MALDI-TOF Technique Research Articles

Automated analysis of synthesized oligorutin and oligoesculin by laccase

The polymerization of phenolic species is an interesting way to generate derivatives with new or improved properties. The structural analysis of these compounds is complex and requires specific analytical techniques. The size exclusion chromatography coupled with an UV detector (SEC-UV) and matrix-assisted laser desorption ionization coupled with a time-of-flight detector (MALDI-TOF) analyses are two methods used to investigate the masses of biopolymers. To analyse the oligomers of rutin and esculin synthesized by the laccase from Trametes versicolor , these two techniques were used and coupled with an automated platform. The conditions of detection by MALDI-TOF were optimized. The parallel synthesis, the preparation of sample, the SEC analysis, and the MALDI spotting were realized automatically on a Chemspeed platform. The MALDI-TOF technique enables the detection of oligomers up to pentamer and nonamer, whereas the SEC-UV permits the detection of about 17-mer for rutin and esculin. The molecular mass distribution is underestimated by MALDI-TOF analysis compared with SEC-UV, but comes to the determination of absolute masses. So these two techniques would be combined to explore the masses and the oligomer distributions.

VipR: variant identification in pooled DNA using R

Motivation: High-throughput-sequencing (HTS) technologies are the method of choice for screening the human genome for rare sequence variants causing susceptibility to complex diseases. Unfortunately, preparation of samples for a large number of individuals is still very cost- and labor intensive. Thus, recently, screens for rare sequence variants were carried out in samples of pooled DNA, in which equimolar amounts of DNA from multiple individuals are mixed prior to sequencing with HTS. The resulting sequence data, however, poses a bioinformatics challenge: the discrimination of sequencing errors from real sequence variants present at a low frequency in the DNA pool.Results: Our method vipR uses data from multiple DNA pools in order to compensate for differences in sequencing error rates along the sequenced region. More precisely, instead of aiming at discriminating sequence variants from sequencing errors, vipR identifies sequence positions that exhibit significantly different minor allele frequencies in at least two DNA pools using the Skellam distribution. The performance of vipR was compared with three other models on data from a targeted resequencing study of the TMEM132D locus in 600 individuals distributed over four DNA pools. Performance of the methods was computed on SNPs that were also genotyped individually using a MALDI-TOF technique. On a set of 82 sequence variants, vipR achieved an average sensitivity of 0.80 at an average specificity of 0.92, thus outperforming the reference methods by at least 0.17 in specificity at comparable sensitivity.Availability: The code of vipR is freely available via: http://sourceforge.net/projects/htsvipr/Contact: altmann@mpipsykl.mpg.de

Validation of two methods for the identification of bacteria directly from blood cultures with the MALDI-TOF technique

Aim We validated two methods for the direct identification of bacteria from positive BACTEC blood culture bottles with the MALDI-TOF technique. Methods 872 blood culture bottles positive with the BACTEC system (BD) were tested. For MALDI-TOF identification two specimen collection and blood cell extraction methods were tested [Method 1: Vacutainer serum seperator tubes (BD); Method 2: MALDI Sepsityper Solutions (Bruker)]. For both methods, bacterial proteins were extracted by the formic acid/ acetonitrile standard method and measured in a MALDI-TOF spectrometer of the FLEX series (Bruker Daltonics). Identification results were compared to routine biochemical methods (VITEK 2, api-system, BioMe´rieux). Results Of 789 blood cultures tested with Method 1, 636 (80%) showed a valid (score value > 1,7) identification result with the MALDI-TOF. Of these valid samples, 630 (99%) were correctly identified to species level in comparison to routine biochemical methods. For Method 2, 83 blood cultures were tested, 75 (80%) were considered as valid, 99% showed a correct result to species level. Discussion The MALDI-TOF technique proved to be a fast and reliable method for the detection of bacteria directly from BACTEC blood culture bottles. Results are available 1–2 days earlier in comparison to conventional methods. We validated two methods for the direct identification of bacteria from positive BACTEC blood culture bottles with the MALDI-TOF technique. 872 blood culture bottles positive with the BACTEC system (BD) were tested. For MALDI-TOF identification two specimen collection and blood cell extraction methods were tested [Method 1: Vacutainer serum seperator tubes (BD); Method 2: MALDI Sepsityper Solutions (Bruker)]. For both methods, bacterial proteins were extracted by the formic acid/ acetonitrile standard method and measured in a MALDI-TOF spectrometer of the FLEX series (Bruker Daltonics). Identification results were compared to routine biochemical methods (VITEK 2, api-system, BioMe´rieux). Of 789 blood cultures tested with Method 1, 636 (80%) showed a valid (score value > 1,7) identification result with the MALDI-TOF. Of these valid samples, 630 (99%) were correctly identified to species level in comparison to routine biochemical methods. For Method 2, 83 blood cultures were tested, 75 (80%) were considered as valid, 99% showed a correct result to species level. The MALDI-TOF technique proved to be a fast and reliable method for the detection of bacteria directly from BACTEC blood culture bottles. Results are available 1–2 days earlier in comparison to conventional methods.

The red wine extract‐induced activation of endothelial nitric oxide synthase is mediated by a great variety of polyphenolic compounds

Phenolic extracts from red wine (RWPs) have been shown to induce nitric oxide (NO)-mediated vasoprotective effects, mainly by causing the PI3-kinase/Akt-dependent activation of endothelial NO synthase (eNOS). RWPs contain several hundreds of phenolic compounds. The aim of the present study was to identify red wine phenolic compounds capable of activating eNOS in endothelial cells using multi-step fractionation. The red wine phenolic extract was fractionated using Sephadex LH-20 and preparative RP-HPLC approaches. The ability of a fraction to activate eNOS was assessed by determining the phosphorylation level of Akt and eNOS by Western blot analysis, and NO formation by electron spin resonance spectroscopy. Tentative identification of phenolic compounds in fractions was performed by MALDI-TOF and HPLC-MS techniques. Separation of RWPs by Sephadex LH-20 generated nine fractions (fractions A to I), of which fractions F, G, H and I caused significant eNOS activation. Fraction F was then subjected to semi-preparative RP-HPLC to generate ten subfractions (subfraction SF1 to SF10), all of which caused eNOS activation. The active fractions and subfractions contained mainly procyanidins and anthocyanins. Isolation of phenolic compounds from SF9 by semi-preparative RP-HLPC lead to the identification of petunidin-O-coumaroyl-glucoside as a potent activator of eNOS.

Magnesium(II) polyporphine: The first electron-conducting polymer with directly linked unsubstituted porphyrin units obtained by electrooxidation at a very low potential

Electrooxidation of magnesium(II) porphine, a totally unsubstituted porphyrin, in acetonitrile solution under potentiostatic or potentiodynamic regime leads to a polymer film at the electrode surface. Polymer deposition takes place at extremely low potential, several hundred mV less positive even compared to the deposition potential for pyrrole or EDOT (at identical monomer concentrations) in the same solvent. Film thickness can be controlled by the passed deposition charge. This material and its THF-soluble fraction have been characterized by various electrochemical methods as well as by UV–visible and IR spectroscopies, XPS, XRD and MALDI-TOF techniques. This analysis has allowed us to conclude that the polymer film is composed by chains of Mg porphine building blocks, with single bonds between the neighboring units. In the course of the potential sweep, this polymer film demonstrates a redox response resembling that of polythiophene-coated electrodes. Namely, the film is electroactive and electronically conducting in two potential ranges (p- and n-doping), which are separated by a broad interval where the film possesses a much higher resistance. The polymer may be switched between all these redox states repeatedly by the change of the potential. The film capacitance in the electroactive potential intervals is proportional to the deposition charge.

Differential characterization of biogenic amine‐producing bacteria involved in food poisoning using MALDI‐TOF mass fingerprinting

Histamine poisoning is caused by the consumption of fish and other foods that harbor bacteria possessing histidine decarboxylase activity. With the aim of preventing histamine formation, highly specific mass spectral fingerprints were obtained from the 16 major biogenic amine-producing enteric and marine bacteria by means of MALDI-TOF MS analysis. All bacterial strains analyzed exhibited specific spectral fingerprints that enabled its unambiguous differentiation. This technique also identified peaks common to certain bacterial groups. Thus, two protein peaks at m/z 4182+/-1 and 8363+/-6 were found to be present in all Enterobacteriaceae species analyzed except for Morganella morganii. Peaks at m/z 3635+/-1 and 7267+/-2 were specific to both M. morganii and Proteus spp. Biogenic amine-forming Proteus spp. exhibited three genus-specific peaks at m/z 3980, 7960+/-1 and 9584+/-2. The genus Photobacterium also showed three genus-specific peaks at m/z 2980+/-1, 4275+/-1 and 6578+/-1. The two histamine-producing Gram-positive bacteria Lactobacillus sp. 30A and Staphylococcus xylosus exhibited a few protein peaks in the 2000-7000 m/z range and could be easily distinguished from biogenic amine-forming Gram-negative bacteria. Clustering based on MALDI-TOF MS also exhibited a good correlation with phylogenetic analysis based on the 16S rRNA gene sequence, validating the ability of the MALDI-TOF technique to establish relationships between microbial strains and species. The approach described in this study leads the way toward the rapid and specific identification of major biogenic amine-forming bacteria based on molecular protein markers with a goal to the timely prevention of histamine food poisoning.

Functional inactivation of triosephosphate isomerase through phosphorylation during etoposide-induced apoptosis in HeLa cells: Potential role of Cdk2

Up-regulation of cyclin-dependent protein kinase 2 (Cdk2) activity has been suggested to be prerequisite for progression of apoptosis induced by various apoptotic stimuli. In this study, we applied a phospho-proteomic technique to screen target molecules of Cdk2 during etoposide-induced apoptosis. For this purpose, phosphoproteins from the cell lysates were enriched by using Fe 3+-IMAC column chromatography and resolved on a high resolution 2D PAGE gel. We identified six phosphoproteins by the use of MALDI-TOF technique. The cellular levels of these phosphoproteins were markedly reduced in the presence of etoposide in HeLa cells transfected with dominant negative mutant construct of Cdk2. Among the six candidate phosphoproteins, human triosephosphate isomerase (TPI), a glycolytic enzyme, was found to be a direct substrate of Cdk2 during etoposide-induced apoptosis. In an in vitro phosphorylation assay, TPI purified by use of a baculoviral expression system was phosphorylated by recombinant Cyclin A/Cdk2 kinase. This led to reduced enzyme activity for the conversion of glucose aldehyde-3-phosphate to dihydroxyacetone phosphate. Such phosphorylation of TPI and a subsequent decrease in its enzyme activity were prevented by treatment with olomoucine, a specific inhibitor of Cdk2. The above findings, taken together, suggest TPI as a potential target protein of Cyclin A/Cdk2. Loss of catalytic activity of TPI as a consequence of phosphorylation of this glycolytic enzyme may disrupt energy production in etoposide-treated HeLa cells, rendering these cells prone to undergo apoptosis.

The influence of organic ligands on the adsorption of cadmium by suspended matter in natural waters studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrochemical methods

Natural water carrying a great amount of suspended particulate matter (SPM) was used as a model system for the study of the competition among organic ligands (dietilentetraamine pentaacetic acid, DTPA, nitrilotriacetic acid, NTA, and citrate, Cit) and natural complexants (SPM) for cadmium adsorption. Speciation diagrams at the pH of the natural sample were obtained by electroanalytical techniques, processing the experimental data with the complexation constants and the mass balance of the system. Results show that the adsorption equilibrium SPM-Cd is completely displaced by DTPA but not completely by NTA or Cit. Furthermore, larger Cit concentrations increase the amount of adsorbed Cd(II). The increment of the complexing capacity may be explained on the basis of SPM-Cit-Cd ternary complexes formation. This hypothesis was supported on the results obtained by applying for the first time the MALDI–TOF technique in a mixture of SPM, Cd(II), organic ligands and their complexes.

Preparation of polyetherols with pyrimidine rings by reaction of 6-aminouracil with excess of propylene carbonate

New method of synthesis of polyetherols with 1,3-pyrimidine rings by the reaction of 6-aminouracil (6-AU) with excess of propylene carbonate (PC) catalyzed by 1,4-diazabicyclo[2.2.2]octane and potassium carbonate is presented. The structures of products were analyzed by IR, 1 H NMR and MALDI ToF techniques. Some physical properties of prepared polyetherols were investigated. Among other things, they show improved thermal stability.

Synthesis of aromatic oligoamide macrocycles

The one-step macrocyclization of monomeric diamine 1 and diacid chloride 2 resulted in a series of eightresidue macrocycles 3 with an oligoamide backbone in which some of the amide hydrogen atoms are replaced with the removable 2,4-dimethoxybenzyl groups. The structures of these macrocycles were established by 1H NMR, HRMS, MALDI-TOF techniques.

In this issue

PROTEOMICSVolume 9, Issue 21 In this issue In this issue First published: 27 October 2009 https://doi.org/10.1002/pmic.200990082AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Triple tag technique turns up tight and loose complexes pp. 4934–4943 The law of unintended consequences is also known as luck – a late snowstorm is good for the person with a snowplow to rent, bad for the one who just put his away. Or the caveman who discovered that a stone hammer could crack a skull as well as a nut. Saade et al. recognized an opportunity in the variable difficulty of releasing proteins from “His-tag” complexes. The affinity of His-tagged proteins for Ni-agarose is very high and displacing the tags requires imidazole. If the pH is in the right range, tagged proteins are not removed by organic solvents, high ionic strength, or denaturants. Thus, if only one member of a complex is His-tagged, other members can be eluted with common methods until the last ones are removed by a pH shift or imidazole. (Is this a case of a silk purse from a sow's ear?). Csk: The>150 x Go-To guy pp. 4944–4961 C-terminal Src kinase, Csk, must be an extremely busy protein – handling tasks in signal transduction and cell communication and cell growth and maintenance (previously recognized) to the regulation of nucleic acid and nucleobase metabolism, protein metabolism, energy pathways, transport, and biogenesis and organization of cells (newly discovered by Yang et al.). These researchers turned up over 150 unique partners of Csk using MALDI-TOF, shotgun LC-MS/MS, immunofluorescence, and other proteomic techniques. Some were direct, some indirect as judged by the presence of one or more Csk-SH2-binding domains. Src Family Kinases, SFKs are normally negatively controlled by Csk. Over-expression and over-activation of SFKs have been associated with cancerous tumors of the colon, liver, pancreas, stomach, breast, ovaries and lung. A formidable list of targets for a Csk master to tackle. Separate the wheat from the chaff before feeding to analyst pp. 4978–4984 Looking in a haystack for a needle is the proverbial impossible task. If you know that you are looking for a steel needle, however, it suddenly becomes much easier – you can use a large magnet to preprocess the haystack. Collect all the ferrous material from the haystack, then search through that for the needle. Renard et al. applied the principle to peptide identification in MS/MS data sets from human (2 types), yeast, and mouse cell lysates. It takes some work to make it work, however. Various relatively simple spectrum preprocessing routines are built into the most popular peptide identification software packages and may not function well following the stand-alone preprocessing package unless both are optimized to work together. The benefits of the preprocessing include cleaner spectra, smaller file sizes and shorter computation times. Volume9, Issue21No. 21 November 2009 RelatedInformation

α-Kinase Anchoring Protein αKAP Interacts with SERCA2A to Spatially Position Ca2+/Calmodulin-dependent Protein Kinase II and Modulate Phospholamban Phosphorylation

The sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) is critical for sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR) and regulating cardiac muscle relaxation. Protein-protein interactions indicated that it exists in complex with Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and its anchoring protein alphaKAP. Confocal imaging of isolated cardiomyocytes revealed the colocalization of CAMKII and alphaKAP with SERCA2a at the SR. Deletion analysis indicated that SERCA2a and CaMKII bind to different regions in the association domain of alphaKAP but not with each other. Although deletion of the putative N-terminal hydrophobic amino acid stretch in alphaKAP prevented its membrane targeting, it did not influence binding to SERCA2a or CaMKII. Both CaMKIIdelta(C) and the novel CaMKIIbeta(4) isoforms were found to exist in complex with alphaKAP and SERCA2a at the SR and were able to phosphorylate Thr-17 on phospholamban (PLN), an accessory subunit and known regulator of SERCA2a activity. Interestingly, the presence of alphaKAP was also found to significantly modulate the Ca(2+)/calmodulin-dependent phosphorylation of Thr-17 on PLN. These data demonstrate that alphaKAP exhibits a novel interaction with SERCA2a and may serve to spatially position CaMKII isoforms at the SR and to uniquely modulate the phosphorylation of PLN.

Partial purification and characterization of a novel antifungal compound against Aspergillus spp. from Synechocystis sp. PCC 6803

The antifungal compound AK-3 is purified and characterized from intracellular metabolites of the unicellular cyanobacteria, Synechocystis sp. PCC 6803. AK-3 clearly had antifungal effects upon growth of the fungi Aspergillus spp. From 2 g of dry cell powder, 4.8 mg of AK-3 was obtained with a yield of 0.24%. Its structure was elucidated by NMR, UV spectra, amino acid analysis by chemical degradation, and MS measurements, including MALDI-TOF and MS-MS techniques. AK-3 contains a high level of antifungal activity compared to itraconazole and is seemingly a new compound based on its amino acid composition. Extraction was performed with an acetic acid/water mixture (pH 3.0) while detection at UV 214 nm found AK-3 did not react with ninhydrin. These results suggest that this compound is a cyclic peptide with high hydrophilicity and a phenolic ring.

C008 Endothelial NO synthase activation by a red Wine Phenolic extract: isolation of active components by bioguided fractionation

Previous studies have shown that Red Wine Phenolic extracts induce nitric oxide (NO)-mediated vasoprotective effects, mainly by causing a redox-sensitive activation of endothelial NO synthase (eNOS). However, Red Wine Phenolic extracts are complex mixtures of a several hundreds of phenolic compounds. Therefore, the aim of the present study was to isolate active phenolic compounds using multi-step bioguided fractionation of the red wine extract. Red Wine Phenolic Extract was submitted to a bioguided fractionation using chromatographic methods. The fractions obtained at each step were tested for their potential to induce the activation of eNOS in endothelial cells. Cultures of porcine coronary artery endothelial cells (P1) were used to determine the phosphorylation level of Akt and endothelial NO synthase by Western blot analysis. Identification of phenolic compounds in each active fraction was performed by MALDI-TOF and HPLC-MS techniques. The first step of fractionation on lipophilic Sephadex® yielded 9 fractions of which 4 of them significantly increased the phosphorylation level of Akt and eNOS in endothelial cells. The active fractions contained mainly procyanidins and some anthocyanins compounds. The fractionation of one of the active fractions by preparative reverse-phase HPLC yielded 11 subfractions ; all of these sub-fractions significantly increased the phosphorylation level of Akt and eNOS. The analysis of the phenolic compounds indicated that these sub-fractions contained mixtures of procyanidin dimers or conjugated anthocyanins. The red wine extract contains several types of phenolic compounds, which are able to enhance the activity of NO synthase in endothelial cells including procyanidins dimers and oligomers as well as several conjugated anthocyanins.

Synthesis and application of oligomers obtained from oxamic acid and ethylene carbonate

Summary — The reaction between oxamic acid and ethylene carbonate in the presence of potassium carbonate catalyst was studied. The products were characterized by 1 H NMR, IR and MALDI ToF techniques. The hydroxyethoxy derivatives of the acid are remarkably thermally stable due to the presence of oxalamidoester group. They were used as components of thermally stable polyurethane

Proteomic Analysis of the Role of STAT5A in the Apoptosis of Human Pre-B Cells : Evidence for a Connection to Daxx and the Oxidative Stress

It is well established that STAT5A and 5B transcription factors (TF) play a major role in hematopoiseis and oncogenesis. In particular, there is a body of evidence suggesting that they might be involved in B lymphocyte survival, proliferation and development as well as in B cell neoplastic transformation. To investigate the role of STAT5A in human precursor B cell survival, we stably transfected a dominant negative form of STAT5A (DN-STAT5A, deleted in its transactivation domain) in the human leukemic Nalm6 pre-B cell line. All clones expressing DN-STAT5A exhibited a lower proliferation rate associated with a higher spontaneous apoptosis and that was massively enhanced upon IL7 stimulation. They also were more sensitive to FAS (CD95)- and at a lesser extent to etoposide-induced cell death than cells transfected with the empty vector (Nalm6neo), suggesting a hitherto unknown link between STAT5A and apoptosis/survival pathways. There was no evidence for changes in the levels of expression of Bcl2, BclxL, Bad or –c-Myc in DN-STAT5A expressing cells whereas Bax and Bim expression was sharply up-regulated in these cells. In order to identify the proteins involved in the connection between Fas and STAT5A, we used a proteomic approach with differential analysis of cells expressing (Nalm6Δ5A749) or not (Nalm6neo) the DN-STAT5A. Statistical analysis of bidimensional (2D) gels and use of MALDI-TOF technique enabled us to identify 7 proteins down-regulated in the Nalm6Δ5A749 cells, including members of heat shock proteins such as hsp27 and hsp70, as well as proteins implied in the control of oxidative stress like glutathione synthetase and transaldolase. By contrast, whereas 6 proteins were shown to be up-regulated, including prohibitin which is a negative regulator of cellular proliferation. Nalm6Δ5A749 cells exhibited enhanced levels of reactive oxygen species in keeping with the idea that oxidative stress might be involved in the increased sensitivity of these cells to apoptotic signals. Although expression of all above mentioned molecules did not change upon Fas stimulation, addition of gluthatione (10mM) resulted in a complete inhibition of Fas-mediated apoptosis in Nalm6Δ5A749 cells. Whether or not prohibitin might be involved in the decreased proliferation rate in DN-STAT5A expressing cells needs further investigation. As Hsp27 has been previously shown to have the potential to sequestrate Daxx, a molecule that is involved in Fas receptor signaling, we investigated the subcellular location of this molecule in Nalm6Δ5A749 cells. We showed that Daxx was essentially expressed in the cytoplasm of these cells whereas it was mainly located in the nucleus in Nalm6neo cells. Our results bring evidence for a role of STAT5A in pre-B cell growth and survival and in particular for a connection between this TF and Fas signaling machinery that might involve Hsp27 and Daxx. They also point out to a so far undescribed link between STAT5 and oxidative stress.

Comparison Of Reaction Pathway And Product Analysis For Hydroxyalkylation Of Parabanic Acid With Oxiranes And Alkylene Carbonates

AbstractReactions of parabanic acid (PA) with ethylene and propylene oxides and carbonates lead to oligomeric products with hydroxyalkyl groups in both cases. The parabanic acid ring undergoes scissions during reactions depending on kind of reagent and reaction conditions. In some cases the trioxoimidazolidine ring remains untouched. Using the MALDI ToF technique the product analysis was performed. The by-products were determined quantitatively by GC. The thermostability of oligomeric products was also determined.

The Analysis of Polystyrene and Polystyrene Aggregates into the Mega Dalton Mass Range by Cryodetection MALDI TOF MS

Mass spectra of atactic polystyrene were collected into the mega-dalton mass range with a matrix-assisted laser desorption ionization time of flight (MALDI TOF) mass spectrometer, which incorporates a cryodetector comprised of an array of 16 superconducting tunnel junctions (STJ). The STJ cryodetector, theoretically, has no loss in signal response at any mass compared with the reduced signal found at high mass when using a conventional secondary-ionization detector. Since ion detection at high m/z is one of the fundamental limitations of mass spectrometry (MS), the cryodetector was used to explore the high m/z limit of the MALDI TOF technique for the analysis of two polymer types. Mass spectra were collected for polystyrene at Mn 170, 400, 900, and 2000 kDa and polymethyl methacrylate (PMMA) at Mn 62.6 kDa and 153.7 kDa. For polystyrene, the data showed a trend toward increased aggregation and charge state with mass. The Mn 2 MDa polystyrene data revealed a peak at m/z 2.2 MegaTh and a charge state analysis revealed that these ions were primarily polystyrene aggregates with a mass of approximately 4 MDa. This aggregate assignment was possible because the cryodetector response allows for the determination of a charge state up to about four. The contribution of each charge state for a selected peak can be determined in this fashion. This analysis revealed the preferential formation of doubly charged even-numbered aggregates over odd-numbered aggregates for high molecular mass polystyrene. A potential mechanism for the aggregation process for doubly charged species is discussed.

Efficient Synthesis of Dendrimers via A Chemoselective Approach

The dendrimer derivatives of the Fréchet-type and 2,2-bis(methylol)propionic acid (bis-MPA) dendrimers were synthesized via an accelerated growth approach, which is based on the combination of click chemistry with traditional esterification/etherification reactions. The required monomer units for one-step generation growth are AB2-monomer (2) and CD2-monomer (4). The synthetic strategy allows the divergent preparation of a fourth-generation dendrimer (2nd generation dendrimer 5 is shown) in only four steps and in multi-gram quantities with an overall yield of 70% from the starting triphenol 1. The obtained dendritic block copolymer contains alternating layers of benzyl ether, triazole and benzyl ether groups. The orthogonality and efficiency of this strategy were demonstrated by monitoring each reaction with NMR and MALDI-TOF techniques.

ERp57 is a potential biomarker for human fertilization capability

Human infertility is a growing concern and while many assisted reproductive technologies exist, their success rates are low. Thus, developing tests, possibly by assessing proteins involved in fertilization, that could predict the outcome of these technologies is of great significance. To identify candidate proteins, we used two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF techniques and detected the ERp57 protein from human testis protein profile. Immunohistochemistry showed that ERp57 was mostly located in spermatogenic cell cytoplasm from spermatocytes to the spermatozoa phases and in Leydig cells of human testes; it was also present at low levels in Sertoli cells. ERp57 was evident in human spermatozoa, primarily in the acrosome and tail; moreover, it appeared to translocate to the equatorial segment after the acrosome reaction. During sperm capacitation, the ERp57 protein underwent post-translational modification. Blocking ERp57 with antibodies significantly inhibited human sperm from penetrating zona-free hamster oocytes in a dose-dependent manner. Finally, expression levels of ERp57 were associated with fertility; they were decreased dramatically in IVF patients with low fertilization rates compared with those with high rates or to fertile sperm donors. Taken together, these results show that ERp57 is a component of human sperm acrosome proteins, which play a critical role in gamete fusion. Furthermore, ERp57 could be a novel phenotype marker for male infertility and has the potential to be used to assess sperm selection for IVF.