Infectivity of DWV Associated to Flower Pollen: Experimental Evidence of a Horizontal Transmission Route

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.     

Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition

Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.     

Design of a cybernetic hand for perception and action

Strong motivation for developing new prosthetic hand devices is provided by the fact that low functionality and controllability—in addition to poor cosmetic appearance—are the most important reasons why amputees do not regularly use their prosthetic hands. This paper presents the design of the CyberHand, a cybernetic anthropomorphic hand intended to provide amputees with functional hand replacement. Its design was bio-inspired in terms of its modular architecture, its physical appearance, kinematics, sensorization, and actuation, and its multilevel control system. Its underactuated mechanisms allow separate control of each digit as well as thumb–finger opposition and, accordingly, can generate a multitude of grasps. Its sensory system was designed to provide proprioceptive information as well as to emulate fundamental functional properties of human tactile mechanoreceptors of specific importance for grasp-and-hold tasks. The CyberHand control system presumes just a few efferent and afferent channels and was divided in two main layers: a high-level control that interprets the user’s intention (grasp selection and required force level) and can provide pertinent sensory feedback and a low-level control responsible for actuating specific grasps and applying the desired total force by taking advantage of the intelligent mechanics. The grasps made available by the high-level controller include those fundamental for activities of daily living: cylindrical, spherical, tridigital (tripod), and lateral grasps. The modular and flexible design of the CyberHand makes it suitable for incremental development of sensorization, interfacing, and control strategies and, as such, it will be a useful tool not only for clinical research but also for addressing neuroscientific hypotheses regarding sensorimotor control.     

Excitation-contraction uncoupling during ischemia in the blood perfused dog heart

The bioluminescent of Ca(2+)-indicator, aequorin, was loaded into the left ventricular apex of blood-perfused hearts from 13 dogs for simultaneous recording of left ventricular pressure and intracellular calcium levels. During a 2 minute period of ischemia, systolic and diastolic pressures significantly decreased. In contrast, these pressure changes were associated with an increase in both systolic and diastolic calcium reaching a maximum diastolic value of 0.59 microM and a systolic value of 1.11 microM. This apparent dissociation between pressure and [Ca2+]i supports the hypothesis that changes in myofilament Ca2+ responsiveness are of major importance in modulating contractility during ischemia in large mammalian hearts.     

Effect of 1,3-1,6 β-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica)

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease.The aim of this study was to investigate the effect of 1,3–1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3–1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection.     

Determining Protein Complex Connectivity Using a Probabilistic Deletion Network Derived from Quantitative Proteomics

Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.