Defining stage-specific activity of potent new inhibitors of Cryptosporidium parvum growth in vitro

Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity

The Role of Action in Affordance Perception Using Virtual Reality

Space perception in virtual reality (VR) is distorted. Does action in conjunction with an avatar\u27s presence improve perception in VR? Participants judged whether a virtual ball was within reach. Condition 1 was perception-only, where the participant was not allowed to move nor could see their arms. Condition 2 was perception with nonvisible action, where the participant could move their real arm to reach but could not see an avatar representation of the arm. Condition 3 was perception with visible action, where the participant could move and see a virtual hand that corresponded to the actual arm movement. Participants overestimated their own reach by about 15% in the avatar condition and the proprioceptive condition. The perception-only condition was the most accurate (only 5% overestimation). Response times were comparable for distances within reach but got longer in Conditions 2 and 3 when the ball was out of reach. The affordance responses (‘yes’ or ‘no’) did not correlate with response time, postural instability, nor with the head leaning forward. Instead, affordance responses mapped onto the mean magnitude of head movements. Specifically, complexity measured by effort-to-compress (ETC), which was lowest at the action boundary in the avatar condition, may helped to differentiate between experimental conditions. Our results point to the lack of expected haptic feedback as a critical variable, and the utility of complex exploration that may have contributed to the difference between the avatar and the perception-only condition

Algorithms to automatically quantify the geometric similarity of anatomical surfaces

We describe new approaches for distances between pairs of 2-dimensional surfaces (embedded in 3-dimensional space) that use local structures and global information contained in inter-structure geometric relationships. We present algorithms to automatically determine these distances as well as geometric correspondences. This is motivated by the aspiration of students of natural science to understand the continuity of form that unites the diversity of life. At present, scientists using physical traits to study evolutionary relationships among living and extinct animals analyze data extracted from carefully defined anatomical correspondence points (landmarks). Identifying and recording these landmarks is time consuming and can be done accurately only by trained morphologists. This renders these studies inaccessible to non-morphologists, and causes phenomics to lag behind genomics in elucidating evolutionary patterns. Unlike other algorithms presented for morphological correspondences our approach does not require any preliminary marking of special features or landmarks by the user. It also differs from other seminal work in computational geometry in that our algorithms are polynomial in nature and thus faster, making pairwise comparisons feasible for significantly larger numbers of digitized surfaces. We illustrate our approach using three datasets representing teeth and different bones of primates and humans, and show that it leads to highly accurate results.Comment: Changes with respect to v1, v2: an Erratum was added, correcting the references for one of the three datasets. Note that the datasets and code for this paper can be obtained from the Data Conservancy (see Download column on v1, v2

NASA aviation safety reporting system

The origins and development of the NASA Aviation Safety Reporting System (ASRS) are briefly reviewed. The results of the first quarter's activity are summarized and discussed. Examples are given of bulletins describing potential air safety hazards, and the disposition of these bulletins. During the first quarter of operation, the ASRS received 1464 reports; 1407 provided data relevant to air safety. All reports are being processed for entry into the ASRS data base. During the reporting period, 130 alert bulletins describing possible problems in the aviation system were generated and disseminated. Responses were received from FAA and others regarding 108 of the alert bulletins. Action was being taken with respect to 70 of the 108 responses received. Further studies are planned of a number of areas, including human factors problems related to automation of the ground and airborne portions of the national aviation system

Telecommunications in the IS Curriculum

A major shift away from centralized data processing to distributed system and an increasing emphasis on the use of IS technology to link individuals and entities inside and outside the orgaxiizatioit has created an imperative to integrate the study of telecoIninuiiicatioris networks into the IS curriculum. The subject of this tutorial is to exploie the appropriate dornain of telecommunications subjects to be included in the IS curriculum md to discuss various approaches to developicg arid teaching courses in telecoininunicatioiis

A scaling law for the cosmological constant from a stochastic model for cosmic structures

A set of scaling laws, based on the stochastic motions of the granular components of astronomical systems, is applied to a cosmological model with a positive cosmological constant. It follows that the mass of the dominant particle in the observable universe must be proportional to the sixth root of the cosmological constant and of the order of the nucleon mass, which is consistent with the Zeldovich scaling law. The approach is a natural way to solve the cosmic coincidence problem. On the other hand, the observed value of the cosmological constant emerges as the result of a scaling law induced by the stochastic mechanism which gives rise to the gravitationally bound systems.Comment: 7 pages, 2 figure

Molecular Beams

Contains reports on five research projects.Lincoln Laboratory (Purchase Order DDL B-00306)United States Air Force (Contract AF19(604)-7400)United States NavyUnited States Arm

Temperature-sensitive Tien Shan tree ring chronologies show multi-centennial growth trends

Two millennia-length juniper ring width chronologies, processed to preserve multi-centennial growth trends, are presented for the Alai Range of the western Tien Shan in Kirghizia. The chronologies average the information from seven near-timberline sampling sites, and likely reflect summer temperature variation. For comparison, chronologies are also built using standard dendrochronological techniques. We briefly discuss some qualities of these "inter-decadal” records, and show the low frequency components removed by the standardization process include a long-term negative trend in the first half of the last millennium and a long-term positive trend since about AD 1800. The multi-centennial scale Alai Range chronologies, where these trends are retained, are both systematically biased (but in an opposite sense) in their low frequency domains. Nevertheless, they represent the best constraints and estimates of long-term summer temperature variation, and reflect the Medieval Warm Period, the Little Ice Age, and a period of warming since about the middle of the nineteenth centur

Antibacterial Gene Transfer Across the Tree of Life

Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group

A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using air-liquid interface (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion \u3e 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions