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Identification of the molecular composition of the cargo transported by individual kinesin motors is critical to an understanding of both motor function and regulation of the proper intracellular placement of numerous cellular components including proteins, RNA, and organelles. In this chapter, we describe methods to identify the motor tail sequences responsible for cargo binding by expression of green fluorescent protein (GFP)-motor tail fusion proteins in mammalian cells. In addition, we detail two complementary approaches to identify specific proteins associated with these targeting sequences: a yeast 2-hybrid screen and affinity chromatography.

Temporin-GHa (GHa) was cloned from Hylarana guentheri, showing a weak antimicrobial activity. In order to improve its bactericidal efficacy, GHaR6R, GHaR7R, GHaR8R and GHaR9W were designed and synthesized. Compared to the parent peptide, the GHa-derived peptides show potent antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA), which is the main pathogen with high morbidity and mortality that causes various infections in humans. These peptides exert bactericidal actions on MRSA by permeabilizing the cytoplasmic membranes and damaging membrane integrity. All of the four peptides exhibit excellent stability under harsh conditions, including extreme temperature and salts. Furthermore, they inhibit the formation of biofilm and eradicate mature biofilm of MRSA. The GHa-derived peptides decrease bacterial surface hydrophobicity, autoaggregation and polysaccharide intercellular adhesion synthesis in concentration-dependent manner. Real-time quantitative reverse transcription PCR analysis revealed that the peptides downregulate the expression of adhesion genes icaADBC involved in biofilm formation. Except for GHaR7R, the other three peptides have low hemolytic toxicity against human erythrocytes. In the presence of human erythrocytes, GHaR7R, GHaR8R and GHaR9W interact with MRSA preferentially. GHaR6R, GHaR8R and GHaR9W show less toxicity toward normal cells HL-7702 and hFOB1.19. These results suggest that the GHa-derived peptides may be promising antimicrobial candidates against MRSA infections.

Histone H3K27 demethylase JMJD3 plays a critical role in gene expression and T cell differentiation. However, the role and mechanisms of JMJD3 in T cell trafficking remain poorly understood. Here, we show that JMJD3 deficiency in [CD4.sup.+] T cells resulted in an accumulation of T cells in the thymus and reduction of T cell number in the secondary lymphoid organs. We identified PDLIM4 as a significantly downregulated target gene in JMJD3- deficient [CD4.sup.+] T cells by gene profiling and ChIP-Seq analyses. We further showed that PDLIM4 functioned as an adaptor protein to interact with sphingosine-1 phosphate receptor 1 (S1P1) and filamentous actin (F-actin), thus serving as a key regulator of T cell trafficking. Mechanistically, JMJD3 bound to the promoter and gene-body regions of the Pdlim4 gene and regulated its expression by interacting with zinc finger transcription factor KLF2. Our findings have identified Pdlim4 as a JMJD3 target gene that affects T cell trafficking by cooperating with S1P1 and have provided insights into the molecular mechanisms by which JMJD3 regulates genes involved in T cell trafficking., Introduction T cell development in the thymus is a multistep process. Early thymic progenitor cells (TPCs) differentiate into T cell receptor-expressing (TCR-expressing) [CD4.sup.+][CD8.sup.+] double-positive (DP) thymocytes in the cortex and [...]

Background: To investigate the relationship between lactate dehydrogenase and apolipoprotein A1 levels and the condition and prognosis of patients with severe pneumonia. Methods: Data was collected from 204 patients with severe pneumonia who were hospitalized from January 1, 2019 to December 1, 2021 in Zhaotong First People's Hospital (respiratory intensive care unit (RICU)), and divided into survival group (160 patients) and death group (44 patients) according to their hospitalization outcome. The relationship between lactate dehydrogenase and apolipoprotein A1 levels and general information, disease, and treatment needs of patients with severe pneumonia was analyzed, and lactate dehydrogenase, apolipoprotein A1, neutrophil-to-lymphocyte ratio, hematocrit, C-reactive protein, calcitoninogen, D-dimer, Acute Physiology and Chronic Health Status Rating System II, and Pneumonia Severity Index scores were compared between the survival and death groups. The value of these indicators in determining the prognosis of patients was analyzed using subject operating characteristic (ROC) curves. Logistic regression was used to analyze the risk factors for death from severe pneumonia. Results: The age and pneumonia type differed significantly between the two groups (P0.05). LDH (Lactate Dehydrogenase) and ApoA1 (Apolipoprotein A1) levels showed significant differences among different age groups with severe pneumonia (P0.05). LDH and ApoA1 levels were higher in severe pneumonia patients with acute exacerbation or MODS (multiple organ dysfunction syndrome), compared to those without (P0.05). LDH and ApoA1, when combined, had a higher predictive value for severe pneumonia mortality (Area Under the Curve, AUC=0.873, P289 U/mL and ApoA1

Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is a RNA helicase required for the translation of 5’ structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5’ structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity. [ABSTRACT FROM AUTHOR]

Many organisms, including the fathead minnow (Pimephales promelas), a toxicological model organism, establish social hierarchies. The social rank of each male in a population is under the control of the hypothalamic-pituitary-gonadal (HPG) axis mainly through regulation of circulating androgen concentrations, which in turn drive the expression of secondary sex characteristics (SSCs). As dominant and subordinate males in an exposure study are initially under different physiological conditions (i.e., differing plasma androgen concentrations), we proposed that they belong to different subpopulations in the context of exposure to compounds that may interact with the HPG axis. Using a meta-analysis of our data from several previously published studies, we corroborated the hypothesis that social status, as indicated by SSCs, results in distinct clusters (eigenvalues >0.8 explaining >80% of variability) with differential expression of plasma vitellogenin, a commonly used biomarker of exposure to contaminants of emerging concern (CEC). Furthermore, we confirmed our predictions that exposure to estrogenic CECs would homogenize plasma vitellogenin response (E1: cluster mean SSC values decreased to 4.33 and 4.86 relative to those of control; E2: decreased to 4.8 and 5.37) across the social hierarchy. In contrast, serotonin-specific reuptake inhibitors expand this response range (cluster mean SSC increased to 5.21 and 6.5 relative to those of control). Our results demonstrated that social hierarchies in male fathead minnows result in heterogeneous responses to chemical exposure. These results represent a cautionary note for the experimental design of single-sex exposure studies. We anticipate our study to be a starting point for the re-evaluation of toxicological data analyses in single sex exposure experiments. [ABSTRACT FROM AUTHOR]

We tested the hypotheses that supplementation of a diet with elemental Mg increases ruminal dissolved H2 (dH2) in rumen fluid, which in turn alters rumen fermentation and microbial community in goats. In a randomised block design, twenty growing goats were allocated to two treatments fed the same basal diet with 1·45 % Mg(OH)2 or 0·6 % elemental Mg. After 28 d of adaptation, we collected total faeces to measure total tract digestibility, rumen contents to analyse fermentation end products and microbial groups, and measured methane (CH4) emission using respiration chambers. Ruminal Mg2+ concentration was similar in both treatments. Elemental Mg supplementation increased dH2 at 2·5 h post morning feeding (+180 %, P<0·001). Elemental Mg supplementation decreased total volatile fatty acid concentration (−8·6 %, P<0·001), the acetate:propionate ratio (−11·8 %, P<0·03) and fungal copy numbers (−63·6 %, P=0·006), and increased propionate molar percentage (+11·6 %, P<0·001), methanogen copy numbers (+47·9 %, P<0·001), dissolved CH4 (+35·6 %, P<0·001) and CH4 emissions (+11·7 %, P=0·03), compared with Mg(OH)2 supplementation. The bacterial community composition in both treatments was overall similar. Ruminal dH2 was negatively correlated with acetate molar percentage and fungal copy numbers (P<0·05), and positively correlated with propionate molar percentage and methanogen copy numbers (P<0·05). In summary, elemental Mg supplementation increased ruminal dH2 concentration, which inhibited rumen fermentation, enhanced methanogenesis and seemed to shift fermentation pathways from acetate to propionate, and altered microbiota by decreasing fungi and increasing methanogens. [ABSTRACT FROM AUTHOR]

The pine aphid Cinara pinitabulaeformis Zhang et Zhang is the main pine pest in China, it causes pine needles to produce dense dew (honeydew) which can lead to sooty mold (black filamentous saprophytic ascomycetes). Although common chemical and physical strategies are used to prevent the disease caused by C. pinitabulaeformis Zhang et Zhang, new strategies based on biological and/or genetic approaches are promising to control and eradicate the disease. However, there is no information about genomics, proteomics or transcriptomics to allow the design of new control strategies for this pine aphid. We used next generation sequencing technology to sequence the transcriptome of C. pinitabulaeformis Zhang et Zhang and built a transcriptome database. We identified 80,259 unigenes assigned for Gene Ontology (GO) terms and information for a total of 11,609 classified unigenes was obtained in the Clusters of Orthologous Groups (COGs). A total of 10,806 annotated unigenes were analyzed to identify the represented biological pathways, among them 8,845 unigenes matched with 228 KEGG pathways. In addition, our data describe propagative viruses, nutrition-related genes, detoxification related molecules, olfactory related receptors, stressed-related protein, putative insecticide resistance genes and possible insecticide targets. Moreover, this study provides valuable information about putative insecticide resistance related genes and for the design of new genetic/biological based strategies to manage and control C. pinitabulaeformis Zhang et Zhang populations. [ABSTRACT FROM AUTHOR]

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins. [ABSTRACT FROM AUTHOR]

Eomesodermin (Eomes) is a T-box transcription factor that has been implicated in the etiology of colorectal cancer and other human malignancies. We screened a panel of human primary colon cancers and patient-matched controls (n = 30) and detected Eomes overexpression at the mRNA and protein level. Similar results were obtained in a panel of rat colon tumors and adjacent normal-looking colonic mucosa (n = 24). In human colon cancer cells, forced overexpression of Eomes enhanced cell viability and protected against staurosporine-induced apoptosis. On the other hand, knocking down Eomes resulted in reduced cell viability, G2/M cell cycle arrest, and apoptosis induction. The apoptotic mechanism centered on the reciprocal downregulation of anti-apoptotic BIRC5 (Survivin) and upregulation of proapoptotic Bcl-2 modifying factor (BMF). In patients with colorectal cancer, high EOMES expression (n = 95) was associated with poor overall survival compared with individuals exhibiting low EOMES levels (n = 80). We conclude from the current investigation, and prior literature, that Eomes has a divergent role in cancer development, with evidence for tumor suppressor and oncogenic functions, depending on stage and tissue context. Further studies are warranted on the apoptotic mechanisms linked to the reciprocal regulation of BMF and BIRC5 in human colorectal cancers characterized by Eomes overexpression. [ABSTRACT FROM AUTHOR]

During eating, the teeth usually endure the sharply temperature changes because of different foods. It is of importance to investigate the heat transfer and heat dissipation behavior of the dentino–enamel junction (DEJ) of human tooth since dentine and enamel have different thermophysical properties. The spatial and temporal temperature distributions on the enamel, dentine, and pulpal chamber of both the human tooth and its discontinuous boundaries, were measured using infrared thermography using a stepped temperature increase on the outer boundary of enamel crowns. The thermal diffusivities for enamel and dentine were deduced from the time dependent temperature change at the enamel and dentine layers. The thermal conductivities for enamel and dentine were calculated to be 0.81 Wm-1K-1 and 0.48 Wm-1K-1 respectively. The observed temperature discontinuities across the interfaces between enamel, dentine and pulp-chamber layers were due to the difference of thermal conductivities at interfaces rather than to the phase transformation. The temperature gradient distributes continuously across the enamel and dentine layers and their junction below a temperature of 42°C, whilst a negative thermal resistance is observed at interfaces above 42°C. These results suggest that the microstructure of the dentin-enamel junction (DEJ) junction play an important role in tooth heat transfer and protects the pulp from heat damage. [ABSTRACT FROM AUTHOR]

Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C) and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme. [ABSTRACT FROM AUTHOR]

N-glycans play important roles in various pathophysiological processes and can be used as clinical diagnosis markers. However, plasma N-glycans change and their pathophysiological significance in the setting of hypercholesterolemia, a major risk factor for atherosclerosis, is unknown. Here, we collected plasma from both hypercholesterolemic patients and cholesterol-fed hypercholesterolemic rabbits, and determined the changes in the whole-plasma N-glycan profile by electrospray ionization mass spectrometry. We found that both the hypercholesterolemic patients and rabbits showed a dramatic change in their plasma glycan profile. Compared with healthy subjects, the hypercholesterolemic patients exhibited higher plasma levels of a cluster of high-mannose and complex/hybrid N-glycans (mainly including undecorated or sialylated glycans), whereas only a few fucosylated or fucosylated and sialylated N-glycans were increased. Additionally, cholesterol-fed hypercholesterolemic rabbits also displayed increased plasma levels of high-mannose in addition to high complex/hybrid N-glycan levels. The whole-plasma glycan profiles revealed that the plasma N-glycan levels were correlated with the plasma cholesterol levels, implying that N-glycans may be a target for treatment of hypercholesterolemia. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3'-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma. [ABSTRACT FROM AUTHOR]

With the use of iTRAQ technique, a multifactorial comparative proteomic study can be performed. In this study, to obtain an overview of ethanol, CYP2E1 and gender effects on liver injury and gain more insight into the underlying molecular mechanism, mouse liver proteomes were quantitatively analyzed using iTRAQ under eight conditions including mice of different genders, wild type versus CYP2E1 knockout, and normal versus alcohol diet. A series of statistical and bioinformatic analyses were explored to simplify and clarify multifactorial comparative proteomic data. First, with the Principle Component analysis, six proteins, CYP2E1, FAM25, CA3, BHMT, HIBADH and ECHS1, involved in oxidation reduction, energy and lipid metabolism and amino acid metabolism, were identified as the most differentially expressed gene products across all of the experimental conditions of our chronic alcoholism model. Second, hierarchical clustering analysis showed CYP2E1 knockout played a primary role in the overall differential protein expression compared with ethanol and gender factors. Furthermore, pair-wise multiple comparisons have revealed that the only significant expression difference lied in wild-type and CYP2E1 knockout mice both treated with ethanol. Third, K-mean clustering analysis indicated that the CYP2E1 knockout had the reverse effect on ethanol induced oxidative stress and lipid oxidation. More importantly, IPA analysis of proteomic data inferred that the gene expressions of two upstream regulators, NRF2 and PPARα, regulated by chronic alcohol feeding and CYP2E1 knockout, are involved in ethanol induced oxidative stress and lipid oxidation. The present study provides an effectively comprehensive data analysis strategy to compare multiple biological factors, contributing to biochemical effects of alcohol on the liver. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with data set identifier of PXD000635. [ABSTRACT FROM AUTHOR]

Recent studies indicate that soluble β-amyloid (sAβ) oligomers, rather than their fibrillar aggregates, contribute to the pathogenesis of Alzheimer's disease (AD), though the mechanisms of their neurotoxicity are still elusive. Here, we demonstrate that sAβ derived from 7PA2 cells exert a much stronger effect on the regulation of a set of functionally validated microRNAs (miRNAs) in primary cultured neurons than the synthetic insoluble Aβ fibrils (fAβ). Synthetic sAβ peptides at a higher concentration present comparable effect on these miRNAs in our neuronal model. Further, the sAβ-induced miR-134, miR-145 and miR-210 expressions are fully reversed by two selective N-methyl-d-aspartate (NMDA) receptor inhibitors, but are neither reversed by insulin nor by forskolin, suggesting an NMDA receptor-dependent, rather than PI3K/AKT or PKA/CREB signaling dependent regulatory mechanism. In addition, the repression of miR-107 expression by the sAβ containing 7PA2 CM is likely involved multiple mechanisms and multiple players including NMDA receptor, N-terminally truncated Aβ and reactive oxygen species (ROS). [ABSTRACT FROM AUTHOR]

DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α)8 barrel fold of the amidohydrolase superfamily and function as dimers. A Zn2+ is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals. [ABSTRACT FROM AUTHOR]

Objective: The α7 nicotinic acetylcholine receptors (nAChRs) play a vital role in the pathophysiology of neuropsychiatric diseases such as Alzheimer’s disease and depression. However, there is currently no suitable positron emission tomography (PET) or Single-Photon Emission Computed Tomography (SPECT) radioligands for imaging α7 nAChRs in brain. Here our aim is to radiosynthesize a novel SPECT radioligand 131I-CHIBA-1001 for whole body biodistribution study and in vivo imaging of α7 nAChRs in brain. Method: 131I-CHIBA-1001 was radiosynthesized by chloramine-T method. Different conditions of reaction time and temperature were tested to get a better radiolabeling yield. Radiolabeling yield and radiochemical purities of 131I-CHIBA-1001 were analyzed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) system. Whole body biodistribution study was performed at different time points post injection of 131I-CHIBA-1001 in KM mice. Monkey subject was used for in vivo SPECT imaging in brain. Result: The radiolabeling yield of 131I-CHIBA-1001 reached 96% within 1.5∼2.0 h at 90∼95°C. The radiochemical purity reached more than 99% after HPLC purification. 131I-CHIBA-1001 was highly stable in saline and fresh human serum in room temperature and 37°C separately. The biodistribution data of brain at 15, 30, and 60 min were 11.05±1.04%ID/g, 8.8±0.04%ID/g and 6.28±1.13%ID/g, respectively. In experimental SPECT imaging, the distribution of radioactivity in the brain regions was paralleled with the distribution of α7 nAChRs in the monkey brain. Moreover, in the blocking SPECT imaging study, the selective α7 nAChR agonist SSR180711 blocked the radioactive uptake in the brain successfully. Conclusion: The CHIBA-1001 can be successfully radiolabeled with 131I using the chloramine-T method. 131I-CHIBA-1001 can successfully accumulate in the monkey brain and image the α7 acetylcholine receptors. 131I-CHIBA-1001 can be a candidate for imagingα7 acetylcholine receptors, which will be of great value for the diagnosis and treatment of mental diseases. [ABSTRACT FROM AUTHOR]

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix which mediate inhibition of axonal regeneration after injury to the central nervous system (CNS). Several neuronal receptors for CSPGs have recently been identified; however, the signaling pathways by which CSPGs restrict axonal growth are still largely unknown. In this study, we applied quantitative phosphoproteomics to investigate the global changes in protein phosphorylation induced by CSPGs in primary neurons. In combination with isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling, strong cation exchange chromatography (SCX) fractionation, immobilized metal affinity chromatography (IMAC) and LC-MS/MS, we identified and quantified 2214 unique phosphopeptides corresponding to 1118 phosphoproteins, with 118 changing significantly in abundance with CSPG treatment. The proteins that were regulated by CSPGs included key components of synaptic vesicle trafficking, axon guidance mediated by semaphorins, integrin signaling, cadherin signaling and EGF receptor signaling pathways. A significant number of the regulated proteins are cytoskeletal and related proteins that have been implicated in regulating neurite growth. Another highly represented protein category regulated by CSPGs is nucleic acid binding proteins involved in RNA post-transcriptional regulation. Together, by screening the overall phosphoproteome changes induced by CSPGs, this data expand our understanding of CSPG signaling, which provides new insights into development of strategies for overcoming CSPG inhibition and promoting axonal regeneration after CNS injury. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The disease is characterized by various cytogenetic and molecular abnormalities with distinct prognoses and gene expression profiles. Emerging evidence has suggested that circulating microRNAs (miRNAs) could serve as noninvasive biomarkers for cancer detection; however, little is known about circulating miRNA profiles in AML patients. In this study, a genome-wide serum miRNA expression analysis was performed using Solexa sequencing for initial screen, followed by validation with real-time PCR assays. The analysis was conducted on training and verification sets of serum samples from 140 newly diagnosed AML patients and 135 normal adult donors. After a two-phase selection and validation process, 6 miRNAs, miR-10a-5p, miR-93-5p, miR-129-5p, miR-155-5p, miR-181b-5p and miR-320d, were found to have significantly different expression levels in AML compared with control serum samples. Furthermore, unsupervised clustering analysis revealed the remarkable ability of the 6-miRNA profile to differentiate between AML patients and normal controls. The areas under the ROC curve for the selected miRNAs ranged from 0.8129 to 0.9531. More importantly, miR-181b-5p levels in serum were significantly associated with overall survival. These data demonstrated that the expression patterns of circulating miRNAs were systematically altered in AML and miR-181b-5p may serve as a predictor for overall survival in AML patients. [ABSTRACT FROM AUTHOR]

Presents the findings of a study by the authors on the role of the carbonyl group in thioester chain length recognition by the medium chain acyl-CoA dehydrogenase from pig kidney. Experimental materials used and methodology adopted; Results; Discussion.

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021–7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR-type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric-acid-positive material (3-deoxy-D-manno-octulosonic acid equivalents) and four neutral sugars: glucose, galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose. Phosphate, glucosamine, glycine, and the fatty acids. 3-hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6-glucosamine disaccharide esterified at C4′ by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non-reducing glucosamine were both acylated by 3-myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3-hydroxymyristic acid. Hydroxyl groups at C4 and C6′ in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS: its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine-sensitized mice, comparable to that of Salmonella. [ABSTRACT FROM AUTHOR]

Abstract: Dimethoate is a widely used organophosphorus insecticide often detected in the environment. A highly effective dimethoate-degrading Paracoccus sp. strain Lgjj-3 was isolated from treatment wastewater. Strain Lgjj-3 can utilize dimethoate as its sole carbon source for growth and degrade an initial concentration of 100 mg l−1 dimethoate to non-detectable levels within 6 h in liquid culture. During the degradation of dimethoate, seven metabolites, including dimethoate carboxylic acid, 2-(hydroxy(methoxy)phosphorylthio)acetic acid, O,O,S-trimethyl thiophosphorothioate, O-methyl O,S-dihydrogen phosphorothioate, phosphorothioic O,O,S-acid, O,O,S-trimethylphosphorothiate and O,O,O-trimethyl phosphoric ester, were successfully detected and identified based on MS/MS and GC–MS analysis. A biochemical degradation pathway of dimethoate by Paracoccus sp. Lgjj-3 is proposed for the first time. Dimethoate is first hydrolyzed by the scission of the amide bond to form dimethoate carboxylic acid, which is subsequently decarboxylated into O,O,S-trimethyl thiophosphorothioate. The produced O,O,S-trimethyl thiophosphorothioate is oxidized to form O,O,S-trimethylphosphorothiate and then further oxidized to form O,O,O-trimethyl phosphoric ester. O,O,S-trimethylphosphorothiate can also be hydrolyzed at the C–O bond to release a CH3 group and cleavaged at the S–C bond through a hydrolytic pathway to form O-methyl O,S-dihydrogen phosphorothioate. Dimethoate carboxylic acid can also be oxidated at the P form the corresponding oxon derivative. Next, hydrolysis can occur at the C–O bond to form 2-(hydroxy(methoxy)phosphorylthio)acetic acid, which can be further hydrolyzed at the S–C bond to form O-methyl O,S-dihydrogen phosphorothioate. Phosphorothioic O,O,S-acid is finally formed by the hydrolysis of the C–O bond of O-methyl O,S-dihydrogen phosphorothioate to lose another CH3 group. [Copyright &y& Elsevier]

Abstract: The interaction between bovine hemoglobin (BHb) and TiO2 colloid was investigated by UV/vis absorption, UV/vis diffuse reflectance spectrum, IR, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. TiO2 effectively quenched the intrinsic fluorescence of BHb via static quenching. The process of binding TiO2 on BHb was a spontaneous molecular interaction procedure. The thermodynamic parameters, ΔH° and ΔS° were estimated to be −78.07kJmol−1, −110.93Jmol−1 K−1 according to the van’ Hoff equation. This indicates that the van der Waals and hydrogen bonds interactions played a major role in stabilizing the TiO2–BHb complex. The effect of TiO2 on the conformation of BHb was analyzed using synchronous fluorescence spectroscopy. [Copyright &y& Elsevier]

Objective: To explore the possibility that local injury to the endometrium in controlled ovarian hyperstimulation cycle improves the incidence of embryo implantation and to analyze the gene expression profile in the endometria of pregnant and nonpregnant patients in in vitro fertilization/embryo transfer (IVF-ET).Design: Prospective study.Setting: A clinical assisted reproductive center of a university hospital.Patient(s): Women undergoing fresh IVF-ET cycles (n = 121), treated with a long protocol for controlled ovarian hyperstimulation, whose endometrium were diagnosed by B-ultrasound showing irregular echo.Intervention(s): Local injury to the endometrium of 60 patients in controlled ovarian hyperstimulation cycle, who were randomly selected from a total of 121 patients. Seven endometrial biopsies samples from day 10 were analyzed by Affymetrix U133 plus 2.0 gene chip.Main Outcome Measure(s): Outcomes of IVF-ET and gene expression assayed by gene chip technology.Result(s): Transfer of the same number of embryos (135 in the experimental and control patients, respectively) resulted in rates of implantation (33.33% vs. 17.78%), clinical pregnancy (48.33% vs. 27.86%), and ongoing or live births per ET (41.67% vs. 22.96%) that were higher in the experimental group compared with controls. Statistically significant differences of the expression level of 218 genes (41 up-regulated and 177 down-regulated) were detected in the endometrial biopsy samples from clinical pregnant patients and nonpregnant patients.Conclusion(s): The results suggested local injury to the endometrium during a COH cycle improved the rates of embryo implantation, clinical pregnancy and live birth in ART. We also demonstrated a statistically significant difference in the messenger RNA (mRNA) expression profiles in the endometrium of pregnant and nonpregnant patients. Further studies on the genes identified herein will assist in predicting implantation competence. [ABSTRACT FROM AUTHOR]