Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila

The dachs gene was first identified almost a century ago based on its requirements for appendage growth, but has been relatively little studied. Here, we describe the phenotypes of strong dachs mutations, report the cloning of the dachs gene, characterize the localization of Dachs protein, and investigate the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. Our results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development.     

Identification by polymerase chain reaction (PCR) of Marshallagia marshalli and Ostertagia gruehneri from Svalbard reindeer

A polymerase chain reaction (PCR) assay to identify two common abomasal nematodes Marshallagia marshalli and Ostertagia gruehneri of Svalbard reindeer was developed. Species-specific PCR primers were designed from internal transcribed spacer (ITS)-2 sequences of rDNA and validated using morphologically identified adult male and female nematodes. Using the species-specific primers, a 110 bp fragment was amplified from M. marshalli and its minor morph Marshallagia occidentalis and a 149 bp fragment was amplified from Ostertagia gruehneri and its minor morph Ostertagia arctica. No PCR products were amplified from the third rare species, Teladorsagia circumcincta, or DNA from the reindeer host. The assay provides a useful tool to estimate species composition for both sexes in this nematode community.     

A flexible multi-dimensional QoS performance measure framework for distributed heterogeneous systems

When users’ tasks in a distributed heterogeneous computing environment (e.g., cluster of heterogeneous computers) are allocated resources, the total demand placed on some system resources by the tasks, for a given interval of time, may exceed the availability of those resources. In such a case, some tasks may receive degraded service or be dropped from the system. One part of a measure to quantify the success of a resource management system (RMS) in such a distributed environment is the collective value of the tasks completed during an interval of time, as perceived by the user, application, or policy maker. The Flexible Integrated System Capability (FISC) measure presented here is a measure for quantifying this collective value. The FISC measure is a flexible multi-dimensional measure such that any task attribute can be inserted and may include priorities, versions of a task or data, deadlines, situational mode, security, application- and domain-specific QoS, and task dependencies. For an environment where it is important to investigate how well data communication requests are satisfied, the data communication request satisfied can be the basis of the FISC measure instead of tasks completed. The motivation behind the FISC measure is to determine the performance of resource management schemes if tasks have multiple attributes that needs to be satisfied. The goal of this measure is to compare the results of different resource management heuristics that are trying to achieve the same performance objective but with different approaches. This research was supported by the DARPA/ITO Quorum Program, by the DARPA/ISO BADD Program and the Office of Naval Research under ONR grant number N00014-97-1-0804, by the DARPA/ITO AICE program under contract numbers DABT63-99-C-0010 and DABT63-99-C-0012, and by the Colorado State University George T. Abell Endowment. Intel and Microsoft donated some of the equipment used in this research. Jong-Kook Kim is pursuing a Ph.D. degree from the School of Electrical and Computer Engineering at Purdue University (expected in August 2004). Jong-Kook received his M.S. degree in electrical and computer engineering from Purdue University in May 2000. He received his B.S. degree in electronic engineering from Korea University, Seoul, Korea in 1998. He has presented his work at several international conferences and has been a reviewer for numerous conferences and journals. His research interests include heterogeneous distributed computing, computer architecture, performance measure, resource management, evolutionary heuristics, and power-aware computing. He is a student member of the IEEE, IEEE Computer Society, and ACM. Debra Hensgen is a member of the Research and Evaluation Team at OpenTV in Mountain View, California. OpenTV produces middleware for set-top boxes in support of interactive television. She received her Ph.D. in the area of Distributed Operating Systems from the University of Kentucky. Prior to moving to private industry, as an Associate Professor in the systems area, she worked with students and colleagues to design and develop tools and systems for resource management, network re-routing algorithms and systems that preserve quality of service guarantees, and visualization tools for performance debugging of parallel and distributed systems. She has published numerous papers concerning her contributions to the Concurra toolkit for automatically generating safe, efficient concurrent code, the Graze parallel processing performance debugger, the SAAM path information base, and the SmartNet and MSHN Resource Management Systems. Taylor Kidd is currently a Software Architect for Vidiom Systems in Portland Oregon. His current work involves the writing of multi-company industrial specifications and the architecting of software systems for the digital cable television industry. He has been involved in the establishment of international specifications for digital interactive television in both Europe and in the US. Prior to his current position, Dr. Kidd has been a researcher for the US Navy as well as an Associate Professor at the Naval Postgraduate School. Dr Kidd received his Ph.D. in Electrical Engineering in 1991 from the University of California, San Diego. H. J. Siegel was appointed the George T. Abell Endowed Chair Distinguished Professor of Electrical and Computer Engineering at Colorado State University (CSU) in August 2001, where he is also a Professor of Computer Science. In December 2002, he became the first Director of the CSU Information Science and Technology Center (ISTeC). ISTeC is a university-wide organization for promoting, facilitating, and enhancing CSU’s research, education, and outreach activities pertaining to the design and innovative application of computer, communication, and information systems. From 1976 to 2001, he was a professor at Purdue University. He received two BS degrees from MIT, and the MA, MSE, and PhD degrees from Princeton University. His research interests include parallel and distributed computing, heterogeneous computing, robust computing systems, parallel algorithms, parallel machine interconnection networks, and reconfigurable parallel computer systems. He has co-authored over 300 published papers on parallel and distributed computing and communication, is an IEEE Fellow, is an ACM Fellow, was a Coeditor-in-Chief of the Journal of Parallel and Distributed Computing, and was on the Editorial Boards of both the IEEE Transactions on Parallel and Distributed Systems and the IEEE Transactions on Computers. He was Program Chair/Co-Chair of three major international conferences, General Chair/Co-Chair of four international conferences, and Chair/Co-Chair of five workshops. He has been an international keynote speaker and tutorial lecturer, and has consulted for industry and government. David St. John is Chief Information Officer for WeatherFlow, Inc., a weather services company specializing in coastal weather observations and forecasts. He received a master’s degree in Engineering from the University of California, Irvine. He spent several years as the head of staff on the Management System for Heterogeneous Networks project in the Computer Science Department of the Naval Postgraduate School. His current relationship with cluster computing is as a user of the Regional Atmospheric Modeling System (RAMS), a numerical weather model developed at Colorado State University. WeatherFlow runs RAMS operationally on a Linux-based cluster. Cynthia Irvine is a Professor of Computer Science at the Naval Postgraduate School in Monterey, California. She received her Ph.D. from Case Western Reserve University and her B.A. in Physics from Rice University. She joined the faculty of the Naval Postgraduate School in 1994. Previously she worked in industry on the development of high assurance secure systems. In 2001, Dr. Irvine received the Naval Information Assurance Award. Dr. Irvine is the Director of the Center for Information Systems Security Studies and Research at the Naval Postgraduate School. She has served on special panels for NSF, DARPA, and OSD. In the area of computer security education Dr. Irvine has most recently served as the general chair of the Third World Conference on Information Security Education and the Fifth Workshop on Education in Computer Security. She co-chaired the NSF workshop on Cyber-security Workforce Needs Assessment and Educational Innovation and was a participant in the Computing Research Association/NSF sponsored Grand Challenges in Information Assurance meeting. She is a member of the editorial board of the Journal of Information Warfare and has served as a reviewer and/or program committee member of a variety of security related conferences. She has written over 100 papers and articles and has supervised the work of over 80 students. Professor Irvine is a member of the ACM, the AAS, a life member of the ASP, and a Senior Member of the IEEE. Timothy E. Levin is a Research Associate Professor at the Naval Postgraduate School. He has spent over 18 years working in the design, development, evaluation, and verification of secure computer systems, including operating systems, databases and networks. His current research interests include high assurance system design and analysis, development of models and methods for the dynamic selection of QoS security attributes, and the application of formal methods to the development of secure computer systems. Viktor K. Prasanna received his BS in Electronics Engineering from the Bangalore University and his MS from the School of Automation, Indian Institute of Science. He obtained his Ph.D. in Computer Science from the Pennsylvania State University in 1983. Currently, he is a Professor in the Department of Electrical Engineering as well as in the Department of Computer Science at the University of Southern California, Los Angeles. He is also an associate member of the Center for Applied Mathematical Sciences (CAMS) at USC. He served as the Division Director for the Computer Engineering Division during 1994–98. His research interests include parallel and distributed systems, embedded systems, configurable architectures and high performance computing. Dr. Prasanna has published extensively and consulted for industries in the above areas. He has served on the organizing committees of several international meetings in VLSI computations, parallel computation, and high performance computing. He is the Steering Co-chair of the International Parallel and Distributed Processing Symposium [merged IEEE International Parallel Processing Symposium (IPPS) and the Symposium on Parallel and Distributed Processing (SPDP)] and is the Steering Chair of the International Conference on High Performance Computing(HiPC). He serves on the editorial boards of the Journal of Parallel and Distributed Computing and the Proceedings of the IEEE. He is the Editor-in-Chief of the IEEE Transactions on Computers. He was the founding Chair of the IEEE Computer Society Technical Committee on Parallel Processing. He is a Fellow of the IEEE. Richard F. Freund is the originator of GridIQ’s network scheduling concepts that arose from mathematical and computing approaches he developed for the Department of Defense in the early 1980’s. Dr. Freund has over twenty-five years experience in computational mathematics, algorithm design, high performance computing, distributed computing, network planning, and heterogeneous scheduling. Since 1989, Dr. Freund has published over 45 journal articles in these fields. He has also been an editor of special editions of IEEE Computer and the Journal of Parallel and Distributed Computing. In addition, he is a founder of the Heterogeneous Computing Workshop, held annually in conjunction with the International Parallel Processing Symposium. Dr. Freund is the recipient of many awards, which includes the prestigious Department of Defense Meritorious Civilian Service Award in 1984 and the Lauritsen-Bennet Award from the Space and Naval Warfare Systems Command in San Diego, California.     

Drug Repurposing: A Systematic Approach to Evaluate Candidate Oral Neuroprotective Interventions for Secondary Progressive Multiple Sclerosis

Objective

To develop and implement an evidence based framework to select, from drugs already licenced, candidate oral neuroprotective drugs to be tested in secondary progressive multiple sclerosis.

Design

Systematic review of clinical studies of oral putative neuroprotective therapies in MS and four other neurodegenerative diseases with shared pathological features, followed by systematic review and meta-analyses of the in vivo experimental data for those interventions. We presented summary data to an international multi-disciplinary committee, which assessed each drug in turn using pre-specified criteria including consideration of mechanism of action.

Results

We identified a short list of fifty-two candidate interventions. After review of all clinical and pre-clinical evidence we identified ibudilast, riluzole, amiloride, pirfenidone, fluoxetine, oxcarbazepine, and the polyunsaturated fatty-acid class (Linoleic Acid, Lipoic acid; Omega-3 fatty acid, Max EPA oil) as lead candidates for clinical evaluation.

Conclusions

We demonstrate a standardised and systematic approach to candidate identification for drug rescue and repurposing trials that can be applied widely to neurodegenerative disorders.     

There is no ‘Conundrum’ of InsP6

Indirect assays have claimed to quantify phytate (InsP₆) levels in human biofluids, but these have been based on the initial assumption that InsP₆ is there, an assumption that our more direct assays disprove. We have shown that InsP₆ does not and cannot (because of the presence of an active InsP₆ phosphatase in serum) exist in mammalian serum or urine. Therefore, any physiological effects of dietary InsP₆ can only be due either to its actions in the gut as a polyvalent cation chelator, or to inositol generated by its dephosphorylation by gut microflora.We are grateful to Dr Vucenik for bringing up a number of interesting points.It is true that we have not quantified the dietary intakes of our human donors any more (but also hardly any less) than has been done by those groups claiming that InsP₆ is present in bodily fluids. As a qualitative observation we should point out that in fact all our donors for ref. [1] do have a regular intake of dietary cereals and indeed, one is a strict vegetarian on a high cereal diet. But it is quantification that reveals this to be a specious issue. The limits of detection in our two relevant publications [1,2] for InsP₆ in plasma and urine were, respectively, around two and three orders of magnitude lower than the levels claimed to be present by Grases et al. [3] in the fluids of experimentally phytate-deprived human subjects. These numbers make the argument that we could not detect any InsP₆ simply because we chose donors on the ‘wrong’ diet untenable.So how have those many claims that InsP₆ is present in body fluids come about? For most of them, the simple answer appears to be that the assays used are indirect and are based entirely on the assumption that InsP₆ is present in the first place. Thus, for example, Valiente and co-workers [4,5] and Chen and co-workers [6,7] measured organic phosphate remaining after a series of fractionations of urine samples and simply assumed it was due to InsP₆, as did March et al. measuring inorganic phosphate after a similar protocol [8]. Grases co-workers [9] have used extensively a less indirect assay, which, after initial ion chromatography and dephosphorylation by a phytase, measures myo-inositol by mass spectrometry, but nevertheless the assay starts with the assumption that InsP₆ is there and that this is what they are quantifying. More recently, direct quantification of InsP₆ in plasma by mass spectrometry has been claimed [10] on the basis that there are peaks in plasma at m/z 624 running near where InsP₆ standards elute in two different HPLC separations [10,11]. But no evidence is presented to show even that these peaks are the same compound, let alone any data to establish firmly that InsP₆ is present, e.g. a minimal requirement of m/z quantified to two decimal places with allowance for C₁₃ content or a full disintegration fingerprint (see also [12]). Any quantified misidentification is likely to have a stochastic element to it, and it is noteworthy that Perelló & Grases have stated [11, p. 255]: ‘…we have found some humans and rats having undetectable [InsP₆], probably depending on their diet or other unknown factors’. In the light of the preceding discussion, we can offer a simpler explanation: the InsP₆ was never there in the first place.In contrast to these claims we have, using two entirely independent specific and sensitive assays with quantified spiking recovery, unambiguously shown that InsP₆ is not present in plasma or urine. This is crucial and central to the whole debate about the actions of dietary InsP₆, because it means that InsP₆ never enters the blood. It is only absorbed after being dephosphorylated, principally to inositol (see [1,2] for further discussion). Ironically, the most direct evidence for this lies in Dr Vucenik''s own data in experiments examining the fate of radioactive InsP₆ fed to animals, in which only inositol was detected in the blood [13]. This particular study was, as Dr Vucenik points out in her letter, conducted on mice. However, exactly the same conclusion (i.e. InsP₆ does not enter the circulation from the gut) is equally clear in her earlier study [14], which she did not cite and which was indeed on rats; does this omission ‘reflect poorly’ on Dr Vucenik''s own ‘report and the author''s credibility in culling scientific data’?In short, dietary InsP₆ can have only two fates: it can stay in the gut, ultimately to be defecated [15], and while it is there it can chelate metal ions to alter their uptake from the gut into the body. This is no ‘straw-man’ and is certainly the most likely explanation for all of the effects of InsP₆ on cultured cells, which comprise the majority of the reports cited by Dr Vucenik. Alternatively, InsP₆ can be converted to inositol (principally by the gut microflora [15]) and be taken up as such into the circulation; were any InsP₆ to get into the blood it would in any case be rapidly dephosphorylated by the phosphatase activity we have shown to be present in human plasma [1].For animal studies, we have raised the possibility [1,2] that it is the inositol so generated (Vitamin Bh, harmless as far as we know) that is the active mediator of any reported beneficial effects of dietary InsP₆. We note that most of the websites touting InsP₆ as a dietary supplement advocate inositol as an important (essential?) co-supplement; that the only human cancer study highlighted as important by Dr Vucenik that we could examine [16] did not administer InsP₆ alone, but only in conjunction with inositol; and that in the few studies where the separate contributions of inositol and InsP₆ have been considered, there are data suggesting that it may be the inositol that matters (e.g. fig. 1 of [17]). Moreover, we are not the only ones to suggest this idea. In the Discussion of their paper (on mice) in which InsP₆ was shown not to enter the blood from the gut [13], Dr Vucenik and her colleagues state: ‘Inositol may be responsible for the antitumor actions observed in both chemopreventitive and efficacy studies of IP₆ … A question remains as to whether the activity of IP₆ in animal models can be replicated by administration of inositol alone because only inositol was detected in plasma and tumor after oral gavage’. Precisely.Finally, returning to InsP₆ itself, which, incidentally, is officially classified by the FDA as a ‘fake’ cancer cure (http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/enforcementactivitiesbyfda/ucm171057.htm), our data lead inevitably to the conclusion that while InsP₆ might impact on the gut environment and thus indirectly on its microflora [2,12], its only plausible direct action on the body will be to inhibit cation uptake from the diet. Although InsP₆ binds trivalent cations with a higher affinity than divalents [18], it is nevertheless comparatively non-specific in this action. Administering chemicals to the diet to manipulate ion uptake is not unknown in modern medicine; for treatment of iron disorders such as haemochromatosis, as an alternative to injection of Desferral, oral administration of the closely related chelator Deferasirox is now sometimes recommended [19]. But Deferasirox is a highly iron-specific chelator, administered under close medical supervision for a directly iron-related pathology. Recommending unmonitored, widespread administration of InsP₆ to address a veritable multitude of different pathologies [20] seems to us to be an entirely different matter.In a well-fed human, where the cation to InsP₆ ratio in the diet is high, InsP₆ may very well do no harm (it is, after all, a natural component of our diet) and there is much evidence to support this idea, as argued by Dr Vucenik. But if InsP₆ is not impacting on cation uptake from the diet to do any harm it is difficult to understand how at exactly the same time it can impact on the same uptake to do good. (See reference [21] for the studies Dr Vucenik requested ‘unequivocally demonstrating the toxicity of pure Ca-Mg-InsP₆ as it occurs naturally’ in humans with low dietary cation uptake.) In the light of the above discussion and our rigorous data, we stand unreservedly by our original closing statement [1]: ‘…that chronically altering cation absorption from the gut by artificially loading the diet with a non-specific chelator … in the hope that it might impact indirectly on cancer or other pathologies seems highly inadvisable’.     

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

Administration of a gonadotropin-releasing hormone antagonist to mares at different times during the luteal phase of the estrous cycle

The GnRH antagonist cetrorelix was given during the early (Days 1-5), mid (Days 6-10 or 5-12) or for the entire (Days 1-16) luteal phase of mares to inhibit the secretion of FSH and LH (Day 0=ovulation). Frequent blood sampling from Day 6 to Day 14 was used to determine the precise time-course of the suppression (cetrorelix given Days 6-10). Cetrorelix treatment caused a decrease in FSH and LH concentrations by 8 and 16 h, respectively, and an obliteration of the response to exogenous GnRH given 24h after treatment onset. Treatment never suppressed gonadotropin concentrations to undetectable levels; e.g. frequent sampling showed that the nadirs reached in FSH and LH were 46.2±6% and 33.1±11%, respectively, of pre-treatment concentrations. Daily FSH concentrations were decreased in all treatment groups but daily LH concentrations were lower only when treatment commenced at the beginning of the luteal phase; progesterone concentrations depended on the time of cetrorelix administration, but the changes suggested a role for LH in corpus luteum function. The inter-ovulatory interval was longer than controls when cetrorelix was given in the mid- or for the entire luteal phase, but was unaffected by treatment in the early phase. Nevertheless, in all groups, FSH concentrations were higher (P<0.05 when compared to Day 0, subsequent ovulation) approximately 6-10 days before this next ovulation. This consistent relationship suggests a stringent requirement for a GnRH-induced elevation of FSH above a threshold at, but only at, this time; i.e. approximately 6-10 days before ovulation.