Alternate Day Fasting Leads to Improved Post-Stroke Motor Recovery in Mice.

BACKGROUND: Caloric restriction promotes neuroplasticity and recovery after neurological injury. In mice, we tested the hypothesis that caloric restriction can act post-stroke to enhance training-associated motor recovery. METHODS: Mice were trained to perform a skilled prehension task. We then induced a photothrombotic stroke in the caudal forelimb area, after which we retrained animals on the prehension task following an 8-day delay. Mice underwent either ad libitum feeding or alternate day fasting beginning 1-day after stroke and persisting for either 7 days or the entire post-stroke training period until sacrifice. RESULTS: Prior studies have shown that post-stroke recovery of prehension can occur if animals receive rehabilitative training during an early sensitive period but is incomplete if rehabilitative training is delayed. In contrast, we show complete recovery of prehension, despite a delay in rehabilitative training, when mice underwent alternate day fasting beginning 1-day post-stroke and persisting for either 7 days or the entire post-stroke training period until sacrifice. Recovery was independent of weight loss. Stroke volumes were similar across groups. CONCLUSIONS: Post-stroke caloric restriction led to recovery of motor function independent of a protective effect on stroke volume. Prehension recovery improved even after ad libitum feeding was reinstituted suggesting that the observed motor recovery was not merely a motivational response. These data add to the growing evidence that post-stroke caloric restriction can enhance recovery.

Rational design of proteasome inhibitors based on the structure of the endogenous inhibitor PI31/Fub1.

Proteasome inhibitors are widely used anticancer drugs. The three clinically approved agents are modified small peptides that preferentially target one of the proteasome's three active sites (ß5) at physiologic concentrations. In addition to these drugs, there is also an endogenous proteasome inhibitor, PI31/Fub1, that enters the proteasome's interior to simultaneously yet specifically inhibit all three active sites. Here, we have used PI31's evolutionarily optimized inhibitory mechanisms to develop a suite of potent and specific ß2 inhibitors. The lead compound strongly inhibited growth of multiple myeloma cells as a standalone agent, indicating the compound's cell permeability and establishing ß2 as a potential therapeutic target in multiple myeloma. The lead compound also showed strong synergy with the existing ß5 inhibitor bortezomib; such combination therapies might help with existing challenges of resistance and severe side effects. These results represent an effective method for rational structure-guided development of proteasome inhibitors.

Squeeze-film properties of synovial fluid and hyaluronate-based viscosupplements.

The rheological properties of synovial fluid and hyaluronate (HA) solutions have been studied using a variety of viscometers and rheometers. These devices measure the viscosity of the fluid's resistance to shearing forces, which is useful when studying the lubrication and frictional properties of movable joints. Less commonly used is a squeeze-film fluid test, mechanistically similar to when two joint surfaces squeeze interposed fluid. In our study, we used squeeze-film tests to determine the rheological response of normal bovine synovial fluid and 10 mg/ml HA-based solutions, Hyalgan/Hyalovet, commercially available 500-700 kDa HA viscosupplements, and a 1000 kDa sodium hyaluronate (NaHy) solution. We found similar rheological responses (fluid thickness, viscosity, viscosity-pressure relationship) for all three fluids, though synovial fluid's minimum squeeze-film thickness was slightly thicker. Squeeze-film loading speed did not affect these results. Different HA concentrations and molecular weights also did not have a significant or consistent effect on the squeeze-film responses. An unexpected result for the HA-solutions was a linear increase in minimum fluid-film thickness with increasing initial fluid-film thickness. This result was attributed to faster gelling of thicker HA-solutions, which formed at a lower squeeze-film strain and higher squeeze-film strain rate compared to thinner layers. Also included is a review of the literature on viscosity measurements of synovial fluid and HA solutions.

Enhanced Spontaneous Motor Recovery After Stroke in Mice Treated With Cerebrolysin.

BACKGROUND: Motor recovery after stroke in humans and in rodent models is time sensitive. Recovery in patients is a result of biological spontaneous recovery via endogenous repair mechanisms and is likely improved by enhancing the synaptic plasticity required for endogenous repair. Cerebrolysin is a polypeptide preparation known to enhance neuroplasticity and may improve recovery in patients. In mice, we tested the hypothesis that Cerebrolysin can act poststroke to enhance both spontaneous and training-associated motor recovery. METHODS: Mice were trained to perform a skilled prehension task. We then induced a photothrombotic stroke in the caudal forelimb area, after which we retrained animals on the prehension task in the presence or absence of Cerebrolysin after a 2-day or 8-day delay. Mice received daily intraperitoneal Cerebrolysin or saline injections starting poststroke day 1 or poststroke day 7. RESULTS: Prior studies showed that poststroke recovery of prehension can occur if animals receive rehabilitative training during an early sensitive period but is incomplete if rehabilitative training is delayed. In contrast, we show complete recovery of prehension, despite a delay in rehabilitative training, when mice receive daily Cerebrolysin administration starting on poststroke day 1 or on poststroke day 8. When Cerebrolysin is given on poststroke day 1, recovery occurred even in the absence of training. Stroke volumes were similar across groups. CONCLUSIONS: Poststroke Cerebrolysin administration leads to recovery of motor function independent of rehabilitative training without a protective effect on stroke volume. This is one of the first demonstrations of training-independent motor recovery in rodent stroke models.

Snagfall the first decade after severe bark beetle infestation of high-elevation forests in Colorado, USA.

The persistence and fall rate of snags (standing dead trees) generated during bark beetle outbreaks have consequences for the behavior, effects, and suppression of potential wildfires, hazard tree and timber salvage operations, wildlife habitat, and numerous ecosystem processes. However, post-beetle snagfall dynamics are poorly understood in most forest types. We tagged standing live and dead lodgepole pine (Pinus contorta), subalpine fir (Abies lasiocarpa), and Engelmann spruce (Picea engelmannii), including beetle-killed pine snags following the peak of a recent mountain pine bark beetle outbreak in watersheds at the Fraser Experimental Forest in northcentral Colorado and sampled snagfall 10 and 12 years later. Bark beetle attacks began in 2003, peaked by 2006, and killed 78% of overstory lodgepole pine in 133 plots distributed across a range of stand and site conditions. Of those snags, only 17% fell between 2007 and 2018. Most snags broke at ground level, due to butt rot, and were oriented downhill. In contrast, snags that tipped up or snapped off above the ground were oriented with the prevailing winds. Equal numbers of snags fell singly and in multiple-tree groups, and equal numbers remained elevated rather than in contact with the ground. Lodgepole pine snagfall was 1.6-times higher on steep slopes (>40%) where dead pine density was higher, compared to flatter sites. Based on our findings and previous research, we estimate that one-half the beetle-killed lodgepole pine in high-elevation forests such as those at Fraser may fall within 15-20 yr of beetle infestation, but that some pine snags are likely to persist for decades longer. Post-outbreak snagfall dynamics create a multiple-decade legacy of bark beetle outbreaks that will persist longer in high-elevation compared to lower-elevation forests.

Evaluating Skilled Prehension in Mice Using an Auto-Trainer.

We describe a method to introduce naïve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that permits the mouse to reach out of its cage and retrieve food pellets. Minimal food restriction is employed to encourage the mice to perform the food retrieval from the slot. As the mice begin to associate coming to the slot for food, the pellets are manually pulled away to stimulate extension and pronation of their paw to grasp and retrieve the pellet through the frontal slot. When the mice begin to reach for the pellets as they arrive at the slot, the behavioral assay can be performed by measuring the rate at which they successfully grasp and retrieve the desired pellet. They are then introduced to an auto-trainer that automates both the process of providing food pellets for the mouse to grasp, and the recording of successful and failed reaching and grasping attempts. This allows for the collection of reaching data for multiple mice with minimal effort, to be used in experimental analysis as appropriate.

Evolutionary Models of Leadership : Tests and Synthesis.

This study tested four theoretical models of leadership with data from the ethnographic record. The first was a game-theoretical model of leadership in collective actions, in which followers prefer and reward a leader who monitors and sanctions free-riders as group size increases. The second was the dominance model, in which dominant leaders threaten followers with physical or social harm. The third, the prestige model, suggests leaders with valued skills and expertise are chosen by followers who strive to emulate them. The fourth proposes that in small-scale, kin-based societies, men with high neural capital are best able to achieve and maintain positions of social influence (e.g., as headmen) and thereby often become polygynous and have more offspring than other men, which positively selects for greater neural capital. Using multiple search strategies we identified more than 1000 texts relevant to leadership in the Probability Sample of 60 cultures from the Human Relations Area Files (HRAF). We operationalized the model with variables and then coded all retrieved text records on the presence or absence of evidence for each of these 24 variables. We found mixed support for the collective action model, broad support for components of the prestige leadership style and the importance of neural capital and polygyny among leaders, but more limited support for the dominance leadership style. We found little evidence, however, of emulation of, or prestige-biased learning toward, leaders. We found that improving collective actions, having expertise, providing counsel, and being respected, having high neural capital, and being polygynous are common properties of leaders, which warrants a synthesis of the collective action, prestige, and neural capital and reproductive skew models. We sketch one such synthesis involving high-quality decision-making and other computational services.

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors.

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

Formation of A Novel Purine Metabolite through CYP3A4 Bioactivation and Glutathione Conjugation.

BACKGROUND: The study of novel sites of metabolism is important in understanding new mechanisms of biotransformation of a particular moiety by metabolic enzymes. This information is valuable in designing metabolically-stable compounds with drug-like properties. It may also provide insights into the existence of active and reactive metabolites. METHODS: We utilized small scale incubations to generate adequate amounts of the metabolite of interest. After purification, LC-MS/MS and Proton Nuclear Magnetic Resonance (1H-NMR) were utilized to unequivocally assign the novel site of glutathione conjugation on the purine ring system. RESULTS: A proposed novel site of glutathione conjugation was investigated on a diaminopurine-containing molecule. It was demonstrated that the formation of the glutathione conjugate at the C-6 position of the purine ring system was due to the bioactivation of the compound to a di-imine intermediate by CYP3A4, followed by the nucleophilic addition of glutathione. CONCLUSION: S-glutathionylation at C-6 position of a purine was proven unequivocally. This previously unreported mechanism constitutes a novel biotransformation for purines.

Paradoxical Motor Recovery From a First Stroke After Induction of a Second Stroke: Reopening a Postischemic Sensitive Period.

BACKGROUND AND OBJECTIVE: Prior studies have suggested that after stroke there is a time-limited period of increased responsiveness to training as a result of heightened plasticity-a sensitive period thought to be induced by ischemia itself. Using a mouse model, we have previously shown that most training-associated recovery after a caudal forelimb area (CFA) stroke occurs in the first week and is attributable to reorganization in a medial premotor area (AGm). The existence of a stroke-induced sensitive period leads to the counterintuitive prediction that a second stroke should reopen this window and promote full recovery from the first stroke. To test this prediction, we induced a second stroke in the AGm of mice with incomplete recovery after a first stroke in CFA. METHODS: Mice were trained to perform a skilled prehension (reach-to-grasp) task to an asymptotic level of performance, after which they underwent photocoagulation-induced stroke in CFA. After a 7-day poststroke delay, the mice were then retrained to asymptote. We then induced a second stroke in the AGm, and after only a 1-day delay, retrained the mice. RESULTS: Recovery of prehension was incomplete when training was started after a 7-day poststroke delay and continued for 19 days. However, a second focal stroke in the AGm led to a dramatic response to 9 days of training, with full recovery to normal levels of performance. CONCLUSIONS: New ischemia can reopen a sensitive period of heightened responsiveness to training and mediate full recovery from a previous stroke.

Fluoxetine Maintains a State of Heightened Responsiveness to Motor Training Early After Stroke in a Mouse Model.

BACKGROUND AND PURPOSE: Data from both humans and animal models suggest that most recovery from motor impairment after stroke occurs in a sensitive period that lasts only weeks and is mediated, in part, by an increased responsiveness to training. Here, we used a mouse model of focal cortical stroke to test 2 hypotheses. First, we investigated whether responsiveness to training decreases over time after stroke. Second, we tested whether fluoxetine, which can influence synaptic plasticity and stroke recovery, can prolong the period over which large training-related gains can be elicited after stroke. METHODS: Mice were trained to perform a skilled prehension task to an asymptotic level of performance after which they underwent stroke induction in the caudal forelimb area. The mice were then retrained after a 1- or 7-day delay with and without fluoxetine. RESULTS: Recovery of prehension after a caudal forelimb area stroke was complete if training was initiated 1 day after stroke but incomplete if it was delayed by 7 days. In contrast, if fluoxetine was administered at 24 hours after stroke, then complete recovery of prehension was observed even with the 7-day training delay. Fluoxetine seemed to mediate its beneficial effect by reducing inhibitory interneuron expression in intact premotor cortex rather than through effects on infarct volume or cell death. CONCLUSIONS: There is a gradient of diminishing responsiveness to motor training over the first week after stroke. Fluoxetine can overcome this gradient and maintain maximal levels of responsiveness to training even 7 days after stroke.

Developing lipid nanoparticle-based siRNA therapeutics for hepatocellular carcinoma using an integrated approach.

Successful siRNA therapeutics requires the optimal integration of multiple components, including an efficient delivery system, a disease indication that is appropriate for siRNA-based therapy, and a potent and nontoxic siRNA against a robust therapeutic target. Although all currently available delivery systems have limitations, it is important to recognize that a careful selection of the disease indication, therapeutic target, and siRNA molecule could partially compensate for deficiencies associated with the delivery system and makes it possible to advance a therapeutic siRNA regimen. In this study, we present the development of siRNA therapeutics for hepatocellular carcinoma using an integrated approach, including the development of an efficient lipid nanoparticle delivery system, the identification of a robust therapeutic target that does not trigger liver toxicity upon target knockdown, and the selection of potent and nonimmunogenic siRNA molecules against the target. The resulting siRNA-containing lipid nanoparticles produced significant antitumor efficacy in orthotopic hepatocellular carcinoma models, and, thus, represent a promising starting point for the development of siRNA therapeutics for hepatocellular carcinoma.

Links among nitrification, nitrifier communities, and edaphic properties in contrasting soils receiving dairy slurry.

Soil biotic and abiotic factors strongly influence nitrogen (N) availability and increases in nitrification rates associated with the application of manure. In this study, we examine the effects of edaphic properties and a dairy (Bos taurus) slurry amendment on N availability, nitrification rates and nitrifier communities. Soils of variable texture and clay mineralogy were collected from six USDA-ARS research sites and incubated for 28 d with and without dairy slurry applied at a rate of ~300 kg N ha(-1). Periodically, subsamples were removed for analyses of 2 M KCl extractable N and nitrification potential, as well as gene copy numbers of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Spearman coefficients for nitrification potentials and AOB copy number were positively correlated with total soil C, total soil N, cation exchange capacity, and clay mineralogy in treatments with and without slurry application. Our data show that the quantity and type of clay minerals present in a soil affect nitrifier populations, nitrification rates, and the release of inorganic N. Nitrogen mineralization, nitrification potentials, and edaphic properties were positively correlated with AOB gene copy numbers. On average, AOA gene copy numbers were an order of magnitude lower than those of AOB across the six soils and did not increase with slurry application. Our research suggests that the two nitrifier communities overlap but have different optimum environmental conditions for growth and activity that are partly determined by the interaction of manure-derived ammonium with soil properties.

Cationic lipids percentage and processing temperature are critical in designing siRNA lipid nanoparticles.

To design a clinically viable small interfering RNA (siRNA) formulation, it is essential to understand the in vivo siRNA delivery mechanism during the product development. However, majority of reported siRNA delivery studies are based on testing only isolated factors, with ambiguous interpretation of often in vitro transfection results. Correlating physicochemical properties with in vivo transfection efficiency thus represents an important step towards rational design of siRNA delivery systems. In this study, design of experiments studies were applied to probe formulation attributes and process parameters, with in vivo activities evaluated as a primary response along with physicochemical properties. Statistical analysis was performed to identify the significance of each input factor towards the in vivo transfection efficiency using a Positive Readout System. The interactions between these factors were also analyzed. Our results indicated that among the formulation factors evaluated, the percentage of cationic lipid is of most significant effect. During the process, temperature stands out as the most significant factor impacting the in vivo activities. These results shed light on our design of siRNA lipid nanoparticle formulations in the early development stage.

A robust in vivo positive-readout system for monitoring siRNA delivery to xenograft tumors.

Delivering small interfering RNA (siRNA) to tumors is the major technical hurdle that prevents the advancement of siRNA-based cancer therapy. One of the difficulties associated with the development of clinically relevant delivery systems is the lack of reliable tools for monitoring siRNA delivery to tumors in vivo. We describe here a novel, positive-readout system where siRNA-mediated target knockdown elicits a rapid and robust increase of reporter activity. Using the positive-readout system, we created (1) ß-galactosidase-based tumor models that allow the detection of target knockdown in 1%-2% of tumor cells and can distinguish between tumor areas where effective target knockdown occurs versus tumor areas that are not accessible to delivery, and (2) luciferase-based tumor models that allow the quantitative assessment of a large number of delivery systems. Using these positive-readout models, we screened a number of literature-described siRNA delivery systems and identified lipid nanoparticles as a promising delivery platform for siRNA-based cancer therapy.

Substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines as multi-targeted inhibitors of insulin-like growth factor-1 receptor (IGF1R) and members of ErbB-family receptor kinases.

This Letter describes the lead discovery, optimization, and biological characterization of a series of substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines as potent inhibitors of IGF1R, EGFR, and ErbB2. The leading compound 11 showed an IGF1R IC(50) of 12 nM, an EGFR (L858R) IC(50) of 31 nM, and an ErbB2 IC(50) of 11 nM, potent activity in cellular functional and anti-proliferation assays, as well as activity in an in vivo pharmacodynamic assay.

Multisite effectiveness trials of treatments for substance abuse and co-occurring problems: have we chosen the best designs?

Multisite effectiveness trials such as those carried out in the National Drug Abuse Treatment Clinical Trials Network (CTN) are a critical step in the development and dissemination of evidence-based treatments because they address how such treatments perform in real-world clinical settings. As Brigham et al. summarized in a recent article (G. S. Brigham, D. J. Feaster, P. G. Wakim, & C. L. Dempsey C. L., 2009), several possible experimental designs may be chosen for such effectiveness trials. These include (a) a new treatment intervention (Tx) is compared to an existing mode of community based treatment as usual (TAU): Tx versus TAU; (b) a new intervention is added to TAU and compared to TAU alone: Tx + TAU versus TAU; or (c) a new intervention is added to TAU and compared to a control condition added to TAU: Tx + TAU versus control + TAU. Each of these designs addresses a different question and has different potential strengths and weaknesses. As of December 2009, the primary outcome paper had been published for 16 of the multisite randomized clinical trials conducted in the CTN, testing various treatments for drug abuse, HIV risk behavior, or related problems. This paper systematically examines, for each of the completed trials, the experimental design type chosen and its original rationale, the main findings of the trial, and the strengths and weaknesses of the design in hindsight. Based on this review, recommendations are generated to inform the design of future effectiveness trials on treatments for substance abuse, HIV risk, and other behavioral health problems.

Imidazo[2,1-b]thiazoles: multitargeted inhibitors of both the insulin-like growth factor receptor and members of the epidermal growth factor family of receptor tyrosine kinases.

The design and enzyme activities of a novel class of imidazo[2,1-b]thiazoles is presented.

Quantifying phosphorus levels in soils, plants, surface water, and shallow groundwater associated with bahiagrass-based pastures.

BACKGROUND, AIM, AND SCOPE: Recent assessments of water quality status have identified eutrophication as one of the major causes of water quality 'impairment' not only in the USA but also around the world. In most cases, eutrophication has accelerated by increased inputs of phosphorus due to intensification of crop and animal production systems since the early 1990 s. Despite substantial measurements using both laboratory and field techniques, little is known about the spatial and temporal variability of phosphorus dynamics across landscapes, especially in agricultural landscapes with cow-calf operations. Critical to determining environmental balance and accountability is an understanding of phosphorus excreted by animals, phosphorus removal by plants, acceptable losses of phosphorus within the manure management and crop production systems into soil and waters, and export of phosphorus off-farm. Further research effort on optimizing forage-based cow-calf operations to improve pasture sustainability and protect water quality is therefore warranted. We hypothesized that properly managed cow-calf operations in subtropical agroecosystem would not be major contributors to excess loads of phosphorus in surface and ground water. To verify our hypothesis, we examined the comparative concentrations of total phosphorus among soils, forage, surface water, and groundwater beneath bahiagrass-based pastures with cow-calf operations in central Florida, USA. MATERIALS AND METHODS: Soil samples were collected at 0-20; 20-40, 40-60, and 60-100 cm across the landscape (top slope, middle slope, and bottom slope) of 8 ha pasture in the fall and spring of 2004 to 2006. Forage availability and phosphorus uptake of bahiagrass were also measured from the top slope, middle slope, and bottom slope. Bi-weekly (2004-2006) groundwater and surface water samples were taken from wells located at top slope, middle slope, and bottom slope, and from the runoff/seepage area. Concentrations of phosphorus in soils, forage, surface water, and shallow groundwater beneath a bahiagrass-based pasture and forage availability at four different landscape positions and soil depth (for soil samples only) in 2004, 2005, and 2006 were analyzed statistically following a two-way analysis of variance using the SAS PROC general linear models model. Where the F-test indicated a significant (p

Reversal of oncogene transformation and suppression of tumor growth by the novel IGF1R kinase inhibitor A-928605.

BACKGROUND: The insulin-like growth factor (IGF) axis is an important signaling pathway in the growth and survival of many cell and tissue types. This pathway has also been implicated in many aspects of cancer progression from tumorigenesis to metastasis. The multiple roles of IGF signaling in cancer suggest that inhibition of the pathway might yield clinically effective therapeutics. METHODS: We describe A-928605, a novel pyrazolo [3,4-d]pyrimidine small molecule inhibitor of the receptor tyrosine kinases (IGF1R and IR) responsible for IGF signal transduction. This compound was first tested for its activity and selectivity via conventional in vitro kinome profiling and cellular IGF1R autophosphorylation. Additionally, cellular selectivity and efficacy of A-928605 were analyzed in an IGF1R oncogene-addicted cell line by proliferation, signaling and microarray studies. Finally, in vivo efficacy of A-928605 was assessed in the oncogene-addicted cell line and in a neuroblastoma model as a single agent as well as in combination with clinically approved therapeutics targeting EGFR in models of pancreatic and non-small cell lung cancers. RESULTS: A-928605 is a selective IGF1R inhibitor that is able to abrogate activation of the pathway both in vitro and in vivo. This novel compound dosed as a single agent is able to produce significant growth inhibition of neuroblastoma xenografts in vivo. A-928605 is also able to provide additive effects when used in combination with clinically approved agents directed against EGFR in non-small cell lung and human pancreatic tumor models. CONCLUSION: These results suggest that a selective IGF1R inhibitor such as A-928605 may provide a useful clinical therapeutic for IGF pathway affected tumors and warrants further investigation.