Aerobic reduction of manganese oxide by Salmonella sp. strain MR4

A new isolate of Salmonella, strain MR4, reduced Mn(IV)O₂ at 2.3 mM under aerobic conditions by about 83% over 24 h. Direct contact of cells to MnO₂ was not necessary as the cell-free spent medium produced a similar amount of Mn(II). Pyruvate (1.6 mM) and oxalate (0.8 mM) were identified in the culture medium and presumed to have a role in Mn(II) production in this microorganism.     

Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-chotesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.     

Proteomic analysis of microvesicles derived from human mesenchymal stem cells

Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC's self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.     

Micropatterning and characterization of electrospun poly(ε‐caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126. © 2010 Wiley Periodicals, Inc.     

Neurocomputational mechanism of controllability inference under a multi-agent setting

Controllability perception significantly influences motivated behavior and emotion and requires an estimation of one’s influence on an environment. Previous studies have shown that an agent can infer controllability by observing contingency between one’s own action and outcome if there are no other outcome-relevant agents in an environment. However, if there are multiple agents who can influence the outcome, estimation of one’s genuine controllability requires exclusion of other agents’ possible influence. Here, we first investigated a computational and neural mechanism of controllability inference in a multi-agent setting. Our novel multi-agent Bayesian controllability inference model showed that other people’s action-outcome contingency information is integrated with one’s own action-outcome contingency to infer controllability, which can be explained as a Bayesian inference. Model-based functional MRI analyses showed that multi-agent Bayesian controllability inference recruits the temporoparietal junction (TPJ) and striatum. Then, this inferred controllability information was leveraged to increase motivated behavior in the vmPFC. These results generalize the previously known role of the striatum and vmPFC in single-agent controllability to multi-agent controllability, and this generalized role requires the TPJ in addition to the striatum of single-agent controllability to integrate both self- and other-related information. Finally, we identified an innate positive bias toward the self during the multi-agent controllability inference, which facilitated behavioral adaptation under volatile controllability. Furthermore, low positive bias and high negative bias were associated with increased daily feelings of guilt. Our results provide a mechanism of how our sense of controllability fluctuates due to other people in our lives, which might be related to social learned helplessness and depression.     

Transmembrane TNF-dependent uptake of anti-TNF antibodies

TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside ‘reverse’ signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.     

Lodopyridones B and C from a marine sediment-derived bacterium Saccharomonospora sp.

HPLC-UV guided isolation of the culture broth of a marine bacterium Saccharomonospora sp. CNQ-490 has led to the isolation of two new natural products, lodopyridones B and C (1 and 2) along with the previously reported lodopyridone A (3). Their chemical structures were established from the interpretation of 2D NMR spectroscopic data and the comparison of NMR data with the lodopyridone A (3). Lodopyridones B and C (1 and 2) possess the thiazole, and chloroquinoline groups which are characteristic features of these molecules. Lodopyridones A–C show weak inhibitory activities on the β-site amyloid precursor protein cleaving enzyme 1 (BACE1).