Low Incubation Temperatures Research Articles

Color of Artificial Light at Night Affects Incubation Behavior in the Great Tit, Parus major

Artificial light at night (ALAN) has been recognized as a biodiversity threat due to the drastic effects it can have on many organisms. In wild birds, artificial illumination alters many natural behaviors that are important for fitness, including chick provisioning. Although incubation is a key determinant of the early developmental environment, studies into the effects of ALAN on bird incubation behavior are lacking. We measured nest temperature in nest boxes of great tits during the incubation period in two consecutive years. Nest boxes were located in eight previously dark field sites that have been experimentally illuminated since 2012 with white, green, or red light, or were left dark. We tested if light treatment affected mean nest temperature, number of times birds leave the nest (off-bout frequency), and off-bout duration during the incubation period. Subsequently, we investigated if incubation behavior is related to fitness. We found that birds incubating in the white light during a cold, early spring had lower mean nest temperatures at the end of incubation, both during the day and during the night, compared to birds in the green light. Moreover, birds incubating in white light took fewer off-bouts, but off-bouts were on average longer. The opposite was true for birds breeding in the green light. Low incubation temperatures and few but long off-bouts can have severe consequences for developing embryos. In our study, eggs from birds that took on average few off-bouts needed more incubation days to hatch compared to eggs from birds that took many off-bouts. Nevertheless, we found no clear fitness effects of light treatment or incubation behavior on the number of hatchlings or hatchling weight. Our results add to the growing body of literature that shows that effects of ALAN can be subtle, can differ due to the spectral composition of light, and can be year-dependent. These subtle alterations of natural behaviors might not have severe fitness consequences in the short-term. However, in the long term they could add up, negatively affecting parent condition and survival as well as offspring recruitment, especially in urban environments where more environmental pollutants are present.

Dormancy attributes in sunflower achenes ( Helianthus annuus L.): II. Sowing date effects

AbstractSunflower (Helianthus annuus L.) seed industry needs nondormant seeds (i.e., seeds with the capacity to germinate in a wide range of environmental conditions) for processing and packaging. Thus, the presence of persistent seed dormancy is an undesirable trait. Dormancy level depends on the genotype and on the maternal environment. The aim of the present work was to study, in a wide range of sunflower genotypes, the effect of variations in sowing date on achene dormancy level and to investigate if these changes are related to variations in (a) embryo or coat‐imposed dormancy, (b) dormancy expression at high or low incubation temperatures, and (c) embryo sensitivity to abscisic acid (ABA). Two experiments were performed. In Experiment I, 20 sunflower genotypes were sown in two contrasting planting dates during three consecutive years, whereas in Experiment II, 1579 genotype was sown in five dates along a 5‐mo period. At harvest and during postharvest storage, fruit structures were incubated at different incubation temperatures in water and ABA. Descriptive multivariate analyses showed that late sowings provoked an enhancement of achene germination at harvest and a faster dormancy release during postharvest storage in all genotypes. However, the magnitude of the response, the temperature at which dormancy was expressed, and the tissue responsible for achene dormancy varied among genotypes. The mechanism that partially explains this dormancy alleviation appears to be the sensitivity of embryo germination to ABA inhibition. Result suggest that sowing date can be managed to obtain less‐dormant sunflower seed lots without significant yield loss.

Axial chlorine coordinated iron-nitrogen-carbon single-atom catalysts for efficient electrochemical CO2 reduction

Iron-nitrogen-carbon single-atom catalysts (Fe-N-C SACs) are promising low-cost catalysts for electrochemical CO2 reduction reaction (CO2RR) to achieve carbon neutrality. However, their relatively low selectivity and activity are related to the strong binding of reaction intermediates (e.g., CO*) on single Fe atoms. Here, combining experimental and theoretical studies, we show that introducing axial chlorine (Cl) atom can modulate the electronic structure of Fe atoms in catalytically active FeN4 sites, which facilitates the desorption of CO* and inhibits the adsorption of H*, resulting in improved activity and selectivity in CO2RR. The Cl modified Fe-N-C SAC embedded in nitrogen-doped carbon nanosheets (FeN4Cl/NC) was synthesized in two steps: pyrolyzing Fe-loaded two-dimensional zeolite imidazole framework nanosheets and low-temperature incubation in hydrochloric acid solution. X-ray absorption spectroscopy results reveal that most atomically dispersed Fe atoms are coordinated with one axial Cl atom at 2.26 Å and four N atoms at 2.02 Å. The optimized FeN4Cl/NC exhibits a CO Faradaic efficiency of 90.5%, a high current density of 10.8 mA cm−2 at a low overpotential of 490 mV, and a high turnover frequency of 1566 h−1, one of the best among recently reported Fe-based CO2RR catalysts. FeN4Cl/NC was further applied as a bifunctional catalyst to construct rechargeable zinc-CO2 batteries, delivering a power density of 0.545 mW cm−2 with excellent stability over 15 h. Tailoring the coordination environment of metal atoms in M-N-C SACs by introducing axial atoms may be further extended as an efficient general approach to design advanced catalysts for various electrochemical applications, such as fuel cells, nitrogen fixation, and lithium-sulfur batteries.

Phytoplankton growth at low temperatures: results from low temperature incubations

Abstract The response of phytoplankton to different temperatures has been studied for decades, and although the general relationship of growth to temperature is known to be exponential, the responses at very low temperatures (from −2 to +2°C) are poorly constrained. To address this uncertainty, we conducted a series of growth experiments with eight species of polar phytoplankton; each was grown in batch culture at temperatures ranging from −1.5 to 4°C, and growth rates determined from changes in cell abundance and chlorophyll. We found that different species responded differently to temperature, although the general response was an increase in growth rate at higher temperatures. The mean response from all taxa was less (ca. 35% less) than predicted from the classic [Eppley, R. W. (1972) Temperature and phytoplankton growth in the sea. Fish. Bull., 7, 1063–1085.] formulation. Maximum growth rates also were most reliable when determined from changes in cell abundance. If the variability in maximum growth rates among taxa is reflective of in situ responses to polar temperature increases, it will be challenging to predict the assemblage response to elevated in situ temperatures in future years.

Divergent incubation temperature effects on thermal sensitivity of hatchling performance in two different latitudinal populations of an invasive turtle.

The incubation temperature for embryonic development affects several aspects of hatchling performance, but its impact on the thermal sensitivity of performance attributes remains poorly investigated. In the present study, Trachemys scripta elegans hatchlings from two different latitudinal populations were collected to assess the effects of different incubation temperatures on the locomotor (swimming speed) and physiological (heart rate) performances, and the thermal sensitivity of these two attributes. The incubation temperature significantly affected the examined physiological traits. Hatchling turtles produced at low incubation temperature exhibited relatively higher cold tolerance (lower body temperatures at which the animals lose the ability to escape from the lethal conditions), and reduced heart rate and swimming speed. Furthermore, the effect of incubation temperature on the thermal sensitivity of swimming speed differed between the low- and high-latitude populations. At relatively high incubation temperatures, the high-latitude hatchling turtles exhibited reduced thermal sensitivities of swimming speed than those of the low-latitude ones. Reduced thermal sensitivity of locomotor performance together with high cold tolerance, exhibited by the high-latitude hatchling turtles potentially reflected local adaptation to relatively colder and more thermally-variable environments.

Evidence of Cold Induced Cytoskeletal Arrest in Hepatocytes of the Western Painted Turtle

The western painted turtle (Chrysemys picta belli) overwinters in ice-covered water bodies and can survive without oxygen (anoxia) for months, during which time metabolic rate decreases so that ATP demand does not exceed anoxic ATP supply. In order to characterize the contribution of the cytoskeleton to overwintering metabolic depression in non-excitable turtle cells, we have optimized a hepatocyte cell culture system. Actin and tubulin cycling are large consumers of cellular ATP pools, and we hypothesized that their cycling activity, and consequently capacity for structural rearrangement, would be downregulated by overwintering conditions. Turtles were acclimated to either 22°C or 4°C for at least 20 days and isolated hepatocytes were plated on either fibronectin or polylysine at varying temperatures, or in cyanide containing media, which acted as a chemical proxy for anoxia. Low incubation temperature, but not cyanide, prevented hepatocytes from forming stable attachment to fibronectin, suggesting inhibition of cell spreading and arrest of actin structural rearrangement by low temperature. Attachment to polylysine, a predominately passive and electrostatic phenomenon, was not inhibited by low temperature or cyanide. Hepatocytes isolated from 4°C acclimated turtles were 30% smaller in 2D area than those of 22°C acclimated turtles. Three-hour cyanide exposure of hepatocytes from 22°C acclimated turtles also resulted in a 30% shrinkage of 2D area, indicating that temperature depression and anoxia may have overlapping inhibitory effects on cell spreading and underlying cytoskeletal turnover. Previous research has shown that cyanide treatment causes reduction of hepatocyte mitochondrial membrane potential. Ongoing work is demonstrating that, regardless of whole animal temperature acclimation, low temperature exposure results in decreased mitochondrial membrane potential as well as increased cytosolic calcium, signaling which may contribute to inhibition of energetically costly actin rearrangement.

Kinetics of Staphylococcus aureus growth and Enterotoxin A production in milk under shaking and static conditions

Numerous studies on bacterial growth or survival predictive models have been conducted since the establishment of predictive microbiology. However, limited research focused on the prediction of bacteria-producing enterotoxins, which are often the causative agents of food-borne diseases. This study aimed to determine an appropriate kinetic model of staphylococcal enterotoxin A (SEA) production in milk after contamination with Staphylococcus aureus. An S. aureus strain producing SEA was inoculated into milk with an initial inoculum concentration of approximately 3.0 log CFU/mL. All samples were incubated for 30–48 h at 20 °C ± 1 °C, 28 °C ± 1 °C, and 36 °C ± 1 °C separately under shaking or static conditions. Duplicate samples were carried out at appropriate intervals to count the number of S. aureus colonies and detect the concentration of SEA. Experimental results showed that the SEA concentration curves under all experimental conditions were sigmoidal and consisted of three phases: lag, exponential, and stationary. Thus, the modified Gompertz model was used to describe the profile of SEA concentration in milk during the incubation. A good fitting accuracy (R2 > 0.97) indicated that the modified Gompertz model was appropriate. In addition to temperature, shaking during incubation also affected the maximal production rate of SEA and the maximal SEA concentrations, and shortened the lag phase at lower incubation temperatures, suggesting that the mechanical movements (e.g., stirring, pumping, and flowing) during the milk processing would increase the risk of SEA occurrence. Besides, the time to detection (TTD) of SEA was found to range from 3 to 24.5 h at temperatures of 36 °C ± 1 °C–20 °C ± 1 °C, at which time the concentrations of S. aureus ranging from 5.0 log CFU/mL–6.9 log CFU/mL at the TTD. This study contributed to understanding the kinetics of SEA production and the possible factors affecting the synthesis of SEA during the manufacturing of liquid foods, such as milk.

Downregulation of epithelial sodium channel (ENaC) activity in human airway epithelia after low temperature incubation

IntroductionThe incubation of airway epithelia cells at low temperatures is a common in vitro experimental approach used in the field of cystic fibrosis (CF) research to thermo-stabilise F508del-CFTR and increase...

Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis

The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.

Soil Nitrogen Mineralization and its Sensitivity to Temperature and Moisture in Temperate Peatlands Under Different Land-Use Management Practices

Abstract The availability of soil nitrogen determines many functions of terrestrial ecosystems, in particular, primary plant productivity, which affects both the plant yield and carbon sequestration. Climate change will lead to shifts in soil temperature and moisture; therefore, to create a more accurate forecast of ecosystem sustainability, it is important to understand how these indicators control nitrogen mineralization in soils. We studied the effect of temperature and moisture on the net ammonification and net nitrification processes in temperate peatlands of central Russia under 4 land-use types: regular plowing for 50 and 100 years, post-agrogenic reforestation for 50 years, and a near-pristine birch forest. During a laboratory incubation experiment, we found that net nitrogen mineralization was significant at all combinations of temperatures and moisture levels. Net nitrification was the main process for nitrogen mineralization, although net ammonification was also possible at low incubation temperatures. The highest rate of net mineralization was typical of forest peatlands, and the lowest rate was typical of arable sites, which were associated with the differences in the quantity, quality and regularity of the incoming plant litter. Despite significant differences in the rate of net nitrification between plots with different land-uses, the temperature sensitivity coefficient of this process was nearly the same for all peatlands and was close to 2; therefore, we did not find evidence of the effect of the organic matter quality on the temperature sensitivity of net nitrification. The moisture level also did not affect the temperature sensitivity of this process. In general, it was found that fluctuations in soil temperature and moisture with climate change will not cause a sharp increase in net nitrification in peatlands, which will prevent the soil from losing nitrogen with runoff or in the form of gaseous compounds, such as N2O.

A dual prokaryotic (<i>E</i>.<i> coli</i>) expression system (pdMAX)

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

Extreme rainfall events and cooling of sea turtle clutches: Implications in the face of climate warming.

Understanding how climate change impacts species and ecosystems is integral to conservation. When studying impacts of climate change, warming temperatures are a research focus, with much less attention given to extreme weather events and their impacts. Here, we show how localized, extreme rainfall events can have a major impact on a species that is endangered in many parts of its range. We report incubation temperatures from the world's largest green sea turtle rookery, during a breeding season when two extreme rainfall events occurred. Rainfall caused nest temperatures to drop suddenly and the maximum drop in temperature for each rain‐induced cooling averaged 3.6°C (n = 79 nests, min = 1.0°C, max = 7.4°C). Since green sea turtles have temperature‐dependent sex determination, with low incubation temperatures producing males, such major rainfall events may have a masculinization effect on primary sex ratios. Therefore, in some cases, extreme rainfall events may provide a “get‐out‐of‐jail‐free card” to avoid complete feminization of turtle populations as climate warming continues.

Strain-Dependent Cheese Spoilage Potential of Clostridium tyrobutyricum.

Clostridium tyrobutyricum, a Gram-positive, anaerobic, spore-forming bacterium, is considered as one of the main causative agents for spoilage of hard and semihard cheeses. Growth of C. tyrobutyricum in cheese is critically influenced by ripening temperature and time, pH, salt and lactic acid concentration, moisture and fat content, and the presence of other microorganisms. Previous studies revealed high intraspecies diversity of C. tyrobutyricum strains and variable tolerance toward pH, temperatures, and salt concentrations. These findings indicate that strain-dependent characteristics may be relevant to assess the risk for cheese spoilage if clostridial contamination occurs. In this study, we aimed to compare the phenotypes of 12 C. tyrobutyricum strains which were selected from 157 strains on the basis of genotypic and proteotypic variability. The phenotypic analysis comprised the assessment of gas production and organic acid concentrations in an experimental cheese broth incubated at different temperatures (37, 20, and 14 °C). For all tested strains, delayed gas production at lower incubation temperatures and a strong correlation between gas production and the change in organic acid concentrations were observed. However, considering the time until gas production was visible at different incubation temperatures, a high degree of heterogeneity was found among the tested strains. In addition, variation among replicates of the same strain and differences due to different inoculum levels became evident. This study shows, that, among other factors, strain-specific germination and growth characteristics should be considered to evaluate the risk of cheese spoilage by C. tyrobutyricum.

Effect of low temperature on abiotic and biotic nitrate reduction by zero-valent Iron

The effect of low temperatures on abiotic and biotic nitrate (NO3−) reduction by zero-valent iron (ZVI) were examined at temperatures below 25 °C. The extent and rate of nitrate removal in batch ZVI reactors were determined in the presence and absence of microorganisms at 3.5, 10, 17, and 25 °C. Under anoxic conditions, NO3− reduction rates in both ZVI-only and ZVI-cell reactors declined as temperature decreased. In ZVI-only reactor, 62% and 17% of initial nitrate concentration were reduced in 6 days at 25 and 3.5 °C, respectively. The reduced nitrate was completely recovered as ammonium ions (NH4+) at both temperatures. The temperature-dependent abiotic reduction rates enabled us to calculate the activation energy (Ea) using the Arrhenius relationship, which was 50 kJ/mol. Nitrate in ZVI-cell reactors was completely removed within 1–2 days at 25 and 10 °C, and 67% of reduction was achieved at 3.5 °C. Only 18–25% of the reduced nitrate was recovered as NH4+ in the ZVI-cell reactors. Soluble iron concentrations (Fe2+ and Fe3+) in the ZVI reactors were also measured as the indicators of anaerobic corrosion. In the ZVI-cell reactors, soluble iron concentrations were 1.7 times higher than that in ZVI-only reactors at 25 °C, suggesting that the enhanced nitrate reduction in the ZVI-cell reactors may be partly due to increased redox activity (i.e., corrosion) on iron surfaces. Anaerobic corrosion of ZVI was also temperature-dependent as substantially lower concentrations of corrosion product were detected at lower incubation temperatures; however, microbially induced corrosion (MIC) of ZVI was much less impacted at lower temperatures than abiotic ZVI corrosion. This study demonstrated that ZVI-supported microbial denitrification is not only more sustainable at lower temperatures, but it becomes more dominant reaction for nitrate removal in microbial-ZVI systems at low temperatures.

Neither altered incubation temperature during fetal development nor preferred rearing temperature improves leg bone characteristics of broilers.

The present study evaluated whether broiler femoral and tibiotarsal characteristics (as assessed at slaughter age) could be improved if birds were reared under their preferred temperature and whether continuous high or low incubation temperature during the fetal period improves bone characteristics of broilers reared under heat stress or thermal preference. Broiler breeder eggs were incubated from day 13 until hatching under cold (36°C), control (37.5°C), or hot (39°C) temperatures. Under these conditions, the eggshell temperatures were 37.4±0.1°C, 37.8±0.15°C, and 38.8±0.3°C, respectively. Then, broiler chicks were reared under control, preferred (determined previously in thermal preference test), or high temperatures. At day 42 of age, the broilers were weighed and euthanized, and femora and tibiotarsi collected to measure weight, length, diaphysis perimeter, breaking strength, maximum flexion, rigidity, ash, phosphorus, and calcium. Rearing under the preferred temperature did not affect broiler body weight or femoral and tibiotarsal characteristics (P>0.05). In contrast, high rearing temperature, decreased the body weight, mineral contents of both bones, femoral breaking strength, and tibiotarsal rigidity (P<0.05). Regarding incubation temperature effects, egg exposure to cold and hot temperatures during the fetal period minimized or avoided a few effects of high rearing temperature, such as those on femoral and tibiotarsal morphological characteristics, mineral composition, and mechanical properties at slaughter age (P<0.05), but not all. In conclusion, rearing under the preferred broiler temperature did not improve the bone characteristics, and the negative effects of high rearing temperature on bone development were minimized but not completely prevented by high or low temperature incubation during the fetal period.

가정간편식 용기용 바이오매스 기반 발포구조체의 특성에 관한 연구

A series of foamed plastic sheets containing biomass (as HMR container) were developed via different foaming process temperatures, and their density, porosity, WVTR, and pore morphology were evaluated. Thermal stability of samples during re-heating the food in oven, change in morphology, density, porosity, and WVTR were investigated using a simulated thermal shock process according to MIL-STD-883E assay. As such, the pore size of samples was generally increased with increasing temperature of the foaming process. It can be explained that as foaming temperature increased, the viscosity of molten resins and the repulsive force against pore expansion decreased. In addition, an increase in the thermal shock cycle reduced the pore size and WVTR, while density increased because high temperature treatment that softened the sheet matrix was followed by a low temperature incubation, which contracted the matrix, thereby changing the physical and morphological properties of samples. However, an insignificant change in density was observed and WVTR tended to be decreased, indicating that as-prepared foamed plastic sheets could be used as a high thermal stable container for HMR application. Therefore, it found that the properties of newly developed HMR containers containing biomass were dependent on the foaming process temperature. Moreover, to better understanding of these newly developed containers, further investigations dealing with foaming process temperature based on various food items and cooking conditions are needed.

Stress induced modifications in photosystem II electron transport, oxidative status, and expression pattern of acc D and rbc L genes in an oleaginous microalga Desmodesmus sp.

The study aimed at understanding the biochemical and molecular level modifications in Desmodesmus sp. under lipid inducing stress conditions. The low-temperature (5 °C) incubation and nitrogen starvation reduced the PS II electron transport in microalga with a maximum reduction of 50–57% in ET0/ABS values. The PS II electron transport recovered in UV treated cultures after an initial reduction of 87–93% in ET0/ABS values. A 2.7–4.4 fold increase in ROS and MDA levels was observed under low-temperature incubation, and nitrogen starvation. The UV treatment caused 1.3–2.4 fold higher ROS and MDA levels than control. The low-temperature incubated, nitrogen starved, and UV treated cultures showed 2.4–4 fold higher acc D gene expression. A higher rbc L gene expression was observed under low-temperature stress. The study showed modifications in PS II electron transport, oxidative status, and expression of acc D and rbc L genes under stress conditions.

Identification of potential peptide markers for the shelf-life of Pacific oysters (Crassostrea gigas) during anhydrous preservation via mass spectrometry-based peptidomics

The shelf-life of live oysters is a quality-concerned issue along with the supply chain of the products. In present study, mass spectrometry-based peptidomics has been employed to investigate changes of endogenous peptides of Pacific oysters (Crassostrea gigas) occurred during anhydrous preservation with the purpose of discovering shelf-life indicators of live oysters. Samples of Crassostrea gigas harvested freshly were acclimated to laboratory environment first and then put under defined anhydrous preservation conditions (being packaged in sterile plastic bags and stored at 4 ± 1 °C in low-temperature incubators). After regular intervals, samples were randomly collected for peptidomics analysis using ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry and potential peptide markers were mined by chemometric tools and verified by a multi-reaction monitoring method with ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry. Approximately 8526 peptides, in total, were characterized and 4 endogenous peptides, SSSTGEVGTYSGTTN, TARNEANVNI, VGIIKGSSSEEA, and TARNEANVNIY, were finally proposed as potential markers for shelf-life evaluating. The proposed markers were applied to real-life samples and expected trends of the markers during anhydrous preservation were observed, implying applicability of the endogenous peptides for shelf-life evaluating. The findings of this study provide favorable evidences of mass spectrometry-based peptidomics in quality assessing for aquaculture products.

CFTR modulator therapy for cystic fibrosis caused by the rare c.3700A>G mutation

BackgroundThe c.3700A>G mutation, a rare cystic fibrosis (CF)-causing CFTR mutation found mainly in the Middle East, produces full-length transcript encoding a missense mutation (I1234V-CFTR), and a cryptic splice site that deletes 6 amino acids in nucleotide binding domain 2 (I1234del-CFTR). MethodsFRT cell models expressing I1234V-CFTR and I1234del-CFTR were generated. We also studied an I1234del-CFTR-expressing gene-edited human bronchial (16HBE14o-) cell model, and primary cultures of nasal epithelial cells from a c.3700A>G homozygous subject. To identify improved mutation-specific CFTR modulators, high-throughput screening was done using I1234del-CFTR-expressing FRT cells. Motivated by the in vitro findings, Trikafta was tested in two c.3700A>G homozygous CF subjects. ResultsFRT cells expressing full-length I1234V-CFTR had similar function to that of wildtype CFTR. I1234del-CFTR showed reduced activity, with modest activation seen with potentiators VX-770 and GLPG1837, correctors VX-809, VX-661 and VX-445, and low-temperature incubation. Screening identified novel arylsulfonyl-piperazine and spiropiperidine-quinazolinone correctors, which when used in combination with VX-445 increased current ~2-fold compared with the VX-661/VX-445 combination. The combination of VX-770 with arylsulfonamide-pyrrolopyridine, piperidine-pyridoindole or pyrazolo-quinoline potentiators gave 2–4-fold greater current than VX-770 alone. Combination potentiator (co-potentiator) efficacy was also seen in gene-edited I1234del-CFTR-expressing human bronchial epithelial cells. In two CF subjects homozygous for the c.3700A>G mutation, one subject had a 27 mmol/L decrease in sweat chloride and symptomatic improvement on Trikafta, and a second subject showed a small improvement in lung function. ConclusionsThese results support the potential benefit of CFTR modulators, including co-potentiators, for CF caused by the c.3700A>G mutation.

Incubation temperature and PCB-126 exposure interactively impair shorebird embryo and post-hatch development

In oviparous wildlife, many critical physiological and behavioural components are strongly influenced by the embryonic and early post-hatch developmental environment. As such, early life stages in these species are highly vulnerable to both natural and anthropogenic stressors. For example, in birds, incubation temperature may influence the rate of egg development while also affecting contaminant metabolism and absorption in body tissues, resulting in potentially multiplicative impacts on embryonic and posthatch development. We tested the hypothesis that cumulative effects of early contaminant exposure and temperature stress can negatively affect avian development and may have interactive effects that are more detrimental than either stressor individually. Using a controlled egg injection and incubation study on killdeer (Charadrius vociferous), eggs were exposed to a known endocrine disruptor, 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126) and incubated at either low (36 °C), intermediate (37.5 °C), or high (39 °C) temperatures. Our results indicated that eggs incubated at low temperature had earlier detection of heartbeat, longer incubation length, lower growth rate post-hatch, and higher post-hatch mortality, compared to eggs incubated under intermediate temperatures. Higher incubation temperatures resulted in shorter incubation length, earlier detection of heart rate and faster righting time. As predicted, embryo and chick mortality were greater in the PCB-dosed birds incubated at intermediate and high temperatures. Incidence of distended yolk sacs (%) also increased with PCB exposure in all temperature groups, with the largest increase in the high temperature group. Overall, our results show that low incubation temperature can cause greater adverse effects than PCB-126 exposure alone, but that negative effects of PCB-126 exposure are exacerbated by high incubation temperatures. These findings suggest that in natural settings, shorebird embryos may be more susceptible to contaminant exposure when incubated at temperatures either below or above the apparent optimum.