Localization of fibronectin in the rat testis : effects of serum and dibutyryl cyclic AMP on fibronectin deposition by peritubular myoid cells in culture

The peritubular zone of the rat testis has an extensive extracellular matrix (ECM). Fibronectin (FN) is distributed primarily in the basal lamina of the seminiferous tubule boundary tissue and is synthesized by peritubular myoid cells. Several extracellular changes are mediated by growth factors and these changes occur at the time of hormone mediated testicular development, particularly in the peritubular zone. The effects of serum or dibutyryl cyclic AMP (cAMP) on FN production by the mesenchymal peritubular myoid cells were evaluated. Rats of various ages (10, 15, 20, 40 and 80 days) were employed for immunofluorescent localization of rat testicular FN in frozen sections. In all age groups tested, FN was primarily present in a broad layer around each seminiferous tubule, and blood vessel, and in variable distribution throughout the interstitial stroma. By day 20 there was no clear distinction in FN staining between the peritubular zone and the interstitial tissue. This indicates an involvement of FN in the ECM developments which occur in the peritubular zone of the testis at this time. The peritubular myoid cells were isolated from 20-22 day old rat testis and cultured on glass coverslips. These cells were grown to confluence with 10% fetal calf serum (FCS) in medium until day 4 and then subcultured to have secondary monocultures maintained with or without serum. By means of immunofluorescence and cytochemistry using avidin-biotin peroxidase complex it was observed that peritubular myoid cells were positive for FN and most of the FN was localized in the perinuclear region. Subcultured peritubular myoid cells maintained for 4 days in medium containing FCS developed an extensive interconnecting FN matrix. In the presence of 0.5 mM cAMP in culture, FN became localized along the filamentous process of peritubular myoid cells and more prominently in the areas of triangulated multi-cell aggregates as well as on the surface of the contracted small spherical cells. The addition of cAMP in the presence of FCS, also caused a noticeable change in the staining pattern; FN was detected along the filamentous process developing into a complex network of cells encased in an extensive matrix. It would appear that the translocation of FN in the cytoplasmic extensions of peritubular myoid cells may be a direct consequence of morphological changes associated with metabolic regulation of cAMP. This may also be related to the puberty associated development of in vivo changes in the ECM produced by peritubular myoid cells.

Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

Enhancing paramedics procedural skills using a cadaveric model

Background Paramedic education has evolved in recent times from vocational post-employment to tertiary pre-employment supplemented by clinical placement. Simulation is advocated as a means of transferring learned skills to clinical practice. Sole reliance of simulation learning using mannequin-based models may not be sufficient to prepare students for variance in human anatomy. In 2012, we trialled the use of fresh frozen human cadavers to supplement undergraduate paramedic procedural skill training. The purpose of this study is to evaluate whether cadaveric training is an effective adjunct to mannequin simulation and clinical placement. Methods A multi-method approach was adopted. The first step involved a Delphi methodology to formulate and validate the evaluation instrument. The instrument comprised of knowledge-based MCQs, Likert for self-evaluation of procedural skills and behaviours, and open answer. The second step involved a pre-post evaluation of the 2013 cadaveric training. Results One hundred and fourteen students attended the workshop and 96 evaluations were included in the analysis, representing a return rate of 84%. There was statistically significant improved anatomical knowledge after the workshop. Students' self-rated confidence in performing procedural skills on real patients improved significantly after the workshop: inserting laryngeal mask (MD 0.667), oropharyngeal (MD 0.198) and nasopharyngeal (MD 0.600) airways, performing Bag-Valve-Mask (MD 0.379), double (MD 0.344) and triple (MD 0.326,) airway manoeuvre, doing 12-lead electrocardiography (MD 0.729), using McGrath(R) laryngoscope (MD 0.726), using McGrath(R) forceps to remove foreign body (MD 0.632), attempting thoracocentesis (MD 1.240), and putting on a traction splint (MD 0.865). The students commented that the workshop provided context to their theoretical knowledge and that they gained an appreciation of the differences in normal tissue variation. Following engagement in/ completion of the workshop, students were more aware of their own clinical and non-clinical competencies. Conclusions The paramedic profession has evolved beyond patient transport with minimal intervention to providing comprehensive both emergency and non-emergency medical care. With limited availability of clinical placements for undergraduate paramedic training, there is an increasing demand on universities to provide suitable alternatives. Our findings suggested that cadaveric training using fresh frozen cadavers provides an effective adjunct to simulated learning and clinical placements.

Posthumous conception in South Australia : the case continues in Re H, AE (No 3) [2013] SASC 196

This editorial considers the issue of posthumous conception as discussed in the case of Re H, AE (No 3) [2013] SASC 196, which involved an application by a South Australian woman to the Supreme Court for authorisation to export her deceased partner's sperm to the Australian Capital Territory, for use in an assisted reproductive technology procedure.

MRI of postmortem specimens of endangered species for comparative brain anatomy

Many of the 5,500 threatened species of vertebrates found worldwide are highly protected and generally unavailable for scientific investigation. Here we describe a noninvasive protocol to visualize the structure and size of brain in postmortem specimens. We demonstrate its utility by examining four endangered species of kiwi (Apteryx spp.). Frozen specimens are thawed and imaged using MRI, revealing internal details of brain structure. External brain morphology and an estimate of brain volume can be reliably obtained by creating 3D models. This method has facilitated a comparison of brain structure in the different kiwi species, one of which is on the brink of extinction. This new approach has the potential to extend our knowledge of brain structure to species that have until now been outside the reach of anatomical investigation.

The synthesis, characterization, X-ray structure and magnetism of dinuclear-based bis[μ-(1,1,3,3-tetracyano-2-ethoxypropenido-κ2N,N′) (1,1,3,3-tetracyano-2-ethoxypropenido-κN)(2,2′-bipyridine)copper(II)] organized in alternating chains via semi-coordinating Cu–N distances

The monoanionic ligand 1,1,3,3 tetracyano-2 ethoxypropenide (tcnoet) is reported with its Cu(II)–bpy complex of formula [Cu2(µ-tcnoet)2(tcnoet)2(bpy)2]. The structure has been determined using X-ray diffraction and features an alternating chain with bridging tcnoet ligands. One ligand acts as a bidentate, dinucleating ligand with one short Cu–N and one medium Cu–N bond, whereas the other tcnoet is largely monodentate, albeit with a very weak interdimer Cu–N bond. Despite the arrangement in dinuclear units, further arranged into linear chains through the non-bridging tcnoet ligand, the compound shows no significant magnetic exchange, as deduced from magnetic susceptibility down to 4 K. Ligand-field, IR and EPR spectra in the solid state and in frozen solution are reported and are consistent with the overall structure.

The Times They Are A Changin': The Effect of Institutional Change on Cooperative Behaviour at 26,000ft over Sixty Years

In 2006, Sir Edmund Hillary lambasted the modern climbing fraternity for abandoning other climbers to a slow frozen death on Everest, claiming that in his day they would never leave someone to die. This followed the controversial death of David Sharp, passed by an estimated 40 climbers who were more interested in the summit than the life of a fellow human being. But was this stinging criticism true or just the faded recollections of a former climbing giant? This book investigates that claim through a narrative analysis, which combines the empirical analysis of Hawley and Salisbury's Himalayan Expedition Database with the anecdotal evidence provided by a plethora of newspaper articles and books. While there is evidence supporting the claim that commercialization is to blame for the breakdown of pro-social behaviour, the results cannot conclude if it is the commercial climber or the operator driving the problem and that the Sherpa are the saving grace.

The doomsday vault: Seed banks, food security, and climate change

"It could easily provide the back-drop for a James Bond movie. Deep inside a mountain near the North Pole, down a fortified tunnel, and behind airlocked doors in a vault frozen to -18 degrees Celsius, scientists are squirreling away millions of seed samples. The samples constitute the very foundation of agriculture, the biological diversity needed so the world's major food crops can adapt to the next pest or disease, or to climate change. It's little wonder that the Svalbard Global Seed Vault has captured the public's imagination more than almost any agricultural topic in recent years. Popular press reports about the ‘Doomsday Vault,’ however, typically mask the complexity of the endeavor and, if anything, underestimate its practical utility." Cary Fowler This chapter considers the use of seed banks to address concerns about intellectual property, climate change and food security. It has a number of themes. First of all, it is interested in the use of ‘Big Science’ projects to address pressing global scientific concerns and Millennium Development Goals. Second, it highlights the increasing use of banks as a means of managing both property and intellectual property across a wide range of fields of agriculture and biotechnology. Third, it considers the linkage of intellectual property, access to genetic resources and benefit sharing. There are a variety of positions in this debate. Some see requirements in respect of access to genetic resources and benefit sharing as an inconvenient burden for science and commerce. Others defend access to genetic resources and benefit sharing as meaningful and productive. Those inclined to somewhat more conspiratorial views suggest that access to genetic resources and benefit sharing are a ruse to facilitate biopiracy. This chapter has a number of components. Section I focuses upon the Consultative Group on International Agricultural Research (CGIAR) network – often raised as a model for Climate Innovation Centres. Section II considers the Svalbard Global Seed Vault – the so-called Doomsday Vault. After a consideration of the World Summit on Food Security in 2009, it is concluded in this chapter that any future international agreement on climate change needs to address intellectual property, plant genetic resources and food security.

Immunization of male bonnet monkeys (M-radiata) with a recombinant FSH receptor preparation affects testicular function and fertility

Immunization of proven fertile adult male monkeys (n = 3) with a recombinant FSH receptor protein preparation (oFSHR-P) (representing amino acids 1-134 of the extracellular domain of the receptor Mr similar to 15KDa) resulted in production of receptor blocking antibodies. The ability of the antibody to bind a particulate FSH receptor preparation and receptors in intact granulosa cells was markedly (by 30-80%) inhibited by FSH. Serum T levels and LH receptor function following immunization remained unchanged. The immunized monkeys showed a 50% reduction (p<0.001) in transformation of spermatogonia(2C) to primary spermatocytes (4C) as determined by flow cytometry and the 4C:2C ratio showed a correlative change (R 0.81, p<0.0007) with reduction in fertility index (sperm counts X motility score). Breeding studies indicated that monkeys became infertile between 242-368 days of immunization when the fertility index was in the range of 123+/-76 to 354+/-42 (compared to a value of 1602+/-384 on day 0). As the effects observed ate near identical to that seen following immunization with FSH it is suggestive that oFSHR-P can substitute for FSH in the development of a contraceptive vaccine.

A role for nocturnal serum testosterone surge in regulating spermatogenesis in the adult non-human primate

Adult male bonnet monkeys exhibit nychthemeral rhythms in testosterone (T) secretion but the precise role of this heightened level of T secretion in regulating spermatogenesis is not known. Intranasal administration of microdoses (500 mu g or 250 mu g/day) of Norethisterone (IN-NET) to adult monkeys (n = 6) at 1600 h each day selectively and completely suppressed the nocturnal surge levels of serum T. Concomitant with this was a significant reduction (P<0.01) in serum LH but not FSH levels. DNA flow cytometric analysis of testicular biopsy tissue showed by week 10 of IN-NET treatment an arrest in meiotic transformation of primary spermatocytes (4C) to round/elongate (1C/HC) spermatids and by week 20 there was a complete absence of 4C, 1C and HC cells (with a relative accumulation in 2C cells). The accumulated meiotic (4C) cells at week 10 showed an increase (>80%, P<0.01) in coefficient of variation and a decrease in intensity of DNA-bound ethidium bromide fluorescence, parameters characteristic of degenerating 'apoptotic' subpopulation of germ cells. While two monkeys exhibited acute oligozoospermia 4 became azoospermic by 20 weeks of IN-NET treatment. A complete, qualitative reversal in the regressive changes in spermatogenesis and near-normal sperm output were apparent at the end of a 20-week recovery phase. These data demonstrate that prolonged, selective suppression of nocturnal surge levels of serum T secretion exerts a primary effect on meiosis in spermatogenesis leading to oligo/azoospermic status in adult bonnet monkeys.

Influence of Elastic-Constants on Mode-I Stress-Intensity Factors in 3-Dimensional Crack Problems

Plates with V-through edge notches subjected to pure bending and specimens with rectangular edge-through-notches subjected to combined bending and axial pull were investigated (under live-load and stress-frozen conditions) in a completely nondestructive manner using scattered-light photoelasticity. Stress-intensity factors (SIFs) were evaluated by analysing the singular stress distributions near crack-tips. Improved methods are suggested for the evaluation of SIFs. The thickness-wise variation of SIFs is also obtained in the investigation. The results obtained are compared with the available theoretical solutions.

EPR observation of phase transitions in calcium cadmium acetate hexahydrate: Using Mn2+ and Cu2+ ions as probe

Results of temperature dependence of EPR spectra of Mn2+ and Cu2+ ions doped calcium cadmium acetate hexahydrate (CaCd(CH3COO)4•6H2O) have been reported. The investigation has been carried out in the temperature range between room temperature ( 300 K) and liquid nitrogen temperature. A I-order phase transition at 146 ± 0.5 K has been confirmed. In addition a new II-order phase transition at 128 ± 1 K has been detected for the first time. There is evidence of large amplitude hindered rotations of CH3 groups which become frozen at 128 K. The incorporation of Cu2+ and Mn2+ probes at Ca2+ and Cd2+ sites respectively provide evidence that the phase transitions are caused by the molecular rearrangements of the common coordinating acetate groups between Ca2+ and Cd2+ sites. In contradiction to the previous reports of a change of symmetry from tetragonal to orthorhombic below 140 K, the symmetry of the host is concluded to remain tetragonal in all the three observed phases between room temperature and liquid nitrogen temperature.

Handling of Two Tropical Australian Sharks to Improve Quality and to Identify the Cause of Tough Texture

Sharks caught in tropical Australian waters occasionally exhibit tough texture. Two species of Carcharinid shark, originally known as the sorrah shark (Carcharinus sorrah) and the black spot shark (Carcharinus tilstoni), compose the majority of the catch. Experiments were conducted to identify the cause of tough texture and to improve the overall quality of the catch. The possibility that a cold shock reaction may occur was investigated by observing the contraction of fillets under a range of temperature conditions before freezing. The effect of on-board handling practices were evaluated using frozen shark fillets, which had been stored prior to freezing in refrigerated seawater at different rigor stages, temperatures and times as trunks. Fillets were analyzed for nucleotides, lactate, thaw pH, sarcomere length and raw and cooked shear force values. The existence of thaw rigor was also investigated. There was little difference in the texture between the individual strips of a fillet exposed to different temperatures but there were significant differences between individual sharks. A cold shock reaction could not be demonstrated in these species. The main influences on texture were of biological origin. The species, sex and size were found to have significant links with texture of fillets. The quality of the fillets deteriorated quicker during the warmer season and were at their worst if the trunks were kept on deck till post-rigor or held in 15 degree C refrigerated seawater before freezing

Pesticide residues in food - acute dietary exposure

Consumer risk assessment is a crucial step in the regulatory approval of pesticide use on food crops. Recently, an additional hurdle has been added to the formal consumer risk assessment process with the introduction of short-term intake or exposure assessment and a comparable short-term toxicity reference, the acute reference dose. Exposure to residues during one meal or over one day is important for short-term or acute intake. Exposure in the short term can be substantially higher than average because the consumption of a food on a single occasion can be very large compared with typical long-term or mean consumption and the food may have a much larger residue than average. Furthermore, the residue level in a single unit of a fruit or vegetable may be higher by a factor (defined as the variability factor, which we have shown to be typically ×3 for the 97.5th percentile unit) than the average residue in the lot. Available marketplace data and supervised residue trial data are examined in an investigation of the variability of residues in units of fruit and vegetables. A method is described for estimating the 97.5th percentile value from sets of unit residue data. Variability appears to be generally independent of the pesticide, the crop, crop unit size and the residue level. The deposition of pesticide on the individual unit during application is probably the most significant factor. The diets used in the calculations ideally come from individual and household surveys with enough consumers of each specific food to determine large portion sizes. The diets should distinguish the different forms of a food consumed, eg canned, frozen or fresh, because the residue levels associated with the different forms may be quite different. Dietary intakes may be calculated by a deterministic method or a probabilistic method. In the deterministic method the intake is estimated with the assumptions of large portion consumption of a ‘high residue’ food (high residue in the sense that the pesticide was used at the highest recommended label rate, the crop was harvested at the smallest interval after treatment and the residue in the edible portion was the highest found in any of the supervised trials in line with these use conditions). The deterministic calculation also includes a variability factor for those foods consumed as units (eg apples, carrots) to allow for the elevated residue in some single units which may not be seen in composited samples. In the probabilistic method the distribution of dietary consumption and the distribution of possible residues are combined in repeated probabilistic calculations to yield a distribution of possible residue intakes. Additional information such as percentage commodity treated and combination of residues from multiple commodities may be incorporated into probabilistic calculations. The IUPAC Advisory Committee on Crop Protection Chemistry has made 11 recommendations relating to acute dietary exposure.