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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There is a need to develop more animal species for assessing toxicity in marine environments. Cyst-based toxicity tests using invertebrates are especially fast, technically simple, cost-effective, and sensitive to a variety of toxicants. Over the past 30 years, a variety of toxicity endpoints have been measured using the marine rotifer Brachionus plicatilis hatched from cysts, including mortality, reproduction, ingestion, swimming, enzyme activity, and gene expression. A consensus has developed that the most ecologically relevant toxicity measurements should be made using more than one species. Furthermore, it has been noted that the rotifer species toxicant sensitivity distribution is much broader than which endpoint is measured. This implies that toxicity should be measured with the simplest, fastest, least expensive test available on as many species as feasible. If a battery of test species is to be used to estimate toxicity, diapause egg-based toxicity tests that do not require culturing of test animals will be key. In this paper, we describe how diapause eggs of a new marine rotifer, Proales similis, can be produced, stored and hatched under controlled conditions to produce animals for toxicity tests. Methods are described for quantifying the toxicity of copper, mercury and cadmium based on mortality, ingestion, reproduction, and diapause egg hatching endpoints. We found that reproduction and ingestion endpoints were generally more sensitive to the metals than mortality or diapause egg hatching. When the copper sensitivity of P. similis was compared to Brachionus manjavacas and B. plicatilis using an ingestion test, similar EC50s were observed. In contrast, the B. rotundiformis ingestion EC50 for copper was about 4× more sensitive. Although diapause egg hatching was not the most sensitive endpoint, it is the most ecologically relevant for assessing sediment toxicity. Our discovery of diapausing eggs in the P. similis life cycle has created a conundrum. We have not observed males or sex in P. similis populations, which is a direct contradiction to the orthodox view of the monogonont rotifer life cycle. Work is needed to clarify how diapause egg production is accomplished by P. similis and whether sexual reproduction is involved., (© 2019 The Authors. Environmental Toxicology published by Wiley Periodicals, Inc.)

Pharmaceutical interventions can slow aging in animals, and have advantages because their dose can be tightly regulated and the timing of the intervention can be closely controlled. They also may complement environmental interventions like caloric restriction by acting additively. A fertile source for therapies slowing aging is FDA approved drugs whose safety has been investigated. Because drugs bind to several protein targets, they cause multiple effects, many of which have not been characterized. It is possible that some of the side effects of drugs prescribed for one therapy may have benefits in retarding aging. We used computationally guided drug screening for prioritizing drug targets to produce a short list of candidate compounds for in vivo testing. We applied the virtual ligand screening approach FINDSITE comb for screening potential anti-aging protein targets against FDA approved drugs listed in DrugBank. A short list of 31 promising compounds was screened using a multi-tiered approach with rotifers as an animal model of aging. Primary and secondary survival screens and cohort life table experiments identified four drugs capable of extending rotifer lifespan by 8-42%. Exposures to 1 µM erythromycin, 5 µM carglumic acid, 3 µM capecitabine, and 1 µM ivermectin, extended rotifer lifespan without significant effect on reproduction. Some drugs also extended healthspan, as estimated by mitochondria activity and mobility (swimming speed). Our most promising result is that rotifer lifespan was extended by 7-8.9% even when treatment was started in middle age.

Rotifers have become widely used in aquatic toxicology as a rapid screening test for toxicity. The commercial availability of diapausing embryos (cysts) have facilitated their popularity because test animals can be obtained without having to master the details of culturing. Other rotifer species have life stages capable of surviving desiccation and also could be used in non-culture systems for toxicity assessment. In this article, we describe a system for toxicity testing in freshwater based on rehydrating desiccated bdelloid rotifers in the genus Philodina. These animals can remain in this anhydrobiotic state for more than one year and then rehydrate within hours to provide animals for toxicity tests. We describe three endpoints: a 1.5 h ingestion test, a 24 h mortality test, and a five day reproductive test. The latter test requires feeding and a method using a dried commercial product is explained. Using desiccated rotifers and dried food in toxicity tests make this system especially attractive because of its flexibility and low threshold of biological expertise required to execute the tests. The use of the Philodina toxicity test is illustrated with four metals: copper, lead, mercury and cadmium. Reproduction generally was the most sensitive endpoint, with EC50s of 0.33, 0.44, 0.60, and 0.12 mg/L, respectively. Ingestion was a close second with EC50s of 0.13, 1.64, 0.64, and 6.26 mg/L, respectively., (© 2017 Wiley Periodicals, Inc.)

Over the last twenty years, many strategies utilizing sol-gel chemistry to integrate biological cells into silica-based materials have been reported. One such strategy, Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition, shows promise as an efficient encapsulation technique due to the ability to vary the silica encapsulation morphology obtained by this process through variation of SG-CViL reaction conditions. In this report, we develop SG-CViL as a tunable, multi-purpose silica encapsulation strategy by investigating the mechanisms governing both silica particle generation and subsequent interaction with phospholipid assemblies (liposomes and living cells). Using Dynamic Light Scattering (DLS) measurements, linear and exponential silica particle growth dynamics were observed which were dependent on deposition buffer ion constituents and ion concentration. Silica particle growth followed a cluster-cluster growth mechanism at acidic pH, and a monomer-cluster growth mechanism at neutral to basic pH. Increasing silica sol aging temperature resulted in higher rates of particle growth and larger particles. DLS measurements employing PEG coated liposomes and cationic liposomes, serving as model phospholipid assemblies, revealed electrostatic interactions promote more stable liposome-silica interactions than hydrogen bonding and facilitate silica coating on suspension cells. However, continued silica reactivity leads to aggregation of silica coated suspensions cells, revealing the need for cell isolation to tune deposited silica thickness. Utilizing these mechanistic study insights, silica was deposited onto adherent HeLa cells under biocompatible conditions with micron scale control over silica thickness, minimal cell manipulation steps, and retained cell viability over several days.

There is great interest in drugs that are capable of modulating multiple aging pathways, thereby delaying the onset and progression of aging. Effective strategies for drug development include the repurposing of existing drugs already approved by the FDA for human therapy. FDA approved drugs have known mechanisms of action and have been thoroughly screened for safety. Although there has been extensive scientific activity in repurposing drugs for disease therapy, there has been little testing of these drugs for their effects on aging. The pool of FDA approved drugs therefore represents a large reservoir of drug candidates with substantial potential for anti-aging therapy. In this paper we employ FINDSITE comb , a powerful ligand homology modeling program, to identify binding partners for proteins produced by temperature sensing genes that have been implicated in aging. This list of drugs with potential to modulate aging rates was then tested experimentally for lifespan and healthspan extension using a small invertebrate model. Three protein targets of the rotifer Brachionus manjavacas corresponding to products of the transient receptor potential gene 7, ribosomal protein S6 polypeptide 2 gene, or forkhead box C gene, were screened against a compound library consisting of DrugBank drugs including 1347 FDA approved, non-nutraceutical molecules. Twenty nine drugs ranked in the top 1 % for binding to each target were subsequently included in our experimental analysis. Continuous exposure of rotifers to 1 µM naproxen significantly extended rotifer mean lifespan by 14 %. We used three endpoints to estimate rotifer health: swimming speed (mobility proxy), reproduction (overall vitality), and mitochondria activity (cellular senescence proxy). The natural decline in swimming speed with aging was more gradual when rotifers were exposed to three drugs, so that on day 6, mean swimming speed of females was 1.19 mm/s for naproxen (P = 0.038), 1.20 for fludarabine (P = 0.040), 1.35 for hydralazine (P = 0.038), as compared to 0.88 mm/s in the control. The average reproduction of control females in the second half of their reproductive lifespan was 1.08 per day. In contrast, females treated with 1 µM naproxen produced 1.4 offspring per day (P = 0.027) and females treated with 10 µM fludarabine or 1 µM hydralazine produced 1.72 (P = <0.001) and 1.66 (P = 0.001) offspring per day, respectively. Mitochondrial activity naturally declines with rotifer aging, but B. manjavacas treated with 1 µM hydralazine or 10 µM fludarabine retained 49 % (P = 0.038) and 89 % (P = 0.002) greater mitochondria activity, respectively, than untreated controls. Our results demonstrate that coupling computation to experimentation can quickly identify new drug candidates with anti-aging potential. Screening drugs for anti-aging effects using a rotifer bioassay is a powerful first step in identifying compounds worthy of follow-up in vertebrate models. Even if lifespan extension is not observed, certain drugs could improve healthspan, slowing age-dependent losses in mobility and vitality.

Environmental temperature greatly affects lifespan in a wide variety of animals, but the exact mechanisms underlying this effect are still largely unknown. A moderate temperature decrease from 22°C to 16°C extends the lifespan of the monogonont rotifer Brachionus manjavacas by up to 163%. Thermodynamic effects on metabolism contribute to this increase in longevity, but are not the only cause. When rotifers are exposed to 16°C for four days and then transfered to 22°C, they survive until day 13 at nearly identical rates as rotifers maintained at 16°C continuously. This persistence of the higher survival for nine days after transfer to 22°C suggests that low temperature exposure alters the expression of genes that affect the rate of aging. The relative persistence of the gene regulation effect suggests that it may play an even larger role in slowing aging than the thermodynamic effects. The life extending effects of these short-term low temperature treatments are largest when the exposure happens early in the life cycle, demonstrating the importance of early development. There is no advantage to lowering the temperature below 16°C to 11° or 5°C. Rotifers exposed to 16°C also displayed increased resistance to heat, starvation, oxidative and osmotic stress. Reproductive rates at 16°C were lower than those at 22°C, but because they reproduce longer, there is no significant change in the lifetime fecundity of females. To investigate which genes contribute to these effects, the expression of specific temperature sensing genes was knocked down using RNAi. Of 12 genes tested, RNAi knockdown of four eliminated the survival enhancing effects of the four-day cold treatment: TRP7, forkhead box C, Y-box factor, and ribosomal protein S6. This demonstrates that active gene regulation is an important factor in temperature mediated life extension, and that these particular genes play an integral role in these pathways. As a thermoresponsive sensor, TRP7 may be responsible for triggering the signaling cascade contributing to temperature mediated life extension. The TRP genes may also provide especially promising candidates for targeted gene manipulations or pharmacological interventions capable of mimicking the effects of low temperature exposure. These results support recent theories of aging that claim rate of aging is determined by an actively regulated genetic mechanism rather than an accumulation of molecular damage., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Many types of tumor, including glioma, melanoma, non-small cell lung, esophageal, and head and neck cancer, among others, are intrinsically resistant to apoptosis induction and poorly responsive to current therapies with proapoptotic agents. In addition, tumors often develop multidrug resistance based on the cellular efflux of chemotherapeutic agents. Thus, novel anticancer agents capable of overcoming these intrinsic or developed tumor resistance mechanisms are urgently needed. We describe a series of 2-aryl-2-(3-indolyl)acetohydroxamic acids that are active against apoptosis- and multidrug-resistant cancer cells as well as glioblastoma neurosphere stemlike cell cultures derived from patients. Thus, the described compounds serve as a novel chemical scaffold for the development of potentially highly effective clinical cancer drugs.

Comparative biogerontology has much to contribute to the study of aging. A broad range of aging rates has evolved to meet environmental challenges, and understanding these adaptations can produce valuable insights into aging. The supra Phylum Lophotrochozoa is particularly understudied and has several groups that have intriguing patterns of aging. Members of the lophotrochozoan phylum Rotifera are particularly useful for aging studies because cohort life tables can be conducted with them easily, and biochemical and genomic tools are available for examining aging mechanisms. This paper reviews a variety of caloric restriction regimens, small molecule inhibitors, and dietary supplements that extend rotifer lifespan, as well as important interactions between caloric restriction and genotype, antioxidant supplements, and TOR and JNK pathways, and the use of RNAi to identify key genes involved in modulating the aging response. Examples of how rapamycin and JNK inhibitor exposure keeps mortality rates low during the reproductive phase of the life cycle are presented, and the ease of conducting life table experiments to screen natural products from red algae for life extending effects is illustrated. Finally, experimental evolution to produce longer-lived rotifer individuals is demonstrated, and future directions to determine the genetic basis of aging are discussed.

Diet has profound effects on animal longevity and manipulation of nutrient sensing pathways is one of the primary interventions capable of lifespan extension. This often is done through caloric restriction (CR) and a variety of CR mimics have been identified that produce life extending effects without adhering to the rigorous CR dietary regimen. Glycerol is a dietary supplement capable mimicking CR by shifting metabolism away from glycolysis and towards oxidative phosphorylation. Glycerol supplementation has a number of beneficial effects, including lifespan extension, improved stress resistance, and enhanced locomotory and mitochondria activity in older age classes. Using rotifers as a model, we show that supplements of 150-300mM glycerol produced 40-50% extension of mean lifespan. This effect was produced by raising glycerol concentration only three times higher than its baseline concentration in rotifer tissues. Glycerol supplementation decreased rotifer reliance on glycolysis and reduced the pro-aging effects of glucose. Glycerol also acted as a chemical chaperone, mitigating damage by protein aggregation. Glycerol treatment improved rotifer swimming performance in older age classes and maintained more mitochondrial activity. Glycerol treatment provided increased resistance to starvation, heat, oxidation, and osmotic stress, but not UV stress. When glycerol was co-administered with the hexokinase inhibitor 2-deoxyglucose, the lifespan extending effect of glycerol was enhanced. Co-administration of glycerol with inhibitors like 2-deoxyglucose can lower their efficacious doses, thereby reducing their toxic side effects., (Copyright © 2014. Published by Elsevier Inc.)

The TOR kinase pathway is central in modulating aging in a variety of animal models. The target of rapamycin (TOR) integrates a complex network of signals from growth conditions, nutrient availability, energy status, and physiological stresses and matches an organism's growth rate to the resource environment. Important remaining problems are the identification of the pathways that interact with TOR and their characterization as additive or synergistic. One of the most versatile stress sensors in metazoans is the Jun-N-terminal kinase (JNK) signaling pathway. JNK is an evolutionarily conserved stress-activated protein kinase that is induced by a range of stressors, including UV irradiation, reactive oxygen species, DNA damage, heat, and bacterial antigens. JNK is thought to interact with the TOR pathway, but its effects on TOR are poorly understood. We used the rotifer Brachionus manjavacas as a model animal to probe the regulation of TOR and JNK pathways and explore their interaction. The effect of various chemical inhibitors was examined in life table and stressor challenge experiments. A survey of 12 inhibitors revealed two, rapamycin and JNK inhibitor, that significantly extended lifespan of B. manjavacas. At 1 μM concentration, exposure to rapamycin or JNK inhibitor extended mean rotifer lifespan by 35% and maximum lifespan by 37%. Exposure to both rapamycin and JNK inhibitor simultaneously extended mean rotifer lifespan by 65% more than either alone. Exposure to a combination of rapamycin and JNK inhibitors conveyed greater protection to starvation, UV and osmotic stress than either inhibitor alone. RNAi knockdown of TOR and JNK gene expression was investigated for its ability to extend rotifer lifespan. RNAi knockdown of the TOR gene resulted in 29% extension of the mean lifespan compared to control and knockdown of the JNK gene resulted in 51% mean lifespan extension. In addition to the lifespan, we quantified mitochondria activity using the fluorescent marker MitoTracker and lysosome activity using LysoTracker. Treatment of rotifers with JNK inhibitor enhanced mitochondria activity nearly 3-fold, whereas rapamycin treatment had no significant effect. Treatment of rotifers with rapamycin or JNK inhibitor reduced lysosome activity in 1, 3 and 8 day old animals, but treatment with both inhibitors did not produce any additive effect. We conclude that inhibition of TOR and JNK pathways significantly extends the lifespan of B. manjavacas. These pathways interact so that inhibition of both simultaneously acts additively to extend rotifer lifespan more than the inhibition of either alone., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Burkholderia mallei , the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the B. mallei bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal in vivo whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time in vivo imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized in vivo imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections.

Mycobacterium tuberculosis (M.tb) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/γc(null) mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34(+) fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45(+)) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-γ, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2-8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB.

Background: A major outbreak of bloody diarrhea associated with Shiga toxin-producing Escherichia coli O104:H4 occurred early in 2011, to which an unusual number of hemolytic uremic syndrome cases were linked. Due to limited information regarding pathogenesis and/or virulence properties of this particular serotype, we investigated the contribution of the aerobactin iron transport system during in vitro and in vivo conditions., Results: A bioluminescent reporter construct was used to perform real-time monitoring of E. coli O104:H4 in a mouse model of infection. We verified that our reporter strain maintained characteristics and growth kinetics that were similar to those of the wild-type E. coli strain. We found that the intestinal cecum of ICR (CD-1) mice was colonized by O104:H4, with bacteria persisting for up to 7 days after intragastric inoculation. MALDI-TOF analysis of heat-extracted proteins was performed to identify putative surface-exposed virulence determinants. A protein with a high similarity to the aerobactin iron receptor was identified and further demonstrated to be up-regulated in E. coli O104:H4 when grown on MacConkey agar or during iron-depleted conditions. Because the aerobactin iron acquisition system is a key virulence factor in Enterobacteriaceae, an isogenic aerobactin receptor (iutA) mutant was created and its intestinal fitness assessed in the murine model. We demonstrated that the aerobactin mutant was out-competed by the wild-type E. coli O104:H4 during in vivo competition experiments, and the mutant was unable to persist in the cecum., Conclusion: Our findings demonstrate that bioluminescent imaging is a useful tool to monitor E. coli O104:H4 colonization properties, and the murine model can become a rapid way to evaluate bacterial factors associated with fitness and/or colonization during E. coli O104:H4 infections.

Animal cells are protected from oxidative damage by an antioxidant network operating as a coordinated system, with strong synergistic interactions. Lifespan studies with whole animals are expensive and laborious, so there has been little investigation of which antioxidant interactions might be useful for life extension. Animals in the phylum Rotifera are particularly promising models for aging studies because they are small (0.1-1 mm), have short, two-week lifespan, display typical patterns of animal aging, and have well characterized, easy to measure phenotypes of aging and senescence. One class of interventions that has consistently produced significant rotifer life extension is antioxidants. Although the mechanism of antioxidant effects on animal aging remains controversial, the ability of some antioxidant supplements to extend rotifer lifespan was unequivocal. We found that exposing rotifers to certain combinations of antioxidant supplements can produce up to about 20% longer lifespan, but that most antioxidants have no effect. We performed life table tests with 20 single antioxidants and none yielded significant rotifer life extension. We tested 60 two-way combinations of selected antioxidants and only seven (12%) produced significant rotifer life extension. None of the 20 three- and four-way antioxidant combinations tested yielded significant rotifer life extension. These observations suggest that dietary exposure of antioxidants can extend rotifer lifespan, but most antioxidants do not. We observed significant rotifer life extension only when antioxidants were paired with trolox, N-acetyl cysteine, L: -carnosine, or EUK-8. This illustrates that antioxidant treatments capable of rotifer life extension are patchily distributed in the parameter space, so large regions must be searched to find them. It furthermore underscores the value of the rotifer model to conduct rapid, facile life table experiments with many treatments, which makes such a search feasible. Although some antioxidants extended rotifer lifespan, they likely did so by another mechanism than direct antioxidation.

Prophylactic administration of CpG oligodeoxynucleotides (CpG ODNs) is known to confer protection against lethal sepsis caused by Burkholderia pseudomallei in the mouse model. The mechanisms whereby CpG regulates the innate immune response to provide protection against B. pseudomallei, however, are poorly characterized. In the present study, we demonstrate that intranasal treatment of mice with Class C CpG, results in recruitment of inflammatory monocytes and neutrophils to the lung at 48 h post-treatment. Mice infected with B. pseudomallei 48 h post-CpG treatment had reduced organ bacterial load and significantly altered cytokine and chemokine profiles concomitant with protection as compared to control animals. CpG administration reduced the robust production of chemokines and pro-inflammatory cytokines in blood, lung and spleen, observed following infection of non-treated animals. Death of control animals coincided with the time of peak cytokine production (day 1-3), while a moderate; sustained cytokine production in CpG-treated animals was associated with survival. In general, CpG treatment resulted in diminished expression of cytokines and chemokines post-infection, though IL-12p40 was released in larger quantities in CpG treated animals. In contrast to CpG-treated animals, the lungs of infected control animals were infiltrated with leukocytes, especially neutrophils, and large numbers of necrotic lesions were observed in lung sections. Therapeutic treatment of B. pseudomallei-infected animals with CpG at 24 h post-infection did not impact survival compared to control animals. In summary, protection of CpG-treated animals was associated with recruitment of inflammatory monocytes and neutrophils into the lungs prior to infection. These responses correspond with early control of bacterial growth, a dampened inflammatory cytokine/chemokine response, reduced lung pathology, and greatly increased survival. In contrast, a delay in recruitment of inflammatory cell populations, despite a robust production of pro-inflammatory cytokines, was associated with poorly controlled bacterial growth, severe lung pathology, and death of control animals.

Background: Colposcopy is widely used in clinical microbicide safety testing but not in preclinical small animal studies. Endoscopic colposcopy could be employed in small animals allowing colposcopy to be used as one component in a multifactorial safety testing paradigm., Study Design: We conducted dose-response studies in mice using 2%, 0.2%, or 0.02% benzalkonium chloride (BZK) as the test compound, and using multiple safety end points that included endoscopic colposcopy, susceptibility to vaginal HSV-2 infection, histology, and entry of inflammatory cells into the vagina., Results: Animals treated with 0.2% or higher BZK experienced vaginal toxicities detectable by all tests used including colposcopy. In contrast, 0.02% BZK produced no significant changes except by histology in which a significant thinning of the vaginal epithelium was seen., Conclusion: Endoscopic colposcopy detected microbicide-elicited changes in the mouse vagina with similar sensitivity to the other endpoints used in these studies and would appear to be useful as part of a multifactorial microbicide safety testing paradigm in mice.

Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during decompensation will provide molecular targets for prevention of heart failure. We have previously shown enhanced ubiquitination (Ub) during the early growth period of pressure overload (PO) hypertrophy near intercalated discs of cardiomyocytes, where integrins are important for mechanotransduction. In this study, we tested the role of integrins upstream of Ub, whether enhanced Ub contributes to survival signaling in early PO, and if loss of this mechanism could lead to decreased ventricular function. The study used a beta(3) integrin (-/-) mouse and a wild-type mouse as a control for in vivo PO by transverse aortic constriction (TAC) and for cultured cardiomyocytes in vitro, stimulated with the integrin-activating peptide RGD. We demonstrate beta(3) integrin mediates transient Ub of targeted proteins during PO hypertrophy, which is necessary for cardiomyocyte survival and to maintain ventricular function. Prosurvival signaling proceeds by initiation of NF-kappaB transcription of the E3 ligase, cIAP1. In PO beta(3)(-/-) mice, absence of this mechanism correlates with increased TUNEL staining and decreased ventricular mass and function by 4 wk. This is the first study to show that a beta(3) integrin/Ub/NF-kappaB pathway contributes to compensatory hypertrophic growth.

In response to an increased hemodynamic load, such as pressure or volume overload, cardiac hypertrophy ensues as an adaptive mechanism. Although hypertrophy initially maintains ventricular function, a yet undefined derailment in this process eventually leads to compromised function (decompensation) and eventually culminates in congestive heart failure (CHF). Therefore, determining the molecular signatures induced during compensatory growth is important to delineate specific mechanisms responsible for the transition into CHF. Compensatory growth involves multiple processes. At the cardiomyocyte level, one major event is increased protein turnover where enhanced protein synthesis is accompanied by increased removal of deleterious proteins. Many pathways that mediate protein turnover depend on a key molecule, mammalian target of rapamycin (mTOR). In pressure-overloaded myocardium, adrenergic receptors, growth factor receptors, and integrins are known to activate mTOR in a PI3K-dependent and/or independent manner with the involvement of specific PKC isoforms. mTOR, described as a sensor of a cell's nutrition and energy status, is uniquely positioned to activate pathways that regulate translation, cell size, and the ubiquitin-proteasome system (UPS) through rapamycin-sensitive and -insensitive signaling modules. The rapamycin-sensitive complex, known as mTOR complex 1 (mTORC1), consists of mTOR, rapamycin-sensitive adaptor protein of mTOR (Raptor) and mLST8 and promotes protein translation and cell size via molecules such as S6K1. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, rapamycin-insensitive companion of mTOR (Rictor), mSin1 and Protor. mTORC2 regulates the actin cytoskeleton in addition to activating Akt (Protein kinase B) for the subsequent removal of proapoptotic factors via the UPS for cell survival. In this review, we discuss pathways and key targets of mTOR complexes that mediate growth and survival of hypertrophying cardiomyocytes and the therapeutic potential of mTOR inhibitor, rapamycin.

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

Ubiquitin conjugation of proteins is critical for cell homeostasis and contributes to both cell survival and death. Here we studied ubiquitination of proteins in pressure overloaded (PO) myocardium in the context of cardiomyocyte survival. Analysis using a feline right ventricular pressure overload (RVPO) model revealed a robust and transient increase in ubiquitination of proteins present in the Triton X-100-insoluble fraction in 24 to 48 h PO myocardium, and confocal micrographs indicate this increase in ubiquitination occurs subsarcolemmaly near the intercalated disc area of cardiomyocytes. The ubiquitination was accompanied by changes in E3 ligases including Cbl, E6AP, Mdm2 and cIAP in the same period of PO, although atrophy-related E3 ligases, MuRF1 and MuRF3 were unaltered. Furthermore, Cbl displayed a substantial increase in both levels of expression and tyrosine phosphorylation in 48 h PO myocardium. Confocal studies revealed enrichment of Cbl at the intercalated discs of 48 h PO cardiomyocytes, as evidenced by its colocalization with N-cadherin. Although apoptosis was observed in 48 h PO myocardium by TUNEL staining, cardiomyocytes showing ubiquitin staining were not positive for TUNEL staining. Furthermore, 48 h PO resulted in the phosphorylation of inhibitor of nuclear factor kappa B (IkappaB), suggesting its ubiquitin-mediated degradation and the nuclear localization of NFkappaB for the expression of specific cell survival factors such as cIAPs. Together these data indicate that increased levels of E3 ligases that regulate cell homeostasis and promote cell survival could ubiquitinate multiple cytoskeletal protein targets and that these events that occur during the early phase of PO may contribute to both cardiomyocyte survival and hypertrophy.

In characterizing ecological risks, considerable consensus building and professional judgments are required to develop conclusions about risk. This is because how to evaluate all the factors that determine ecological risk is not well defined and is subject to interpretation. Here we report on the application of a procedure to weigh the evidence of ecological risk and develop conclusions about risk that will incorporate the strengths and weaknesses of the assessment. The procedure was applied to characterize ecological risk of chemical contamination in nearshore areas adjacent to the Portsmouth Naval Shipyard, located at the mouth of the Great Bay Estuary, New Hampshire and Maine, USA. Measures of exposure and effect were used to interpret the magnitude of risk to the assessment endpoints of pelagic species, epibenthic species, the benthic community, eelgrass plants, the salt marsh community, and avian receptors. The evidence of chemical exposure from water, sediment, and tissue and the evidence of biological effects to representative pelagic, epibenthic, benthic, eelgrass, salt marsh, and avian species were weighed to characterize ecological risk. Individual measures were weighted by the quality and reliability of their data and risk was estimated from the preponderance, magnitude, extent, and strength of causal relationships between the data on exposure and effects. Relating evidence of risk to hypothesized pathways of exposure made it possible to estimate the magnitude of risk from sediment and water and express the confidence associated with the findings. Systematically weighing the evidence of risk rendered conclusions about risk in a manner that was clearly defined, objective, consistent, and did not rely solely on professional judgment.

Chorio-allantoic membranes (CAMs) were collected and analyzed for chlorinated hydrocarbons as part of a wildlife toxicology demonstration project at Naval Air Station (NAS) Whidbey Island, Washington, USA. Concentrations of DDT, DDE, DDD, Aroclor 1254, and Aroclor 1260 were found at concentrations below 0.4 ppm for 13 of 14 samples. The low correlations among DDT and its metabolites in CAMs suggest herons are not being exposed to a consistent source of these compounds. Comparison of chlorinated hydrocarbon data for CAMs from three Puget Sound heron colonies, NAS Whidbey, Samish Island and Dumas Bay, indicates contaminant burdens in herons from NAS Whidbey and Samish Island are significantly lower than burdens in herons from Dumas Bay.

Our technique of reconstruction of the anterior and posterior cruciate ligament in both acute and chronic tears, using both the semitendinosus and gracilis tendons, is described in detail. Combined rotatory instabilities were carefully evaluated and corrected during the same operation. Reefing of the posteromedial capsular ligament was done for anteromedial rotatory instability, and for significant valgus instability, the O'Donoghue medial reconstruction was performed. The Ellison procedure was initially done for associated anterolateral rotatory instability, but has been replaced by the Losee procedure. This combination of anterior cruciate ligament reconstruction with the appropriate extraarticular procedures has produced our most stable knee joints. Posterior cruciate ligament reconstruction was also performed through an anterior incision using both the semitendinosus and gracilis tendons. When both cruciates were found to be incompetent, the gracilis was used to reconstruct the posterior cruciate and the semitendinosus for the anterior cruciate. In addition, all associated rotatory instabilities were corrected at the same operation. This technique has been performed on 372 patients (342 anterior and 30 posterior cruciate ligament tears) by one surgeon over a period of five years. The average follow-up was 22 months. Objective comparison of the reconstructed knee with the sound knee showed 84% good results.

A synthetic energy source, 1,3-butanediol-1,3-dioctanoate (BDDO), and corn oil were fed at the 10% level in diets for chicks recovering from Newcastle disease virus at two levels of severity. There were little differences in plasma lipid concentrations between corn oil and BDDO groups. Liver lipid analyses showed significantly higher (P less than or equal to .01) triglyceride concentration for the BDDO group as compared with corn oil. In both trials, total liver lipid content was higher in all components for the BDDO group. Triglycerides and total lipids were notably lower (P less than or equal to .01) in skin of the BDDO group as compared with corn oil. Liver and skin lipid variations were attributed to different metabolic routes for BDDO and corn oil. The data suggest that BDDO tends to allow liver synthesis of triglycerides while suppressing lipid storage in the skin.

A review of charts and x-ray films of individuals who sustained ankle sprains was carried out to determine the presence of calcaneonavicular coalition. Coalitions were present in 63% of 215 patients with 223 ankle sprains. In 130 ankle sprains recorded as occurring directly in athletics, 65% of the x-ray films showed some degree of calcaneal navicular abnormality. This study found a much higher percentage of coalitions than anticipated in either the general population or the injured athletic population and reinforced the belief that there may be an underlying anatomic predisposition to ankle sprains.

Myositis ossificans traumatica, although it is one of a number of heterotopic bone formation entities, is a specific lesion with a clearly recognized etiology and natural evolutionary pattern. Early conservative treatment with rest, elevation, and immobilization minimizes additional trauma and decreases the likelihood of incapacitating bone formation. Subsequently, active range of motion exercises progressing to resistive exercises usually effect a satisfactory recovery and return to full athletic participation. With this treatment regimen, few athletes will be left with significant functional impairment. However, surgery does have a definite role in the treatment of this condition and is indicated in those athletes who develop a large mass of mature lamellar bone which is painful and is associated with muscle weakness and a significant loss of joint motion. Four patients are presented in which surgery resulted in satisfactory recovery and return to full participation in football.

Secondary reconstruction of the torn anterior cruciate ligament has been accomplished by semitendinosus tenodesis in 78 athletes (average age, 20 years; range, 13 to 45 years old). This series was limited to patients who had isolated tears of the ligament or tears of the anterior cruciate ligament associated with tears of one or both menisci. Meniscectomies were performed in 71 knees and reefing of the posteromedial capsular ligament in 35 knees. After an average of 11 months of follow-up (range, 6 to 29 months postoperatively), 67 (86%) of the patients have obtained significant improvement (0 to 1+ on a scale of 0 to 3+) in anteroposterior and anteromedial rotational instability of the joint. The viability and function of the reconstructed ligament was confirmed by direct visualization in three patients at 12, 25 and 26 months postoperatively. Reefing of the posteromedial capsular ligament appears to increase anteromedial rotation stability. This series will be expanded and the follow-up evaluations will continue over a period of years in an effort to determine whether or not semitendinosus tenodesis is superior to other methods of intraarticular reconstruction for a torn anterior cruciate ligament.

1,3-Butanediol-1,3-dioctanoate (BDDO), a synthetic source of energy, has been shown to be equal to corn oil when fed to chicks recovering from moderate and severe Newcastle disease virus infections. Body weight increments of chicks fed diets containing 10% BDDO were equal to or greater than those of chicks fed 10% corn oil, both with restricted feeding regimens. Kilocalories of metabolizable energy required to produce 100 g of body weight increment over a basal group was used as a means of quantitating energy demand. BDDO was comparable to corn oil as an energy source with no adverse effects. Liver/body weight ratios were greater in the BDDO-fed chicks. Circadian rhythmicity of liver size and liver glycogen content was demonstrated. Chicks fed BDDO had total liver glycogen content threefold that of the corn oil controls, which was attributed to stimulation of insulin secretion. Catch-up growth in the chick following the growth depression of disease appears to be as well facilitated by a synthetic source as by a natural one.

Reconstruction of the anterior cruciate ligament using the semitendinosus and gracilis tendons combined with the appropriate extraarticular procedures has been performed by the authors in 482 cases. In 321 cases both the semitendinosus and gracilis tendons were used and in 161 the semitendinosus alone. This retrospective study was done to determine if the use of these two tendons resulted in any significant loss of hamstring strength. Evaluation of quadriceps strength was also done as a measure of postoperative rehabilitation. Fifty-one patients with an average follow-up time of 26.2 months were tested on the Cybex machine (Cybex Co., Ronkonkoma, New York) by two examiners using the same technique. The examiners had not participated in the surgery or rehabilitation of these patients. In the reconstructed knee in which both semitendinosus and gracilis were used, hamstring strength was found to average 99% compared to the normal knee. When the semitendinosus alone was used there was no difference (102%) from the normal knee. Quadriceps strength in both groups averaged 96% in comparison to the normal quadriceps. These results confirm that no significant loss of hamstring strength occurred when the semitendinosus and gracilis tendons were used to construct the anterior cruciate ligament. Further, quadriceps strength of 96% as compared to the normal knee indicated a very acceptable degree of postoperative rehabilitation in this series.