From protoplasts to gene clusters

Advances in protoplast technology underpinned many crucial developments in our understanding of the molecular biology of filamentous fungi. This review follows one of these developments, namely the discovery and analysis of difuran toxin gene clusters. Our understanding of the biosynthetic pathway of the agriculturally important toxin, aflatoxin, has been dramatically enhanced by the use of protoplasts and protoplast-based gene transformation methods. Since the identification of the first pathway genes by complementation of mutants with transforming DNA, transformation has continued to play a critical role in the elucidation of gene function and regulation. But despite the wealth of knowledge accumulated so far some fundamental questions remain to be answered. How did these gene clusters evolve? What is the biological role of aflatoxin? The discovery of homologues of aflatoxin genes in other fungal species such as the pine needle pathogen Dothistroma septosporum may help to shed some light on these questions.     

Network slicing to improve multicasting in HPC clusters

In high performance computing (HPC) resources’ extensive experiments are frequently executed. HPC resources (e.g. computing machines and switches) should be able to handle running several experiments in parallel. Typically HPC utilizes parallelization in programs, processing and data. The underlying network is seen as the only non-parallelized HPC component (i.e. no dynamic virtual slicing based on HPC jobs). In this scope we present an approach in this paper to utilize software defined networking (SDN) to parallelize HPC clusters among the different running experiments. We propose to accomplish this through two major components: A passive module (network mapper/remapper) to select for each experiment as soon as it starts the least busy resources in the network, and an SDN-HPC active load balancer to perform more complex and intelligent operations. Active load balancer can logically divide the network based on experiments’ host files. The goal is to reduce traffic to unnecessary hosts or ports. An HPC experiment should multicast, rather than broadcast to only cluster nodes that are used by the experiment. We use virtual tenant network modules in Opendaylight controller to create VLANs based on HPC experiments. In each HPC host, virtual interfaces are created to isolate traffic from the different experiments. The traffic between the different physical hosts that belong to the same experiment can be distinguished based on the VLAN ID assigned to each experiment. We evaluate the new approach using several HPC public benchmarks. Results show a significant enhancement in experiments’ performance especially when HPC cluster experiences running several heavy load experiments simultaneously. Results show also that this multi-casting approach can significantly reduce casting overhead that is caused by using a single cast for all resources in the HPC cluster. In comparison with InfiniBand networks that offer interconnect services with low latency and high bandwidth, HPC services based on SDN can provide two distinguished objectives that may not be possible with InfiniBand: The first objective is the integration of HPC with Ethernet enterprise networks and hence expanding HPC usage to much wider domains. The second objective is the ability to enable users and their applications to customize HPC services with different QoS requirements that fit the different needs of those applications and optimize the usage of HPC clusters.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro: III. DNA synthesis of lymphocytes in clusters

The lymphocytes which engage in DNA synthesis during the in vitro immune response to PPD (purified protein derivative of tuberculin) were studied by scintillation counting and in autoradiographs prepared from cultures of macrophages and immune T-lymphocyte-enriched lymphocytes. The lymphocytes in these cultures were located in three compartments: lymphocytes in macrophage-lymphocyte clusters, lymphocytes attached to macrophages but not involved in clusters, and not macrophage-attached lymphocytes. One of the cluster cells, the central lymphocyte, which is attached directly to the macrophage, was identified as the only DNA-synthesizing lymphocyte in the cluster early in the culture period. In cultures extended beyond 20 hr the increase in percentage of DNA-synthesizing lymphocytes in the cluster and macrophage-attached compartments exceeded the increase in the compartment of not macrophage-attached lymphocytes. The total amount of radiolabeled thymidine incorporated into lymphocytes in a blast transformation assay was directly proportional to the number of macrophage-lymphocyte clusters produced by the same lymphocytes in a cluster assay.     

Using repeated measurements to validate hierarchical gene clusters

MOTIVATION: Hierarchical clustering is a common approach to study protein and gene expression data. This unsupervised technique is used to find clusters of genes or proteins which are expressed in a coordinated manner across a set of conditions. Because of both the biological and technical variability, experimental repetitions are generally performed. In this work, we propose an approach to evaluate the stability of clusters derived from hierarchical clustering by taking repeated measurements into account. RESULTS: The method is based on the bootstrap technique that is used to obtain pseudo-hierarchies of genes from resampled datasets. Based on a fast dynamic programming algorithm, we compare the original hierarchy to the pseudo-hierarchies and assess the stability of the original gene clusters. Then a shuffling procedure can be used to assess the significance of the cluster stabilities. Our approach is illustrated on simulated data and on two microarray datasets. Compared to the standard hierarchical clustering methodology, it allows to point out the dubious and stable clusters, and thus avoids misleading interpretations. AVAILABILITY: The programs were developed in C and R languages.     

Caffeine clusters as transmitters of actinomycin antibiotics to DNA in solution

Using the screening model of hypochromism, we showed that caffeine forms regular clusters consisting of 8–12 molecules. Addition of 7-aminoactinomycin D (7AAMD, a fluorescent analogue of actinomycin D) to the clusters leads to its sorption on the cluster surface. Photoexcitation of 7AAMD leads to its desorption from the surface into the aqueous phase and emission of a quantum. Fluorescence of 7AAMD in the presence of caffeine clusters is quenched by dinitrophenol more weakly than without clusters (the quenching constants are ～ 85 and ～280 M^?1, respectively) due to decreased steric availability of the antibiotic to the quencher. Addition of 7AAMD-caffeine complexes to DNA leads to a long-wavelength shift in the excitation spectrum and an increase in the fluorescence intensity along with a shift of the fluorescence spectrum to the short-wavelength area. This fact reflects redistribution of the antibiotic from the caffeine surface to the hydrophobic areas inside DNA.     

Caffeine clusters as transmitters of actinomycin antibiotics to DNA in solutions

Using the screening model of hypochromism, we showed that caffeine forms regular clusters consisting of 8-12 molecules. Addition of 7-aminoactinomycin D (7AAMD, a fluorescent analogue of actinomycin D) to the clusters leads to its sorption on the cluster surface. Photoexcitation of 7AAMD leads to its desorption from the surface into the aqueous phase and emission of a quantum. Fluorescence of 7AAMD in the presence of caffeine clusters is quenched by dinitrophenol more weakly than without clusters (the quenching constants are approximately 85 and approximately 280 M(-1), respectively) due to decreased steric availability of the antibiotic to the quencher. Addition of 7AAMD-caffeine complexes to DNA leads to a long-wavelength shift in the excitation spectrum and an increase in the fluorescence intensity along with a shift of the fluorescence spectrum to the short-wavelength area. This fact reflects redistribution of the antibiotic from the caffeine surface to the hydrophobic areas inside DNA. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. IV. Synergy of lymphocytes in the formation of clusters.

T-lymphocyte-enriched lymph node lymphocytes from guinea pigs immunized with Mycobacterium tuberculosis produce clusters with macrophages when cultivated on monolayers of syngeneic purified protein derivative of tuberculin (PPD)-pulsed peritoneal macrophages. The clusters consist of a macrophage with a central lymphocyte attached to it, and several peripheral lymphocytes attached to the central one. By mechanical manipulation immune lymphocytes incubated on monolayers of PPD-pulsed macrophages were separated into those which adhered firmly to the macrophages after 4 hr of culture and those which did not adhere. While neither of the two populations was able to produce significant numbers of clusters alone, they did so in combination. The number of macrophage-lymphocyte clusters which are produced in a culture depends not only on the absolute number of immune lymphocytes in the culture, but also on the concentration of lymphocytes per area of the macrophage monolayer, with high concentrations resulting in high numbers of clusters. Autoradiographic studies showed that the DNA-synthesizing lymphocytes physically associated with macrophages were located mainly inside the clusters in cultures with high concentrations of lymphocytes but mainly outside the clusters in cultures with low concentrations of lymphocytes.     

Investigating causation in cancer clusters

Questions concerning clusters of cancer cases frequently arise in public health practice. The process of investigating any such cluster requires awareness that such case groupings can easily occur by chance and that any search for biologically meaningful causes will be severely constrained by various methodologic difficulties. These include (1) the long and probably variable latent periods between causative events and cancer diagnosis, (2) the limited numbers of cases available for study in any given cluster situation, and (3) the clinical non-specificity of cancer cases whereby no readily available means are at hand to identify the specific causes for any particular case. Evaluation of any given cluster should involve careful consideration of such limitations, together with a preliminary assessment of the specific cases involved and their community or workplace setting, before more intensive study is undertaken. Received: 22 February 1996 / Accepted in revised form: 13 June 1996     

Three-iron clusters in iron-sulfur proteins

Contents. 1. Introduction and history. 2. Characteristic spectroscopic features of 3Fe clusters. 1. General considerations. 2. M?ssbauer spectroscopy. 3. Magnetic circular dichroism (MCD) spectroscopy. 4. Electron paramagnetic resonance (EPR) spectroscopy. 5. Resonance Raman (RR) spectroscopy. 6. Extended X-ray fine-structure (EXAFS) spectroscopy. 3. Results of X-Ray diffraction studies. 4. Proteins containing or showing features characteristic of 3Fe clusters 1. Overview. 2. Ferredoxin I of Azotobacter vinelandii. 3. Ferredoxin II of Desulfovibrio gigas. 4. Aconitase from beef heart. 5. Other observations and considerations relevant to 3Fe clusters or cluster interconversions 1. Oxidative degradation of [4Fe-4S] clusters to 3Fe clusters. 2. Extrusion studies on 3Fe clusters. 3. Reconstitution of 3Fe clusters. 4. Disposition of iron ligands in cluster interconversions. 6. Do all 3Fe clusters have the same structure? Evidence for [3Fe-4S] clusters. 7. Are 3Fe clusters artifacts or biologically significant structures?     

Hydrophobic clusters in protein structures

Globular proteins fold such that the hydrophobic groups are packed inside forming hydrophobic clusters, and the hydrophilic groups are present on the surface. In this article we analyze clusters of hydrophobic groups of atoms in 781 protein structures selected from the PDB. Our analysis showed that every structure consists of two types of clusters: at least one large cluster that forms the hydrophobic core and probably dictates the protein fold; and numerous smaller clusters, which might be involved in the stabilization of the fold. We also analyzed the preference of the hydrophobic groups in each of the amino acids toward forming hydrophobic clusters. We find that hydrophobic groups from the hydrophilic amino acids also contribute toward cluster formation. Proteins 2008. © 2008 Wiley‐Liss, Inc.