapoptotic activity of HLXB9 by phosphorylating HLXB9 at Ser-78/Ser-80 (pHLXB9). Although HLXB9 is found in the nucleus and cytoplasm, pHLXB9 is predominantly nuclear. Both pHLXB9 and active GSK-3β are elevated in β cells with menin knockdown, in MEN1-associated β cell tumors (insulinomas), and also in human sporadic insulinomas. Pharmacologic inhibition of GSK-3β blocked cell proliferation in three different rodent insulinoma cell lines by arresting the cells in G₂/M phase and caused apoptosis. Taken together, these data suggest that the combination of GSK-3β and pHLXB9 forms a therapeutically targetable mechanism of insulinoma pathogenesis. Our results reveal that GSK-3β and pHLXB9 can serve as novel targets for insulinoma treatment and have implications for understanding the pathways associated with β cell proliferation.     

Lipophilic phosphonium-lanthanide compounds with magnetic, luminescent, and tumor targeting properties

Multifunctional phosphonium-lanthanide compounds that simultaneously possess paramagnetism, luminescence, and tumor mitochondrial targeting properties were prepared by use of a facile method. These compounds were fully characterized by use of ¹H, ¹³C, ³¹P NMR, FT-IR, and elemental analyses. The thermal properties of these compounds including melting points and decomposition temperatures were investigated using DSC and TGA analyses. In addition, the paramagnetism, luminescence, and tumor targeting properties of these multifunctional compounds were confirmed by respective use of SQUID, fluorescence, and cell cytotoxicity studies. All compounds exhibited paramagnetism at room temperature, which could provide target delivery of these compounds to parts of the body containing tumor cells using a strong external magnetic field. In addition, these compounds display two major characteristic emissions originating from Dy^3 +, which can be utilized for imaging tumor cells. The IC₅₀ values of these compounds measured against normal breast cell line (Hs578Bst) are significantly greater than those measured against the corresponding carcinoma breast cell line (Hs578T), clearly indicating the selective tumor targeting properties of these compounds. Confocal fluorescence microscopy studies were used to confirm the yellowish-green fluorescence corresponding to the emission of dysprosium thiocyanate anion within cancer cells upon exposure of cancer cell lines such as human pancreatic carcinoma cell line (MIAPaCa-2) and human breast carcinoma (MDA-MB-231) to a solution of these phosphonium-dysprosium compounds.     

Anthraquinone derivatives from the mangrove-derived fungus Phomopsis sp. PSU-MA214

One new tetrahydroanthraquinone derivative, (2R,3S)-7-ethyl-1,2,3,4-tetrahydro-2,3,8-trihydroxy-6-methoxy-3-methyl-9,10-anthracenedione (1), together with five known anthraquinones (2–6), two known phenylethyl alcohols (7–8) and one known butanamide (9), were isolated from the mangrove-derived fungus Phomopsis sp. PSU-MA214. Their structures were established by spectroscopic evidence. Compound 1 is a rare ethyltetrahydroanthraquinone and exhibited weak cytotoxicity against breast cancer (MCF-7) cell lines and antibacterial activity against the standard Staphylococcus aureus ATCC25923 and methicillin-resistant S. aureus SK1.     

Synergistic effects of multiple natural products in pancreatic cancer cells

Pancreatic cancer (PC) remains the fourth most common cause of cancer related death in the United States. Therefore, novel strategies for the prevention and treatment are urgently needed. Numerous dietary and pharmacological agents have been proposed as alternative strategies for the prevention and/or treatment of PC. Isoflavone is a prominent flavonoid found in soy products and has been proposed to be responsible for lowering the incidence of PC in Asians. Similarly, curcumin, an active ingredient of turmeric, that inhibits growth of malignant neoplasms, has a promising role in the prevention and/or treatment of PC. Here we examined whether isoflavone together with curcumin could elicit a greater inhibition of growth of PC cells than either agent alone, and also sought to determine the molecular mechanism of action. We found that the inhibition of cell growth and induction of apoptosis was significantly greater in the combination group than that could be achieved by either agent alone. These changes were associated with decreased Notch-1 expression and DNA binding activity of NF-kappaB and its target genes such as Cyclin D1, Bcl-2, and Bcl-xL. Moreover, we found that the combination of four natural agents at lower concentration was much more effective. Collectively, our results suggest that diet containing multiple natural products should be preferable over single agents for the prevention and/or treatment of PC. The superior effects of the combinatorial treatment could partly be attributed to the inhibition of constitutive activation of Notch-1 and NF-kappaB signaling pathways.     

Topological and Functional Characterization of the ssSPTs,Small Activating Subunits of Serine Palmitoyltransferase

The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase (hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met²⁵ in ssSPTa and Val²⁵ in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1^S331F/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered.