Distal Cluster Research Articles

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

BackgroundPhotosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.ResultsWe adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.ConclusionsOur results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

Structure and Substrate-Induced Conformational Changes of the Secondary Citrate/Sodium Symporter CitS Revealed by Electron Crystallography

The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into its overall structural organization. Here, we present the three-dimensional map of dimeric CitS obtained with electron crystallography. Each monomer has 13 a-helical transmembrane segments; six are organized in a distal helix cluster and seven in the central dimer interface domain. Based on structural analyses and comparison to VcINDY, we propose a molecular model for CitS, assign the helices, and demonstrate the internal structural symmetry. We also present projections of CitS in several conformational states induced by the presence and absence of sodium and citrate as substrates. Citrate binding induces a defined movement of a helices within the distal helical cluster. Based on this, we propose a substrate translocation site and conformational changes that are in agreement with the transport model of ‘‘alternating access’’.

The clinical impact of chromosomal rearrangements with breakpoints upstream of the SOX9gene: two novel de novo balanced translocations associated with acampomelic campomelic dysplasia

BackgroundThe association of balanced rearrangements with breakpoints near SOX9 [SRY (sex determining region Y)-box 9] with skeletal abnormalities has been ascribed to the presumptive altering of SOX9 expression by the direct disruption of regulatory elements, their separation from SOX9 or the effect of juxtaposed sequences.Case presentationWe report on two sporadic apparently balanced translocations, t(7;17)(p13;q24) and t(17;20)(q24.3;q11.2), whose carriers have skeletal abnormalities that led to the diagnosis of acampomelic campomelic dysplasia (ACD; MIM 114290). No pathogenic chromosomal imbalances were detected by a-CGH. The chromosome 17 breakpoints were mapped, respectively, 917–855 kb and 601–585 kb upstream of the SOX9 gene. A distal cluster of balanced rearrangements breakpoints on chromosome 17 associated with SOX9-related skeletal disorders has been mapped to a segment 932–789 kb upstream of SOX9. In this cluster, the breakpoint of the herein described t(17;20) is the most telomeric to SOX9, thus allowing the redefining of the telomeric boundary of the distal breakpoint cluster region related to skeletal disorders to 601–585 kb upstream of SOX9. Although both patients have skeletal abnormalities, the t(7;17) carrier presents with relatively mild clinical features, whereas the t(17;20) was detected in a boy with severe broncheomalacia, depending on mechanical ventilation. Balanced and unbalanced rearrangements associated with disorders of sex determination led to the mapping of a regulatory region of SOX9 function on testicular differentiation to a 517–595 kb interval upstream of SOX9, in addition to TESCO (Testis-specific enhancer of SOX9 core). As the carrier of t(17;20) has an XY sex-chromosome constitution and normal male development for his age, the segment of chromosome 17 distal to the translocation breakpoint should contain the regulatory elements for normal testis development.ConclusionsThese two novel translocations illustrate the clinical variability in carriers of balanced translocations with breakpoints near SOX9. The translocation t(17;20) breakpoint provides further evidence for an additional testis-specific SOX9 enhancer 517 to 595 kb upstream of the SOX9 gene.

High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations

AbstractTo identify genomic alterations in chronic lymphocytic leukemia (CLL), we performed single-nucleotide polymorphism–array analysis using Affymetrix Version 6.0 on 353 samples from untreated patients entered in the CLL8 treatment trial. Based on paired-sample analysis (n = 144), a mean of 1.8 copy number alterations per patient were identified; approximately 60% of patients carried no copy number alterations other than those detected by fluorescence in situ hybridization analysis. Copy-neutral loss-of-heterozygosity was detected in 6% of CLL patients and was found most frequently on 13q, 17p, and 11q. Minimally deleted regions were refined on 13q14 (deleted in 61% of patients) to the DLEU1 and DLEU2 genes, on 11q22.3 (27% of patients) to ATM, on 2p16.1-2p15 (gained in 7% of patients) to a 1.9-Mb fragment containing 9 genes, and on 8q24.21 (5% of patients) to a segment 486 kb proximal to the MYC locus. 13q deletions exhibited proximal and distal breakpoint cluster regions. Among the most common novel lesions were deletions at 15q15.1 (4% of patients), with the smallest deletion (70.48 kb) found in the MGA locus. Sequence analysis of MGA in 59 samples revealed a truncating mutation in one CLL patient lacking a 15q deletion. MNT at 17p13.3, which in addition to MGA and MYC encodes for the network of MAX-interacting proteins, was also deleted recurrently.

EPR Spectroscopic Studies of the Fe–S Clusters in the O2-Tolerant [NiFe]-Hydrogenase Hyd-1 from Escherichia coli and Characterization of the Unique [4Fe–3S] Cluster by HYSCORE

The unusual [4Fe-3S] cluster proximal to the active site plays a crucial role in allowing a class of [NiFe]-hydrogenases to function in the presence of O(2) through its unique ability to undergo two rapid, consecutive one-electron transfers. This property helps to neutralize reactive oxygen species. Mechanistic details and the role of the medial and distal clusters remain unresolved. To probe the Fe-S relay, continuous wave and pulse electron paramagnetic resonance (EPR) studies were conducted on the O(2)-tolerant hydrogenase from Escherichia coli (Hyd-1) and three variants with point mutations at the proximal and/or medial clusters. Reduction potentials of the proximal ([4Fe-3S](5+/4+/3+)) and medial ([3Fe-4S](+/0)) clusters were determined by potentiometry. The medial [3Fe-4S](+/0) reduction potential is exceptionally high, implicating a mechanistic role in O(2)-tolerance. Numerous experiments establish that the distal cluster has a ground state S > 1/2 in all three variants and indicate that this is also the case for native Hyd-1. Concurrent with the Hyd-1 crystal structure, EPR data for the 'superoxidized' P242C variant, in which the medial cluster is 'magnetically silenced', reveal two conformations of the proximal [4Fe-3S](5+) cluster, and X-band HYSCORE spectroscopy shows two (14)N hyperfine couplings attributed to one conformer. The largest, A((14)N) = [11.5,11.5,16.0] ± 1.5 MHz, characterizes the unusual bond between one Fe (Fe(4)) and the backbone amide-N of cysteine-20. The second, A((14)N) = [2.8,4.6,3.5] ± 0.3 MHz, is assigned to N(C19). The (14)N hyperfine couplings are conclusive evidence that Fe(4) is a valence-localized Fe(3+) in the superoxidized state, whose formation permits an additional electron to be transferred rapidly back to the active site during O(2) attack.

Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner

The activation of Smoothened (Smo) requires phosphorylation at three clusters of Serine residues in Drosophila Hedgehog (Hh) signaling. However, the mechanism by which phosphorylation promotes Smo conformational change and subsequently activates Smo in response to Hh gradient remains unclear. Here, we show that the conformational states of Smo are determined by not only the amount but also the position of the negative charges provided by phosphorylation. By using a Smo phospho-specific antibody, we demonstrate that Smo is differentially phosphorylated at three clusters of serine residues in response to levels of Hh activity. Mutating the first cluster, compared to mutating the other clusters, impairs Smo activity more severely, whereas mutating the last cluster prohibits C-terminus dimerization. In addition, phosphorylation of the membrane proximal cluster promotes phosphorylation of the distal cluster. We propose a zipper-lock model in which the gradual phosphorylation at these clusters induces a gradual conformational change in the Smo cytoplasmic tail, which promotes the interaction between Smo and Costal2 (Cos2). Moreover, we show that Hh regulates both PKA and CK1 phosphorylation of Smo. Thus, the differential phosphorylation of Smo mediates the thresholds of Hh activity.

Effects of Cluster Thinning and Preflowering Leaf Removal on Growth and Grape Composition in cv. Sangiovese

Crop-regulation techniques applied as preflowering defoliation (D), early cluster thinning at preflowering (ECT), and cluster thinning at lag-phase of berry growth (LCT) were tested over three seasons on high-yielding <i>Vitis vinifera</i> L. Sangiovese and compared to non-defoliated, unthinned control vines. Treatment severity consisted of removing primary leaves and any laterals developed from nodes 1 to 6 in D and of thinning 50% of clusters chosen from among distal clusters or those inserted on weak shoots in ECT and LCT plots. Although yield per vine was not as reduced in D (−32%) as in ECT and LCT treatments (−45%) as compared to the control, D vines also had largely improved sugar and total anthocyanin concentrations and the highest total phenolics. Yield components were also markedly affected by treatments: D vines had smaller clusters and berries, leading to improved cluster looseness and to higher relative skin and seed growth. While all crop-regulating treatments led to an increase in the final leaf-to-fruit ratio, parameters of technological maturity were essentially uncoupled, as equally high Brix levels corresponded to the highest titratable acidity in D and, conversely, to lowest titratable acidity and highest pH in ECT and LCT. Overall results showed that different final yield-grape composition patterns can be reached depending on the technique used for crop regulation as a primary consequence of a diversified degree of compensation triggered on single-yield components.

Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells

Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for F-actin-we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function-associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse.

Phylogenetic and functional conservation of the NKR-P1F and NKR-P1G receptors in rat and mouse

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3+ NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3+ and NKR-P1B+ NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D− and NKR-P1D+ NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.

The Distal VH Gene Cluster of the Igh Locus Contains Distinct Regulatory Elements with Pax5 Transcription Factor-Dependent Activity in Pro-B Cells

V(H)-DJ(H) recombination of the immunoglobulin heavy chain (Igh) locus is temporally and spatially controlled during early B cell development, and yet no regulatory elements other than the V(H) gene promoters have been identified throughout the entire V(H) gene cluster. Here, we discovered regulatory sequences that are interspersed in the distal V(H) gene region. These conserved repeat elements were characterized by the presence of Pax5 transcription factor-dependent active chromatin by binding of the regulators Pax5, E2A, CTCF, and Rad21, as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B cell development. The pro-B cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal V(H)-DJ(H) recombination at the Igh locus.

Effects of Leaf Position on Blade and Petiole Mineral Nutrient Concentration of Tempranillo Grapevine ( Vitis vinifera L.)

Concentrations of N, P, K, Ca, Mg, Fe, Mn, Zn, Cu, and B were monitored throughout the season in leaf blades and petioles sampled at three different positions on the shoot of Tempranillo ( Vitis vinifera L.) grapevines. Petioles had higher concentrations of K, Mg, and Zn, whereas N, P, Ca, and micronutrients other than Zn were more abundant in leaf blades. Seasonal trends were identified for macronutrients, with decreasing patterns for N, P, and K, while Ca and Mg increased through the season. Minor differences in nutrient concentration were observed in leaf blades and petioles opposite basal and distal clusters. These results provide guidelines for the sampling of representative tissues for nutritional diagnosis of Tempranillo after veraison. However, differences among leaves opposite basal clusters and those placed four nodes beyond the second cluster suggest that these younger leaves are not useful for nutritional diagnosis using reference concentrations obtained for basal leaves.

Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3.

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein−ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein−carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

Enzymatic Ring-Opening Mechanism of Verdoheme by the Heme Oxygenase: A Combined X-ray Crystallography and QM/MM Study

The least understood mechanism during heme degradation by the enzyme heme oxygenase (HO) is the third step of ring opening of verdoheme to biliverdin, a process which maintains iron homeostasis. In response to this mechanistic uncertainty, we launched a combined study of X-ray crystallography and theoretical QM/MM calculations, designed to elucidate the mechanism. The air-sensitive ferrous verdoheme complex of HmuO, a heme oxygenase from Corynebacterium diphtheriae, was crystallized under anaerobic conditions. Spectral analysis of the azide-bound verdoheme-HmuO complex crystals assures that the verdoheme group remains intact during the crystallization and X-ray diffraction measurement. The structure offers the first solid evidence for the presence of a water cluster in the distal pocket of this catalytically critical intermediate. The subsequent QM/MM calculations based on this crystal structure explore the reaction mechanisms starting from the FeOOH-verdoheme and FeHOOH-verdoheme complexes, which mimic, respectively, the O(2)- and H(2)O(2)-supported degradations. In both mechanisms, the rate-determining step is the initial O-O bond breaking step, which is either homolytic (for FeHOOH-verdoheme) or coupled to electron and proton transfers (in FeOOH-verdoheme). Additionally, the calculations indicate that the FeHOOH-verdoheme complex is more reactive than the FeOOH-verdoheme complex in accord with experimental findings. QM energies with embedded MM charges are close to and yield the same conclusions as full QM/MM energies. Finally, the calculations highlight the dominant influence of the distal water cluster which acts as a biocatalyst for the conversion of verdoheme to biliverdin in the two processes, by fixing the departing OH and directing it to the requisite site of attack, and by acting as a proton shuttle and a haven for the highly reactive OH(-) nucleophile.

Sugar accumulation in 'Zweigelt' grapes as affected by "Traubenwelke"

Traubenwelke (berry shrivel, grape wilting), a physiological disorder during berry ripening, is a severe threat to grape production in numerous winegrowing areas worldwide. Investigations in Austrian viticulture, focusing on the particularly sensitive grape cultivar 'Blauer Zweigelt' ( Vitis vinifera L.), showed that afflicted clusters exhibited insufficient colouring, loss of turgor, low sugar content and high acidity. The aim of this study was to determine the chronological disease development on the basis of the process of sugar accumulation in both unhealthy and healthy clusters in relation to the emergence of visual symptoms. Sugar accumulation in afflicted clusters stopped shortly after veraison, well (approx. 20 days) before visible symptoms were detectable, and subsequently the degree of ripeness remained unchanged until harvest. The results suggested that development of Traubenwelke initiated before veraison. Distal clusters were more frequently affected than proximal clusters, whereas the position of the shoot on the vine did not impact frequency of occurrence. The gas exchange of leaves positioned opposite to healthy clusters was not significantly different than that of leaves opposite to unhealthy clusters.

Electrochemical applications. How click chemistry brought biomimetic models to the next level: electrocatalysis under controlled rate of electron transfer

This tutorial review discusses the immobilization of alkyne-terminated cytochrome c oxidase models on azide-functionalized self-assembled monolayers (SAM) coated gold electrodes that was made possible by click chemistry. The rate of electron delivery from the electrode to the model could be tuned by changing the nature of the SAM. Biologically relevant electron transfer rates (2-4 s(-1)) were obtained on slow SAMs allowing the model to turn over catalytically under steady-state conditions. Hence, click chemistry was a crucial tool to demonstrate, through electrocatalytic studies: (1) the role played by several features present in the distal side of the model, such as the Cu(B)-Tyr244 pair, the distal pocket, and the stabilizing role of a distal water cluster; (2) the reversible inhibition of O(2) reduction by H(2)S.

Coping with cadmium exposure in various ways: the two Helicid snails Helix pomatia and Cantareus aspersus share the metal transcription factor‐2, but differ in promoter organization and transcription of their Cd‐metallothionein genes

Gastropods are able to withstand fluctuating availabilities of nonessential trace elements such as cadmium by induction of Cd-specific metallothionein isoform (Cd-MT) expression. As in other species, the induction mechanism involves the binding of metal-regulatory transcription factors (MTF-1 or MTF-2) to metal responsive elements (MREs) in the MT promoter regions. Cd-dependent transcription of Cd-MT genes was assessed by quantitative real time PCR in two helicid gastropods, Helix pomatia and Cantareus aspersus, over a period of eight days. The promoter regions of the Cd-MT genes of the two species were sequenced and compared regarding the position of MREs and other relevant potential transcription factor binding sites (TFBs). Cd-MT gene transcription is induced after Cd exposure in Helix pomatia and Cantareus aspersus, showing a transient peak in Helix pomatia, contrasting with a persistent induction rate in Cantareus aspersus. Since the existence of MTF-2 was verified in both species, differing transcription patterns of Cd-MT genes must be due to functional differences in their metal-responsive promoter regions. Both promoters contain a proximal cluster of three MREs overlapping with TFBs for the transcriptional regulator Sp1. In contrast to Cantareus aspersus, however, the Cd-MT gene of Helix pomatia hosts an additional distal MRE overlapping with a Sp1 binding site and a CACCC box. Inhibitory effects of MRE overlapping Sp1 binding sites were observed in other MT genes. We therefore suggest that transient Cd-MT transcription upon Cd(2+) exposure in Helix pomatia may be the result of an inhibitory action of the distal MRE cluster.

Immobilization of the hyperthermophilic hydrogenase from Aquifex aeolicus bacterium onto gold and carbon nanotube electrodes for efficient H2 oxidation

The electrochemistry of membrane-bound [NiFe] hydrogenase I ([NiFe]-hase I) from the hyperthermophilic bacterium Aquifex aeolicus was investigated at gold and graphite electrodes. Direct and mediated H(2) oxidation were proved to be efficient in a temperature range of 25-70 degrees C, describing a potential window for H(2) oxidation similar to that of O(2)-tolerant hydrogenases. Search for enhancement of current densities and enzyme stability was achieved by the use of carbon nanotube coatings. We report high catalytic currents for H(2) oxidation up to 1 mA cm(-2), 10 times higher than at the bare electrode. Interestingly, high stability of the direct catalytic process was observed when encapsulating A. aeolicus [NiFe]-hase I into a carboxylic functionalized single walled carbon nanotube network. This suggests a peculiar interaction between the enzyme and the electrode material. The parameters that governed the orientation of the enzyme before electron transfer were thus investigated using self-assembled-monolayer gold electrodes. No control of the orientation by the charge or the hydrophobicity of the interface was demonstrated. This behavior was explained on the basis of a structural comparison between A. aeolicus [NiFe]-hase I and Desulfovibrio fructosovorans [NiFe] hydrogenase, which revealed the absence of acidic residues and an additional loop in the environment of the [4Fe-4S] distal cluster in A. aeolicus [NiFe]-hase I. Finally, the effect of inhibitors on the direct oxidation of H(2) by A. aeolicus [NiFe]-hase I encapsulated in a single walled carbon nanotube network was investigated. No inhibition by CO and tolerance toward O(2) were observed. Discussion of the reasons for such tolerance was undertaken on the basis of structural comparison with hydrogenases from aerobic bacteria.

Chemical induction of silent biosynthetic pathway transcription in Aspergillus niger

Manipulation of the fungal epigenome is hypothesized to be an effective method for accessing natural products from silent biosynthetic pathways. A library of epigenetic modifiers was tested using the fungus Aspergillus niger to determine the impact of small-molecule inhibitors on reversing the transcriptional suppression of biosynthetic genes involved in polyketide (PKS), non-ribosomal peptide (NRPS), and hybrid PKS-NRPS (HPN) production. Examination of expressed sequence tag libraries from A. niger demonstrated that >70% of its PKS-, NRPS-, and HPN-encoding gene clusters were transcriptionally suppressed under standard laboratory culture conditions. Using a chemical epigenetic methodology, we showed that treatment of A. niger with suberoylanilide hydroxamic acid and 5-azacytidine led to the transcriptional upregulation of many secondary-metabolite-encoding biosynthetic gene clusters. Chemical epigenetic modifiers exhibited positional biases for upregulating chromosomally distal gene clusters. In addition, a phylogenetic-based preference was noted in the upregulation of reducing clade I PKS gene clusters, while reducing clade IV PKS gene clusters were largely unaffected. Manipulating epigenetic features in fungi is a powerful method for accessing the products of silent biosynthetic pathways. Moreover, this approach can be readily incorporated into modern microbial screening operations.

Hip joint centre location: An ex vivo study

The human hip joint is normally represented as a spherical hinge and its centre of rotation is used to construct femoral anatomical axes and to calculate hip joint moments. The estimate of the hip joint centre (HJC) position using a functional approach is affected by stereophotogrammetric errors and soft tissue artefacts. The aims of this study were (1) to assess the accuracy with which the HJC position can be located using stereophotogrammetry and (2) to investigate the effects of hip motion amplitude on this accuracy. Experiments were conducted on four adult cadavers. Cortical pins, each equipped with a marker cluster, were implanted in the pelvis and femur, and eight skin markers were attached to the thigh. Recordings were made while an operator rotated the hip joint exploiting the widest possible range of motion. For HJC determination, a proximal and a distal thigh skin marker cluster and two recent analytical methods, the quartic sphere fit (QFS) method and the symmetrical centre of rotation estimation (SCoRE) method, were used. Results showed that, when only stereophotogrammetric errors were taken into account, the analytical methods performed equally well. In presence of soft tissue artefacts, HJC errors highly varied among subjects, methods, and skin marker clusters (between 1.4 and 38.5 mm). As expected, larger errors were found in the subject with larger soft tissue artefacts. The QFS method and the distal cluster performed generally better and showed a mean HJC location accuracy better than 10 mm over all subjects. The analysis on the effect of hip movement amplitude revealed that a reduction of the amplitude does not improve the HJC location accuracy despite a decrease of the artefact amplitude.

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.