Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn[superscript III][subscript 2]-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn[superscript III][subscript 2]-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min[superscript –1] mg[superscript –1]). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from Mn[superscript III][subscript 2]-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·[over β [subscript 2]]. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ~1250 nmol min[superscript –1] mg[superscript –1] using a 1:1 mixture of NrdE:Mn[superscript III][subscript 2]-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (~1 μM), NrdE is a monomer (α) and Mn[superscript III][subscript 2]-Y· NrdF is a dimer (β[subscript 2]). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.Massachusetts Institute of Technology. Biophysical Instrumentation Facility (NSF-007031

Efficient Task Offloading Algorithm for Digital Twin in Edge/Cloud Computing Environment

In the era of Internet of Things (IoT), Digital Twin (DT) is envisioned to empower various areas as a bridge between physical objects and the digital world. Through virtualization and simulation techniques, multiple functions can be achieved by leveraging computing resources. In this process, Mobile Cloud Computing (MCC) and Mobile Edge Computing (MEC) have become two of the key factors to achieve real-time feedback. However, current works only considered edge servers or cloud servers in the DT system models. Besides, The models ignore the DT with not only one data resource. In this paper, we propose a new DT system model considering a heterogeneous MEC/MCC environment. Each DT in the model is maintained in one of the servers via multiple data collection devices. The offloading decision-making problem is also considered and a new offloading scheme is proposed based on Distributed Deep Learning (DDL). Simulation results demonstrate that our proposed algorithm can effectively and efficiently decrease the system's average latency and energy consumption. Significant improvement is achieved compared with the baselines under the dynamic environment of DTs

Observation on A-PRF promoting regeneration of osteochondral defects in rabbit knee joints

Objective·To explore the role of advanced platelet-rich fibrin (A-PRF) in osteochondral regeneration.Methods·Bone-marrow mesenchymal stem cells (BMSCs) and knee joint chondrocytes were obtained from New Zealand rabbits. A-PRF was obtained by low-speed centrifugation of the heart blood of rabbits. The histological structure of A-PRF was observed by an optical microscope. The release of growth factors in A-PRF was detected by ELISA, including platelet-derived growth factor, transforming growth factor-β, insulin-like growth factor, vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor. A-PRF's cytotoxicity and capability for promoting the proliferation of rabbit BMSCs were detected by live/dead double staining and MTT methods. The effect of A-PRF on the gene expression of type Ⅱ collagen, aggrecan, alkaline phosphatase (ALP) and osteocalcin (OCN) in rabbit BMSCs was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Transwell chambers were used to determine the effect of A-PRF on the migration ability of rabbit BMSCs and the chondrocytes. Rabbit knee osteochondral defect models were established, and 18 rabbits were randomly divided into 3 groups. The A-PRF group (n=6) was implanted with A-PRF in the defect, the A-PRF+BMSCs group (n=6) was implanted with rabbit BMSCs on A-PRF, and the control group (n=6) did not undergo implantation. The rabbits were sacrificed 12 weeks after surgery and the knee joint specimens were stained with hematoxylin-eosin (H-E), toluidine blue and safranin O/fast green. Based on the surface morphology and histology of the knee joints, the International Cartilage Repair Society (ICRS) scoring system was used for macroscopic and histological scoring.Results·A-PRF had a loose network structure and can slowly release growth factors. No cytotoxicity to rabbit BMSCs was observed after adding A-PRF, and the the capability for promoting the proliferation of rabbit BMSCs was significantly increased at 24, 48 and 72 h after adding A-PRF (all P<0.05). Chondrogenesis-related gene Ⅱ collagen and aggrecan, as well as osteogenesis-related genes ALP and OCN were significantly up-regulated (all P<0.05). After adding A-PRF, the migration abilities of rabbit BMSCs and chondrocytes were significantly enhanced (both P<0.05), and the migration ability of rabbit BMSCs was significantly higher than that of chondrocytes (P=0.025). The joint surface morphology in the rabbit knee joint defect models was observed. It can be seen that the defects in the A-PRF group and the A-PRF+BMSCs group were basically restored, while the the defects in the control group were only covered by soft tissue. In the ICRS macroscopic score, there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group, but the scores of the two groups were all significantly higher than those of the control group (all P<0.05). According to the histological results, both the A-PRF group and the A-PRF+BMSCs group formed osteochondral repair, but the cartilage in the A-PRF group was more mature, while the control group formed fibrous repair. In the ICRS histological score, there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group, but the scores of both the groups were significantly higher than those of the control group (both P<0.05).Conclusion·Autologous A-PRF has good biocompatibility and the capability for promoting the proliferation of BMSCs. It can promote the repair of cartilage and subchondral bone both in vitro and in vivo

Diphthamide Biosynthesis: Characterization And Mechanistic Studies Of An Unconventional Radical Sam Enzyme Phdph2

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps. The first step is the formation of a C-C bond between the histidine residue and the 3-amino-3carboxylpropyl group of S-adenosylmethionine (SAM), which is catalyzed by four enzymes Dph1-Dph4 in eukaryotic or only one enzyme Dph2 in archaea; the second step is the trimethylation of the amino group by Dph5; and the last step is an ATP depended amidation of the carboxyl group by an unknown enzyme. We have recently found that in an archaeal species Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe- 4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM enzymes, biochemical evidence suggests that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Further evidence shows that the 3-amino-3-carboxypropyl radical does not undergo hydrogen ion reaction, which was observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer. The successful reconstitution of the first step of diphthamide biosynthesis provides the substrate for the second step. We then reconstituted the second step using P. horikoshii PhDph5 in vitro. The results demonstrate that PhDph5 is sufficient to catalyze the mono-, di-, and trimethylation of PhEF2. Interestingly, the trimethylated product from the PhDph5-catalyzed reaction can easily eliminate the trimethylamino group even in the very mild reaction conditions. This unexpected finding on the diphthamide biosynthesis pathway may suggest that the last amidation step occurs very quickly in cells to avoid the elimination reaction or the amidation step occurs before the trimethylation step

Review of Performance and Application of Giant Magnetostrictive Materials under the Multi-field Coupling

The Giant magnetostrictive materials（GMM） have some distinct advantages such as large magnetostrictive coefficient,fast dynamic response,and high electromechanical conversion efficiency,which can be used into power conversion among electric,magnetic,mechanics and heat,etc. Based on multi- field coupling theory,a brief description about the characteristics of GMM is given,comparing with the piezoelectric ceramic material,and the relationships between these characteristics and magnetic field intensity,temperature,frequency is stated. Then,various mathematical models with different features are stressed,along with the multi- field coupling effect. Meanwhile,the foreign and domestic researches and applications of GMM are introduced,including the operation principle,structure and basic performance of GMM devices. Finally,the development of GMM in the future is discussed

Enzyme-Substrate Binding Kinetics Indicate that Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer

Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) that are formed on DNA upon exposure of cells to ultraviolet light. The light-driven electron-transfer mechanism by which photolyase catalyzes the CPD monomerization after the enzyme-substrate complex has formed has been studied extensively. However, much less is understood about how photolyase recognizes CPDs on DNA. It has been clearly established that photolyase, like many other DNA-repair proteins, requires flipping of the CPD site into an extrahelical position. Photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base. In this paper, we perform direct measurements of photolyase binding to CPD-containing undecamer DNA that has been labeled with a fluorophore. We find that the association constant of ∼2 × 106 M-1 is independent of the location of the CPD on the undecamer DNA. The binding kinetics of photolyase are best described by two rate constants. The slower rate constant is ∼104 M-1 s-1 and is most likely due to steric interference of the fluorophore during the binding process. The faster rate constant is on the order of 2.5 × 105 M-1 s-1 and reflects the binding of photolyase to the CPD on the DNA. This result indicates that photolyase finds and binds to a CPD lesion 100-4000 times slower than other DNA-repair proteins. In light of the existing literature, we propose a mechanism in which photolyase recognizes a CPD that is flipped into an extrahelical position via a three-dimensional search

Correction: Inflammatory markers in postoperative delirium (POD) and cognitive dysfunction (POCD): A meta-analysis of observational studies.

[This corrects the article DOI: 10.1371/journal.pone.0195659.]

Inflammatory markers in postoperative delirium (POD) and cognitive dysfunction (POCD): A meta-analysis of observational studies

<div><p>Background</p><p>The aim of this study was to summarize and discuss the similarities and differences in inflammatory biomarkers in postoperative delirium (POD) and cognitive dysfunction (POCD).</p><p>Methods</p><p>A systematic retrieval of literature up to June 2017 in PubMed, Embase, the Cochrane Library, the China National Knowledge Infrastructure database, and the Wanfang database was conducted. Extracted data were analyzed with STATA (version 14). The standardized mean difference (SMD) and the 95% confidence interval (95% CI) of each indicator were calculated using a random effect model. We also performed tests of heterogeneity, sensitivity analysis, assessments of bias, and meta-regression in this meta-analysis.</p><p>Results</p><p>A total of 54 observational studies were included. By meta-analysis we found significantly increased C-reactive protein (CRP) (9 studies, SMD 0.883, 95% CI 0.130 to 1.637, <i>P</i> = 0.022 in POD; 10 studies, SMD -0.133, 95% CI -0.512 to 0.246, <i>P</i> = 0.429 in POCD) and interleukin (IL)-6 (7 studies, SMD 0.386, 95% CI 0.054 to 0.717, <i>P</i> = 0.022 in POD; 16 studies, SMD 0.089, 95% CI -0.133 to 0.311, <i>P</i> = 0.433 in POCD) concentrations in both POD and POCD patients. We also found that the SMDs of CRP and IL-6 from POCD patients were positively correlated with surgery type in the meta-regression (CRP: Coefficient = 1.555365, <i>P</i> = 0.001, 10 studies; IL-6: Coefficient = -0.6455521, <i>P</i> = 0.086, 16 studies).</p><p>Conclusion</p><p>Available evidence from medium-to-high quality observational studies suggests that POD and POCD are indeed correlated with the concentration of peripheral and cerebrospinal fluid (CSF) inflammatory markers. Some of these markers, such as CRP and IL-6, play roles in both POD and POCD, while others are specific to either one of them.</p></div

Meta-regression between the SMDs of peripheral CRP and IL-6 Levels before surgery and the potential sources of heterogeneity.

<p>Meta-regression between the SMDs of peripheral CRP and IL-6 Levels before surgery and the potential sources of heterogeneity.</p