A new technique for isolating pathogenic fungi from mixed flora inocula

The development of differential and selective media for the isolation of fungi pathogenic for man is briefly reviewed. A new primary culture technique for medical mycology is presented. This involves the use of an appropriate paper carrier impregnated with suitable concentrations of cycloheximide and chloramphenicol. The carrier, when properly placed on the surface of an agar medium inoculated with a mixed-flora specimen, favors the isolation of a wide spectrum of pathogenic and opportunistic fungi. Evaluation systems for the technique included artificial mixtures of bacteria and fungi, pooled clinical materials artificially seeded with fungi, and routine clinical laboratory specimens. Test fungi were grouped according to a selection pattern based on the extent of their outgrowth within the bacterial inhibition zone (BIZ) established by the carrier on a battery of media. Two main advantages of the antibiotics-impregnated carrier system over the antibiotics-fortified medium system are: (1) the diffusion gradient of cycloheximide allows for the selective outgrowth throughout the BIZ of pathogenic fungi resistant to it, and allows the more susceptible fungal pathogens and opportunists to grow out up to their levels of tolerance to the antibiotic within the BIZ; (2) the system is economically advantageous.     

The fine structure of the polymorphic spermatozoa of Fusitriton oregonensis (Mollusca: Gastropoda), with notes on the cytochemistry of the internal secretions

Summary The prosobranch Fusitriton oregonensis exhibits an unusual form of sperm polymorphism. The viable, eupyrene sperm are attached in groups of about fifty to worm-shaped, apyrene, carrier sperm. There is a second apyrene sperm, which is lancet-shaped and has a different internal organization than the carrier, but does not transport eupyrene sperm.The eupyrene sperm are filiform (185 m long), with a conical acrosome, elongate nucleus and midpiece. They contain large stores of glycogen in the principal piece, together with an unusually high proportion of protein. The latter is due to a complex interconnecting system of fibres that supports the tail internally. A distinct annulus is located, characteristically, at the junction between midpiece and principal piece.The carrier sperm has a core of about 112 axonemes that arise from basal bodies in the anterior end and extend through its entire length of 36 m. The basal bodies have unstriated rootlets that are embedded in a granular cap. Large membrane-bound yolk bodies are arranged along the length of the carrier sperm, on either side of the median axonemal core. Dense bodies, which may be indigestible residues formed from the degeneration of the nucleus, are excreted by exocytosis. Individual carrier sperm are capable of corkscrew propulsion, resembling that of spirochaetes.The lancet sperm is three times as long as the carrier. The sixteen or so axonemes, which are arranged peripherally like a cage enclosing the cytoplasm, originate from a dense centriolar plate in the anterior end. The cytoplasm is filled with secretions including small yolk granules, dense bodies (also excreted), clear vesicles, and a membranated granular secretion that resembles mucus. The possible functions of the lancet and carrier sperm are discussed.     

Computational design of a CNT carrier for a high affinity bispecific anti-HER2 antibody based on trastuzumab and pertuzumab Fabs

This is a preliminary cross multidisciplinary theoretical-computational approach for the design of a drug delivery system based on immunoconjugated carbon nanotube against HER2- overexpressing cancer cells. This drug delivery system allows the release of an encapsulated cytotoxic cocktail in a controlled manner under pulsed radio frequency (RF) irradiation. Our effort is focused on the computational aided design of a high affinity bispecific anti-HER2 antibody and an opening mechanism of the carbon nanotube (CNT) based cytotoxic carrier for controlling multiple drug release. We study the main interactions between the antibody and the antigen by a computational scanning mutagenesis approach of trastuzumab and pertuzumab fragment antigen binding (Fab) structures in order to enhance their binding affinity. Then, each Fab fragments is joined by a polypeptide linker which should be stable enough to avoid the “open form” of antibody. On the other hand, we also conjugate the engineered antibody to functionalized CNTs (f-CNTs), which encapsulate the inhibitors of the HER2/PI3K/Akt/mTOR signaling pathway. We take advantage of the fact that f-CNT converts the RF radiation absorption into heat release. A pulsed laser at 13.45 MHz increments the temperature around 40 °C for triggering the nano-caps destabilization, which allows the switching of the opening mechanism of the drug carrier. Nano-caps will be a dual pH/temperature responsive in order to take advantage of lysosome characteristic (acidic pH) and heat release from the carrier. Nano-caps are functionalized with organic amide moieties, which hydrolyze quickly at an acidic pH into primary amines, and protonated amines generate repulsion interactions with other charged species, which trigger the cytotoxics release.

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Kinetics of irreversible inhibition of choline transport in synaptosomes by ethylcholine mustard aziridinium

Summary Ethylcholine mustard aziridinium (ECMA) inhibits choline transport in synaptosomes at a half-maximal concentration of about 20 m. The rate of inhibition falls off rapidly after 10 min and the concentration dependency reaches a plateau at about 100 m. The inhibition is not removed by washing the synaptosomes, and choline and hemicholinium-3 protect the carrier against attack by the mustard. Choline efflux, particularly that stimulated by choline in the medium (transactivation) is also inhibited by the aziridinium compound. Similarly choline influx activated by preloaded internal choline is inhibited by ECMA. The mustard can enter the synaptosomes in an active form but most of the carrier is alkylated when facing the outside. Prior depolarization of the synaptosomes causes an increase in the rate of inhibition by ECMA which is proportionally about the same as the increase in choline influx also caused by depolarization. At low ECMA concentrations the rate of inhibition is that of a first-order reaction with the carrier but at high ECMA concentrations the translocation of the carrier to the outward-facing conformation controls the rate of inhibition. Using a model of choline transport with some simplifying assumptions it is possible to estimate the amount of carrier; cholinergic synaptosomes carry about six times the concentration of carrier found in noncholinergic ones. In noncholinergic synaptosomes the carrier faces predominately out, the reverse in cholinergic ones. The rate constant of carrier translocation is increased by combination with choline some six- to sevenfold to about 3.5 min^–1. The rate constant of ECMA attack on the carrier is about 440m ^–1 sec^–1.     

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing¹. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.Download video file.^{(86M, mov)}     

Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

The effect of pyridoxal 5-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC₅₀ of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (K _i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [³H]pyridoxal 5-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5-phosphate. These results indicate that pyridoxal 5-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.     

Laccase production in semi-solid cultures of Phanerochaete chrysosporium

Cultures of Phanerochaete chrysosporium, operating with an inert carrier (nylon sponge) and a non-inert carrier (barley straw), were employed in order to study laccase production during semi-solid state conditions. Manganese (IV) oxide, added to the cultures increased laccase activity 16-fold especially in barley straw cultures, in which a maximum laccase activity of 360 U/l (one unit is defined as 1 mol of 2,2-azino-di-[3-ethyl-benzothiazoline-(6)-sulphonicacid] oxidized per minute) was achieved.     

MPI code encapsulating using parallel CORBA object

This paper describes a technique that allows an MPI code to be encapsulated into a component. Our technique is based on an extension to the Common Object Request Broker Architecture (CORBA) from the OMG (Object Management Group). The proposed extensions do not modify the CORBA core infrastructure (the Object Request Broker) so that it can fully coexist with existing CORBA applications. An MPI code is seen as a new kind of CORBA object that hides most of the cumbersome problems when dealing with parallelism. Such a technique can be used to connect MPI codes to existing CORBA software infrastructures which are now being developed in the framework of several research and development projects such as JACO3^*, JULIUS^** or TENT^*** from DLR. To illustrate the concept of parallel CORBA object, we present a virtual reality application that is made of the coupling of a light simulation application (radiosity) and a visualisation tool using VRML and Java.     

Elucidation of the structure of the lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype 5 using strong alkaline degradation of the lipopolysaccharide

The products of the strong alkaline degradation of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 5 were separated by anion-exchange HPLC and studied by electrospray ionization mass spectrometry and NMR spectroscopy.It was found that two major products have the same inner core region and lipid A carbohydrate backbone but different outer core regions.The difference is in the position of a rhamnose residue,which is substituted with either an additional glucose residue or a disaccharide remainder of the degraded O-polysaccharide.The site and the configuration of the linkage between the O-polysaccharide and the core were determined and,together with published data,the structure of the so-called biological repeating unit of the O-antigen was defined.The glycosidic linkage of the 2-acetamido-2,6-dideoxy D-glucose (N-acetyl-D-quinovosamine) residue is when it links the O-polysaccharide to the core and when it connects the interior repeating units of the O-polysaccharide to each other.     

The mitochondrial tricarboxylate carrier

The tricarboxylate carrier has recently been purified from rat liver mitochondria by three distinct scientific groups using different methods. A 37–38-kDa protein has been prepared by silca gel 60 chromatography by our group (Claeys and Azzi, 1989; Glerumet al., 1990). The specific citrate transport activity of this preparation is not significantly different from that measured in mitochondria and it is inhibitable by 1,2,3-benzenetricarboxylic acid. Bisacciaet al. (1990) have reported the isolation of a 30-kDa protein by Celite 535 chromatography, and Kaplan's group (Kaplanet al., 1990) have isolated a 32.5-kDa protein by Matrex Orange, Matrex Blue, and Affi-Gel chromatography. Peptide mapping has failed to support any structural homologies between the 37–38-kDa and the 30–32.5-kD proteins. The 38-kD protein is N-terminally blocked. The peptides obtained by several cleavage procedures have been partially sequenced. Their sequence information has been used to obtain different cDNA clones by a dual approach, the polymerase chain reaction and screening of a ZAP cDNA library. The largest cDNA which could be isolated is 2,986 bp in length and contains a 1071-bp-long open reading frame and an unusually long 3 untranslated region, both of which have been completely sequenced. The protein sequence of the carrier from the first in-frame methionine is 322 amino acids in length and exhibits a molecular mass of 35,546. Comparison of the protein sequence to the sequences of the four members of the mitochondrial carrier protein family (ADP/ATP carrier, phosphate carrier, 2-oxoglutarate/malate carrier, and uncoupling protein) does not reveal significant similarity (cf. Walkeret al., 1987). A tripartite internal homology, which is a characteristic of these proteins, is not present in the sequence of the tricarboxylate carrier protein. The mRNA for the tricarboxylate carrier is expressed in rat liver and brain, but not in rat heart.     

Redox behavior of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The redox properties of the copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b were investigated. The ESR spectrum of the pMMO-containing membranes from M. trichosporium OB3b indicated a typical type II copper (II) signal (g = 2.24, A = 18.4 mT, g = 2.06, ₂= 0.84). By anaerobic addition of excess amounts of duroquinol, an optimum reductant of pMMO, the ESR spectra indicated that the copper cluster in membranes was reduced and successively oxidized by dioxygen, a substrate of pMMO. The result suggests that the copper is the active site of pMMO or an electron carrier. During the titration, the intensity of the type II copper signal decreased with decreasing potential and the multiple hyperfine structure at g = 2.06 appeared clearly. Although the copper signal did not change by treatment of the EDTA-treated membranes with duroquinol and dioxygen, the copper signal intensity decreased with decreasing potential in the redox titration. These results suggest that some redox mediators play a role as an electron carrier between the active site and a reductant, and the presence of at least two types of copper sites in pMMO- containing membranes. On the basis of the ESR spectra of the EDTA-treated membranes and the as-isolated membranes, it is concluded that one type of the copper sites functions as the active site of pMMO (A-site), and the other type of copper sites plays a role as an electron carrier (E-site)     

Selective transport of Li+ across lipid bilayer membranes mediated by an ionophore of novel design (ETH1644)

Summary The neutral noncyclic, lithium-selective ionophore ETH1644, which is structurally different from previously available ionophores of this type, is a selective carrier of Li^– in lipid bilayer membranes of various lipid composition. The ionophore forms a 21 carrier/cation complex, and the rate-limiting step in the overall transport process is the diffusion of the carrier/ion complex across the membrane.The selectivity sequence for lithiumvs. other ions normally found in biological systems is: Li⁺ (1)>Na⁺ (0.017)K⁺ (0.017) >Cl^– (0.001), Ca²⁺ and Mg²⁺ are impermeant. At neutral pH protons do not interfere with the Li⁺-carrying ability of this ionophore. On the basis of structural differences and supported by conductance data, it is argued that the improved selectivity of Li⁺ over the other alkali cations is due more to a decrease in the affinities of the ionophore for the latter cations that to an increase of its affinity to Li⁺. This ionophore can also act as a carrier of biogenic amines (catecholes, indoles and derivatives), with the structure of the permeant species and mechanism of permeation similar to that observed with the alkali cations. The selectivity sequence is: tryptamine (18.1)>phenylethylamine (11.6)> tyramine (2.4)>Li⁺(1)>serotonin (0.34)>epinephrine (0.09) >dopamine (0.05)>norepinephrine (0.02), showing the ionophore to be more selective to Li⁺ than to any of the neurotransmitters studies.     

Clinical analysis of germline copy number variation in DMD using a non-conjugate hierarchical Bayesian model

Background

Detection of copy number variants (CNVs) is an important aspect of clinical testing for several disorders, including Duchenne muscular dystrophy, and is often performed using multiplex ligation-dependent probe amplification (MLPA). However, since many genetic carrier screens depend instead on next-generation sequencing (NGS) for wider discovery of small variants, they often do not include CNV analysis. Moreover, most computational techniques developed to detect CNVs from exome sequencing data are not suitable for carrier screening, as they require matched normals, very large cohorts, or extensive gene panels.

Methods

We present a computational software package, geneCNV (http://github.com/vkozareva/geneCNV), which can identify exon-level CNVs using exome sequencing data from only a few genes. The tool relies on a hierarchical parametric model trained on a small cohort of reference samples.

Results

Using geneCNV, we accurately inferred heterozygous CNVs in the DMD gene across a cohort of 15 test subjects. These results were validated against MLPA, the current standard for clinical CNV analysis in DMD. We also benchmarked the tool’s performance against other computational techniques and found comparable or improved CNV detection in DMD using data from panels ranging from 4,000 genes to as few as 8 genes.

Conclusions

geneCNV allows for the creation of cost-effective screening panels by allowing NGS sequencing approaches to generate results equivalent to bespoke genotyping assays like MLPA. By using a parametric model to detect CNVs, it also fulfills regulatory requirements to define a reference range for a genetic test. It is freely available and can be incorporated into any Illumina sequencing pipeline to create clinical assays for detection of exon duplications and deletions.

     

Electrical properties and structural transitions in the mitochondrion

Charge carrier generation and transport in the mitochondrial lipoprotein system has been investigated by electrical conductivity, low frequency dielectric relaxation, and thermoelectric power. A parallel study was conducted on morphological/structural changes by DTA and NMR. The results obtained confirm the need to consider concurrently free charge carrier processes and polarization phenomena. All techniques show a transition at the same temperature. The steady state conductivity is correlated with main chain segmental reorientations of the phospholipid moiety below the transition and with an interfacial polarization process above it. The Seebeck coefficient provides a useful new aid to characterizing the charge carriers, confirming that they are electronic. The terminal cytochrome oxidase component was investigated separately but it largely reflected ionic impurities characteristic of the isolation process, so that the results were of no intrinsic value.     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

Isolation of a protein coded for by the permease gene of theLac operon ofEscherichia coli

Conclusions We can only presume from the results of our investigations, that the peak area of¹⁴C enrichment having neither galactosidase nor triansacetylase activity, and which is not present in the chromatograph of the permease-less (200 P) mutant, must be the protein expression of the permease (y⁺) gene ofE. coli. This protein is apparently cytoplasmic. There are, however, several alternatives to the hypothesis that this product is the membrane carrier itself. Indeed, if it is the carrier, we must explain why it appears only in the cytoplasm, and not in the membrane fraction. Had the permease been dislodged from the membrane during osmotic lysis and the subsequent buffer extraction, we should presumably have detected some residual¹⁴C enrichment in the membrane fractions. This we could not find. Moreover, accumulated evidence to date leads us to surmise that transport carriers are located in the cell membrane [4, 5, 6, 7]. What, then, is the protein coded for by the permease gene? There is the possibility that, as the galactoside transport system is an active, energy-requiring system, what we have separated is that portion of this system from which the active transport derives its energy. Alternatively, we may have located the protein (enzyme) system responsible for the synthesis of a non-protein carrier molecule. If the carrier is of a lipid or phospholipid nature (4, 6), the added¹⁴C and³H phenylalanine might not label the carrier itself but would be incorporated into an ancillary system such as that described above. All of these possibilities must certainly be explored, but it is at least evident that the techniques outlined in this report can be used successfully to isolate physically, and to characterize, cellular components linked to transport functions.The major items of equipment used in this study were provided from a grant to Prof. A. J. Birch of this department by the Nuffield Foundation.     

The significance of pericentric inversions of chromosome 2

Summary Thirteen new cases of a pericentric inversion 2 collected from different laboratories are reported. In addition 41 cases of a pericentric inversion 2 were reviewed from the literature. The pooled data were analysed using Weinberg's proband method to evaluate the risk of a carrier for either children with congenital anomalies or reproductive wastage. In the corrected sample of 166 lifeborn offspring of carriers of a pericentric inversion 2 there were five who showed phenotypic anomalies and two died a few hours after delivery. The reported anomalies are heterogeneous and probably reflect the basic risk of any couple for abnormal lifeborn offspring. There has been no observation of a lifeborn who inherited an unbalanced recombination of a parental pericentric inversion 2. A carrier of a pericentric inversion 2 obviously has an increased risk for reproductive wastage. This is indicated by (1) an increase of the rate of spontaneous abortions and (2) an increase of the rate of index patients ascertained because of previous miscarriages. The risk of a carrier of a pericentric inversion 2 for a spontaneous abortion or a stillbirth may be about twice the basic risk of the general population.