Mitotic spindle disassembly in human cells relies on CRIPT having hierarchical redox signals.

Swift and complete spindle disassembly in late mitosis is essential for cell survival, yet how it happens is largely unknown in mammalian cells. Here we used real-time live cell microscopy and biochemical assays to show that the primordial dwarfism (PD)-related cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization with a nucleolar concentration during interphase but was distributed to spindles and underwent redox modifications to form disulfide bonds in CXXC pairs during mitosis. Then, it directly interacted with, and might transfer a redox response to, tubulin subunits via a putative redox exchange among cysteine residues to induce microtubule depolymerization. Expression of CRIPT proteins with mutations of these cysteine residues blocked spindle disassembly, generating two cell types with long-lasting metaphase spindles or spindle remnants. Live-cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase might share a common CRIPT-bearing redox-controlled mechanism.

Fluoranthene determination based on a rapid and sensitive syringe extraction and solid-phase fluorescence technique.

A rapid and sensitive strategy was proposed for the detection of fluoranthene (FL), which is a polycyclic aromatic hydrocarbon (PAH), in water samples. In this work, syringe solid-phase extraction (SPE) combined with solid-phase fluorescence spectrometry was used to determine FL in PAHs polluted environmental samples. The fluorescence signals were directly monitored on the membrane surface after FL was enriched by syringe SPE. Under the optimal conditions, the proposed method showed a linear relationship in the concentration range 2-50 µg/L with a correlation coefficient (R2 ) of 0.998, and the limit of detection was 0.143 µg/L. The recoveries varied from 93.47% to 109.81% in the actual samples, with the relative standard deviations (n = 3) ranging from 2.06% to 6.32%. According to the results, the established method can be applied in the field of rapid detection as it is fast, simple, portable, and highly sensitive, and has strong anti-interference.

Toxin inhibition in C. crescentus VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding.

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo.

Clinical significance of traditional clinical parameters and inflammatory biomarkers for the prognosis of patients with spinal chondrosarcoma: a retrospective study of 150 patients in a single center.

BACKGROUND: To investigate the clinical significance of five inflammatory biomarkers and conventional clinical parameters in prognostic prediction of spinal chondrosarcoma. METHODS: Univariate and multivariate analyses were performed to investigate independent prognostic factors for recurrence and death of patients with spinal chondrosarcoma. Disease-free survival (DFS) and overall survival (OS) were estimated by Kaplan-Meier curve, and differences were analyzed by log-rank test. The optimal cutoff values for NLR, PLR, LMR, and CAR were determined by X-tile program. RESULTS: The optimal cutoff value for NLR, PLR, LMR, AGR, and CAR was 2.7, 200, 3.0, 1.5, and 0.2, respectively. Of the 150 patients included, recurrence was detected in 105 patients, and death occurred in 78 patients. Multivariate analysis indicated that Tomita I-III, total resection, and CAR < 0.2 were significantly associated with longer DFS. Meanwhile, preoperative Frankel score D-E, total resection, and CAR < 0.2 were favorable prognostic factors for OS. Subtype analysis showed that only total resection was an independent prognostic factor for DFS of recurrent spinal chondrosarcoma. CONCLUSION: Total resection could significantly reduce the recurrence rate of spinal chondrosarcoma and improve OS of chondrosarcoma patients. Tomita classification I-III was a favorable factor for DFS, and preoperative Frankel score A-C was an adverse prognostic factor for OS. CAR was the most robust prognostic indicator with a discriminatory ability as compared with other inflammatory indicators. These slides can be retrieved under Electronic Supplementary Material.

Development of a novel model for predicting survival of patients with spine metastasis from colorectal cancer.

OBJECTIVE: To develop a novel nomogram for predicting survival of patients with spine metastasis from colorectal cancer (SMCRC) based on the clinical characteristics and prognostic factors. METHODS: Included in this study were 93 SMCRC patients who received treatments in our institute between 2006 and 2017, whose clinical data were analyzed retrospectively by univariate and multivariate analysis to identify independent variables that could predict prognosis. A nomogram for survival prediction was established on the basis of preoperative independent factors, and then subjected to bootstrap re-samples for internal validation. The discrimination was measured by concordance index (C-index). We used ROC analysis with the corresponding AUROC to compare the prediction accuracy of Changzheng Nomogram with three existing prognostic systems (Tomita, Tokuhashi and Bauer). RESULTS: The high and median degrees of primary tumor differentiation, primary tumor surgery, carcinoembryonic antigen ≤ 5 ng/ml, no visceral metastases and ECOG-PS (0-2) were favorable prognostic factors for CRC metastases in the spine. These five preoperative independent factors were identified and entered into the nomogram with the C-index of 0.786 (0.739-0.833). The calibration curves for probability of 12- and 24-month overall survival (OS) showed good agreement between the predictive risk and the actual risk, and calibration was assessed. Compared with the previous prognostic systems, Changzheng Nomogram reported in this study showed higher accuracy in predicting OS of patients with SMCRC spinal metastases (p < 0.05). CONCLUSION: By using this novel predictive model, clinicians could more precisely estimate the survival outcome of individual patients by evaluating clinical characteristics and identify subgroups of patients who are in need of a specific individual treatment strategy. These slides can be retrieved under Electronic Supplementary Material.

Analysis of Mucosa-Associated Microbiota in Colorectal Cancer.

BACKGROUND The aim of this study was to compare the microbiota community structure, assess differences in intestinal bacterial types, and identify metagenomic biomarkers for disparate stages of colorectal cancer formation. MATERIAL AND METHODS A total of 160 individuals were recruited: 61 cases with non-tumor colon were regarded as the normal group, 47 cases with histology-substantiated colorectal adenomas were regarded as the adenoma group, and 52 cases with invasive adenocarcinomas were regarded as the cancer group. Biopsy on the mucosa was performed on each subject. USEARCH was used to process the sequences data and generate OTUs. Gut mucosal microbiota from healthy controls, adenoma patients, and carcinoma patients were analyzed. RESULTS Principal coordinate analysis of unweighted and weighted UniFrac distance showed a separation in composition of microbiota in the 3 groups. Bacteria with potential tumorigenesis, like Bacteroides fragilis and Fusobacterium, were more common in the carcinoma group, while some SCFA (short chain fatty acids) - producing microbes were enriched in the normal group. The commensal Escherichia were more abundant in adenoma patients. CONCLUSIONS Our study provides insights into possible function of gut microbiota in diagnosis and treatment of colorectal cancer. Some bacteria, such as Butyricicoccus, E. coli, and Fusobacterium, can be used as potential biomarkers for normal, adenoma, and cancer groups, respectively.

Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri.

The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg(2+) -dependent ribonuclease and has been shown to target initiator tRNA(fMet) in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892-899. © 2016 Wiley Periodicals, Inc.

Synthesis and antifungal activity of novel triazole compounds containing piperazine moiety.

Design and synthesis of triazole library antifungal agents having piperazine side chains, analogues to fluconazole were documented. The synthesis highlighted utilization of the click chemistry on the basis of the active site of the cytochrome P450 14α-demethylase (CYP51). Their structures were characterized by (1)H-NMR, (13)C-NMR, MS and IR. The influences of piperazine moiety on in vitro antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi.

DOTA functionalized adsorbent DOTA@Sludge@Chitosan derived from recycled shrimp shells and sludge and its application for lead and chromium removal from water.

DOTA@Sludge@Chitosan was synthesized by a facile treatment using DOTA (1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid) to modify dry sludge and chitosan in an acidic solution. The performance of developed DOTA@Sludge@Chitosan was investigated for the adsorptive removal of Cr6+ and Pb2+ from water. Characterization studies showed that the materials possess a large surface area (52.009 m2/g), pore volume (0.069 cm3/g), and abundant functional groups of amino and hydroxyl. The prepared material showed a synergetic effect due to carboxylic acid and sludge, effectively removing Cr6+ and Pb2+. It reached 329.4 mg/g (Pb2+) and 273.3 mg/g (Cr6+) at 20 °C, much higher than commercial activated carbon. The regeneration of the adsorbent was tested for six adsorption and desorption cycles. The results demonstrate that the DOTA@Sludge@Chitosan adsorbent well-maintained high adsorption capacity attributed to its stability, making it a promising adsorbent for heavy metals removal from industrial effluent.

Protein expression, crystallization and preliminary X-ray crystallographic analysis of the isolated Shigella flexneri VapC toxin.

Upon release from the stable complex formed with its antitoxin VapB, the toxin VapC (MvpT) of the Gram-negative pathogen Shigella flexneri is capable of globally down-regulating translation by specifically cleaving initiator tRNA(fMet) in the anticodon region. Recombinant Shigella flexneri VapC(D7A) harbouring an active-site mutation was overexpressed in Escherichia coli, purified to homogeneity and crystallized by the vapour-diffusion technique. A preliminary X-ray crystallographic analysis shows that the crystals diffracted to at least 1.9 Å resolution at a synchrotron X-ray source and belonged to the trigonal space group in the hexagonal setting, H3, with unit-cell parameters a = b = 120.1, c = 52.5 Å, α = ß = 90, γ = 120°. The Matthews coefficient is 2.46 Å(3) Da(-1), suggesting two molecules per asymmetric unit and corresponding to a solvent content of 50.0%.

Complete genome sequencing and comparative genomic analyses of a new spotted-fever Rickettsia heilongjiangensis strain B8.

Rickettsia heilongjiangensis, a tick-borne obligate intracellular bacterium and causative agent of spotted fever in China, has attracted increasing concern regarding its capability in causing human rickettsiosis. Here, we conducted a genomic analysis of a new R. heilongjiangensis strain B8 (B8) isolated from the serum of a patient who had been bitten by a Haemaphysalis longicornis tick in Anhui Province, China. The present study sought to identify exclusive genes that might be associated with the pathogenicity of B8 using comparative genomics. Specifically, the sequences of B8 were assembled into one circular chromosome of 1,275,081 bp and predicted to contain 1447 genes. Comparative genome analyses were performed based on the genome of B8 and 28 spotted fever group (SFG) rickettsial genomes deposited in NCBI. Phylogenomic analyses indicated the B8 strain was clustered within the R. heilongjiangensis species; however, a sum of 112 and 119 B8-unique genes was identified when compared with R. heilongjiangensis and R. japonica strains, respectively. Functional annotation analyses revealed that these B8-unique genes were mainly annotated to defence mechanisms, lipid transport and metabolism, cell wall/membrane/envelope biogenesis. These data indicate B8 rather represents a previously undescribed human-pathogenic SFG rickettsia lineage, which may be an intermediate lineage of R. heilongjiangensis and R. japonica. Overall, this study isolated a new strain of R. heilongjiangensis in East-Central China for the first time, and provided potential B8-unique genetic loci that could be used for the discrimination of B8 from other R. heilongjiangensis and closely related SFG Rickettsial strains.

Efficient adsorption of heavy metals from wastewater on nanocomposite beads prepared by chitosan and paper sludge.

Chitosan was commonly used with inorganic salt and organic compounds to prepare adsorption material for water treatment. Different materials were mixed for the preparation, leading to a high cost for water treatment. Sludge from papermaking has abundant fiber and inorganic salt, which can reduce the addition of raw materials in preparing the adsorption material and thus lower the cost. This work used recycled industrial paper sludge to prepare adsorption material to remove heavy metals from wastewater. The adsorption properties of the prepared sludge-chitosan material for Cu2+ and Cr3+ in wastewater were investigated. The impacts of adsorption time, pH, and initial concentrations of Cu2+ and Cr3+ on adsorption amount were studied and optimized. The saturated adsorption capacity of sludge-chitosan material for Cu2+ and Cr3+ can reach 114.6 and 110.3 mg/g. The adsorption kinetics satisfied the pseudo-second-order model, indicating two modes, physical diffusion, and chem-sorption, in the heavy metal adsorption by the sludge-chitosan materials. Physical distribution has little Effect on chemical adsorption. The materials can be applied to treating Cu2+ and Cr3+ containing wastewater with the proposed cheap and readily available sludge-chitosan material. The results confirmed that sludge-chitosan material possessed good regeneration performance and was an ideal adsorbent.

Erratum: Metformin induces cell cycle arrest, apoptosis and autophagy through ROS/JNK signaling pathway in human osteosarcoma: Erratum.

[This corrects the article DOI: 10.7150/ijbs.33787.].

Rapid determination of trace Cu2+ by an in-syringe membrane SPE and membrane solid-phase spectral technique.

A new in-syringe membrane SPE and solid-phase visible spectral method was proposed for the rapid extraction and visible spectral determination of trace Cu2+. The chelation and membrane SPE can be accomplished in a syringe. The yellow Cu(DDTC)2 complex was separated using a polyethersulfone membrane from the sample solution. Then, the complex can be detected directly on the polyethersulfone membrane utilizing solid-phase visible absorbance spectra without elution. The proposed method simplified the experimental procedure and improved the sensitivity to the µg L-1 level. Furthermore, this method is environmentally friendly since it avoids the use of organic solvents. After the investigation of the influence of different variables on the membrane SPE procedure, water and blood plasma were analyzed to validate the proposed method. A LOD of 0.04 µg L-1 and recoveries of 96.0-103.7% confirmed that the present work can be applied for the determination of trace Cu2+ in water and blood plasma samples.

Flash nanoprecipitation with Gd(III)-based metallosurfactants to fabricate polylactic acid nanoparticles as highly efficient contrast agents for magnetic resonance imaging.

Polylactic acid (PLA) nanoparticles coated with Gd(III)-based metallosurfactants (MS) are prepared using a simple and rapid one-step method, flash nanoprecipitation (FNP), for magnetic resonance imaging (MRI) applications. By co-assembling the Gd(III)-based MS and an amphiphilic polymer, methoxy poly(ethylene glycol)-b-poly(ϵ-caprolactone) (mPEG-b-PCL), PLA cores were rapidly encapsulated to form biocompatible T1 contrast agents with tunable particle size and narrow size distribution. The hydrophobic property of Gd(III)-based MS were finely tuned to achieve their high loading efficiency. The size of the nanoparticles was easily controlled by tuning the stream velocity, Reynolds number and the amount of the amphiphilic block copolymer during the FNP process. Under the optimized condition, the relaxivity of the nanoparticles was achieved up to 35.39 mM-1 s-1 (at 1.5 T), which is over 8 times of clinically used MRI contrast agents, demonstrating the potential application for MR imaging.

Gadolinium complexes of macrocyclic diethylenetriamine-N-oxide pentaacetic acid-bisamide as highly stable MRI contrast agents with high relaxivity.

Gadolinium(iii) complexes are generally considered efficient magnetic resonance imaging (MRI) contrast agents (CAs) and widely used in clinical applications. High relaxivity and stability are two essential criteria for a Gd(iii)-complex to be used as a MRI-CA. One crucial strategy to achieve high relaxivity for small molecular Gd(iii)-based MRI contrast agents is to increase the hydration number q. Meanwhile, metal complexes with macrocyclic ligands have been proved to inherit high thermodynamic stability and kinetic inertness. Herein, a series of macrocyclic ligands based on diethylenetriamine-N-oxide pentaacetic acid-bisamide were synthesized. Among them, cyclo-DTPA-NO-C6O2 (3d) was the strongest ligand for Gd(iii) as confirmed by experimental results. The hydration number of the Gd-cyclo-DTPA-NO-C6O2 (4d) complex was characterized by luminescence measurements to be 3 and the coordination structure was confirmed with computational simulations. Consequently, the relaxivity of this complex (14.3 mM-1 s-1, 1.5 T, 25 °C) is about triple that of commercial MRI CAs. The conditional stability constant of the Gd(iii) complex, pGd, calculated from spectrophotometric titration studies, was comparable to that of one of the most stable commercial MRI-CAs, Gd-DTPA (Magnevist®). Meanwhile, the kinetic inertness of the complex was even higher than that of Gd-DTPA thanks to its macrocyclic coordination structure.

Prognostic Significance of Preoperative Inflammatory Biomarkers and Traditional Clinical Parameters in Patients with Spinal Metastasis from Clear Cell Renal Cell Carcinoma: A Retrospective Study of 95 Patients in a Single Center.

PURPOSE: The purpose of this retrospective study was to identify preoperative inflammatory biomarkers and clinical parameters and evaluate their prognostic significance in patients with spinal metastasis from clear cell renal cell carcinoma (CCRCC). PATIENTS AND METHODS: Correlations of overall survival (OS) with traditional clinical parameters and inflammatory indicators including the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), albumin-globulin ratio (AGR), and C-reactive protein to albumin ratio (CRP/Alb ratio) were analyzed in 95 patients with spinal metastasis from CCRCA using the Kaplan-Meier method to identify potential prognostic factors. Factors with P values ≤ 0.1 were subjected to multivariate analysis by Cox regression analysis. P values ≤ 0.05 were considered statistically significant. RESULTS: The 95 patients included in this study were followed up by a mean of 48.8 months (median 51 months; range 6-132 months), during which 21 patients died, with a death rate of 22.1%. The statistical results indicated that patients with total piecemeal spondylectomy (TPS), targeted therapy, NLR < 3.8 and PLR < 206.9 had a significantly longer OS rate. CONCLUSION: TPS and targeted therapy could significantly prolong the OS of patients with spinal metastasis from CCRCC. In addition, NLR and PLR are robust and convenient prognostic indicators that have a discriminatory ability superior to other inflammatory biomarkers.

Metformin induces cell cycle arrest, apoptosis and autophagy through ROS/JNK signaling pathway in human osteosarcoma.

Metformin, an ancient drug commonly used for treating type II diabetes, has been associated to anti-cancer capacity in a variety of developing cancers, though the mechanism remains elusive. Here, we aimed to examine the inhibitory effect of metformin in osteosarcoma. Herein, we demonstrated that metformin treatment blocked proliferation progression by causing accumulation of G2/M phase in U2OS and 143B cells. Furthermore, metformin treatment triggered programmed cell death process in osteosarcoma cell lines. Further research indicated the induction of apoptosis and autophagy triggered by metformin could remarkably attenuate after the treatment of ROS scavenger NAC and JNK inhibitor SP600125. Additionally, our results showed that NAC-suppressed JNK/c-Jun signaling pathway could have been activated through metformin treatment. Lastly, metformin could inhibit osteosarcoma growth under safe dose in vivo. Thus, we propose that metformin could induce cell cycle arrest as well as programmed cell death, including apoptosis and autophagy, through ROS-dependent JNK/c-Jun cascade in human osteosarcoma. This metformin-induced pathway provides further insights into its antitumor potential molecular mechanism and illuminates potential cancer targets for osteosarcoma.

The antifungal activity of essential oil from Melaleuca leucadendra (L.) L. grown in China and its synergistic effects with conventional antibiotics against Candida.

To identify the natural antifungal agents, the antifungal activities of Melaleuca leucadendra (L.) L. essential oil (ML-EO) from Fujian Province of China were assayed. Treatment of ML-EO in combination with the front-line using antibiotics against Candida led to synergistic effects. Electron microscopy analysis on the oil treated C. albicans cells revealed the formation of mesosome-like structures, suggesting well the membrane damage caused by the essential oil. The Griess assay by monitoring NO production in LPS-stimulated RAW264.7 cells indicated the potent anti-inflammatory activity of ML-EO. In comparison with the marked essential oil of Melaleuca alternifolia EO, ML-EO had almost the same chemical components. In general, the antifungal activity of ML-EO and its synergistic interactions with conventional antibiotics were able to lead the development of new treatment strategies on Candida infection.

N-Benzylanilines as Fatty Acid Synthesis Inhibitors against Biofilm-related Methicillin-resistant Staphylococcus aureus.

Bacterial fatty acid synthase system is a well validated target for the development of novel antimicrobial agents. This study reports the synthesis of Schiff bases and their reductive N-benzylanilines. Most N-benzylanilines were active against Gram-positive bacteria, among which compound 4k performed best against both S. aureus and MRSA with the MIC value at 0.5 mg/L. Moreover, we identified the strong antibacterial activity for compound 4k against 19 clinical MRSA strains isolated from different specimen, which indicated its potential in clinical application. In vitro biofilm inhibition and microscopy assay revealed compound 4k inhibits biofilm formation and eradicates preformed biofilm effectively. The size-exclusion chromatography and docking study indicated that compound 4k mimics the binding mode of triclosan with saFabI. The efficiency of the protein-inhibitor interaction was evaluated by measuring NADPH reduction using trans-2-octenoyl-CoA as substrate. Overall, our data demonstrate that N-benzylaniline is a promising scaffold for anti-staphylococcal drug development.