Perforin gene defects in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, rapidly fatal, autosomal recessive immune disorder characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. Linkage analyses indicate that FHL is genetically heterogeneous and linked to 9q21.3-22, 10q21-22, or another as yet undefined locus. Sequencing of the coding regions of the perforin gene of eight unrelated 10q21-22-linked FHL patients revealed homozygous nonsense mutations in four patients and missense mutations in the other four patients. Cultured lymphocytes from patients had defective cytotoxic activity, and immunostaining revealed little or no perforin in the granules. Thus, defects in perforin are responsible for 10q21-22-linked FHL. Perforin-based effector systems are, therefore, involved not only in the lysis of abnormal cells but also in the down-regulation of cellular immune activation.     

How a DNA polymerase clamp loader opens a sliding clamp

Processive chromosomal replication relies on sliding DNA clamps, which are loaded onto DNA by pentameric clamp loader complexes belonging to the AAA+ family of adenosine triphosphatases (ATPases). We present structures for the ATP-bound state of the clamp loader complex from bacteriophage T4, bound to an open clamp and primer-template DNA. The clamp loader traps a spiral conformation of the open clamp so that both the loader and the clamp match the helical symmetry of DNA. One structure reveals that ATP has been hydrolyzed in one subunit and suggests that clamp closure and ejection of the loader involves disruption of the ATP-dependent match in symmetry. The structures explain how synergy among the loader, the clamp, and DNA can trigger ATP hydrolysis and release of the closed clamp on DNA.     

A participatory farming system approach for sustainable broomrape (Orobanche spp.) management in the Near East and North Africa

Broomrapes (Orobanche spp.) are aggressive and damaging parasitic weeds which have a tremendous impact on agriculture in East Africa, the Mediterranean region and the Middle East. Despite the availability of technologies to control broomrapes in economically important crops, Orobanche infestation continues to increase, threatening the livelihoods of millions of farmers. Many of the technologies developed have not been effectively disseminated and there has been little or zero adoption by farmers—who continue to use ineffective management practices that exacerbate the problem. The adaptation and dissemination of appropriate management practices are major priorities in broomrape control. However, such work must take into consideration the specific socio-economic characteristics of individual farming systems. Orobanche is a community threat and effective management requires a community-based integrated management approach. Recognizing the central role of farmers in parasitic weed management, a technical cooperation project (TCP) involving FAO, ICARDA and seven countries in the Near East and North Africa (NENA) region was implemented to improve the dissemination of knowledge and skills by using a farmer field school approach: a form of education that uses experiential learning methods to build farmers’ expertise. This paper reviews conventional Orobanche research and development approaches, and highlights weaknesses in the management of the parasitic weed using these approaches as opposed to participatory approaches. The benefits and challenges of participatory farming system approaches in relation to integrated broomrape management (IBM) are also discussed. Lessons learned from achieving community ownership of, and institutional support for, IBM could be applied to other sectors (e.g. public health) in which there is a need for institutional learning and reform. Recommendations are made that include regional collaboration within the framework of a proposed Near East and North Africa Orobanche Management Network (NENAOMAN).     

Combination of Trabectedin and Gemcitabine for Advanced Soft Tissue Sarcomas: Results of a Phase I Dose Escalating Trial of the German Interdisciplinary Sarcoma Group (GISG)

Background: Evaluation of the potential efficacy and safety of combination therapies for advanced soft tissue sarcomas (STS) has increased substantially after approval of trabectedin and pazopanib. Trabectedin’s introduction in Europe in 2007 depended mainly on its activity in so-called L-sarcomas (liposarcoma and leiomyosarcoma); combination of trabectedin with other chemotherapies used in STS seems of particular interest. Methods: We initiated within the German Interdisciplinary Sarcoma Group (GISG) a phase I dose escalating trial evaluating the combination of trabectedin and gemcitabine in patients with advanced and/or metastatic L-sarcomas (GISG-02; ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01426633","term_id":"NCT01426633"}}NCT01426633). Patients were treated with increasing doses of trabectedin and gemcitabine. The primary endpoint was to determine the maximum tolerated dose. Results: Five patients were included in the study. Two patients were treated on dose level 1 comprising trabectedin 0.9 mg/m² on day 1 and gemcitabine 700 mg/m² on days 1 + 8, every 3 weeks. Due to dose-limiting toxicity (DLT) in both patients (elevated transaminases and thrombocytopenia), an additional three patients were treated on dose level −1 with trabectedin 0.7 mg/m² plus gemcitabine 700 mg/m². Of these three patients, two demonstrated another DLT; therefore, the trial was stopped and none of the dose levels could be recommended for phase II testing. Conclusion: The GISG-02 phase I study was stopped with the conclusion that the combination of gemcitabine and trabectedin is generally not recommended for the treatment of patients with advanced and/or metastatic leiomyosarcoma or liposarcoma. Also, this phase I study strongly supports the necessity for careful evaluation of combination therapies.     

Detection of nodavirus in barramundi, Lates calcarifer (Bloch), using recombinant coat protein-based ELISA and RT–PCR

The coat protein encoded by the nodavirus RNA2 gene originally isolated from greasy grouper, Epinephelus tauvina , was cloned, expressed as a recombinant polyhistidine-tailed fusion protein and characterized by immunoblot analysis. The purified recombinant protein was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) to detect body exudate and plasma antibodies against the coat protein in both experimentally infected and commercial barramundi. In addition, the nucleotide sequence was employed to develop a RT–PCR detection assay based on the T4 region. The results showed that the virus could be detected as early as 3 days post-infection by RT–PCR while antibodies against the recombinant coat protein were detectable on day 6 post-infection. Among 112 commercial barramundi samples collected from October 1999 to April 2000, 9% showed positive ELISA results which were further verified by Western blot.     

Tracking bowfin with acoustic telemetry: Insight into the ecology of a living fossil

Little is known about the spatial ecology and behaviour of bowfin (Amia calva), despite the fact that it is an important freshwater carnivore, the last living member of the Amiiformes and effectively a living fossil. In the summer of 2013, acoustic telemetry transmitters were surgically implanted in ten bowfin captured in Toronto Harbour on Lake Ontario. Using a stationary acoustic telemetry array that covered most of the 18‐km² harbour, the residency and movement patterns of bowfin were tracked from their release until November 2014. Detected bowfin ranged in size from 562 to 725 mm total length and included six males and three females (one female was not detected). Bowfin showed high site fidelity with most fish detections concentrated in embayments and within the Toronto Islands, areas characterised by relatively high stable water temperatures and submerged vegetative cover. Statistical modelling revealed that bowfin residency was significantly affected by season, body size, site‐specific estimates of vegetative cover and an interaction between body size and season. Bowfin residency increased with vegetative cover and was highest for large fish during the winter and fall months. Despite the overall high site fidelity exhibited by individuals, several bowfin were mobile over the spring and summer months and moved 5.2–12.9 km among telemetry receivers in the inner and outer harbours. The results of this study provide insight into the seasonal habitat preference, home range size and activity level of this unique fish.     

Characterization of stages of Hepatozoon americanum and of parasitized canine host cells

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in "onion skin" cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a "tail" in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.     

Acellular Dermal Matrix for Surgical Repair of Ventral Hernia in Horses

The aim of the present study was to evaluate acellular dermal matrix (ADM) of rat origin for the repair of ventral hernia in horses. The skin from rats, to be used as a graft, was de-epithelialized using hypertonic solution and further decellularized with 0.5% sodium dodecyl sulfate and 0.25% tri-(n-butyl)-phosphate. Under general anesthesia, the hernial ring was exposed and repaired with the ADM graft using inlay graft technique. Blood samples were collected at postimplantation days 0, 15, and 30 and were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis to assess the serum protein concentration of the animals, as well as for gelatin zymography for the identification of matrix metalloproteinases. All animals had an uneventful recovery without clinical signs of wound dehiscence, infection, or recurrence of hernias during 6-month follow-up period. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of concentration of the serum proteins revealed that this was increased at day 15 and had decreased again at day 30. Gelatin zymography showed only one major band of 92 kDa in the serum of all the horses with the implant, but the relative amount of 92 kDa was higher at day 15 as compared with day 0 and day 30. It may be concluded that ADM of rat origin can be used safely in horses for repair of ventral hernia.     

Multiple statistical approaches of community fingerprint data reveal bacterial populations associated with general disease suppression arising from the application of different organic field management strategies

Multiple statistical analyses of terminal restriction fragment length polymorphism (T-RFLP) data were used to screen and identify bacterial populations involved in general disease suppression in an organically managed soil. Prior to sampling three different management strategies (i.e. mixed hay (H), tilled fallowing and open-field vegetables production) were used during the transition from conventional to organic farming, with and without compost amendment. The H transition strategy consistently led to the lowest damping-off disease incidence on two different crops in separate greenhouse and field experiments. Bacterial population structure in bulk soil and the rhizosphere of both crops was characterized using T-RFLP analyses of amplified 16S rDNA sequences. First, principal component analysis (PCA) revealed changes in the relative abundance of bacterial terminal restriction fragments (TRF) in response to transition strategy and/or compost amendment in eight different experimental contexts. In each context, a different subset of TRF substantially contributed to the variation along the first two principal components. However, terminal restriction fragment M148 contributed significantly to the observed variation in 6 out of the 8 experiments, and moderately in the remaining 2 experiments. As a second approach, nonparametric analyses of variance revealed that the relative abundance of TRF differed among treatments. While the responsive subsets identified varied somewhat by experimental context, M137, M139 and M141 were more abundant in samples from the H transition strategy in multiple experimental contexts. Subsequent correlation analyses revealed that TRF associated with disease suppressive treatments (i.e. H with and without compost) were frequently negatively correlated with damping-off disease incidence. As a group, these TRF were disproportionately associated with lower disease levels further indicating their role in disease suppression. Interestingly, in silico analysis of the bacterial 16S rDNA sequence database revealed that the TRF identified in this study (e.g. M137, M139, M141, and M148) might correspond to well-characterized genera of bacterial biological control agents.