Incubation Temperature Research Articles

The thermal sensitivity of growth and survival in a wild reptile with temperature‐dependent sex determination

The Charnov‐Bull hypothesis is the leading explanation for the evolution of environmental sex determination (ESD), which includes temperature‐dependent sex determination (TSD), the most common form of ESD. Charnov‐Bull predicts a sex‐by‐incubation temperature interaction for fitness, matching offspring sex with thermal conditions that increase parental fitness. However, there is no general explanation for how the sex‐by‐temperature interaction arises. Two competing explanations for the interaction lie in the survival to maturity hypothesis (SM) and the Trivers–Willard extension (TW). Under SM, the sex that matures later is produced under optimal incubation regimes as the late‐maturing sex accrues more mortality by maturation, while TW suggests that males are always produced under optimal incubation regimes as male mating success is more sensitive to condition (general health, vigor) than female fecundity. In a system where females mature later than males, as in the painted turtle Chrysemys picta, SM and TW generate opposite predictions for the effect of incubation temperature on juvenile survival. We incubated C. picta eggs under either female‐promoting temperatures (28 ± 3 °C) or male‐promoting temperatures (25 ± 3 °C), then released the hatchlings into their natal pond. We used a Bayesian capture–mark–recapture approach to follow their survival over two growing seasons. We found a 2% depression of biweekly survival in individuals incubated under the cooler temperature, providing subtle support for SM. Incubation treatments did not influence growth. Large‐scale field experiments such as this one will be necessary for understanding TSD evolution, and we underline general principles to execute such experiments successfully.

First report of leaf blight caused by Nigrospora hainanensis on Euonymus japonicus in China.

Euonymus japonicus Thunb., belonging to the family Celastreace and native to East Asia, is a widely cultivated evergreen ornamental woody plant with important ecological and economic values. In May 2023, serious leaf blight of E. japonicus occurred in the campus green space at Guiyang University, Guizhou Province, China (26°55'85"N, 106°78'04"E). Early symptom appeared as small, circular light brown spots on the edges or tips of the leaves. Then, the spot developed visible necrosis, initially light brown to dark brown halos with clear margins. Subsequently, severely infected leaves appear totally wilt, and significantly decrease their ornamental values. In a 0.07-ha field, the disease incidence reached to 40-55%. To identify the pathogen, ten typical symptomatic E. japonicus leaves were collected. They were initially immersed in 75% ethanol for 3 min, and by sodium hypochlorite (4% NaClO) solution for 45 s, and ultimately rinsed with sterile distilled water (dH2O) five times for not less than 1 min each time, then, placed the leaves on potato dextrose agar (PDA) medium and cultured for 5 days at 25°C in constant temperature incubator. Cultures were purified to yield eight isolates. Early colonies are white and regularly rounded, gradually turning dark brown to black with fluffy mycelium. Conidia were single celled, smooth, black, spherical or ellipsoidal. The conidia size of the representative strain, GY-2 and GY-3, was averagely 12.3-17.3 µm × 10.8-17 µm (n = 50). The conidiogenous cells were monoblastic, hyaline, globose or ampulliform. Morphology-based identification revealed the strain as Nigrospora spp. (Wang et al., 2017). For further confirmation, PCR of GY-2 and GY-3 DNA was performed with the primers ITS1/ITS4 (White et al., 1990), Bt2a-F/Bt2b-R (Glass and Don-aldson 1995), and TEF1-728F/TEF1-986R (Carbone and Kohn 1999). Sequences of the ITS region, TUB and TEF1 genes from the strain GY-2 and GY-3 were deposited in GenBank. (GY-2: OR999377, PP112221 and PP150467; GY-3: PP406871, PP421045 and PP421046, respectively). BLAST analysis showed GY-2 100%, 100%, and 98.36%; GY-3 99.43%, 98.21% and 100% (ITS region, TEF1, and TUB) identity to N. hainanensis sequences (accession numbers. NR_153480.1, KY019415.1, and KY019464.1; KX986094.1, OP611475.1, and KY019597.1). Additionally, tandem sequences of ITS, TUB and TEF1 constructed by MEGA 7.0 confimed the homology through the phylogenetic tree. Pathogenicity tests were conducted on healthy plants grown, each 5 mm diameter of active growing mycelium plug of isolate GY-2 was attached to 15 leaves from five healthy 2-year-old E. japonicu plants. The same number of leaves in the control group were treated with non-inoculated plugs only. All the plants were incubated at 25°C and 75% relative humidity with a 16-h/8-h photoperiod. After 10 days, no symptoms appeared on the leaves of the control group. In contrast, symptomatic blight appeared on all leaves inoculated with GY-2. Pathogenicity tests were performed five times. Pure strains were re-isolated from diseased leaves and, confirmed to be N. hainanensis based on the above methods. Recently, Nigrospora oryzae was reported as causal agent of leaf spots on Euonymus japonicus in China (Xu et al., 2023). To our knowledge, this study is the first report of N. hainanensis causing leaf blight on E. japonicu. Identification of the etiological agent may provide assistance for sustainable management in the future.

Effect of temperature on the incubation, growth, survival, and presence of skeletal deformities in larvae of the clownfish Amphiprion ocellaris (Pomacentridae) under rearing conditions

The results of the effect of five different incubation temperatures on the development of clownfish (Amphiprion ocellaris) larvae are presented. This ornamental species is widely marketed worldwide and is reared in captivity at CIAD to meet the demand in the domestic market. The experimental work was carried out over 30 days post-hatching to observe the temperature's effect on the development of the larvae's vertebral column and caudal complex. The eggs were incubated at 26, 28, 30, 32, and 34°C. The percentage of hatching, survival, growth, and the presence of skeletal malformations were evaluated. Hatching and survival percentages did not show significant differences between treatments. Regarding growth, there were significant differences between treatments, with organisms showing slower growth at 26°C and larger sizes at 34°C. The highest percentage of malformations occurred in larvae cultured at 34°C, with lordosis being the most frequently observed deformity among the treatments. Therefore, it can be concluded that the optimal temperature range for cultivating and harvesting quality A. ocellaris organisms is between 28 and 30°C.

Serial block-face scanning electron microscopy of adherent cells on thin plastic substrate

Abstract Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. Here, we present a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. A standard sample preparation and embedding protocol for thin section electron microscopy provided already sufficient resolution and image quality to visualize larger structures. To improve resolution and image quality of SBF imaging, we stepwise tested modifications of the protocol, such as the moderate increase of the heavy metal content of the sample. Modifications of the embedding by either the reduction of the resin layer (minimal embedding) or the addition of silver colloid to the resin were evaluated at high and low vacuum imaging conditions. The optimized sample preparation protocol is very similar to the standard preparation protocol for thin section electron microscopy, so that the samples can also be used for this application. The protocol applies a higher concentration of osmium tetroxide, a higher temperature for heavy metal incubation and an additional lead en bloc staining. In summary, the presented workflow provides a generic and adaptable solution for studying adherent cells by SBF SEM.

Purification and characterization of glutaminase and urease-free L-asparaginase from Bacillus atrophaeus with acrylamide reduction potential

L-asparaginase (L-asnase) is a versatile enzyme with uses in food industry and medicine. Current study aimed to isolate L-asnase producing microorganism/s without urease and glutaminase and optimize L-asnase production. First, screening and isolation of L-asnase-producing bacterial strains that did not produce glutaminase and urease from chicken gizzards were performed. For this purpose, the enzyme producing bacteria were screened on the agar medium supplied with substrate and phenol red indicator dye. Among the isolated bacteria, 1 isolate showed L-asnase free of glutaminase and urease. The selected strain was identified by biochemical, morphological and 16s rRNA sequencing. The selected strain was identified as Bacillus atrophaeus by 16S rRNA sequencing. The effects of incubation temperature (30°C) and time (72 hours), medium pH (8.0) and nutritional sources (glucose and NaNO3) on L-asnase production were determined. L-asnase was purified with acetone, and its molecular weight was determined to be 42 kDa by SDS-Page. Enzyme kinetics were also calculated, and it was determined that Vmax was 43 μmol/mL/min and Km was 2.7 mM. L-asnase activity was highest at 40 ℃ and the optimal pH was 8.0. L-asnase activity was stimulated by Mn2+, Mg2+, and Ca2+ but inhibited by Co2+, Na+, Zn2+, and Hg2+. L-asnase was utilized to treat potato chips before they were fried in order to assess its capacity to mitigate acrylamide. The result was an 80% reduction in acrylamide concentration when compared to the untreated control. Based on these findings, it appears that L-asnase could have potential use in the food industry.

Effects of inclusion of the blend of essential oils, organic acids, curcumin, tannins, vitamin E, and zinc in the maternal diet, and of incubation temperature on early and late development of quail

The maternal diet and egg incubation temperature are some of the factors that can influence the embryonic development and performance of the newly chicks at 15 d of age. This study evaluated the effects of adding a blend of organic acids, essential oils, curcumin, tannins, vitamin E, and zinc microencapsulated in to the diet of female quails (Coturnix coturnix japonica) on their productive, reproductive performance and redox parameters of their eggs and the interaction of maternal diet × incubation temperature on embryo (E16 and E18) and chicks development. At 98 d of age, 64 female quails with a mean body weight of 150 g ± 0.5 were distributed into two treatments: a Basal diet or a diet supplemented with blend (Sannimix). The eggs from each female were incubated at 37.5°C (Control) and 38.5°C (High Temperature) throughout the incubation period. After hatching, chicks were distributed in a 2 (maternal diet) × 2 (incubation temperature) factorial design. Female quails supplemented with Sannimix showed better productive and reproductive performance and produced higher-quality embryos. Their offspring had greater weight at hatch and at 15 d of age. The eggs and offspring of supplemented with Sannimix female quails showed better oxidative stability. At E16 and E18, High Temperature increased yolk sac utilization and gene expression of the growth hormone receptor (GHR). At E16, embryos from supplemented with Sannimix female quail had higher expression of insulin-like growth factor type I (IGFI) and heat shock protein 70 kDa genes. At 15 d of age, highest expression of the GHR and IGFI genes was observed in chicks from female quails fed the Sannimix diet, regardless of incubation temperature. Regarding the maternal diet × incubation temperature an improved result was observed for chicks from female quails fed with Sannimix even when eggs are exposed to High Temperature during the incubation. The supplementation of quail diets with blend Sannimix improves productive and reproductive performance, egg quality and their embryos, as well as their offspring quality.

Integration of Myrica rubra-based N-doped carbon dots with Fe3S4 as excellent peroxidase mimics for colorimetric assay and smartphone-based intelligent sensing of p-aminophenol in waters.

To realize the reutilization of waste Myrica rubra in the analytical field, we synthesized Myrica rubra-based N-doped carbon dots (MN-CDs) and further anchored them onto the surface of Fe3S4 to fabricate Fe3S4@MN-CD nanocomposites. The as-fabricated nanocomposites possessed higher peroxidase-mimetic activity than its two precursors, resulting from the synergistic effect between them, and could catalyze colorless 3,3',5,5'-tetramethylbenzidine (TMB) into deep blue oxTMB with a strong 652-nm absorption. Under optimized conditions (initial solution pH, 3.5; incubation temperature, 35 ℃; Fe3S4@MN-CD concentration, 50µg mL-1, and 652-nm absorption), Fe3S4@MN-CDs were employed for colorimetric assay of p-aminophenol (p-AP) with wide linear range (LR, 2.9-100 µM), low detection limit (LOD, 0.87 µM), and satisfactory recoveries (86.3-105%) in environmental waters. Encouragingly, this colorimetric assay provided the relative accuracy of 97.0-99.4% as compared withconventional HPLC-UV detection. A portable smartphone-based colorimetric application was developed by combining the Fe3S4@MN-CD-based visually chromogenic reaction with a "Thing Identify" APP software. Besides, we engineered an image-capturing device feasible for field use, in which the internal-compact sealing prevented external light source from entering photography chamber, thereby reducing light interference, and also the bottom light source enhanced the intensity of blue imaging. This colorimetric platform exhibited satisfactory LR (1-500 µM), low LOD (0.3 µM), and fortification recoveries (86.6-99.6%). In the chromogenic reaction catalyzed by Fe3S4@MN-CDs, ·O2- played a key role in concomitant with the participation of •OH and h+. Both the colorimetric assay and smartphone-based intelligent sensing show great promising in on-site monitoring of p-AP under field conditions.

Gene expression dynamics during temperature-dependent sex determination in a sea turtle

Fifty years ago, researchers discovered a link between ambient temperature and the sex of turtle embryos. More recently, significant progress has been made in understanding the influence of temperature on freshwater turtles. However, our understanding of the key genetic factors in other turtle groups, such as sea turtles, remains limited. To address this gap, we conducted RNA-seq analyses on embryonic tissues from the sea olive ridley turtle during the thermosensitive period (stages 21–26) at temperatures known to produce males (26 °C) and females (33 °C). Our findings revealed that incubation temperatures primarily influence genes with broad expression across tissues due to differential cell division rates and later have an effect regulating gonad-specific transcripts. This effect is mostly related to gene activation rather than transcription repression. We performed transcriptome analyses following shifts in incubation temperatures of bi-potential gonads. This approach allowed us to identify genes that respond rapidly and may be closer to the beginning of the temperature-sensing pathway. Notably, we observed swift adaptations in the expression levels of chromatin modifiers JARID2 and KDM6B, as well as the splicing factor SRSF5, and transcription regulators THOC2, DDX3X and CBX3, but little impact in the overall gonad-specific pathways, indicating that temperature-sensing genes may change rapidly but the rewiring of the gonad's developmental fate is complex and resilient. Author summarySea turtles, one of the most iconic creatures of our oceans, confront a troubling reality of endangerment, a peril magnified by the looming specter of climate change. This climatic shift is gradually increasing the temperature of the nesting beaches thus causing dramatic male/female population biases. Conservation efforts will need genetic and molecular information to reverse the negative effects of climate change on the populations. In this study, we conducted the first transcriptomic analysis of embryonic tissues, including gonads, brain, liver, and mesonephros, in the olive ridley sea turtle during the critical thermosensitive period spanning stages 21–26. We examined both male-producing (26 °C) and female-producing (33 °C) temperatures and found that incubation temperatures influence temperature-sensitive genes that are either expressed globally or specifically associated with the gonads. These findings indicate that incubation temperatures predominantly sway genes with broad expression patterns due to differential cell division rates. This natural process was opted in the gonads to drive sex determination. We also identified genes that are rapidly capable of sensing temperature changes and that could play a role in the activation of the sex determination pathway. Overall, our study sheds light on the intricate interplay between temperature and gene expression during sea turtle development, revealing dynamic changes in the transcriptome and highlighting the involvement of key genetic players in sex determination.

Larval feeding activity and use of embryonic resources determine juvenile performance of the common prawn Palaemonserratus

Phenotypic links are the potential for “carryover” of effects of experience during one life history stage into performance and selection at subsequent stages. They reflect plastic responses to the environment experienced during an early phase on the phenotype of subsequent phases. We are studying these effects by following individuals of the shrimp Palaemon serratus from the embryonic (eggs carried by females) through the larval phase (pelagic) to the juvenile phase (benthic). In experiment 1, we investigated the effects of larval prey concentration (10, 4 and 2 Artemia/mL) and larval incubation temperature (16 and 22 °C) on larval performance (metamorphosis rate, developmental duration and growth) and then on juvenile performance (survival and Specific Growth Rate, SGR, at 18 and 24 °C in 14 days). In experiment 2, we investigated the effects of embryonic incubation temperature (larval biomass and lipid content of newly hatched larvae from embryos incubated at 12 and 18 °C) and larval prey concentration on larval performance and then on juvenile performance. In both experiments, the larvae plastically increased their development time in response to the reduction in temperature and prey concentration, whereas their survival decreased with temperature and prey concentration. The quantity of lipids available at hatching decreased with decreasing embryonic incubation temperature, which reduced the larval performance, particularly with a low concentration of prey. Survival at 14 days post-metamorphosis was significantly reduced when the embryos were incubated at 12 °C compared with those incubated at 18 °C, regardless of the subsequent larval incubation conditions, revealing phenotypic links between overconsumption of embryonic yolk reserves and post-metamorphic fitness. Overall, juveniles had a better SGR at 24 than at 18 °C, and even better when incubated under stressful embryo-larval conditions (temperature and prey concentration). This study highlighted phenotypic links between developmental stages and over developmental periods of several months.

Asparaginase treatment to mitigate acrylamide formation in wheat and rye cookies

The Maillard reaction between asparagine and a carbonyl source can lead to acrylamide formation, which is classified as "probably carcinogenic to humans". This study aimed to investigate the impact of varying amounts, incubation times, and temperatures of asparaginases on reducing acrylamide in wheat and rye cookies while preserving sensory attributes. To determine if different asparaginases reduce acrylamide without negative effects on cookie quality, acrylamide was quantified using ELISA, and color, texture and sensory attributes were assessed. Adding asparaginases resulted in a reduction of acrylamide by up to 85%. Incubating cookie dough for 10 or 30 min at 60 °C or 90 °C did not impact acrylamide formation. Acrylamide concentrations were higher by an average of 78 μg/kg for an incubation temperature of 90 °C compared to 60 °C. Rye cookies exhibited higher acrylamide concentrations by 490 μg/kg compared to wheat due to higher free asparagine in rye flour. Asparaginases showed minimal effects on cookie color and texture, with no changes in sensory evaluation. Acrylamide can be reduced using asparaginases, providing a simple and effective means of ensuring safe food for consumers.

First report of Fusarium commune Causing Bulb Rot on Lily (Lilium brownii var. Viridulum Baker) in Hunan Province of China.

Lily (Lilium brownii var. Viridulum Baker) is a well-known edible plant with large, white and sweet bulb scales that has important medicinal value (Zhou et al. 2021) and is grown mainly in the Hebei, Shanxi and Henan provinces of China. In May 2021, a case of bulb rot was discovered in a 3.33 hm2 plantation in Huaihua, Hunan Province, affecting 20% of the area (27°59'30″N, 110°32'20″E). The disease is most severe during the rainy season in May and June. In the early stage, irregular brown spots appeared on the lily scales, the necrosis was depressed and gradually enlarges, and in the later stage, the scales were scattered from the base of the disc and slough off. Ten samples were taken randomly from different plants in the plantation area to isolate the pathogens. After washing with sterile water, they were cut into small pieces and sterilised with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s, rinsed three times with sterile water and dried on sterile filter paper, then placed on a water agar plate and incubated in the dark in a constant temperature incubator at 28℃ for 3 to 5 days. After 2 days, the mycelium at the edge of the colony was transferred to a PDA plate and incubated for 3-5 days at 28°C in the dark to obtain pure fungal isolates. Eighteen purified fungal isolates were obtained, of which sixteen looked like Fusarium (88.9% isolation rate) and three representative isolates (BHBR2, BHBR3 and BHBR5) were selected for further study. The surface of this fungus was white with dense aerial mycelium. Some had an orange centre in the medium. Microconidia were oval in shape and appeared either straight or slightly curved. These microconidia were colourless, had 0-1 septa and measured 3.334 to 14.724 × 2.216 to 5.385 μm (n=100). Macroconidia were predominantly three-septate, crescent-shaped structures that were thin-walled and slightly curved. Cells at the apex and base were similarly curved. Macroconidia measured 17.956 to 32.150 × 2.788 to 4.492 μm (n=100). The mitochondrial small subunit (mtSSU) and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the NMS1/NMS2 and TEF-R/TEF-F primers to verify the identity of the pathogens (Stewart et al. 2006). The sequences were submitted to GenBank (BHBR2: mtSSU, PP273435; TEF, OR900976; BHBR3: mtSSU, PP277729; TEF, OR900977; BHBR5: mtSSU, PP277728; TEF, OR900978). A concatenated phylogenetic tree of the two genes was constructed and analysis showed that BHBR2, BHBR3 and BHBR5 were significantly clustered with Fusarium commune. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as Fusarium commune. We carried out pathogenicity tests using two methods, one in which 6 × 6 mm fungal blocks were inoculated on lily (L. brownie var. viridulum Baker) scales and controls inoculated with sterile blocks, and the other in which strain BHBR2 was selected to carry out pathogenicity tests on bulbs of live plants soaked with 50 ml of a 1 × 106 conidial suspension and bulbs of control plants soaked with sterile water, all in three replicates. They were placed in a growth chamber at 28°C and 80% relative humidity, and the scales were moistened with moistened sterile filter paper. After 3 days of rearing treated scales, lesions appeared on lily scales inoculated with mycelial blocks and expanded with time, whereas no lesions appeared on lily scales inoculated with sterile blocks. One month later, whole plants soaked in the spore suspension wilted, while the control plants grew well. The pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. This confirms Koch's hypothesis. Fusarium commune has been shown to be the most important pathogenic fungus causing root rot in Alfalfa (Medicago sativa) (Yang et al. 2022) and blueberry (Vaccinium uliginosum L.) (Li et al. 2023) in China. To our knowledge, this is the first report of Fusarium commune causing lily bulb rot in the world, which will lay the foundation for future control of lily bulb rot.

Bio-inspired synthesis of silver nanoparticles using Salsola imbricata and its application as antibacterial additive in glass ionomer cement

Nanotechnology has gained immense popularity and observed rapid development due to the remarkable physio-chemical properties of nanoparticles (NPs) and related nanomaterials. The green production of NPs has many benefits over traditional techniques because the current procedures are expensive, time-consuming, and involve harmful substances that limit their applicability. This study aimed to use a novel green source, the Salsola imbricata (SI) plant, which is commonly found in Central Asia and known for its medicinal properties as a reducing and stabilizing agent for the synthesis of AgNPs. The current study also utilized efficient statistical design, the Plackett–Burman Design (PBD) of Experiment method to synthesize the NPs. The characterization of NPs was carried out using UV-Vis spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM). The PBD results showed that only two out of four factors i.e. AgNO3 concentration and incubation time, were significant for the synthesis of SI-AgNPs. While remaining factors, incubation temperature and plant extract: AgNO3 ratio were non-significant. The SEM analysis result showed that SI-AgNPs had a size of 20–50 nm. The SI-AgNPs demonstrated strong antibacterial activity against oral pathogens such as S. mutans and Lactobacillus acidophilus, with the highest efficacy observed at a concentration of 2 mg ml−1. The addition of SI-AgNPs in glass ionomer cement significantly increased the antibacterial activity of GIC against S. mutans. Based on the results of the current study, the plant based AgNPs can be further evaluated in detail as alternate antimicrobial agent either alone or in combination with other antimicrobial agents for different dental applications.

Powdered water kefir: Effect of spray drying and lyophilization on physical, physicochemical, and microbiological properties

This study addresses the challenge of optimizing powdered water kefir's fermentation and preservation processes to enhance its physical-chemical, microbiological, and technological characteristics. The main objective was to determine the best fermentation conditions and evaluate the efficacy of different drying methods. The optimal fermentation conditions were 5 % kefir grains, 10 % brown sugar, and an incubation temperature of 25 °C. Remarkably, the microbiological analysis revealed high abundances of Zymomonas mobilis (grains: 94.31 % and beverage: 91.68 %), Sporolactobacillus spathodeae (grains: 3.00 % and beverage: 5.42 %), and Liquorilactobacillus satsumensis (grains: 1.47 % and beverage: 0.62 %) among bacteria, and Lachancea fermentati (grains: 95.54 % and beverage: 67.53 %), Wickerhamomyces anomalus (grains: 3.00 % and beverage: 26.77 %) among fungi. The study innovatively demonstrates that lyophilization preserves the viability of these microorganisms, making it a promising method for producing stable, probiotic-rich powdered kefir. Although spray drying resulted in a logarithmic reduction of 3 logs CFU/g, it maintained sufficient microorganism counts, proving its viability as an alternative drying method. These methods retain the ideal physical-chemical properties and expand the accessibility and practical applications of water kefir. This research underscores the potential for powdered water kefir to deliver health benefits conveniently and versatilely, paving the way for broader industrial and academic applications.

A comparison of fixation and immunofluorescence protocols for successful reproducibility and improved signal in human left ventricle cardiac tissue.

Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh-frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised.

Improved photoelectrochemical performance of TiO2-in-MIL-101(Cr)@CDs@AgNPs and application for the detection of ultralow level AβO

TiO2 was successfully synthesized within the internal pores of MIL-101(Cr), leading to the formation of a TiO2-in-MIL-101(Cr) composite. This heterojunction structure significantly improved the photogenerated electron-hole separation within the metal-organic framework (MOF) composite. Subsequently, carbon dots (CDs) and silver nanoparticles (AgNPs) were introduced to further modify the TiO2-in-MIL-101(Cr), resulting in a multimodified MIL-101(Cr) composite. The integration of CDs enhanced the light absorption capabilities of the composite, thereby boosting the photocurrent. Meanwhile, AgNPs not only enhanced the absorption of visible light through the local surface plasmon resonance effect (LSPR) but also facilitated efficient electron transport. This multimodal modification strategy led to a remarkable enhancement in the photoelectrochemical (PEC) performance of MIL-101(Cr), with the photocurrent density (J) increasing by approximately a factor of 19. To further expand the functionality of this composite, AβO capture DNA (cDNA) and CuS-binding aptamer were grafted onto the TiO2-in-MIL-101(Cr)@CDs@AgNPs, creating a sandwich-like structure. Based on this multimodified MIL-101(Cr) composite, an "on-off-on" type PEC biosensor was constructed. The p-type semiconductor CuS served as a signal amplifier, capturing photogenerated electrons and effectively reducing the photocurrent. When cDNA hybridized with AβO, the Apt-CuS moiety detached from the MIL-101(Cr) composite, leading to the restoration of the photocurrent. The PEC biosensor for the detection of AβO exhibited optimal performance at a pH of 7.00, an incubation temperature of 37 °C, and an incubation time of 45 min. Under these conditions, the biosensor demonstrated a wide detection range of 5 fM to 1 μM, with an ultralow detection limit of 4.36 fM. This method exhibits high sensitivity, robust anti-interference capabilities, and holds great promise for applications in tracing the detection of AβO in human serum.

Embryonic and larval development of yellow perch (Perca flavescens) and its sensitivity to incubation temperature.

The ontogenetic development in teleost fish is sensitive to temperature, and the developmental rate has a direct relationship with the environmental temperature within a species' thermal tolerance limit. Temperature determines time to and survival at hatching. Yellow perch is a North American species of ecological and commercial importance, and its phenology is vulnerable to climate change. The embryonic development of yellow perch was comparable to closely related members of the family Percidae. Developmental progression was fastest at 18°C and slowest at 12°C, with medial progression at 15°C. Time to hatch and swim-up, feeding onset, and exogenous feeding phases were different across all incubation temperatures regardless of a gradual post-hatch warming of the 12 and 15°C groups to a common garden temperature of 18°C. Incubation temperature may lower the rate of survival to hatch at 15°C and had complex impacts on developmental abnormalities. Temperature had significant effects on the development rate, time of hatch, survival, and incidence of developmental abnormalities. Early ontogenetic thermal history in ectotherms is an important factor determining phenotypic variation. It will be important to link the thermally induced changes in development described here to the physiological and morphological differences and to link the developmental abnormalities to functional performance.

Optimization Production of an Endo-β-1,4-Xylanase from Streptomyces thermocarboxydus Using Wheat Bran as Sole Carbon Source

Xylanases, key enzymes for hydrolyzing xylan, have diverse industrial applications. The bioprocessing of agricultural byproducts to produce xylanase through fermentation approaches is gaining importance due to its significant potential to reduce enzyme production costs. In this work, the productivity of Streptomyces thermocarboxydus TKU045 xylanase was enhanced through liquid fermentation employing wheat bran as the sole carbon source. The maximum xylanase activity (25.314 ± 1.635 U/mL) was obtained using the following optima factors: 2% (w/v) wheat bran, 1.4% (w/v) KNO3, an initial pH of 9.8, an incubation temperature of 37.3 °C, and an incubation time of 2.2 days. Xylanase (Xyn_TKU045) of 43 kDa molecular weight was isolated from the culture supernatant and was biochemically characterized. Analysis through liquid chromatography with tandem mass spectrometry revealed a maximum amino acid identity of 19% with an endo-1,4-β-xylanase produced by Streptomyces lividans. Xyn_TKU045 exhibited optimal activity at pH 6, with remarkable stability within the pH range of 6.0 to 8.0. The enzyme demonstrated maximum efficiency at 60 °C and considerable stability at ≤70 °C. Mg2+, Mn2+, Ba2+, Ca2+, 2-mercaptoethanol, Tween 20, Tween 40, and Triton X-100 positively influenced Xyn_TKU045, while Zn2+, Fe2+, Fe3+, Cu2+, and sodium dodecyl sulfate exhibited adverse impact. The kinetic properties of Xyn_TKU045 were a Km of 0.628 mg/mL, a kcat of 75.075 s−1 and a kcat/Km of 119.617 mL mg−1s−1. Finally, Xyn_TKU045 could effectively catalyze birchwood xylan into xylotriose and xylobiose as the major products.

Biosynthesis and characterization of silver nanoparticles from Cedrela toona leaf extracts: An exploration into their antibacterial, anticancer, and antioxidant potential

Abstract This research work aims to synthesize environmentally benign and cost-effective metal nanoparticles. In this current research scenario, the leaf extract of Cedrela toona was used as a reducing agent to biosynthesize silver nanoparticles (AgNPs). The synthesis of AgNPs was confirmed by the color shift of the reaction mixture, i.e., silver nitrate and plant extract, from yellow to dark brown colloidal suspension and was established by UV-visible analysis showing a surface plasmon resonance band at 434 nm. Different experimental factors were optimized for the formation and stability of AgNPs, and the optimum conditions were found to be 1 mM AgNO3 concentration, a 1:9 ratio of extract/precursor, and an incubation temperature of 70°C for 4 h. The Fourier transform infrared spectroscopy spectra indicated the presence of phytochemicals in the leaf extract that played the role of bioreducing agents in forming AgNPs. X-ray diffraction patterns confirmed the presence of AgNPs with a mean size of 25.9 nm. The size distribution and morphology of AgNPs were investigated by scanning electron microscopy, which clearly highlighted spherical nanoparticles with a size distribution of 22–30 nm with a mean average size of 25.5 nm. Moreover, prominent antibacterial activity was found against Enterococcus faecalis (21 ± 0.5 mm), Bacillus subtilis (20 ± 0.9 mm), Pseudomonas aeruginosa (18 ± 0.3 mm), Staphylococcus aureus (16 ± 0.7 mm), Klebsiella pneumoniae (16 ± 0.3 mm), and Escherichia coli (14 ± 0.7 mm). In addition, antioxidant activity was determined by DPPH and ABTS assays. Higher antioxidant activity was reported in AgNPs compared to the plant extract in both DPPH (IC50 = 69.62 µg·ml−1) and ABTS assays (IC50 = 47.90 µg·ml−1). Furthermore, cytotoxic activity was also investigated by the MTT assay against MCF-7 cells, and IC50 was found to be 32.55 ± 0.05 µg·ml−1. The crux of this research is that AgNPs synthesized from the Cedrela toona leaf extract could be employed as antibacterial, antioxidant, and anticancer agents for the treatment of bacterial, free radical-oriented, and cancerous diseases.

Germinated VD20 rice – a local rice variety in Vietnam: Effect of process conditions

Rice is one of important staple foods, which mainly consumed by human in daily life. One of the methods which could enhance the nutritional profile as well as functional of rice is germination process. VD20 rice, a local rice from Tien Giang province (Vietnam), was used in this study. This investigation was aimed to study the effect of ratio of rice to soaking solution (1:1-1:3), pH of soaking solution (distilled water, pH = 3-5), and time of soaking (3-6 hours) on the germination rate, moisture content of grain and total polyphenol content of germinated rice. Besides that, the impact of germination conditions, including incubation time (16-22 hours) and temperature (31-37oC), on the quality of germinated rice was also elucidated. The obtained results showed the ratio of soaking water to rice is 2.5:1 for the highest polyphenol content and germination rate. The longer the soaking time, the polyphenol content gradually decreases but the germination rate gradually increases over time. Soaking solutions with a pH of 4 for 6 hours were the most apporiate conditions for soaking VD20 rice. During the germination process, temperature and incubation time significant affected the polyphenol content and germination rate. At a temperature of 37℃ for 20 hours, the polyphenol content was highest (33.85 mgGAE/100 g) and gradually decreases with incubation time, but the germination rate gradually increases. Germination process of VD20 enhance the nutrional quality of rice, which could be seen in the increasing of phenolic compounds content. However, it also affected by various factors, as know as, soaking conditions and incubation conditions, which could further study to ultilize and optimize. The obtained product also have the promising application as the nutraceutical food.

Investigating the impact of temperature on growth rate of the root rot fungus, Gymnopus fusipes.

Gymnopus fusipes is an understudied root rot pathogen associated with multiple tree species and is linked to episodes of oak decline across the United Kingdom and Europe. Although the reported distribution of G. fusipes is broad, many observations rely solely on visual identification of fruiting bodies, which can be unreliable, and lack confirmation by molecular and/or isolation data to verify this broad ecological range. Given the paucity of information regarding the true ecological distribution of G. fusipes, it is difficult to predict and model the potential distribution of the species under both current and future climate scenarios. In this study, to determine the growth capabilities of G. fusipes across a range of ecologically relevant temperatures, five geographically diverse isolates of G. fusipes were grown at five different temperatures ranging from 4-37°C, to determine the optimal temperature for G. fusipes growth, and to establish whether geographically diverse isolates exhibit local adaptation to temperature tolerance. Incubation temperature had a significant effect on G. fusipes growth rate, with 25°C representing the optimum (P<0.001). Isolates had differing growth rates at each of the temperatures, with an isolate from the UK having the highest overall growth rate across all five temperatures tested (P<0.001), and at the optimum, increased by a mean value of over 4915 mm2. Local adaptation to temperature tolerance was not found in the isolates tested. These data demonstrate the optimal incubation temperature for future laboratory studies on G. fusipes and provide the first data on the growth rate of this pathogen across ecologically relevant climate ranges that may inform land managers, modellers, and policy makers in predicting the current and potentially future geographical limits of this widespread root rot pathogen.