Higher Incubation Temperatures Research Articles

Effects of incubation temperature on asparagus bud sprouting, abscisic acid content and sex

Dormancy in asparagus (Asparagus officinalis) spears depends on the growing temperature. It is crucial to understand the mechanism of the induction and break of dormancy related with bud sprouting of asparagus. During periods with low temperatures, the contents of several components; e.g. plant hormones in asparagus buds change and the plants acquire dormancy. Similarly, higher temperatures suppress sprouting. In this study, we investigated the effects of incubation temperatures on the sprouting rate of buds, growth of spears, ABA concentration and sex. During 22 days of low-temperature incubation, sprouting was not inhibited at 25°C, but it was almost inhibited at 16°C. The ABA concentrations of white bud clusters were higher at 16°C than at 25°C at the end of the incubation period, but they were not significantly different between male and female plants. The ABA concentrations were higher in brown than in white bud clusters, and they were similar among brown bud clusters at both 25 and 16°C. In the high-temperature incubation experiment, sprouting was slightly suppressed and growth rate of spears was significantly decreased at 38°C compared to those at 25°C. The ABA concentration of white bud clusters decreased gradually during the incubation at both 38 and 25°C, but that of brown bud clusters increased at both temperatures. Moreover, the ABA levels in both white and brown bud clusters from male plants fluctuated and they were higher compared to their levels in bud clusters from female plants. These results suggest that sprouting varied with the incubation temperature and may or may not be regulated by ABA.

A highly stable laccase obtained by swapping the second cupredoxin domain

The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2–9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50–70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.

Responses of developmental and physiological traits to manipulated incubation conditions in broiler embryos at hypoxic high altitude

Abstract. The effects of hypoxia at increased altitude levels on the cardio-respiratory development of broiler embryos are distinct in comparison with those at sea level. The aim of the study was to investigate the effects of high incubation temperature (H) and oxygen supplementation (O) during hypoxic high altitude (HA) on developmental and physiological traits of embryos and hatching performance of embryonated hatching eggs in broilers at different embryonic stages. A total of 1280 eggs obtained from broiler breeders laid at sea level were used. Eggshell quality characteristics were measured for 20 eggs. The rest of the 1260 eggs were divided into seven incubation condition (IC) groups (180 eggs per group) including a control group at 37.8 ∘C and 21 % O2; O groups, with daily 1 h 23.5 % O2 supplementation at 37.8 ∘C as O0−11, O12−21, and O18−21; H groups at 38.5 ∘C high incubation temperature at 21 % O2 as H0−11, H12−21, and H18−21 from days 0 to 11, 12 to 21, and 18 to 21 of incubation, respectively. All groups were incubated in three different incubators at hypoxic HA. The effect of IC was determined on eggshell temperature, hatching performance, embryo development, right ventricular (RV) to total ventricular (TV) ratio, and blood parameters. The highest egg water loss and embryonic mortality and the lowest hatchability were in the H0−11 group, which depended on increased eggshell temperature during incubation. On day 18 of incubation, due to the decreased egg water loss in the O12−21 and O18−21 groups, there was an increase in hatchability in fertile eggs similar to the middle and late H groups. Towards the end of incubation, embryo/chick weights were not different and RV and TV weights increased in the treated groups, and the RV ∕ TV ratio changed between 15 and 26 %. At hatching, yolk sac weight increased in H0−11 and H12−21 groups. The O groups had the lowest serum tri-iodothyronine (T3) concentration as distinct from H groups. The serum thyroxine (T4) concentration increased in the treated groups, dependent on sex of the embryo. Blood hemoglobin concentration of O groups decreased relative to other groups. The hematocrit value was the lowest in the O12−21 and highest in the H12−21 groups. The H and O treatments during pre-hatch hypoxic HA condition can be positively evaluated on physiological traits of embryos after half of incubation depended on the timing of the IC exposure to the hatching eggs obtained from broiler breeders at sea level.

MiRNAs regulate acute transcriptional changes in broiler embryos in response to modification of incubation temperature

MicroRNAs are post-transcriptional regulators that play critical roles in diverse biological processes. We hypothesize that miRNAs may be involved in regulating transcriptome responses to changes in embryonic incubation temperature in chickens affecting differentiation and proliferation processes during tissue development. Therefore, we conducted comparative transcriptome profiling of miRNAs to examine altered expression in breast and hind muscle of embryos and day 35 chickens experiencing high (38.8 °C), control (37.8 °C), or low (36.8 °C) embryonic incubation temperature during embryonic day (ED) 7–10 or ED10–13. The results revealed differential expression of miRNAs due to modification of embryonic incubation temperature in a muscle type-specific and a developmental stage-specific manner. The immediate effects of thermal change observed in embryos were substantial compared to the subtle long-term effects in chickens at day 35 post-hatch. Upregulation of miR-133 in breast muscle and downregulation of miR-199a-5p, miR-1915, and miR-638 in hind muscle post ED7–10 high-temperature treatment are functionally associated with myogenesis and body size. ED10–13 low-temperature treatment led to downregulation of let-7, miR-93, and miR-130c that are related to proliferation and differentiation. The results provide insight into the dynamics of miRNA expression at variable embryonic incubation temperatures during developmental processes and indicate a major regulatory role of miRNAs in acute responses to modified environmental conditions that affect remodelling of cells and tissues.

Comparative catalytic and bacteriostatic properties of silver nanoparticles biosynthesized using three kinds of polysaccharide

Silver nanoparticles (Ag NPs) were biosynthesized using konjac glucomannan (KGM), κ-carrageenan (CRG) and inulin as reducing and capping agent through one-spot method. The structure and morphology of Ag NPs were investigated using UV-visible spectroscopy, transmission electron microscopy, Fourier transform infrared and X-ray powder diffraction. The catalysis and antibacterial properties were also evaluated. Results showed that Ag NPs fabrication was influenced by the incubation temperature and polysaccharide category. CRG displayed a stronger reduction of Ag+ than KGM, and the lowest was inulin. A High incubation temperature promoted the reduction of Ag NPs. As for Ag100 NPs, the kapp of Ag NPs based on CRG, inulin, and KGM were 5.80×10-2 s-1, 2.14×10-2 s-1, and 2.16×10-2 s-1 respectively. Bacteriostatic test showed excellent antimicrobial activity of Ag NPs for Escherichia coli and Staphylococcus aureus. Thus, natural polysaccharide could be effective reducing and stabilizing agents for Ag NPs biosynthesis. The excellent antibacterial and catalytic properties may facilitate their use in medical, food, and biological applications.

Germination and seed conservation of a pioneer species, Tecoma stans (Bignoniaceae), from tropical dry forest of Colombia

Seed germination and seed longevity under different environmental conditions are fundamental to understand the ecological dynamics of a species, since they are decisive for its success within the ecosystem. Taking this into account, seed germination and seed storage behavior of a pioneer species of tropical dry forest (Tecoma stans) were studied in the laboratory, to establish the effect of different environmental conditions on a local tree population. Two seed lots collected in July 2011, from Cali (Colombia), were evaluated under three alternating temperatures (20/30, 20/25, 25/30 ºC; 16/8 h) and four light qualities (12-hour white light photoperiod, darkness, and 15 minutes of red light -R and far red light -FR). Final germination was recorded for all treatments; for white light treatment the daily germination was recorded to calculate mean germination rate, mean germination time, and two synchronization indices. To assess the effect of light quality on physiological variables, a destructive germination test was carried out. For this test, another seed lot was evaluated under the same light conditions using an alternating temperature of 20/30 °C - 16/8 h, recording germination during six days for every treatment. In addition, seeds were stored at two different moisture contents (7.7, 4.1 %) and three storage temperatures (20, 5, -20 ºC), during two time periods (one and three months); a seed germination test was conducted for each treatment. Four replicates of 35 seeds per treatment were used for all experiments. Germination was high (GP > 90 %) with all alternating temperatures under white light, whereas under R, FR, and darkness germination was evenly successful at low temperatures, but at higher temperature, half of the seeds entered into secondary dormancy (GP= 45-65 %). However, mean germination rate and synchronization under R and FR decreased significantly in comparison to white light treatment and, consequently, mean germination time increased. Seed storage behavior of this species is orthodox due to the high germination (GP > 90 %) obtained under all treatments. In conclusion, T. stans seeds have a negative germination response at high incubation temperatures in the absence of white light, entering into a secondary dormancy. In contrast, an environment with a lower temperature and without white light delays the germination, but at the end seeds are able to reach the same germination values. This seed dependence on incident light in limiting conditions suggests a physiological mechanism on the seed tissues of this species, probably mediated by phytochromes. Finally, the orthodox seed storage behavior of T. stans is a reason to include this species in ex situ seed conservation programs for restoration and recovery of the tropical dry forest; however, long-term studies should be conducted in order to evaluate the maintenance of this characteristic throughout longer periods of time. Rev. Biol. Trop. 66(2): 918-936. Epub 2018 June 01.

Effect of temperature and pH on the community dynamics of coagulase-negative staphylococci during spontaneous meat fermentation in a model system

Coagulase-negative staphylococci (CNS) contribute to the product quality of fermented meats. In spontaneously fermented meats, CNS communities are variable and difficult to predict, as their compositions depend on a superposed combination of different processing factors. To partially disentangle this superposition, a meat model system was used to study the influence of temperature and pH on the CNS community dynamics. Therefore, cured pork mince was prepared that was divided into three batches of different initial acidity levels, namely pH 5.7, pH 5.5, and pH 5.3. These three batches were incubated at three different temperatures, namely 23 °C, 30 °C, and 37 °C. Hence, the experimental set-up resulted in nine combinations of different temperature and initial pH values. Samples were analysed after 3 and 14 days to monitor pH, colony counts, and species diversity of the CNS communities, based on mannitol-salt-phenol-red agar (MSA) medium. At conditions of mild acidity (pH 5.7) and low temperature (23 °C), as often encountered during artisan-type meat fermentations, a co-prevalence of Staphylococcus xylosus, Staphylococcus equorum, and Staphylococcus saprophyticus occurred. At the same initial pH but higher incubation temperatures (30 °C and 37 °C), Staphylococcus lugdunensis became the prevailing CNS species, besides S. saprophyticus (30 °C) and the coagulase-positive species Staphylococcus aureus (37 °C). When the initial pH was set at 5.5, S. saprophyticus was the prevailing CNS species at both 23 °C and 30 °C, but it was replaced by Staphylococcus epidermidis and Staphylococcus simulans at 37 °C after 3 and 14 days, respectively. At the most acidic conditions (pH 5.3), CNS counts declined and many of the MSA isolates were of non-staphylococcal nature. Among others, Staphylococcus carnosus (23 °C), Staphylococcus warneri (30 °C), and S. epidermidis (37 °C) were found. Overall, the results of the present study indicated that the processing factors temperature and pH had a clear impact on the shaping of staphylococcal communities during meat fermentation.

Short communication: High incubation temperature in bovine mammary epithelial cells reduced the activity of the mTOR signaling pathway

Hyperthermia alters utilization of AA in protein synthesis and cell-signaling activity in bovine mammary cells. Essential AA and insulin regulate translation of proteins by controlling the activity of mammalian target of rapamycin (mTOR) signaling pathway. The objectives of this study were to evaluate (1) the effects of incubation temperature on the mTOR signaling pathway and transcription of AA transporters in a bovine mammary alveolar cell line (MAC-T) and (2) the combined effects of incubation temperature and insulin on the mTOR signaling pathway in this cell line. Cells were cultured in medium with 10% fetal bovine serum at 37°C and 5% CO2. In experiment 1, cells were subjected to 37°C (control) or 41.5°C (high incubation temperature; HT) for 12 h. In experiment 2, cells were assigned to 1 of 4 treatments as a 2 × 2 factorial arrangement, including 2 cell culture temperatures (control and HT) and absence or presence of 1.0 μg/mL of insulin. Proteins were harvested and separated by gel electrophoresis. In experiment 1, gene expression of AA transporters (SLC1A1, SLC1A5, SLC3A2, SLC7A1, SLC7A5, and SLC36A1) were evaluated, and changes of ≥2 fold were deemed significantly different. In experiments 1 and 2, immunoblotting was used to identify total and site-specific phosphorylated forms of protein kinase B (Akt1; Ser473), p70 S6 kinase (S6K1; Thr389), ribosomal protein S6 (rpS6; Ser235/236), and eukaryotic elongation factor 2 (eEF2; Thr56). Phosphorylated and total forms of Akt1, S6K1, rpS6, and eEF2 were quantified and expressed as the ratio of phosphorylated to total protein. In experiment 1, HT resulted in a ≥2-fold increase expression of SLC1A1 and SLC3A2. High incubation temperature reduced the phosphorylated to total ratio of Akt1 and rpS6 and increased the phosphorylated to total ratio of eEF2. In experiment 2, we found no temperature by insulin interactions on phosphorylation state of the protein factors of interest. High incubation temperature reduced the phosphorylated to total ratio of Akt1. The addition of insulin increased the phosphorylated to total ratio of Akt1, S6K1, and rpS6. In summary, HT reduced the activity of the mTOR signaling pathway and increased the expression of AA transporters. High incubation temperature possibly reduced protein translation by reducing the mTOR signaling pathway activity in an effort to adapt to thermal stress. These results may help explain the direct effect of elevated temperature on AA metabolism and protein translation in heat-stressed animals.

Stability of dissolved percarbonate and its implications for groundwater remediation

Efforts to improve the understanding of oxidant stability are of great practical significance to the design of an in-situ chemical oxidation (ISCO) system for soil and groundwater remediation. In this study, the stability of an emerging ISCO oxidant sodium percarbonate (SPC) was investigated. Although the dry solid form of SPC is relatively stable, dissolved SPC decomposes much faster than H2O2. SPC had higher oxidation efficiency for the dye Orange G than inactivated or alkaline-activated H2O2. Both OH− and HCO3−/CO3a2−, generated from SPC dissolution, activated the peroxide content of SPC and thus promoted its decomposition and pollutant oxidation. Higher incubation temperature and longer incubation period lead to faster SPC decomposition. Decomposed SPC had lower pollutant oxidation capability.

Thermophilic endospores associated with migrated thermogenic hydrocarbons in deep Gulf of Mexico marine sediments

Dormant endospores of thermophilic bacteria (thermospores) can be detected in cold marine sediments following high-temperature incubation. Thermospores in the cold seabed may be explained by a dispersal history originating in deep biosphere oil reservoir habitats where upward migration of petroleum fluids at hydrocarbon seeps transports viable cells into the overlying ocean. We assessed this deep-to-shallow dispersal hypothesis through geochemical and microbiological analyses of 111 marine sediments from the deep water Eastern Gulf of Mexico. GC-MS and fluorescence confirmed the unambiguous presence of thermogenic hydrocarbons in 71 of these locations, indicating seepage from deeply sourced petroleum in the subsurface. Heating each sediment to 50 °C followed by 16S rRNA gene sequencing revealed several thermospores with a cosmopolitan distribution throughout the study area, as well as thermospores that were more geographically restricted. Among the thermospores having a more limited distribution, 12 OTUs from eight different lineages were repeatedly detected in sediments containing thermogenic hydrocarbons. A subset of these were significantly correlated with hydrocarbons (p < 0.05) and most closely related to Clostridiales previously detected in oil reservoirs from around the world. This provides evidence of bacteria in the ocean being dispersed out of oil reservoirs, and suggests that specific thermospores may be used as model organisms for studying warm-to-cold transmigration in the deep sea.

Breeding sex ratio and population size of loggerhead turtles from Southwestern Florida.

Species that display temperature-dependent sex determination are at risk as a result of increasing global temperatures. For marine turtles, high incubation temperatures can skew sex ratios towards females. There are concerns that temperature increases may result in highly female-biased offspring sex ratios, which would drive a future sex ratio skew. Studying the sex ratios of adults in the ocean is logistically very difficult because individuals are widely distributed and males are inaccessible because they remain in the ocean. Breeding sex ratios (BSR) are sought as a functional alternative to study adult sex ratios. One way to examine BSR is to determine the number of males that contribute to nests. Our goal was to evaluate the BSR for loggerhead turtles (Caretta caretta) nesting along the eastern Gulf of Mexico in Florida, from 2013–2015, encompassing three nesting seasons. We genotyped 64 nesting females (approximately 28% of all turtles nesting at that time) and up to 20 hatchlings from their nests (n = 989) using 7 polymorphic microsatellite markers. We identified multiple paternal contributions in 70% of the nests analyzed and 126 individual males. The breeding sex ratio was approximately 1 female for every 2.5 males. We did not find repeat males in any of our nests. The sex ratio and lack of repeating males was surprising because of female-biased primary sex ratios. We hypothesize that females mate offshore of their nesting beaches as well as en route. We recommend further comparisons of subsequent nesting events and of other beaches as it is imperative to establish baseline breeding sex ratios to understand how growing populations behave before extreme environmental effects are evident.

Lamellar-cubic transition of a dihydrazide derivative and its effect on the gel stability.

N,N'-Bis(4-n-alkyloxybenzoyl)hydrazine (4D16) was demonstrated to show three different aggregates, i.e. a crystalline cubic phase and two kinds of lamellar structure with layer spacings of 34.20 Å (termed the L1 structure) and 40.85 Å (L2 structure) depending on the type of solvents. Lamellar (L1)-crystalline cubic transition during heating was confirmed for 4D16 showing the L1 structure. 4D16 organogels in cyclohexane and benzene exhibited either a mixture of the L1 structure and the crystalline cubic phase or only one of the two structures. 4D16 gels prepared at a higher concentration or a lower incubation temperature consisted of more lamellar L1 structures compared to those obtained at a lower concentration or a higher incubation temperature. Annealing of the as-prepared 4D16 gels at certain temperatures for different time periods caused gradual lamellar L1-cubic transition, and thus increased the content of the cubic phase in the gels, which showed lower Tgel compared to those of the as-prepared ones. The existence of the cubic phase in 4D16 gels in cyclohexane and benzene destabilized the gels.

TaqMan Multiplex Real-Time qPCR assays for the detection and quantification of Barley yellow dwarf virus, Wheat dwarf virus and Wheat streak mosaic virus and the study of their interactions

The phytopathogenic fungus Sclerotinia sclerotiorum forms dormant structures (termed sclerotia) that germinate myceliogenically under certain environmental conditions. During myceliogenic germination, sclerotia produce hyphae, which can infect leaves or stems of host plants directly from the ground; this is termed basal infection. This study determined which abiotic conditions were most important for promoting myceliogenic germination of sclerotia in vitro. A high sclerotium hydration level and low incubation temperature (15°C) improved mycelial growth in the presence of a nutrient source. Sclerotia incubated without a nutrient source on moist sand, vigorously myceliogenically germinated most frequently (63%) when they had been previously imbibed and then conditioned at −20°C. By far the most consistent amount of vigorous myceliogenic germination (&gt;75%) was produced when sclerotia were heat-dried before being submerged in water. The hyphae of these sclerotia were shown to infect and proliferate on leaves of intact Brassica napus plants. This research provides a better understanding of the abiotic conditions that are likely to increase the risk of basal infection by S. sclerotiorum.

Effect of Temperature on the Morphometrical and Physical Parameters of Erythrocytes and Polymorphonuclear Leucocytes in Carassius gibelio (Bloch)

Dynamics of the morphometric and physical properties of hemocytes of the Prussian carp Carassius gibelio (Bloch) under the influence of a temperature factor has been studied with atomic force microscopy in experiments in vitro. It is found that, at a low incubation temperature (5°C), as opposed to room temperature (20°C), morphometric parameters change in erythrocytes; at a high temperature (40°C) they change in polymorphonuclear leucocytes. The low incubation temperature reduces the adhesion and elasticity of polymorphonuclear leucocytes and erythrocytes of C. gibelio, whereas a high incubation temperature leads to a decrease in adhesion in polymorphonuclear white blood cells.

Computer-Assisted Recovery of Threatened Plants: Keys for Breaking Seed Dormancy of Eryngium viviparum.

Many endangered plants such as Eryngium viviparum (Apiaceae) present a poor germination rate. This fact could be due to intrinsic and extrinsic seed variability influencing germination and dormancy of seeds. The objective of this study is to better understand the physiological mechanism of seed latency and, through artificial intelligence models, to determine the factors that stimulate germination rates of E. viviparum seeds. This description could be essential to prevent the disappearance of endangered plants. Germination in vitro was carried out under different dormancy breaking and incubation procedures. Percentages of germination, viability and E:S ratio were calculated and seeds were dissected at the end of each assay to describe embryo development. The database obtained was modeled using neurofuzzy logic technology. We have found that the most of Eryngium seeds (62.6%) were non-viable seeds (fully empty or without embryos). Excluding those, we have established the germination conditions to break seed dormancy that allow obtaining a real germination rate of 100%. Advantageously, the best conditions pointed out by neurofuzzy logic model for embryo growth were the combination of 1 mg L−1 GA3 (Gibberellic Acid) and high incubation temperature and for germination the combination of long incubation and short warm stratification periods. Our results suggest that E. viviparum seeds present morphophysiological dormancy, which reduce the rate of germination. The knowledge provided by the neurofuzzy logic model makes possible not just break the physiological component of dormancy, but stimulate the embryo development increasing the rate of germination. Undoubtedly, the strategy developed in this work can be useful to recover other endangered plants by improving their germination rate and uniformity favoring their ex vitro conservation.

Sex determination mode does not affect body or genital development of the central bearded dragon (Pogona vitticeps)

BackgroundThe development of male- or female-specific phenotypes in squamates is typically controlled by either temperature-dependent sex determination (TSD) or chromosome-based genetic sex determination (GSD). However, while sex determination is a major switch in individual phenotypic development, it is unknownhow evolutionary transitions between GSD and TSD might impact on the evolution of squamate phenotypes, particularly the fast-evolving and diverse genitalia. Here, we take the unique opportunity of studying the impact of both sex determination mechanisms on the embryological development of the central bearded dragon (Pogona vitticeps). This is possible because of the transitional sex determination system of this species, in which genetically male individuals reverse sex at high incubation temperatures. This can trigger the evolutionary transition of GSD to TSD in a single generation, making P. vitticeps an ideal model organism for comparing the effects of both sex determination processes in the same species.ResultsWe conducted four incubation experiments on 265 P. vitticeps eggs, covering two temperature regimes (“normal” at 28 °C and “sex reversing” at 36 °C) and the two maternal sexual genotypes (concordant ZW females or sex-reversed ZZ females). From this, we provide the first detailed staging system for the species, with a focus on genital and limb development. This was augmented by a new sex chromosome identification methodology for P. vitticeps that is non-destructive to the embryo. We found a strong correlation between embryo age and embryo stage. Aside from faster growth in 36 °C treatments, body and external genital development was entirely unperturbed by temperature, sex reversal or maternal sexual genotype. Unexpectedly, all females developed hemipenes (the genital phenotype of adult male P. vitticeps), which regress close to hatching.ConclusionsThe tight correlation between embryo age and embryo stage allows the precise targeting of specific developmental periods in the emerging field of molecular research on P. vitticeps. The stability of genital development in all treatments suggests that the two sex-determining mechanisms have little impact on genital evolution, despite their known role in triggering genital development. Hemipenis retention in developing female P. vitticeps, together with frequent occurrences of hemipenis-like structures during development in other squamate species, raises the possibility of a bias towards hemipenis formation in the ancestral developmental programme for squamate genitalia.

Cold Hardiness of Overwintering Larvae of Sphenoptera sp. (Coleoptera: Buprestidae) in Western China.

An undetermined species of Sphenoptera sp. is an important pest of Artemisia ordosica Krasch, and recently, an outbreak of this insect has spread throughout Ningxia, Inner Mongolia, Shanxi, and other regions in western China. The cold hardiness of overwintering larvae of Sphenoptera sp. was determined by measuring their supercooling point (SCP) and their mortality at sub-zero temperatures. Additionally, quantitative changes in sugars and low molecular weight sugar alcohols in larvae were determined following exposure of larvae to low temperatures. Mean SCP of overwintering larvae (i.e., collected in January) was -30.2 ± 0.60°C. The mortality rate of larvae approached 100% at -30°C, whereas mortality rates of larvae in the higher temperature treatments were generally less than 25%. Five sugars and sugar alcohols (i.e., glycerol, fructose, glucose, inositol, and trehalose) were detected in larvae. When larvae were exposed to low temperatures for 4 h, inositol, trehalose and total content was generally higher at the highest incubation temperature (-10°C) than at lower incubation temperatures. Following a longer exposure (30 d), content of fructose, glucose, trehalose and total content generally increased with decreasing temperature down to -25°C. Thus it appears that Sphenoptera sp. larvae are freeze avoidant, and their cold hardiness may be achieved by accumulation low molecular weight sugars and sugar alcohols.

Rapid optimized flow cytometric crossmatch (FCXM) assays: The Halifax and Halifaster protocols

The flow cytometric crossmatch (FCXM) assay, which detects the presence of donor specific HLA antibodies in patient sera, is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time, we developed and validated two modified crossmatch procedures, namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60% and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform, reduced wash times, increased serum to cell suspension volume ratio, shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification, employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ± 2.63% vs 69 ± 19.06%), reduce cell isolation time (by ∼40%) and conserve FCXM assay reagents. Importantly, linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98–0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally, a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7% and 96.8% specificity and sensitivity, respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care.

Hormonal and thermal induction of sex reversal in the bearded dragon (Pogona vitticeps, Agamidae)

In amniotes (mammals, reptiles and birds) we can find two major sex-determining modes: environmental sex determination (ESD), largely in the form of temperature-dependent sex determination, and genotypic sex determination (GSD). It is evident that in reptiles there were multiple transitions between these two modes, but their directions are often difficult to reconstruct. However, it seems that GSD to ESD transitions were much less frequent. Such a transition demands production of sex-reversed individuals, i.e. individuals with a mismatch between gonadal and genotypic sex, which have to be viable and fertile in spite of lacking sex-specific parts of genomes linked to Y or W chromosomes. Up to now, there is only a single well-documented transition from the ancestral GSD to ESD among amniotes: a laboratory experiment with the bearded dragon, Pogona vitticeps. The gonadal females with male-typical ZZ genotype induced by high incubation temperatures in this species possessed masculinized morphology and behaviour. However, it is not clear whether these masculinized phenotypic traits can be attributed to the lack of female-specific W chromosome, or to extreme incubation temperatures. In this contribution, we describe the results of the experiments aiming to develop a system allowing disentangling the effects of temperature and genotype by hormonal manipulations, specifically by the application of fadrozole and exogenous estradiol (E2) to developing eggs in a captive bred population of the bearded dragon. We documented that both high incubation temperature and E2 application at normal incubation temperature can induce development of ZZ sex-reversed females in this laboratory population and that these females are able to lay eggs. On the other hand, the application of fadrozole led either to high hatchling mortality or to development of individuals with enlarged hemipenes, but did not induce sex reversal. The developed system will be useful in future studies to fully utilize the very valuable bearded dragon model for general understanding of potential limits and constraints of GSD to ESD transitions.

Rapid detection of cryptococcal antigen by a flow assay

Cryptococcosis is a life-threatening infection caused by &lt;em&gt;Cryptococcus neoformans&lt;/em&gt; and &lt;em&gt;C.&lt;/em&gt; &lt;em&gt;gattii&lt;/em&gt;. Tests for quick detection of the cryptococcal antigen are needed. This study compares the performance of a lateral flow assay (LFA) to the latex agglutination method. Thirty-five cryopreserved positive samples (sera and cerebrospinal fluids) plus three negative sera for control have been examined. LFA does not need high-temperature incubation or enzyme pre-treatment. All the results, except for one serum, agree with previous obtained with latex agglutination method. LFA has an important clinical utility for its rapidity and sensitivity, and it also can be used as a point-of-care test.