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Despite recent progress in the treatment of rheumatoid arthritis (RA), many patients still fail to achieve remission or low disease activity. An imbalance between auto-reactive effector T cells (Teff) and regulatory T cells (Treg) may contribute to joint inflammation and damage in RA. Therefore, restoring this balance is a promising approach for the treatment of inflammatory arthritis. Accordingly, our group has previously shown that the combination of TGF-β-releasing microparticles (MP), rapamycin-releasing MP, and IL-2-releasing MP (TRI MP) can effectively increase the ratio of Tregs to Teff in vivo and provide disease protection in several preclinical models. In this study TRI MP was evaluated in the collagen-induced arthritis (CIA) model. Although this formulation has been tested previously in models of destructive inflammation and transplantation, this is the first model of autoimmunity for which this therapy has been applied. In this context, TRI MP effectively reduced arthritis incidence, the severity of arthritis scores, and bone erosion. The proposed mechanism of action includes not only reducing CD4+ T cell proliferation, but also expanding a regulatory population in the periphery soon after TRI MP administration. These changes were reflected in the CD4+ T cell population that infiltrated the paws at the onset of arthritis and were associated with a reduction of immune infiltrate and inflammatory myeloid cells in the paws. TRI MP administration also reduced the titer of collagen antibodies, however the contribution of this reduced titer to disease protection remains uncertain since there was no correlation between collagen antibody titer and arthritis score., Competing Interests: The authors declare the following competing interests: S.R.L. is an inventor on patent 14/372,977 entitled “Controlled Release Formulations for the Induction and Proliferation of Blood Cells” which was issued to the University of Pittsburgh on 04/30/20. This patent covers the TRI MP formulation used in this manuscript. All other authors declare they have no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Current drug-eluting stents have abluminal polymer coating; however, thrombus formation in these compared with that in uniformly coated stents remains controversial. We evaluated thrombus formation and early endothelialization after using abluminal biodegradable polymer-coated sirolimus- (BP-SES), and everolimus-eluting stents (BP-EES) versus a durable polymer-coated everolimus-eluting stent (DP-EES) in an in vivo setting. BP-SES, BP-EES, and DP-EES (n = 6 each) were implanted in coronary arteries of 12 mini-pigs that were then sacrificed after 7 and 10 days. Stents were stained with hematoxylin and eosin, and a combined Verhoeff and Masson trichrome stain. Areas of fibrin deposition were digitally detected and measured with off-line morphometric software. Stents were investigated for re-endothelialization by transmission electron microscopy. At 7 days, histological analysis revealed the lowest area of fibrin deposition in BP-SES (BP-SES vs. BP-EES vs. DP-EES; 0.10 ± 0.06 mm2 vs. 0.15 ± 0.07 mm2 vs. 0.19 ± 0.06 mm2, p = 0.0004). At 10 days, the area of fibrin deposition was significantly greater in DP-EES (0.13 ± 0.04 mm2 vs. 0.14 ± 0.05 mm2 vs. 0.19 ± 0.08 mm2, p = 0.007). Endothelial cells in BP-SES demonstrated a significantly greater number of tight junctions than those in DP-EES according to by transmission electron microscopy for both days (p<0.05). Various parameters, including an inflammatory reaction and neointimal formation, were comparable among the groups at 7 and 10 days. An abluminal biodegradable polymer-coated SES showed the least fibrin deposition and greatest endothelial cell recovery at an early stage following implantation in the coronary arteries of mini-pigs., Competing Interests: Gaku Nakazawa is a consultant for Abbott Vascular Japan, Terumo Corporation, and Japan Medical Device Engineering, and is in receipt of research grants from Abbott Vascular, Terumo Corporation, Boston Scientific, and Japan Medical Device Engineering; Junko Souba, Atsushi Isobe and Hitomi Hagiwara are employees of Terumo Corporation; Yuji Ikari is in receipt of research grants from Boston Scientific, Abbott Vascular, Kaneka Medics, Asahi Intech, Terumo Corp. All other authors have no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: Data concerning the effect of current smoking on solely new-generation drug-eluting stents (DES) are limited. We investigated the impact of current smoking on 2-year clinical outcomes between durable-polymer (DP)-coated DES (zotarolimus-eluting [ZES] and everolimus eluting [EES]) and biodegradable-polymer (BP)-coated biolimus-eluting stent (BES) in acute myocardial infarction (AMI) patients after successful percutaneous coronary intervention (PCI)., Methods: Finally, a total of 8357 AMI patients with current smoking underwent successful PCI with new-generation DES (ZES, EES, and BES) were enrolled and divided into three groups as ZES (n = 3199), EES (n = 3987), and BES group (n = 1171). The primary endpoint was the occurrence of major adverse cardiac events (MACE) defined as all-cause death (cardiac death [CD] or non-cardiac death), recurrent AMI (re-MI), any revascularization (target lesion revascularization [TLR], target vessel revascularization [TVR], and non-TVR). The secondary endpoint was the incidence of definite or probable stent thrombosis (ST)., Results: The 2-year adjusted hazard ratio (HR) of MACE for ZES vs. EES (1.055; 95% confidence interval [CI], 0.843-1.321; p = 0.638), ZES vs. BES (HR, 0.885; 95% CI, 0.626-1.251; p = 0.488), EES vs. BES (HR, 0.889; 95% CI, 0.633-1.250; p = 0.499), and ZES/EES vs. BES (HR, 0.891; 95% CI, 0.648-1.126; p = 0.480) were similar. The occurrence of ST after adjustment were also comparable. In addition, the 2-year adjusted HR for all-cause death, CD, re-MI, TLR, TVR, and non-TVR were not different., Conclusions: In this study, DP-DES and BP-DES showed comparable safety and efficacy during 2-year follow-up periods. Therefore, DP-DES or BP-DES are equally acceptable in AMI patients with current smoking undergoing PCI., Competing Interests: The authors have declared that no competing interests exist.

Endosseous titanium (Ti) implant failure due to poor biocompatibility of implant surface remains a major problem for osseointegration. Improving the topography of Ti surface may enhance osseointegration, however, the mechanism remains unknown. To investigate the effect of modified Ti surface on osteogenesis, we loaded rapamycin (RA) onto nano-hydroxyapatite (HAp) coated Ti surface which was acid-etched, alkali-heated and HAp coated sequentially. Sodium hyaluronate (SH) was employed as an intermediate layer for the load of RA, and a steady release rate of RA was maintained. Cell vitality of MC3T3-E1 was assessed by MTT. Osteogenesis of MC3T3-E1 on this modified Ti surface was evaluated by alkaline phosphatase (ALP) activity, mineralization and related osteogenesis genes osteocalcin (OCN), osteopontin (OPN), Collagen-I and Runx2. The result revealed that RA/SH-loaded nano-HAp Ti surface was innocent for cell vitality and even more beneficial for cell osteogenesis in vitro. Furthermore, osteogenesis of MC3T3-E1 showed significant association with the mammalian target of rapamycin (mTOR) phosphorylation by RA, which required further study about the mechanism. The approach to this modified Ti surface presented in this paper has high research value for the development of Ti-based implant., Competing Interests: The authors have declared that no competing interests exist.

Target of rapamycin complex 1 (TORC1) phosphorylates autophagy-related Atg13 and represses autophagy under nutrient-rich conditions. However, when TORC1 becomes inactive upon nutrient depletion or treatment with the TORC1 inhibitor rapamycin, Atg13 dephosphorylation occurs rapidly, and autophagy is induced. At present, the phosphatases involved in Atg13 dephosphorylation remain unknown. Here, we show that two protein phosphatase 2A (PP2A) phosphatases, PP2A-Cdc55 and PP2A-Rts1, which are activated by inactivation of TORC1, are required for sufficient Atg13 dephosphorylation and autophagy induction after TORC1 inactivation in budding yeast. After rapamycin treatment, dephosphorylation of Atg13, activation of Atg1 kinase, pre-autophagosomal structure (PAS) formation and autophagy induction are all impaired in PP2A-deleted cells. Conversely, overexpression of non-phosphorylatable Atg13 suppressed defects in autophagy in PP2A mutant. This study revealed that the orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1., Competing Interests: The authors have declared that no competing interests exist.

Infection with the white spot syndrome virus (WSSV) induces a metabolic shift in shrimp that resembles the "Warburg effect" in mammalian cells. This effect is triggered via activation of the PI3K-Akt-mTOR pathway, and it is usually accompanied by the activation of other metabolic pathways that provide energy and direct the flow of carbon and nitrogen. Here we show that unlike the glutamine metabolism (glutaminolysis) seen in most cancer cells to double deaminate glutamine to produce glutamate and the TCA cycle intermediate α-ketoglutarate (α-KG), at the WSSV genome replication stage (12 hpi), although glutaminase (GLS) expression was upregulated, only glutamate was taken up by the hemocytes of WSSV-infected shrimp. At the same time, we observed an increase in the activity of the two enzymes that convert glutamate to α-KG, glutamate dehydrogenase (GDH) and aspartate aminotransferase (ASAT). α-ketoglutarate concentration was also increased. A series of inhibition experiments suggested that the up-regulation of GDH is regulated by mTORC2, and that the PI3K-mTORC1 pathway is not involved. Suppression of GDH and ASAT by dsRNA silencing showed that both of these enzymes are important for WSSV replication. In GDH-silenced shrimp, direct replenishment of α-KG rescued both ATP production and WSSV replication. From these results, we propose a model of glutamate-driven anaplerosis that fuels the TCA cycle via α-KG and ultimately supports WSSV replication.

FKBP22, an Escherichia coli-encoded PPIase (peptidyl-prolyl cis-trans isomerase) enzyme, shares substantial identity with the Mip-like pathogenic factors, caries two domains, exists as a dimer in solution and binds some immunosuppressive drugs (such as FK506 and rapamycin) using its C-terminal domain (CTD). To understand the effects of these drugs on the structure and stability of the Mip-like proteins, rFKBP22 (a chimeric FKBP22) and CTD+ (a CTD variant) have been studied in the presence and absence of rapamycin using different probes. We demonstrated that rapamycin binding causes minor structural alterations of rFKBP22 and CTD+. Both the proteins (equilibrated with rapamycin) were unfolded via the formation of intermediates in the presence of urea. Further study revealed that thermal unfolding of both rFKBP22 and rapamycin-saturated rFKBP22 occurred by a three-state mechanism with the synthesis of intermediates. Intermediate from the rapamycin-equilibrated rFKBP22 was formed at a comparatively higher temperature. All intermediates carried substantial extents of secondary and tertiary structures. Intermediate resulted from the thermal unfolding of rFKBP22 existed as the dimers in solution, carried an increased extent of hydrophobic surface and possessed relatively higher rapamycin binding activity. Despite the formation of intermediates, both the thermal and urea-induced unfolding reactions were reversible in nature. Unfolding studies also indicated the considerable stabilization of both proteins by rapamycin binding. The data suggest that rFKBP22 or CTD+ could be exploited to screen the rapamycin-like inhibitors in the future.

The importance of neovascularization for primary and metastatic tumor growth fostered numerous clinical trials of angiogenesis inhibitors either alone or in combination with conventional antineoplastic therapies. One challenge with the use of molecularly targeted agents has been the disconnection between size reduction and tumor biologic behavior, either when the drug is efficacious or when tumor resistance emerges. Here, we report the synthesis and characterization of (64)Cu-NOTA-bevacizumab as a PET imaging agent for imaging intratumoral VEGF content in vivo. (64)Cu-NOTA-bevacizumab avidly accumulated in 786-O renal carcinoma xenografts with lower levels in host organs. RAD001 (everolimus) markedly attenuated (64)Cu-NOTA-bevacizumab accumulation within 786-O renal carcinoma xenografts. Tumor tissue and cellular molecular analysis validated PET imaging, demonstrating decreases in total and secreted VEGF content and VEGFR2 activation. Notably, (64)Cu-NOTA-bevacizumab PET imaging was concordant with the growth arrest of RAD001 tumors. These data suggest that immunoPET targeting of angiogenic factors such as VEGF could be a new class of surrogate markers complementing the RECIST criteria in patients receiving molecularly targeted therapies.

Background: The ability to disrupt the function of a specific protein on a rapid time scale provides a powerful tool for biomedical research. Specific proteases provide a potential method to selectively cleave a chosen protein, but rapid control of protease activity is difficult., Methodology/principal Findings: A heterologous expression system for rapid target-directed proteolysis in mammalian cells was developed. The system consists of an inducible NIa protease from the tobacco etch virus (TEVp) and a chosen protein into which a TEVp substrate recognition sequence (TRS) has been inserted. Inducible activity was conferred to the TEVp using rapamycin-controlled TEVp fragment complementation. TEVp activity was assayed using a FRET-based reporter construct. TEVp expression was well tolerated by mammalian cells and complete cleavage of the substrate was possible. Cleavage at 37 degrees C proceeded exponentially with a time constant of approximately 150 minutes. Attempts to improve cleavage efficiency were hampered by substantial background activity, which was attributed to inherent affinity between the TEVp fragments. A second TEVp assay, based on changes in inactivation of a modified K(V)3.4 channel, showed that functional properties of a channel can be using altered using an inducible TEVp system. Similar levels of background activity and variability were observed in both electrophysiological and FRET assays., Conclusions/significance: The results suggested that an optimum level of TEVp expression leading to sufficient inducible activity (with minimal background activity) exists but the variability in expression levels between cells makes the present system rather impractical for single cell experiments. The system is likely to be more suitable for experiments in which the cell-to-cell variability is less of an issue; for example, in experiments involving large populations of cells.