A life-like virtual cell membrane using discrete automata

A framework is presented that captures the discrete and probabilistic nature of molecular transport and reaction kinetics found in a living cell as well as formally representing the spatial distribution of these phenomena. This particle or agent-based approach is computationally robust and complements established methods. Namely it provides a higher level of spatial resolution than formulations based on ordinary differential equations (ODE) while offering significant advantages in computational efficiency over molecular dynamics (MD). Using this framework, a model cell membrane has been constructed with discrete particle agents that respond to local component interactions that resemble flocking or herding behavioural cues in animals. Results from simulation experiments are presented where this model cell exhibits many of the characteristic behaviours associated with its biological counterpart such as lateral diffusion, response to osmotic pressure gradients, membrane growth and cell division. Lateral diffusion rates and estimates for the membrane modulus of elasticity derived from these simple experiments fall well within a biologically relevant range of values. More importantly, these estimates were obtained by applying a simple qualitative tuning of the model membrane. Membrane growth was simulated by injecting precursor molecules into the proto-cell at different rates and produced a variety of morphologies ranging from a single large cell to a cluster of cells. The computational scalability of this methodology has been tested and results from benchmarking experiments indicate that real-time simulation of a complete bacterial cell will be possible within 10 years.     

Treatment of lesions of the rotator cuff

The impingement syndrome and tendinopathy of the rotator cuff are the most common causes (complaints) of pain and disability of the shoulder. The aim of this study is to evaluate the efficacy of a specific rehabilitative protocol, integrated with the administration of a nutritional supplement, in the conservative rehabilitative treatment, as well as in post-surgery, of patients with lesions of the rotator cuff. Two groups with syndrome of the rotator cuff were formed to follow different therapeutic courses, in relation to the choice of each subject to undergo the conservative treatment (Arm A) or the surgical one (Arm B). In Arm A the study included the association of therapy with ESWT (shock waves) with the proprioceptive Multi Joint System, for rehabilitating joint movement and muscle strength of the shoulder, and a specific nutritional supplement to reduce the pain and conserve the cartilage tissue. Between February 2009 and June 2009, we enrolled 30 subjects (randomized into three homogenous groups A1, A2, A3), average age 45±10 years, with rotator cuff syndrome with calcification of the shoulder, diagnosed through clinical examination and investigative instruments (X-ray, echography or NMR). In Arm B, from September 2009 to January 2010, we enrolled 50 patients (randomized into two groups, B1 and B2), 24 male (average age 58.4: min 28 and max 78) and 26 females (average age 59.5: min 30 and max 80), who had undergone rotator cuff operations and acromionplasty for non-massive lesions without important gleno-humeral instability, with either open or arthroscopic procedures. The analysis of the results of Arm A highlights that in terms of reducing pain the main benefits were found in Group A1 where the supplement was given. From the analysis of the data of Arm B, in both groups an improvement of the first 4 items evaluated was evident. In Group B1, 84 percent of the patients declared to be satisfied and improved and 16 percent were dissatisfied; in Group B2, where the nutritional supplement was given, 92 percent were satisfied and 8 percent were dissatisfied. In conclusion, we retain that in cases of rotator cuff syndrome, an integrated rehabilitative approach, whether conservative or post-surgical, directed at taking total control of the patient, must observe particular attention to the optimization of the articular tissular metabolic balance in order to favour better functional recovery.     

Role of Hematopoietic Cells in the Maintenance of Chronic Human Torquetenovirus Plasma Viremia

Many aspects of the life cycle of torquetenoviruses (TTVs) are essentially unexplored. In particular, it is still a matter of speculation which cell type(s) replicates the viruses and maintains the generally high viral loads found in the blood of infected hosts. In this study, we sequentially measured the TTV loads in the plasma of four TTV-positive leukemia patients who were strongly myelosuppressed and then transplanted with haploidentical hematopoietic stem cells. The findings provide clear quantitative evidence for an extremely important role of hematopoietic cells in the maintenance of TTV viremia.Torquetenoviruses (TTVs) are small naked DNA viruses distinguished by a circular single-stranded DNA genome of only 3.8 kb, classified within the newly established family Anelloviridae (7). TTVs have been found in several animal species but do not appear capable of interspecies transmission. Due to their extensive genetic heterogeneity, human TTVs have been operatively subdivided into 5 genogroups and more than 40 genotypes (4). A remarkable feature of these TTVs is their presence in the plasma of nearly all people, regardless of geographical origin, age, and health status, raising many questions about their life cycle and possible pathological implications (2, 5). Plasma loads of TTVs vary extensively in both healthy and diseased individuals, usually ranging between 10³ and 10⁷ DNA copies per ml of plasma. However, some patients, including those with selected inflammatory or neoplastic disorders, transplant recipients, and human immunodeficiency virus-infected individuals, have a tendency to carry especially high burdens of TTVs (1, 6, 13, 22-24).By studying the dynamics of TTV viremia in individuals treated with alpha interferon for hepatitis C, the kinetics of virus replication was found to be quite high, with numbers of virions released into plasma and cleared from it daily on the same order of magnitude as other chronic plasma viremia-inducing viruses, such as the hepatitis B, hepatitis C, and human immunodeficiency viruses (16). Yet, due to considerable difficulties encountered in propagating TTVs in culture and in distinguishing the virions passively adsorbed onto the cells from the ones replicating inside cells, the tissue or tissues where these large numbers of TTV virions originate have yet to be established. Given that the amino acid compositions of the capsid protein believed to mediate viral adsorption to cells are quite diverse in different TTVs (2, 3, 9), it is also possible that permissive cells vary depending on the TTV considered. Relevant studies are limited. Short-term cultures of phytohemagglutinin-stimulated peripheral lymphocytes, but not resting lymphocytes were found to permit a measurable level of TTV replication (15, 18), indicative of at least a moderate degree of lymphotropism. On the other hand, the detection of replicative forms of TTV DNA in several tissues, including bone marrow, peripheral blood mononuclear cells, and liver, has suggested that TTVs might be polytropic in nature (2, 21).In 1999, Kanda et al. (10), researching TTV plasma of bone marrow transplant recipients with a qualitative PCR, noticed that 5 out of 6 previously positive patients tested negative in a sampling collected during the myelosuppressed period and became positive again after graft reconstitution, leading them to suggest that TTV might replicate mainly in hematopoietic cells. In the present study, we further developed this observation by measuring the TTV load in sequential plasma samples obtained from four TTV-positive leukemia patients undergoing hematopoietic stem cell transplantation. This procedure basically consists of a myeloablative conditioning regimen (chemotherapy plus radiotherapy) followed by reinfusion of a positively selected CD34⁺ stem cell population. The findings are of interest because, in addition to confirming the decrease of TTV load observed by Kanda et al., they shed light on the kinetics of the effect, thus providing a better insight onto the role of hematopoietic cells in the maintenance of TTV viremia and on the life cycle of TTV in general.Table ​Table11 summarizes the main characteristics of the patients selected for the study. They were treated with 10 Gy total-body irradiation (TBI) on day 0 and received 5 mg/kg/day thiotepa on days 2 and 3, 40 mg/m²/day fludarabine on days 3 to 7, and 1.2 mg/kg/day antithymocyte globulin on days 4 to 8, and then, on day 10, they received the indicated numbers of positively selected CD34⁺ hematopoietic stem cells from HLA-haploidentical donors. Peripheral blood samples were collected for TTV studies immediately before TBI and at selected times for the next 30 days, and plasma was stored in aliquots at −80°C until DNA extraction. The assay used for TTV quantification was a previously described highly sensitive TaqMan real-time PCR having the potential to detect and quantitate all hitherto recognized genetic forms of the virus (15, 16). All samples from each patient were assayed in a single run and in triplicate, and at least two independent DNA extractions for each sample were examined. The DNA extracts obtained at time zero were also typed with a previously described panel of five distinct PCR assays (12), each specific for one of the genogroups into which TTVs are subdivided. At the start of the study, the patients had viral loads ranging from 4.7 to 6.8 log copies per ml of plasma and harbored between 1 and 3 TTV genogroups (Table ​(Table1).1). In particular, all carried genogroup 1, which is highly represented in our area (12), and two carried one or two further genogroups. Consistent with previous findings (12), the patient who harbored three genogroups was the one with the highest viral load. As shown by Fig. ​Fig.1,1, in all four patients, TBI was followed by a steady decline of TTV viremia that continued for at least 22 days and progressively brought the virus to levels very close to the detection limit of the detection/quantitation method used, corresponding to values ranging between 0.003 (patient 3) and 0.00009 (patient 1) of the loads present prior to TBI. However, in no instance did the viral loads go below the limit of sensitivity of the assay (2 × 10² TTV DNA copies per ml of plasma). Although the size of the study does not permit firm conclusions on this aspect, it is noteworthy that the extent of decline was unrelated to the type and number of infecting TTV genogroup(s) originally present in the patients.Open in a separate windowFIG. 1.Plasma TTV loads and WBC counts in the peripheral blood of the 4 patients (Pt. 1 to 4) enrolled in the study. The arrow indicates the day the patients were infused with CD34⁺ hematopoietic stem cells from HLA-haploidentical donors. The horizontal broken line represents the lower limit of sensitivity of the TTV detection method used.

TABLE 1.

Relevant parameters of the patients enrolled

A microsatellite-based genetic linkage map of the waterflea, Daphnia pulex: On the prospect of crustacean genomics

We describe the first genetic linkage map for Daphnia pulex using 185 microsatellite markers, including 115 new markers reported in this study. Our approach was to study the segregation of polymorphisms in 129 F2 progeny of one F1 hybrid obtained by crossing two genetically divergent lineages of Daphnia isolated from two Oregon populations. The map spanned 1206 Kosambi cM and had an average intermarker distance of 7 cM. Linkage groups ranged in size from 7 to 185 cM and contained 4 to 27 markers. The map revealed 12 linkage groups corresponding to the expected number of chromosomes and covers approximately 87% of the genome. Tests for random segregation of alleles at individual loci revealed that 21% of the markers showed significant transmission ratio distortion (primarily homozygote deficiency) likely due to markers being linked to deleterious recessive alleles. This map will become the anchor for the physical map of the Daphnia genome and will serve as a starting point for mapping single and quantitative trait loci affecting ecologically important phenotypes. By mapping 342 tentative orthologous gene pairs (Daphnia/Drosophila) into the Daphnia linkage map, we facilitate future comparative projects.     

Costimulation via CD55 on human CD4+ T cells mediated by CD97

Decay-accelerating factor (CD55) is a complement regulatory protein, which is expressed by most cells to protect them from complement-mediated attack. CD55 also binds CD97, an EGF-TM7 receptor constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells upon activation. Early results suggested that CD55 could further enhance T cell proliferation induced by phorbol ester treatment. The present study demonstrates that coengagement of CD55, using either cross-linking mAbs or its natural ligand CD97, and CD3 results in enhanced proliferation of human peripheral blood CD4(+) T cells, expression of the activation markers CD69 and CD25, and secretion of IL-10 and GM-CSF. Recently, an increase in T cell responsiveness in CD55(-/-) mice was shown to be mediated by a lack of complement regulation. In this study, we show that direct stimulation of CD55 on CD4(+) T cells with CD97 can modulate T cell activation but does not interfere with CD55-mediated complement regulation. Our results support a multifaceted role for CD55 in human T cell activation, constituting a further link between innate and adaptive immunity.     

Two 2[5H]-Furanones as Possible Signaling Molecules in Lactobacillus helveticus

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.     

Multilocus genetic analyses differentiate between widespread and spatially restricted cryptic species in a model ascidian

Elucidating the factors that shape species distributions has long been a fundamental goal in ecology and evolutionary biology. In spite of significant theoretical advancements, empirical studies of range limits have lagged behind. Specifically, little is known about how the attributes that allow species to expand their ranges and become widespread vary across phylogenies. Here, we studied the ascidian Botryllus schlosseri, a worldwide invasive species that is also characterized by marked genetic subdivision. Our study includes phylogenetic and population genetic data based on mitochondrial and nuclear genes, as well as polymorphic microsatellites for B. schlosseri colonies sampled from the southern and northern coasts of Europe and the eastern and western coasts of North America. We demonstrate that this well-known model organism comprises three highly divergent and probably reproductively isolated cryptic species (A, D and E), with two more (B and C) being suggested by data retrieved from GenBank. Among these, species A, recovered in all of the surveyed regions, is by far the most common and widespread. By contrast, species B-E, occurring mostly in sites from northern Europe, are considerably more geographically restricted. These findings, along with inferences made on transport opportunity, suggest that divergent evolutionary histories promoted differences in invasive potential between B. schlosseri sibling species, indicating that attributes that facilitate dramatic shifts in range limits can evolve more easily and frequently than previously thought. We propose environmental disturbance as a selective force that could have shaped the evolution of invasiveness in the B. schlosseri complex.     

Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov

Sebacinales was described in 2004 and is currently recognized as the earliest diverging lineage of mycorrhizal Basidiomycota. In addition, recent research has demonstrated that no other known fungal order harbours a broader spectrum of mycorrhizal types. Yet because of the character poor morphology of these inconspicuous fungi, a reliable systematic framework for Sebacinales is still out of reach. In order to increase the body of comparative data on Sebacinales, we followed a polyphasic approach using a sampling of seven diverse Sebacinales strains, including several isolates of Australian orchid mycorrhizae, Piriformospora indica, and a multinucleate rhizoctonia isolated from a pot culture of Glomus fasciculatum (Williams 1985) with clover. We performed molecular phylogenetic analyses from candidate barcoding regions [rDNA: internal transcribed spacer (ITS)1-5.8-ITS2, 28S; translation elongation factor 1-α (TEF)], enzymatic profiling, genome size estimation by quantitative polymerase chain reaction (PCR), and karyotype analysis using pulsed field gel electrophoresis. Here, we report significant differences in the physiological and molecular parameters inferred from these morphologically very similar strains. Particularly, our results indicate that intron sequences of the TEF gene are useful markers for Sebacinales at the species level. As a first taxonomic consequence, we describe Piriformospora williamsii as a new member of the so far monotypic genus Piriformospora and show that this genus contains still undescribed species that were recently discovered as endophytes of field-collected specimens of Anthyllis, Medicago, and Lolium in Germany.