Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.     

Dynamic partitioning into lipid rafts controls the endo-exocytic cycle of the alphaL/beta2 integrin, LFA-1, during leukocyte chemotaxis

Cell migration entails the dynamic redistribution of adhesion receptors from the cell rear toward the cell front, where they form new protrusions and adhesions. This process may involve regulated endo-exocytosis of integrins. Here we show that in primary neutrophils unengaged alphaL/beta2 integrin (LFA-1) is internalized and rapidly recycled upon chemoattractant stimulation via a clathrin-independent, cholesterol-sensitive pathway involving dynamic partitioning into detergent-resistant membranes (DRM). Persistent DRM association is required for recycling of the internalized receptor because 1) >90% of endocytosed LFA-1 is associated with DRM, and a large fraction of the internalized receptor colocalizes intracellularly with markers of DRM and the recycling endocytic compartment; 2) a recycling-defective mutant (alphaL/beta2Y735A) dissociates rapidly from DRM upon being endocytosed and is subsequently diverted into a late endosomal pathway; and 3) a dominant negative Rab11 mutant (Rab11S25N) induces intracellular accumulation of endocytosed alphaL/beta2 and prevents its enrichment in chemoattractant-induced lamellipodia. Notably, chemokine-induced migration of neutrophils over immobilized ICAM-1 is abrogated by cholesterol-sequestering agents. We propose that DRM-associated endocytosis allows efficient retrieval of integrins, as they detach from their ligands, followed by polarized recycling to areas of the plasma membrane, such as lamellipodia, where they establish new adhesive interactions and promote outside-in signaling events.     

A simple mathematical model of the interaction between intracranial pressure and cerebral hemodynamics

Ursino, Mauro, and Carlo Alberto Lodi. A simplemathematical model of the interaction between intracranial pressure andcerebral hemodynamics. J. Appl.Physiol. 82(4): 1256-1269, 1997.A simplemathematical model of intracranial pressure (ICP) dynamics oriented toclinical practice is presented. It includes the hemodynamics of thearterial-arteriolar cerebrovascular bed, cerebrospinal fluid (CSF)production and reabsorption processes, the nonlinear pressure-volumerelationship of the craniospinal compartment, and a Starling resistormechanism for the cerebral veins. Moreover, arterioles are controlledby cerebral autoregulation mechanisms, which are simulated by means ofa time constant and a sigmoidal static characteristic. The model isused to simulate interactions between ICP, cerebral blood volume, andautoregulation. Three different related phenomena are analyzed: thegeneration of plateau waves, the effect of acute arterial hypotensionon ICP, and the role of cerebral hemodynamics during pressure-volume index (PVI) tests. Simulation results suggest the following:1) ICP dynamics may become unstablein patients with elevated CSF outflow resistance and decreasedintracranial compliance, provided cerebral autoregulation is efficient.Instability manifests itself with the occurrence of self-sustainedplateau waves. 2) Moderate acutearterial hypotension may have completely different effects on ICP,depending on the value of model parameters. If physiological compensatory mechanisms (CSF circulation and intracranial storage capacity) are efficient, acute hypotension has only negligible effectson ICP and cerebral blood flow (CBF). If these compensatory mechanismsare poor, even modest hypotension may induce a large transient increasein ICP and a significant transient reduction in CBF, with risks ofsecondary brain damage. 3) The ICPresponse to a bolus injection (PVI test) is sharply affected, viacerebral blood volume changes, by cerebral hemodynamics andautoregulation. We suggest that PVI tests may be used to extractinformation not only on intracranial compliance and CSF circulation,but also on the status of mechanisms controlling CBF.

     

Relation between fitness tests and match performance in elite Italian soccer referees

This study examined the relation between field-test results and match performance in elite Italian soccer referees. Subjects (n = 22) were all experienced elite-level referees enrolled in the Commissione Arbitri Nazionali (CAN) and thus officiating in the Serie A and B Italian championships. Referees were, on separate occasions, tested for fitness (50-m, 200-m, and 12-minute run tests) and observed a minimum of 1 and a maximum of 3 times (n = 39) during Serie A matches. Match analyses were performed considering 11 match activity categories. Analyses of correlations were performed considering 50-m, 200-m, and 12-minute run test performances as independent variables and total distance, maximal speed distance (runs performed at speeds faster than 24 km.h-1), and high-intensity activity distance (runs performed at speeds faster than 18 km.h-1, high intensity activity [HIA]) as dependent variables. Statistical significance was set at p 相似文献   

Identification of trophic interactions between <Emphasis Type="Italic">Macrolophus caliginosus</Emphasis> (Heteroptera: Miridae) and <Emphasis Type="Italic">Myzus persicae</Emphasis> (Homoptera: Aphididae) using real time PCR

Understanding food web interactions in native or agricultural ecosystems is an important step towards establishing sustainable pest management strategies. While the role of generalist predators as biological control agents is increasingly appreciated, the study of trophic interactions between individual predator species and their prey provides practical difficulties. Recently, different approaches have been suggested to determine prey items from predator guts using molecular methods. Macrolophus caliginosus is a generalist predator active in herbaceous agro-ecosystems. We developed a system to identify the DNA of its prey after ingestion, using Myzus persicae as a model. Esterase (MpEST) and cytochrome oxidase I (MpCOI) genes were targeted in the aphid, while M. caliginosus COI gene was used as control for predator DNA. Real time PCR proved to be specific and sensitive enough to detect prey DNA upon ingestion after feeding experiments. The system provided a linear amplification response with only 10 fg of prey genomic DNA as template. The detection system of MpCOI gene was more sensitive than MpEST, while the detection period was similar for both genes. Possibilities for using the system in ecological and biosafety studies with regard to sustainable pest management are discussed.

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at − 5 °C, and then cooled slowly to − 30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation–dehydration, vitrification, encapsulation–vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants.     

Sexual selection and maintenance of sex: evidence from comparisons of rates of genomic accumulation of mutations and divergence of sex-related genes in sexual and hermaphroditic species of Caenorhabditis

Several hypotheses have been proposed to explain the persistenceof dioecy despite the reproductive advantages conferred to hermaphrodites,including greater efficiency at purging deleterious mutationsin the former. Dioecy can benefit from both mutation purgingand accelerated evolution by bringing together beneficial mutationsin the same individual via recombination and shuffling of genotypes.In addition, mathematical treatment has shown that sexual selectionis also capable of mitigating the cost of maintaining separatesexes by increasing the overall fitness of sexual populations,and genomic comparisons have shown that sexual selection canlead to accelerated evolution. Here, we examine the advantagesof dioecy versus hermaphroditism by comparing the rate of evolutionin sex-related genes and the rate of accumulation of deleteriousmutations using a large number of orthologs (11,493) in thedioecious Caenorhabditis remanei and the hermaphroditic Caenorhabditisbriggsae. We have used this data set to estimate the deleteriousmutation rate per generation, U, in both species and find thatalthough it is significantly higher in hermaphrodites, bothspecies are at least 2 orders of magnitude lower than the valuerequired to explain the persistence of sex by efficiency atpurging deleterious mutations alone. We also find that genesexpressed in sperm are evolving rapidly in both species; however,they show a greater increase in their rate of evolution relativeto genes expressed in other tissues in C. remanei, suggestingstronger sexual selection pressure acting on these genes indioecious species. Interestingly, the persistence of a signalof rapid evolution of sperm genes in C. briggsae suggests arecent evolutionary origin of hermaphrodism in this lineage.Our results provide empirical evidence of increased sexual selectionpressure in dioecious animals, supporting the possibility thatsexual selection may play an important role in the maintenanceof sexual reproduction.     

Revisiting Street Intersections Using Slot-Based Systems

Since their appearance at the end of the 19th century, traffic lights have been the primary mode of granting access to road intersections. Today, this centuries-old technology is challenged by advances in intelligent transportation, which are opening the way to new solutions built upon slot-based systems similar to those commonly used in aerial traffic: what we call Slot-based Intersections (SIs). Despite simulation-based evidence of the potential benefits of SIs, a comprehensive, analytical framework to compare their relative performance with traffic lights is still lacking. Here, we develop such a framework. We approach the problem in a novel way, by generalizing classical queuing theory. Having defined safety conditions, we characterize capacity and delay of SIs. In the 2-road crossing configuration, we provide a capacity-optimal SI management system. For arbitrary intersection configurations, near-optimal solutions are developed. Results theoretically show that transitioning from a traffic light system to SI has the potential of doubling capacity and significantly reducing delays. This suggests a reduction of non-linear dynamics induced by intersection bottlenecks, with positive impact on the road network. Such findings can provide transportation engineers and planners with crucial insights as they prepare to manage the transition towards a more intelligent transportation infrastructure in cities.