The Cre/loxP-Based Recombinant HBV cccDNA System In Vitro and In Vivo.

Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.

Considerations for protein and amino acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism.

The concept of a standardized reference diet (SRD) is used in laboratory model organisms to ensure nutritional control between studies and laboratories. Although models using the genetically identical, all female parthenogenetic marbled crayfish (Procambarus virginalis) are growing in popularity, research into nutrition in this species still has many knowledge gaps. To fast track the development of a SRD in terms of protein and amino acids (SRDprotein) for this species, we first analyzed the composition of its body amino acids to determine the ideal protein concept (IPC) of indispensable amino acids in wild-caught P. virginalis (which had an unusually high preponderance of leucine and arginine). Then, we strategically evaluated three common clusters of types of fish feed: (1) ornamental fish feed (SER) fortified with a naturally occurring alga (Spirulina). This type of feed was protein-high in arginine and leucine (SER + SPI) that fulfils the species' IPC for iso-protein (~ 40%), iso-phosphorus (~ 0.8%) and near iso-energetic (~ 475 kcal 100 g-1); (2) freeze-dried live feed consisting of chironomid larvae (CHI) fortified with Spirulina (CHI + SPI) that fulfils the IPC for iso-protein (~ 46%), iso-phosphorus (~ 0.7%) and near iso-energetic (~ 405 kcal 100 g-1); and (3) a commercially standardized 'starter diet' for carnivorous fish larvae (FISH) and post-larval shrimps (SHRIMP) with iso-protein (~ 56%) and iso-phosphorus (~ 1.6%). A total of six diets, embracing a diverse range of proteinaceous feeds, were used in a 100-day ad libitum feeding and growth trial. The FISH group outperformed all the other groups (p < 0.05) and our exploratory multivariate analysis revealed an ideal demand of > 44% protein (tailored to deliver high arginine 3% and leucine 4%, followed by the usual lysine > 3.5% and methionine 1.2%) but also the lowest carbohydrate level (21%). For SRDprotein, our findings show that the FISH diet is ideal and suggest the possibilities of using a CHI + SPI diet for further optimization (more economic use of protein and phosphorus).

Effects of aging and reproductive history on bone parameters of common marmoset (Callithrix jacchus) mandibles.

To understand effects of aging and reproductive history in the bones of common marmosets (Callithrix jacchus), mandibles from 79 males and 66 females were analyzed. Dry bone specimen was prepared from dissected mandible, and analyzed using a dual-energy x-ray absorptiometry (DXA) measurement system in terms of bone weight (BnW), bone area (AREA), bone mineral content (BMC), bone mineral density (BMD, ratio of BMC to AREA) and bone mineral ratio (BMR, ratio of BMC to BnW). The mandible bones became porous and thicker with age. The age-related changes in BnW, AREA and BMC showed inflection points at around 1.5-2 Y and 13-15 Y. The period before 1.5-2 Y corresponds to the growth phase, the period between the inflection points is the aging phase, followed by senescence after the second inflection point. BMD increased until 1.5-2 Y and gradually decreased thereafter in males, with a more dramatic decrease in females, probably because of pregnancy and lactation. BMR was stable after reaching its peak by 1 Y, unlike the other parameters we analyzed. BMD of parous female tended to be lower than that of nulliparous female aged 2-5 Y. This study identified some of the particular effects of aging and reproductive history on characteristics of mandible bones in common marmoset.

Three-dimensional shape-conformation performances of wound dressings tested in a robotic sacral pressure ulcer phantom.

Effective exudate retention by dressings requires close and intimate dressing-wound contact, immediately and continuously after the dressing application. Any dressing-wound spaces may allow for build-up of non-retained fluids, causing exudate pooling which forms a favourable environment for pathogen growth. Maceration may follow if the pooled exudates spread to peri-wound skin. Dressings with a claimed 3D-shape-conformation technology are commercially available; however, their effectiveness in minimising dressing-wound gaps has never been scientifically investigated. We present a novel bioengineering methodology for testing the effectiveness of such 3D-shape-conformation dressings, using our recently reported robotic phantom system of a sacral pressure ulcer. By means of 3D laser scanning and bespoke software, we reconstructed dressing shapes after simulated use and calculated the goodness-of-fit between each dressing (swelled to near-saturation) and the corresponding wound geometry. Two dressing sizes (10 × 10 cm and 12.5 × 12.5 cm) and two wound depths (2.5 or 2 cm) were considered. All the tested dressings were far from reaching good contact with the (simulated) wounds: Approximately one-third of the wound volume and nearly half of the wound surface were not in contact with the swelled dressings. Our present findings question whether 3D-shape-conformation dressings are effective, by revealing their swelling behaviour which was previously unknown.

Red-tailed phascogales: A review of their biology and importance as model marsupial species.

There are many limitations when using traditional laboratory species. Limits on variation, may result in limited outcomes, at both the species and individual level, due to different individuals/species having diverse physiological processes, or differing molecular and genetic mechanisms. By using a variety of model species, we will be able to develop creative solutions to biological problems and identify differences of which we were not previously aware. The laboratory mouse has been a suitable model species for various mammalian studies, however most are bred specifically for laboratory research with limited variability due to selective breeding. Marsupial models offer unique research opportunities compared to eutherian models. We believe that there should be an expansion in marsupial model species, and the introduction of the red-tailed phascogale (Phascogale calura), a dasyurid marsupial, should be one of them. Phascogales are easily managed in captivity, and there are now multiple studies involving their development, reproduction, nutrition, behavior and immune system, which can serve as a baseline for future studies. The addition of the phascogale as a model species will improve future mammalian studies by introducing variability and offer alternate solutions to biological problems, particularly in the areas of genetics, nutrition, immunology, the neuro-endocrine system, and ageing, due to their semelparous reproductive strategy and hence, subsequent predictive physiology. In this review, we provide information based on existing research on red-tailed phascogales to support their inclusion as a model species.

Hallucinations on demand: the utility of experimentally induced phenomena in hallucination research.

Despite the desire to delve deeper into hallucinations of all types, methodological obstacles have frustrated development of more rigorous quantitative experimental techniques, thereby hampering research progress. Here, we discuss these obstacles and, with reference to visual phenomena, argue that experimentally induced phenomena (e.g. hallucinations induced by flickering light and classical conditioning) can bring hallucinations within reach of more objective behavioural and neural measurement. Expanding the scope of hallucination research raises questions about which phenomena qualify as hallucinations, and how to identify phenomena suitable for use as laboratory models of hallucination. Due to the ambiguity inherent in current hallucination definitions, we suggest that the utility of phenomena for use as laboratory hallucination models should be represented on a continuous spectrum, where suitability varies with the degree to which external sensory information constrains conscious experience. We suggest that existing strategies that group pathological hallucinations into meaningful subtypes based on hallucination characteristics (including phenomenology, disorder and neural activity) can guide extrapolation from hallucination models to other hallucinatory phenomena. Using a spectrum of phenomena to guide scientific hallucination research should help unite the historically separate fields of psychophysics, cognitive neuroscience and clinical research to better understand and treat hallucinations, and inform models of consciousness. This article is part of the theme issue 'Offline perception: voluntary and spontaneous perceptual experiences without matching external stimulation'.

Psychosis-Relevant Effects of Intravenous Delta-9-Tetrahydrocannabinol: A Mega Analysis of Individual Participant-Data from Human Laboratory Studies.

INTRODUCTION: There is increasing interest in the relationship between cannabinoids and psychosis. While individual human laboratory studies have been critical in demonstrating that cannabinoids (e.g., delta-9-tetrahydrocannabinol [THC]) can induce acute transient psychosis-like effects in healthy human volunteers, combining data from multiple studies offers a fine-grained view of these effects. METHODS: THC-induced psychosis-relevant effects were examined using a data repository of 10 double-blind, randomized, placebo-controlled, crossover studies with 400 i.v. THC infusions in healthy human volunteers. The Positive and Negative Syndrome scale was used to measure psychotomimetic effects. The profile of symptoms, frequency of a response, its relationship to THC dose and substance use, latent structure in Positive and Negative Syndrome scale response, and the relationships between psychotomimetic and perceptual alteration symptoms were evaluated. RESULTS: Clinically meaningful increases in positive symptoms were noted in 44.75% infusions; conceptual disorganization, hallucinations, blunted affect, somatic concern, motor retardation, and poor attention were the items most frequently altered by THC. The increase in Positive and Negative Syndrome scale positive symptoms was positively associated with THC dose (beta = 11.13, SE = 4.94, Wald χ 2 = 19.88, P < .001) and negatively associated with frequent cannabis use (beta = -0.575, SE = 0.14, Wald χ 2 = 18.13, P < .001). Furthermore, positive symptoms were strongly correlated with Clinician Administered Dissociative States Scale perceptual alterations score (rs = 0.514, P < .001). CONCLUSION: Intravenous administration of THC consistently induces psychotomimetic effects that include symptoms across Positive and Negative Syndrome scale domains. Moreover, healthy individuals who frequently use cannabis have a blunted psychotomimetic response.

Effect of Delaying Replacement of Parenteral Nutrition Intravenous Administration Sets: Preclinical Experiments and a Dynamic Laboratory Model of Microbial Colonization.

BACKGROUND: Recommendations prescribe daily intravenous administration set (IVAS) replacement for parenteral nutrition (PN) comprising intravenous fat emulsions (IVFE) due to risk of micro-organism growth and resultant central-line associated bloodstream infections (CLABSIs), but system disconnection for this practice may allow contamination and CLABSIs. MATERIALS AND METHODS: Laboratory experiments and model development were used to simulate PN administration after contamination from healthcare workers' hands. This study observed the growth of micro-organisms known to cause CLABSIs in a variety of PN and other IV fluids and developed a model to investigate the effect of delaying IVAS replacement on microbial growth for up to 7 days. RESULTS: Micro-organisms grew at different rates and were affected by solution type. In static experiments, growth was supported in IVFE and all-in-one PN, but suppressed in 50% glucose. Growth patterns were consistent over time for Staphylococcus epidermidis, Staphylococcus aureus, and Candida albicans in IVFE, all-in-one PN, and 0.9% sodium chloride in both static and dynamic experiments. C. albicans grew exponentially to clinically significant numbers in all-in-one PN and IVFE IVAS after 30 hours, but negligible growth of S. epidermidis or S. aureus occurred for 7 days. CONCLUSION: All-in-one PN and IVFE support the C. albicans growth after minimal initial contamination, with micro-organisms migrating from the fluid bag to the central venous access device. Improved aseptic nontouch technique during clinical practice is vital to prevent contamination. Daily IVAS replacement of for all-in-one PN and IVFE should continue until the safety of prolonging IVAS replacement is confirmed by randomized trials.

Greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) as a resistant model host for Nosema pyrausta (Microsporidia: Nosematidae).

Galleria mellonella fed 3 million Nosema pyrausta spores per larva showed 0 and 5% infestation rate at 30 °C and 24 °C, respectively. N. pyrausta virulence did not increase after passage through G. mellonella for three generations. When larvae were pretreated with phenylthiourea, Bacillus thuringiensis or combination of both, infection rates were 11%, 15% and 22%, respectively. Injection of untreated and potassium hydroxide-primed spores resulted in approximately 10% and 50% infection, respectively. G. mellonella is resistant to high dosages of N. pyrausta spores, serving as a prospective model of insect resistance to microsporidia, while host immunosuppression and/or spore activation increases success of the pathogen.

A cadaver study into the number of fasciotomies required to decompress the anterior compartment in forearm compartment syndrome.

PURPOSE: There is no typical approach for decompression of forearm compartment syndrome, due to contradictory considerations regarding the characteristics of forearm anterior compartment deep fascia. The main purpose of this study was to determine how many fasciae should be opened to fully decompress the forearm anterior compartment. Further, the compliance of the deep anterior compartment was also investigated, to strengthen our results. METHODS: An experimental study of a laboratory model of acute forearm compartment syndrome was performed. A deep forearm injection of egg white was undertaken to create an acute forearm compartment syndrome in sixteen non-embalmed human forearms from six male and two female donors. The pressure in the superficial and deep anterior compartments was recorded four times, both before and after each fasciotomy and the compliance of the deep anterior compartment was calculated for each step. RESULTS: The first incision of the superficial lamina of the deep fascia was not sufficient to decrease the elevated compartment pressure in the superficial and deep anterior compartments. Whereas the pressures decreased to near-baseline levels, following the fasciotomy of the intermuscular septum observed posterior to the flexor carpi radialis. The last incision of the deep lamina of the deep anterior fascia had no noticeable impact. These observations supported the hypothesis of high compliance of the deep anterior compartment. CONCLUSION: Two successive incisions were necessary to decompress the anterior compartment: the incision of the superficial lamina of the deep fascia and the incision of the intermuscular septum.

Zika virus transmission to mouse ear by mosquito bite: a laboratory model that replicates the natural transmission process.

BACKGROUND: Zika disease has transformed into a serious global health problem due to the rapid spread of the arbovirus and alarming severity including congenital complications, microcephaly and Guillain-Barré syndrome. Zika virus (ZIKV) is primarily transmitted to humans through the bite of an infective mosquito, with Aedes aegypti being the main vector. METHODS: We successfully developed a ZIKV experimental transmission model by single infectious Ae. aegypti bite to a laboratory mouse using circulating Brazilian strains of both arbovirus and vector. Mosquitoes were orally infected and single Ae. aegypti were allowed to feed on mouse ears 14 days post-infection. Additionally, salivary gland (SG) homogenates from infected mosquitoes were intrathoracically inoculated into naïve Ae. aegypti. Mosquito and mouse tissue samples were cultured in C6/36 cells and processed by quantitative real-time PCR. RESULTS: A total of 26 Ae. aegypti were allowed to feed individually on mouse ears. Of these, 17 mosquitoes fed, all to full engorgement. The transmission rate of ZIKV by bite from these engorged mosquitoes to mouse ears was 100%. The amount of virus inoculated into the ears by bites ranged from 2 × 102-2.1 × 1010 ZIKV cDNA copies and was positively correlated with ZIKV cDNA quantified from SGs dissected from mosquitoes post-feeding. Replicating ZIKV was confirmed in macerated SGs (2.45 × 107 cDNA copies), mouse ear tissue (1.15 × 103 cDNA copies, and mosquitoes 14 days post-intrathoracic inoculation (1.49 × 107 cDNA copies) by cytopathic effect in C6/36 cell culture and qPCR. CONCLUSIONS: Our model illustrates successful transmission of ZIKV by an infectious mosquito bite to a live vertebrate host. This approach offers a comprehensive tool for evaluating the development of infection in and transmission from mosquitoes, and the vertebrate-ZIKV interaction and progression of infection following a natural transmission process.

Microsurgical Simulation Exercise for Surgical Training.

OBJECTIVE: Initial training for orthopedic surgical residents (postgraduate years 1-5) in microsurgery using the turkey wing model and evaluation of their proficiency. DESIGN: Residents were given a questionnaire on their comfort level with microsurgery and microsurgical knowledge, followed by a lecture on the subject. They watched a surgical dissection and repair of the turkey wing's neurovasculature. Residents performed the dissection and repairs of the artery, vein, and nerve. A postquestionnaire was administered following the simulation exercise. Their performances on repairs were graded and results compared by academic year. SETTING AND PARTICIPANTS: A total of 21 orthopedic surgery residents were recruited from Stony Brook University Medical Center, Stony Brook, NK. RESULTS: This training activity resulted in significant improvements in both microsurgical knowledge (41%) and comfort (37%). Senior residents scored significantly higher than juniors on 6 microsurgical parameters. The largest effect was in nerve repair showing 4 parameters that differed significantly between groups. CONCLUSION: Microsurgical techniques require extensive training to master. The turkey wing model for repair of the artery, vein, and nerve represents a realistic simulation of a human hand artery, vein, and nerve. It provides an inexpensive method for residents to practice on real tissue for improving microsurgical technique.

Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function.

In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10(9)/l. The in vitro model yielded median platelet counts at 51×10(9)/l (range 26-93×10(9)/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia.

Lizard tail spinal cord: a new experimental model of spinal cord injury without limb paralysis.

Spinal cord injury (SCI) is a well-known devastating lesion that sadly is very resistant to all treatment attempts. This fact has stimulated the exploration of multiple regenerative strategies that are examined at both the basic and clinical level. For laboratory research, differentin vivomodels are used, but each has many important limitations. The main limitation of these models is the high level of animal suffering related to the inflicted neurologic injury. It has caused a growing tendency to limit the injury, but this, in turn, produces incomplete SCI models and uncertainties in the neuroregeneration interpretation. To overcome such limitations, a new experimental SCI model is proposed. Geckos have been extensively examined as a potential animal model of SCI. Their spinal cord extends into the tail and can be transected without causing the typical neurologic consequences observed in rat models. In this study, we compared the gecko tail SCI model with the rat model of thoracic SCI. Anatomic and histologic analyses showed comparability between the gecko and rat in diameter of spinal canal and spinal cord, as well as applicability of multiple staining techniques (hematoxylin and eosin, immunostaining, and scanning and transmission electron microscopy). We tested the suitability ofin vivostudy with 3 prototype implants for the reconstruction of SCI: a multichannel sponge, a multilaminar tube, and a gel cylinder. These were compared with a spinal cord excision (control). A 20-wk observation revealed no adverse effects of SCI on the animals' well-being. The animals were easily housed and observed. Histologic analysis showed growth of nervous tissue elements on implant surface and implant cellular colonization. The study showed that the gecko SCI model can be used as a primary model for the assessment of SCI treatment methods. It provides a platform for testing multiple solutions with limited animal suffering before performing tests on mammals. Detailed results of the experimental conditions and testing techniques are provided.-Szarek, D., Marycz, K., Lis, A., Zawada, Z., Tabakow, P., Laska, J., Jarmundowicz, W. Lizard tail spinal cord: a new experimental model of spinal cord injury without limb paralysis.

Design and development of a simple laboratory model to detect (15)N enrichment in cyanobacterial biomass and extra cellular ammonia using (15)N gas.

A laboratory scale working model that could detect the (15)N enrichment in cyanobacterial biomass and extracellular ammonia, using (15)N gas under in vitro conditions was designed and fabricated. Using the model, (15)N enrichment of 0.48% atom excess was detected in the cyanobacterial biomass on the 30 d after inoculation. The (15)N enrichment increased linearly in the extracellular ammoniacal fraction from the 20 d onward. The model would prove to be a useful tool to quantify the extent of (15)N enrichment under in vitro conditions using (15)N gas.