The effect of sex, age, and location on carnivory in Utah black bears (Ursus americanus).

Ungulates are important to the diet of bears because they are high in protein, and the level of dietary protein strongly influences bear size. The size a bear obtains as an adult influences important life history characteristics, such as age of reproduction and reproductive success; therefore, it is important to know what foods are available to bears and how they are utilizing them. We tested hypotheses concerning the effect of age, sex, and location on black bear carnivory. We collected hair and vestigial premolar teeth from 49 Utah black bears, Ursus americanus according to the Utah Division of Wildlife Resources hunt unit. Hunt units differed in habitat quality and local ungulate density. We analyzed a vestigial premolar for the age of the bears and used analysis of the δ13C and δ15N values of the hairs of each bear to infer the degree of carnivory. δ15N of black bear hairs was positively correlated with increased availability of ungulates. There was a positive relationship between the δ15N of bear hairs and age in hunt units with the highest ungulate densities only. The δ15N and δ13C of black bear hairs were positively correlated, suggesting that bears are more carnivorous at higher altitudes. This study demonstrates the value of stable isotope analysis in understanding the feeding ecology of bears over broad geographic ranges. It demonstrates that ungulate availability is important to the feeding ecology of black bears in the Intermountain West.

Corrigendum: Efficient decellularization of whole porcine kidneys improves reseeded cell behavior (2016 Biomed. Mater. 11 025003).

The manuscript 'Efficient decellularization of whole porcine kidneys improves reseeded cell behavior' (Poornejad et al 2016 Biomedical Materials 11: 025003) describes our efforts to improve the process for recellularization of porcine kidneys. We obtained what we believed to be an immortalized cell line of human renal cortical tubular epithelium (RCTE) cells from the Feinberg School of Medicine, Northwestern University to conduct our reseeding experiments. The RCTE cells that were provided to us were later discovered to actually be Madin-Darby Canine Kidney (MDCK) epithelial cells. A published erratum pertaining to this issue has been published (Caralt et al 2017 American Journal of Transplantation 17: 1429). Despite being of canine origin, MDCK cells are a distal tubule epithelial cell line that behave similarly to human RCTE cells. The conclusions regarding reseeding as reported in our paper are still sound.

Baseline effects of lysophosphatidylcholine and nerve growth factor in a rat model of sciatic nerve regeneration after crush injury.

Schwann cells play a major role in helping heal injured nerves. They help clear debris, produce neurotrophins, upregulate neurotrophin receptors, and form bands of Büngner to guide the healing nerve. But nerves do not always produce enough neurotrophins and neurotrophin receptors to repair themselves. Nerve growth factor (NGF) is an important neurotrophin for promoting nerve healing and lysophosphatidylcholine (LPC) has been shown to stimulate NGF receptors (NGFR). This study tested the administration of a single intraneural injection of LPC (1 mg/mL for single LPC injection and 10 mg/mL for multiple LPC injections) at day 0 and one (day 7), two (days 5 and 7), or three (days 5, 7, and 9) injections of NGF (160 ng/mL for single injections and 80 ng/mL for multiple injections) to determine baseline effects on crushed sciatic nerves in rats. The rats were randomly divided into four groups: control, crush, crush-NGF, and crush-LPC-NGF. The healing of the nerves was measured weekly by monitoring gait; electrophysiological parameters: compound muscle action potential (CMAP) amplitudes; and morphological parameters: total fascicle areas, myelinated fiber counts, fiber densities, fiber packing, and mean g-ratio values at weeks 3 and 6. The crush, crush-NGF, and crush-LPC-NGF groups statistically differed from the control group for all six weeks for the electrophysiological parameters but only differed from the control group at week 3 for the morphological parameters. The crush, crush-NGF, and crush-LPC-NGF groups did not differ from each other over the course of the study. Single injections of LPC and NGF one week apart or multiple treatments of NGF at 5, 7 and 9 days post-injury did not alter the healing rate of the sciatic nerves during weeks 1-6 of the study. These findings are important to define the baseline effects of NGF and LPC injections, as part of a larger effort to determine the minimal dose regimen of NGF to regenerate peripheral nerves.

The impact of decellularization agents on renal tissue extracellular matrix.

The combination of patient-specific cells with scaffolds obtained from natural sources may result in improved regeneration of human tissues. Decellularization of the native tissue is the first step in this technology. Effective decellularization uses agents that lyse cells and remove all cellular materials, leaving intact collagenous extracellular matrices (ECMs). Removing cellular remnants prevents an immune response while preserving the underlying structure. In this study, the impact of five decellularization agents (0.1 N NaOH, 1% peracetic acid, 3% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 0.05% trypsin/EDTA) on renal tissue was examined using slices of porcine kidneys. The NaOH solution induced the most efficient cell removal, and resulted in the highest amount of cell viability and proliferation after recellularization, although it also produced the most significant damage to collagenous fiber networks, glycosaminoglycans (GAGs) and fibroblast growth factor (FGF). The SDS solution led to less severe damage to the ECM structure but it resulted in lower metabolic activity and less proliferation. Peracetic acid and Triton X-100 resulted in minimum disruption of ECMs and the most preserved GAGs and FGF. However, these last two agents were not as efficient in removing cellular materials as NaOH and SDS, especially peracetic acid, which left more than 80% of cellular material within the ECM. As a proof of principle, after completing the comparison studies using slices of renal ECM, the NaOH process was used to decellularize a whole kidney, with good results. The overall results demonstrate the significant effect of cell lysing agents and the importance of developing an optimized protocol to avoid extensive damage to the ECM while retaining the ability to support cell growth.

Rapid separation of bacteria from blood-review and outlook.

The high morbidity and mortality rate of bloodstream infections involving antibiotic-resistant bacteria necessitate a rapid identification of the infectious organism and its resistance profile. Traditional methods based on culturing the blood typically require at least 24 h, and genetic amplification by PCR in the presence of blood components has been problematic. The rapid separation of bacteria from blood would facilitate their genetic identification by PCR or other methods so that the proper antibiotic regimen can quickly be selected for the septic patient. Microfluidic systems that separate bacteria from whole blood have been developed, but these are designed to process only microliter quantities of whole blood or only highly diluted blood. However, symptoms of clinical blood infections can be manifest with bacterial burdens perhaps as low as 10 CFU/mL, and thus milliliter quantities of blood must be processed to collect enough bacteria for reliable genetic analysis. This review considers the advantages and shortcomings of various methods to separate bacteria from blood, with emphasis on techniques that can be done in less than 10 min on milliliter-quantities of whole blood. These techniques include filtration, screening, centrifugation, sedimentation, hydrodynamic focusing, chemical capture on surfaces or beads, field-flow fractionation, and dielectrophoresis. Techniques with the most promise include screening, sedimentation, and magnetic bead capture, as they allow large quantities of blood to be processed quickly. Some microfluidic techniques can be scaled up. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:823-839, 2016.

Using Hemolysis as a Novel Method for Assessment of Cytotoxicity and Blood Compatibility of Decellularized Heart Tissues.

Developing patient-specific transplantable organs is a promising response to the increasing need of more effective therapies for patients with organ failure. Advances in tissue engineering strategies have demonstrated favorable results, including the use of decellularized hearts as scaffolds for cardiac engineering; however, there is a need to establish methods to characterize the cytotoxicity and blood compatibility of cardiac extracellular matrix (cECM) scaffolds created by decellularization. In this study, porcine hearts were decellularized in an automated perfusion apparatus utilizing sodium dodecyl sulfate (SDS) detergent. Residual SDS was measured by a colorimetric assay. Phosphate-buffered saline, distilled water (DW), and Triton X-100 washes were used to remove SDS. The efficiency of detergent removal was measured as a function of time. It was observed that using Triton-X 100 can nearly double the rate of SDS removal. An assay based on human blood hemolysis was developed to measure the remaining cytotoxicity of the cECM. The results from the hemolysis cytotoxicity assay were consistent with a standard live/dead assay using MS1 endothelial cells incubated with the cECM. This study demonstrated an effective, reliable, and relatively inexpensive method for determining the cytotoxicity and blood compatibility of decellularized cECM scaffolds.

Efficient decellularization of whole porcine kidneys improves reseeded cell behavior.

Combining patient-specific cells with the appropriate scaffold to create functional kidneys is a promising technology to provide immunocompatible kidneys for the 100,000+ patients on the organ waiting list. For proper recellularization to occur, the scaffold must possess the critical microstructure and an intact vascular network. Detergent perfusion through the vasculature of a kidney is the preferred method of decellularization; however, harsh detergents could be damaging to the microstructure of the renal tissue and may undesirably solubilize the endogenous growth and signaling factors. In this study, automated decellularization of whole porcine kidneys was performed using an improved method that combined physical and chemical steps to efficiently remove cellular materials while producing minimal damage to the collagenous extracellular matrix (ECM). Freezing/thawing, incremental increases in flow rate under constant pressure, applying osmotic shock to the cellular membranes, and low concentrations of the detergent sodium dodecyl sulfate (SDS) were factors used to decrease SDS exposure time during the decellularization process from 36 to 5 h, which preserved the microstructure while still removing 99% of the DNA. The well-preserved glycosaminoglycans (GAGs) and collagen fibers enhanced cell-ECM interactions. Human renal cortical tubular epithelium (RCTE) cells grew more rapidly when cultured on the ECM obtained from the improved decellularization process and also demonstrated more in vivo-like gene expression patterns. The optimized, automated process that resulted from this work is now used routinely in our laboratory to rapidly decellularize porcine kidneys and could be adapted to other large organs (e.g. heart, liver, and lung).

Current Cell-Based Strategies for Whole Kidney Regeneration.

Chronic kidney diseases affect thousands of people worldwide. Although hemodialysis alleviates the situation by filtering the patient's blood, it does not replace other kidney functions such as hormone release or homeostasis regulation. Consequently, orthotopic transplantation of donor organs is the ultimate treatment for patients suffering from end-stage renal failure. Unfortunately, the number of patients on the waiting list far exceeds the number of donors. In addition, recipients must remain on immunosuppressive medications for the remainder of their lives, which increases the risk of morbidity due to their weakened immune system. Despite recent advancements in whole organ transplantation, 40% of recipients will face rejection of implanted organs with a life expectancy of only 10 years. Bioengineered patient-specific kidneys could be an inexhaustible source of healthy kidneys without the risk of immune rejection. The purpose of this article is to review the pros and cons of several bioengineering strategies used in recent years and their unresolved issues. These strategies include repopulation of natural scaffolds with a patient's cells, de-novo generation of kidneys using patient-induced pluripotent stem cells combined with stepwise differentiation, and the creation of a patient's kidney in the embryos of other mammalian species.

Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix.

MRI evaluation of spontaneous intervertebral disc degeneration in the alpaca cervical spine.

Animal models have historically provided an appropriate benchmark for understanding human pathology, treatment, and healing, but few animals are known to naturally develop intervertebral disc degeneration. The study of degenerative disc disease and its treatment would greatly benefit from a more comprehensive, and comparable animal model. Alpacas have recently been presented as a potential large animal model of intervertebral disc degeneration due to similarities in spinal posture, disc size, biomechanical flexibility, and natural disc pathology. This research further investigated alpacas by determining the prevalence of intervertebral disc degeneration among an aging alpaca population. Twenty healthy female alpacas comprised two age subgroups (5 young: 2-6 years; and 15 older: 10+ years) and were rated according to the Pfirrmann-grade for degeneration of the cervical intervertebral discs. Incidence rates of degeneration showed strong correlations with age and spinal level: younger alpacas were nearly immune to developing disc degeneration, and in older animals, disc degeneration had an increased incidence rate and severity at lower cervical levels. Advanced disc degeneration was present in at least one of the cervical intervertebral discs of 47% of the older alpacas, and it was most common at the two lowest cervical intervertebral discs. The prevalence of intervertebral disc degeneration encourages further investigation and application of the lower cervical spine of alpacas and similar camelids as a large animal model of intervertebral disc degeneration.

Automation of Pressure Control Improves Whole Porcine Heart Decellularization.

Whole heart decellularization combined with patient-specific cells may prove to be an extremely valuable approach to engineer new hearts. Mild detergents are commonly used in the decellularization process, but are known to denature and solubilize key proteins and growth factors and can therefore be destructive to the extracellular matrix (ECM) during the decellularization process. In this study, the decellularization of porcine hearts was accomplished in 24 h with only 6 h of sodium dodecyl sulfate exposure and 98% DNA removal. Automatically controlling the pressure during decellularization reduced the detergent exposure time while still completely removing immunogenic cell debris. Stimulation of macrophages was greatly reduced when comparing native tissue samples to the processed ECM. Complete cell removal was confirmed by analysis of DNA content. General collagen and elastin preservation was demonstrated. Glycosaminoglycans and collagen quantification both showed no significant differences in content after decellularization. The compression elastic modulus of the ECM after decellularization was lower than native at low strains, but there was no significant difference at high strains. Polyurethane casts of the vasculature of native and decellularized hearts demonstrated that the microvasculature network was preserved after decellularization. A static blood thrombosis assay using bovine blood was also developed. Finally, the recellularization potential of the ECM samples was demonstrated by reseeding cardiac fibroblasts and endothelial cells on the myocardium and endocardium samples.

Freezing/Thawing without Cryoprotectant Damages Native but not Decellularized Porcine Renal Tissue.

Whole organ decellularization of porcine renal tissue and recellularization with a patient's own cells would potentially overcome immunorejection, which is one of the most significant problems with allogeneic kidney transplantation. However, there are obstacles to achieving this goal, including preservation of the decellularized extracellular matrix (ECM), identifying the proper cell types, and repopulating the ECM before transplantation. Freezing biological tissue is the best option to avoid spoilage; however, it may damage the structure of the tissue or disrupt cellular membranes through ice crystal formation. Cryoprotectants have been used to repress ice formation during freezing, although cell toxicity can still occur. The effect of freezing/thawing on native (n = 10) and decellularized (n = 10) whole porcine kidneys was studied without using cryoprotectants. Results showed that the elastic modulus of native kidneys was reduced by a factor of 22 (P < 0.0001) by freezing/thawing or decellularization, while the elastic modulus for decellularized ECM was essentially unchanged by the freezing/thawing process (p = 0.0636). Arterial pressure, representative of structural integrity, was also reduced by a factor of 52 (P < 0.0001) after freezing/thawing for native kidneys, compared to a factor of 43 (P < 0.0001) for decellularization and a factor of 4 (P < 0.0001) for freezing/thawing decellularized structures. Both freezing/thawing and decellularization reduced stiffness, but the reductions were not additive. Investigation of the microstructure of frozen/thawed native and decellularized renal tissues showed increased porosity due to cell removal and ice crystal formation. Orcein and Sirius staining showed partial damage to elastic and collagen fibers after freezing/thawing. It was concluded that cellular damage and removal was more responsible for reducing stiffness than fibril destruction. Cell viability and growth were demonstrated on decellularized frozen/thawed and non-frozen samples using human renal cortical tubular epithelial (RCTE) cells over 12 d. No adverse effect on the ability to recellularize after freezing/thawing was observed. It is recommended that porcine kidneys be frozen prior to decellularization to prevent contamination, and after decellularization to prevent protein denaturation. Cryoprotectants may still be necessary, however, during storage and transportation after recellularization.

Strategies and processes to decellularize and recellularize hearts to generate functional organs and reduce the risk of thrombosis.

Heart failure is one of the leading causes of death in the United States. Current therapies, such as heart transplants and bioartificial hearts, are helpful, but not optimal. Decellularization of porcine whole hearts followed by recellularization with patient-specific human cells may provide the ultimate solution for patients with heart failure. Great progress has been made in the development of efficient processes for decellularization, and the design of automated bioreactors. Challenges remain in selecting and culturing cells, growing the cells on the decellularized scaffolds without contamination, characterizing the regenerated organs, and preventing thrombosis. Various strategies have been proposed to prevent thrombosis of blood-contacting devices, including reendothelization and the creation of nonfouling surfaces using surface modification technologies. This review discusses the progress and remaining challenges involved with recellularizing whole hearts, focusing on the prevention of thrombosis.

Preliminary results of combining low frequency low intensity ultrasound and liposomal drug delivery to treat tumors in rats.

Ultrasound is a convenient trigger for site-specific drug delivery in cancer therapy. Nano-sized liposomes formulated from soy phosphatidyl choline, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] and alpha-tocopherol were loaded with Doxorubicin (Dox) using a pH gradient. The liposomal suspension was infused through the tail vein of BDIX rats possessing bilateral intradermal DHD/K12 tumors on their hind legs. Then 20-kHz ultrasound was applied to only one of the tumors for 15 minutes. This therapy was repeated weekly for 4 weeks. The results showed that in five of six rats, the tumors regressed to non-measurable size within 4 weeks. A paired comparison of the normalized size of the insonated and non-insonated tumors in the same rat indicated that the insonated tumors were smaller (p < 0.0001, n = 6 rats, 21 pairs). This observation has significant potential for non-invasive site-specific therapy of solid tumors.

Distribution of doxorubicin in rats undergoing ultrasonic drug delivery.

Ultrasound (US) increases efficacy of drugs delivered from micelles, but the pharmacokinetics have not been studied previously. In this study, US was used to deliver doxorubicin (Dox) sequestered in micelles in an in vivo rat model with bilateral leg tumors. One of two frequencies with identical mechanical index and intensity was delivered for 15 min to one tumor immediately after systemic injection of micellar Dox. Pharmacokinetics in myocardium, liver, skeletal muscle, and tumors were measured for 1 week. When applied in combination with micellar Dox, the ultrasoincated tumor had higher Dox concentrations at 30 min, compared to bilateral noninsonated controls. Initially, concentrations were highest in heart and liver, but within 24 h they decreased significantly. From 24 h to 7 days, concentrations remained highest in tumors, regardless of whether they received US or not. Comparison of insonated and noninsonated tumors showed 50% more Dox in the insonated tumor at 30 min posttreatment. Four weekly treatment produced additional Dox accumulation in the myocardium but not in liver, skeletal leg muscle, or tumors compared to single treatment. Controls showed that neither US nor the empty carrier impacted tumor growth. This study shows that US causes more release of drug at the targeted tumor.

Role of frequency and mechanical index in ultrasonic-enhanced chemotherapy in rats.

PURPOSE: The therapeutic effect of ultrasound and micellar-encapsulated doxorubicin was studied in vivo using a tumor-bearing rat model with emphasis on how tumor growth rate is affected by ultrasonic parameters such as frequency and intensity. METHODS: This study employed ultrasound of two different frequencies (20, 476 kHz) and two pulse intensities, but identical mechanical indices and temporal average intensities. Ultrasound was applied weekly for 15 min to one of two bilateral leg tumors (DHD/K12/TRb colorectal epithelial cell line) in the rat model immediately after intravenous injection of micelle-encapsulated doxorubicin. This therapy was applied weekly for 6 weeks. RESULTS: Results showed that tumors treated with drug and ultrasound displayed, on average, slower growth rates than non-insonated tumors (P = 0.0047). However, comparison between tumors that received 20 or 476-kHz ultrasound treatments showed no statistical difference (P = 0.9275) in tumor growth rate. CONCLUSION: Application of ultrasound in combination with drug therapy was effective in reducing tumor growth rate, irrespective of which frequency was employed.

Using stable isotopes to unravel and predict the origins of great cormorants (Phalacrocorax carbo sinensis) overwintering at Kinmen.

The Food and Agricultural Organization of the United Nations and the World Organization for Animal Health have called for a better understanding of the role that migrating birds may play in spreading H5N1 highly pathogenic avian influenza (HPAI). Bird banding, traditionally used in studies of migration, is limited by low recapture rates. Telemetry can only be applied to larger species and a limited number of birds. We show that analyses of multiple stable isotopes (delta(13)C, delta(15)N, delta(18)O and deltaD) can provide an understanding of the number of breeding populations represented at large congregations of wintering birds, probable locations of these breeding populations, and which breeding populations do not contribute migrants to a wintering site. As Asia is thought to be the origin of many HPAI strains and the center of their evolution, and as bird migration is poorly understood in this part of the world, we recommend that, in addition to banding, satellite, and VHF telemetry, the stable isotope analysis of migration patterns should become a part of long-term surveillance studies.

Towards a physiologically based diagnosis of anorexia nervosa and bulimia nervosa.

Diagnosis of anorexia nervosa (AN) and bulimia nervosa (BN), while including such physiological data as weight and the reproductive status of the individual, are primarily based on questionnaires and interviews that rely on self-report of both body-related concerns and eating-related behaviors. While some key components of eating disorders are psychological and thus introspective in nature, reliance on self-report for the assessment of eating-related behaviors and nutritional status lacks the objectivity that a physiologically based measure could provide. The development of a more physiologically informed diagnosis for AN and BN would provide a more objective means of diagnosing these disorders, provide a sound physiological basis for diagnosing subclinical disorders and could also aid in monitoring the effectiveness of treatments for these disorders. Empirically supported, physiologically based methods for diagnosing AN and BN are reviewed herein as well as promising physiological measures that may potentially be used in the diagnosis of AN and BN.

An objective means of diagnosing anorexia nervosa and bulimia nervosa using 15N/14N and 13C/12C ratios in hair.

An objective means based on the carbon and nitrogen stable isotope analysis of five hairs per individual is presented for distinguishing between individuals with anorexia nervosa and/or bulimia nervosa from non-clinical individuals (i.e. clinically normal controls). Using discriminant analysis, an algorithm has been developed that provides both sensitivity and specificity of 80% in making diagnoses of individuals with these eating disorders. With further refinements, the results suggest that it may be also possible to distinguish between individuals with anorexia or bulimia. Finally, the study shows the value of conducting blind tests and using larger sample sizes of both control and treatment groups. Both groups are needed to validate the diagnostic value of a method and to provide measures of sensitivity and specificity of any diagnostic test.

Treatment of biofilm infections on implants with low-frequency ultrasound and antibiotics.

Medical implants are sometimes colonized by biofilm-forming bacteria, which are very difficult to treat effectively. The combination of gentamicin and ultrasonic exposure for 24 hours was previously shown to reduce the viability of Escherichia coli biofilms in vivo. This article shows that such treatment for 48 hours reduced viable E coli bacteria to nearly undetectable levels. However, when Pseudomonas aeruginosa biofilms were implanted and treated for 24 and 48 hours, no significant ultrasonic-enhanced reduction of viable bacteria was observed. The difference in response of these 2 organisms is attributed to greater impermeability and stability of the outer membrane of P aeruginosa.