Caenorhabditis elegans dnj-14, the orthologue of the DNAJC5 gene mutated in adult onset neuronal ceroid lipofuscinosis, provides a new platform for neuroprotective drug screening and identifies a SIR-2.1-independent action of resveratrol

Adult onset neuronal lipofuscinosis (ANCL) is a human neurodegenerative disorder characterized by progressive neuronal dysfunction and premature death. Recently, the mutations that cause ANCL were mapped to the DNAJC5 gene, which encodes cysteine string protein alpha. We show here that mutating dnj-14, the Caenorhabditis elegans orthologue of DNAJC5, results in shortened lifespan and a small impairment of locomotion and neurotransmission. Mutant dnj-14 worms also exhibited age-dependent neurodegeneration of sensory neurons, which was preceded by severe progressive chemosensory defects. A focussed chemical screen revealed that resveratrol could ameliorate dnj-14 mutant phenotypes, an effect mimicked by the cAMP phosphodiesterase inhibitor, rolipram. In contrast to other worm neurodegeneration models, activation of the Sirtuin, SIR-2.1, was not required, as sir-2.1; dnj-14 double mutants showed full lifespan rescue by resveratrol. The Sirtuin-independent neuroprotective action of resveratrol revealed here suggests potential therapeutic applications for ANCL and possibly other human neurodegenerative diseases

Cytochemical techniques and energy-filtering transmission electron microscopy applied to the study of parasitic protozoa

The study of parasitic protozoa plays a major role in cell biology, biochemistry and molecular biology. Numerous cytochemical techniques have been developed in order to unequivocally identify the nature of subcellular compartments. Enzyme and immuno-cytochemistry allow the detection of, respectively, enzymatic activity products and antigens in particular sites within the cell. Energy-filtering transmission electron microscopy permits the detection of specific elements within such compartments. These approaches are particularly useful for studies employing antimicrobial agents where cellular compartments may be destroyed or remarkably altered and thus hardly identified by standard methods of observation. In this regard cytochemical and spectroscopic techniques provide valuable data allowing the determination of the mechanisms of action of such compounds

The Typical Flight Performance of Blowflies: Measuring the Normal Performance Envelope of Calliphora vicina Using a Novel Corner-Cube Arena

Despite a wealth of evidence demonstrating extraordinary maximal performance, little is known about the routine flight performance of insects. We present a set of techniques for benchmarking performance characteristics of insects in free flight, demonstrated using a model species, and comment on the significance of the performance observed. Free-flying blowflies (Calliphora vicina) were filmed inside a novel mirrored arena comprising a large (1.6 m1.6 m1.6 m) corner-cube reflector using a single high-speed digital video camera (250 or 500 fps). This arrangement permitted accurate reconstruction of the flies' 3-dimensional trajectories without the need for synchronisation hardware, by virtue of the multiple reflections of a subject within the arena. Image sequences were analysed using custom-written automated tracking software, and processed using a self-calibrating bundle adjustment procedure to determine the subject's instantaneous 3-dimensional position. We illustrate our method by using these trajectory data to benchmark the routine flight performance envelope of our flies. Flight speeds were most commonly observed between 1.2 ms−1 and 2.3 ms−1, with a maximum of 2.5 ms−1. Our flies tended to dive faster than they climbed, with a maximum descent rate (−2.4 ms−1) almost double the maximum climb rate (1.2 ms−1). Modal turn rate was around 240°s−1, with maximal rates in excess of 1700°s−1. We used the maximal flight performance we observed during normal flight to construct notional physical limits on the blowfly flight envelope, and used the distribution of observations within that notional envelope to postulate behavioural preferences or physiological and anatomical constraints. The flight trajectories we recorded were never steady: rather they were constantly accelerating or decelerating, with maximum tangential accelerations and maximum centripetal accelerations on the order of 3 g

The Great American Biotic Interchange: Dispersals, Tectonics, Climate, Sea Level and Holding Pens

The biotic and geologic dynamics of the Great American Biotic Interchange are reviewed and revised. Information on the Marine Isotope Stage chronology, sea level changes as well as Pliocene and Pleistocene vegetation changes in Central and northern South America add to a discussion of the role of climate in facilitating trans-isthmian exchanges. Trans-isthmian land mammal exchanges during the Pleistocene glacial intervals appear to have been promoted by the development of diverse non-tropical ecologies

The what and where of adding channel noise to the Hodgkin-Huxley equations

One of the most celebrated successes in computational biology is the Hodgkin-Huxley framework for modeling electrically active cells. This framework, expressed through a set of differential equations, synthesizes the impact of ionic currents on a cell's voltage -- and the highly nonlinear impact of that voltage back on the currents themselves -- into the rapid push and pull of the action potential. Latter studies confirmed that these cellular dynamics are orchestrated by individual ion channels, whose conformational changes regulate the conductance of each ionic current. Thus, kinetic equations familiar from physical chemistry are the natural setting for describing conductances; for small-to-moderate numbers of channels, these will predict fluctuations in conductances and stochasticity in the resulting action potentials. At first glance, the kinetic equations provide a far more complex (and higher-dimensional) description than the original Hodgkin-Huxley equations. This has prompted more than a decade of efforts to capture channel fluctuations with noise terms added to the Hodgkin-Huxley equations. Many of these approaches, while intuitively appealing, produce quantitative errors when compared to kinetic equations; others, as only very recently demonstrated, are both accurate and relatively simple. We review what works, what doesn't, and why, seeking to build a bridge to well-established results for the deterministic Hodgkin-Huxley equations. As such, we hope that this review will speed emerging studies of how channel noise modulates electrophysiological dynamics and function. We supply user-friendly Matlab simulation code of these stochastic versions of the Hodgkin-Huxley equations on the ModelDB website (accession number 138950) and http://www.amath.washington.edu/~etsb/tutorials.html.Comment: 14 pages, 3 figures, review articl

A randomized, double-blind comparison of OROS® hydromorphone and controlled-release morphine for the control of chronic cancer pain

<p>Abstract</p> <p>Background</p> <p>Long-acting opioid formulations are advocated for maintaining pain control in chronic cancer pain. OROS^®hydromorphone is a sustained-release formulation of hydromorphone that requires dosing once daily to maintain therapeutic concentrations. The objective of this study was to demonstrate the clinical equivalence of immediate-release and sustained-release formulations of hydromorphone and morphine for chronic cancer pain.</p> <p>Methods</p> <p>200 patients with cancer pain (requiring ≤ 540 mg/d of oral morphine) participated in this double-blind, parallel-group trial. Patients were randomized to receive hydromorphone or morphine (immediate-release for 2–9 days, sustained-release for 10–15 days). Efficacy was assessed with the Brief Pain Inventory (BPI), investigator and patient global evaluations, Eastern Cooperative Oncology Group performance status, and the Mini-Mental State Examination. The primary endpoint was the 'worst pain in the past 24 hours' item of the BPI, in both the immediate-release and sustained-release study phases, with treatments deemed equivalent if the 95% confidence intervals (CI) of the between-group differences at endpoint were between -1.5 and 1.5. No equivalence limits were defined for secondary endpoints.</p> <p>Results</p> <p>Least-squares mean differences (95% CI) between groups were 0.2 (-0.4, 0.9) in the immediate-release phase and -0.8 (-1.6, -0.01) in the sustained-release phase (intent-to-treat population), indicating that the immediate-release formulations met the pre-specified equivalence criteria, but that the lower limit of the 95% CI (-1.6) was outside the boundary (-1.5) for the sustained-release formulations. BPI 'pain now PM' was significantly lower with OROS^®hydromorphone compared with controlled-release morphine (least-squares mean difference [95% CI], -0.77 [-1.49, -0.05]; <it>p </it>= 0.0372). Scores for other secondary efficacy variables were similar between the two sustained-release treatments. At endpoint, > 70% of investigators and patients rated both treatments as good to excellent. The safety profiles of hydromorphone and morphine were similar and typical of opioid analgesics.</p> <p>Conclusion</p> <p>Equivalence was demonstrated for immediate-release formulations of hydromorphone and morphine, but not for the sustained-release formulations of OROS^®hydromorphone and controlled-release morphine. The direction of the mean difference between the treatments (-0.8) and the out-of-range lower limit of the 95% CI (-1.6) were in favor of OROS^®hydromorphone.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: NCT0041054</p

Changes in magnetic resonance mammography due to hormone replacement therapy

BACKGROUND: The aim of the present article is to investigate effects of hormone replacement therapy (HRT) on contrast medium enhancement patterns in postmenopausal patients during magnetic resonance mammography (MRM). MATERIALS AND METHODS: Two hundred and fifteen patients receiving hormonal medication were divided into four groups: 150 patients with 1 MRM during HRT (group A), 13 patients with 2 MRMs under HRT (group B), 30 patients with 1 MRM during HRT and 1 MRM after HRT withdrawal (group C), and 22 women with 1 MRM after HRT withdrawal (group D). Dynamic MRM was performed at 1.5 Tesla. Signal intensity changes were characterized by five time curves: minimal enhancement (type I), weak continuous enhancement (type II), strong continuous enhancement (type III), and a steep initial slope followed by a plateau phenomenon (type IV) or a washout effect (type V). RESULTS: Of all 193 patients under HRT (group A + group B + group C), 60 patients (31.1%) showed curve type I, 88 patients (45.6%) showed type II and 45 patients (23.3%) showed type III. There were significant differences to 52 patients after HRT withdrawal (group C + group D) (P < 0.0001), with 42 patients (80.8%) for curve type I, 8 patients (15.4%) for type II, and 2 patients (3.8%) for type III. In both MRM sessions in group B, 69% of the patients showed identical curve types without significant differences (P = 0.375). In group C, 28 of 30 patients (93%) dropped to lower curve types with significant differences in curve types during and after HRT (P < 0.0001). CONCLUSION: The majority of patients receiving postmenopausal HRT showed bilateral symmetrical, continuous enhancement without evidence of a plateau phenomenon or a washout effect due to HRT in MRM. Hormonal effects could be proven and were reproducible and reversible

Immunogenicity and Protective Capacity of a Virosomal Respiratory Syncytial Virus Vaccine Adjuvanted with Monophosphoryl Lipid A in Mice

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation

Autism as a disorder of neural information processing: directions for research and targets for therapy

The broad variation in phenotypes and severities within autism spectrum disorders suggests the involvement of multiple predisposing factors, interacting in complex ways with normal developmental courses and gradients. Identification of these factors, and the common developmental path into which theyfeed, is hampered bythe large degrees of convergence from causal factors to altered brain development, and divergence from abnormal brain development into altered cognition and behaviour. Genetic, neurochemical, neuroimaging and behavioural findings on autism, as well as studies of normal development and of genetic syndromes that share symptoms with autism, offer hypotheses as to the nature of causal factors and their possible effects on the structure and dynamics of neural systems. Such alterations in neural properties may in turn perturb activity-dependent development, giving rise to a complex behavioural syndrome many steps removed from the root causes. Animal models based on genetic, neurochemical, neurophysiological, and behavioural manipulations offer the possibility of exploring these developmental processes in detail, as do human studies addressing endophenotypes beyond the diagnosis itself

Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9.

The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease